Posts Tagged ‘Eukaryotic’

Platelet Endothelial Aggregation Receptor-1 (PEAR1) Gene to be most strongly associated with Dual Antiplatelet Therapy Response: Genetic Determinants of Variable Response to Aspirin (alone and in combination with Clopidogrel)

Reporter: Aviva Lev-Ari, PhD, RN

4 Genetic Variation in PEAR1 is Associated with Platelet Aggregation and Cardiovascular Outcomes

Joshua P. Lewis1Kathleen Ryan1Jeffrey R. O’Connell1Richard B. Horenstein1,Coleen M. Damcott1Quince Gibson1Toni I. Pollin1Braxton D. Mitchell1Amber L. Beitelshees1Ruth Pakzy1Keith Tanner1Afshin Parsa1Udaya S. Tantry2Kevin P. Bliden2Wendy S. Post3Nauder Faraday3William Herzog4Yan Gong5Carl J. Pepine6Julie A. Johnson5Paul A. Gurbel2 and Alan R. Shuldiner7*

Author Affiliations

1University of Maryland School of Medicine, Baltimore, MD

2Sinai Hospital of Baltimore, Baltimore, MD

3Johns Hopkins University School of Medicine, Baltimore, MD

4Sinai Hospital of Baltimore & Johns Hopkins University School of Medicine, Baltimore, MD

5University of Florida College of Pharmacy, Gainesville, FL

6University of Florida College of Medicine, Gainesville, FL

7University of Maryland School of Medicine & Veterans Administration Medical Center, Baltimore, MD

* University of Maryland School of Medicine & Veterans Administration Medical Center, Baltimore, MD ashuldin@medicine.umaryland.edu


Background-Aspirin or dual antiplatelet therapy (DAPT) with aspirin and clopidogrel is standard therapy for patients at increased risk for cardiovascular events. However, the genetic determinants of variable response to aspirin (alone and in combination with clopidogrel) are not known.

Methods and Results-We measured ex-vivo platelet aggregation before and after DAPT in individuals (n=565) from the Pharmacogenomics of Antiplatelet Intervention (PAPI) Study and conducted a genome-wide association study (GWAS) of drug response. Significant findings were extended by examining genotype and cardiovascular outcomes in two independent aspirin-treated cohorts: 227 percutaneous coronary intervention (PCI) patients, and 1,000 patients of the International VErapamil SR/trandolapril Study (INVEST) GENEtic Substudy (INVEST-GENES). GWAS revealed a strong association between single nucleotide polymorphisms on chromosome 1q23 and post-DAPT platelet aggregation. Further genotyping revealed rs12041331 in the platelet endothelial aggregation receptor-1 (PEAR1) gene to be most strongly associated with DAPT response (P=7.66×10-9). In Caucasian and African American patients undergoing PCI, A-allele carriers of rs12041331 were more likely to experience a cardiovascular event or death compared to GG homozygotes (hazard ratio = 2.62, 95%CI 0.96-7.10, P=0.059 and hazard ratio = 3.97, 95%CI 1.10-14.31, P=0.035 respectively). In aspirin-treated INVEST-GENES patients, rs12041331 A-allele carriers had significantly increased risk of myocardial infarction compared to GG homozygotes (OR=2.03, 95%CI 1.01-4.09, P=0.048).

Conclusions – Common genetic variation in PEAR1 may be a determinant of platelet response and cardiovascular events in patients on aspirin, alone and in combination with clopidogrel.

Clinical Trial Registration Information-clinicaltrials.gov; Identifiers:NCT00799396 and NCT00370045




Circulation: Cardiovascular Genetics.2013; 6: 184-192 Published online before print February 7, 2013,doi: 10.1161/​CIRCGENETICS.111.964627




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The immune response mechanism is the holy grail of the human defense system for health.   IDO, indolamine 2, 3-dioxygenase, is a key gene for homeostasis of immune responses and producing an enzyme catabolizing the first rate-limiting step in tryptophan degradation metabolism. The hemostasis of immune system is complicated.  In this review, the  properties of IDO such as basic molecular genetics, biochemistry and genesis will be discussed.

IDO belongs to globin gene family to carry oxygen and heme.  The main function and genesis of IDO comes from the immune responses during host-microbial invasion and choice between tolerance and immunegenity.  In human there are three kinds of IDOs, which are IDO1, IDO2, and TDO, with distinguished mechanisms and expression profiles. , IDO mechanism includes three distinguished pathways: enzymatic acts through IFNgamma, non-enzymatic acts through TGFbeta-IFNalpha/IFNbeta and moonlighting acts through AhR/Kyn.

The well understood functional genomics and mechanisms is important to translate basic science for clinical interventions of human health needs. In conclusion, overall purpose is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.

The first part of the review concerns the basic science information gained overall several years that lay the foundation where translational research scientist should familiar to develop a new technology for clinic. The first connection of IDO and human health came from a very natural event that is protection of pregnancy in human. The focus of the translational medicine is treatment of cancer or prevention/delay cancer by stem cell based Dendritic Cell Vaccine (DCvax) development.

Table of Contents:

  • Abstract

1         Introduction: IDO gene encodes a heme enzyme

2        Location, location, location

3        Molecular genetics

4        Types of IDO:

4.1       IDO1,

4.2       IDO2,

4.3       IDO-like proteins

5        Working mechanisms of IDO

6        Infection Diseases and IDO

7. Conclusion

  1. 1.     Indoleamine 2, 3-dioxygenase (IDO) gene encodes a heme enzyme

IDO is a key homeostatic regulator and confined in immune system mechanism for the balance between tolerance and immunity.  This gene encodes indoleamine 2, 3-dioxygenase (IDO) – a heme enzyme (EC= that catalyzes the first rate-limiting step in tryptophan catabolism to N-formyl-kynurenine and acts on multiple tryptophan substrates including D-tryptophan, L-tryptophan, 5-hydroxy-tryptophan, tryptamine, and serotonin.

The basic genetic information describes indoleamine 2, 3-dioxygenase 1 (IDO1, IDO, INDO) as an enzyme located at Chromosome 8p12-p11 (5; 6) that active at the first step of the Tryptophan catabolism.    The cloned gene structure showed that IDO contains 10 exons ad 9 introns (7; 8) producing 9 transcripts.

After alternative splicing only five of the transcripts encode a protein but the other four does not make protein products, three of transcripts retain intron and one of them create a nonsense code (7).  Based on IDO related studies 15 phenotypes of IDO is identified, of which, twelve in cancer tumor models of lung, kidney, endometrium, intestine, two in nervous system, and one HGMD- deletion.

  1. 2.     Location, Location and Location

The specific cellular location of IDO is in cytosol, smooth muscle contractile fibers and stereocilium bundle. The expression specificity shows that IDO is present very widely in all cell types but there is an elevation of expression in placenta, pancreas, pancreas islets, including dendritic cells (DCs) according to gene atlas of transcriptome (9).  Expression of IDO is common in antigen presenting cells (APCs), monocytes (MO), macrophages (MQs), DCs, T-cells, and some B-cells. IDO present in APCs (10; 11), due to magnitude of role play hierarchy and level of expression DCs are the better choice but including MOs during establishment of three DC cell subset, CD14+CD25+, CD14++CD25+ and CD14+CD25++ may increase the longevity and efficacy of the interventions.

IDO is strictly regulated and confined to immune system with diverse functions based on either positive or negative stimulations. The positive stimulations are T cell tolerance induction, apoptotic process, and chronic inflammatory response, type 2 immune response, interleukin-12 production (12).  The negative stimulations are interleukin-10 production, activated T cell proliferation, T cell apoptotic process.  Furthermore, there are more functions allocating fetus during female pregnancy; changing behavior, responding to lipopolysaccharide or multicellular organismal response to stress possible due to degradation of tryptophan, kynurenic acid biosynthetic process, cellular nitrogen compound metabolic process, small molecule metabolic process, producing kynurenine process (13; 14; 15).

IDO plays a role in a variety of pathophysiological processes such as antimicrobial and antitumor defense, neuropathology, immunoregulation, and antioxidant activity (16; 17; 18; 19).


 3.     Molecular Genetics of IDO:

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3' untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database. (reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3′ untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database.
(reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

Molecular genetics data from earlier findings based on reporter assay results showed that IDO promoter is regulated by ISRE-like elements and GAS-sequence at -1126 and -1083 region (20).  Two cis-acting elements are ISRE1 (interferon sequence response element 1) and interferon sequence response element 2 (ISRE2).

Analyses of site directed and deletion mutation with transfected cells demonstrated that introduction of point mutations at these elements decreases the IDO expression. Removing ISRE1 decreases the effects of IFNgamma induction 50 fold and deleting ISRE1 at -1126 reduced by 25 fold (3). Introducing point mutations in conserved t residues at -1124 and -1122 (from T to C or G) in ISRE consensus sequence NAGtttCA/tntttNCC of IFNa/b inducible gene ISG4 eliminates the promoter activity by 24 fold (21).

ISRE2 have two boxes, X box (-114/1104) and Y Box 9-144/-135), which are essential part of the IFNgamma response region of major histocompatibility complex class II promoters (22; 23).  When these were removed from ISRE2 or introducing point mutations at two A residues of ISRE2 at -111 showed a sharp decrease after IFNgamma treatment by 4 fold (3).

The lack of responses related to truncated or deleted IRF-1 interactions whereas IRF-2, Jak2 and STAT91 levels were similar in the cells, HEPg2 and ME180 (3). Furthermore, 748 bp deleted between these elements did not affect the IDO expression, thus the distance between ISRE1 and ISRE2 elements have no function or influence on IDO (3; 24)

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

4.     There are three types of IDO in human genome:

IDO was originally discovered in 1967 in rabbit intestine (25). Later, in 1990 the human IDO gene is cloned and sequenced (7).  However, its importance and relevance in immunology was not created until prevention of allocation of fetal rejection and founding expression in wide range of human cancers (26; 27).

There are three types of IDO, pro-IDO like, IDO1, and IDO2.  In addition, another enzyme called TDO, tryptophan 2, 3, dehydrogenase solely degrade L-Trp at first-rate limiting mechanism in liver and brain.

4.1.  IDO1:

IDO1 mechanism is the target for immunotherapy applications. The initial discovery of IDO in human physiology is protection of pregnancy (1) since lack of IDO results in premature recurrent abortion (28; 26; 29).   The initial rate-limiting step of tryptophan metabolism is catalyzed by either IDO or tryptophan 2, 3-dioxygenase (TDO).

Structural studies of IDO versus TDO presenting active site environments, conserved Arg 117 and Tyr113, found both in TDO and IDO for the Tyr-Glu motif, but His55 in TDO replaced by Ser167b in IDO (30; 2). As a result, they are regulated with different mechanisms (1; 2) (30).  The short-lived TDO, about 2h, responds to level of tryptophan and its expression regulated by glucorticoids (31; 32).  Thus, it is a useful target for regulation and induced by tryptophan so that increasing tryptophan induces NAD biosynthesis. Whereas, IDO is not activated by the level of Trp presence but inflammatory agents with its interferon stimulated response elements (ISRE1 and ISRE2) in its (33; 34; 35; 36; 3; 10) promoter.

TDO promoter contains glucorticoid response elements (37; 38) and regulated by glucocorticoids and other available amino acids for gluconeogenesis. This is how IDO binds to only immune response cells and TDO relates to NAD biosynthesis mechanisms. Furthermore, TDO is express solely in liver and brain (36).  NAD synthesis (39) showed increased IDO ubiquitous and TDO in liver and causing NAD level increase in rat with neuronal degeneration (40; 41).  NAM has protective function in beta-cells could be used to cure Type1 diabetes (40; 42; 43). In addition, knowledge on NADH/NAD, Kyn/Trp or Trp/Kyn ratios as well as Th1/Th2, CD4/CD8 or Th17/Threg are equally important (44; 40).

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

4.2. IDO2:

The third type of IDO, called IDO2 exists in lower vertebrates like chicken, fish and frogs (45) and in human with differential expression properties. The expression of IDO2 is only in DCs, unlike IDO1 expresses on both tumors and DCs in human tissues.  Yet, in lower invertebrates IDO2 is not inhibited by general inhibitor of IDO, D-1-methyl-tryptophan (1MT) (46).   Recently, two structurally unusual natural inhibitors of IDO molecules, EXIGUAMINES A and B, are synthesized (47).  LIP mechanism cannot be switch back to activation after its induction in IDO2 (46).

Crucial cancer progression can continue with production of IL6, IL10 and TGF-beta1 to help invasion and metastasis.  Inclusion of two common SNPs affects the function of IDO2 in certain populations.  SNP1 reduces 90% of IDO2 catalytic activity in 50% of European and Asian descent and SNP2 produce premature protein through inclusion of stop-codon in 25% of African descent lack functional IDO2 (Uniport).

4.3. IDO-like proteins: The Origin of IDO:

Knowing the evolutionary steps will helps us to identify how we can manage the regulator function to protect human health in cancer, immune disorders, diabetes, and infectious diseases.

Bacterial IDO has two types of IDOs that are group I and group II IDO (48).  These are the earliest version of the IDO, pro-IDO like, proteins with a quite complicated function.  Each microorganism recognized by a specific set of receptors, called Toll-Like Receptors (TLR), to activate the IDO-like protein expression based on the origin of the bacteria or virus (49; 35).   Thus, the genesis of human IDO originates from gene duplication of these early bacterial versions of IDO-like proteins after their invasion interactions with human host.  IDO1 only exists in mammals and fungi.

Fungi also has three types of IDO; IDOa, IDO beta, and IDO gamma (50) with different properties than human IDOs, perhaps multiple IDO is necessary for the world’s decomposers.

All globins, haemoglobins and myoglobins are destined to evolve from a common ancestor, which  is only 14-16kDa (51) length. Binding of a heme and being oxygen carrier are central to the enzyme mechanism of this family.  Globins are classified under three distinct origins; a universal globin, a compact globin, and IDO-like globin (52) IDO like globin widely distributed among gastropodic mollusks (53; 51).  The indoleamine 2, 3-dioxygenase 1–like “myoglobin” (Myb) was discovered in 1989 in the buccal mass of the abalone Sulculus diversicolor (54).

The conserved region between Myb and IDO-like Myb existed for at least 600 million years (53) Even though the splice junction of seven introns was kept intact, the overall homolog region between Myb and IDO is only about 35%.

No significant evolutionary relationship is found between them after their amino acid sequence of each exon is compared to usual globin sequences. This led the hint that molluscan IDO-like protein must have other functions besides carrying oxygen, like myoglobin.   Alignment of S. cerevisiae cDNA, mollusk and vertebrate IDO–like globins show the key regions for controlling IDO or myoglobin function (55). These data suggest that there is an alternative pathways of myoglobin evolution.  In addition, understanding the diversity of globin may help to design better protocols for interventions of diseases.

Mechanisms of IDO:

The dichotomy of IDO mechanism lead the discovery that IDO is more than an enzyme as a versatile regulator of innate and adaptive immune responses in DCs (66; 67; 68). Meantime IDO also involve with Th2 response and B cell mediated autoimmunity showing that it has three paths, short term (acute) based on enzymatic actions, long term (chronic) based on non-enzymatic role, and moonlighting relies of downstream metabolites of tryptophan metabolism (69; 70).

IFNgamma produced by DC, MQ, NK, NKT, CD4+ T cells and CD8+ T cells, after stimulation with IL12 and IL8.  Inflammatory cytokine(s) expressed by DCs produce IFNgamma to stimulate IDO’s enzymatic reactions in acute response.  Then, TDO in liver and tryptophan catabolites act through Aryl hydrocarbon receptor induction for prevention of T cell proliferation. This mechanism is common among IDO, IDO2 (expresses in brain and liver) and TDO expresses in liver) provide an acute response for an innate immunity (30). When the pDCs are stimulated with IFNgamma, activation of IDO is go through Jak, STAT signaling pathway to degrade Trp to Kyn causing Trp depletion. The starvation of tryptophan in microenvironment inhibits generation of T cells by un-read t-RNAs and induce apoptosis through myc pathway.  In sum, lack of tryptophan halts T cell proliferation and put the T cells in apoptosis at S1 phase of cell division (71; 62).

The intermediary enzymes, functioning during Tryptophan degradation in Kynurenine (Kyn) pathway like kynurenine 3-hydroxylase and kynureninase, are also induced after stimulation with liposaccaride and proinflammatory cytokines (72). They exhibit their function in homeostasis through aryl-hydrocarbon receptor (AhR) induction by kynurenine as an endogenous signal (73; 74).  The endogenous tumor-promoting ligand of AhR are usually activated by environmental stress or xenobiotic toxic chemicals in several cellular processes like tumorigenesis, inflammation, transformation, and embryogenesis (Opitz ET. Al, 2011).

Human tumor cells constitutively produce TDO also contributes to production of Kyn as an endogenous ligand of the AhR (75; 27).  Degradation of tryptophan by IDO1/2 in tumors and tumor-draining lymph nodes occur. As a result, there are animal studies and Phase I/II clinical trials to inhibit the IDO1/2 to prevent cancer and poor prognosis (NewLink Genetics Corp. NCT00739609, 2007).

 IDO mechanism for immune response

Systemic inflammation (like in sepsis, cerebral malaria and brain tumor) creates hypotension and IDO expression has the central role on vascular tone control (63).  Moreover, inflammation activates the endothelial coagulation activation system causing coagulopathies on patients.  This reaction is namely endothelial cell activation of IDO by IFNgamma inducing Trp to Kyn conversion. After infection with malaria the blood vessel tone has decreases, inflammation induce IDO expression in endothelial cells producing Kyn causing decreased trp, lower arterial relaxation, and develop hypotension (Wang, Y. et. al 2010).  Furthermore, existing hypotension in knock out Ido mice point out a secondary mechanism driven by Kyn as an endogenous ligand to activate non-canonical NfKB pathway (63).

Another study also hints this “back –up” mechanism by a significant outcome with a differential response in pDCs against IMT treatment.  Unlike IFN gamma conditioned pDC blocks T cell proliferation and apoptosis, methyl tryptophan fails to inhibit IDO activity for activating naïve T cells to make Tregs at TGF-b1 conditioned pDCs (77; 78).

 Indoleamine-Pyrrole 2,3,-Dioxygenase; IDO dioxygenase; Indeolamine-2,3

The second role of the IDO relies on non-enzymatic action as being a signal molecule. Yet, IDO2 and TDO are devoid of this function. This role mainly for maintenance of microenvironment condition. DCs response to TGFbeta-1 exposure starts the kinase Fyn induce phosphorylation of IDO-associated immunoreceptor tyrosine–based inhibitory motifs (ITIMs) for propagation of the downstream signals involving non-canonical (anti-inflammatory) NF-kB pathway for a long term response. When the pDCs are conditioned with TGF-beta1 the signaling (68; 77; 78) Phospho Inositol Kinase3 (PIK-3)-dependent and Smad independent pathways (79; 80; 81; 82; 83) induce Fyn-dependent phosphorylation of IDO ITIMs.  A prototypic ITIM has the I/V/L/SxYxxL/V/F sequence (84), where x in place of an amino acid and Y is phosphorylation sites of tyrosines (85; 86).

