Posts Tagged ‘cardiac arrhythmias’

Human Genetics and Childhood Diseases

Larry H. Bernstein, Md, FCAP, Curator




Publication Roundup: HGMD

HGMD®, the Human Gene Mutation Database is used by scientists around the world to find information on reported genetic mutations. The papers below use the database to advance our understanding of disease, DNA dynamics, and more.

Local DNA dynamics shape mutational patterns of mononucleotide repeats in human genomes
First author: Albino Bacolla

Scientists in the US and UK published results in Nucleic Acids Research of a detailed analysis of single-base substitutions and indels in the human genome. Their findings show that certain base positions are more susceptible to mutagenesis than others. They used HGMD Professional to find mutations in specific genomic regions for analysis; the paper includes charts showing mutation patterns, germline SNPs, and more from HGMD data.

High prevalence of CDH23 mutations in patients with congenital high-frequency sporadic or recessively inherited hearing loss
First author: Kunio Mizutari

This Orphanet Journal of Rare Diseases paper from scientists in Japan sequenced 72 patients with unexplained hearing loss, finding several CDH23 mutations, some of which were novel. Mutations in the gene have been linked to Usher syndrome and other forms of hereditary hearing loss. The scientists used HGMD to find all known CDH23 mutations within nearly 70 coding regions.

Mutation analyses and prenatal diagnosis in families of X-linked severe combined immunodeficiency caused by IL2Rγ gene novel mutation
First author: Q.L. Bai

In Genetics and Molecular Research, scientists report the utility of mutation analysis of the interleukin-2 receptor gamma gene to assess carrier status and perform prenatal diagnosis for X-linked severe combined immunodeficiency. They studied two high-risk families, along with 100 controls, to evaluate the approach. Sequence variation was determined using HGMD Professional and an X-SCID database, and a new mutation was discovered in the project.

Impact of glucocerebrosidase mutations on motor and nonmotor complications in Parkinson’s disease
First author: Tomoko Oeda

Researchers from three hospitals in Japan published this Neurobiology of Aging report that may help stratify Parkinson’s disease patients by prognosis. They sequenced mutations in the GBA gene in 215 patients, finding that those who had mutations associated with Gaucher disease suffered dementia and psychosis much earlier than those who didn’t. The team found previously reported GBA mutations using HGMD Professional.

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort
First author: Elisabet Selga

In this PLoS One publication, scientists from a number of institutions in Spain examined genetic variation among patients with Brugada syndrome, a rare genetic cardiac arrhythmia. They sequenced 14 genes in 55 patients, identifying 61 variants and finding the subset that appear pathogenic. Variants were filtered against a number of databases, including HGMD.



Local DNA dynamics shape mutational patterns of mononucleotide repeats in human genomes

Albino Bacolla1Xiao Zhu2Hanning Chen3Katy Howells4David N. Cooper4 and Karen M. Vasquez1

Nucl. Acids Res. (26 May 2015) 43(10): 5065-5080.

Single base substitutions (SBSs) and insertions/deletions are critical for generating population diversity and can lead both to inherited disease and cancer. Whereas on a genome-wide scale SBSs are influenced by cellular factors, on a fine scale SBSs are influenced by the local DNA sequence-context, although the role of flanking sequence is often unclear. Herein, we used bioinformatics, molecular dynamics and hybrid quantum mechanics/molecular mechanics to analyze sequence context-dependent mutagenesis at mononucleotide repeats (A-tracts and G-tracts) in human population variation and in cancer genomes. SBSs and insertions/deletions occur predominantly at the first and last base-pairs of A-tracts, whereas they are concentrated at the second and third base-pairs in G-tracts. These positions correspond to the most flexible sites along A-tracts, and to sites where a ‘hole’, generated by the loss of an electron through oxidation, is most likely to be localized in G-tracts. For A-tracts, most SBSs occur in the direction of the base-pair flanking the tracts. We conclude that intrinsic features of local DNA structure, i.e. base-pair flexibility and charge transfer, render specific nucleotides along mononucleotide runs susceptible to base modification, which then yields mutations. Thus, local DNA dynamics contributes to phenotypic variation and disease in the human population.


Changes in human genomic DNA in the form of base substitutions and insertions/deletions (indels) are essential to ensure population diversity, adaptation to the environment, defense from pathogens and self-recognition; they are also a critical source of human inherited disease and cancer. On a genome-wide scale, base substitutions result from the combined action of several factors, including replication fidelity, lagging versus leading strand DNA synthesis, repair, recombination, replication timing, transcription, nucleosome occupancy, etc., both in the germline and in cancer (14). On a much finer scale [(over a few base pairs (bp)], rates of base substitutions may be strongly influenced by interrelationships between base–protein and base–base interactions. For example, the mutator role of activation-induced deaminase (AID) in B-cells during class-switch recombination and somatic hypermutation (5) targets preferentially cytosines within WRC (W: A|T; R: A|G) sequences (6), whereas apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) overexpression displays a preference for base substitutions at cytosines in TCW contexts (7). Other examples, such as the induction of C→T transitions at CG:CG dinucleotides by cytosine-5-methylation and the role of UV light in promoting base substitutions at pyrimidine dimers have been well documented (reviewed in (4,8)). More recently, complex patterns of base substitution at guanosines in cancer genomes have been found to correlate with changes in guanosine ionization potentials as a result of electronic interactions with flanking bases (9), suggesting a role for electron transfer and oxidation reactions in sequence-dependent mutagenesis. However, despite these advances, the increasing number of sequence-dependent patterns of mutation noted in genome-wide sequencing studies has met with a lack of understanding of most of the underlying mechanisms (10). Thus, a picture is emerging in which mutations are often heavily dependent on sequence-context, but for which our comprehension is limited.

Mononucleotide repeats comprise blocks of identical base pairs (A|T or C|G; hereafter referred to as A-tracts and G-tracts) and display distinct features: they are abundant in vertebrate genomes; mutations within the tracts occur more frequently than the genome-wide average; mutations generally increase with increasing tract length; length instability is a hallmark of mismatch repair-deficiency in cancers; and sequence polymorphism within the general population has been linked to phenotypic diversity (1115). Thus, mononucleotide repeats appear ideal for addressing the question of sequence-dependent mutagenesis since base pairs within the tracts are flanked by identical neighbors. Both historic and recent investigations concur with the conclusion that a major source of mononucleotide repeat polymorphism is the occurrence of slippage (i.e. repeat misalignment) during semiconservative DNA replication, which gives rise to the addition or deletion of repeat units (11,12). An additional and equally important source of mutation has recently been suggested to arise from errors in DNA replication by translesion synthesis DNA polymerases, such as pol η and pol κ (13), also on slipped intermediates, leading to single base substitutions.

A key question that remains unanswered in these studies and which is relevant to the issue of sequence context-dependent mutagenesis is whether all base pairs within mononucleotide repeats display identical susceptibility to single base changes and whether indels (which are consequent to DNA breakage) occur randomly within the tracts.

Herein, we combine bioinformatics analyses on mononucleotide repeat variants from the 1000 Genomes Project and cancer genomes with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations to address the question of sequence-dependent mutagenesis within these tracts. We show that mutations along both A-tracts and G-tracts are highly non-uniform. Specifically, both base substitutions and indels occur preferentially at the first and last bp of A-tracts, whereas they are concentrated between the second and third G:C base pairs in G-tracts. These positions coincide with the most flexible base pairs for A-tracts and with the preferential localization of a ‘hole’ that results when one electron is lost due to an oxidation reaction anywhere along G-tracts. Thus, despite the uniformity of sequence composition, mutations occur in a sequence-dependent context at homopolymeric runs according to a hierarchy that is imposed by both local DNA structural features and long-range base–base interactions. We also show that the repair processes leading to base substitution must differ between A- and G-tracts, since in the former, but not in the latter, base substitutions occur predominantly in the direction of the base immediately flanking the tracts. Additional sequence-dependent patterns of mutation are likely to arise from studies of more heterogeneous sequence combinations, possibly involving other aspects intrinsic to the structure of DNA.



Mononucleotide repeat variation is defined by tract length and flanking base composition

We define mononucleotide repeats in the GRCh37/hg19 (hg19) human genome assembly as uninterrupted runs of A:T and G:C base pairs (hereafter referred to as A-tracts and G-tracts, respectively) from 4 to 13 base pairs in length (Figure 1A). We retrieved a total of 48,767,945 A-tracts and 13,633,781 G-tracts, both of which displayed a biphasic distribution with an inflection point between tract lengths of 8 and 9 (bp) and with the number of runs declining with length more dramatically for G-tracts than for A-tracts (Figure 1B), as noted previously (29). Both the number of short tracts and the extent of decline varied with flanking base composition, TA[n]T runs being two- to three-fold more abundant than CA[n]Cs (Supplementary Figure S1A) and AG[n]As declining the most rapidly (Supplementary Figure S1B). Thus, mononucleotide runs exist as a collection of separate pools of sequences in extant human genomes, each maintained at distinctive rates of sequence stability, as determined by factors such as bp composition (A:T versus G:C), tract length and flanking sequence composition.

Figure 1.

Mononucleotide repeat variation, evolutionary conservation and association with transcription. (A) The search algorithm was designed to retrieve runs of As or Ts (A-tracts) and Gs or Cs (G-tracts) length n (n = 4 to 13), along with their 5′ (n = 0) and 3′ (n = n + 1) nearest neighbors from hg19. Tract bases were numbered 5′ to 3′ with respect to the purine-rich sequence. The panel exemplifies the nomenclature for A- and G-tracts of length 4. (B) Logarithmic plot of the number of A-tracts (closed circles) and G-tracts (open circles) in hg19 as a function of length. (C) Normalized fractions of polymorphic tracts (F SNV) (number of SNVs divided by both hg19 number of tracts and n) from the 1KGP for A-tracts (closed circles) and G-tracts (open circles). (D) Radial plot of SNVs in the 1KGP at the 5′ and 3′ nearest neighbors of A-tracts. Periphery, tract length; horizontal axis, scale for the fraction of SNVs (F SNV). (E) Radial plot of SNVs in the 1KGP at the 5′ and 3′ nearest neighbors of G-tracts. (F) Percent difference in the numbers of A-tracts (closed circles) and G-tracts (open circles) between syntenic regions of hg19 and HN genomes. (G) The exponents of Benjamini-corrected P-values for A-tract-containing genes enriched in transcription-factor binding sites plotted as a function of A-tract length (triangles); each value represents the median of the top 11 USCS_TFBS terms. The percent A-tracts (closed circles) and G-tracts (open circles) intersecting genomic regions pulled-down by chromatin immunoprecipitation using antibodies against transcription factors are plotted as a function of tract length. (H) List of gene enrichment terms with a Benjamini-corrected P-value of <0.05 in common between genes containing A- and G-tracts of lengths 4–13, excluding the UCSC_TFBS terms.

 We examined the extent of sequence variation in the human population by mapping 38,878,546 single nucleotide variants (SNVs) from 1092 haplotype-resolved genomes (the 1000 Genomes Project, 1KGP) (30) to the hg19 A- and G-tracts. The normalized fractions of polymorphic tracts (F SNV) were greater for G-tracts than A-tracts and both displayed Gaussian-type distributions, with maxima of 0.067 for G-tracts of length 8 and 0.017 for A-tracts of length 9 (Figure 1C). CA[n]C and AG[n]A runs displayed the highest F SNV values for A- and G-tracts, respectively (Supplementary Figure S1C and D), with F SNV values for AG[n]As attaining ∼0.10 at length 8. We conclude that flanking base composition influences the rates of SNV within mononucleotide runs and, as a consequence, their representation in the reference human genome.

F SNV values at the flanking 5′ and 3′ bp were similar between A- and G-tracts, except for minor differences for the least represented (i.e. longest) tracts and did not exceed 0.02 (Supplementary Figure S1E). These fractions are expected to be greater than at more distant positions from the tracts, based on previous data (29). SNVs at G-tracts, but not at A-tracts, were more frequent than at flanking base pairs. F SNVs for base pairs flanking short (≤8 bp) tracts were at least twice as high as those flanking long tracts; F SNVs also displayed distinct sequence preference with most (∼0.1) variants occurring at Ts 3′ of G-tracts (Figure 1D and E). In summary, SNVs at mononucleotide runs do not increase monotonically with length but peak at 8–9 bp. This behavior mirrors the genomic distributions, both with respect to the total number of tracts (Figure 1B) and the subsets flanked by specific-sequence combinations (Supplementary Figure S1A–D). Variation at flanking base pairs also displayed a biphasic pattern centered at a length of 8–9 bp, with a greater chance of variation adjacent to G- than A-tracts and with characteristic sequence preferences.

Long tracts are evolutionarily conserved and associated with high transcription

To assess whether more variable monosatellite runs (Figure 1C) might have undergone a greater reduction in number in extant humans relative to extinct hominids, we compared the number of A- and G-tracts between syntenic regions of five individuals comprising hg19 and three Neanderthal (HN) specimens (31). The difference between hg19 and HN was very small (<±2%) for the short tracts, but it displayed more negative values in hg19 with increasing tract length, which reached a maximum of −11.8 and −32.7% for A- and G-tracts, respectively, of length 9. Beyond this threshold, the numbers of tracts converged for A-tracts, whereas they were more abundant in hg19 for G-tracts >11 bp (Figure 1F). In summary, the largest difference in the number of mononucleotide runs between hg19 and HN sequences was centered at 9 bp for both A- and G-tracts, suggesting that the length distributions (Figure 1A and Supplementary Figure S1A and B) reflect distinct rates of evolutionary gains and losses due to differential sequence mutability (Figure 1C) as a function of length and flanking sequence composition (12).

The fact that long (>9 bp) mononucleotide runs display low variability in the human population (Figure 1C) and sequence conservation during evolutionary divergence (Figure 1F) raises the possibility that they might serve functional roles. Through gene enrichment analyses, we found that genes containing A- and G-tracts were enriched for genes associated with the term ‘UCSC_TFBS’, which pertains to transcripts harboring frequent transcription factor binding sites (32,33). For A-tract-containing genes, the median P-values for the top 11 UCSC_TFBS terms decreased from 2.95E-26 for tracts of length 4 to 5.22E-241 for tracts of length 13 (Figure 1G). The percent of A-tracts intersecting genomic fragments amplified from chromatin immunoprecipitation using transcription-factor binding antibodies (32,33) also increased from 8.7 to 9.9 from length 6 to 13, whereas it was constant (mean ± SD, 22.4 ± 1.1) for G-tracts (Figure1G). For gene classes excluding ‘UCSC_TFBS’, a search for categories enriched at P < 0.05 and common to all A- and G-tract-containing genes returned a set of 25 terms, 22 of which were associated with high levels of tissue-specific gene expression (Figure 1H). In summary, these analyses extend prior work (14) supporting a role for mononucleotide tracts in enhancing gene expression, a function that for A-tracts appears to increase with increasing tract length.

Repeat variability is highly skewed

Next we addressed whether bp along A- and G-tracts display equal probability and type of variation. In the 1KGP dataset, the number of SNVs at each position along both A- and G-tracts of length 4 was within a two-fold difference (144,000–240,000); for both types of sequence, transitions (i.e. A→G and G→A) were the predominant (51–78%) type of base substitution (Supplementary Figure S2A and B). However, with increasing length, the number of SNVs decreased up to 30-fold more drastically for G-tracts than for A-tracts, with increasing numbers of transversions (A→T and G→C|T) being predominant. Normalizing the data for the number of tracts genome-wide revealed that the extent of SNV varied by up to 10-fold, depending upon tract length and bp position. Specifically, the highest degree of variation was observed at the first and last A within the A-tracts (i.e. A1 and An), which underwent up to 61% A→T and 43% A→C transversions, respectively, at length 9 (Figure 2A). Likewise, for G-tracts, the most polymorphic sites were G3, followed by G2, for mid-size tracts of 8–10 bp, with 44% G→C transversions at G3 for tracts of length 8 (Figure2B). Thus, the extent of SNV at mononucleotide runs is grossly skewed in human genomes, both along the sequence itself and across tract length, which must account for the bell-shape behavior in F SNV for the tracts as a whole (Figure 1C).

Figure 2.

Population variation spectra. (A) Variation spectra of A-tracts. Percent (number of SNVs at each position divided by the number of tracts in hg19 × 100) of A→T (black), A→C (red) and A→G (green) SNVs in the 1KGP dataset (left). Percent SNVs at A1 as a function of tract length (right). (B) Variation spectra of G-tracts. As in panel A with G→T (black), G→C (red) and G→A (cyan) (left). Percent SNVs at G3 as a function of tract length (right). (C) Percent A→T, A→C and A→G transitions at each position along A-tracts (stars) preceded and followed by a T (TA[n]T, left), C (CA[n]C), center) and G (GA[n]G, right) as a function of tract length. (D) Percent G→T, G→C and G→A transitions at each position along G-tracts (stars) preceded and followed by a T (TG[n]T, left), C (CG[n]C), center) and A (AG[n]A, right) as a function of tract length. (E) Percent transitions at base pairs (stars) preceding or following A-tracts (left) and G-tracts (right) as a function of tract length (n). *, mutated position.

We assessed whether SNV hypervariability was associated with specific combinations of nearest neighbors. For A-tracts flanked 5′ by a T, C or G, the highest percentage of SNVs was observed at A1 when preceded by a T, which reached 7.9% for TA[n] tracts of length 9 (Supplementary Figure S2C). By contrast, for 3′ T, C or G, the greatest effect was elicited by a C, with the highest percentage (7.1%) of SNVs at An for A[n]C tracts of length 9 (Supplementary Figure S2D). Therefore, flanking base pairs play a critical role both in the spectra and frequencies of SNVs at A-tracts. More detailed plots along A-tracts either preceded (Supplementary Figure S2E), followed (Supplementary Figure S2F) or preceded and followed (Figure 2C) by a T, C or G revealed the dramatic and long-range (up to 9–10 bp for the longest tracts, higher than the value of 4 bp predicted by mathematical models of slippage (11)) influence of flanking base pairs on variation spectra, in which up to 95% of the changes were in the direction of the base flanking the tract. Because the number of A-tracts preceded or followed by a specific base varies by up to three-fold (Supplementary Figure S2G), we conclude that for A-tracts, the overall mutation fractions and spectra are the result of at least three variables; length, position along the tract, and base composition of the 5′ and 3′ nearest-neighbors.

For G-tracts flanked 5′ by a T, C or A, high percentages (10–12%) of SNVs were observed at G1 for tracts preceded by a C, an effect that decreased with increasing tract length (Supplementary Figure S3A). This result, together with an exceedingly low number of G→A transitions at G1 for tracts not preceded by a C (Supplementary Figure S3C) relative to all tracts (Supplementary Figure S2B), is consistent with the known high mutability of CG:CG dinucleotides as a result of cytosine-5 methylation (9). The hypermutability at G2 was observed preferentially for tracts preceded by an A, and to a lesser extent T, whereas that at G3 was insensitive to flanking sequence composition. Likewise, G-tracts flanked 3′ by a T, C or A did not display marked sequence-dependent effects (Supplementary Figure S3B). Detailed plots of the SNV spectra along G-tracts either preceded (Supplementary Figure S3D), followed (Supplementary Figure S3E), or preceded and followed (Figure 2D) by a T, C or A revealed a noticeable effect only for 5′ T in association with G→T substitutions at G1for tracts of length ≥8. Thus, despite a consistent over-representation of G-tracts flanked 5′ by a T (Supplementary Figures S3F and S1B), which must account for the high absolute number of SNVs at G1 for TG[n] relative to AG[n] and CG[n] (Supplementary Figure S3G), nearest-neighbor base composition seems to play a lesser role in SNV spectra at G-tracts than at A-tracts.

With respect to SNVs at the flanking 5′ and 3′ nearest positions, no B→A or H→G substitutions (Figure 1A) were found above a length threshold of 9 for A-tracts and 8 for G-tracts (Figure 2E, gray shading) out of 5969 SNVs, implying that tract expansion by recruiting flanking base pairs is disfavored at these lengths. In summary, base substitution along mononucleotide repeats is strongly skewed towards the edges of A-tracts and within the 5′ half of G-tracts, with frequencies that peak at midsize lengths (8–9 bp). For A-tracts ≥7 bp, base substitution occurred almost exclusively in the direction of the flanking nearest-neighbors. Finally, base substitution at flanking bases did not contribute to tract expansion for mononucleotide runs longer than 8–9 bp.

Insertions and deletions display length and positional preference

In addition to SNVs, mononucleotide runs are polymorphic in length as a result of indels. Herein, we consider separately two types of indels: one in which tract length changes by ±1 and flanking bp composition is not altered (slippage); the other comprising all other cases involving the addition or removal of 1–200 bp (indels). Slippage is a widely accepted mutational mechanism (1112,34), whereby DNA replication errors at reiterated DNA motifs cause changes in the number of motifs (most often +/−1). The normalized fractions of slippage in the 1KGP dataset peaked at lengths of 8 bp for A-tracts and 9 bp for G-tracts (Figure 3A), generating bell-shaped curves similar to those observed for SNVs (Figure1C) and with no differences in the highest fraction of ‘slipped’ tracts, which peaked at ∼0.02. By contrast, +1 slippage occurred more frequently than −1 slippage at A-tracts (Figure 3B). These results support recent studies on microsatellite repeats (12) and contrast with previous conclusions that slippage increases monotonically with tract length, and that the extent of slippage differs between A- and G-tracts (35,36).

Figure 3.

Population insertions and deletions. (A) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage in the 1KGP dataset as a function of tract length. Data were obtained by dividing the number of events by both the number of hg19 tracts and tract length (n). (B) Ratio of the number of +1 to −1 slippage for A-tracts (closed circles) and G-tracts (open circles). (C) Indels at A-tracts. For positions along the tracts (‘Tract’), ‘F Indel’ is the ratio between the number of indels and the number of tracts in hg19 multiplied by tract length. For the positions immediately flanking the tracts genomic coordinates (‘Before tract’ and ‘After tract’), ‘F Indel’ is the ratio between the number of indels and the number of tracts in hg19. (D) Indels at G-tracts, calculated as described in panel C. (E) Heatmap representation of insertions along A-tracts. The percent insertions (i.e. the number of insertions at each position divided by the number of tracts in hg19) (y-axis) plotted as a function of location (x-axis) from position 0 (insertion between the bp 5′ to the tract and the first bp of the tract) to position n + 1 (insertion between the bp 3′ to the last bp of the tract and the following bp) (see Figure 1A) and as a function of tract length (z-axis). (F) Heatmap representation of insertions along G-tracts.