Smad independent pathway stimulates SHP and PIK3 induce both SHP and IDO phosphorylation. Then, formed SHP-IDO complex can induce non-canonical (non-inflammatory) NF-kB pathway (64; 79; 80; 82) by phosphorylation of kinase IKKa to induce nuclear translocation of p52-Relb towards their targets.  Furthermore, the SHP-IDO complex also may inhibit IRAK1 (68). SHP-IDO complex activates genes through Nf-KB for production of Ido1 and Tgfb1 genes and secretion of IFNalpha/IFNbeta.  IFNa/IFNb establishes a second short positive feedback loop towards p52-RelB for continuous gene expression of IDO, TGFb1, IFNa and IFNb (87; 68).  However, SHP-IDO inhibited IRAK1 also activates p52-RelB.  Nf-KB induction at three path, one main and two positive feedback loops, is also critical.  Finally, based on TGF-beta1 induction (76) cellular differentiation occurs to stimulate naïve CD4+ T cell differentiation to regulatory T cells (Tregs).  In sum, TGF-b1 and IFNalpha/IFNbeta stimulate pDCs to keep inducing naïve T cells for generation of Treg cells at various stages, initiate, maintain, differentiate, infect, amplify, during long-term immune responses (67; 66).

Moonlighting function of Kyn/AhR is an adaptation mechanism after the catalytic (enzymatic) role of IDO depletes tryptophan and produce high concentration of Kyn induce Treg and Tr1 cell expansion leading Tregs to use TGFbeta for maintaining this environment (67; 76). In this role, Kyn pathway has positive-feedback-loop function to induce IDO expression.

In T cells, tryptophan starvation induces Gcn2-dependent stress signaling pathway, which initiates uncharged Trp-tRNA binding onto ribosomes. Elevated GCN2 expression stimulates elF2alfa phosphorylation to stop translation initiation (88). Therefore, most genes downregulated and LIP, an alternatively initiated isoform of the b/ZIP transcription factor NF-IL6/CEBP-beta (89).

This mechanism happens in tumor cells based on Prendergast group observations. As a result, not only IDO1 propagates itself while producing IFNalpha/IFNbeta, but also demonstrates homeostasis choosing between immunegenity by production of TH17or tolerance by Tregs. This mechanism acts like a see-saw. Yet, tolerance also can be broken by IL6 induction so reversal mechanism by SOC-3 dependent proteosomal degradation of the enzyme (90).  All proper responses require functional peripheral DCs to generate mature DCs for T cells to avoid autoimmunity (91).

Niacin (vitamin B3) is the final product of tryptophan catabolism and first molecule at Nicotinomic acid (NDA) Biosynthesis.  The function of IDO in tryptophan and NDA metabolism has a great importance to develop new clinical applications (40; 42; 41).  NAD+, biosynthesis and tryptophan metabolisms regulate several steps that can be utilize pharmacologically for reformation of healthy physiology (40).

IDO for protection in Microbial Infection with Toll-like Receptors

The mechanism of microbial response and infectious tolerance are complex and the origination of IDO based on duplication of microbial IDO (49).  During microbial responses, Toll-like receptors (TLRs) play a role to differentiate and determine the microbial structures as a ligand to initiate production of cytokines and pro-inflammatory agents to activate specific T helper cells (92; 93; 94; 95). Uniqueness of TLR comes from four major characteristics of each individual TLR by ligand specificity, signal transduction pathways, expression profiles and cellular localization (96). Thus, TLRs are important part of the immune response signaling mechanism to initiate and design adoptive responses from innate (naïve) immune system to defend the host.

TLRs are expressed cell type specific patterns and present themselves on APCs (DCs, MQs, monocytes) with a rich expression levels (96; 97; 98; 99; 93; 100; 101; 102; 87). Induction signals originate from microbial stimuli for the genesis of mature immune response cells.  Co-stimulation mechanisms stimulate immature DCs to travel from lymphoid organs to blood stream for proliferation of specific T cells (96).  After the induction of iDCs by microbial stimuli, they produce proinflammatory cytokines such as TNF and IL-12, which can activate differentiation of T cells into T helper cell, type one (Th1) cells. (103).

Utilizing specific TLR stimulation to link between innate and acquired responses can be possible through simple recognition of pathogen-associated molecular patterns (PAMPs) or co-stimulation of PAMPs with other TLR or non-TLR receptors, or even better with proinflammatory cytokines.   Some examples of ligand- TLR specificity shown in Table1, which are bacterial lipopeptides, Pam3Cys through TLR2 (92; 104; 105).  Double stranded (ds) RNAs through TLR3 (106; 107), Lipopolysaccharide (LPS) through TLR4, bacterial flagellin through TLR5 (108; 109), single stranded RNAs through TLR7/8 (97; 98), synthetic anti-viral compounds imiquinod through TLR 7 and resiquimod through TLR8, unmethylated CpG DNA motifs through TLR9 (Krieg, 2000).

IDO action

Then, the specificity is established by correct pairing of a TLR with its proinflammatory cytokines, so that these permutations influence creation and maintenance of cell differentiation. For example, leading the T cell response toward a preferred Th1 or Th2 response possible if the cytokines TLR-2 mediated signals induce a Th2 profile when combined with IL-2 but TLR4 mediated signals lean towards Th1 if it is combined with IL-10 or Il-12, (110; 111)  (112).

TLR ligand TLR Reference
Lipopolysaccharide, LPS TLR4 (96).  (112).
Lipopeptides, Pam3Cys TLR2 (92; 104; 105)
Double stranded (ds) RNAs TLR3 (106; 107)
Bacterial flagellin TLR5 (108; 109)
Single stranded RNAs TLR7/8 (97; 98)
Unmethylated CpG DNA motifs TLR9 (Krieg, 2000)
Synthetic anti-viral compounds imiquinod and resiquimod TLR7 and TLR8 (Lee J, 2003)

Furthermore, if the DCs are stimulated with IL-6, DCs relieve the suppression of effector T cells by regulatory T cells (113).

The modification of IDO+ monocytes manage towards specific subset of T cell activation with specific TLRs are significantly important (94).

The type of cell with correct TLR and stimuli improves or decreases the effectiveness of stimuli. Induction of IDO in monocytes by synthetic viral RNAs (isRNA) and CMV was possible, but not in monocyte derived DCs or TLR2 ligand lipopeptide Pam3Cys since single- stranded RNA ligands target TLR7/8 in monocytes derive DCs only (Lee J, 2003).  These data show that TLRs has ligand specificity, signal transduction pathways, expression profiles and cellular localization so design of experiments should follow these rules.


Overall our purpose of this information is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.  This first part of the review concerns the basic science information gained overall several years that lay the foundation that translational research scientist should familiar to develop a new technology for clinic. The first connection of IDO and human health came from a very natural event that is protection of pregnancy in human. The focus of the translational medicine is treatment of cancer or prevention/delay cancer by stem cell based Dendritic Cell Vaccine (DCvax) development.


1. Biochemistry of tryptophan in health and disease. BenderDA. 1983, Mol Aspects Med , pp. 6:101–197.

2. Molecular insights into substrate recognition and catalysis by indolamine 2,3-dioxygenase. Forouhar, F., Anderson, R., Mowat, C.F, et al. 2006, PNAS, pp. vol. 104, no:2, 473-478.

3. Importance of the Two Interferon-stimulated Response Element. Konan KV, Taylor, MW. 1996, J. Biol. Chem.-, pp. 19140-5.

4. induction of indolamine 2,3 dioxygenase: A mechanism of the anti-tumor activity of interferon gamma. Ozaki, Y., Edelstein, M.P., Duch, D.S. 1998, PNAS USA., pp. vol:85, 1242-1246.

5. Localization of the human indoleamine 2,3-dioxygenase (IDO) gene to the pericentromeric region of human chromosome 8. . Burkin, D. J., Kimbro, K. S., Barr, B. L., Jones, C., Taylor, M. W., Gupta, S. L. 1993, Genomics , pp. 17: 262-263.

6. Localization of indoleamine 2,3-dioxygenase gene (INDO) to chromosome 8p12-p11 by fluorescent in situ hybridization. Najfeld, V., Menninger, J., Muhleman, D., Comings, D. E., Gupta, S. L. 1993, Cytogenet. Cell Genet. , pp. 64: 231-232.

7. Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA. . Dai, W., Gupta, S. L. 1990, Biochem. Biophys. Res. Commun. , pp. 168: 1-8.

8. Gene structure of human indoleamine 2,3-dioxygenase. Kadoya, A., Tone, S., Maeda, H., Minatogawa, Y., Kido, R. 1992, Biochem. Biophys. Res. Commun. , pp. 189: 530-536.

9. A gene atlas of th emouse and human protein-encoding transcriptomes. Andrew I. Su, Tim Wiltshire, Serge Batalov , Hilmar Lapp , Keith A. Ching , David Block, Jie Zhang , Richard Soden , Mimi Hayakawa , Gabriel Kreiman , Michael P. Cooke , John R. Walker , and John B. Hogenesch. 2004, PNAS, pp. vol. 101, no. 166062-6067 (10.1073/pnas.0400782101).

10. Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. Hwu P, Du MX, Lapointe R, Do M, Taylor MW, Young HA. 2000, J. Immunol, pp. 164:3596–3599.

11. Inhibition of T cell proliferation by acrophage tryptophan catabolism. Munn, D.H. et al. 1999, J. Exp. Med., p. 189:1363.

12. HeLa cells cocultured with peripheral blood lymphocytes acquire an immuno-inhibitory phenotype through up-regulation of indoleamine 2,3-dioxygenase activity. Logan, G. J., Smyth, C. M. F., Earl, J. W., Zaikina, I., Rowe, P. B., Smythe, J. A., Alexander, I. E. 2002, Immunology, pp. 105:478-487.

13. Indoleamine 2,3-Dioxygenase – Is It an Immun Suppressor? Soliman H, Mediaville-Varela M, Antonia S. 2010, Cancer J. , pp. 16:354-359.

14. Targeting the immunoregulatory indoleamine 2,3-dioxygenase pathway in immunotherapy. Johnson BA, III, Baban B, Mellor AL. 2009, Immunotherapy. , pp. 645–661.

15. Indoleamine 2,3-dioxygenase and regulation of T cell immunity. AL., Mellor. 2005, Biochem Biophys Res Commun. , pp. 338(1):20–24.

16. Fallarino, F., Grohmann, U., Hwang, K. W., Orabona, C., Vacca, C., Bianchi, R., Belladonna, M. L., Fioretti, M. C.Modulation of tryptophan catabolism by regulatory T cells. Fallarino, F., Grohmann, U., Hwang, K. W., Orabona, C., Vacca, C., Bianchi, R., Belladonna, M. L., Fioretti, M. C., Alegre, M.-L., Puccetti, P. 2003, Nature Immun., pp. 4: 1206-1212.

17. CTLA-4-Ig regulates tryptophan catabolism in vivo. Grohmann, U., Orabona, C., Fallarino, F., Vacca, C., Calcinaro, F., Falorni, A., Candeloro, P., Belladonna, M. L., Bianchi, R., Fioretti, M. C., Puccetti, P. 2002, Nature Immun. , pp. 3: 1097-1101.

18. Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy. Grohmann, U., Volpi, C., Fallarino, F., Bozza, S., Bianchi, R., Vacca, C., Orabona, C., Belladonna, M. L., Ayroldi, E., Nocentini, G., Boon, L., Bistoni, F., Fioretti, M. C., Romani, L., Riccardi, C., Puccetti, P. 2007, Nature Med., pp. 13:579-586.

19. Cells expressing indoleamine 2,3-dioxygenase inhibit T cell responses. Mellor, A. L., Keskin, D. B., Johnson, T., Chandler, P., Munn, D. H. 2002, J. Immun. , pp. 168: 3771-3776.

20. Chon, SY, Hassanain, HH, Piine, R., and Gupta, SL. 1995, J. Interferon Cytokine Res. , pp. 15, 517-526.

21. Levy, ED, KEsler, DS, Pine, R., Reich, N, and Darnell, JE.Jr et al. 1988, Genes Dev, pp. 2,383-393.

22. Benoist, C. and Manthis, D. 1990, Annu. Rev of Immunol., pp. 8, 681-715.

23. Dorn, A, Durand, B., Marling, C., Meur, M.L., Beoist, C., and Mathis, D. 1987, PNAS USA, pp. 34, 6249-6253.

24. Konan, K.V. Ph.D. Thesis. Transcriptional Regulation of the Indolamine 2,3-oxygenase Gene. s.l. : Indiana University, Bloominigton, 1995.

25. Tryptophan pyrrolase of rabbit intestine: D- and L–tryptophan cleaving enzyme or enzymes. Yamamoto, S., and Hayashi, O. 1967, J Biol Chem, pp. 242: 5260-5266.

26. Prevention of allogeneic fetal rejection by tryptophan catabolism. Munn, DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, Brown C, Mellor AL. 1998, Science, pp. 281:1191–3.

27. Evidence for a tumoral immune resistance mechanismbased on tryptophan degradation by indoleamine 2,3-dioxygenase. Uyttenhove, C. et al. 2003, Nature Med. 9,, pp. 1269–1274 .

28. Pregnancy: success and failure within the Th1/Th2/Th3 paradigm. Raghupathy, R. 2001., Seminars in Immunology, pp. Volume 13, Issue 4, Pages 219–227.

29. Why is the fetal allograft not rejected? Davies, C. J. March 2007 , J ANIM SCI , pp. vol. 85 no. 13 suppl E32-E35 .

30. Exploring the mechanism of tryptoophan 2,3-dioxygenase. Thackray, S., Mowat, C.G., Chapman, K. 2008, Biochem. Society Transaction., pp. 36, 1120-1123.

31. The new life of a centenarian: signalling functions of NAD(P). Berger F, Ramírez-Hernández MH, Ziegler M. 2004, Trends Biochem Sci , pp. 29:111–118 .

32. Biochemistry of tryptophan in health and disease. DA, Bender. 1983, Mol Aspects Med, pp. 6:101–197.

33. Poliovirus induces indoleamine-2,3-dioxygenase and quinolinic acid synthesis in macaque brain. Heyes MP, Saito K, Jacobowitz D, Markey SP, Takikawa O, Vickers JH. 1992, FASEB J., pp. 6:2977–2989.

34. Sanni LA, Thomas SR, Tattam BN, Moore DE, Chaudhri G, Stocker R, Hunt NH 1998Dramatic changes in oxidative tryptophan metabolism along the kynurenine pathway in experimental cerebral and noncerebral malaria. . Sanni LA, Thomas SR, Tattam BN, Moore DE, Chaudhri G, Stocker R, Hunt NH. 1998, Am J Pathol, pp. 152:611–619.

35. Induction of pulmonary indoleamine 2,3-dioxygenase by intraperitoneal injection of bacterial lipopolysaccharide. . Yoshida R, Hayaishi O. 1978, Proc Natl Acad Sci USA , pp. 75:3998–4000.

36. Induction of indoleamine 2,3-dioxygenase in mouse lung during virus infection. . Yoshida R, Urade Y, Tokuda M, Hayaishi O. 1979, Proc Natl Acad Sci USA , pp. 76:4084–4086.

37. Induction of pulmonary indoleamine 2,3-dioxygenase by intraperitoneal injection of bacterial lipopolysaccharide. Yoshida R, Hayaishi. 1978, PNAS USA, pp. 3998-4000.

38. Sequence of human 2,3-dioxygenase (TDO2): presence of a glucorticoid response-like element composed of a GTT repeat and intronic CCCCT repeat. Comings DE, Muhleman D, Dietz G, Sherman M, Forest. 1995, Genomics, pp. 29:390-396165.

39. Studies on the biosynthesis of Nicotinamide adenine inucleotide. II.Arole of picolinic carboxylase in the Biosynthesisofnicotinamideadeninedinucleotidefromtryptophan in mammals. Ikeda M, Tsuji H, Nakamura S, Ichiyama A, Nishizuka Y, HayaishiO. 1965, J. Biol. Chem. , pp. 240: 1395-1401.

40. The Secret Life of NAD+: An Old Metabolite Controlling New Metabolic Signaling Pathways. Houtkooper R.H., Carles Cantó C. , Wanders, R.J. and Auwerx, J. 2010, Endocrine Reviews , pp. vol. 31 no. 2 194-223, doi: 10.1210/er.2009-0026.

41. Stimulation of Nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy. Sasaki Y, Araki T, Milbrandt J. 2006, J Neurosci , pp. 26: 8484–8491.

42. European Nicotinamide Diabetes Intervention Trial (ENDIT): a randomised controlled trial of intervention before the onset of type 1 diabetes. Gale EA, Bingley PJ, Emmett CL, CollierT. 2004, Lancet., pp. 363:925–931.

43. Safety of high-dose nicotinamide: a review. Knip M, Douek IF, Moore WP, Gillmor HA, McLean AE, Bingley PJ, Gale EA. 2000, Diabetologia, pp. 43:1337–1345.

44. Large supplements of nicotinic acid and nicotinamide increase tissue NAD and poly(ADP-ribose) levels but do not affect diethylnitrosamine-induced altered hepatic foci in Fischer-344 rats. JacksonTM, Rawling JM, Roebuck BD, Kirkland JB. 1995, J Nutr , p. 125:1455.

45. Characterization and evolution of vertebrate indelamine 2,3-dihydrogenases IDOs from monotremes and marsupials. Yuasa, HJ, Ball, HJ, Ho, YF, Austin, CJ, et al. 2009, Comp. Biochem. Physiol. B. Biochem.. Mol. Biol., pp. 153 (2): 137-144.

46. Novel tryptophan catabolic enzyme IDO2 is the preferred biochemical target of the antitumor indolamine 2,3-dihydrogenase inhibitor compound D-1 methyl-tryptophan. Metz, R., Duhadaway, JB, Kamasani, U, Laury-Kleintop, L., Muller, AJ, Prendergast, GC. 2007, Cancer Res., pp. 67 (15): 7082-7087.

47. Total synthesis of exiguamines A and B inspired by catechollamine chemistry. Sofiyev, V, Lumb, JP, Volgraf, M., Trauner, D. 2012, Chemistry., pp. 18 (16): 4999-5005.

48. Molecular evolution of bacterial indolamine 2,3-dioxygenase. Yuasa, H J, Ushigoe, A, Ball, HJ. 2011, Gene., pp. 484 (1) : 22-31.

49. Infectious tolerance and the long-term acceptance of transplant tissue. Waldman, H., Adams, E., Fairchild, P., and Cobbold, S. 2006, J. Immunol., pp. 212:301-313.

50. Molecular evolution and characterizationof fungal indolamine 2,3-dioxygenases. Yuasa, HJ and Ball, HJ. 2012, J. Mol. Eval., pp. 72 (2): 160-168.

51. convergent evolution. The gene structure of Sulculus 41 kDa myoglobin is homologous with tht of human indolamine dioxygenase. Suzuki, T, Imai, K. 1996, Biochim. Biophys. Acta., pp. 1308(1):41-48.

52. Evolutionof myoglobin. Suzuki, T., Imai, K. 1998, Cell Mol Life Sci, pp. 54(9):979-1004.

53. A myoglobin evolved from indolamine 2,3-dioxygenase, trtptophan-degrading enzyme. Suzuki, T., Kawamichi, H., Imai, K. 1998, Comp Biochem Phisiol. Mol. Biol., pp. 121(2):117-128.

54. Do molluscs possess indolamine 2,3-dioxygenase? Yuasa, HJ and Suzuki, T. 2005, Comp. Biochem. Physiol. B. Biochem. Mol. Biol. , pp. (3) 445-454.

55. Comparison studies of the indolamine dioxygenase-like myoglobin from the abalone Sulculus diversicolor. Suzuki, T., Imai, K. 1997, Comp. Biohem. Phsiol B Biochem Mol Biol, pp. 117 (4)599-604.

56. Orchestration of the immune response by dendritic cells. Buckwalter MR, Albert ML. 2009, Curr Biol., pp. 19(9):355–361.

57. Dendritic cells and the control of immunity. Banchereau J, Steinman RM. 1998, Nature., pp. 245–52.

58. IDO expression by dendritic cells: tolerance and tryptophan catabolism. . Munn DH, Mellor AL. 2004, Nat Rev Immunol. , pp. 762–74.

59. Monocyte and Macrophage. Gordon, S. and Taylor, P.R. 2005, NATURE REVIEWS | IMMUNOLOGY , pp. vol:5, 953-964.

60. Blood monocytes consist of two principal subsets with distinct migratory properties. Geissmann F, Jung S, Littman DR. 2003, Immunity. , pp. 19:71–82.

61. Identification of a novel cell type in peripheral lymphoid organs of mice. I Morphology, quantitation, tissue distribution. . Steinman RM, Cohn ZA. 1973, J Exp Med., pp. 137(5):1142–1162.

62. T cell apoptosis by tryptophan catabolism. Fallarino F, Grohmann U, Vacca C, Bianchi R, Orabona C, Spreca A, Fioretti MC, Puccetti P. 2002, Cell Death Differ , pp. 9:1069–1077.

63. Kynurenine is a novel endothelium derived relaxing factor produced during inflammation. Wang, et al. 2010, Nat. Med., pp. 16(3): 279-285.

64. Activation of the noncanonical NF-kB pathway by HIV controls a Dendritic cell immunoregulatory phenotype. Manches, O. Fernandez, V.M.,, Plumas, J., Chaperot, L., and Bhardwaj, N. 2012, PNAS, pp. vol: 109, 14122-14127.

65. B cells inhibit induction of T cell-dependent tumor immunity. Qin, Z., Richter, G., Schuler, T., Ibe, S., Cao, X, Blakenstein, T. 1998, Nat. Med, p. 4:627.

66. Different partners, Opposite Outcmes: A new perspective of immunobiology of Indolamine 2,3 dioxygenase. Orabona, C., Pallotta, M.T., Grohman, U. 2012, Molecular Medicine., pp. 18:834-842.

67. Indolamine 2,3-dioxygenase: From catalyst to signaling function. Fallarino, F., Grohman, U., and Puccetti, P. 2012, Eurepean J. of Immunol. , pp. 42:1932-1937.

68. IDO: more than an enzyme. Chen, W. 2011, Nature Immonology, pp. 809-811.

69. Indolamine2,3-dehydrogenase in lung dendritic cells promotes Th2 responses and allergic inflammation. Xu, H., Oriss, T.B., Fei, M., Henry, A.C., Melgert, B.N., Chen, L., Mellor, A.L. 2008, PNAS USA, pp. 105: 6690-6695.

70. The immunoregulatory enzyme IDO paradoxically drives B-cellmediated autoimmunity. Scott, G.N., DuHadaway, J., Pigott, E., Ridge, N., Prendergast, G.C., Muller, A.J., Mandik-Nayak, L. 2009, J. Immunol., pp. 182:7509-7517.

71. Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Lee GK, Park HJ, Macleod M, Chandler P, Munn DH, Mellor AL. 2002, Immunology , pp. 107:452–460.

72. Enzymology of NAD+ homeostasis in man. . Magni G, Amici A, Emanuelli M, Orsomando G, Raffaelli N, Ruggieri S. 2004, Cell Mol Life Sci , pp. 61:19–34.

73. Kynurenine pathway enzymes in dendritic cells initiate tolerogenesis in the absence of functional IDO. . Belladonna ML, Grohmann U, Guidetti P, Volpi C, Bianchi R, Fioretti MC, Schwarcz R, Fallarino F, Puccetti P. 2006, J Immunol. , pp. ;177:130–7.

74. An indogenous tumour promoting ligand of the human aryl hydrocarbon receptor. Opitz, et. al. 2011, pp. doi: 10.1038/nature10491,.

75. Inhibition of indoleamine 2,3-dioxygenase, animmunoregulatorytarget of the cancer suppression gene Bin1, potentiates cancer chemotherapy. Muller, A. J. et al. 2005, Nature Med. , pp. 11, 312–319 .

76. TGF-b; a master of all T cell trades. Li, M.O., Fravell, R.A. 2008, Cell. , pp. 134: 392-404.

77. Palotta, M.T. et al. 2011, Nat. Immunol., pp. 12:870-878.

78. Chen, W. et al. 2003, J. Exp. Immunol., p. 198: 1875.

79. Smads: transcriptional activators of TGF-beta responses. . Derynck R, Zhang Y, Feng XH. 1998, Cell , pp. 95 (6): 737–40. doi:10.1016/S0092-8674(00)81696-7.PMID 9865691. .

80. Smad transcription factors. Massagué J, Seoane J, Wotton D. 2005, Genes Dev, pp. 19 (23): 2783–810. doi:10.1101/gad.1350705. PMID .

81. A structural basis for mutational inactivation of the tumour suppressor Smad4. Shi Y, Hata A, Lo RS, Massagué J, Pavletich NP. 1997, Nature., pp. 388 (6637): 87–93.doi:10.1038/40431. PMID 9214508.

82. Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads. Itoh F, Asao H, Sugamura K, Heldin CH, ten Dijke P, Itoh S. 2001, EMBO J., pp. 20 (15): 4132– doi:10.1093/emboj/20.15.4132. PMC 149146. PMID 11483516.

83. SMAD_Signaling_Network. http://www.sabiosciences.com. [Online] 2013. http://www.sabiosciences.com/pathway.php?sn=SMAD_Signaling_Network.

84. Immune inhibitory receptors. Revetch, J.V., and Lanier, L.L. 2000, Science., pp. 290:84-89.

85. Soc3 drives proteasomal degradation of indolamine 2,3-dioxygenase (IDO) and antagonizes IDO-dependent tolerogenesis. Orabona, C., Pallotta, M., Volpi, C., et al. 2008, PNAS USA, pp. 105: 20828-20833.

86. Cutting edge; silencing supressor of cytokine signaling3 expression in dendritic cells turns CD28-Ig from immune adjuvant to supressant. Orabona, C.,, Belladonna, M.L., et all. 2005, J. Immunol., pp. 174: 6582-6586.

87. Molecular signatures of T-cell inhibition in HIV-1 infection. Larsson, M., Shankar. E.M, Che, K.F., Ellegard, R., Barathan, M., Velu, V., and Kamarulzaman, A. 2013, Retrovirology, p. 10:31.

88. TGF-beta and CD4+CD25+ regulatory cells. Huber, S. and Schramn, C. 2006, Front. Bioscie., pp. 11:1014-1023.

89. Immune Escape as a fundemental trait of cancer; focus on IDO. Prendergast, G.C. 2008, Oncogene., pp. 27, 3889-3900.

90. Il-6 inhibits the tolerogenic functionof CD8+ dendritic cells expressing indolamine 2,3-dioxygenase. Grohman, U., Fallarino, F., et al. 2001, J. Immunol., pp. 167:708-714.

91. Avoiding horror autotoxicus: Th eimportance of dentritic cells in peripheral T cell tolerance. Steinman, R.M., and Nussenzweig, M.C. 2002, PNAS, pp. no:1, 351-358.

92. Dendritic-cell function in Toll-like receptor- and MyD88-knockout mice . Kaisho, T., Akira, S. 2001, Trends Immunol , pp. 22,78-83.

93. Innate sensing of self and non-self RNAs by Toll-like receptors. Sioud, M. 2006., Trends Mol Med., pp. 12:67–76.

94. Impaired expression of indoleamine 2, 3-dioxygenase in monocyte-derived dendritic cells in response to Toll-like receptor-7/8 ligands. Furset, G., Fløisand, Y. and Sioud, M. 2008, Immunology., pp. 123(2): 263–271, doi: 10.1111/j.1365-2567.2007.02695.x.

95. Toll-;ike receptor 9 mediated induction of the immunorepressor pathway of tryptophan metabolism. Fallarino, F., and Puccetti, P. 2006, Eur. J. of Imm., pp. 36:8-11.

96. Toll-like receptors and host defense against microbial pathogens: bringing specificity to the innate immune system. . Netea MG, der Graaf C, Van der Meer JWM, Kullberg BJ. 2004, J Leukoc Biol. , pp. 75:749–55.

97. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. . Heil F, Hemmi H, Hochrein H, et al. 2004, Science. , pp. 303:1526–9.

98. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. . Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. 2004., Science. , pp. 303:1529–31. .

99. The role of CpG motifs in innate immunity. Krieg, A.M. 2000., Curr Opin Immunol., pp. 12:35–43.

100. Anendogenous tumour-promoting ligand of the human aryl hydrocarbon receptor. Opitz, C.A., Litzenburger, U.M., Sahm, F., Ott,M., Tritschler, I., Trump, S. 2011, Nature, pp. vol 478; 197-203.

101. Impaired impression of Indolamine 2,3-deoxygenase in monocyte derived DCs in response to TLR-7/8. Furset, G., Floisand, Y., Sioud, M. 2007, Immunology, pp. 263-271.

102. Activationof the noncanonical NF-kB pathway by HIV controls a Dendritic cell immunoregulatory phenotype. Manches, O. Fernandez, V.M.,, Plumas, J., Chaperot, L., and Bhardwaj, N. 2012, PNAS, pp. vol: 109, 14122-14127.

103. Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo . de Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Baetselier, P., Urbain, J., Leo, O., Moser, M. 1996, J. Exp. Med., pp. 184,1413-1424.

104. Subsets of dendritic cell precursors express different Toll-like receptors and respond to different microbial antigens . Kadowaki, N., Ho, S., Antonenko, S., de Waal Malefyt, R., Kastelein, R. A., Bazan, F., Liu, Y-J. 2001, J. Exp. Med., pp. 194,863-869 .

105. TRAF6 is a critical factor for dendritic cell maturation and development . Kobayashi, T., Walsh, P. T., Walsh, M. C., Speirs, K. M., Chiffoleau, E., King, C. G., Hancock, W. W., Caamano, J. H., Hunter, C. A., Scott, P., Turka, L. A., Choi, Y. 2003, Immunity , pp. 19,353-363 .

106. Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA. Sadik CD, Bachmann M, Pfeilschifter J, Mühl H. 2009, Nucleic Acids Res. , pp. 37(15):5041-56. doi: 10.1093/nar/gkp525. Epub 2009 Jun 18.

107. Triggering of the dsRNA sensors TLR3, MDA5, and RIG-I induces CD55 expression in synovial fibroblasts. Karpus ON, Heutinck KM, Wijnker PJ, Tak PP, Hamann J. 2012, PLoS One., p. 7(5):e35606. doi: 10.1371/journal.pone.0035606. Epub 2012 May 10.

108. The structure of the TLR5-flagellin complex: a new mode of pathogen detection, conserved receptor dimerization for signaling. Lu J, Sun PD. 2012, Sci Signal., p. 5(216):pe11. doi: 10.1126/scisignal.2002963. .

109. Flagellin/Toll-like receptor 5 response was specifically attenuated by keratan sulfate disaccharide via decreased EGFR phosphorylation in normal human bronchial epithelial cells. Shirato K, Gao C, Ota F, Angata T, Shogomori H, Ohtsubo K, Yoshida K, Lepenies B, Taniguchi N. 2013, Biochem Biophys Res Commun., pp. doi:pii: S0006-291X(13)00779-1. 10.1016/j.bbrc.2013.05.009. [Epub ahead of print].

110. Differential induction of interleukin-10 and interleukin-12 in dendritic cells by microbial Toll-like receptor activators and skewing of T-cell cytokine profiles Infect. Qi, H., Denning, T. L., Soong, L. 2003, Immun. , pp. 71,3337-3342 .

111. Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D.Activation of Toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10 . Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D., Modlin, R. L. 2000, J. Immunol. , pp. 165,3804-3810.

112. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human dendritic cells . Re, F., Strominger, J. L. 2001, J. Biol. Chem. , pp. 276,37692-37699.

113. Pasare, C., Medzhitov, R. (2003) Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Pasare, C., Medzhitov, R. 2003, Science , pp. 299,1033-1036 .



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Observations on Finding the Genetic Links in Common Disease: Whole Genomic Sequencing Studies

Author: Larry H Bernstein, MD, FCAP

In this article I will address the following article by Dr. SJ Williams.

Finding the Genetic Links in Common Disease:  Caveats of Whole Genome Sequencing Studies


In the November 23, 2012 issue of Science, Jocelyn Kaiser reports (Genetic Influences On Disease  Remain Hidden in News and  Analysis) on the difficulties that many genomic studies are encountering correlating genetic variants to high risk of type 2 diabetes and heart disease. American Society of  Human Genetics annual 2012 meeting, results of DNA sequencing studies reporting on genetic variants and links to high risk type 2 diabetes and heart disease, part of an international effort to determine the genetic events contributing to complex, common diseases like diabetes.
The key point is that these disease links are challenged by the identification of genetic determinants that do not follow Mendelian Genetics.  There are many disease associated gene variants, and they have not been deleted as a result of natural selection.  In the case of diabetes (type 2), the genetic risk is a low as 26%.

Gene-wide-association studies (GAWS) have identified single nucleotide polymorphisms (SNPs) with associations for common diseases, most of these individually carry only only 20-40% of risk. This is not sufficient for prediction
and use in personalized  treatment.

What is the implication of this.  Researchers have gone to exome-sequencing and  to whole genome sequencing for answers. SNPs can be easily done  by microarray, and in a clinic setting. GWAS is difficult and has inherent complexity, and it has had high cost of use. But the cost of the technology has been dropping precipitously. Technology is being redesigned for more rapid diagnosis and use in clinical research and personalized medicine.  It appears that this is not  yet a game changer.

My own thinking is that the answer doesn’t  fully lie in the genome sequencing, but that it must turn on the very large weight of importance in the regulatory function in the genome, that which was once “considered” dark matter.  In the regulatory function you have a variety of interactions and adaptive changes to the proximate environment, and this is a key to the nascent study of metabolomics.

Three projects highlighted are:
1.  National Heart, Lung and Blood Institute Exome Sequencing Project (ESP)[2]: heart, lung, blood

  • A majority of variants linked to any disease are rare
  • Groups of variants in the same gene confirmed a link between
    APOC3 and risk for early-onset heart attack

2.  T2D-GENES Consortium
3.  GoT2D

  • SNP and PAX4 gene association for type 2 diabetes in East Asians
  • No new rare variants above 1.5% frequency for diabetes


The unsupported conclusion from this has been

  1. the common disease-common variant hypothesis, which predicts that common disease-causing genetic variants exist in all human populations, but   (common unexplained complexity?) each individual variant will necessarily only have a small effect on disease susceptibility (i.e. a low associated relative risk).
  1. the common disease, many rare variants hypothesis, which postulates that disease is caused by multiple strong-effect variants, (an alternative complexity situation?) Dickson et al. (2010)  PLoS Biol 2010 8(1):e1000294

The reality is that it has been difficult to associate any variant with prediction of risk, but an alternative approach appears to be intron sequencing and missing information on gene-gene interactions.

Jocelyn Kaiser’s Science article notes this in a brief interview with Harry Dietz of Johns Hopkins University where he suspects that “much of the missing heritability lies in gene-gene interactions”.

Oliver Harismendy and Kelly Frazer and colleagues’ recent publication in Genome Biology  http://genomebiology.com/content/11/11/R118 support this notion.  The authors used targeted resequencing
of two endocannabinoid metabolic enzyme genes (fatty-acid-amide hydrolase (FAAH) and monoglyceride lipase (MGLL) in 147 normal weight and 142 extremely obese patients.

English: The human genome, categorized by func...

English: The human genome, categorized by function of each gene product, given both as number of genes and as percentage of all genes. (Photo credit: Wikipedia)

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Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis

Curator: Aviva Lev-Ari, PhD, RN

UPDATED on 6/13/2013

with a CASE of  Anti-Ro Antibodies and Reversible Atrioventricular Block

N Engl J Med 2013; 368:2335-2337 June 13, 2013 DOI: 10.1056/NEJMc1300484

As an Introduction to the Genetics of Conduction Disease, we selected the following article which represents the MOST comprehensive review of the Human Cardiac Conduction System presented to date:

Circulation.2011; 123: 904-915 doi: 10.1161/​CIRCULATIONAHA.110.942284

The Cardiac Conduction System

  1. David S. Park, MD, PhD;
  2. Glenn I. Fishman, MD

+Author Affiliations

  1. From the Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, NY.
  1. Correspondence to Glenn I. Fishman, MD, Leon H. Charney Division of Cardiology, New York University School of Medicine, 522 First Ave, Smilow 801, New York, NY 10016. E-mail glenn.fishman@med.nyu.edu

Key Words:

The human heart beats 2.5 billion times during a normal lifespan, a feat accomplished by cells of the cardiac conduction system (CCS). The functional components of the CCS can be broadly divided into the impulse-generating nodes and the impulse-propagating His-Purkinje system. Human diseases of the conduction system have been identified that alter impulse generation, impulse propagation, or both. CCS dysfunction is primarily due to acquired conditions such as myocardial ischemia/infarct, age-related degeneration, procedural complications, and drug toxicity. Inherited forms of CCS disease are rare, but each new mutation provides invaluable insight into the molecular mechanisms governing CCS development and function. Applying a multidisciplinary approach, which includes human genetic screening, biophysical analysis, and transgenic mouse technology, has yielded a broad array of gene families involved in maintaining normal CCS physiology (Figure 1). In this review, we discuss gene families that have been implicated in human CCS diseases of rhythm, conduction block, accessory conduction, and development (Table). We also investigate evolving therapeutic strategies that may serve as adjuvant or replacement therapy to current implantable pacemakers.