With respect to indels, the normalized fractions were low (<1 × 10−3) along short (4–6 bp) A- and G-tracts, but rose to a plateau for longer tracts as reported earlier (11); this plateau was 10-fold higher for G-tracts (∼0.03) than for A-tracts (∼0.003) (Figure 3C and D). Indels also occurred more frequently (up to six-fold for A-tracts of length 11) at nearest-neighboring base pairs (‘Before tract’ and ‘After tract’ in Figure 3C and D) than along the tracts. Thus, contrary to SNVs and slippage, indels increased to a plateau with mononucleotide tract length.

We analyzed in detail the locations of insertions along the tracts and the flanking positions with respect to the 5′ to 3′ orientation of the tracts (Figure 1A). The normalized fractions demonstrated that insertions peaked at the 3′, and to a lesser extent 5′, ends of the longest A-tracts (Figure 3E), but remained low. For G-tracts, insertions occurred most efficiently at two locations (G2–3 and G5) (Figure 3F), they increased with tract length (up to ∼0.04), and attained ∼10-fold higher values than for A-tracts. In conclusion, insertion sites at A- and G-tracts followed the patterns observed for SNVs (Figure 2A and B), suggesting that factors associated with local DNA dynamics sensitize specific bases along the tracts to genetic alteration, inducing both SBS and indels.

Base pair flexibility and charge localization map to sites of sequence changes

To elucidate elements of intrinsic DNA dynamics that may be responsible for the biases in SNV and insertion sites, we performed molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations on model A[6], A[9], G[6] and G[9] duplex DNA fragments. We focused on water bridge coordination (Figure 4A), bp step flexibility, and for the G[6] and G[9], charge localization, as these properties are known to impact the susceptibility of DNA to base damage, repair and mutation. The fractions of one water coordination increased along the A[9] and A[6] structures in a 5′ to 3′ direction, irrespective of flanking sequence composition, in concert with a decrease in minor groove width (Figure 4B and Supplementary Figure S4A) as predicted (37). Vstep, a measure of bp structural fluctuation, displayed a prominent peak of ∼40 Å3deg3 at the 5′-TA-3′ step for both structures (Figure 4C and Supplementary Figure S4B), which together with low water occupancy points to 5′-TA-3′ being a preferred location for base modification and mutation. In the G[9] and G[6] structures water coordination involved mostly two-water bridges due to wide (∼14 Å) minor grooves (Figure 4Dand Supplementary Figure S4C), whereas flexibility was modest (∼20–22 Å3deg3, Figure 4E and Supplementary Figure S4D). Thus, bp dynamics are likely to impact mutations at A-tracts to a greater extent than at G-tracts. Guanine has the lowest ionization potential (IP) of all four bases and IP further decreases at guanine runs, rendering them targets for electron loss, charge localization, oxidation and eventually mutation (4,38). Because after electron loss the ensuing charge (hole) can migrate along the DNA double-helix and relocalize at specific guanines, we addressed whether the preferred sites of mutation along G-tracts, i.e. G2–3 and G5, would also be preferred sites for charge localization. The QM/MM determinations indicated that whereas for the short G[6] fragment the difference in the density-derived atomic partial charges (DDAPC) (i.e. the hole) localized most often (∼50%) to the first position (Figure 4F), for the long G[9] fragment charge localization shifted downstream (mostly to the second, but also to positions 6–7, Figure 4G). Importantly, the charge was found exclusively around the guanine rings (Figure 4H). Thus, the two main sites of sequence change along G-tracts, i.e. G2–3 and G5, coincide with positions where charge localization and hence one-electron oxidation reactions is predicted to occur most frequently. In summary, bp flexibility at A-tracts and charge transfer at G-tracts likely represent intrinsic DNA features underlying the bias in SNV and insertions at mononucleotide runs in human genomes.

Figure 4.

MD and QM/MM simulations. (A) Molecular modeling of one (left) and two (right) minor groove water bridge coordination. (B) Fraction of one-water bridge occupancy (left axis) at A[9] DNA sequences flanked 5′ and 3′ by a T (black circles), C (red circles) or G (green circles). Minor groove widths (right axis), as determined from intrastrand phosphate-to-phosphate distances. (C) Vstep for A[9] DNA sequences, determined as the product of the square root of the eigenvalues (λi) described by the six bp step parameters shift, slide, rise, tilt, roll and twist; i.e. Vstep=6i=1λi−−√. (D) Fraction of one- (black circles) and two-water (red circles) bridge occupancy (left axis) at G[9] DNA sequences. Minor groove widths (right axis), as assessed from intrastrand phosphate-to-phosphate distances. (E) Vstep for G9 DNA sequences. (F) Average charge redistribution (open circles and right axis) for G[6] DNA structures upon vertical ionization, examined by calculating the difference on the density-derived atomic partial charges (DDAPC) for the neutral and negatively charged states. Histogram of the number of instances (left axis) in which the largest charge redistribution occurred at a specific position along the G[6] structures. (G) DDAPC for G[9] DNA structures (open circles and right axis) and histogram of the number of instances (left axis) in which the largest charge redistribution occurred at a specific position. (H) VMD rendering of a G[9] DNA structure displaying hole localization at G2. Capped base pairs were removed for clarity.

Position and orientation along nucleosome core particles modulate sequence variation

DNA wrapped around histones in nucleosomes is subject to local deformation (39), which may impact mutation. Thus, we analyzed the 1KGP SNVs at A- and G-tracts predicted to overlap with well-positioned nucleosome core particles (NCPs) (16). In hg19, the percentage of tracts that overlap with NCPs decreased moderately from ∼90% at length of 4 to 81% and 71% for A- and G-tracts of length 13, respectively (Figure 5A), suggesting that mononucleotide runs are not depleted in NCPs in human genomes as previously proposed (40). A-tracts of lengths 4–8 base pairs displayed distinctive peaks along the NCP surface in phase with the helical repeat of DNA (10.5 bp) and with minor grooves facing toward the inner protein core (lengths 4–5) (16) (Figure 5B and Supplementary Figure S5A). A-tracts of length of 9–13 bp exhibited only half (six) the peaks evident for the shorter tracts. For the G-tracts, only small peaks with no clear minor groove-inward-facing regions were detected (Supplementary Figure S5B).

Figure 5.

Positioning along nucleosome core particles. (A) Percent of A-tract (open circles) and G-tract (closed circles) base pairs in hg19 overlapping with well-positioned NCP genomic coordinates as a function of tract length. (B) Counts of base pairs in hg19 A-tracts of length 5 overlapping with NCPs genomic regions as a function of distance from the histone octamer dyad axis. Minor groove-inward-facing regions (gray) were derived from the X-ray crystal structure of NCP147 (41). (C) Percent SNVs in the 1KGP dataset (left axis) at every bp along A-tracts of length 5 for tracts centered at maxima (black) and minima (gray) along NCPs (Figure 5B). Percent increase (right axis) of SNVs at minima relative to maxima (green). P-values for paired t-tests: 0.013 (*), 0.002 (**) and 4.7 × 10−6 (***). (D) Whisker plots of%SNVs (left axis) at A1 for A-tracts of length 5 centered at maxima and minima (black) along NCPs (Figure 5B). Percent difference (right axis) in the number of A-tracts of length 5 in hg19 preceded by C, T or G (red) between those centered at minima and those centered at maxima (Figure5B). (E) C-containing/G-containing ratios (see text) for G-tracts of length 5 in hg19 as a function of distance from the NCP dyad axis (black) and location of core histones (maroon and green). Peaks correspond to negative iSAT (i.e. tilt parameters multiplied by the corresponding sin θ) values (gray) (39). Ratios of%SNV at G1 (upshifted by 0.5 for clarity) between C-containing (5′-CCCCCG-3′ sequences on the hg19 forward strand) and G-containing (5′-CGGGGG-3′ sequences on the hg19 forward strand) (Figure 1A) CG[5] tracts mapping NCP Chip-seq genomic intervals (red) fitted by a non-parametric local regression (loess; sampling proportion, 0.100; polynomial degree, 3). (F) VMD rendering (top) of TATTT residues 34–38 (yellow) and the complementary AAATA residues 672–753 (pink) from the 1EQZ pdb nucleosomal crystal structure, corresponding to peak area from −40 to −36 in Figure 5E. The switch in G-tract (lengths of 5 and 7) orientation along NCPs (bottom) serves to position the C-containing strand on the outside (yellow) and, correspondingly, the G-containing strand on the inside (pink).

 To assess if tract-positioning along NCPs influences SNVs, we selected A-tracts of lengths 5, 7 and 9 bp and G-tracts of lengths 5 and 7 bp whose central positions coincided with either the maxima or minima (41) (Figure 5B and Supplementary Figure S5A and B) and conducted pair-wiset-tests (330 total) between permutations of ‘categories’, including ‘tracts centered at maxima versus minima’, ‘position along the tracts’, ‘flanking sequence composition’, ‘specific NCP locations’ and ‘tract orientation’. For A-tracts, 79/207 (38%) significant pairs were found, 68 (86%) of which were related to differences between tracts centered at maxima versus minima, with a preponderance (63%) of tests displaying increased %SNVs at minima (Supplementary Figure S5C and E). For example, %SNVs at length 5 bp were greater at minima than at maxima at each position along the A-tracts (Figure 5C). A→C substitutions at A1 were more abundant at maxima than at minima (mean ± SD, 18.7 ± 0.7% at max and 17.6 ± 0.8% at min; P-value 0.001), whereas A→T substitutions at the same position displayed the opposite trend (mean ± SD, 18.4 ± 0.5% at max and 19.8 ± 1.1% at min; P-value 0.0005) (Figure 5D). A-tracts of length 7 also exhibited a similar pattern at A7 (Supplementary Figure S5H). The percentages of CA[5] and A[7]C tracts in hg19 centered at maxima were greater than at minima and the reverse was observed for the TA[5] and A[7T] tracts (Figure 5D and Supplementary Figure S5H). Thus, we conclude that positioning along the NCP surface of both the double-helical grooves and junctions with flanking base pairs influence SNVs along A-tracts. However, this influence is complex and for the most part, difficult to predict.

For G-tracts, most pairwise comparisons (18/34, 53%) indicated SNV variation according to sequence orientation (Supplementary Figure S5F and G). In hg19, the ratio of the numbers of G-tracts of lengths 5 and 7 for which the C-containing strand coincided with the forward sequence (downstream example sequence in Figure 1A) to the numbers of G-tracts for which the G-containing strand coincided with the forward sequence (upstream example sequence in Figure 1A) (C-containing/G-containing ratios) displayed a prominent 10.5-bp oscillation in phase with iSAT (Figure 5E), a measure of ‘inside’ and ‘outside’ bases, according to the bp step tilt parameter (39). Analysis of the helical path of a 146-bp DNA fragment wrapped around histones showed that the oscillation in the C-containing/G-containing ratios corresponds to a preference for guanine bases to face the protein core (Figure 5F). We analyzed the subset of G-tracts preceded by a 5′ C (i.e. CG[5]) to assess whether SNVs at G1, the position known to be mutable due to CpG methylation also oscillated with the C-containing/G-containing ratios. Oscillation in SNV-C-containing/SNV-G-containing values was evident, with peaks aligning to the hg19 troughs (Figure 5E) implying that the cytosines facing the protein surface harbor more variants than those facing away. We conclude that A- and G-tracts display preferential positioning (the former) and orientation (the latter) along NCPs, which in turn modulate the rate of sequence variation.

Mutations associated with human disease

Knowing that the first and last As of long A-tracts and G2–3 in G-tracts are the major sites of SNV in the human population, we addressed whether these features are also discernible in mutated mononucleotide tracts associated with human genetic disease. We collected 9,450,456 unique SBSs (both SBSs and SNVs refer to single base changes) from sequenced cancer genomes and normalized the percent mutations along A- and G-tracts to enable a direct comparison with the 1KGP dataset. For A-tracts (Figure 6A and Supplementary Figure S6A), SBSs displayed the same trend as the 1KGP data (Figure 2A) with respect to the bell-shape increase in mutations at A1 and An and the mutation spectra, although the susceptibility to mutation as a function of tract length attained greater values (6.36% for length 11 in cancer versus 4.15% for length 9 in the 1KGP datasets at A1). The first and last 3 bp also harbored more SBSs than in the 1KGP dataset for tracts >7 bp, a feature that we found to be due exclusively to a large cancer dataset (42) containing high-level microsatellite instability (MSI) samples (Supplementary Figure S6B and C), which are known to result from mismatch-repair deficiency (15). Thus, A-tracts display similar patterns of base substitution between the germline and somatic cancer tissues. For G-tracts, mutation spectra were characterized by G→T transversions at tract lengths >7, particularly at G1, the most frequently mutated position for tracts lengths up to 11 bp (Figure 6B and Supplementary Figure S6D). This trend persisted even when the high rates of methylation-mediated deamination mutations at the CG dinucleotide were removed (Supplementary Figure S6E). Thus, mutation patterns in cancer genomes contrast with those observed in the germline, both with respect to the most mutable position (G1 versus G2–3) and the types of base substitution (G→T in cancer genomes versus G→T and G→C in the germline).

Figure 6.

Mutation patterns in cancer genomes. (A) Mutation spectra for SBSs at A-tracts. Percent values were obtained by dividing the total number of SBSs at each position by the number of tracts in hg19 and then multiplying by 3.2516 to equalize the percentage of A-tracts of length 4 between the cancer genomes and the 1KGP datasets. (B) Mutation spectra for SBSs at G-tracts in cancer genomes. Percent values were obtained as in (A) using a multiplication factor of 3.7419. (C) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage, obtained by dividing the number of events by both the number of tracts in hg19 and tract length. (D) Indels at A-tracts, calculated as described in Figure 3C. (E) Indels at G-tracts, calculated as described in Figure3C. (F) Heatmap representation of insertions along G-tracts, as described in Figure 3E.

 With respect to slippage, the fractions for A-tracts elicited an excess at lengths 9 and 10 bp relative to the 1KGP dataset, which was also due to the MSI-containing dataset. For G-tracts, the fractions peaked at length 8, as for the 1KGP dataset (Figures 3A and 6C), implying that the propensity to undergo slippage is indistinguishable between the germline and soma. Indels were also more abundant at flanking base pairs than along the tracts (Figure 6D and E), particularly for G-tracts of length >7, similar to the 1KGP dataset (Figure 3C and D). Detailed analyses of insertions revealed that both G1 and the preceding position were the most significant sites of mutation (F-values up to 0.08 at G1 for tracts of length 8) (Figure 6F). Thus, the 5′ end of long G-tracts is the most susceptible site for both SBSs and insertions in cancer genomes, in contrast to the germline where these occur within the runs, typically at G2–3.

We also extracted the mutated A- and G-tracts from the Human Gene Mutation Database (HGMD), a collection of >150,000 germline gene mutations associated with human inherited disease. A total of 1519 genes were mutated at A- or G-tracts out of a total of 3972 (38%); 3480 SBSs and 2866 slippage events were noted within these tracts, 85 and 46% of which were predicted to be disease-causing, respectively (Figure 7A and Supplementary Table S1). Ranking genes by the number of literature reports indicated that among the top 10 entries three were associated with cancer (BRCA1, BRCA2 and APC), two with hemophilia (F8 and F9), four with debilitating lesions of the skin (COL71A), muscle (DMD), lung (CFTR) and kidney (PKD1), with one causing hypercholesterolemia (LDLR) (Figure 7B). Thus, mutations within A- and G-tracts carry a high social burden by contributing to some of the most common human pathological conditions.

Figure 7.

Mutation patterns in HGMD and model for sequence context-dependent changes. (A) Number of germline SBSs and slippage events (Slip.) at A- and G-tracts in HGMD. Gene alterations were classified as disease-causing mutation (DM), likely disease-causing mutation (DM?), disease-associated and putatively functional polymorphism (DFP), disease-associated polymorphism with additional supporting functional evidence (DP) and invitro/laboratory orinvivo functional polymorphism (FP). Codon changes (SIFT predictor) were classified as damaging (d), null (n), tolerated (t) and low-confidence prediction (l). (B) The 10 most commonly reported genes in HGMD with mutations at A- and G-tracts. Various mutated tracts were generally reported for the same gene in different reports. (C) Mutation spectra for SBSs at A- (left) and G-tracts (right) in HGMD. Percent values were obtained by dividing the total number of SBSs at each position by the number of tracts in hg19 exons. A|G→T (black), A|G→C (red), A→G (green), G→A (cyan). (D) Normalized fractions of A-tracts (closed circles) and G-tracts (open circles) displaying +/−1 bp slippage, obtained by dividing the total number of events by the number of tracts in hg19 exons and by tract length. (E) Model for sequence context-dependent changes at A-tracts (left) and G-tracts (right). *, site of base modification.

 For both A- and G-tracts, SBSs occurred mostly at tract lengths of 4–7, with patterns more similar to those in the 1KGP than in the cancer datasets, both with respect to the location of the most mutable positions (first and last As and first/second Gs) and the types of base substitution (A→T and G→H) (Figure 7C and Supplementary Figure S6F). Likewise, slippage events peaked at tract lengths of 7–9 as observed in the 1KGP dataset (Figure 7D). In summary, the patterns of both SBSs and slippage in the HGMD dataset followed the trend observed in the 1KGP dataset, suggesting that germline variants at mononucleotide repeats leading to either population variation or human inherited disease may have arisen through similar mechanisms.

Why are specific A:T and G:C base pairs within A- and G-tracts more susceptible to sequence changes than their identical neighbors? For A-tracts, bp flexibility may play a role. Chemical damage to DNA, such as by hydroxyl radicals has been shown to be proportional to the geometrical solvent-accessible surface of the atomic groups, which increases with DNA flexibility (43). Along A-tracts flexibility is restricted, but it is high at both the 5′ and 3′ junctions. Thus, the fact that the highest rates of mutation coincide with the highest degree of flexibility at the 5′-TA-3′ bp step is consistent with the view that this position may be susceptible to DNA damage as a result of flexibility. Other sources of DNA dynamics are also likely to be relevant, such as sugar flexibility at the junctions, which increases with tract length (44). Chemical modification at these junctions may then lead to base substitution and indels, the latter as a result of strand breaks.

With respect to SNV mutation spectra, these were found mostly in the direction of flanking base composition above a length of 7–8 bp. We interpret this behavior in terms of DNA slippage along A-tracts when attempts are made during translesion synthesis (TLS) to bypass a damaged site (Figure 7Ei). Two scenarios may be considered to account for A→T transitions at A1. In the first, the last tract-template base would loop out into the polymerase active site permitting base-pairing and strand elongation (Figure 7Eii) using the tract-flanking base as a template (34,4546). In the second (Figure 7Eiii), slippage would occur behind the polymerase, prompting extension past the newly created A*:T mispair generated by primer/template misalignment. Either pathway would yield a common intermediate (Figure 7Eiv) that contains the base complementary to the junction across from the damaged site upon slippage resolution (34). Following DNA synthesis (S) and/or repair (R) (Figure 7Ev and vi), this mispair will generate a base change that is always identical to the tract-flanking base.

For G-tracts, the high rates of G→T transversions at G1 in cancer genomes are also consistent with preferred chemical attack at this site due to high flexibility (Figure 7F top). Direct chemical attack at a guanine is known to result in stable products, such as 8-oxo-G and Fapy-G, both of which are known to yield G→T transversions (4750). Thus, G1 may be the most susceptible site for such reactions for G-tracts of lengths ≥7 (Figure 7Fright), which in cancer genomes would become a mutation hotspot. In the germline, SNVs peaked inside G-tract base pairs, while mutational spectra were insensitive to flanking base composition; these events are inconsistent with a role for template misalignment and slippage as noted for A-tracts. Rather, the correspondence between hotspot mutations at G2–3 and G5 and the QM/MM simulations suggest a role for charge transfer. A large body of work during the past 20 years using computational, theoretical chemistry and biophysical techniques on short oligonucleotides, has shown that guanine is the most easily oxidizable base in DNA and that indeed a guanine radical cation can be generated through long-range hole transfer from an oxidant via one-electron oxidation mechanisms (5155). GGG triplets were found to act as the most effective traps in hole transfer by both experimental and theoretical work (5659), demonstrating that the resulting guanine radical cation (or its neutral deprotonated form) became rather delocalized, but it preferentially centered at the first and second G. These well-established patterns of chemical reactivity are consistent with our experimental observation of high mutation frequencies at G1 for short G-tracts and the results from QM/MM simulations on G6. For longer tracts, the downstream shift in mutation hotspots, i.e., G2–3 and G5, also correlate well with the charge localization predicted from QM/MM simulations, which explicitly included solvent effects and structural fluctuations. Thus, in conjunction with the constrained density functional theory (60), both the neutral and oxidized forms of a guanine nucleobase can be reliably constructed to infer the accurate determination of mutational patterns of mononucleotide repeats in human genomic DNA.

The compact organization of the sperm genome (61), and presumably low levels of oxidative stress in the germline, may enable guanine oxidization through one-electron oxidation reactions rather than by direct chemical attack, thereby favoring the formation of radical cations. A charge injected at G1 by electron loss would then migrate to neighboring guanines and localize at sites of low IP, such as G2 (Figure 7F left). Guanine radical cations are known to readily undergo further chemical modification leading to products such as 8-oxo-G, oxazolone, imidazolone, guanidinohydantoin, and spiroiminodyhydantoin (62) (M in Figure 7F), to yield G→T, G→C and G→A substitutions (4,63). Our model is in line with recent observations in which mutations at guanines within short G-runs (1–4 bp) correlate with sequence-dependent IPs at the target guanine in cancer genomes (9). Interestingly, these correlations were not observed in the germline (9). We interpret these composite observations as follows. The IP values for G-runs have been shown to decrease asymptotically with tract length, although the absolute values vary according to the methods and assumptions used (we obtained a value of 5.43 eV for both G[6] and G[9]) (64,65). We suggest that short G-runs with high IPs undergo one-electron oxidation reactions in the oxidative environment of cancer cells but would be refractory to such a mechanism in the germline (Figure 7Fright yellow and left white sectors). As length increases and IP values fall, G-runs would be attacked directly by oxidants abundant in tumor cells (Figure 7F orange sector), whereas oxidation will be limited to electron loss in the germline environment (Figure 7F left yellow sector).