Figure 1.

View larger version:

Figure 1.

Cardiac conduction system cell. Genes identified in human cardiac conduction system disease are highlighted.


Genetic Basis of Conduction System Disease

Diseases of Automaticity

The human sinoatrial node (SAN) is a crescent-shaped, intramural structure with its head located subepicardially at the junction of the right atrium and the superior vena cava and its tail extending 10 to 20 mm along the crista terminalis.26 The SAN has complex 3-dimensional tissue architecture with central and peripheral components made up of distinct ion channel and gap junction expression profiles.27 Central and peripheral cells have different action potential characteristics and conduction properties (Figure 2).27Experimental and computational models have demonstrated that SAN heterogeneity is necessary to maintain normal automaticity and impulse conduction.28,,30

Figure 2.

Figure 2.

Electrophysiological heterogeneity of the sinoatrial node (SAN). The central SAN, the site of dominant pacemaking, is electronically insulated from the hyperpolarizing atrial myocardium through the differential expression of connexins and ion channels. Peripheral SAN cells are electrophysiologically intermediate between central cells and atrial cardiomyocytes. SR indicates sarcoplasmic reticulum.

Pacemaker automaticity is due to spontaneous diastolic depolarization of phase 4, which depolarizes the membrane to threshold potential generating rhythmic action potentials. The current paradigm of SAN automaticity has been modeled as 2 clocks that function in concert, the “membrane voltage clock” and the “calcium clock.” The membrane voltage clock is produced by the net disequilibrium between the decay of outward potassium currents (IK) and the activation of inward currents that include, but are not limited to, background sodium-sensitive current (Ib Na), L- and T-type calcium currents (ICa,L,ICa,T), sustained inward (Ist) current, and hyperpolarization-activated current (If) (Figure 2).27,31,,33

The subsarcolemmal calcium clock contributes to SAN diastolic depolarization through the spontaneous, rhythmic release of Ca2+ from the sarcoplasmic reticulum (SR) via the ryanodine type 2 receptor (RYR2).34 The local intracellular calcium (Cai) elevations drive the sodium-calcium exchange current (INCX) to substitute 1 intracellular Ca2+ for 3 extracellular Na+. The net gain in positive charge results in membrane depolarization.35The elevation of intracellular Ca2+ occurs in the latter third of diastolic depolarization and is sensitive to β-adrenergic stimulation.36

Human mutations affecting the voltage clock

  • (SCN5A and HCN4),

  • calcium clock (RYR2 and CASQ2), or both mechanisms

  • (ANKB) have been identified that negatively affect sinus node function.37,38

Diseases of Conduction BlockConduction block can occur at any level of the CCS and can manifest as sinoatrial exit block, atrioventricular block, infra-Hisian block, or bundle branch block. Impaired conduction can be caused by ion channel defects that alter action potential shape or by defective coupling between cardiomyocytes. Inherited defects in cardiac conduction have been linked to mutations in SCN5A and SCN1B (both affect phase 0) and KCNJ2 (affects phase 3 and 4). 

The cardiac sodium channel consists of the pore-forming α-subunit (encoded by SCN5A) and a modulatory β-subunit (encoded by SCN1B). The α-subunit contains a voltage sensor that allows for rapid activation in response to membrane depolarization. After depolarization, the sodium channel undergoes a period of inactivation, in which it is refractory to further impulses. SCN5A requires membrane repolarization to relieve the inactivated state. The inward rectifier potassium channel, Kir2.1, encoded by KCNJ2, maintains the resting membrane potential. Therefore, proper functioning of Nav1.5 and Kir2.1 is necessary for normal cardiac excitability.


Progressive cardiac conduction defect, or Lev-Lenègre disease, is characterized by age-related, fibrosclerotic degeneration of the His-Purkinje system.6 Impulse propagation through the proximal ventricular conduction system progressively declines, resulting in bundle branch blocks and eventually complete atrioventricular block. An inherited form of Lev-Lenègre disease is associated with loss of function mutations in SCN5A and can exist alone or as overlap syndromes with Brugada or long QT syndrome 3.6 Inherited progressive cardiac conduction defect is associated with a high risk of complete atrioventricular block and Stoke-Adams syncope without ventricular dysrhythmia.7 Schott et al8 identified a mutation in SCN5A that cosegregates with Lenègre disease in a large French family. Affected individuals had variable degrees of conduction block requiring pacemaker implantation in 4 family members because of syncope or complete heart block. Linkage analysis and candidate gene sequencing identified a T>C substitution at position +2 of the donor splice site of intron 22 (IVS22+2 T>C), which results in a mutant lacking the voltage-sensitive segment.8 Functional analysis demonstrated no transient inward sodium current in response to depolarization, consistent with a loss-of-function mutation.6


The majority of patients with Brugada and conduction disease do not have SCN5Amutations. Therefore, modifiers of Nav1.5 expression or function have become the target of candidate gene sequencing approaches. Watanabe et al9 identified SCN1B mutations in 3 families with conduction disease with or without Brugada syndrome. Coexpression of mutant β-subunits with Nav1.5 resulted in diminished sodium current.


Mutations in KCNJ2 have been found in a rare autosomal dominant condition called Andersen-Tawil syndrome, characterized by periodic paralysis, dysmorphic features, polymorphic ventricular tachycardia, and cardiac conduction disease.10,11 ECG evaluation of 96 patients with Andersen-Tawil syndrome from 33 unrelated kindreds revealed conduction defects at multiple levels from the atrioventricular node to the distal conduction system.55 Cardiomyocytes expressing a dominant-negative subunit of Kir2.1 exhibited a 95% reduction in IK1, resulting in significant action potential prolongation. Mouse models of Andersen-Tawil syndrome exhibited a slower heart rate and significant slowing of conduction.56,57

Diseases of Accessory Conduction

Wolff-Parkinson-White (WPW) syndrome is characterized by preexcitation of ventricular myocardium via an accessory pathway (bundle of Kent) that bypasses the normal slow conduction through the atrioventricular node. Ventricular preexcitation is common, with a disease prevalence of 1.5 to 3 per 1000 people.22,58 Histological evaluation of Kent bundles resected from human subjects displayed features of typical ventricular myocytes with expression of connexin 43 (Cx43).59 The expression of high-conductance gap junctions in bypass tracts enables them to preexcite ventricular myocardium, manifesting as a short PR and a slurred QRS complex, or “delta wave,” on the ECG. The vast majority of WPW cases are sporadic, and the underlying mechanism remains unknown; however, rare inherited forms have been reported. Vidaillet et al60 determined that 3.4% of probands with WPW had 1 or more first-degree relatives with accessory conduction.


A familial form of WPW with an autosomal dominant mode of transmission was identified in 2 families. Thirty-one affected individuals had evidence of preexcitation and cardiac hypertrophy. A missense mutation in PRKAG2 was identified that results in a constitutively active form of the γ2 regulatory subunit of AMP-activated protein kinase.22,23 Under normal conditions, AMP-activated protein kinase responds to energy-depleted states by increasing glucose uptake and promoting glycolysis. Transgenic mice expressing a heart-restricted, constitutively active mutant, PRKAG2N488I, recapitulated the human WPW phenotype of cardiac hypertrophy, preexcitation, and conduction defects. The predominant histological finding was ventricular myocyte engorgement with glycogen-laden vacuoles. The disruption of the annulus fibrosus by vacuolated ventricular myocytes resulted in the preexcitation phenotype.61 Using a mouse model of reversible glycogen-storage defect, Wolf et al62 demonstrated that the cardiomyopathy and CCS degeneration seen in PRKAG2N488I mice were reversible processes.


Lalani et al24 reported a novel WPW syndrome associated with microdeletion of the bone morphogenetic protein-2 (Bmp2) region within 20p12.3 that is characterized by variable cognitive deficits and dysmorphic features. The BMPs are members of the transforming growth factor-β superfamily and play a critical role in cardiac development. Mice with cardiac deletion of BMP receptor type Ia (Bmpr1a) were embryonic lethal before E18.5 because of abnormal development of trabecular and compact myocardium, interventricular septum, and endocardial cushion.63 More restricted deletion of Bmpr1a in the atrioventricular canal resulted in defective atrioventricular valve formation and maturation defects in the annulus fibrosus, resulting in preexcitation.64,65


Diseases of CCS Development

Congenital heart disease is the most common form of birth defect, affecting 1% to 2% of live births.66 Arrhythmias may result from defective CCS specification/patterning, malformation or displacement of the conduction system, altered hemodynamics, prolonged hypoxic states, or postoperative sequelae.67,68 Developmentally, the conduction system derives from myocardial precursor cells within the fetal heart.69,,71The process by which conduction cells are specified or recruited into a “conduction” versus “working myocyte” lineage is determined by the regional expression of transcription factors.69,,74 The main transcription factors identified in human CCS development are the T-box and homeobox factors.


Holt-Oram syndrome is an autosomal dominant condition characterized by preaxial radial ray limb deformities (defects of the radius, carpal bones, and/or thumbs) and cardiac septation defects. The septal defects are typically ostium secundum atrial septal defects, muscular ventricular septal defects, and atrioventricular canal defects. Patients with Holt-Oram syndrome manifest variable degrees of CCS dysfunction, such as sinus bradycardia and atrioventricular block, even in the absence of overt structural heart disease. In 1997, Basson et al18 screened 2 families with Holt-Oram syndrome and identified mutations in the T-box transcription factor, TBX5. The T-box transcription factors can function as transcriptional activators or repressors and are known to be critical regulators of cardiac specification and differentiation. Seven TBX family members are expressed in the developing heart, 3 of which (TBX1, TBX5, TBX20) have been linked to human congenital heart disease.75

Mice deficient in Tbx5 were embryonic lethal at E10.5 because of arrested development of the atria and left ventricle. Tbx5+/− mice recapitulated the upper limb and cardiac manifestations of human Holt-Oram syndrome, including the conduction abnormalities.72,76 Significant maturation defects in the atrioventricular canal and ventricular conduction system were present.76 Moskowitz et al76 demonstrated thatTbx5+/− mice have maturation failure of the atrioventricular canal manifesting as persistent atrioventricular rings around the tricuspid and mitral valves. Patterning defects were noted in the His bundle and bundle branches, including complete absence of right bundle branch formation in some cases. Expression of CCS-enriched markers, such as atrial natriuretic factor and Cx40, were found to be significantly downregulated, implicating TBX5 as a transcriptional activator of these genes. TBX5 and the homeobox transcription factor NKX2-5 were found to act synergistically in upregulating atrial natriuretic factor and Cx40 expression.76

Conduction Disease Associated With Neuromuscular Disorders

Neuromuscular disorders represent a diverse collection of diseases that commonly present with cardiac involvement. Mutations have been identified in genes involved in the cytoskeleton, nuclear envelope, and mitochondrial function. Cardiac involvement typically manifests as dilated or hypertrophic cardiomyopathy, atrioventricular conduction defects, and atrial and ventricular dysrhythmias.82


Mutations affecting the nuclear envelope have been associated with significant CCS dysfunction. The inner membrane of the nuclear envelope is a highly organized structure, composed of integral membrane proteins and nuclear cytoskeletal proteins that function together in higher-order chromatin structure and transcriptional regulation. The lamins (A, B, and C) are an integral part of an intermediate filament network that imparts structural rigidity to the inner nuclear membrane. Emerin, a member of the nuclear lamina-associated protein family, putatively mediates anchoring of chromatin to the cytoskeleton. Mutations in emerin (EMD) or lamin A/C (LMNA) result in X-linked Emery-Dreifuss muscular dystrophy and autosomal dominant Emery-Dreifuss muscular dystrophy,20respectively. Individuals with Emery-Dreifuss muscular dystrophy develop progressive skeletal muscle weakness in the first decade of life and cardiac involvement (dilated cardiomyopathy and atrioventricular block) in the second decade.82,83

Arimura et al84 engineered a mouse model of autosomal dominant Emery-Dreifuss muscular dystrophy by knocking-in an Lmna missense mutation (H222P) previously identified from a family with typical autosomal dominant Emery-Dreifuss muscular dystrophy. The mouse model faithfully recapitulated the human disease with LmnaH222P/H222P mice exhibiting locomotive defects, dilated cardiomyopathy, and CCS dysfunction. Telemetric evaluation of the mutant mice revealed PR prolongation and QRS complex widening. A similar CCS defect was seen in mice haploinsufficient in the Lmna gene. Lmna+/− mice exhibited sinus bradycardia with variable degrees of atrioventricular block. Histological evaluation of these mice revealed nuclear deformation and apoptosis in atrioventricular node cells.85 Another engineered mouse line expressing LmnaN195K, known to cause autosomal dominant dilated cardiomyopathy with conduction disease in humans,86 exhibited high-grade atrioventricular block and complete heart block. Biochemical evaluation revealed reduced expression and mislocalization of Cx40 and Cx43 in mutant atrial tissue.87 Desmin staining also revealed structural defects of the sarcomere and intercalated discs.87

Genome-wide expression profiling of Lmna H222P mouse hearts revealed significant increases in mitogen-activated protein kinase (MAPK) signaling pathways.88Hyperactivation of MAPK pathways is associated with cardiomyopathy and CCS dysfunction. A significant increase of the activated forms of 2 MAPKs, JNK and ERK1/2, was noted in mutant hearts that predated the onset of overt or molecularly defined cardiomyopathy.88 Treatment of Lmna H222P mice with an inhibitor of ERK phosphorylation abrogated the increase in biomarkers of cardiomyopathy and restored ejection fraction to normal levels. These findings directly link MAPK hyperactivation to the cardiomyopathic phenotype in Lmna H222P mice.89

On the basis of the phenotypes of these mouse models, lamin A/C appears to maintain the functional integrity of the CCS in 2 ways: (1) protection of the nucleus against mechanical stress and (2) maintenance of proper chromatin organization to ensure accurate gene expression, such as in connexin expression and MAPK signaling pathways.83

Future Directions

Linkage analysis with positional cloning has been a highly effective means of identifying gene mutations within kindreds of monogenic disease. More than 1000 genes have been identified with this approach, including those in this review. With the sequencing of the human genome, the promise of identifying genetic causes of complex, multifactorial diseases is becoming more of a reality. One major step in this direction was the development of genome-wide association studies.94

The genome-wide association study is a test of association between a disease and genetic markers that span the entire genome. The technique relies on the fact that variance at one locus predicts with high probability variance of an adjacent locus because of linkage disequilibrium. In other words, there is nonrandom cosegregation of a series of genetic markers that are close together in the genome. This cluster of linked markers is known as a haplotype. The first study of haplotype structure within 4 populations (Yoruban, Northern/Western Europeans, Chinese, and Japanese) was published in Naturein 2005 by the International HapMap Consortium. Their work reported that individual genetic markers (single nucleotide polymorphisms) predict adjacent markers typically with a resolution of ≈30 000 bp. Considering that the human genome is ≈3×109 bp, they projected that <500 000 single nucleotide polymorphisms would be needed to survey the entire genome for all common genetic variants.94,95

Genome-wide association studies have now been used to identify genetic variants that influence ECG parameters in different populations. Intermediate parameters, such as heart rate or PR interval, were used as surrogate markers of disease for 2 reasons: (1) They have an association with cardiovascular morbidity and atrial fibrillation, and (2) they have tighter associations with gene variants than the actual disease. Holm et al96reported several genome-wide associations using a cutoff P value <1.6×10−9. One locus harboring MYH6 was associated with heart rate, 4 loci (TBX5SCN10ACAV1, andARHGAP24) were associated with PR interval, and 4 loci (TBX5SCN10A6p21, and10q21) were associated with QRS duration. They went on to test these associations with individuals manifesting different arrhythmias in an Icelandic and Norwegian population. Correlations were found between atrial fibrillation and TBX5 and CAV1 (P=4.0×10−5 andP=0.00032, respectively), between advanced atrioventricular block and TBX5 (P=0.0067), and between pacemaker implantation and SCN10A (P=0.0029).

Similar loci were identified by 2 additional independent genome-wide association studies in a European population and an Indian Asian population. Pfeufer et al97 reported 9 loci that were highly associated with PR interval (P<5×10−8) from a meta-analysis of the CHARGE Consortium with >28 000 European subjects. One locus had associations with 2 sodium channels (SCN10A and SCN5A), and 6 loci were near genes involved in cardiac development (CAV1-CAV2NKX2-5SOX5WNT11MEIS1and TBX5-TBX3). Of these,SCN10ASCN5ACAV1-CAV2NKX2-5, and SOX5 were found to be associated with atrial fibrillation. Chambers et al98 also reported the association between SCN10A and PR interval in 6543 Indian Asians. Physiological testing of Scn10a-deficient mice revealed shortened PR intervals in knockout mice with no significant difference in all other ECG and echocardiographic parameters.

The discovery of novel gene families associated with human conduction and arrhythmic diseases with the use of genome-wide association studies is well under way. Identification of SCN10A by 3 independent groups studying different populations confirms the fidelity of this approach. Further experiments confirming the significance of these associations will need to be performed. In addition to identifying novel gene targets, this technique will also aid in the discovery of new associations with noncoding regions in which new epigenetic modifiers and transcriptional/translational regulators, such as microRNAs, will be identified.

Therapeutic Strategies

The current standard of care for symptomatic bradycardia due to conduction system disease is the implantation of an electronic pacemaker. Despite their success, electronic pacemakers have limitations, which include lead complications, finite battery life, potential for infection, lack of autonomic responsiveness, and size restriction in younger patients. These limitations have spurred on the development of biological pacemakers, the premise of which is to restore pacemaking activity with the use of viral-based or stem cell–based gene delivery systems.99 The identification and characterization of genes involved in generating pacemaker currents have allowed biological pacemaker technology to become a reality.

The restoration of sinus pacing rates can be achieved by modulating inward and outward currents to establish or increase the slope of diastolic depolarization in cardiac tissue. Increasing inward currents and/or decreasing outward currents increase the slope of diastolic depolarization and therefore the pacing rate. Genes that have been investigated or are under current investigation include the following: (1) β2-adrenergic receptor,100,101(2) dominant-negative Kir2.1 mutants,102 (3) adenylate cyclase type VI (ACVI),103,104and (4) HCN channels.105 The β2-adrenergic receptor and adenylate cyclase type VI both increase cAMP levels, leading to activation of endogenous HCN channels and calcium clock mechanisms. Although initial animal models using the β2-adrenergic receptor showed promise with transient increases in heart rate, the potential for proarrhythmia and the inability of this approach to establish de novo pacemaker activity limited its efficacy.101

Another approach focused on modifying ionic currents that convert working myocardial cells, which have relatively stable diastolic potentials, into cells with phase 4 diastolic depolarization. It was postulated that atrial and ventricular myocytes have the potential for automaticity, but that hyperpolarizing currents, such as IK1, prevent diastolic depolarization by stabilizing the resting membrane potential. Miake et al102 confirmed this hypothesis when they demonstrated that adenoviral delivery of a dominant-negative Kir2.1 construct into the left ventricle of guinea pigs resulted in conversion of quiescent myocytes into pacemaker cells. Unfortunately, significant action potential prolongation limited the clinical utility of this treatment strategy.102

Rosen and colleagues105,106 demonstrated that automaticity could be induced in quiescent myocardium with the use of heterologous expression of HCN channels that produce the pacemaker current If. Qu and Plotnikov et al demonstrated that stable autonomous rhythms could be generated when adenovirus encoding HCN2 was injected into the left atrium105 or left bundle branch106 of a canine heart. To bypass the limitations of viral-based systems, such as host immune response, several groups reported the successful use of cell-based delivery systems. Plotnikov et al107 reported the successful implantation of human mesenchymal stem cells expressing HCN2 in the left ventricle of a canine model of atrioventricular block. Dogs maintained stable ectopic pacemaker activity for >6 weeks without the use of immunosuppression.107 Human mesenchymal stem cells electronically couple to host myocardium through gap junctions; therefore, conditions with significant gap junction remodeling may affect the efficacy of this method.

Although standalone biological pacemakers may be far into the future, adjuvant biological pacemakers may find real-world utility for current deficiencies of electronic pacemakers, such as limited battery life and device infections. For example, biological preparations used in conjunction with device therapy may be used to extend battery life, decreasing the frequency of generator changes. Transient injectable pacemakers may also function as bridge therapy after lead extraction of an infected device. The need for adjuvant biological pacemakers is clear, but continued refinement of gene- and cell-based delivery systems will be necessary to make this technology a reality.99


Although rare, inherited arrhythmias have become an invaluable tool in identifying the genetic determinants of CCS function. Each new mutation enhances our understanding and appreciation of the biochemical and structural complexity needed for cardiac impulse generation and propagation. This methodology is hampered, however, by the relative scarcity of inherited conditions affecting the CCS. The addition of genome-wide association studies has broadened this search for novel genes beyond rare familial afflictions to include common, multifactorial conditions. It is hoped that this exciting new frontier will bring to light the complex interplay of genes and genetic/epigenetic modifiers that influence the prevalence of common diseases. These genetic screens will ultimately yield a bevy of new gene targets for pharmaceutical or gene-based therapeutics of the future.

Sources of Funding

Studies in the Fishman laboratory are supported by National Institutes of Health grants HL64757, HL081336, and HL82727 and a New York State STEM award (to Dr Fishman) and a Heart Rhythm Foundation Fellowship (to Dr Park).

Genetics of Atrioventricular Conduction Disease in Humans.

Benson DW.


Division of Cardiology, ML7042, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. woody.benson@cchmc.org


Atrioventricular (AV) conduction disease (block) describes impairment of the electrical continuity between the atria and ventricles. Classification of AV block has utilized biophysical characteristics, usually the extent (first, second, or third degree) and site of block (above or below His bundle recording site). The genetic significance of this classification is unknown. In young patients, AV block may result from injury or be the major cardiac manifestation of neuromuscular disease. However, in some cases, AV block has unknown or idiopathic cause. In such cases, familial clustering has been noted and published pedigrees show autosomal dominant inheritance; associated heart disease is common (e.g., congenital heart malformation, cardiomyopathy). The latter finding is not surprising given the common origin of working myocytes and specialized conduction system elements. Using genetic models incorporating reduced penetrance (disease absence in some individuals with disease gene), variable expressivity (individuals with disease gene have different phenotypes), and genetic heterogeneity (similar phenotypes, different genetic cause), molecular genetic causes of AV block are being identified. Mutations identified in genes with diverse functions (transcription, excitability, and energy homeostasis) for the first time provide the means to assess risk and offer insight into the molecular basis of this important clinical condition previously defined only by biophysical characteristics.


Additional Studies on Genetic Congenital AV Block

1) 12738236
Na+ channel mutation leading to loss of function and non-progressive cardiac conduction defects.
BACKGROUND: We previously described a Dutch family in which congenital cardiac conduction disorder has clinically been identified. The ECG of the index patient showed a first-degree AV block associated with extensive ventricular conduction delay. Sequencing of the SCN5A locus coding for the human cardiac Na+ channel revealed a single nucleotide deletion at position 5280, resulting in a frame-shift in the sequence coding for the pore region of domain IV and a premature stop codon at the C-terminus. METHODS AND RESULTS: Wild type and mutant Na+ channel proteins were expressed in Xenopus laevis oocytes and in mammalian cells. Voltage clamp experiments demonstrated the presence of fast activating and inactivating inward currents in cells expressing the wild type channel alone or in combination with the beta1 subinut (SCN1B). In contrast, cells expressing the mutant channels did not show any activation of inward current with or without the beta1 subunit. Culturing transfected cells at 25 degrees C did not restore the Na+ channel activity of the mutant protein. Transient expression of WT and mutant Na+ channels in the form of GFP fusion proteins in COS-7 cells indicated protein expression in the cytosol. But in contrast to WT channels were not associated with the plasma membrane. CONCLUSIONS: The SCN5A/5280delG mutation results in the translation into non-function channel proteins that do not reach the plasma membrane. This could explain the cardiac conduction defects in patients carrying the mutation.
2) 12956334
The genetic origin of atrioventricular conduction disturbance in humans.
Atrioventricular (AV) conduction disturbance (block) describes impairment of the electrical continuity between the atria and ventricles. Clinical classification of AV block has utilized biophysical characteristics, usually the extent (1st, 2nd, 3rd degree) and site of block (above or below His bundle recording site). The genetic significance of this classification is not known. In some casesAV block occurrence is associated with intrauterine exposure to maternal antibody (anti-Ro, anti-La), and other cases are associated with injury (e.g. surgery). Based on familial clustering of idiopathic AV block, a genetic cause has also been suspected. Published pedigrees show autosomal dominant inheritance, and associated heart disease is common (e.g. congenital heart malformation, cardiomyopathy, etc.). The latter finding is not unexpected given the common origin of working myocytes and elements of the specialized conduction system. Using genetic models incorporating reduced penetrance (presence of disease genotype in absence of phenotype), variable expressivity (presence of a disease genotype with variable phenotypes) and genetic heterogeneity (similar phenotypes, different disease genotypes), molecular genetic causes of AV block are being identified. These findings are significant as they provide insight into the molecular basis of a clinical condition previously defined only by biophysical characteristics.
3) 15372490
Genetics of atrioventricular conduction disease in humans.
Atrioventricular (AV) conduction disease (block) describes impairment of the electrical continuity between the atria and ventricles. Classification of AV block has utilized biophysical characteristics, usually the extent (first, second, or third degree) and site of block (above or below His bundle recording site). The genetic significance of this classification is unknown. In young patients, AV block may result from injury or be the major cardiac manifestation of neuromuscular disease. However, in some cases, AV blockhas unknown or idiopathic cause. In such cases, familial clustering has been noted and published pedigrees show autosomal dominant inheritance; associated heart disease is common (e.g., congenital heart malformation, cardiomyopathy). The latter finding is not surprising given the common origin of working myocytes and specialized conduction system elements. Using genetic models incorporating reduced penetrance (disease absence in some individuals with disease gene), variable expressivity (individuals with disease gene have different phenotypes), and genetic heterogeneity (similar phenotypes, different genetic cause), molecular genetic causes of AV block are being identified. Mutations identified in genes with diverse functions (transcription, excitability, and energy homeostasis) for the first time provide the means to assess risk and offer insight into the molecular basis of this important clinical condition previously defined only by biophysical characteristics.


Anti-Ro Antibodies and Reversible Atrioventricular Block

N Engl J Med 2013; 368:2335-2337 June 13, 2013DOI: 10.1056/NEJMc1300484

To the Editor:

Transplacental transfer of anti-Ro antibodies is a well-known cause of conduction defects and permanent atrioventricular block in newborns.1 In adults, conduction disturbances related to these antibodies are rare.2

We report a case of a 26-year-old woman with no history of this condition who was admitted to the hospital through the emergency department after having several syncopal episodes. Electrocardiography (ECG) performed while the patient was at rest showed complete atrioventricular block and ventricular escape rhythm associated with left bundle-branch block (Figure 1AFIGURE 1Electrocardiographic Findings.). Laboratory evaluation included a positive test for antinuclear antibodies (with the HEp-2 cell substrate) at a titer of 1:320, with a speckled pattern and specificity for extractable nuclear antigens, including antibodies against Ro52 confirmed by means of immunoblot and enzyme-linked immunosorbent assays (first measurement of antibodies, 1.2 U per milliliter). No clinical manifestations of rheumatologic disease were present. Other causes of reversible atrioventricular block were ruled out. The patient had no history of cardiac surgery, ablation procedures, or drug use. There was no evidence of infiltrative diseases (e.g., sarcoidosis or amyloidosis) or myocardial ischemia, nor was there clinical suspicion of infectious diseases that cause conduction disturbances (e.g., Lyme disease or Chagas’ disease). Levels of electrolytes and thyrotropin were normal. Transthoracic echocardiography and magnetic resonance imaging were unremarkable.

During the first 4 days after admission, the patient had varying degrees of atrioventricular block. An electrophysiological study showed a mildly prolonged HV interval of 62 msec during sinus rhythm (normal values, 35 to 55 msec) and a pathologic response to atrial pacing, with atrioventricular block occurring after the deflection of the bundle of His during continuous stimulation at a fixed cycle length of 490 msec (Figure 1B). Intravenous methylprednisolone was initiated at a dose of 1 mg per kilogram of body weight per day, and 1:1 atrioventricular conduction was subsequently maintained on surface ECG. A second electrophysiological study during treatment showed normal atrioventricular conduction.

Maintenance immunosuppressive therapy with azathioprine (at a dose of 100 mg daily) and methylprednisolone (at a dose of 4 mg daily) was initiated and continued for 12 months. Serial anti-Ro (SS-A) levels fluctuated during follow-up and became negative after 1 year. Because of the uncertainty of the outcome, a backup pacemaker was implanted. The patient remained completely asymptomatic for 12 months with sustained normal atrioventricular conduction.

In this case of atrioventricular block in an adult patient with positive anti-Ro antibodies, we used electrophysiological testing to localize the conduction defect below the atrioventricular node. This finding, together with left bundle-branch block detected on ECG, suggests specific involvement of the Purkinje fibers. The pathogenesis of cardiac conduction disturbances in adults with positive anti-Ro (SS-A) antibodies remains unclear.3 Experimental studies suggest that anti-Ro antibodies interact with calcium channels and cause reversible inhibition of calcium currents. In fetal hearts, the internalization of these channels leads to apoptosis and fibrosis of the conduction tissue. The presence of a fully developed sarcoplasmic reticulum and the apparent lack of antibody-induced apoptosis in adult cardiomyocytes may explain the differential susceptibility of adult hearts to anti-Ro antibodies2 and, conceivably, the reversibility of the conduction disease in such persons.

Irene Santos-Pardo, M.D.
Melania Martínez-Morillo, M.D.
Roger Villuendas, M.D.
Antoni Bayes-Genis, M.D., Ph.D.
Hospital Universitari Germans Trias i Pujol, Badalona, Spain



Chameides L, Truex RC, Vetter V, Rashkind WJ, Galioto FM Jr, Noonan JA. Association of maternal systemic lupus erythematosus with congenital complete heart block. N Engl J Med 1977;297:1204-1207
Full Text | Web of Science | Medline

Lazzerini PE, Capecchi PL, Laghi-Pasini F. Anti-Ro/SSA antibodies and cardiac arrhythmias in the adult: facts and hypotheses. Scand J Immunol 2010;72:213-222
CrossRef | Web of Science | Medline

Costedoat-Chalumeau N, Georgin-la-Vialle S, Amoura Z, Piette J-C. Anti-SSA/Ro and anti-SSB/La antibody-mediated congenital heart block. Lupus 2005;14:660-664
CrossRef | Web of Science | Medline



New Research on the Genetics of Conduction Disease

Heart failure clinics


conduction diseases (CD) include defects in impulse generation and conduction. Patients with CD may manifest a wide range of clinical presentations, from asymptomatic to potentially life-threatening arrhythmias. The pathophysiologic mechanisms underlying CD are diverse and may have implications for diagnosis, treatment, and prognosis. Known causes of functional CD include cardiac ion channelopathies or defects in modifying proteins, such as cytoskeletal proteins. Progress in molecular biology and genetics along with development of animal models has increased the understanding of the molecular mechanisms of these disorders. This article discusses the genetic basis for CD and its clinical implications.
(Beinart et al. 2010)
Beinart R, Ruskin J, et al. (2010). The genetics of conduction disease. Heart Fail Clin 6 (2): 201-14.
PMID: 20347788  DOI: 10.1016/j.hfc.2009.11.006  PII: S1551-7136(09)00108-1
PLoS genetics


(Curran and Mohler 2012)
Curran J and Mohler PJ (2012). Defining the Pathways Underlying the Prolonged PR Interval in Atrioventricular Conduction Disease. PLoS Genet. 8 (12): e1003154.
PMID: 23236297  DOI: 10.1371/journal.pgen.1003154  PII: PGENETICS-D-12-02668
BMC medical genetics


BACKGROUND: Mutations in the gene encoding the nuclear membrane protein lamin A/C have been associated with at least 7 distinct diseases including autosomal dominant dilated cardiomyopathy withconduction system disease, autosomal dominant and recessive Emery Dreifuss Muscular Dystrophy, limb girdle muscular dystrophy type 1B, autosomal recessive type 2 Charcot Marie Tooth, mandibuloacral dysplasia, familial partial lipodystrophy and Hutchinson-Gilford progeria.METHODS: We used mutation detection to evaluate the lamin A/C gene in a 45 year-old woman with familial dilated cardiomyopathy and conduction system disease whose family has been well characterized for this phenotype 1.RESULTS: DNA from the proband was analyzed, and a novel 2 base-pair deletion c.908_909delCT in LMNA was identified.CONCLUSIONS: Mutations in the gene encoding lamin A/C can lead to significant cardiac conductionsystem disease that can be successfully treated with pacemakers and/or defibrillators. Genetic screening can help assess risk for arrhythmia and need for device implantation.
(MacLeod et al. 2003)
MacLeod HM, Culley MR, et al. (2003). Lamin A/C truncation in dilated cardiomyopathy with conduction disease. BMC Med. Genet. 4: 4.
PMID: 12854972  DOI: 10.1186/1471-2350-4-4
Heart (British Cardiac Society)


(MacRae 2012)
MacRae CA (2012). Pattern recognition: combining informatics and genetics to re-evaluate conduction disease. Heart 98 (17): 1263-4.
PMID: 22875820  DOI: 10.1136/heartjnl-2012-302408  PII: heartjnl-2012-302408
The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology


Atrioventricular (AV) conduction disease (block) describes impairment of the electrical continuity between the atria and ventricles. Classification of AV block has utilized biophysical characteristics, usually the extent (first, second, or third degree) and site of block (above or below His bundle recording site). The genetic significance of this classification is unknown. In young patients, AV block may result from injury or be the major cardiac manifestation of neuromuscular disease. However, in some cases, AV block has unknown or idiopathic cause. In such cases, familial clustering has been noted and published pedigrees show autosomal dominant inheritance; associated heart disease is common (e.g., congenital heart malformation, cardiomyopathy). The latter finding is not surprising given the common origin of working myocytes and specialized conduction system elements. Using genetic models incorporating reduced penetrance (disease absence in some individuals with diseasegene), variable expressivity (individuals with disease gene have different phenotypes), and genetic heterogeneity (similar phenotypes, different genetic cause), molecular genetic causes of AV block are being identified. Mutations identified in genes with diverse functions (transcription, excitability, and energy homeostasis) for the first time provide the means to assess risk and offer insight into the molecular basis of this important clinical condition previously defined only by biophysical characteristics.
(Benson 2004) – ORIGINAL FIRST PAPER on the Subject
Benson DW (2004). Genetics of atrioventricular conduction disease in humans. Anat Rec A Discov Mol Cell Evol Biol 280 (2): 934-9.
PMID: 15372490  DOI: 10.1002/ar.a.20099
SOURCE for REFERENCES Listed below:
  1. 1.
    1. Benson DW,
    2. Wang DW,
    3. Dyment M,
    4. Knilans TK,
    5. Fish FA,
    6. Strieper MJ,
    7. Rhodes TH,
    8. George AL Jr.

    . Congenital sick sinus syndrome caused by recessive mutations in the cardiac sodium channel gene (SCN5A). J Clin Invest. 2003;112:1019–1028.

  2. 2.
    1. Makiyama T,
    2. Akao M,
    3. Tsuji K,
    4. Doi T,
    5. Ohno S,
    6. Takenaka K,
    7. Kobori A,
    8. Ninomiya T,
    9. Yoshida H,
    10. Takano M,
    11. Makita N,
    12. Yanagisawa F,
    13. Higashi Y,
    14. Takeyama Y,
    15. Kita T,
    16. HorieM

    . High risk for bradyarrhythmic complications in patients with Brugada syndrome caused by SCN5A gene mutations. J Am Coll Cardiol. 2005;46:2100–2106.

  3. 3.
    1. Groenewegen WA,
    2. Firouzi M,
    3. Bezzina CR,
    4. Vliex S,
    5. van Langen IM,
    6. Sandkuijl L,
    7. Smits JP,
    8. Hulsbeek M,
    9. Rook MB,
    10. Jongsma HJ,
    11. Wilde AA

    . A cardiac sodium channel mutation cosegregates with a rare connexin40 genotype in familial atrial standstill. Circ Res. 2003;92:14–22.

  4. 4.
    1. Veldkamp MW,
    2. Wilders R,
    3. Baartscheer A,
    4. Zegers JG,
    5. Bezzina CR,
    6. Wilde AA

    .Contribution of sodium channel mutations to bradycardia and sinus node dysfunction in LQT3 families. Circ Res. 2003;92:976–983.

  5. 5.
    1. Probst V,
    2. Allouis M,
    3. Sacher F,
    4. Pattier S,
    5. Babuty D,
    6. Mabo P,
    7. Mansourati J,
    8. VictorJ,
    9. Nguyen JM,
    10. Schott JJ,
    11. Boisseau P,
    12. Escande D,
    13. Le Marec H

    . Progressive cardiac conduction defect is the prevailing phenotype in carriers of a Brugada syndrome SCN5A mutation. J Cardiovasc Electrophysiol. 2006;17:270–275.