These models (template misalignment for A-tracts and charge transfer for G-tracts) suggest a more complex scenario for mechanisms underlying mononucleotide repeat polymorphism in the human population than recently proposed (13), in which nucleotide misincorporation by error-prone polymerases is proposed as a primary source of mutations at both A- and G-tracts. As already stated, the directionality of SNVs toward tract-flanking bases in A-tracts and the hotspot mutations at G2–3, supports multiple and distinct mechanisms of base substitution at mononucleotide repeats.

Our analyses highlight additional information, including the lack of mutations in the direction of tract-base composition for base pairs flanking long tracts, the association with gene expression and the preference of guanines for the inner NCP surface, and extend prior observations (12) such as the bell-shape character of base substitution and slippage, whose mechanisms remain to be fully clarified. Finally, we document the contribution of mononucleotide mutagenesis to key aspects of human pathology beyond the well-established MSI instability in cancer (15), including hemophilia and tissue degeneration. Our collective work supports the conclusion that as the human genome undergoes evolutionary diversification and along the way suffers disease-associated mutations, oxidation reactions including charge transfer may play a prominent role.


Supplementary Data are available at NAR Online.



Mutation analyses and prenatal diagnosis in families of X-linked severe combined immunodeficiency caused by IL2Rγ gene novel mutation

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Genet. Mol. Res. 14 (2): 6164 – 6172   DOI: 10.4238/2015.June.9.2
Severe combined immunodeficiency diseases (SCIDs) are a group of primary immunodeficiency diseases characterized by a severe lack of T cells (or T cell dysfunction) caused by various gene abnormalities and accompanied by B cell dysfunction (WHO, 1992; Buckley et al., 1997). The incidence rates in infants were 1/75,000-1/10,0000 (WHO, 1992), but no morbidity statistics are available in China. The 2 genetic modes of SCID include X-linked recessive and autosomal recessive genetic inheritance. X-linked severe combined immunodeficiency (X-SCID) is the most common form, accounting for 50-60% of SCID cases (Noguchi et al., 1993). Immune system abnormalities in patients with X-SCID include T-B+NK-, in which T cells (CD3+) and natural killer (NK) cells (CD16+/CD56+) are absent or significantly reduced, and the number of B cells (CD19+) is normal or increased, causing reduced immunoglobulin production and class switching disorder (Buckley, 2004; Fischer et al., 2005). The IL- 2Rg gene mutation has been confirmed to be a major cause of X-SCID (Noguchi et al., 1993). In recent years, great progress has been made in understanding the pathogenesis of primary immunodeficiency disease and its application in clinical treatment, particularly regarding the development of critical care medicine and immune reconstruction technology. With timely control of infection and early bone marrow or stem cell transplantation, X-SCID patients can be treated, prolonging survival time. Therefore, early diagnosis of X-SCID is very important for patient treatment. Gene diagnosis has become a better early diagnosis or differential diagnosis method. In addition, familial X-SCID brings a great psychological burden to the relatives of patients. Ordinary chromosome analysis and immunological evaluation cannot be used for female carrier identification and fetal diagnosis, and gene diagnosis is the most effective method of carrier detection and prenatal diagnosis. In this study, we detected mutations in 2 families with X-SCID and identified 2 novel mutations, confirming the X-SCID pedigrees. Prenatal diagnosis was performed for the pregnant fetus in the mother of one of the probands based on gene diagnosis. Female individuals in this family were subjected to carrier detection.
IL2Rg gene mutation test Direct sequencing of 1-8 exons and the flanking region of the IL2Rg gene by PCR in family 1 showed that the 3rd exon of the proband contained the c.361-363delGAG heterozygous deletion mutation, which led to deletion of the 121st amino acid glutamate (p.E121del) in its coding product. There were no sequence variations in other coding regions or in the shear zone. The proband’s mother carried the same heterozygous mutation, while his father did not carry the mutation site (Figure 2a, b, c). This mutation was not observed in any cases of the control group, and this family was identified as an X-SCID family. The c.510-511insGAACT insertion heterozygous mutation was present in the 4th exon of the proband’s mother in family 2. This mutation was a 5-base repeat of GAACT, resulting in a change in amino acid 173 from tryptophan into a stop codon (p.W173X). While there were no sequence variations in other coding regions or in the shear zone, the patient’s father did not carry the mutation (see Figure 2d, e). We did not find this mutation in the healthy control group. We presumed that the 4th exon of the deceased child in family 2 contained the c.510-511insGAACT insertion mutation, leading to X-SCID symptoms, and thus we speculated that this family was an X-SCID pedigree. Prenatal diagnosis We verified the chorionic villus status of the fetus in family 1 using the PowerPlex 16 HS System kit. The results of prenatal diagnosis showed that the fetal tissue contained no maternal contamination and that this fetus was female. The results of prenatal diagnosis showed that there was no c.361-363delGAG (p.E121del) heterozygous mutation in the female fetus of family 1.
Figure 2. Sequencing graph of IL2Rg gene in 2 pedigrees with X-chain severe combined immunodeficiency. a.-c. Family 1. a. Normal control (rectangle indicates 3 edentulous bases of this patient). b. Proband carrying the c.361- 363delGAG (p.E121del) mutation (arrow indicates deletion of fragment connection sites). c. The proband’s mother contained a c.361-363delGAG (p.E121del) heterozygous mutation (arrow). d.-e. Family 2. d. The proband’s mother carried the c.510-511insGAACT (p.W173X) heterozygous mutation (arrow indicates that the reverse sequencing graph was positive). e. Normal control (rectangular box indicates 2 normal copies of GAACT (the mutation fragment was 3 copies). Carrier detection results For the c.361-363delGAG (p.E121del) site, the gene analysis results of the female individual in family 1 showed that I2 (proband’s grandmother) was a heterozygous carrier and that II3 (proband’s aunt) was a non-carrier and had no mutations.
IL-2 can combine with the IL-2 receptor (IL-2R) of the immune cell membrane. IL-2R is composed of 3 subunits, including the IL-2Ra chain (CD25), IL-2Rb chain (CD122), and IL- 2Rg chain (CD132). IL-2Rg functional units in common with IL-4, IL-7, IL-9, IL-15, IL-21, and other cytokine receptors, and these regions are referred to as the total chain (Li et al., 2000). The IL-2Rg chain can maintain the integrity of the IL-2R complex and is required for the internalization of the IL-2/IL-2R complex; it is also the link that contacts the cell membrane surface factor region and downstream cell signal transduction molecules. Therefore, the integrity of the IL-2Rg chain is vital for the immune function of an organism (Malka et al., 2008; Shi et al., 2009).
Mutations in the IL2Rg gene, which encodes IL-2Rg, were identified to be a major cause of X-SCID in 1993 (Noguchi et al., 1993). The IL2Rg gene is located on chromosome X q21.3-22, is 37.5 kb length, and contains 8 exons, which encode 369 IL-2Rg amino acids. The IL2Rg chain exhibits varying structural regions, such as the signal peptide [amino acids (AA) 1-22], extracellular domain (AA 23-262), transmembrane region (AA 263-283), and intracellular region (AA 284-369). The WSXWS motif is located in the extracellular region (AA 237-241), while Box 1 is located in the intracellular region (AA 286-294).
By the end of 2013, the Human Gene Mutation Database contained a total of 200 mutations in the IL2Rg gene (HGMD Professional 2013.4). The most common mutation types in the IL2Rg gene were the missense or nonsense mutations, which result from single base changes. A total of 100 missense or nonsense mutations have been identified, followed by insertion or deletion mutations in a total of 50 species. The 3rd most common type of mutations includes shear mutations in approximately 30 species. Eight exons contained mutations, and mutations in 3rd or 4th exons were the highest, accounting for a total mutation rate of 43% (86/200). According to the X-SCID gene database (IL2RGbase) (http://research.nhgri., the gene mutations in IL2Rg mainly occurred in the extracellular region of the IL2Rg chain (Fugmann et al., 1998). Zhang et al. (2013) reported that the IL2Rg gene mutations in 10 patients with X-SCID in China were located in the extracellular region. Two mutations reported in our study were also located in the extracellular region. The mutation of IL2Rg gene in family 1 was a codon mutation in the 3rd exon, resulting in a 3-base deletion. The c.361-363delGAG (p.E121del) mutation was located in the extracellular area of the IL- 2Rg subunit, and we inferred that the 121 glutamate deletion caused by the mutation would lead to changes in the structure of the peptide chain, affecting signal transmission and resulting in serious symptoms. The mutation of family 2 was a GAACT repeat of ILR2g gene; this repeat of 5 bases resulted in 173 codon changes from tryptophan into a stop codon. Generation of the peptide chain with the mutation lacked 196 amino acids compared to the normal chain, including the intracellular, transmembrane, and some extracellular regions, directly affecting the structure and function of receptors and causing disease. No studies have been reported regarding these 2 mutations. We combined with the mutation characteristics and clinical manifestations and diagnosed family 1 as X-SCID pedigrees. Although the patient in family 2 was deceased, it can be speculated that the 2 deceased patients in family 2 were X-SCID pedigrees caused by c.510-511insGAACT (W173X).
Prenatal diagnosis can accurately identify fetal situations and be used to avoid birth defects, which can also ease the anxiety of the pregnant mother. Gene diagnosis for pedigrees of patients based on DNA samples has advanced recently, particularly with the application of high-throughput sequencing technology (Alsina et al., 2013). We can now perform gene analysis for varied clinical infectious diseases for differential diagnosis. However, the effectiveness of prenatal diagnosis for pedigrees in which the proband is dead remains unclear. Because the gene mutations in the proband is unknown in these cases, the patient’s situation was only inferred by his mother’s genotypes. However, we considered that for the deceased, if we can define the mother was a pathogenic gene carrier, even if the proband is not X-SCID, the woman also has a risk of having X-SCID children and this pedigree may be X-linked recessive inheritance. Prenatal diagnosis may provide a choice for preventing the birth of patients in these families in the premise of informed consent.
Gene diagnosis of IL2Rg can also be used for carrier detection of suspected females in the family.
In the present study, we performed carrier detection of the patient’s grandmother and aunt in family 1 and determined that the patient’s pathogenic mutations were from his grandmother. His aunt did not inherit the pathogenic gene, and thus she was a non-carrier and her fertility will not be affected. In this study, we used direct sequencing of PCR products and identified IL2Rg gene mutations in 2 pedigrees with X-SCID. We found 2 unreported mutations in the IL2Rg gene, and prenatal diagnosis and carrier detection were conducted in 1 X-SCID family. Because the incidence rate of X-SCID is extremely low, it is difficult to promote the widespread use and application of genetic diagnosis. However, this study may provide some implications for the diagnosis of infants with immunodeficiency, and gene diagnosis techniques such as conventional or high-throughput sequencing should be used as soon as possible during pregnancy, which can be used to guide treatment. This method can also provide reliable prenatal diagnosis and carrier detection service for these families.
MEF2A gene mutations and susceptibility to coronary artery disease in the Chinese population
J. Li1 , H.-X. Chen2 , J.-G. Yang3 , W. Li3 , R. Du3 and L. Tian3       DOI
Coronary artery disease (CAD) has high morbidity and mortality rates worldwide. Thus, the pathogenesis of CAD has long been the focus of medical studies. Myocyte enhancer factor 2A (MEF2A) was first discovered as a CAD-related gene by Wang (2005) and Wang et al. (2003, 2005). Three mutation points in exon 7 of MEF2A were subsequently identified by Bhagavatula et al. (2004); however, Altshuler and Hirschhorn (2005) and Weng et al. (2005) predicted that the MEF2A gene lacked mutations. Zhou et al. (2006a,b) analyzed the mutations and polymorphisms in exons 7 and 11 of the MEF2A gene in the Han population in Beijing, and various rare mutations were found in exon 11 rather than in exon 7. The clinical significance of specific 21-bp deletions in MEF2A was also explored, and previous studies have shown mixed results. In this study, polymerase chain reaction-singlestrand conformation polymorphism (PCR-SSCP) and DNA sequencing were used to detect exon 11 of the MEF2A gene in samples collected from 210 CAD patients and 190 healthy controls and to investigate the function of the MEF2A gene in CAD pathogenesis and their correlation.
CAD, a common disease in China, is induced by multiple factors, such as genetics, the environment, and lifestyle. Thus, a multi-faceted approach is necessary in the study of CAD pathogenesis, particularly in molecular biology research, which is important for developing comprehensive treatment of CAD based on gene therapy. The MEF2A gene was first identified as a CAD-related gene through linkage analysis of a large family with CAD (9 of 13 patients developed MI) in 2003.
In this study, we found the following mutations: 1) codon 451G/T (147191) heterozygous or homozygous mutation; 2) loss of 1 (Q), 2 (QQ), 3 (QQP), 6 (425QQQQQQ430), and 7 (424QQQQQQQ430) amino acids (147108-147131); and 3) codon 435G/A (147143) heterozygous mutation. Among these mutations, the synonymous mutation at locus 147191 was confirmed by reference to the National Center for Biotechnology Information (NCBI) database to be a single nucleotide polymorphism, which was also demonstrated in our study by the extensive presence of this polymorphism in healthy controls. However, the heterozygous mutation at locus 147143 was only found in the genomes of CAD patients, and was therefore identified as a mutation.
Given that MEF2A is a CAD-related gene, the results of various studies are controversial among several countries. Weng et al. (2005) screened gene mutations in exon 11 of the MEF2A gene from 300 CAD patients and 1500 healthy controls. They hypothesized that the changes in 5-12 CAG repeats are genetic polymorphisms and that the 21-base deletion in exon 11 of the MEF2A gene did not induce autosomal dominant genetic CAD. Gonzalez et al. (2006) suggested that the CAG repeat polymorphism was independent of MI susceptibility in Spanish patients. Kajimoto et al. (2005) reported that the CAG repeat sequence was not correlated with MI susceptibility in Japanese patients. Horan et al. (2006) also found that the CAG repeat sequence was not associated with the susceptibility to early-onset familial CAD in an Irish population. Hsu et al. (2010) identified no correlation between the CAG repeat sequence and CAD susceptibility in the Taiwanese population. Dai et al. (2010) found that the structural change in exon 11 was not related to CAD in the Chinese Han population. Lieb et al. (2008) and Guella et al. (2009) hypothesized that MEF2A was independent of CAD. However, Yuan et al. (2006) and Han et al. (2007) suggested that the CAG repeat sequence was correlated with CAD because 9 CAG repeats was an independent predictor of CAD. Elhawari et al. (2010) and Maiolino et al. (2011) suggested that MEF2A is a susceptibility gene for CAD. Dai et al. (2013) showed that mutations in exon 12 are associated with the early onset of CAD in the Chinese population. Liu et al. (2012) failed to demonstrate a correlation between the CAG repeat sequence and CAD through case-control analysis, systematic review, and meta-analysis, but found that the 21- base deletion in exon 11 was strongly associated with CAD, and that genetic variations in MEF2A may be a relatively rare, but specific, pathogenic gene for CAD/MI. Kajimoto et al. (2005) reported 4-15 CAG repeats. However, only 4-11 CAG repeats were observed in our study, possibly because of genetic differences in patients in this study. Eleven CAG repeats were observed in most samples from the control group, and the proportion of 10, 9, and 8 repeats exceeded 1%. The heterozygous mutation at 147143, as well as the 4 and 5 CAG repeats, was only observed in CAD patients. Thus, we speculated that the CAG repeat sequence is correlated with CAD susceptibility, and the presence of 4 or 5 repeats may be a risk factor for CAD, which was inconsistent with the results obtained by Han et al. (2007). The inconsistency in these results may be explained by the differences in subjects and sample sizes among studies.
Impact of glucocerebrosidase mutations on motor and nonmotor complications in Parkinson’s disease

Homozygous and compound heterozygous mutations in GBA encoding glucocerebrosidase lead to Gaucher disease (GD). A link between heterozygous GBAmutations and Parkinson’s disease (PD) has been suggested ( Bembi et al., 2003,Goker-Alpan et al., 2004, Halperin et al., 2006, Machaczka et al., 1999, Neudorfer et al., 1996, Tayebi et al., 2001 and Tayebi et al., 2003). In 2009, a 16-center worldwide analysis of GBA revealed that heterozygous GBA mutation carriers have a strong risk of PD ( Sidransky et al., 2009).

In addition, heterozygote GBA mutations not only carry a risk for PD development but also the possibility of some risk burden on the progression of PD clinical course. In cross-sectional analyses of GBA mutations in PD patients, earlier disease onset, increased cognitive impairment, a greater family history of PD, and more frequent pain were reported in patients with mutations, compared with no mutations ( Chahine et al., 2013,Clark et al., 2007, Gan-Or et al., 2008, Kresojevic et al., 2015, Lwin et al., 2004, Malec-Litwinowicz et al., 2014, Mitsui et al., 2009, Neumann et al., 2009, Nichols et al., 2009,Seto-Salvia et al., 2012, Sidransky et al., 2009, Swan and Saunders-Pullman, 2013 and Wang et al., 2012). Recently, a few prospective studies have investigated clinical features of PD with GBA and showed a more rapid progression of motor impairment and cognitive decline in GBA mutation cases than in PD controls ( Beavan et al., 2015, Brockmann et al., 2015 and Winder-Rhodes et al., 2013). However, in terms of motor complications such as wearing-off and dyskinesia, no studies exist in the longitudinal course of PD with GBA mutations.

Here, we conducted a multicenter retrospective cohort analysis, and the data were investigated by survival time analysis to show the impact of GBA mutations on PD clinical course. We also investigated regional cerebral blood flow (rCBF) and cardiac sympathetic nerve degeneration of subjects with GBA mutations, compared with matched PD controls.

3.1. Subjects

Among the 224 eligible PD patients (the subjects were not related to each other), 9 subjects were excluded from the analysis (4 due to multiple system atrophy findings on subsequent brain MRI and 5 because of insufficient clinical information). Therefore, 215 PD patients [female, 52.1%; age, 66.7 ± 10.8 (mean ± standard deviation)] were analyzed. For non-PD healthy controls, 126 patients’ spouses (female, 58.7%; age, 67.3 ± 10.3) without a family history of PD or GD were enrolled.

3.2. GBA mutations and risk ratios for PD

In the PD subjects, we identified 10 nonsynonymous and 2 synonymous GBA variants. Within the nonsynonymous variants, 7 mutations were previously reported in GD [R120W, L444P-A456P-V460 (RecNciI), L444P, D409H, A384D, D380N, and444L(1447-1466 del 20, insTG)] as GD-associated mutations. Three nonsynonymous mutations have never been reported in GD patients [I(-20)V, I489V, and there was one novel mutation (Y11H)].

GD-associated GBA mutations were found in 19 of the 215 (8.8%) PD patients but none in the healthy controls. The risk of PD development relative to these GD-associated mutations was estimated as an OR of 25.1 [95% confidence interval (CI), 1.50–420,p = 0.0001] with 0-cell correction. The nonsynonymous mutations that were not reported in GD patients had no association with PD development (p = 0.506; OR, 1.3; 95% CI, 0.7–2.6) ( Table 1). Four subjects had double mutations. For subsequent analyses, 2 subjects with double mutations of I (-20)V and K466K were adopted to the group of mutations unreported in GD, and 2 subjects with double mutations of R120W and I(-20)V, and of R120W and L336L were adopted to the group of GD-associated mutations.

Table 1.Frequency of glucocerebrosidase gene allele in Parkinson’s disease patients and controls

Allele name PD (n = 215) Controls (n = 126) p Odds ratio (95% CI)
GD-associated mutations
 R120W 7a 0 0.050 9.1 (0.5–160.8)
 RecNciI (L444P-A456P-V460) 4 0
 L444P 4 0
 D409H 1 0
 A384D 1 0
 D380N 1 0
444L(1447-1466 del 20, insTG) 1 0
 Subtotal, n (%) 19 (8.8%) 0 (0%) <0.001 25.1 (1.5–419.8)b
Nonsynonymous mutations not reported in GD
 I(-20)V 27a 13 0.603 1.3 (0.6–2.5)
 I489V 3 0
 Y11Hc 0 1
 Subtotal, n (%) 30 (14.0%) 14 (11.1%) 0.506 1.3 (0.7–2.6)
Synonymous, n
 K466K 2a 1
 L336L 1a 0
Allele names refer to the processed protein (excluding the 39-residue signal peptide).

Key: CI, confidence interval; GD, Gaucher disease; PD, Parkinson’s disease.

a Four subjects had double mutations; 2 of I(-20)V and K466K, 1 of I(-20)V and R120W, and 1 of R120W and L336L.
b Odds ratio was calculated by adding 0.5 to each value.
c Novel mutation.
3.3. Clinical features of PD patients by GBA mutation groups

The clinical features of PD patients with GD-associated mutations, those with mutations unreported in GD, and those without mutations are shown in Table 2. In the GD-associated mutation group, females, those with a family history and those with dementia (DSM IV) were significantly more frequent than those in the no-mutation group (p = 0.047, 0.012, and 0.020, respectively). The age of PD onset was lower in patients with GD-associated mutations (55.2 ± 9.9 years ± standard deviation), compared with those without mutations (59.3 ± 11.5), although the statistical difference was not significant. There were no differences in clinical manifestations between subjects with mutations unreported in GD and those without mutations, except for dopamine agonist dosage (p = 0.026) ( Table 2).

Table 2.Epidemiological and clinical features of PD patients with Gaucher disease–associated GBA mutations, those with mutations previously unreported in GD and those without mutations

Variables Total n = 215 Mutation (-) GD-associated mutations

Mutations unreported in GD

167 19a pb 29c pd
Sex Female, n (%) 83 (49.7) 14 (73.7) 0.047 15 (51.7) ns
Age Mean (SD) 67.0 (10.8) 62.2 (10.7) 0.063e 67.5 (11.2) nsf
Disease duration (y) Mean (SD) 7.7 (5.5) 6.9 (4.6) nsf 7.2 (4.9) nsf
Onset age Mean (SD) 59.3 (11.5) 55.2 (9.9) ns 60.3 (11.8) ns
Family history Yes, n (%) 17 (11.0)g 6 (31.6) 0.012 0 (0.0) ns
Dementia (DSM-IV) Yes, n (%) 29 (17.4) 9 (47.4) 0.020 5 (17.2) ns
MMSE Mean (SD) 25.8 (5.4)h 23.3 (7.7) nsf 27.0 (3.4)i nsf
Onset symptom (tremor vs. others) Tremor, n (%) 78 (46.8) 9 (47.4) ns 15 (51.7) ns
Modified H-Y on (<3 vs. ≥3) ≥3, n (%) 82 (49.1) 14 (73.7) 0.042 16 (55.2) ns
UPDRS part 3 Mean (SD) 23.6 (12.2)j 28.5 (13.8) nsf 21.9 (8.7) nsf
Wearing off Yes, n (%) 70 (41.9) 9 (47.4) ns 13 (44.8) ns
Dyskinesia Yes, n (%) 49 (29.3) 8 (42.1) ns 8 (27.6) ns
Mood disorder Yes, n (%) 43 (25.7) 8 (42.1) ns 7 (24.1) ns
Orthostatic hypotension symptom Yes, n (%) 21 (12.6) 5 (26.3) ns 7 (24.1) ns
Psychosis history Yes, n (%) 59 (35.3) 10 (52.6) ns 7 (24.1) ns
ICD history Yes, n (%) 8 (4.8) 1 (5.3) ns 1 (3.4) ns
Stereotactic brain surgery for PD Yes, n (%) 4 (2.4) 0 (0.0) ns 0 (0.0) ns
Agonist LED mg/d Mean (SD) 92.8 (114.2) 72.1 (137.7) nse 163.7 (155.6) 0.026e
Levodopa LED mg/d Mean (SD) 400.7 (184.2) 456.7 (206.9) nsf 369.2 (230.3) nse
Total LED mg/d Mean (SD) 496.4 (233.7) 537.9 (258.9) nsf 525.7 (287.4) nsf
Categorical data were examined by Fisher’s exact test.

Key: DSM-IV, Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition; GBA, glucocerebrosidase gene; GD, Gaucher disease; H-Y, Hoehn and Yahr; ICD, impulse control disorder; LED, levodopa equivalent dose; ns, not significant; MMSE, Mini-Mental State Examination; PD, Parkinson’s disease; SD, standard deviation; UPDRS, Unified Parkinson’s Disease Rating Scale.

a Including a double-mutation subject (with a mutation unreported in GD).
b GD-associated mutations versus mutation (-).
c Two subjects with double mutation, including GD-associated mutations, were assigned to GD-associated mutation group.
d Other mutations versus mutation (-).
e Examined by Student t test after Levene’s test for equality of variances.
f Examined by Mann-Whitney U-test because of non-Gaussian distribution.
g    n = 155 due to 10 missing data.
h    n = 164 due to 3 missing data.
i     n = 28 due to 1 missing datum.
j     n = 165 due to 2 missing data.

3.4. Survival time analyses to develop dementia, psychosis, dyskinesia, and wearing-off

Time to develop clinical outcomes (dementia, psychosis, dyskinesia, and wearing-off) was compared in 19 subjects with GD-associated mutations, 29 with mutations unreported in GD, and 167 without mutation. The median observation time was 6.0 years. The subjects with GD-associated mutations showed a significantly earlier development of dementia and psychosis, compared with subjects without mutation (p < 0.001 and p = 0.017) ( Supplementary Table e-1, Fig. 1A and B). We rereviewed the clinical record of the subject who showed early dementia (defined by DSM IV) ( Fig. 1A) and made sure it did not satisfy the criteria of DLB ( McKeith et al., 2005).

Kaplan–Meier curves of dementia and psychosis in Parkinson's disease (PD) ...
Fig. 1.

Kaplan–Meier curves of dementia and psychosis in Parkinson’s disease (PD) patients with Gaucher disease (GD)-associated glucocerebrosidase gene (GBA) mutations and those without mutations. PD patients with GD-associated GBA mutations and those without GBA mutations were compared to investigate the time taken to develop dementia (A) and psychosis (B). Because of insufficient information in several patients, the numbers in each analysis were different. The patients with and without mutations were 17 and 165 (A), 18 and 165 (B) against a total of 19 and 167. DSM IV, Diagnostic and Statistical Manual of Mental Disorders, revised fourth edition. p-Values were calculated by log-rank tests.

The associations of GBA mutations and these symptoms were estimated as HRs, adjusting for sex and age at PD onset. HRs were 8.3 for dementia (95% CI, 3.3–20.9; p < 0.001) and 3.1 for psychosis (95% CI, 1.5–6.4; p = 0.002). The time until development of wearing-off and dyskinesia complications was not statistically significant, with HRs of 1.5 (95% CI, 0.8–3.1; p = 0.219) and 1.9 (95% CI, 0.9–4.1; p = 0.086) ( Table 3).

Table 3.Hazard ratios of GBA pathogenic mutations for clinical symptoms

Model Clinical feature Hazard ratio 95% CI p
1 Dementia (DSM-IV) 8.3 3.3–20.9 <0.001
2 Psychosis 3.1 1.5–6.4 0.002
3 Wearing-off 1.5 0.8–3.1 0.219
4 Dyskinesia 1.9 0.9–4.1 0.086
Each model was adjusted for sex and age at onset.

Key: CI, confidence interval; DSM-IV; The Diagnostic and Statistical Manual of Mental Disorders part 1IV; GBA, glucocerebrosidase.

Subjects with mutations unreported in GD did not show significant differences in time to develop all 4 outcomes, compared with no mutation subjects. Therefore, subjects with GD-unreported mutations were regarded as subjects without GBA mutations in further analyses.

3.5. rCBF on SPECT in patients with GD-associated GBA mutations

We conducted pixel-by-pixel comparisons of rCBF on SPECT between PD subjects with mutations (cases) and sex-, age-, and disease duration-matched PD subjects without any mutations in GBA (controls). Four controls were adopted for each case (except for a 34-year-old female case who was matched to a control), and in total 12 cases (female 50%, age at SPECT mean ± standard error (SE); 58.9 ± 3.3 years, disease duration at SPECT 7.3 ± 1.5 years) and 45 controls (female 64.4%, age at SPECT mean ± SE; 61.0 ± 1.3 years, disease duration at SPECT 7.1 ± 0.7 years) were analyzed. As a result, a significantly lower rCBF was seen in the cases compared to the controls in the bilateral parietal cortex, including the precuneus ( Fig. 2).

Regional cerebral blood flow in the group with GD-associated mutations compared ...
Fig. 2.

Regional cerebral blood flow in the group with GD-associated mutations compared with the matched Parkinson’s disease group without mutations. Regions with lower regional cerebral blood flow in the group with GD-associated mutations displayed on an anatomic reference map. Abbreviation: GD, Gaucher disease.

3.6. H/M ratios on MIBG scintigraphy in patients with GD-associated GBA mutations

Cardiac MIBG scintigraphy visualizes catecholaminergic terminals in vivo that are reduced as well as brain dopaminergic neurons in PD patients. We also investigated MIBG scintigraphy between 16 cases (female 68.8%, age at examination mean ± SE; 60.2 ± 2.6 years, disease duration at examination 6.2 ± 1.2 years) and sex-, age- and disease duration-matched 61 controls [(63.8 %, age 62.0 ± 1.1 years, disease duration 5.5 ± 0.6 years) (1:4 except for 1 young 34-year-old female case who was matched to a control)]. In the results, both early and late H/M ratios declined in both groups and did not show any significant differences (p = 0.309 and 0.244) ( Supplementary Table e-2).

4. Discussion

4.1. Contributions of GD-associated GBA mutations to the development of PD

In the analysis of 215 PD patients and 126 non-PD controls, we identified 10 nonsynonymous heterozygous GBA mutations, including 1 novel mutation. Among these mutations, 7 were GD-associated, and the patients carrying these mutations represented 8.8% of the PD cohort. No significant association was found between the GD-unreported mutations and PD development, which suggests that only the GD-associated mutations are a genetic risk for PD. According to a worldwide multicenter analysis of 1883 fully sequenced PD patients, 7% of the GD-associated mutations are found in non-Ashkenazi Jewish PD patients ( Sidransky et al., 2009). Although the mutation frequency in the present study was similar to previous results, the OR of GD-associated heterozygous mutations (25.1) was significantly greater than the OR (5.43) of other ethnic cohorts (Sidransky et al., 2009) and was consistent with an OR of 28.0 from a previous Japanese report ( Mitsui et al., 2009). These results, taken together, suggest the possibility thatGBA mutations are at a distinct risk for PD in the Japanese population. However, a larger Japanese cohort study is required to confirm this.

4.2. Cross-sectional clinical figures of PD with GBA mutations

Before the survival time analyses, we investigated clinical features at enrollment between mutation groups. The lower onset age, more frequent family history and dementia, and worse disease severity of PD in patients with GBA mutations, compared with those without mutations, were consistent with previous cross-sectional case-control reports ( Anheim et al., 2012, Brockmann et al., 2011, Chahine et al., 2013, Lesage et al., 2011, Li et al., 2013, Mitsui et al., 2009, Neumann et al., 2009, Seto-Salvia et al., 2012 and Sidransky et al., 2009). In contrast, female-predominance (73.7%, p = 0.047) in patients with mutations observed in the present study is inconsistent ( Neumann et al., 2009 and Seto-Salvia et al., 2012).

4.3. Impact of GBA mutations on the clinical course of PD

To investigate the impact of GBA mutations on the clinical course of PD, a prospective-designed study over a long period is preferred. Although there has been a few longitudinally designed study to date, follow-up clinical data for a median of 6 years of 121 PD cases from a community-based incident cohort was recently reanalyzed; results demonstrate that progression to dementia defined by DSM IV (HR 5.7) and Hoehn and Yahr stage 3 (HR 3.2) are significantly earlier in 4 GBA mutation-carrier patients compared with 117 patients with wild-type GBA ( Winder-Rhodes et al., 2013). A 2-year follow-up clinical report of 28 heterozygous GBA carriers who were recruited from relatives of GD-patients shows slight but significant deterioration of cognition and smelling, compared to healthy controls ( Beavan et al., 2015). Brockmann et al. (2015)assessed motor and nonmotor symptoms including cognitive and mood disturbances for 3 years in 20 PD patients with GBA mutations and showed a more rapid disease progression of motor impairment and cognitive decline in GBA mutation cases comparing to sporadic PD controls. The current long-term retrospective cohort study up to 12 years reinforced these results. It revealed that dementia and psychosis developed significantly earlier in subjects with GD-associated mutations compared with those without mutation, and the HRs of GBA mutations were estimated at 8.3 for dementia and 23.1 for psychosis, with adjustments for sex and PD onset age. In contrast, the results showed no significant difference in developing wearing-off and dyskinesia.

In this study, we also investigated whether GD-unreported mutations affected the clinical course of PD. In both cross-sectional and survival time analyses, the mutations unreported in GD carried no increased burden on clinical symptoms such as dementia, psychosis, wearing-off, and dyskinesia.

4.4. Reduced rCBF in PD with GBA mutations compared with matched PD controls

We found a significantly decreased rCBF, reflecting decreased synaptic activity, in the bilateral parietal cortex including the precuneus, in subjects with GD-associated mutations compared with matched subjects without mutations. The pattern of reduced rCBF was very similar to the pattern of H215O positron-emission tomography that Goker-Alpan (2012) reported, showing decreased resting rCBF in the lateral parietal association cortex and the precuneus bilaterally in GD subjects with parkinsonism (7 subjects with homozygous or compound heterozygous GBA mutations), compared with 11 PD without GBA mutations. Results suggest that PD with heterozygous GBAmutations and GD patients presenting parkinsonism had a common reduced pattern of rCBF. Interestingly, in their study, rCBF in the precuneus—but not in the lateral parietal cortex—correlated with IQ, suggesting that the involvement of the precuneus is critical for defining GBA-associated patterns.

4.5. Reduced cardiac MIBG H/M ratios as well as matched PD controls

We also showed that cardiac MIBG H/M ratios in subjects with GD-associated mutations were lower than the cutoff point for PD discrimination (Sawada et al., 2009), suggesting that postganglionic sympathetic nerve terminals to the epicardium were denervated, as well as in PD without mutations.

4.6. Mechanisms of impact on PD clinical course by GD-associated GBA mutations

Experimental studies suggesting a bidirectional pathogenic loop between α-synuclein and glucocerebrosidase have been accumulated (Fishbein et al., 2014, Gegg et al., 2012, Mazzulli et al., 2011, Noelker et al., 2015, Schondorf et al., 2014 and Uemura et al., 2015). Loss of glucocerebrosidase function compromises α-synuclein degradation in lysosome, whereas aggregated α-synuclein inhibits normal lysosomal function of glucocerebrosidase. The pathogenic loop may facilitate neurodegeneration in GD-associated PD brain, resulting in early development of dementia or psychosis as shown in the present study. Several recent researches propose the possibility that the similar mechanism as in PD with GBA mutations exists even in idiopathic PD brain ( Alcalay et al., 2015, Chiasserini et al., 2015, Gegg et al., 2012 and Murphy et al., 2014). On the other hand, the impacts of GD-associated GBA mutations for the development of motor complications such as wearing-off and dyskinesia were not statistically significant, suggesting other pathophysiological mechanisms in the striatal circuit brought out after long-term therapy especially by l-dopa.

4.7. Limitations

Our study has several limitations. In the design of the study, we assumed that the sample size was 215 (PD patients) for survival time analyses and investigated 224 PD patients. We assumed that the mutation prevalence would be 9.4%, and in fact, we found 19 patients with mutations (8.5%) of the 224 patients. Based on these figures, we estimated the risk ratios of heterozygous GBA mutations for the risk of PD development and PD clinical symptoms as ORs in the cross-sectional multivariate analyses, although the 95% CIs were broad. More of subject numbers will be needed to determine robust risk ratios.

Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort

PLOS   Published: July 14, 2015   DOI:

Brugada syndrome (BrS) was identified as a new clinical entity in 1992 [1]. Six years later, the first genetic basis for the disease was identified, with the discovery of genetic variations inSCN5A [2]. Nowadays, more than 300 pathogenic variations in this first gene are known to be associated with BrS [3]. SCN5A encodes for the α subunit of the cardiac voltage-dependent sodium channel (Nav1.5), which is responsible for inward sodium current (INa), and thus plays an essential role in phase 0 of the cardiac action potential (AP). Genetic variations in this gene can explain around 20–25% of BrS cases [3].

Since BrS was classified as a genetic disease, several other genes have been described to confer BrS-susceptibility [47]. Pathogenic variations have been mainly described in: 1) genes encoding proteins that modulate Nav1.5 function, and 2) other calcium and potassium channels and their regulatory subunits. All these proteins participate, either directly or indirectly, in the development of the cardiac AP. Although the incidence of pathogenic variations in these BrS-associated genes is low [6], it is considered that, among all of them, they could provide a genetic diagnosis for up to an extra 5–10% of BrS cases. Hence, altogether, a genetic diagnosis can be achieved approximately in 35% of clinically diagnosed BrS patients.
Other types of genetic abnormalities have been suggested to explain the remaining percentage of undiagnosed patients. Indeed, multiplex ligation-dependent probe amplification (MLPA) has allowed the detection of large-scale gene rearrangements involving one or several exons ofSCN5A in BrS cases. However, the low proportion of BrS patients carrying large genetic imbalances identified to date suggests that this type of rearrangements will provide a genetic diagnosis for a modest percentage of BrS cases [810].
BrS has been associated with an increased risk of sudden cardiac death (SCD), despite the reported variability in disease penetrance and expressivity [11]. The prevalence of BrS is estimated at about 1.34 cases per 100 000 individuals per year, with a higher incidence in Asia than in the United States and Europe [12]. However, the dynamic nature of the typical electrocardiogram (ECG) and the fact that it is often concealed, hinder the diagnosis of BrS. Therefore, an exhaustive genetic testing and subsequent family screening may prove to be crucial in identifying silent carriers. A large percentage of these pathogenic variation carriers are clinically asymptomatic, and may be at risk of SCD, which is, sometimes, the first manifestation of the disease [13].
In the present work, we aimed to determine the spectrum and prevalence of genetic variations in BrS-susceptibility genes in a Spanish cohort diagnosed with BrS, and to identify variation carriers among relatives, which would enable the adoption of preventive measures to avoid SCD in their families.

Study population 

Table 1. Demographics of the 55 Spanish BrS patients included in the study.

The table shows the demographic characteristics of all the patients included in the study. Numbers in parentheses represent the relative percentages for each condition. T1 ECG refers to Type 1 BrS diagnostic electrocardiogram (ECG), obtained either spontaneously, or after drug challenge. The information regarding both the electrophysiological studies (EPS) and the treatment was not available for all the patients. Two of the patients that didn’t receive any treatment died, and were not taken into account for the calculations of percentages (+2 dead). ICD, intracardiac cardioverter defibrillator.


Table 2. Characteristics of the Spanish BrS patients carrying rare genetic variations.

The table shows the clinical characteristics of the probands who carried rare genetic variations in SCN5A, SCN2B, or RANGRF. All of them are potentially pathogenic except that found in RANGRF, which is of unknown significance (see discussion). All the potentially pathogenic variations (PPVs) that had been previously reported, except p.P1725L and p.R1898C, had been identified in BrS patients. p.P1725L had been associated with Long QT Syndrome and p.R1898C was found in Exome Variant Server with a MAF of 0.0079%. No rare variations were identified in the control population. Patient’s age is expressed in years. Bold identifies the patients carrying variations that had not been described previously. M, male; F, female; S, syncope; ICD, intracardiac cardioverter defibrillator; UK, unknown; EPS, electrophysiological studies (+, positive response;-, negative response; N/P, not performed). The two patients who carried two PPVs each are identified by a and b, respectively.

Sequencing of genes associated with BrS

We performed a genetic screening of 14 genes (SCN5A, CACNA1C, CACNB2, GPD1L,SCN1B, SCN2B, SCN3B, SCN4B, KCNE3, RANGRF, HCN4, KCNJ8, KCND3, and KCNE1L), which allowed the identification of 61 genetic variations in our cohort. Of these, 20 were classified as potentially pathogenic variations (PPVs), one variation of unknown significance, and 40 common or synonymous variants considered benign.

The 20 PPVs were found in 18 of the 55 patients (32.7% of the patients, 83.3% males; Table 2). Sixteen patients (88.9%) carried one PPV, and two patients (11.1%) carried two different PPVs each. Nineteen out of the 20 PPVs identified were localized in SCN5A and one in SCN2B.

The vast majority of the PPVs identified were missense (70%). We also detected 2 nonsense variations (10%), 3 insertions or deletions causing frameshifts (15%), and one splicing variation (5%). The three frameshifts (p.R569Pfs*151, p.E625Rfs*95 and p.R1623Efs*7) were identified in SCN5A. These were not found in any of the databases consulted (see Methods), and were thus considered potentially pathogenic (see below). The other 16 rare variations identified inSCN5A had been previously described, and hence were also considered potentially pathogenic. Fourteen of them had been identified in BrS patients. Of these, 6 had also been identified in individuals diagnosed with other cardiac electric diseases (i.e. Sick Sinus Syndrome, Long QT Syndrome, Sudden Unexplained Nocturnal Death Syndrome or Idiopathic Ventricular Fibrillation [2,15,16,20,21,25]). The other 2, p.P1725L and p.R1898C, had only been associated with Long QT Syndrome or found in Exome Variant Server with a MAF of 0.0079%, respectively. Furthermore, we identified a variation in SCN2B (c.632A>G in exon 4 of the gene, resulting in p.D211G) which was considered pathogenic. This patient was included within our cohort, but the functional characterization of channels expressing SCN2B p.D211G was object of a previous study from our group [7]. We also identified a nonsense variation in RANGRFwhich has been formerly reported as rare genetic variation of unknown significance [29].

Additionally, we screened the relatives of those probands carrying a PPV. We analysed a total of 129 relatives, 69 of which (53.5%) were variation carriers. Genotype-phenotype correlations evidenced that 8 of the families displayed complete penetrance (S3 Table). Additionally, no relatives were available for one of the probands carrying a PPV, thus hampering genotype-phenotype correlation assessment. The other 12 families showed incomplete penetrance.


MLPA analysis

The 37 patients with negative results after the genetic screening of the 14 BrS-associated genes underwent MLPA analyses of SCN5A. This technique did not reveal any large exon deletion or duplication in this gene for any of the patients.


SCN5A p.R569Pfs*151 (c.1705dupC), a novel PPV

A 41-year-old asymptomatic male presented a type 3 BrS ECG which was suggestive of BrS. Flecainide challenge unmasked a type 1 BrS ECG (Fig 1A, left), which was also spontaneously observed sometimes during medical follow up. Sequencing of SCN5A revealed a duplication of a cytosine at position 1705 (c.1705dupC; Fig 1A, right), which originated a frameshift that lead to a truncated Nav1.5 channel (p.R569Pfs*151). The proband’s sister also carried this duplication, but had never presented signs of arrhythmogenesis. The proband’s twin daughters were also variation carriers, displayed normal ECGs and, to date, are asymptomatic (Fig 1A, middle). Thus, p.R569Pfs*151 represents a novel genetic alteration in the Nav1.5 channel that could potentially lead to BrS, but with incomplete penetrance.


Fig 1. Characteristics of the probands carrying non-reported potentially pathogenic variations (PPVs) in SCN5A and their families.

Left: Electrocardiograms of the probands: (A) patient carrying the p.R569Pfs*151 variation, showing the ST elevation characteristic of BrS in V1 at the time of the flecainide test; (B) patient carrying the p.E625Rfs*95 variation, showing the spontaneous ST elevation characteristic of BrS in V1 and V2; and (C) patient carrying the p.R1623Efs*7 variation, showing the spontaneous ST elevation characteristic of BrS in V1 and V2. Middle: Family pedigrees. Open symbols designate clinically normal subjects, filled symbols mark clinically affected individuals and question marks identify subjects without an available clinical diagnosis. Plus signs indicate the carriers of the PPVs and minus signs, non-carriers. The crosses mark deceased individuals and arrows identify the proband. Right: Detail of the electropherograms obtained after SCN5Asequence analysis of a control subject (left panels) and of the probands (right panels).