  6. 6.
    1. Probst V,
    2. Kyndt F,
    3. Potet F,
    4. Trochu JN,
    5. Mialet G,
    6. Demolombe S,
    7. Schott JJ,
    8. Baro I,
    9. Escande D,
    10. Le Marec H

    . Haploinsufficiency in combination with aging causes SCN5A-linked hereditary Lenegre disease. J Am Coll Cardiol. 2003;41:643–652.

  7. 7.
    1. Kyndt F,
    2. Probst V,
    3. Potet F,
    4. Demolombe S,
    5. Chevallier JC,
    6. Baro I,
    7. Moisan JP,
    8. Boisseau P,
    9. Schott JJ,
    10. Escande D,
    11. Le Marec H

    . Novel SCN5A mutation leading either to isolated cardiac conduction defect or Brugada syndrome in a large French family.Circulation. 2001;104:3081–3086.

  8. 8.
    1. Schott JJ,
    2. Alshinawi C,
    3. Kyndt F,
    4. Probst V,
    5. Hoorntje TM,
    6. Hulsbeek M,
    7. Wilde AA,
    8. Escande D,
    9. Mannens MM,
    10. Le Marec H

    . Cardiac conduction defects associate with mutations in SCN5A. Nat Genet. 1999;23:20–21.

  9. 9.
    1. Watanabe H,
    2. Koopmann TT,
    3. Le Scouarnec S,
    4. Yang T,
    5. Ingram CR,
    6. Schott JJ,
    7. Demolombe S,
    8. Probst V,
    9. Anselme F,
    10. Escande D,
    11. Wiesfeld AC,
    12. Pfeufer A,
    13. Kaab S,
    14. Wichmann HE,
    15. Hasdemir C,
    16. Aizawa Y,
    17. Wilde AA,
    18. Roden DM,
    19. Bezzina CR

    . Sodium channel beta1 subunit mutations associated with Brugada syndrome and cardiac conduction disease in humans. J Clin Invest. 2008;118:2260–2268.

  10. 10.
    1. Andelfinger G,
    2. Tapper AR,
    3. Welch RC,
    4. Vanoye CG,
    5. George AL Jr.,
    6. Benson DW

    .KCNJ2 mutation results in Andersen syndrome with sex-specific cardiac and skeletal muscle phenotypes. Am J Hum Genet. 2002;71:663–668.

  11. 11.
    1. Plaster NM,
    2. Tawil R,
    3. Tristani-Firouzi M,
    4. Canun S,
    5. Bendahhou S,
    6. Tsunoda A,
    7. Donaldson MR,
    8. Iannaccone ST,
    9. Brunt E,
    10. Barohn R,
    11. Clark J,
    12. Deymeer F,
    13. George AL Jr.,
    14. Fish FA,
    15. Hahn A,
    16. Nitu A,
    17. Ozdemir C,
    18. Serdaroglu P,
    19. Subramony SH,
    20. Wolfe G,
    21. Fu YH,
    22. Ptacek LJ

    . Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen’s syndrome. Cell. 2001;105:511–519.

  12. 12.
    1. Schulze-Bahr E,
    2. Neu A,
    3. Friederich P,
    4. Kaupp UB,
    5. Breithardt G,
    6. Pongs O,
    7. IsbrandtD

    . Pacemaker channel dysfunction in a patient with sinus node disease. J Clin Invest.2003;111:1537–1545.

  13. 13.
    1. Milanesi R,
    2. Baruscotti M,
    3. Gnecchi-Ruscone T,
    4. DiFrancesco D

    . Familial sinus bradycardia associated with a mutation in the cardiac pacemaker channel. N Engl J Med.2006;354:151–157.

  14. 14.
    1. Nof E,
    2. Luria D,
    3. Brass D,
    4. Marek D,
    5. Lahat H,
    6. Reznik-Wolf H,
    7. Pras E,
    8. Dascal N,
    9. Eldar M,
    10. Glikson M

    . Point mutation in the HCN4 cardiac ion channel pore affecting synthesis, trafficking, and functional expression is associated with familial asymptomatic sinus bradycardia. Circulation. 2007;116:463–470.

  15. 15.
    1. Bhuiyan ZA,
    2. van den Berg MP,
    3. van Tintelen JP,
    4. Bink-Boelkens MT,
    5. Wiesfeld AC,
    6. Alders M,
    7. Postma AV,
    8. van Langen I,
    9. Mannens MM,
    10. Wilde AA

    . Expanding spectrum of human RYR2-related disease: new electrocardiographic, structural, and genetic features.Circulation. 2007;116:1569–1576.

  16. 16.
    1. Postma AV,
    2. Denjoy I,
    3. Kamblock J,
    4. Alders M,
    5. Lupoglazoff JM,
    6. Vaksmann G,
    7. Dubosq-Bidot L,
    8. Sebillon P,
    9. Mannens MM,
    10. Guicheney P,
    11. Wilde AA

    . Catecholaminergic polymorphic ventricular tachycardia: RYR2 mutations, bradycardia, and follow up of the patients. J Med Genet. 2005;42:863–870.

  17. 17.
    1. Postma AV,
    2. Denjoy I,
    3. Hoorntje TM,
    4. Lupoglazoff JM,
    5. Da Costa A,
    6. Sebillon P,
    7. Mannens MM,
    8. Wilde AA,
    9. Guicheney P

    . Absence of calsequestrin 2 causes severe forms of catecholaminergic polymorphic ventricular tachycardia. Circ Res. 2002;91;e21–e26.

  18. 18.
    1. Basson CT,
    2. Bachinsky DR,
    3. Lin RC,
    4. Levi T,
    5. Elkins JA,
    6. Soults J,
    7. Grayzel D,
    8. Kroumpouzou E,
    9. Traill TA,
    10. Leblanc-Straceski J,
    11. Renault B,
    12. Kucherlapati R,
    13. Seidman JG,
    14. Seidman CE

    . Mutations in human TBX5 [corrected] cause limb and cardiac malformation in Holt-Oram syndrome. Nat Genet. 1997;15:30–35.

  19. 19.
    1. Schott JJ,
    2. Benson DW,
    3. Basson CT,
    4. Pease W,
    5. Silberbach GM,
    6. Moak JP,
    7. MaronBJ,
    8. Seidman CE,
    9. Seidman JG

    . Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science. 1998;281:108–111.

  20. 20.
    1. Bonne G,
    2. Di Barletta MR,
    3. Varnous S,
    4. Becane HM,
    5. Hammouda EH,
    6. Merlini L,
    7. Muntoni F,
    8. Greenberg CR,
    9. Gary F,
    10. Urtizberea JA,
    11. Duboc D,
    12. Fardeau M,
    13. Toniolo D,
    14. Schwartz K

    . Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy. Nat Genet. 1999;21:285–288.

  21. 21.
    1. Le Scouarnec S,
    2. Bhasin N,
    3. Vieyres C,
    4. Hund TJ,
    5. Cunha SR,
    6. Koval O,
    7. MarionneauC,
    8. Chen B,
    9. Wu Y,
    10. Demolombe S,
    11. Song LS,
    12. Le Marec H,
    13. Probst V,
    14. Schott JJ,
    15. AndersonME,
    16. Mohler PJ

    . Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease. Proc Natl Acad Sci U S A. 2008;105:15617–15622.

  22. 22.
    1. Gollob MH,
    2. Green MS,
    3. Tang AS,
    4. Gollob T,
    5. Karibe A,
    6. Ali Hassan AS,
    7. Ahmad F,
    8. Lozado R,
    9. Shah G,
    10. Fananapazir L,
    11. Bachinski LL,
    12. Roberts R

    . Identification of a gene responsible for familial Wolff-Parkinson-White syndrome. N Engl J Med.2001;344:1823–1831.

  23. 23.
    1. Arad M,
    2. Benson DW,
    3. Perez-Atayde AR,
    4. McKenna WJ,
    5. Sparks EA,
    6. Kanter RJ,
    7. McGarry K,
    8. Seidman JG,
    9. Seidman CE

    . Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy. J Clin Invest.2002;109:357–362.

  24. 24.
    1. Lalani SR,
    2. Thakuria JV,
    3. Cox GF,
    4. Wang X,
    5. Bi W,
    6. Bray MS,
    7. Shaw C,
    8. Cheung SW,
    9. Chinault AC,
    10. Boggs BA,
    11. Ou Z,
    12. Brundage EK,
    13. Lupski JR,
    14. Gentile J,
    15. Waisbren S,
    16. PursleyA,
    17. Ma L,
    18. Khajavi M,
    19. Zapata G,
    20. Friedman R,
    21. Kim JJ,
    22. Towbin JA,
    23. Stankiewicz P,
    24. Schnittger S,
    25. Hansmann I,
    26. Ai T,
    27. Sood S,
    28. Wehrens XH,
    29. Martin JF,
    30. Belmont JW,
    31. PotockiL

    . 20p12.3 Microdeletion predisposes to Wolff-Parkinson-White syndrome with variable neurocognitive deficits. J Med Genet. 2009;46:168–175.

  25. 25.
    1. Mahadevan M,
    2. Tsilfidis C,
    3. Sabourin L,
    4. Shutler G,
    5. Amemiya C,
    6. Jansen G,
    7. NevilleC,
    8. Narang M,
    9. Barcelo J,
    10. O’Hoy K,
    11. Leblond S,
    12. Earle-Macdonald J,
    13. De Jong PJ,
    14. WieringaB,
    15. Korneluk RG

    . Myotonic dystrophy mutation: an unstable CTG repeat in the 3′ untranslated region of the gene. Science. 1992;255:1253–1255.

  26. 26.
    1. Anderson KR,
    2. Ho SY,
    3. Anderson RH

    . Location and vascular supply of sinus node in human heart. Br Heart J. 1979;41:28–32.

  27. 27.
    1. Lei M,
    2. Zhang H,
    3. Grace AA,
    4. Huang CL

    . SCN5A and sinoatrial node pacemaker function. Cardiovasc Res. 2007;74:356–365.

  28. 28.
    1. Lei M,
    2. Jones SA,
    3. Liu J,
    4. Lancaster MK,
    5. Fung SS,
    6. Dobrzynski H,
    7. Camelliti P,
    8. MaierSK,
    9. Noble D,
    10. Boyett MR

    . Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking. J Physiol. 2004;559:835–848.

  29. 29.
    1. Zhang H,
    2. Holden AV,
    3. Kodama I,
    4. Honjo H,
    5. Lei M,
    6. Varghese T,
    7. Boyett MR

    .Mathematical models of action potentials in the periphery and center of the rabbit sinoatrial node. Am J Physiol. 2000;279;H397–H421.

  30. 30.
    1. Dobrzynski H,
    2. Li J,
    3. Tellez J,
    4. Greener ID,
    5. Nikolski VP,
    6. Wright SE,
    7. Parson SH,
    8. Jones SA,
    9. Lancaster MK,
    10. Yamamoto M,
    11. Honjo H,
    12. Takagishi Y,
    13. Kodama I,
    14. Efimov IR,
    15. Billeter R,
    16. Boyett MR

    . Computer three-dimensional reconstruction of the sinoatrial node.Circulation. 2005;111:846–854.

  31. 31.
    1. Liu J,
    2. Noble PJ,
    3. Xiao G,
    4. Abdelrahman M,
    5. Dobrzynski H,
    6. Boyett MR,
    7. Lei M,
    8. NobleD

    . Role of pacemaking current in cardiac nodes: insights from a comparative study of sinoatrial node and atrioventricular node. Prog Biophys Mol Biol. 2008;96:294–304.

  32. 32.
    1. Liu J,
    2. Dobrzynski H,
    3. Yanni J,
    4. Boyett MR,
    5. Lei M

    . Organisation of the mouse sinoatrial node: structure and expression of HCN channels. Cardiovasc Res.2007;73:729–738.

  33. 33.
    1. Chen PS,
    2. Joung B,
    3. Shinohara T,
    4. Das M,
    5. Chen Z,
    6. Lin SF

    . The initiation of the heart beat. Circ J. 2010;74:221–225.

  34. 34.
    1. Huser J,
    2. Blatter LA,
    3. Lipsius SL

    . Intracellular Ca2+ release contributes to automaticity in cat atrial pacemaker cells. J Physiol. 2000;524();415–422.

  35. 35.
    1. Bogdanov KY,
    2. Vinogradova TM,
    3. Lakatta EG

    . Sinoatrial nodal cell ryanodine receptor and Na(+)-Ca(2+) exchanger: molecular partners in pacemaker regulation. Circ Res. 2001;88:1254–1258.

  36. 36.
    1. Vinogradova TM,
    2. Bogdanov KY,
    3. Lakatta EG

    . β-Adrenergic stimulation modulates ryanodine receptor Ca(2+) release during diastolic depolarization to accelerate pacemaker activity in rabbit sinoatrial nodal cells. Circ Res. 2002;90:73–79.

  37. 37.
    1. Lakatta EG,
    2. Maltsev VA,
    3. Vinogradova TM

    . A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart’s pacemaker. Circ Res. 2010;106:659–673.

  38. 38.
    1. Joung B,
    2. Ogawa M,
    3. Lin SF,
    4. Chen PS

    . The calcium and voltage clocks in sinoatrial node automaticity. Korean Circ J. 2009;39:217–222.

  39. 39.
    1. Ruan Y,
    2. Liu N,
    3. Priori SG

    . Sodium channel mutations and arrhythmias. Nat Rev Cardiol. 2009;6:337–348.

  40. 40.
    1. Smits JP,
    2. Koopmann TT,
    3. Wilders R,
    4. Veldkamp MW,
    5. Opthof T,
    6. Bhuiyan ZA,
    7. Mannens MM,
    8. Balser JR,
    9. Tan HL,
    10. Bezzina CR,
    11. Wilde AA

    . A mutation in the human cardiac sodium channel (E161K) contributes to sick sinus syndrome, conduction disease and Brugada syndrome in two families. J Mol Cell Cardiol. 2005;38:969–981.

  41. 41.
    1. Butters TD,
    2. Aslanidi OV,
    3. Inada S,
    4. Boyett MR,
    5. Hancox JC,
    6. Lei M,
    7. Zhang H

    .Mechanistic links between Na+ channel (SCN5A) mutations and impaired cardiac pacemaking in sick sinus syndrome. Circ Res. 2010;107:126–137.

  42. 42.
    1. Verheijck EE,
    2. van Kempen MJ,
    3. Veereschild M,
    4. Lurvink J,
    5. Jongsma HJ,
    6. BoumanLN

    . Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution. Cardiovasc Res. 2001;52:40–50.

  43. 43.
    1. Fedorov VV,
    2. Schuessler RB,
    3. Hemphill M,
    4. Ambrosi CM,
    5. Chang R,
    6. Voloshina AS,
    7. Brown K,
    8. Hucker WJ,
    9. Efimov IR

    . Structural and functional evidence for discrete exit pathways that connect the canine sinoatrial node and atria. Circ Res. 2009;104:915–923.

  44. 44.
    1. Herrmann S,
    2. Stieber J,
    3. Ludwig A

    . Pathophysiology of HCN channels. Pflugers Arch. 2007;454:517–522.

  45. 45.
    1. Biel M,
    2. Wahl-Schott C,
    3. Michalakis S,
    4. Zong X

    . Hyperpolarization-activated cation channels: from genes to function. Physiol Rev. 2009;89:847–885.

  46. 46.
    1. Stieber J,
    2. Herrmann S,
    3. Feil S,
    4. Loster J,
    5. Feil R,
    6. Biel M,
    7. Hofmann F,
    8. Ludwig A

    . The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci U S A.2003;100:15235–15240.

  47. 47.
    1. Ludwig A,
    2. Herrmann S,
    3. Hoesl E,
    4. Stieber J

    . Mouse models for studying pacemaker channel function and sinus node arrhythmia. Prog Biophys Mol Biol. 2008;98:179–185.

  48. 48.
    1. Herrmann S,
    2. Stieber J,
    3. Stockl G,
    4. Hofmann F,
    5. Ludwig A

    . HCN4 provides a ‘depolarization reserve’ and is not required for heart rate acceleration in mice. EMBO J.2007;26:4423–4432.

  49. 49.
    1. Hoesl E,
    2. Stieber J,
    3. Herrmann S,
    4. Feil S,
    5. Tybl E,
    6. Hofmann F,
    7. Feil R,
    8. Ludwig A

    .Tamoxifen-inducible gene deletion in the cardiac conduction system. J Mol Cell Cardiol.2008;45:62–69.

  50. 50.
    1. Rubenstein DS,
    2. Lipsius SL

    . Mechanisms of automaticity in subsidiary pacemakers from cat right atrium. Circ Res. 1989;64:648–657.

  51. 51.
    1. Ju YK,
    2. Allen DG

    . How does beta-adrenergic stimulation increase the heart rate? The role of intracellular Ca2+ release in amphibian pacemaker cells. J Physiol.1999;516();793–804.

  52. 52.
    1. Rizzi N,
    2. Liu N,
    3. Napolitano C,
    4. Nori A,
    5. Turcato F,
    6. Colombi B,
    7. Bicciato S,
    8. Arcelli D,
    9. Spedito A,
    10. Scelsi M,
    11. Villani L,
    12. Esposito G,
    13. Boncompagni S,
    14. Protasi F,
    15. Volpe P,
    16. PrioriSG

    . Unexpected structural and functional consequences of the R33Q homozygous mutation in cardiac calsequestrin: a complex arrhythmogenic cascade in a knock in mouse model. Circ Res. 2008;103:298–306.

  53. 53.
    1. Liu N,
    2. Colombi B,
    3. Memmi M,
    4. Zissimopoulos S,
    5. Rizzi N,
    6. Negri S,
    7. Imbriani M,
    8. Napolitano C,
    9. Lai FA,
    10. Priori SG

    . Arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia: insights from a RyR2 R4496C knock-in mouse model. Circ Res.2006;99:292–298.

  54. 54.
    1. Mohler PJ,
    2. Schott JJ,
    3. Gramolini AO,
    4. Dilly KW,
    5. Guatimosim S,
    6. duBell WH,
    7. SongLS,
    8. Haurogne K,
    9. Kyndt F,
    10. Ali ME,
    11. Rogers TB,
    12. Lederer WJ,
    13. Escande D,
    14. Le Marec H,
    15. Bennett V

    . Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature. 2003;421:634–639.