SCN5A p.E625Rfs*95 (c.1872dupA), a novel PPV

A 51-year-old asymptomatic male was diagnosed with BrS since he presented a spontaneous ST segment elevation in leads V1 and V2 characteristic of type 1 BrS ECG (Fig 1B, left). The sequencing of SCN5A evidenced an adenine duplication at position 1872 (c.1872dupA, Fig 1B, right). This genetic variation results in a truncated Nav1.5 channel (p.E625Rfs*95). The genetic analysis of the proband’s relatives proved that only her mother carried the variation (Fig 1B, middle). She was asymptomatic, but a BrS ECG was unmasked upon ajmaline challenge. The proband’s sister was found dead in her crib at 6 months of age, which suggests that her death might be compatible with BrS. Therefore, the p.E625Rfs*95 variation in the Nav1.5 channel represents a novel genetic alteration potentially causing BrS.

SCN5A p.R1623Efs*7 (c.4867delC), a novel PPV

The proband, a 31-year-old male, was admitted to hospital after suffering a syncope. His baseline 12-lead ECG showed a ST segment elevation in leads V1 and V2 that strongly suggested BrS type 1 (Fig 1C, left). A deletion of the cytosine at position 4867 (c.4867delC) was observed upon SCN5A sequencing (Fig 1C, right). This base deletion leads to a frameshift that originates a truncated Nav1.5 channel (p.R1623Efs*7). Genetic screening of his parents and sisters evidenced that none of them carried this novel variation (Fig 1C, middle). None of them had presented any signs of arrhythmogenicity, nor had a BrS ECG. Nevertheless, in uterogenetic analysis of one of his daughters proved that she had inherited the variation. She died when she was 1 year of age of non-arrhythmogenic causes. Hence, the p.R1623Efs*7 variation in the Nav1.5 channel is a novel genetic alteration originated de novo in the proband that could potentially lead to BrS.

Synonymous and common genetic variations portrayal

In our cohort, we identified 40 single nucleotide variations which were common genetic variants and/or synonymous variants (S2 Table). Twenty-nine had a minor allele frequency (MAF) over 1%, and were thus considered common genetic variants.

We also identified 11 variants with MAF less than 1%. Of them, 9 were synonymous variants, what made us assume that they were not disease-causing. Four of these synonymous variants were not found in any of the databases consulted, and thus their MAF was considered to be less than 1%. Each of these synonymous variations was identified in 1 patient of the cohort. A similar proportion of individuals carrying these novel variations was detected upon sequencing of 300 healthy Spanish individuals (600 alleles). The remaining 2 variants were missense, and although they had either a MAF of less than 1% or an unknown MAF according to the Exome Variant Server and dbSNP websites, they were common in our cohort (29.2 and 50%, respectively; S2 Table), and a similar MAF was detected in a Spanish cohort of healthy individuals (26.7% and 48.8%, respectively).

Influence of phenotype and age on PPV discovery

To assess if a connection existed between the probands’ phenotype and the PPV detection yield, we classified the patients in our cohort according to their ECG (spontaneous or induced type 1), the presence of BrS cases within their families, and the presence/absence of symptoms. Even though the overall PPV detection yield was 32.7%, it was even higher for symptomatic patients (Fig 2). Indeed, in this group of patients, having a family history of BrS was identified as a factor for increased PPV discovery yield. In the case of absence of BrS in the family, the variation discovery yield was almost double for those patients having a spontaneous type 1 BrS ECG than for patients with drug-induced type 1 ECG (45.5% vs 25%, respectively). In addition, we identified a PPV in 44.4% of the asymptomatic patients who presented family history of BrS and a spontaneous type 1 BrS ECG. When the patient presented drug-induced type 1 ECG or in the absence of family history of BrS, the PPV discovery yield was of around 15%.


Fig 2. Influence of the phenotype on PPV discovery yield.

Bar graph comparing the PPV detection yield in 8 different clinical categories (stated below the graph). Each bar shows the total number of patients for each clinical category divided in those with a PPV (black) and those without an identified PPV (white). The number of patients (in brackets) and percentages are given. Pos, positive; Neg, negative; Spont, spontaneous type 1 BrS ECG; Drug, drug-induced type 1 BrS ECG; n, number of patients.

We also investigated the role of age on the PPV occurrence. No significant age differences were observed between variation carriers and non-carriers (38.6±10.3 and 43.5±14.4, respectively, p = 0.16). However, the PPV discovery yield was higher for patients with ages between 30 and 50 years: out of the total of patients carrying a PPV, 83.3% of the patients were in this age range, while 11.1% were younger and 5.6% were older patients (Fig 3A, upper panel). The PPV discovery yield was significantly higher for symptomatic than for asymptomatic patients (42.3% vs 24.1%, respectively; Fig 3A, lower panels).


Fig 3. Influence of the age on PPVs discovery yield.

(A) Pie charts showing the distribution of patients in the overall population as well as in the categories of symptomatic and asymptomatic patients regarding PPV discovery. The percentage and the number of patients (in brackets) are given for each group. The small pie charts correspond to the age distribution of patients with an identified PPV. (B) Bar graphs of the PPV detection yields obtained for each of the age groups (< 30 years, 30–50 years and > 50 years). Numbers inside each bar correspond to the number of patients carrying a PPV for each category and the percentages represent the variation detection yield.

Noteworthy, in the 30–50 age range, 52.9% (9/17) of the symptomatic patients and 35.3% (6/17) of asymptomatic patients carried one PPV (Fig 3B, middle). Additionally, 40% (2/5) of the symptomatic young patients (< 30 years) were variation carriers, while no PPVs were identified in asymptomatic patients within this age range.

Overall, 55 unrelated Spanish patients clinically diagnosed with BrS were included in our study.Table 1 shows the demographics of this cohort, and Table 2 and S1 Table show the clinical and genetic characteristics of all the patients included in the study. The mean age at clinical diagnosis was of 41.9±13.3 years. Although the majority of patients were males (74.5%), their age at diagnosis was not different than that of females (41.8±12.1 years and 42.3±16.3 years, respectively; p = 0.92). A type 1 BrS ECG was present spontaneously in 37 patients (67.3%), and drug challenge revealed a type 1 BrS ECG for the remaining 18 patients (32.7%). Almost half of the patients had experienced symptoms, including 2 SCD and 4 aborted SCD. Patients who had not previously experienced any signs of arrhythmogenicity despite having a BrS ECG were considered asymptomatic. Comparison of symptomatic vs asymptomatic patients evidenced a similar percentage of males (73.1% and 75.9%, respectively). However, the mean age at diagnosis was different between the two groups of patients (37.7±14.3 and 45.7±11.4, respectively; p<0.05).


To the best of our knowledge, this is the first comprehensive genetic evaluation of 14 BrS-susceptibility genes and MLPA of SCN5A in a Spanish cohort. Well delimited BrS cohorts from Japan, China, Greece and even Spain have been genetically studied [24,3032]. Additionally, an international compendium of BrS genetic variations identified in more than 2100 unrelated patients from different countries was published in 2010 [3]. However, all these studies screenedSCN5A exclusively. In 2012, Crotti et al. reported the spectrum and prevalence of genetic variations in 12 BrS-susceptibility genes in a BrS cohort [5]. However, this study included patients of different ethnicity. Here, we report the analysis of 14 genes which has been conducted on a well-defined BrS cohort of the same ethnicity.

Our results confirm that SCN5A is still the most prevalent gene associated with BrS. Indeed,SCN5A-mediated BrS in our cohort (30.9%) is higher than the proportion described in other European reports [3,23], where a potentially causative variation is identified in only 20–25% of BrS patients. The reason for this discrepancy is unclear but could point towards a higher prevalence of SCN5A PPVs in the Spanish population or to selection bias. Additionally, we identified a genetic variation in SCN2B (c.632A>G, which results in p.D211G). We have formerly published the comprehensive electrophysiological characterization of this variation, and showed that indeed this variation could be responsible of the phenotype of the patient, thus linking SCN2B with BrS for the first time [7]. Also, we identified a variation in RANGRF. This variation (c.181G>T leading to p.E61X) had been previously reported in a Danish atrial fibrillation cohort [33]. Surprisingly, the authors reported an incidence of 0.4% for this variation in the healthy Danish population, which brought into question its pathogenicity. Our finding of this variation in an asymptomatic patient displaying a type 2 BrS ECG also points toward considering it as a rare genetic variation with a potential modifier effect on the phenotype but not clearly responsible for the disease [29].

No PPVs were identified in the other genes tested. Certainly, it is well accepted that the contribution of these genes to the disease is minor, and thus should only be considered under special circumstances [13,34]. In addition, recent studies have questioned the causality of variations identified in some of these minority genes [35].

We also used the MLPA technique for the detection of large exon duplications and/or deletions in SCN5A in patients without PPVs, and no large rearrangements were identified. This is in accordance with previous reports, which revealed that such imbalances are uncommon [810].

Kapplinger et al. [3] reported a predominance of PPVs in transmembrane regions of Nav1.5. Indeed, it has been proposed that most rare genetic variations in interdomain linkers may be considered as non-pathogenic [36]. In contrast, PPVs identified in this study are mainly located in extracellular loops and cytosolic linker regions of Nav1.5 (Fig 4). Additionally, 2 of our non-previously reported frameshifts are located in the DI-DII linker. These 2 genetic variations lead to truncated proteins, which would lack around 75% of the protein sequence, and thus are presupposed to be pathogenic.


Fig 4. Nav1.5 channel scheme showing the relative position of the SCN5A PPVs identified in our cohort.

Open symbols indicate already described variations and closed symbols locate novel variations reported in this study. DI to DIV designate the 4 domains of the protein, and numbers 1–6 identify the different segments within each domain. Crosses mark the voltage sensor.

In our cohort, we have identified 40 synonymous or common genetic variations, 4 of which have not been previously reported. These variations are gradually becoming more and more important in the explanation of certain phenotypes of genetic diseases. Only a few common variations identified here are already published as phenotypic modifiers [37,38]. The effect of these and other common variants identified in our cohort on BrS phenotype should be further studied.

Unexpectedly, almost 40% (7/18) of the PPV carriers did not present signs of arrhythmogenicity. We also performed genotype-phenotype correlations of the PPVs identified in the families (S3 Table). These studies uncovered relatives, most of whom were young individuals, who carried a familial variation but had never exhibited any clinical manifestations of the disease. This is in agreement with Crotti et al. and Priori et al. [5,23], who postulated that a positive genetic testing result is not always associated with the presence of symptoms. Indeed, the existence of asymptomatic patients carrying genetic variations described to cause a severe Nav1.5 channel dysfunction has been reported [39]. The identification of silent carriers is of paramount importance since it allows the adoption of preventive measures before any lethal episode takes place. Unknown environmental factors, medication and modifier genes have been suggested to influence and/or predispose to arrhythmogenesis [11]. Hence, this group of patients has to be cautiously followed in order to avoid fatal events.

Our studies on the connection between patients’ phenotype and the PPV detection yield highlighted the presence of symptoms as a factor for an increased variation discovery yield. Within the group of symptomatic individuals, a PPV was identified in a higher proportion of patients displaying a spontaneous type 1 BrS ECG than for patients showing a drug-induced ECG. Likewise, within the asymptomatic patients with family history of BrS, those who presented spontaneous type 1 BrS ECG carried a PPV more often than those with a drug-induced ECG (Fig 2). Referring to age, the vast majority (17/20, 85%) of the PPVs were identified in patients around their fourth decade of age (30–50 years). This is in accordance with the accepted mean age of disease manifestation. Moreover, in this age range, more than 50% of the patients who presented symptoms carried a variation that could be pathogenic (Fig 3). Importantly, 35.3% of asymptomatic patients of around 40 years of age also carried one of such variations. These data highlight the importance of performing a genetic test even in the absence of clinical manifestations of the disease, and particularly when in the 30–50 years range, which is in accordance with consensus recommendations [13,34].

In conclusion, we have analysed for the first time 14 BrS-susceptibility genes and performed MLPA of SCN5A in a Spanish BrS cohort. Our cohort showed male prevalence with a mean age of disease manifestation around 40 years. BrS in this cohort was almost exclusivelySCN5A-mediated. The mean PPV discovery yield in our Spanish BrS patients is higher than that described for other BrS cohorts (32.7% vs 20–25%, respectively), and is even higher for patients in the 30–50 years age range (up to 53% for symptomatic patients). All these evidences support the genetic testing, at least of SCN5A, in all clinically well diagnosed BrS patients.


Study Limitations

First of all, drug challenge tests were not performed for all the relatives who were asymptomatic variation carriers. This fact hampered their clinical diagnosis and represents an impediment to definitely assess the link between PPVs and BrS. These patients are nowadays under follow-up.

New PPVs have been identified in our cohort. The clinical information available for the families suggests that these new variations could be pathogenic. Still, in vitro studies of these variations are required in order to evaluate their functional effects and verify their pathogenic role. Additionally, genotyping in an independent cohort would help reduce the likelihood of type I (false positive) error in genetic variant discovery.

We have to acknowledge that the study set is relatively small. Consequently, the classification of patients according to the different clinical categories rendered rather small sub-groups, which may lead to over-interpretation of the results. Future studies will be directed to the genetic screening of additional Spanish BrS patients, which will probably reinforce the significance of the tendencies observed here.


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Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine

Introduction to e-Series A: Cardiovascular Diseases, Volume Four Part 2: Regenerative Medicine

Author and Curator: Larry H Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN

This document is entirely devoted to medical and surgical therapies that have made huge strides in

  • simplification of interventional procedures,
  • reduced complexity, resulting in procedures previously requiring surgery are now done, circumstances permitting, by medical intervention.

This revolution in cardiovascular interventional therapy is regenerative medicine.  It is regenerative because it is largely driven by

  • the introduction into the impaired vasculature of an induced pleuripotent cell, called a stem cell, although
  • the level of differentiation may not be a most primitive cell line.

There is also a very closely aligned development in cell biology that extends beyond and including vascular regeneration that is called synthetic biology.  These developments have occurred at an accelerated rate in the last 15 years. The methods of interventional cardiology were already well developed in the mid 1980s.  This was at the peak of cardiothoracic bypass surgery.

Research on the endothelial cell,

  • endothelial cell proliferation,
  • shear flow in small arteries, especially at branch points, and
  • endothelial-platelet interactions

led to insights about plaque formation and vessel thrombosis.

Much was learned in biomechanics about the shear flow stresses on the luminal surface of the vasculature, and there was also

  • the concomitant discovery of nitric oxide,
  • oxidative stress, and
  • the isoenzymes of nitric oxide synthase (eNOS, iNOS, and nNOS).

It became a fundamental tenet of vascular biology that

  • atherogenesis is a maladjustment to oxidative stress not only through genetic, but also
  • non-genetic nutritional factors that could be related to the balance of omega (ω)-3 and omega (ω)-6 fatty acids,
  • a pro-inflammatory state that elicits inflammatory cytokines, such as, interleukin-6 (IL6) and c-reactive protein(CRP),
  • insulin resistance with excess carbohydrate associated with type 2 diabetes and beta (β) cell stress,
  • excess trans- and saturated fats, and perhaps
  • the now plausible colonic microbial population of the gastrointestinal tract (GIT).

There is also an association of abdominal adiposity,

  • including the visceral peritoneum, with both T2DM and with arteriosclerotic vessel disease,
  • which is presenting at a young age, and has ties to
  • the effects of an adipokine, adiponectin.

Much important work has already been discussed in the domain of cardiac catheterization and research done to

  • prevent atheroembolization.and beyond that,
  • research done to implant an endothelial growth matrix.

Even then, dramatic work had already been done on

  • the platelet structure and metabolism, and
  • this has transformed our knowledge of platelet biology.

The coagulation process has been discussed in detailed in a previous document.  The result was the development of a

  • new class of platelet aggregation inhibitors designed to block the activation of protein on the platelet surface that
  • is critical in the coagulation cascade.

In addition, the term long used to describe atherosclerosis, atheroma notwithstanding, is “hardening of the arteries”.  This is particularly notable with respect to mid-size arteries and arterioles that feed the heart and kidneys. Whether it is preceded by or develops concurrently with chronic renal insufficiency and lowered glomerular filtration rate is perhaps arguable.  However, there is now a body of evidence that points to

  • a change in the vascular muscularis and vessel stiffness, in addition to the endothelial features already mentioned.

This has provided a basis for

  • targeted pharmaceutical intervention, and
  • reduction in salt intake.

So we have a  group of metabolic disorders, which may alone or in combination,

  • lead to and be associated with the long term effects of cardiovascular disease, including
  • congestive heart failure.

This has been classically broken down into forward and backward failure,

  • depending on decrease outflow through the aorta (ejection fraction), or
  • decreased venous return through the vena cava,

which involves increased pulmonary vascular resistance and decreased return into the left atrium.

This also has ties to several causes, which may be cardiac or vascular. This document, as the previous, has four pats.  They are broadly:

  1. Stem Cells in Cardiovascular Diseases
  2. Regenerative Cell and Molecular Biology
  3. Therapeutics Levels In Molecular Cardiology
  4. Research Proposals for Endogenous Augmentation of circulating Endothelial Progenitor Cells (cEPCs)

As in the previous section, we start with the biology of the stem cell and the degeneration in cardiovascular diseases, then proceed to regeneration, then therapeutics, and finally – proposals for augmenting therapy with circulating endogenous endothelial progenitor cells (cEPCs).



stem cells























Key pathways involving NO

Key pathways involving NO





stem cell lin28

stem cellLlin28

1479-5876-10-175-1-l  translational research with feedback loops

Tranlational Research -Lab to Bedside



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Curation, HealthCare System in the US, and Calcium Signaling Effects on Cardiac Contraction, Heart Failure, and Atrial Fibrillation, and the Relationship of Calcium Release at the Myoneural Junction to Beta Adrenergic Release

Curation, HealthCare System in the US, and Calcium Signaling Effects on Cardiac Contraction, Heart Failure, and Atrial Fibrillation, and the Relationship of Calcium Release at the Myoneural Junction to Beta Adrenergic Release

Curator and e-book Contributor: Larry H. Bernstein, MD, FCAP
Curator and BioMedicine e-Series Editor-in-Chief: Aviva Lev Ari, PhD, RN


Content Consultant to Six-Volume e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC

This portion summarises what we have covered and is now familiar to the reader.  There are three related topics, and an extension of this embraces other volumes and chapters before and after this reading.  This approach to the document has advantages over the multiple authored textbooks that are and have been pervasive as a result of the traditional publication technology.  It has been stated by the founder of ScoopIt, that amount of time involved is considerably less than required for the original publications used, but the organization and construction is a separate creative process.  In these curations we amassed on average five articles in one curation, to which, two or three curators contributed their views.  There were surprises, and there were unfulfilled answers along the way.  The greatest problem that is being envisioned is the building a vision that bridges and unmasks the hidden “dark matter” between the now declared “OMICS”, to get a more real perspective on what is conjecture and what is actionable.  This is in some respects unavoidable because the genome is an alphabet that is matched to the mino acid sequences of proteins, which themselves are three dimensional drivers of sequences of metabolic reactions that can be altered by the accumulation of substrates in critical placements, and in addition, the proteome has functional proteins whose activity is a regulatory function and not easily identified.  In the end, we have to have a practical conception, recognizing the breadth of evolutionary change, and make sense of what we have, while searching for more.

We introduced the content as follows:

1. We introduce the concept of curation in the digital context, and it’s application to medicine and related scientific discovery.

Topics were chosen were used to illustrate this process in the form of a pattern, which is mostly curation, but is significantly creative, as it emerges in the context of this e-book.

  • Alternative solutions in Treatment of Heart Failure (HF), medical devices, biomarkers and agent efficacy is handled all in one chapter.
  • PCI for valves vs Open heart Valve replacement
  • PDA and Complications of Surgery — only curation could create the picture of this unique combination of debate, as exemplified of Endarterectomy (CEA) vs Stenting the Carotid Artery (CAS), ischemic leg, renal artery stenosis.

2. The etiology, or causes, of cardiovascular diseases consist of mechanistic explanations for dysfunction relating to the heart or vascular system. Every one of a long list of abnormalities has a path that explains the deviation from normal. With the completion of the analysis of the human genome, in principle all of the genetic basis for function and dysfunction are delineated. While all genes are identified, and the genes code for all the gene products that constitute body functions, there remains more unknown than known.

3. Human genome, and in combination with improved imaging methods, genomics offers great promise in changing the course of disease and aging.

4. If we tie together Part 1 and Part 2, there is ample room for considering clinical outcomes based on individual and organizational factors for best performance. This can really only be realized with considerable improvement in information infrastructure, which has miles to go.


Curation is an active filtering of the web’s  and peer reviewed literature found by such means – immense amount of relevant and irrelevant content. As a result content may be disruptive. However, in doing good curation, one does more than simply assign value by presentation of creative work in any category. Great curators comment and share experience across content, authors and themes.
Great curators may see patterns others don’t, or may challenge or debate complex and apparently conflicting points of view.  Answers to specifically focused questions comes from the hard work of many in laboratory settings creatively establishing answers to definitive questions, each a part of the larger knowledge-base of reference. There are those rare “Einstein’s” who imagine a whole universe, unlike the three blindmen of the Sufi tale.  One held the tail, the other the trunk, the other the ear, and they all said this is an elephant!
In my reading, I learn that the optimal ratio of curation to creation may be as high as 90% curation to 10% creation. Creating content is expensive. Curation, by comparison, is much less expensive.  The same source says “ is my content marketing testing “sandbox”. In sharing, he says that comments provide the framework for what and how content is shared.

Healthcare and Affordable Care Act

We enter year 2014 with the Affordable Care Act off to a slow start because of the implementation of the internet signup requiring a major repair, which is, unfortunately, as expected for such as complex job across the US, and with many states unwilling to participate.  But several states – California, Connecticut, and Kentucky – had very effective state designed signups, separate from the federal system.  There has been a very large rush and an extension to sign up. There are many features that we can take note of:

1. The healthcare system needed changes because we have the most costly system, are endowed with advanced technology, and we have inexcusable outcomes in several domains of care, including, infant mortality, and prenatal care – but not in cardiology.

2. These changes that are notable are:

  • The disparities in outcome are magnified by a large disparity in highest to lowest income bracket.
  • This is also reflected in educational status, and which plays out in childhood school lunches, and is also affected by larger class size and cutbacks in school programs.
  • This is not  helped by a large paralysis in the two party political system and the three legs of government unable to deal with work and distraction.
  • Unemployment is high, and the banking and home construction, home buying, and rental are in realignment, but interest rates are problematic.

3.  The  medical care system is affected by the issues above, but the complexity is not to be discounted.

  •  The medical schools are unable at this time to provide the influx of new physicians needed, so we depend on a major influx of physicians from other countries
  • The technology for laboratories, proteomic and genomic as well as applied medical research is rejuvenating the practice in cardiology more rapidly than any other field.
  • In fields that are imaging related the life cycle of instruments is shorter than the actual lifetime use of the instruments, which introduces a shortening of ROI.
  • Hospitals are consolidating into large consortia in order to maintain a more viable system for referral of specialty cases, and also is centralizing all terms of business related to billing.
  • There is reduction in independent physician practices that are being incorporated into the hospital enterprise with Part B billing under the Physician Organization – as in Partners in Greater Boston, with the exception of “concierge” medical practices.
  • There is consolidation of specialty laboratory services within state, with only the most specialized testing going out of state (Quest, LabCorp, etc.)
  • Medicaid is expanded substantially under the new ACA.
  • The federal government as provider of services is reducing the number of contractors for – medical devices, diabetes self-testing, etc.
  • The current rearrangements seeks to provide a balance between capital expenses and fixed labor costs that it can control, reduce variable costs (reagents, pharmaceutical), and to take in more patients with less delay and better performance – defined by outside agencies.

Cardiology, Genomics, and calcium ion signaling and ion-channels in cardiomyocyte function in health and disease – including heart failure, rhythm abnormalities, and the myoneural release of neurotransmitter at the vesicle junction.

This portion is outlined as follows:

2.1 Human Genome: Congenital Etiological Sources of Cardiovascular Disease

2.2 The Role of Calcium in Health and Disease

2.3 Vasculature and Myocardium: Diagnosing the Conditions of Disease

Genomics & Genetics of Cardiovascular Disease Diagnoses

actin cytoskeleton

wall stress, ventricular workload, contractile reserve

Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

calcium and actin skeleton, signaling, cell motility

hypertension & vascular compliance

Genetics of Conduction Disease

Ca+ stimulated exostosis: calmodulin & PKC (neurotransmitter)

complications & MVR

disruption of Ca2+ homeostasis cardiac & vascular smooth muscle

synaptotagmin as Ca2+ sensor & vesicles

atherosclerosis & ion channels

It is increasingly clear that there are mutations that underlie many human diseases, and this is true of the cardiovascular system.  The mutations are mistakes in the insertion of a purine nucleotide, which may or may not have any consequence.  This is why the associations that are being discovered in research require careful validation, and even require demonstration in “models” before pursuing the design of pharmacological “target therapy”.  The genomics in cardiovascular disease involves very serious congenital disorders that are asserted early in life, but the effects of and development of atherosclerosis involving large and medium size arteries has a slow progression and is not dominated by genomic expression.  This is characterized by loss of arterial elasticity. In addition there is the development of heart failure, which involves the cardiomyocyte specifically.  The emergence of regenerative medical interventions, based on pleuripotent inducible stem cell therapy is developing rapidly as an intervention in this sector.

Finally, it is incumbent on me to call attention to the huge contribution that research on calcium (Ca2+) signaling has made toward the understanding of cardiac contraction and to the maintenance of the heart rhythm.  The heart is a syncytium, different than skeletal and smooth muscle, and the innervation is by the vagus nerve, which has terminal endings at vesicles which discharge at the myocyte junction.  The heart specifically has calmodulin kinase CaMK II, and it has been established that calmodulin is involved in the calcium spark that triggers contraction.  That is only part of the story.  Ion transport occurs into or out of the cell, the latter termed exostosis.  Exostosis involves CaMK II and pyruvate kinase (PKC), and they have independent roles.  This also involves K+-Na+-ATPase.  The cytoskeleton is also discussed, but the role of aquaporin in water transport appears elsewhere, as the transport of water between cells.  When we consider the Gibbs-Donnan equilibrium, which precedes the current work by a century, we recall that there is an essential balance between extracellular Na+ + Ca2+ and the intracellular K+ + Mg2+, and this has been superceded by an incompletely defined relationship between ions that are cytoplasmic and those that are mitochondrial.  The glass is half full!


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Heart, Vascular Smooth Muscle, Excitation-Contraction Coupling (E-CC), Cytoskeleton, Cellular Dynamics and Ca2 Signaling

Heart, Vascular Smooth Muscle, Excitation-Contraction Coupling (E-CC), Cytoskeleton, Cellular Dynamics and Ca2 Signaling

Author and Curator: Larry H Bernstein, MD, FCAP

Author and Cardiovascular Three-volume Series, Editor: Justin Pearlman, MD, PhD, FACC, and

Curator: Aviva Lev-Ari, PhD, RN


AP, action potential; ARVD2, arrhythmogenic right ventricular cardiomyopathy type 2; CaMKII, Ca2+/calmodulim-dependent protein kinase II; CICR, Ca2+ induced Ca2+ release;CM, calmodulin; CPVT, catecholaminergic polymorphic ventricular tachycardia;  ECC, excitation–contraction coupling; FKBP12/12.6, FK506 binding protein; HF, heart failure; LCC, L-type Ca2+ channel;  P-1 or P-2, phosphatase inhibitor type-1 or type-2; PKA, protein kinase A; PLB, phosphoplamban; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; RyR1/2, ryanodine receptor type-1/type-2; SCD, sudden cardiac death; SERCA, sarcoplasmic reticulum Ca2+ ATPase; SL, sarcolemma; SR, sarcoplasmic reticulum.

This is Part V of a series on the cytoskeleton and structural shared thematics in cellular movement and cellular dynamics.

The Series consists of the following articles:

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP


Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN


Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
 and Aviva Lev-Ari, PhD, RN


Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets


Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN


Part V: Heart, Vascular Smooth Muscle, Excitation-Contraction Coupling (E-CC), Cytoskeleton, Cellular Dynamics and Ca2 Signaling

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN


Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

Aviva Lev-Ari, PhD, RN


Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

In the first part, we discussed common MOTIFs across cell-types that are essential for cell division, embryogenesis, cancer metastasis, osteogenesis, musculoskeletal function, vascular compliance, and cardiac contractility.   This second article concentrates on specific functionalities for cardiac contractility based on Ca++ signaling in excitation-contraction coupling.  The modifications discussed apply specifically to cardiac muscle and not to skeletal muscle.  Considering the observations described might raise additional questions specifically address to the unique requirements of smooth muscle, abundant in the GI tract and responsible for motility in organ function, and in blood vessel compliance or rigidity. Due to the distinctly different aspects of the cardiac contractility and contraction force, and the interactions with potential pharmaceutical targets, there are two separate articles on calcium signaling and cardiac arrhythmias or heart failure (Part 2 and Part 3).  Part 2 focuses on the RYANODINE role in cardiac Ca(2+) signaling and its effect in heart failure.  Part 3 takes up other aspects of heart failure and calcium signaling with respect to phosporylation/dephosphorylation. I add a single review and classification of genetic cardiac disorders of the same cardiac Ca(2+) signaling and the initiation and force of contraction. Keep in mind that the heart is a syncytium, and this makes a huge difference compared with skeletal muscle dynamics. In Part 1 there was some discussion of the importance of Ca2+ signaling on innate immune system, and the immunology will be further expanded in a fourth of the series.


This second article on the cardiomyocyte and the Ca(2+) cycling between the sarcomere and the cytoplasm, takes a little distance from the discussion of the ryanodine that precedes it.  In this discussion we found that there is a critical phosphorylation/dephosphorylation balance that exists between Ca(+) ion displacement and it occurs at a specific amino acid residue on the CaMKIId, specific for myocardium, and there is a 4-fold increase in contraction and calcium release associated with this CAM kinase (ser 2809) dependent exchange.  These events are discussed in depth, and the research holds promise for therapeutic application. We also learn that Ca(2+) ion channels are critically involved in the generation of arrhythmia as well as dilated and hypertrophic cardiomyopathy.  In the case of arrhythmiagenesis, there are two possible manners by which this occurs.  One trigger is Ca(2+) efflux instability.  The other is based on the finding that when the cellular instability is voltage driven, the steady-state wave­length (separation of nodes in space) depends on electrotonic coupling between cells and the steepness of APD and CV restitution. The last article is an in depth review of the genetic mutations that occur in cardiac diseases.  It is an attempt at classifying them into reasonable groupings. What are the therapeutic implications of this? We see that the molecular mechanism of cardiac function has been substantially elucidated, although there are contradictions in experimental findings that are unexplained.  However, for the first time, it appears that personalized medicine is on a course that will improve health in the population, and the findings will allow specific targets designed for the individual with a treatable impairment in cardiac function that is identifiable early in the course of illness. This article is a continuation to the following articles on tightly related topics: Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton     Larry H Bernstein, MD, FCAP Part II:  Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility    Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease    Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
 and  Aviva Lev-Ari, PhD, RN Part  IV:  The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN  http:/ Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Part V:  Heart Smooth Muscle and Cardiomyocyte Cells: Excitation-Contraction Coupling & Ryanodine Receptor (RyR) type-1/type-2 in Cytoskeleton Cellular Dynamics and Ca2+ Signaling

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN Part VI:  Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD Curator: Aviva Lev-Ari, PhD, RN and Advanced Topics in Sepsis and the Cardiovascular System at its End Stage Larry H Bernstein, MD, FCAP

The Role of Protein Kinases and Protein Phosphatases in the Regulation of Cardiac Sarcoplasmic Reticulum Function

EG Kranias, RC Gupta, G Jakab, HW Kim, NAE Steenaart, ST Rapundalo Molecular and Cellular Biochemistry 06/1988; 82(1):37-44. · 2.06 Impact Factor Canine cardiac sarcoplasmic reticulum is phosphorylated by

  • adenosine 3,5-monophosphate (cAMP)-dependent and
  • calcium calmodulin-dependent protein kinases on
  • a proteolipid, called phospholamban.

Both types of phosphorylation are associated with

  •  an increase in the initial rates of Ca(2+) transport by SR vesicles
  • which reflects an increased turnover of elementary steps of the calcium ATPase reaction sequence.

The stimulatory effects of the protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase, which

  • can dephosphorylate both the CAMP-dependent and the calcium calmodulin-dependent sites on phospholamban.

Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by protein kinases and protein phosphatases. calcium release calmodulin + ER Ca(2+) and contraction

Regulation of the Cardiac Ryanodine Receptor Channel by Luminal Ca2+ involves Luminal Ca2+ Sensing Sites

I Györke, S Györke.   Biophysical Journal 01/1999; 75(6):2801-10. · 3.65 Impact factor  http:// The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca(2+) was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 microM to 2 mM Ca(2+) (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 microM to 20 mM Ca(2+) on the luminal (trans) side of the channel and with Cs+ as the charge carrier. Under conditions of low [trans Ca(2+)] (20 microM), increasing [cis Ca(2+)] from 0.1 to 10 microM caused a gradual increase in channel open probability (Po). Elevating [cis Ca(2+)] [cytosolic] above 100 microM resulted in a gradual decrease in Po. Elevating trans [Ca(2+)] [luminal] enhanced channel activity (EC50 approximately 2.5 mM at 1 microM cis Ca2+) primarily by increasing the frequency of channel openings. The dependency of Po on trans [Ca2+] [luminal] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid. Elevated luminal Ca(2+)

  1. enhanced the sensitivity of the channel to activating cytosolic Ca(2+), and it
  2. essentially reversed the inhibition of the channel by high cytosolic Ca(2+).

Potentiation of Po by increased luminal Ca(2+) occurred irrespective of whether the electrochemical gradient for Ca(2+) supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca(2+) through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca(2+) acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca2+ are mediated by distinct Ca(2+)-sensitive site(s) at the luminal face of the channel or associated protein. F1.large  calcium movement and RyR2 receptor

Protein phosphatases Decrease Sarcoplasmic Reticulum Calcium Content by Stimulating Calcium Release in Cardiac Myocytes

D Terentyev, S Viatchenko-Karpinski, I Gyorke, R Terentyeva and S Gyorke Texas Tech University Health Sciences Center, Lubbock, TX J Physiol 2003; 552(1), pp. 109–118. Phosphorylation/dephosphorylation of Ca2+ transport proteins by cellular kinases and phosphatases plays an important role in regulation of cardiac excitation–contraction coupling; furthermore,

  • abnormal protein kinase and phosphatase activities have been implicated in heart failure.

However, the precise mechanisms of action of these enzymes on intracellular Ca2+ handling in normal and diseased hearts remains poorly understood. We have investigated

  •   the effects of protein phosphatases PP1 and PP2A on spontaneous Ca(2+) sparks and SR Ca(2+) load in myocytes permeabilized with saponin.

Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca(2+) sparks followed by a nearly complete disappearance of events, which were accompanied by depletion of the SR Ca(2+) stores, as determined by application of caffeine. These changes in

  •  Ca(2+) release and
  • SR Ca(2+) load

could be prevented by the inhibitors of PP1 and PP2A phosphatase activities okadaic acid and calyculin A. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1-medited RyR dephosphorylation in our permeabilized myocytes preparations was confirmed biochemically by quantitative immunoblotting using a phosphospecific anti-RyR antibody. Our results suggest that

  •  increased intracellular phosphatase activity stimulates
  • RyR mediated SR Ca(2+) release
    • leading to depleted SR Ca(2+) stores in cardiac myocytes.

In heart muscle cells, the process of excitation–contraction (EC) coupling is mediated by

  •  Ca(2+) influx through sarcolemmal L-type Ca(2+) channels
  • activating Ca(2+) release channels (ryanodine receptors, RyRs) in the sarcoplasmic reticulum (SR).

Once activated, the RyR channels allow Ca(2+) to be released from the SR into the cytosol to induce contraction. This mechanism is known as Ca(2+)-induced calcium release (CICR) (Fabiato, 1985; Bers, 2002).  During relaxation, most of the Ca(2+) is resequestered into the SR by the Ca(2+)-ATPase. The amount of Ca(2+) released and the force of contraction depend on

  •  the magnitude of the Ca(2+) trigger signal,
  • the functional state of the RyRs and
  • the amount of Ca(2+) stored in the SR.

F1.large  calcium movement and RyR2 receptor Ca(2+) and contraction calcium release calmodulin + ER Reversible phosphorylation of proteins composing the EC coupling machinery plays an important role in regulation of cardiac contractility (Bers, 2002). Thus, during stimulation of the b-adrenergic pathway, phosphorylation of several target proteins, including

  • the L-type Ca(2+) channels,
  • RyRs and
  • phospholamban,

by protein kinase A (PKA) leads to an overall increase in SR Ca2+ release and contractile force in heart cells (Callewaert et al. 1988, Spurgeon et al. 1990; Hussain & Orchard, 1997; Zhou et al. 1999; Song et al. 2001; Viatchenko-Karpinski & Gyorke, 2001). PKA-dependent phosphorylation of the L-type Ca(2+) channels increases the Ca2+ current (ICa), increasing both

  • the Ca2+ trigger for SR Ca2+ release and
  • the SR Ca(2+) content

(Callewaert et al. 1988; Hussain & Orchard, 1997; Del Principe et al. 2001). Phosphorylation of phospholamban (PLB) relieves the tonic inhibition dephosphorylated PLB exerts on the SR Ca(2+)-ATPase (SERCA) resulting in enhanced SR Ca(2+) accumulation and enlarged Ca(2+) release (Kranias et al. 1985; Simmermann & Jones, 1998). With regard to the RyR, despite clear demonstration of phosphorylation of the channel in biochemical studies (Takasago et al. 1989; Yoshida et al. 1992), the consequences of this reaction to channel function have not been clearly defined. RyR phosphorylation by PKA and Ca(2+)–calmodulin dependent protein kinase (CaMKII) has been reported to increase RyR activity in lipid bilayers (Hain et al. 1995; Marx et al. 2000; Uehara et al. 2002). Moreover, it has been reported that in heart failure (HF), hyperphosphorylation of RyR causes

  •  the release of FK-506 binding protein (FKBP12.6) from the RyR,
    • rendering the channel excessively leaky for Ca(2+) (Marx et al. 2000).

However, other studies have reported no functional effects (Li et al. 2002) or even found phosphorylation to reduce RyR channel steady-state open probability (Valdivia et al. 1995; Lokuta et al. 1995).  The action of protein kinases is opposed by dephosphorylating phosphatases. Three types of protein phosphatases (PPs), referred to as PP1, PP2A and PP2B (calcineurin), have been shown to influence cardiac performance (Neumann et al. 1993; Rusnak & Mertz, 2000). Overall, according to most studies phosphatases appear to downregulate SR Ca(2+) release and contractile performance (Neumann et al. 1993; duBell et al. 1996, 2002; Carr et al. 2002; Santana et al. 2002). Furthermore, PP1 and PP2A activities appear to be increased in heart failure (Neumann, 2002; Carr et al. 2002). However, again the precise mode of action of these enzymes on intracellular Ca(2+) handling in normal and diseased hearts remains poorly understood.  In the present study, we have investigated the effects of protein phosphatases PP1 and PP2A on local Ca(2+) release events, Ca(2+) sparks, in cardiac cells. Our results show that

  •  phosphatases activate RyR mediated SR Ca(2+) release
    • leading to depletion of SR Ca(2+) stores.

These results provide novel insights into the mechanisms and potential role of protein phosphorylation/dephosphorylation in regulation of Ca(2+) signaling in normal and diseased hearts. F2.large   RyR and calcium


Effects of PP1 and PP2A on Ca2+ sparks and SR Ca(2+) content.

[1]  PP1 caused an early transient potentiation of Ca2+ spark frequency followed by a delayed inhibition of event occurrence. [2]  PP1 produced similar biphasic effects on the magnitude and spatio-temporal characteristics of Ca(2+) sparks Specifically, during the potentiatory phase (1 min after addition of the enzyme), PP1 significantly increased

  • the amplitude,
  • rise-time,
  • duration and
  • width of Ca(2+) sparks;

during the inhibitory phase (5 min after addition of the enzyme),

  •  all these parameters were significantly suppressed by PP1.

The SR Ca(2+) content decreased by 35 % or 69 % following the exposure of myocytes to either 0.5 or 2Uml_1 PP1, respectively (Fig. 1C). Qualitatively similar results were obtained with phosphatase PP2A. Similar to the effects of PP1, PP2A (5Uml_1) produced a transient increase in Ca(2+) spark frequency (~4-fold) followed by a depression of event occurrence and decreased SR Ca(2+) content (by 82 % and 65 %, respectively). Also similar to the action of PP1, PP2A increased

  •  the amplitude and
  • spatio-temporal spread (i.e. rise-time, duration and width) of Ca(2+) sparks at 1 min
  • and suppressed the same parameters at 5 min of exposure to the enzyme (Table 1).

Together, these results suggest that phosphatases enhance spark-mediated SR Ca2+ release, leading to decreased SR Ca(2+) content. Preventive effects of calyculin A and okadaic acid Preventive effects of ryanodine

PP1-mediated RyR dephosphorylation

F3.large  cardiomyocyte SR F3.large  cardiomyocyte SR F2.large   RyR and calcium coupled receptors coupled receptors The cardiac RyR is phosphorylated at Ser-2809 (in the rabbit sequence) by both PKA and CAMKII (Witcher et al. 1991; Marx et al. 2000). Although additional phosphorylation sites may exist on the RyR (Rodriguez et al. 2003), but Ser-2809 is believed to be the only site that is phosphorylated by PKA, and RyR hyperphosphorylation at this site has been reported in heart failure (Marx et al. 2000).  To test whether indeed phosphatases dephosphorylated the RyR in our permeabilized myocyte experiments we performed quantitative immunoblotting using an antibody that specifically recognizes the phosphorylated form of the RyR at Ser-2809 (Rodriguez et al. 2003). Myocytes exhibited a significant level of phosphorylation under baseline conditions. Maximal phosphorylation was 201 % of control. When exposed to 2Uml_1 PP1, RyR phosphorylation was 58 % of the control basal condition. Exposing to a higher PP1 concentration (10Uml_1) further reduced RyR phosphorylation to 22% of control. Thus, consistent with the results of our functional measurements,

  •  PP1 decreased RyR phosphorylation in cardiac myocytes.

Figure 1. Effects of PP1 on properties of Ca(2+) sparks and SR Ca(2+) content in rat permeabilized myocytes    see . A, spontaneous Ca(2+) spark images recorded under reference conditions, and 1 or 5 min after exposure of the cell to 2Uml_1 PP1. Traces below the images are Ca(2+) transients induced by application of 10 mM caffeine immediately following the acquisition of sparks before (3 min) and after (5 min) application of PP1 in the same cell. The Ca(2+) transients were elicited by a whole bath application of 10 mM caffeine. B, averaged spark frequency at early (1 min) and late (5 min) times following the addition of either 0.5 or 2Uml_1 of PP1 to the bathing solution. C, averaged SR Ca(2+) content for 0.5 or 2Uml_1 of PP1 measured before and 5 min after exposure to the enzyme. Data are presented as means ± S.E.M. of 6 experiments in different cells. Figure 2. Effects of PP2A on properties of Ca2+ sparks and SR Ca2+ content in rat permeabilized myocytes   see . A, spontaneous Ca(2+) spark images recorded under reference conditions, and 1 or 5 min after exposure of the cell to 5Uml_1 PP2A. Traces below the images are Ca(2+) transients induced by application of 10 mM caffeine immediately following the acquisition of sparks before (3 min) and after (5 min) application of PP2A in the same cell. B and C, averaged spark frequency (B) and SR Ca(2+) content (C) for the same conditions as in A. Data are presented as means ± S.E.M. of 6 experiments in different cells.


In the present study, we have investigated the impact of physiologically relevant exogenous protein phosphatases PP1 and PP2A on RyR-mediated SR Ca(2+) release (measured as Ca(2+) sparks) in permeabilized heart cells. Our principal finding is that

  • phosphatases stimulated RyR channels lead to depleted SR Ca(2+) stores.

These results have important ramifications for understanding the mechanisms and role of protein phosphorylation/dephosphorylation in

  •  modulation of Ca(2+) handling in normal and diseased heart.

Modulation of SR Ca2+ release by protein phosphorylation/dephophorylation

Since protein dephosphorylation clearly resulted in increased functional activity of the Ca(+)release channel, our results imply that a reverse, phosphorylation reaction should reduce RyR activity. If indeed such effects take place, why do they not manifest in inhibition of Ca(+)sparks? One possibility is that enhanced Ca(+) uptake by SERCA

  •  masks or overcomes the effects phosphorylation may have on RyRs.