  55. 55.
    1. Zhang L,
    2. Benson DW,
    3. Tristani-Firouzi M,
    4. Ptacek LJ,
    5. Tawil R,
    6. Schwartz PJ,
    7. George AL,
    8. Horie M,
    9. Andelfinger G,
    10. Snow GL,
    11. Fu YH,
    12. Ackerman MJ,
    13. Vincent GM

    .Electrocardiographic features in Andersen-Tawil syndrome patients with KCNJ2 mutations: characteristic T-U-wave patterns predict the KCNJ2 genotype. Circulation.2005;111:2720–2726.

  56. 56.
    1. Zaritsky JJ,
    2. Redell JB,
    3. Tempel BL,
    4. Schwarz TL

    . The consequences of disrupting cardiac inwardly rectifying K(+) current (I(K1)) as revealed by the targeted deletion of the murine Kir2.1 and Kir2.2 genes. J Physiol. 2001;533:697–710.

  57. 57.
    1. McLerie M,
    2. Lopatin AN

    . Dominant-negative suppression of I(K1) in the mouse heart leads to altered cardiac excitability. J Mol Cell Cardiol. 2003;35:367–378.

  58. 58.
    1. Light PE

    . Familial Wolff-Parkinson-White syndrome: a disease of glycogen storage or ion channel dysfunction? J Cardiovasc Electrophysiol. 2006;17():S158–S161.

  59. 59.
    1. Peters NS,
    2. Rowland E,
    3. Bennett JG,
    4. Green CR,
    5. Anderson RH,
    6. Severs NJ

    . The Wolff-Parkinson-White syndrome: the cellular substrate for conduction in the accessory atrioventricular pathway. Eur Heart J. 1994;15:981–987.

  60. 60.
    1. Vidaillet HJ Jr.,
    2. Pressley JC,
    3. Henke E,
    4. Harrell FE Jr.,
    5. German LD

    . Familial occurrence of accessory atrioventricular pathways (preexcitation syndrome). N Engl J Med. 1987;317:65–69.

  61. 61.
    1. Arad M,
    2. Moskowitz IP,
    3. Patel VV,
    4. Ahmad F,
    5. Perez-Atayde AR,
    6. Sawyer DB,
    7. WalterM,
    8. Li GH,
    9. Burgon PG,
    10. Maguire CT,
    11. Stapleton D,
    12. Schmitt JP,
    13. Guo XX,
    14. Pizard A,
    15. Kupershmidt S,
    16. Roden DM,
    17. Berul CI,
    18. Seidman CE,
    19. Seidman JG

    . Transgenic mice overexpressing mutant PRKAG2 define the cause of Wolff-Parkinson-White syndrome in glycogen storage cardiomyopathy. Circulation. 2003;107:2850–2856.

  62. 62.
    1. Wolf CM,
    2. Arad M,
    3. Ahmad F,
    4. Sanbe A,
    5. Bernstein SA,
    6. Toka O,
    7. Konno T,
    8. Morley G,
    9. Robbins J,
    10. Seidman JG,
    11. Seidman CE,
    12. Berul CI

    . Reversibility of PRKAG2 glycogen-storage cardiomyopathy and electrophysiological manifestations. Circulation.2008;117:144–154.

  63. 63.
    1. Gaussin V,
    2. Van de Putte T,
    3. Mishina Y,
    4. Hanks MC,
    5. Zwijsen A,
    6. Huylebroeck D,
    7. Behringer RR,
    8. Schneider MD

    . Endocardial cushion and myocardial defects after cardiac myocyte-specific conditional deletion of the bone morphogenetic protein receptor ALK3.Proc Natl Acad Sci U S A. 2002;99:2878–2883.

  64. 64.
    1. Stroud DM,
    2. Gaussin V,
    3. Burch JB,
    4. Yu C,
    5. Mishina Y,
    6. Schneider MD,
    7. Fishman GI,
    8. Morley GE

    . Abnormal conduction and morphology in the atrioventricular node of mice with atrioventricular canal targeted deletion of Alk3/Bmpr1a receptor. Circulation.2007;116:2535–2543.

  65. 65.
    1. Gaussin V,
    2. Morley GE,
    3. Cox L,
    4. Zwijsen A,
    5. Vance KM,
    6. Emile L,
    7. Tian Y,
    8. Liu J,
    9. HongC,
    10. Myers D,
    11. Conway SJ,
    12. Depre C,
    13. Mishina Y,
    14. Behringer RR,
    15. Hanks MC,
    16. Schneider MD,
    17. Huylebroeck D,
    18. Fishman GI,
    19. Burch JB,
    20. Vatner SF

    . Alk3/Bmpr1a receptor is required for development of the atrioventricular canal into valves and annulus fibrosus. Circ Res.2005;97:219–226.

  66. 66.
    1. Hoffman JI,
    2. Kaplan S

    . The incidence of congenital heart disease. J Am Coll Cardiol. 2002;39:1890–1900.

  67. 67.
    1. Walsh SR,
    2. Tang T,
    3. Wijewardena C,
    4. Yarham SI,
    5. Boyle JR,
    6. Gaunt ME

    .Postoperative arrhythmias in general surgical patients. Ann R Coll Surg Engl.2007;89:91–95.

  68. 68.
    1. Walsh EP,
    2. Cecchin F

    . Arrhythmias in adult patients with congenital heart disease.Circulation. 2007;115:534–545.

  69. 69.
    1. Gourdie RG,
    2. Mima T,
    3. Thompson RP,
    4. Mikawa T

    . Terminal diversification of the myocyte lineage generates Purkinje fibers of the cardiac conduction system.Development. 1995;121:1423–1431.

  70. 70.
    1. Cheng G,
    2. Litchenberg WH,
    3. Cole GJ,
    4. Mikawa T,
    5. Thompson RP,
    6. Gourdie RG

    .Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage. Development. 1999;126:5041–5049.

  71. 71.
    1. Moorman AF,
    2. Christoffels VM

    . Development of the cardiac conduction system: a matter of chamber development. Novartis Found Symp. 2003;250:25–34;discussion 34–43, 276–279.

  72. 72.
    1. Bruneau BG,
    2. Nemer G,
    3. Schmitt JP,
    4. Charron F,
    5. Robitaille L,
    6. Caron S,
    7. Conner DA,
    8. Gessler M,
    9. Nemer M,
    10. Seidman CE,
    11. Seidman JG

    . A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell. 2001;106:709–721.

  73. 73.
    1. Christoffels VM,
    2. Smits GJ,
    3. Kispert A,
    4. Moorman AF

    . Development of the pacemaker tissues of the heart. Circ Res. 2010;106:240–254.

  74. 74.
    1. Moskowitz IP,
    2. Kim JB,
    3. Moore ML,
    4. Wolf CM,
    5. Peterson MA,
    6. Shendure J,
    7. NobregaMA,
    8. Yokota Y,
    9. Berul C,
    10. Izumo S,
    11. Seidman JG,
    12. Seidman CE

    . A molecular pathway including Id2, Tbx5, and Nkx2-5 required for cardiac conduction system development.Cell. 2007;129:1365–1376.

  75. 75.
    1. Stennard FA,
    2. Harvey RP

    . T-box transcription factors and their roles in regulatory hierarchies in the developing heart. Development. 2005;132:4897–4910.

  76. 76.
    1. Moskowitz IP,
    2. Pizard A,
    3. Patel VV,
    4. Bruneau BG,
    5. Kim JB,
    6. Kupershmidt S,
    7. RodenD,
    8. Berul CI,
    9. Seidman CE,
    10. Seidman JG

    . The T-box transcription factor Tbx5 is required for the patterning and maturation of the murine cardiac conduction system. Development.2004;131:4107–4116.

  77. 77.
    1. Ranganayakulu G,
    2. Elliott DA,
    3. Harvey RP,
    4. Olson EN

    . Divergent roles for NK-2 class homeobox genes in cardiogenesis in flies and mice. Development.1998;125:3037–3048.

  78. 78.
    1. Tanaka M,
    2. Chen Z,
    3. Bartunkova S,
    4. Yamasaki N,
    5. Izumo S

    . The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development. Development. 1999;126:1269–1280.

  79. 79.
    1. Jay PY,
    2. Harris BS,
    3. Maguire CT,
    4. Buerger A,
    5. Wakimoto H,
    6. Tanaka M,
    7. KupershmidtS,
    8. Roden DM,
    9. Schultheiss TM,
    10. O’Brien TX,
    11. Gourdie RG,
    12. Berul CI,
    13. Izumo S

    . Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system. J Clin Invest.2004;113:1130–1137.

  80. 80.
    1. Wakimoto H,
    2. Kasahara H,
    3. Maguire CT,
    4. Izumo S,
    5. Berul CI

    . Developmentally modulated cardiac conduction failure in transgenic mice with fetal or postnatal overexpression of DNA nonbinding mutant Nkx2.5. J Cardiovasc Electrophysiol.2002;13:682–688.

  81. 81.
    1. Kasahara H,
    2. Wakimoto H,
    3. Liu M,
    4. Maguire CT,
    5. Converso KL,
    6. Shioi T,
    7. Huang WY,
    8. Manning WJ,
    9. Paul D,
    10. Lawitts J,
    11. Berul CI,
    12. Izumo S

    . Progressive atrioventricular conduction defects and heart failure in mice expressing a mutant Csx/Nkx2.5 homeoprotein. J Clin Invest. 2001;108:189–201.

  82. 82.
    1. Hsu DT

    . Cardiac manifestations of neuromuscular disorders in children. Paediatr Respir Rev. 2010;11:35–38.

  83. 83.
    1. Holaska JM

    . Emerin and the nuclear lamina in muscle and cardiac disease. Circ Res. 2008;103:16–23.

  84. 84.
    1. Arimura T,
    2. Helbling-Leclerc A,
    3. Massart C,
    4. Varnous S,
    5. Niel F,
    6. Lacene E,
    7. FromesY,
    8. Toussaint M,
    9. Mura AM,
    10. Keller DI,
    11. Amthor H,
    12. Isnard R,
    13. Malissen M,
    14. Schwartz K,
    15. Bonne G

    . Mouse model carrying H222P-Lmna mutation develops muscular dystrophy and dilated cardiomyopathy similar to human striated muscle laminopathies. Hum Mol Genet. 2005;14:155–169.

  85. 85.
    1. Wolf CM,
    2. Wang L,
    3. Alcalai R,
    4. Pizard A,
    5. Burgon PG,
    6. Ahmad F,
    7. Sherwood M,
    8. Branco DM,
    9. Wakimoto H,
    10. Fishman GI,
    11. See V,
    12. Stewart CL,
    13. Conner DA,
    14. Berul CI,
    15. Seidman CE,
    16. Seidman JG

    . Lamin A/C haploinsufficiency causes dilated cardiomyopathy and apoptosis-triggered cardiac conduction system disease. J Mol Cell Cardiol.2008;44:293–303.

  86. 86.
    1. Fatkin D,
    2. MacRae C,
    3. Sasaki T,
    4. Wolff MR,
    5. Porcu M,
    6. Frenneaux M,
    7. Atherton J,
    8. Vidaillet HJ Jr.,
    9. Spudich S,
    10. De Girolami U,
    11. Seidman JG,
    12. Seidman C,
    13. Muntoni F,
    14. MuehleG,
    15. Johnson W,
    16. McDonough B

    . Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy and conduction-system disease. N Engl J Med. 1999;341:1715–1724.

  87. 87.
    1. Mounkes LC,
    2. Kozlov SV,
    3. Rottman JN,
    4. Stewart CL

    . Expression of an LMNA-N195K variant of A-type lamins results in cardiac conduction defects and death in mice.Hum Mol Genet. 2005;14:2167–2180.

  88. 88.
    1. Muchir A,
    2. Pavlidis P,
    3. Decostre V,
    4. Herron AJ,
    5. Arimura T,
    6. Bonne G,
    7. Worman HJ

    .Activation of MAPK pathways links LMNA mutations to cardiomyopathy in Emery-Dreifuss muscular dystrophy. J Clin Invest. 2007;117:1282–1293.

  89. 89.
    1. Muchir A,
    2. Shan J,
    3. Bonne G,
    4. Lehnart SE,
    5. Worman HJ

    . Inhibition of extracellular signal-regulated kinase signaling to prevent cardiomyopathy caused by mutation in the gene encoding A-type lamins. Hum Mol Genet. 2009;18:241–247.

  90. 90.
    1. Morgenlander JC,
    2. Nohria V,
    3. Saba Z

    . EKG abnormalities in pediatric patients with myotonic dystrophy. Pediatr Neurol. 1993;9:124–126.

  91. 91.
    1. Berul CI,
    2. Maguire CT,
    3. Aronovitz MJ,
    4. Greenwood J,
    5. Miller C,
    6. Gehrmann J,
    7. Housman D,
    8. Mendelsohn ME,
    9. Reddy S

    . DMPK dosage alterations result in atrioventricular conduction abnormalities in a mouse myotonic dystrophy model. J Clin Invest. 1999;103;R1–R7.

  92. 92.
    1. Wakimoto H,
    2. Maguire CT,
    3. Sherwood MC,
    4. Vargas MM,
    5. Sarkar PS,
    6. Han J,
    7. ReddyS,
    8. Berul CI

    . Characterization of cardiac conduction system abnormalities in mice with targeted disruption of Six5 gene. J Interv Card Electrophysiol. 2002;7:127–135.

  93. 93.
    1. Mahadevan MS,
    2. Yadava RS,
    3. Yu Q,
    4. Balijepalli S,
    5. Frenzel-McCardell CD,
    6. BourneTD,
    7. Phillips LH

    . Reversible model of RNA toxicity and cardiac conduction defects in myotonic dystrophy. Nat Genet. 2006;38:1066–1070.

  94. 94.
    1. Hardy J,
    2. Singleton A

    . Genomewide association studies and human disease. N Engl J Med. 2009;360:1759–1768.

  95. 95.
    A haplotype map of the human genome. Nature. 2005;437:1299–1320.
  96. 96.
    1. Holm H,
    2. Gudbjartsson DF,
    3. Arnar DO,
    4. Thorleifsson G,
    5. Thorgeirsson G,
    6. Stefansdottir H,
    7. Gudjonsson SA,
    8. Jonasdottir A,
    9. Mathiesen EB,
    10. Njolstad I,
    11. Nyrnes A,
    12. Wilsgaard T,
    13. Hald EM,
    14. Hveem K,
    15. Stoltenberg C,
    16. Lochen ML,
    17. Kong A,
    18. Thorsteinsdottir U,
    19. Stefansson K

    . Several common variants modulate heart rate, PR interval and QRS duration. Nat Genet. 2010;42:117–122.

  97. 97.
    1. Pfeufer A,
    2. van Noord C,
    3. Marciante KD,
    4. Arking DE,
    5. Larson MG,
    6. Smith AV,
    7. Tarasov KV,
    8. Muller M,
    9. Sotoodehnia N,
    10. Sinner MF,
    11. Verwoert GC,
    12. Li M,
    13. Kao WH,
    14. KottgenA,
    15. Coresh J,
    16. Bis JC,
    17. Psaty BM,
    18. Rice K,
    19. Rotter JI,
    20. Rivadeneira F,
    21. Hofman A,
    22. Kors JA,
    23. Stricker BH,
    24. Uitterlinden AG,
    25. van Duijn CM,
    26. Beckmann BM,
    27. Sauter W,
    28. Gieger C,
    29. LubitzSA,
    30. Newton-Cheh C,
    31. Wang TJ,
    32. Magnani JW,
    33. Schnabel RB,
    34. Chung MK,
    35. Barnard J,
    36. SmithJD,
    37. Van Wagoner DR,
    38. Vasan RS,
    39. Aspelund T,
    40. Eiriksdottir G,
    41. Harris TB,
    42. Launer LJ,
    43. Najjar SS,
    44. Lakatta E,
    45. Schlessinger D,
    46. Uda M,
    47. Abecasis GR,
    48. Muller-Myhsok B,
    49. EhretGB,
    50. Boerwinkle E,
    51. Chakravarti A,
    52. Soliman EZ,
    53. Lunetta KL,
    54. Perz S,
    55. Wichmann HE,
    56. Meitinger T,
    57. Levy D,
    58. Gudnason V,
    59. Ellinor PT,
    60. Sanna S,
    61. Kaab S,
    62. Witteman JC,
    63. Alonso A,
    64. Benjamin EJ,
    65. Heckbert SR

    . Genome-wide association study of PR interval. Nat Genet.2010;42:153–159.

  98. 98.
    1. Chambers JC,
    2. Zhao J,
    3. Terracciano CM,
    4. Bezzina CR,
    5. Zhang W,
    6. Kaba R,
    7. Navaratnarajah M,
    8. Lotlikar A,
    9. Sehmi JS,
    10. Kooner MK,
    11. Deng G,
    12. Siedlecka U,
    13. ParasramkaS,
    14. El-Hamamsy I,
    15. Wass MN,
    16. Dekker LR,
    17. de Jong JS,
    18. Sternberg MJ,
    19. McKenna W,
    20. Severs NJ,
    21. de Silva R,
    22. Wilde AA,
    23. Anand P,
    24. Yacoub M,
    25. Scott J,
    26. Elliott P,
    27. Wood JN,
    28. Kooner JS

    . Genetic variation in SCN10A influences cardiac conduction. Nat Genet.2010;42:149–152.

  99. 99.
    1. Cho HC,
    2. Marban E

    . Biological therapies for cardiac arrhythmias: can genes and cells replace drugs and devices? Circ Res. 2010;106:674–685.

  100. 100.
    1. Edelberg JM,
    2. Aird WC,
    3. Rosenberg RD

    . Enhancement of murine cardiac chronotropy by the molecular transfer of the human beta(2) adrenergic receptor cDNA. J Clin Invest. 1998;101:337–343.

  101. 101.
    1. Edelberg JM,
    2. Huang DT,
    3. Josephson ME,
    4. Rosenberg RD

    . Molecular enhancement of porcine cardiac chronotropy. Heart. 2001;86:559–562.

  102. 102.
    1. Miake J,
    2. Marban E,
    3. Nuss HB

    . Gene therapy: biological pacemaker created by gene transfer. Nature. 2002;419:132–133.

  103. 103.
    1. Ruhparwar A,
    2. Kallenbach K,
    3. Klein G,
    4. Bara C,
    5. Ghodsizad A,
    6. Sigg DC,
    7. Karck M,
    8. Haverich A,
    9. Niehaus M

    . Adenylate-cyclase VI transforms ventricular cardiomyocytes into biological pacemaker cells. Tissue Eng Part A. 2010;16:1867–1872.

  104. 104.
    1. Sastry A,
    2. Arnold E,
    3. Gurji H,
    4. Iwasa A,
    5. Bui H,
    6. Hassankhani A,
    7. Patel HH,
    8. Feramisco JR,
    9. Roth DM,
    10. Lai NC,
    11. Hammond HK,
    12. Narayan SM

    . Cardiac-directed expression of adenylyl cyclase VI facilitates atrioventricular nodal conduction. J Am Coll Cardiol. 2006;48:559–565.