In addition, the potential inhibitory influence of protein phosphorylation on RyR activity in myocytes could be countered by feedback mechanisms  involving changes in luminal Ca(2+)(Trafford et al. 2002; Gyorke et al. 2002). In particular, reduced open probability of RyRs would be expected to lead to

  •  increased Ca2+ accumulation in the SR;
  • and increased intra-SR [Ca(2+)], in turn would
  • increase activity of RyRs at their luminal Ca(2+) regulatory sites

as demonstrated for the RyR channel inhibitor tetracaine (Gyorke et al. 1997; Overend et al. 1997). Thus

  • potentiation of SERCA
  • combined with the intrinsic capacity of the release mechanism to self-regulate

could explain at least in part why PKA-mediated protein phoshorylation results in maintained potentiation of Ca(2+) sparks despite a potential initial decrease in RyR activity.

Role of altered RyR Phosphorylation in Heart Failure

Marx et al. (2000) have proposed that  enhanced levels of circulating catecholamines lead to increased phosphorylation of RyR in heart failure.  Based on biochemical observations as well as on studying properties of single RyRs incorporated into artificial lipid bilayers, these investigators have hypothesized that

  •  hyperphosphorylation of RyRs contributes to pathogenesis of heart failure
    • by making the channel excessively leaky due to dissociation of FKBP12.6 from the channel.

We show that the mode of modulation of RyRs by phosphatases does not support this hypothesis as

  • dephosphorylation caused activation instead of

Interestingly, our results provide the basis for a different possibility in which

  •  dephophosphorylation of RyR rather than its phosphorylation causes depletion of SR Ca(2+) stores by stimulating RyRs in failing hearts.

It has been reported that PP1 and PP2 activities are increased in heart failure (Huang et al. 1999; Neumann et al. 1997; Neuman, 2002). Furthermore,  overexpression of PP1 or ablation of the endogenous PP1 inhibitor, l-1, results in

  • depressed contractile performance and heart failure (Carr et al. 2002).

Our finding that PP1 causes depletion of SR Ca(2+) stores by activating RyRs could account for, or contribute to, these results.


1 DelPrincipe F, Egger M, Pignier C & Niggli E (2001). Enhanced E-C coupling efficiency after beta-stimulation of cardiac myocytes. Biophys J 80, 64a. 2 Gyorke I & Gyorke S (1998). Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites. Biophys J 75, 2801–2810. 3 Gyorke S, Gyorke I, Lukyanenko V, Terentyev D, Viatchenko-Karpinski S & Wiesner TF (2002). Regulation of sarcoplasmic reticulum calcium release by luminal calcium in cardiac muscle. Front Biosci 7, d1454–d1463. 4 Gyorke I, Lukyanenko V & Gyorke S (1997). Dual effects of tetracaine on spontaneous calcium release in rat ventricular myocytes. J Physiol 500, 297–309. 5 MacDougall LK, Jones LR & Cohen P (1991). Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban. Eur J Biochem 196, 725–734. 6 Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N & Marks AR (2000). PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 101, 365–376. 7 Rodriguez P, Bhogal MS & Colyer J (2003). Stoichiometric phosphorylation of cardiac ryanodine receptor on serine-2809 by calmodulin-dependent kinase II and protein kinase A. J Biol Chem (in press).

The δC Isoform of CaMKII Is Activated in Cardiac Hypertrophy and Induces Dilated Cardiomyopathy and Heart Failure

T Zhang, LS Maier, ND Dalton, S Miyamoto, J Ross, DM Bers, JH Brown.  University of California, San Diego, La Jolla, Calif; and Loyola University, Chicago, Ill. Circ Res. 2003;92:912-919. Recent studies have demonstrated that transgenic (TG) expression of either Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) or CaMKIIδB, both of which localize to the nucleus, induces cardiac hypertrophy. However,

  •  CaMKIV is not present in heart, and
  • cardiomyocytes express not only the nuclear CaMKIIδB
    • but also a cytoplasmic isoform, CaMKII δC.

In the present study, we demonstrate that

  1.  expression of the δC isoform of CaMKII is selectively increased and
  2. its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload.

To determine whether enhanced activity of this cytoplasmic δC isoform of CaMKII can lead to phosphorylation of Ca(2+) regulatory proteins and induce hypertrophy, we generated TG mice that expressed the δC isoform of CaMKII.  Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca2(2+) handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and

  • CaMKII is found associated with the RyR  from the CaMKII TG mice.
  • Phosphorylation of phospholamban is increased specifically at the CaMKII but not at the PKA phosphorylation site.

These findings are the first to demonstrate that CaMKIIδC can mediate phosphorylation of Ca(2+) regulatory proteins in vivo and provide evidence for the involvement of CaMKIIδC activation in the pathogenesis of dilated cardiomyopathy and heart failure.  Multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaM kinases or CaMKs) are transducers of Ca2+ signals that phosphorylate a wide range of substrates and thereby affect Ca(2+)-mediated cellular responses.1 The family includes CaMKI and CaMKIV, monomeric enzymes activated by CaM kinase kinase,2,3 and CaMKII, a multimer of 6 to 12 subunits activated by autophosphorylation.1 The CaMKII subunits α, β, γ, and δ show different tissue distributions,1 with

  • the δ isoform predominating in the heart.4–7
  • Splice variants of the δ isoform, characterized by the presence of a second variable domain,4,7 include δB, which contains a nuclear localization signal (NLS), and
  • δC, which does not. CaMKII composed of δB subunits localizes to the nucleus, whereas CaMKIIδC localizes to the cytoplasm.4,8,9

CaMKII has been implicated in several key aspects of acute cellular Ca(2+) regulation related to cardiac excitation-contraction (E-C) coupling. CaMKII

  • phosphorylates sarcoplasmic reticulum (SR) proteins including the ryanodine receptors (RyR2) and
  • phospholamban (PLB).10–14

Phosphorylation of RyR has been suggested to alter the channel open probability,14,15 whereas phosphorylation of PLB has been suggested to regulate SR Ca(2+) uptake.14 It is also likely that CaMKII phosphorylates the L-type Ca(2+) channel complex or an associated regulatory protein and thus

  1. mediates Ca(2+) current (ICa) facilitation.16-18 and
  2. the development of early after-depolarizations and arrhythmias.19

Thus, CaMKII has significant effects on E-C coupling and cellular Ca(2 +) regulation. Nothing is known about the CaMKII isoforms regulating these responses.  Contractile dysfunction develops with hypertrophy, characterizes heart failure, and is associated with changes in cardiomyocyte (Ca2+) homeostasis.20  CaMKII expression and activity are altered in the myocardium of rat models of hypertensive cardiac hypertrophy21,22 and heart failure,23 and

  • in cardiac tissue from patients with dilated cardiomyopathy.24,25

Several transgenic mouse models have confirmed a role for CaMK in the development of cardiac hypertrophy, as originally suggested by studies in isolated neonatal rat ventricular myocytes.9,26–28 Hypertrophy develops in transgenic mice that overexpress CaMKIV,27 but this isoform is not detectable in the heart,4,29 and CaMKIV knockout mice still develop hypertrophy after transverse aortic constriction (TAC).29  Transgenic mice overexpressing calmodulin developed severe cardiac hypertrophy,30 later shown to be associated with an increase in activated CaMKII31; the isoform of CaMKII involved in hypertrophy could not be determined from these studies. We recently reported that transgenic mice that overexpress CaMKIIδB, which is highly concentrated in cardiomyocyte nuclei, develop hypertrophy and dilated cardiomyopathy.32 To determine whether

  • in vivo expression of the cytoplasmic CaMKIIδC can phosphorylate cytoplasmic Ca(2+) regulatory proteins and
  • induce hypertrophy or heart failure,

we generated transgenic (TG) mice that expressed the δC isoform of CaMKII under the control of the cardiac specific α-myosin heavy chain (MHC) promoter. Our findings implicate CaMKIIδC in the pathogenesis of dilated cardiomyopathy and heart failure and suggest that

  • this occurs at least in part via alterations in Ca(2+) handling proteins.33

Ca(2+) and contraction RyR yuan_image3  Ca++ exchange yuan_image3  Ca++ exchange


 Expression and Activation of CaMKIIδC Isoform After TAC

To determine whether CaMKII was regulated in pressure overload–induced hypertrophy, CaMKIIδ expression and phosphorylation were examined by Western blot analysis using left ventricular samples obtained at various times after TAC.  A selective increase (1.6-fold) in the lower band of CaMKIIδwas observed as early as 1 day and continuously for 4 days (2.3-fold) and 7 days (2-fold) after TAC (Figure 1A).  To confirm that CaMKIIδC was increased and determine whether this occurred at the transcriptional level, we performed semiquantitative RT-PCR using primers specific for the CaMKIIδC isoform. These experiments revealed that

  • mRNA levels for CaMKIIδC were increased 1 to 7 days after TAC (Figure 1B).

In addition to examining CaMKII expression, the activation state of CaMKII was monitored by its autophosphorylation, which confers Ca2-independent activity.

Figure 1. Expression and activation of CaMKII δC isoform after TAC.

see A, Western blot analysis of total CaMKII in left ventricular (LV) homogenates obtained at indicated times after TAC. Cardiomyocytes transfected with CaMKIIδB and δC (right) served as positive controls and molecular markers. Top band (58 kDa) represents CaMKIIδB plus δ9, and the bottom band (56 kDa) corresponds to CaMKIIδC. *P0.05 vs control. B, Semiquantitative RT-PCR using primers specific for CaMKIIδC isoform (24 cycles) and GAPDH (19 cycles) using total RNA isolated from the same LV samples. C, Western blot analysis of phospho-CaMKII in LV homogenates obtained at various times after TAC. Three bands seen for each sample represent CaMKIIγ subunit (uppermost), CaMKIIδB plus δ9 (58 kDa), and CaMKIIδC (56 kDa). Quantitation is based on the sum of all of the bands. *P0.05 vs control.

 Figure 2. Expression and activation of CaMKII in CaMKIIδC transgenic mice.

see A, Transgene copy number based on Southern blots using genomic DNA isolated from mouse tails (digested with EcoRI). Probe (a 32P-labeled 1.7-kb EcoRI-SalI -MHC fragment) was hybridized to a 2.3-kb endogenous fragment (En) and a 3.9-kb transgenic fragment (TG). Transgene copy number was determined from the ratio of the 3.9-kb/2.3-kb multiplied by 2. B, Immunocytochemical staining of ventricular myocytes isolated from WT and CaMKIIδTG mice. Myocytes were cultured on laminin-coated slides overnight. Transgene was detected by indirect immunofluorescence staining using rabbit anti-HA antibody (1:100 dilution) followed by FITC-conjugated goat antirabbit IgG antibody (1:100 dilution). CaMKIIδB localization to the nucleus in CaMKIIδB TG mice (see Reference 32) is shown here for comparative purpose. C, Quantitation of the fold increase in CaMKIIδprotein expression in TGL and TGM lines. Different amounts of ventricular protein (numbers) from WT control, TG () and their littermates () were immunoblotted with an anti-CaMKIIδ antibody. Standard curve from the WT control was used to calculate fold increases in protein expression in TGL and TGM lines. D, Phosphorylated CaMKII in ventricular homogenates was measured by Western blot analysis (n5 for each group). **P0.01 vs WT.

 Generation and Identification of CaMKIIδC Transgenic Mice

TG mice expressing HA-tagged rat wild-type CaMKIIδC under the control of the cardiac-specific α-MHC promoter were generated as described in Materials and Methods. By Southern blot analysis, 3 independent TG founder lines carrying 3, 5, and 15 copies of the transgene were identified. They were designated as TGL (low copy number), TGM (medium copy number), and TGH (high copy number), The founder mice from the TGH line died at 5 weeks of age with marked cardiac enlargement.  The other two lines showed germline transmission of the transgene. The transgene was expressed only in the heart. Although CaMKII protein levels in TGL and TGM hearts were increased 12- and 17-fold over wild-type (WT) controls (Figure 2C), the amount of activated CaMKII was only increased 1.7- and 3-fold in TGL and TGM hearts (Figure 2D). The relatively small increase in CaMKII activity in the TG lines probably reflects the fact that the enzyme is not constitutively activated and that the availability of Ca2/CaM, necessary for activation of the overexpressed CaMKII, is limited. Importantly,

  • the extent of increase in active CaMKII in the TG lines was similar to that elicited by TAC.

 Cardiac Overexpression of CaMKIIδC Induces Cardiac Hypertrophy and Dilated Cardiomyopathy

There was significant enlargement of hearts from CaMKIIδC TGM mice by 8 to 10 weeks [see  (Figure 3A) and from TGL mice by 12 to 16 weeks. Histological analysis showed ventricular dilation (Figure 3B), cardiomyocyte enlargement (Figure 3C), and mild fibrosis (Figure 3D) in CaMKIIδC TG mice. Quantitative analysis of cardiomyocyte cell volume from 12-week-old TGM mice gave values of 54.7 + 0.1 pL for TGM (n = 96) versus 28.6 + 0.1 pL for WT littermates (n=94; P0.001). Ventricular dilation and cardiac dysfunction developed over time in proportion to the extent of transgene expression. Left ventricular end diastolic diameter (LVEDD) was increased by 35% to 45%, left ventricular posterior wall thickness (LVPW) decreased by 26% to 29% and fractional shortening decreased by 50% to 60% at 8 weeks for TGM and at 16 weeks for TGL. None of these parameters were significantly altered at 4 weeks in TGM or up to 11 weeks in TGL mice, indicating that heart failure had not yet developed.  Contractile function was significantly decreased. Figure 6. Dilated cardiomyopathy and dysfunction in CaMKIIδC TG mice at both whole heart and single cell levels.  [see Fig 6:] C, Decreased contractile function in ventricular myocytes isolated from 12-week old TGM and WT controls presented as percent change of resting cell length (RCL) stimulated at 0.5 Hz. Representative trace and mean values are shown. *P0.05 vs WT. Figure 7. Phosphorylation of PLB in CaMKIIδC TG mice.  [see Fig 7:] Thr17 and Ser16 phosphorylated PLB was measured by Western blots using specific anti-phospho antibodies. Ventricular homogenates were from 12- to 14-week-old WT and TGM mice (A) or 4 to 5-week-old WT and TGM mice (B). Data were normalized to total PLB examined by Western blots (data not shown here). n = 6 to 8 mice per group; *P0.05 vs WT.

 Cardiac Overexpression of CaMKIIδC Results in Changes in the Phosphorylation of Ca2 Handling Proteins

To assess the possible involvement of phosphorylation of Ca2cycling proteins in the phenotypic changes observed in the CaMKIIC TG mice, we first compared PLB phosphorylation state in homogenates from 12- to 14-week-old TGM and WT littermates. Western blots using antibodies specific for phosphorylated PLB showed a 2.3-fold increase in phosphorylation of Thr17 (the CaMKII site) in hearts from TGM versus WT (Figure 7A). Phosphorylation of PLB at the CaMKII site was also increased 2-fold in 4- to 5-week-old TGM mice (Figure 7B). Significantly, phosphorylation of the PKA site (Ser16) was unchanged in either the older or the younger TGM mice (Figures 7A and 7B). (see  To demonstrate that the RyR2 phosphorylation changes observed in the CaMKII transgenic mice are not secondary to development of heart failure, we performed biochemical studies examining RyR2 phosphorylation in 4- to 5-week-old TGM mice. At this age, most mice showed no signs of hypertrophy or heart failure (see Figure 6B) and there was no significant increase in myocyte size (21.3 + 1.3 versus 27.7 + 4.6 pL; P0.14). Also, twitch Ca2 transient amplitude was not yet significantly depressed, and mean δ [Ca2+]i (1 Hz) was only 20% lower (192 + 36 versus 156 + 13 nmol/L; P0.47) versus 50% lower in TGM at 13 weeks.33  The in vivo phosphorylation of RyR2, determined by back phosphorylation, was significantly (2.10.3-fold; P0.05) increased in these 4- to 5-week-old TGM animals (Figure 8C), an increase equivalent to that seen in 12- to 14-week-old mice. We also performed the RyR2 back-phosphorylation assay using purified CaMKII rather than PKA. RyR2 phosphorylation at the CaMKII site was also significantly increased (2.2 + 0.3-fold; P0.05) in 4- to 5-week-old TGM mice (Figure 8C).  ( The association of CaMKII with the RyR2 is consistent with a physical interaction between this protein kinase and its substrate. The catalytic subunit of PKA and the phosphatases PP1 and PP2A were also present in the RyR2 immunoprecipitates, but not different in WT versus TG mouse hearts (Figure 8D). These data provide further evidence that

  • the increase in RyR2 phosphorylation, which precedes development of failure in the 4- to 5-week-old CaMKIIδC TG hearts, can be attributed to the increased activity of CaMKII.


  1. CaMKII is involved in the dynamic modulation of cellular
  2. Ca2 regulation and has been implicated in the development of cardiac hypertrophy and heart failure.14
  3. Published data from CaMK-expressing TG mice demonstrate that forced expression of CaMK can induce cardiac hypertrophy and lead to heart failure.27,32

However, the CaMK genes expressed in these mice are neither the endogenous isoforms of the enzyme nor the isoforms likely to regulate cytoplasmic Ca(2+) handling, because they localize to the nucleus.

  1.  the cytoplasmic cardiac isoform of CaMKII is upregulated at the expression level and is in the active state (based on autophosphorylation) after pressure overload induced by TAC.
  2.  two cytoplasmic CaMKII substrates (PLB and RyR) are phosphorylated in vivo when CaMKII is overexpressed and its activity increased to an extent seen under pathophysiological conditions.
  3. CaMKIIδ is found to associate physically with the RyR in the heart.
  4.  heart failure can result from activation of the cytoplasmic form of CaMKII and this may be due to altered Ca(2+) handling.

 Differential Regulation of CaMKIIδ Isoforms in Cardiac Hypertrophy

  1.  The isoform of CaMKII that predominates in the heart is the δ isoform.4–7 Neither the α nor the β isoforms are expressed and there is only a low level of expression of the γ isoforms.39
  2. Both δB and δC splice variants of CaMKIIδ are present in the adult mammalian myocardium36,40 and expressed in distinct cellular compartments.4,8,9

We suggest that the CaMKIIδ isoforms are differentially regulated in pressure-overload–induced hypertrophy, because the expression of CaMKIIδC is selectively increased as early as 1 day after TAC. Studies using RT-PCR confirm that

  • CaMKIIδC is regulated at the transcriptional level in response to TAC. In addition,
  • activation of both CaMKIIδB and CaMKIIδC, as indexed by autophosphorylation, increases as early as 2 days after TAC.
  • Activation of CaMKIIδB by TAC is relevant to our previous work indicating its role in hypertrophy.9,32
  • The increased expression, as well as activation of the CaMKIIδC isoform, suggests that it could also play a critical role in both the acute and longer responses to pressure overload.

In conclusion, we demonstrate here that CaMKIIδC can phosphorylate RyR2 and PLB when expressed in vivo at levels leading to 2- to 3-fold increases in its activity. Similar increases in CaMKII activity occur with TAC or in heart failure. Data presented in this study and in the accompanying article33 suggest that altered phosphorylation of Ca(2+) cycling proteins is a major component of the observed decrease in contractile function in CaMKIIδC TG mice. The occurrence of increased CaMKII activity after TAC, and of RyR and PLB phosphorylation in the CaMKIIδC TG mice suggest that

  • CaMKIIδC plays an important role in the pathogenesis of dilated cardiomyopathy and heart failure.

These results have major implications for considering CaMKII and its isoforms in exploring new treatment strategies for heart failure.

Cardiac Electrophysiological Dynamics From the Cellular Level to the Organ Level

Daisuke Sato and Colleen E. Clancy Department of Pharmacology, University of California – Davis, Davis, CA. Biomedical Engineering and Computational Biology 2013:5: 69–75 Abstract: Cardiac alternans describes contraction of the ventricles in a strong-weak-strong-weak sequence at a constant pacing fre­quency. Clinically, alternans manifests as alternation of the T-wave on the ECG and predisposes individuals to arrhythmia and sudden cardiac death. In this review, we focus on the fundamental dynamical mechanisms of alternans and show how alternans at the cellular level underlies alternans in the tissue and on the ECG. A clear picture of dynamical mechanisms underlying alternans is important to allow development of effective anti-arrhythmic strategies. The cardiac action potential is the single cellular level electrical signal that triggers contraction of the heart.1 Under normal conditions, the originating activation signal comes from a small bundle of tissue in the right atrium called the sinoatrial node (SAN). The action potentials generated by the SAN initiate an excitatory wave that, in healthy tissue, propagates smoothly through a well-defined path and causes excitation and contraction in the ventricles. In disease states, the normal excitation pathway is disrupted and a variety of abnormal rhythms can occur, including cardiac alternans, a well-known precursor to sudden cardiac death. Cardiac alternans was initially documented in 1872 by a German physician, Ludwig Traube.2 He observed contraction of the ventricles in a strong-weak-strong-weak sequence even though the pacing frequency was constant. Clinically, alternans mani­fests as alternation of the T-wave on the ECG, typi­cally in the microvolt range. It is well established that individuals with microvolt T-wave alternans are at much higher risk for arrhythmia and sudden cardiac death. A clear picture of physio­logical mechanisms underlying alternans is important to allow development of effective anti-arrhythmic drugs. It is also important to understand dynamical mechanisms because while the cardiac action poten­tial is composed of multiple currents, each of which confers specific properties, revelation of dynamical mechanisms provides a unified fundamental view of the emergent phenomena that holds independently of specific current interactions. The ventricular myocyte is an excitable cell pro­viding the cellular level electrical activity that under­lies cardiac contraction. Under resting conditions, the membrane potential is about -80 mV. When the cell is stimulated, sodium (Na) channels open and the membrane potential goes above 0 mV. Then, a few ms later, the inward current L-type calcium (Ca) current activates and maintains depolarization of the mem­brane potential. During this action potential plateau, several types of outward current potassium (K) chan­nels also activate. Depending on the balance between inward and outward currents, the action potential duration (APD) is determined.The diastolic interval (DI) that follows cellular repolarization describes the duration the cell resides in the resting state until the next excitation. During the DI, channels recover with kinetics determined by intrinsic time constants. APD restitution defines the relationship between the APD and the previous DI (Fig. 1 top panel). In most cases1, the APD becomes longer as the previous DI becomes longer due to recovery of the L-type Ca channel (Fig. 1, bottom panel), and thus the APD restitution curve has a positive slope. Figure 1. (Top): APD and DI. (Bottom): The physiological mechanism of APD alternans involves recovery from inactivation of ICaL.  [see]

 Action Potential Duration Restitution

In 1968 Nolasco and Dahlen showed graphically that APD alternans occurs when the slope of the APD res­titution curve exceeds unity. Why is the steepness of the slope important? As shown graphically in Figure 2, APD alternans amplitude is multiplied by the slope of the APD restitution curve in each cycle. When the slope is larger than one, then the alternans amplitude will be amplified until the average slope reaches 1 or the cell shows a 2:1 stimulus to response ratio.  The one-dimensional mapping between APD and DI fails to explain quasi-periodic oscillation of the APD. Figure 2. APD restitution and dynamical mechanism of APD alternans.   [see]

Calcium Driven Alternans

A strong-weak-strong-weak oscillation in contrac­tion implies that the Ca transient (CaT) is alternating. Until 1999 it was assumed that if the APD is alternat­ing then the CaT alternates because the CaT follows APD changes. However, Chudin et al showed that CaT can alternate even when APD is kept constant during pacing with a periodic AP clamp waveform.14 This implies that the intracellular Ca cycling has intrinsic nonlinear dynamics. A critical component in this process is the sarcoplasmic reticulum (SR), a subcellular organelle that stores Ca inside the cell. When Ca enters a cell through the L-type Ca channel (or reverse mode Na-Ca exchanger (NCX) ryanodine receptors open and large Ca releases occur from the SR (Ca induced Ca release). The amount of Ca release steeply depends on SR Ca load. This steep relation between Ca release and SR Ca load is the key to induce CaT alternans.  A one-dimensional map between Ca release and SR calcium load can be constructed to describe the relationship21 similar to the map used in APD restitution.

 Subcellular Alternans

A number of experimental and computational stud­ies have been undertaken to identify molecular mechanisms of CaT alternans by identifying the specific components in the calcium cycling process critical to formation of CaT alternans. These compo­nents include SR Ca leak and load, Ca spark frequency and amplitude, and rate of SR refilling. For example, experiments have shown that alternation in diastolic SR Ca is not required for CaT alternans.24 In addition, stochastic openings of ryanodine receptors (RyR) lead to Ca sparks that occur randomly, not in an alternating sequence that would be expected to underlie Ca altern-ans. So, how do local random sparks and constant dia­stolic SR calcium load lead to global CaT alternans? Mathematical models with detailed representations of subcellular Ca cycling have been developed in order to elucidate the underlying mechanisms. Model­ing studies have shown that even when SR Ca load is not changing, RyRs, which are analogous to ICaL in APD alternans, recover gradually from refractoriness. As RyR availability increases (for example during a long diastolic interval) a single Ca spark from a RyR will be larger in amplitude and recruit neighboring Ca release units to generate more sparks. The large resultant CaT causes depletion of the SR and when complete recovery of RyRs does not occur prior to the arrival of the next stimulus, the subsequent CaT will be small. This process results in an alternans of CaT amplitude from beat-to-beat.

 Coupling Between the Membrane Potential and Subcellular Calcium Dynamics

Importantly, the membrane voltage and intracellu­lar Ca cycling are coupled via Ca sensitive channels such as the L-type Ca channel and the sodium-calcium exchanger (NCX). The membrane voltage dynamics and the intracellular Ca dynamics are bi-directionally coupled. One direction is from voltage to Ca. As the DI becomes longer, the CaT usually becomes larger since the recovery time for the L-type Ca channel in increased and the SR Ca release becomes larger. The other direction is from Ca to voltage. Here we consider two major currents, NCX and ICaL. As the CaT becomes larger, forward mode NCX becomes larger and pro­longs APD. On the other hand, as the CaT becomes larger, ICaL becomes smaller due to Ca-induced inacti­vation, and thus, larger CaT shortens the APD. There­fore, depending on which current dominates, larger CaT can prolong or shorten APD. If a larger CaT pro­longs (shortens) the APD, then the coupling is positive (negative). The coupled dynamics of the membrane voltage and the intracellular Ca cycling can be cate­gorized by the instability of membrane voltage (steep APD restitution), instability of the intracellular Ca cycling (steep relation between Ca release versus SR Ca load), and the coupling (positive or negative). If the coupling is positive, alternans is electromechani­cally concordant (long-short-long-short APD cor­responds to large-small-large-small CaT sequence) regardless of the underlying instability mechanism. On the other hand, if the coupling is negative, alternans is electromechanically concordant in a voltage-driven regime. However, if alternans is Ca driven, alternans becomes electromechanically discordant (long-short-long-short APD corresponds to small-large-small-large CaT sequence). It is also possible to induce quasi- periodic oscillation of APD and CaT when volt­age and Ca instabilities contribute equally.

 Alternans in Higher Dimensions

Tissue level alternans in APD and CaT also occur and here we describe how the dynamical mechanism of alternans at the single cell level determines the phenomena in tissue. Spatially discordant alternans (SDA) where APDs in different regions of tissue alternate out-of-phase, is more arrhythmogenic since it causes large gradients of refractoriness and wave-break, which can initiate ventricular tachycardia and ventricular fibrillation. How is SDA induced? As the APD is a function of the previous DI, con­duction velocity (CV) is also function of the previ­ous DI (CV restitution) since the action potential propagation speed depends on the availability of the sodium channel. As the DI becomes shorter, sodium channels have less time to recover. Therefore, in general, as the DI becomes shorter, the CV becomes slower. When tissue is paced rapidly, action poten­tials propagate slowly near the stimulus, and thenac-celerate downstream as the DI becomes longer. This causes heterogeneity in APD (APD is shorter near the stimulus). During the following tissue excitation, APD becomes longer and the CV becomes faster at the pacing site then gradually APD becomes shorter and the CV becomes slower. The interaction between steep APD restitution and steep CV restitution creates SDA. This mechanism applies only when the cel­lular instability is voltage driven. When the cellular instability is Ca driven, the mechanism of SDA formation is different. If the volt­age-Ca coupling is negative, SDA can form without steep APD and CV restitution. The mechanism can be understood as follows. First, when cells are uncou­pled, alternans of APD and Ca are electromechanically discordant. If two cells are alternating in opposite phases, once these cells are coupled by voltage, due to electrotonic coupling, the membrane voltage of both cells is synchronized and thus APD becomes the same. This synchronization of APD amplifies the difference of CaT between two cells (Fig. 5 in). In other words it desynchronizes CaT. This instability mechanism is also found in subcellular SDA. In the case where the instability is Ca driven and the coupling is positive, there are several interest­ing distinctive phenomena that can occur. First, the profile of SDA of Ca contains a much steeper gra­dient at the node (point in space where no alternans occurs–cells downstream of the node are alternating out of phase with those upstream of the node) com­pared to the case of voltage driven SDA. Thus, the cellular mechanism of instability can be identified by evaluating the steepness of the alternans amplitude gradient in space around the node. When the cellular instability is voltage driven, the steady-state wave­length (separation of nodes in space) depends on electrotonic coupling between cells and the steepness of APD and CV restitution, regardless of the initial conditions. However, if the cellular instability is Ca driven, the location of nodes depends on the pacing history, which includes pacing cycle length and other parameters affected by pacing frequency. In this case, once the node is formed, the location of the node may be fixed, especially when Ca instability is strong. Such an explanation may apply to recent experimen­tal results. Summary In this review, we described how the origin of alternans at the cellular level (voltage driven, Ca drive, coupling between voltage and Ca) affects the formation of spatially discordant alternans at the tissue level. Cardiac alternans is a multi-scale emergent phenomenon. Channel properties determine the instability mechanism at the cellular level. Alternans mechanisms at cellular level determine SDA patterns at the tissue level. In order to understand alternans and develop anti-arrhythmic drug and therapy, multi-scale modeling of the heart is useful, which is increasingly enabled by emerging technologies such as general-purpose computing on graphics processing units (GPGPU) and cloud computing.

English: Diagram of contraction of smooth musc...

English: Diagram of contraction of smooth muscle fiber (Photo credit: Wikipedia)

Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs receptors voltage gated Ca(2) channel Marks-Wehrens Model and multiphosphorylation  site model ncpcardio0419-f4   calcium leak

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Demonstration of a diagnostic clinical laboratory neural network agent applied to three laboratory data conditioning problems

Izaak Mayzlin                                                                        Larry Bernstein, MD

Principal Scientist, MayNet                                            Technical Director

Boston, MA                                                                          Methodist Hospital Laboratory, Brooklyn, NY

Our clinical chemistry section services a hospital emergency room seeing 15,000 patients with chest pain annually.  We have used a neural network agent, MayNet, for data conditioning.  Three applications are – troponin, CKMB, EKG for chest pain; B-type natriuretic peptide (BNP), EKG for congestive heart failure (CHF); and red cell count (RBC), mean corpuscular volume (MCV), hemoglobin A2 (Hgb A2) for beta thalassemia.  Three data sets have been extensively validated prior to neural network analysis using receiver-operator curve (ROC analysis), Latent Class Analysis, and a multinomial regression approach.  Optimum decision points for classifying using these data were determined using ROC (SYSTAT, 11.0), LCM (Latent Gold), and ordinal regression (GOLDminer).   The ACS and CHF studies both had over 700 patients, and had a different validation sample than the initial exploratory population.  The MayNet incorporates prior clustering, and sample extraction features in its application.   Maynet results are in agreement with the other methods.

Introduction: A clinical laboratory servicing a hospital with an  emergency room seeing 15,000 patients with chest pain to produce over 2 million quality controlled chemistry accessions annually.  We have used a neural network agent, MayNet, to tackle the quality control of the information product.  The agent combines a statistical tool that first performs clustering of input variables by Euclidean distances in multi-dimensional space. The clusters are trained on output variables by the artificial neural network performing non-linear discrimination on clusters’ averages.  In applying this new agent system to diagnosis of acute myocardial infarction (AMI) we demonstrated that at an optimum clustering distance the number of classes is minimized with efficient training on the neural network. The software agent also performs a random partitioning of the patients’ data into training and testing sets, one time neural network training, and an accuracy estimate on the testing data set. Three examples to illustrate this are – troponin, CKMB, EKG for acute coronary syndrome (ACS); B-type natriuretic peptide (BNP), EKG for the estimation of ejection fraction in congestive heart failure (CHF); and red cell count (RBC), mean corpuscular volume (MCV), hemoglobin A2 (Hgb A2) for identifying beta thalassemia.  We use three data sets that have been extensively validated prior to neural network analysis using receiver-operator curve (ROC analysis), Latent Class Analysis, and a multinomial regression approach.

In previous studies1,2 CK-MB and LD1 sampled at 12 and 18 hours postadmission were near-optimum times used to form a classification by the analysis of information in the data set. The population consisted of 101 patients with and 41 patients without AMI based on review of the medical records, clinical presentation, electrocardiography, serial enzyme and isoenzyme  assays, and other tests. The clinical or EKG data, and other enzymes or sampling times were not used to form a classification but could be handled by the program developed. All diagnoses were established by cardiologist review. An important methodological problem is the assignment of a correct diagnosis by a “gold standard” that is independent of the method being tested so that the method tested can be suitably validated. This solution is not satisfactory in the case of myocardial infarction because of the dependence of diagnosis on a constellation of observations with different sensitivities and specificities. We have argued that the accuracy of diagnosis is  associated with the classes formed by combined features and has greatest uncertainty associated with a single measure.

Methods:  Neural network analysis is by MayNet, developed by one of the authors.  Optimum decision points for classifying using these data were determined using ROC (SYSTAT, 11.0), LCM (Latent Gold)3, and ordinal regression (GOLDminer)4.   Validation of the ACS and CHF study sets both had over 700 patients, and all studies had a different validation sample than the initial exploratory population.  The MayNet incorporates prior clustering, and sample extraction features in its application.   We now report on a new classification method and its application to diagnosis of acute myocardial infarction (AMI).  This method is based on the combination of clustering by Euclidean distances in multi-dimensional space and non-linear discrimination fulfilled by the Artificial Neural Network (ANN) trained on clusters’ averages.   These studies indicate that at an optimum clustering distance the number of classes is minimized with efficient training on the ANN. This novel approach to ANN reduces the number of patterns used for ANN learning and works also as an effective tool for smoothing data, removing singularities,  and increasing the accuracy of classification by the ANN. The studies  conducted involve training and testing on separate clinical data sets, which subsequently achieves a high accuracy of diagnosis (97%).

Unlike classification, which assumes the prior definition of borders between classes5,6, clustering procedure includes establishing these borders as a result of processing statistical information and using a given criteria for difference (distance) between classes.  We perform clustering using the geometrical (Euclidean) distance between two points in n-dimensional space, formed by n variables, including both input and output variables. Since this distance assumes compatibility of different variables, the values of all input variables are linearly transformed (scaled) to the range from 0 to 1.

The ANN technique for readers accustomed to classical statistics can be viewed as an extension of multivariate regression analyses with such new features as non-linearity and ability to process categorical data. Categorical (not continuous) variables represent two or more levels, groups, or classes of correspondent feature, and in our case this concept is used to signify patient condition, for example existence or not of AMI.

The ANN is an acyclic directed graph with input and output nodes corresponding respectively to input and output variables. There are also “intermediate” nodes, comprising so called “hidden” layers.  Each node nj is assigned the value xj that has been evaluated by the node’s “processing” element, as a non-linear function of the weighted sum of values xi of nodes ni, connected with nj by directed edges (ni, nj).

xj = f(wi(1),jxi(1) + wi(2),jxi(2) + … + wi(l),jxi(l)),

where xk is the value in node nk and wk,j is the “weight” of the edge (nk, nj).  In our research we used the standard function f(x), “sigmoid”, defined as f(x)=1/(1+exp(-x)).  This function is suitable for categorical output and allows for using an efficient back-propagation algorithm7 for calculating the optimal values of weights, providing the best fit for learning set of data, and eventually the most accurate classification.

Process description:  We implemented the proposed algorithm for diagnosis of AMI. All the calculations were performed on PC with Pentium 3 Processor applying the authors’ unique Software Agent Maynet. First, using the automatic random extraction procedure, the initial data set (139 patients) was partitioned into two sets — training and testing.  This randomization also determined the size of these sets (96 and 43, respectively) since the program was instructed to assign approximately 70 % of data to the training set.

The main process consists of three successive steps: (1) clustering performed on training data set, (2) neural network’s training on clusters from previous step, and (3) classifier’s accuracy evaluation on testing data.

The classifier in this research will be the ANN, created on step 2, with output in the range [0,1], that provides binary result (1 – AMI, 0 – not AMI), using decision point 0.5.

In this demonstartion we used the data of two previous studies1,2 with three patients, potential outliers, removed (n = 139). The data contains three input variables, CK-MB, LD-1, LD-1/total LD, and one output variable, diagnoses, coded as 1 (for AMI) or 0 (non-AMI).

Results: The application of this software intelligent agent is first demonstrated here using the initial model. Figures 1-2 illustrate the history of training process. One function is the maximum (among training patterns) and lower function shows the average error. The latter defines duration of training process. Training terminates when the average error achieves 5%.

There was slow convergence of back-propagation algorithm applied to the training set of 96 patients. We needed 6800 iterations to achieve the sufficiently small (5%) average error.

Figure 1 shows the process of training on stage 2. It illustrates rapid convergence because we deal only with 9 patterns representing the 9 classes, formed on step 1.

Table 1 illustrates the effect of selection of maximum distance on the number of classes formed and on the production of errors. The number of classes increased with decreasing distance, but accuracy of classification does not decreased.

The rate of learning is inversely related to the number of classes. The use of the back-propagation to train on the entire data set without prior processing is slower than for the training on patterns.

     Figures 2 is a two-dimensional projection of three-dimensional space of input variables CKMB and LD1 with small dots corresponding to the patterns and rectangular as cluster centroids (black – AMI, white – not AMI).

     We carried out a larger study using troponin I (instead of LD1) and CKMB for the diagnosis of myocardial infarction (MI).  The probabilities and odds-ratios for the TnI scaled into intervals near the entropy decision point are shown in Table 2 (N = 782).  The cross-table shows the frequencies for scaled TnI results versus the observed MI, the percent of values within MI, and the predicted probabilities and odds-ratios for MI within TnI intervals.  The optimum decision point is at or near 0.61 mg/L (the probability of MI at 0.46-0.6 mg/L is 3% and the odds ratio is at 13, while the probability of MI at 0.61-0.75 mg/L is 26% at an odds ratio of 174) by regressing the scaled values.

     The RBC, MCV criteria used were applied to a series of 40 patients different than that used in deriving the cutoffs.  A latent class cluster analysis is shown in Table 3.  MayNet is carried out on all 3 data sets for MI, CHF, and for beta thalassemia for comparison and will be shown.

Discussion:  CKMB has been heavily used for a long time to determine heart attacks. It is used in conjunction with a troponin test and the EKG to identify MI but, it isn’t as sensitive as is needed. A joint committee of the AmericanCollege of Cardiology and European Society of Cardiology (ACC/ESC) has established the criteria for acute, recent or evolving AMI predicated on a typical increase in troponin in the clinical setting of myocardial ischemia (1), which includes the 99th percentile of a healthy normal population. The improper selection of a troponin decision value is, however, likely to increase over use of hospital resources.  A study by Zarich8 showed that using an MI cutoff concentration for TnT from a non-acute coronary syndrome (ACS) reference improves risk stratification, but fails to detect a positive TnT in 11.7% of subjects with an ACS syndrome8. The specificity of the test increased from 88.4% to 96.7% with corresponding negative predictive values of 99.7% and 96.2%. Lin et al.9 recently reported that the use of low reference cutoffs suggested by the new guidelines results in markedly increased TnI-positive cases overall. Associated with a positive TnI and a negative CKMB, these cases are most likely false positive for MI. Maynet relieves this and the following problem effectively.

Monitoring BNP levels is a new and highly efficient way of diagnosing CHF as well as excluding non-cardiac causes of shortness of breath. Listening to breath sounds is only accurate when the disease is advanced to the stage in which the pumping function of the heart is impaired. The pumping of the heart is impaired when the circulation pressure increases above the osmotic pressure of the blood proteins that keep fluid in the circulation, causing fluid to pass into the lung’s airspaces.  Our studies combine the BNP with the EKG measurement of QRS duration to predict whether a patient has a high or low ejection fraction, a measure to stage the severity of CHF.

We also had to integrate the information from the hemogram (RBC, MCV) with the hemoglobin A2 quantitation (BioRad Variant II) for the diagnosis of beta thalassemia.  We chose an approach to the data that requires no assumption about the distribution of test values or the variances.   Our detailed analyses validates an approach to thalassemia screening that has been widely used, the Mentzer index10, and in addition uses critical decision values for the tests that are used in the Mentzer index. We also showed that Hgb S has an effect on both Hgb A2 and Hgb F.  This study is adequately powered to assess the usefulness of the Hgb A2 criteria but not adequately powered to assess thalassemias with elevated Hgb F.


1.  Adan J, Bernstein LH, Babb J. Lactate dehydrogenase isoenzyme-1/total ratio: accurate for determining the existence of myocardial infarction. Clin Chem 1986;32:624-8.

2. Rudolph RA, Bernstein LH, Babb J. Information induction for predicting acute myocardial infarction.  Clin Chem 1988;34:2031- 2038.

3. Magidson J. “Maximum Likelihood Assessment of Clinical Trials Based on an Ordered Categorical Response.” Drug Information Journal, Maple Glen, PA: Drug Information Association 1996;309[1]: 143-170.

4. Magidson J and Vermoent J.  Latent Class Cluster Analysis. in J. A. Hagenaars and A. L. McCutcheon (eds.), Applied Latent Class Analysis. Cambridge: CambridgeUniversity Press, 2002, pp. 89-106.

5. Mkhitarian VS, Mayzlin IE, Troshin LI, Borisenko LV. Classification of the base objects upon integral parameters of the attached network. Applied Mathematics and Computers.  Moscow, USSR: Statistika, 1976: 118-24.

6.Mayzlin IE, Mkhitarian VS. Determining the optimal bounds for objects of different classes. In: Dubrow AM, ed. Computational Mathematics and Applications. MoscowUSSR: Economics and Statistics Institute. 1976: 102-105.

7. RumelhartDE, Hinton GE, Williams RJ. Learning internal representations by error propagation. In:

RumelhartDE, Mc Clelland JL, eds. Parallel distributed processing.   Cambridge, Mass: MIT Press, 1986; 1: 318-62.

8. Zarich SW, Bradley K, Mayall ID, Bernstein, LH. Minor Elevations in Troponin T Values Enhance Risk Assessment in Emergency Department Patients with Suspected Myocardial Ischemia: Analysis of Novel Troponin T Cut-off Values.  Clin Chim Acta 2004 (in press).

9. Lin JC, Apple FS, Murakami MM, Luepker RV.  Rates of positive cardiac troponin I and creatine kinase MB mass among patients hospitalized for suspected acute coronary syndromes.  Clin Chem 2004;50:333-338.

10.Makris PE. Utilization of a new index to distinguish heterozygous thalassemic syndromes: comparison of its specificity to five other discriminants.Blood Cells. 1989;15(3):497-506.

Acknowledgements:   Jerard Kneifati-Hayek and Madeleine Schlefer, Midwood High School, Brooklyn, and Salman Haq, Cardiology Fellow, Methodist Hospital.

Table 1. Effect of selection of maximum distance on the number of classes formed and on the accuracy of recognition by ANN

ClusteringDistanceFactor F(D = F * R)  Number ofClasses  Number of Nodes inThe HiddenLayers  Number ofMisrecognizedPatterns inThe TestingSet of 43 Percent ofMisrecognized  2414135  1, 02, 03, 01, 02, 03, 0

3, 2

3, 2






Figure 1.

Figure 2.

Table 2.  Frequency cross-table, probabilities and odds-ratios for scaled TnI versus expected diagnosis

Range Not MI MI N Pct in MI Prob by TnI Odds Ratio
< 0.45 655 2 657 2 0 1
0.46-0.6 7 0 7 0 0.03 13
0.61-0.75 4 0 4 0. 0.26 175
0.76-0.9 13 59 72 57.3 0.82 2307
> 0.9 0 42 42 40.8 0.98 30482
679 103 782 100


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