  105. 105.
    1. Qu JH,
    2. Plotnikov AN,
    3. Danilo P,
    4. Shlapakova I,
    5. Cohen IS,
    6. Robinson RB,
    7. RosenMR

    . Expression and function of a biological pacemaker in canine heart. Circulation.2003;107:1106–1109.

  106. 106.
    1. Plotnikov AN,
    2. Sosunov EA,
    3. Qu JH,
    4. Shlapakova IN,
    5. Anyukhovsky EP,
    6. Liu LL,
    7. Janse MJ,
    8. Brink PR,
    9. Cohen IS,
    10. Robinson RB,
    11. Danilo P,
    12. Rosen MR

    . Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation. 2004;109:506–512.

  107. 107.
    1. Plotnikov AN,
    2. Shlapakova I,
    3. Szabolcs MJ,
    4. Danilo P,
    5. Lorell BH,
    6. Potapova IA,
    7. LuZJ,
    8. Rosen AB,
    9. Mathias RT,
    10. Brink PR,
    11. Robinson RB,
    12. Cohen IS,
    13. Rosen MR

    . Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart. Circulation. 2007;116:706–713.

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Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing[1]

Curator and Reporter: Stephen J. Williams, Ph.D.

Genomic instability is considered a hallmark and necessary for generating the mutations which drive tumorigenesis. Multiple studies had suggested that there may be multiple driver mutations and a plethora of passenger mutations driving a single tumor.  This diversity of mutational spectrum is even noticed in cultured tumor cells (refer to earlier post Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single Human Cell).  Certainly, intratumor heterogeneity has been a concern to clinicians in determining the proper personalized therapy for a given cancer patient, and has been debated if multiple biopsies of a tumor is required to acquire a more complete picture of a tumor’s mutations.  In the New England Journal of Medicine, lead author Dr. Marco Gerlinger in the laboratory of Dr. Charles Swanton of the Cancer Research UK London Research Institute, and colleagues reported the results of a study to determine if intratumoral differences exist in the mutational spectrum of primary and metastatic renal carcinomas, pre- and post-treatment with the mTOR (mammalian target of rapamycin) inhibitor, everolimus (Afinitor®)[1].

The authors compared exome sequencing of multiregion biopsies from four patients with metastatic renal-cell carcinoma who had been enrolled in the Personalized RNA Interference to Enhance the Delivery of Individualized Cytotoxic and Targeted Therapeutics clinical trial of everolimus (E-PREDICT) before and after cytoreductive surgery.

Biopsies taken:

  • Multiregion spatial biopsy of primary tumor (representing 9 regions of the tumor)
  • Chest-wall metastases
  • Perinephric metastases
  • Germline DNA as control

Multiple platforms were used to determine aberrations as follows:

  1. Illumina Genome Analyzer IIx and Hiseq: for sequencing and mutational analysis
  2. Illumina Omni 2.5: for SNP (single nucleotide polymorphism)-array-based allelic imbalance detection for chromosomal imbalance and ploidy analysis
  3. Affymetrix Gene 1.0 Array: for mRNA analysis

A phylogenetic reconstruction of all somatic mutations occurring in primary disease and associated metastases was  performed to determine the clonal evolution of the metastatic disease given the underlying heterogeneity of the tumor.  Basically the authors wanted to know if the mutational spectra of one metastasis could be found in biopsies taken from the underlying primary tumor or if the mutational landscape of metastases had drastically changed.


Multiregion exon-capture sequencing of DNA from pretreatment biopsy samples of the primary tumor, chest wall metastases, and perinephrous metastasis revealed 128 mutations classified as follows:

  • 40 ubiquitous mutations
  • 59 mutations shared by several but not all regions
  • 29 mutations unique to specific regions
  • 31 mutations shared by most primary tumor regions
  • 28 mutations shared by most metastatic regions

The authors mapped these mutations out with respect to their location, in order to determine how the metastatic lesions evolved from the primary tumor, given the massive heterogeneity in the primary tumor.  Construction of this “phylogenetic tree” (see Merlo et. al[2]) showed that the disease evolves in a branched not linear pattern, with one branch of clones evolving into a metastatic disease while another branch of clones and mutations evolve into the primary disease.

One of the major themes of the study is shown by results that an average of 70 somatic mutations were found in a single biopsy (a little more than just half of all tumor mutations) yet only 34% of the mutations in multiregion biopsies were detected in all tumor regions.

This indicated to the authors that “a single biopsy was not representative of the mutational landscape of the entire bulk tumor”. In addition, microarray studies concluded that gene-expression signatures from a single biopsy would not be able to predict outcome.

Everolimus therapy did not change the mutational landscape.  Interestingly, allelic composition and ploidy analyses revealed an extensive intratumor heterogeneity, with ploidy heterogeneity in two of four tumors and 26 of 30 tumor samples containing divergent allelic-imbalances.  This strengthens the notion that multiple clones with diverse genomic instability exist in various regions of the tumor.

 The intratumor heterogeneity reveals a convergent tumor evolution with associated heterogeneity in target function

Genes commonly mutated in clear cell carcinoma[3, 4] (and therefore considered the prevalent driver mutations for renal cancer) include:

Only VHL mutations were found in all regions of a given tumor, however there were three distinct SETD2 mutations (frameshift, splice site, missense) which were located in different regions of the tumor.

SETD2 trimethylates histones at various lysine residues, such as lysine residue 36 (H3K36).  The trimethylation of H3K36 is found on many actively transcribed genes.  Immunohistochemistry showed trimethylated H3K36 was reduced in cancer cells but positive in most stromal cells and in SETD2 wild-type clear-cell carcinomas.

Interestingly most regions of the primary tumor, except one, contained a kinase-domain activating mutation in mTOR.  Immunohistochemistry analysis of downstream target genes of mTOR revealed that mTOR activity was enhanced in regions containing this mutation.  Therefore the intratumoral heterogeneity corresponded to therapeutic activity, leading to the impression that a single biopsy may result in inappropriate targeted therapy.   Additional downstream biomarkers of activity confirmed both the intratumoral heterogeneity of mutational spectrum as well as an intratumoral heterogeneity of therapeutic-target function.

The authors conclude that “intratumor heterogeneity can lead to underestimation of the tumor genomics landscape from single tumor biopsies and may present major challenges to personalized-medicine and biomarker development”.

In an informal interview with Dr. Swanton, he had stressed the importance of performing these multi-region biopsies and the complications that intratumoral heterogeneity would present for personalized medicine, biomarker development, and chemotherapy resistance.

Q: Your data clearly demonstrates that multiple biopsies must be done to get a more complete picture of the tumor’s mutational landscape.  In your study, what percentage of the tumor would be represented by the biopsies you had performed?

Dr. Swanton: Realistically this is a very difficult question to answer, the more biopsies we sequence, the more we find, in the near term it may be very difficult to ever formally address this in large metastatic tumours

Q:  You have very nice data which suggest that genetic intratumor heterogeneity complicates the tumor biomarker field? do you feel then that quests for prognostic biomarkers may be impossible to attain?

Dr. Swanton: Not necessarily although heterogeneity is likely to complicate matters

Identifying clonally dominant lesions may provide better drug targets

Predicting resistance events may be difficult given the potential impact of tumour sampling bias and the concern that in some tumours a single biopsy may miss a relevant subclonal mutation that may result in resistance

Q:  Were you able to establish the degree of genomic instability among the various biopsies?

Dr. Swanton:  Yes, we did this by allelic imbalance analysis and found that the metastases were more genomically unstable than the primary region from which the metastasis derived

Q: I was actually amazed that there was a heterogeneity of mTOR mutations and SETD2 after everolimus therapy?   Is it possible these clones obtained a growth advantage?

Dr. Swanton: We think so yes, otherwise we wouldn’t identify recurrent mutations in these “driver genes”

Dr. Swanton will present his results at the 2013 AACR meeting in Washington D.C. (http://www.aacr.org/home/scientists/meetings–workshops/aacr-annual-meeting-2013.aspx)

The overall points of the article are as follows:

  • Multiple biopsies of primary tumor and metastases are required to determine the full mutational landscape of a patients tumor
  • The intratumor heterogeneity will have an impact on the personalized therapy strategy for the clinician


  • Metastases arising from primary tumor clones will have a greater genomic instability and mutational spectrum than the tumor from which it originates


  • Tumors and their metastases do NOT evolve in a linear path but have a branched evolution and would complicate biomarker development and the prognostic and resistance outlook for the patient

A great video of Dr. Swanton discussing his research can be viewed here


Everolimus: an inhibitor of mTOR

The following information was taken from the New Medicine Oncology Database (http://www.nmok.net)




Approved/Filed Indications

Novartis PharmaCurrent as of: August 30, 2012 Generic Name: Everolimus
Brand Name: Afinitor
Other Designation: RAD001, RAD001C
RAD001, an ester of the macrocytic immunosuppressive agent sirolimus (rapamycin), is an inhibitor of mammalian target of rapamycin (mTOR) kinase.Administration Route: intravenous (IV) • PO • solid organ transplant
• renal cell carcinoma (RCC), metastatic after failure of treatment with sunitinib, sorafenib, or sunitinib plus sorafenib
• renal cell carcinoma, advanced, refractory to treatment with vascular endothelial growth factor (VEGF)-targeted therapy
• treatment of progressive neuroendocrine tumors (NET) of pancreatic origin (PNET) in patients with inoperable, locally advanced or metastatic disease

Marker Designation
Gene Location

Marker Description


5’-AMP-activated Protein Kinase (AMPK)AMPK beta 1 (beta1 non-catalytic subunit) • HAMPKb (beta1 non-catalytic subunit) • MGC17785 (beta1 non-catalytic subunit) • AMPK2 (alpha1 catalytic subunit) • PRKAA (alpha1 catalytic subunit) • AMPK alpha 1 (alpha1 catalytic subunit) • AMPKa1 ( AMPK is a member of a metabolite-sensing protein kinase family found in all eukaryotes. It functions as a cellular fuel sensor and its activation strongly suppresses cell proliferation in non-malignant cells and cancer cells. AMPK regulates the cell cycle by upregulating the p53-p21 axis and modulating the TSC2-mTOR (mammalian target of rapamycin) pathway. The AMPK signaling network contains a number of tumor suppressor genes including LKB1, p53, TSC1 and TSC2, and modulates growth factor signaling involving proto-oncogenes including PI3K, Akt and ERK. AMPK activation is therefore therapeutic target for cancer (Motoshima H, etal, J Physiol, 1 Jul 2006; 574(Pt 1): 63–71).AMPK is a protein serine/threonine kinase consisting of a heterotrimeric complex of a catalytic alpha subunit and regulatory ß and gamma subunits. AMPK is activated by increased AMP/ATP ratio, under conditions such as glucose deprivation, hypoxia, ischemia and heat shock. It is also activated by several hormones and cytokines. AMPK inhibits ATP-consuming cellular events, protein synthesis, de novo fatty acid synthesis, and generation of mevalonate and the downstream products in the cholesterol synthesis pathway (Motoshima H, etal, J Physiol, 1 Jul 2006; 574(Pt 1): 63–71). – ovarian cancer
– brain cancer
– liver cancer
– leukemia
– colon cancer
CREB regulated transcription coactivator 2 (CRTC2)TOR complex 2 (TORC2, mTORC2) • RP11-422P24.6 • transducer of regulated cAMP response element-binding protein (CREB)2 • transducer of CREB protein 2 • TOR1Location: 1q21.3 The mammalian target of rapamycin (mTOR) exists in two complexes, TORC1 and TORC2, which are differentially sensitive to rapamycin. cAMP response element-binding protein (CREB) regulated transcription coactivator 2 (CRTC2) or TORC2 is a multimeric kinase composed of mTOR, mLST8, mSin1, and rictor. The complex is insensitive to acute rapamycin exposure and functions in controlling cell growth and actin cytoskeletal assembly.TORC2 controls gene silencing, telomere length maintenance, and survival under DNA-damaging conditions. It is primaily located in the cytoplasm but also shuttles into the nucleus (Schonbrun M, etal, Mol Cell Biol, Aug 2009;29(16):4584-94). – brain cancer
Hypoxia inducible factor 1 alpha (HIF1A)HIF1-alpha (HIF-1 alpha) • HIF-1A • PASD8 • MOP1 • bHLHe78Location: 14q21-q24 The alpha subunit of the hypoxia inducible factor 1 (HIF-1alpha) is a 826 amino acid antigen consisting of a basic helix-loop-helix (bHLH)-PAS domain at its N-terminus. HIF-1alpha is rapidly degraded by the proteasome under normal conditions, but is stabilized by hypoxia resulting in the transactivation of several proangiogenic genes. HIF-1alpha is responsible for inducing production of new blood vessels as needed when tumors outgrow existing blood supplies. HIF-1alpha serves as a transcriptional factor that regulates gene expression involved in response to hypoxia and promotes angiogenesis.HIF-1alpha is a proangiogenic transcription factor induced primarily by tumor hypoxia that is critically involved in tumor progression, metastasis and overall tumor survival. HIF-1alpha functions as a survival factor that is required for tumorigenesis in many types of malignancies, and is expressed in a majority of metastases and late-stage tumors. HIF-1alpha is overexpressed in brain, breast, colon, endometrial, head and neck, lung, ovarian, and pancreatic cancer, and is associated with increased microvessel density and/or VEGF expression – prostate cancer
– bladder cancer
– nasopharyngeal cancer
– head and neck cancer
– kidney cancer
– pancreatic cancer
– endometrial cancer
– breast cancer
Mammalian target of rapamycin (mTOR)FK506 binding protein 12-rapamycin associated protein 1 • RAFT1 • FK506 binding protein 12-rapamycin associated protein 2 • FRAP • FRAP1 • FRAP2 • RAPT1 • FKBP-rapamycin associated protein • FKBP12-rapamycin complex-associated protein 1 • rapamycin target protein • TOR • FLJ44809 • MTORC1 • MTORC2 • RPTOR • RAPTOR • KIAA1303 • mammalian target of rapamycin complex 1Location: 1p36.22 The mammalian target of rapamycin (mTOR) is a large serine/threonine protein (Mr 300,000) having heat repeats, and protein-protein interaction domains at its amino terminus, and a protein kinase domain at its carboxy terminus. mTOR is a member of the phosphoinositide 3-kinase (PI3K)-related kinase (PIKK) family and a central modulator of cell growth. It regulates cell growth, proliferation and survival by impacting on protein synthesis and transcription. mTOR is present in two multi-protein complexes, a rapamycin-sensitive complex, TOR complex 1 (TORC1), defined by the presence of Raptor and a rapamycin insensitive complex, TOR complex 2 (TORC2), with Rictor, Protor and Sin1. Rapamycin selectively inhibits mTORC1 by binding indirectly to the mTOR/Raptor complex via FKBP12, resulting in inhibition of p70S6kinase but not the mTORC2 substrate AKTSer473. Selective inhibition of p70S6K attenuates negative feedback loops to IRS1 and TORC2 resulting in an increase in pAKT which may limit the activity of rapamycin.In a hypoxic environment the increase in mass of solid tumors is dependent on the recruitment of mitogens and nutrients. As a function of nutrient levels, particularly essential amino acids, mTOR acts as a checkpoint for ribosome biogenesis and cell growth. Ribosome biogenesis has long been recognized in the clinics as a predictor of cancer progression; increase in size and number of nucleoli is known to be associated with the most aggressive tumors and a poor prognosis. In bacteria, ribosome biogenesis is independently regulated by amino acids and energy charge. The mTOR pathway is controlled by intracellular ATP levels, independent of amino acids, and mTOR itself is an ATP sensor (Kozma SC, etal, AACR02, Abs. 5628). – breast cancer
– pancreatic cancer
– multiple myeloma
– liver cancer
– brain cancer
– prostate cancer
– kidney cancer
– lymphoma
Signal transducer and activator of transcription 3 (STAT3)Stat-3 • acute-phase response factor (APRF) • FLJ20882 • HIESLocation: 17q21 Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT protein family. STAT3, plays a critical role in hematopoiesis. STAT3 is located in the cytoplasm and translocated to the nucleus after tyrosine phosphorylation. In response to cytokines and growth and other activation factors, STAT family members are phosphorylated by the receptor associated kinases and then form homo- or heterodimers, which translocate to the cell nucleus where they act as transcription activators. – multiple myeloma
– hematologic malignancy
– lymphoma
Sonic hedgehog homolog (SHH)Shh • HHG1 • HHG-1 • holoprosencephaly 3 (HPE3) • HLP3 • SMMCILocation: 7q36 Sonic hedgehog, a secreted hedgehog ligand, is a human homolog of the Drosophila segment polarity gene hedgehog, cloned by investigators at Harvard University (Marigo V, etal, Genomics, 1 Jul 1995;28 (1):44-51).The mammalian sonic hedgehog (Shh) pathway controls proliferation of granule cell precursors in the cerebellum and is essential for normal embryonic development. Shh signaling is disrupted in a variety of malignancies. – pancreatic cancer
– CNS cancer


1.         Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P et al: Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. The New England journal of medicine 2012, 366(10):883-892.

2.         Merlo LM, Pepper JW, Reid BJ, Maley CC: Cancer as an evolutionary and ecological process. Nature reviews Cancer 2006, 6(12):924-935.

3.         Varela I, Tarpey P, Raine K, Huang D, Ong CK, Stephens P, Davies H, Jones D, Lin ML, Teague J et al: Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 2011, 469(7331):539-542.

4.         Dalgliesh GL, Furge K, Greenman C, Chen L, Bignell G, Butler A, Davies H, Edkins S, Hardy C, Latimer C et al: Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature 2010, 463(7279):360-363.

Other Articles related to this topic appeared on this Open Access Online Scientific Journal, including the following:

AMPK Is a Negative Regulator of the Warburg Effect and Suppresses Tumor Growth In Vivo

Genomics of bronchial epithelial dysplasia

Genomics in Medicine- Tomorrow’s Promise

Prostate Cancer: Androgen-driven “Pathomechanism” in Early-onset Forms of the Disease

CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC

CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics and Computational Genomics – Part IIB

Genome-Wide Detection of Single-Nucleotide and Copy-Number Variation of a Single Human Cell

Directions for Genomics in Personalized Medicine

LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer Personalized Treatment: Part 2

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1

Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders @ http://pharmaceuticalintelligence.com

In Focus: Targeting of Cancer Stem Cells

Modulating Stem Cells with Unread Genome: microRNAs

What can we expect of tumor therapeutic response?


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Genomics of Bronchial Epithelial Dysplasia

Curator: Larry H Bernstein, MD, FCAP


C. Walker, LJ Robertson, MW Myskow, N. Pendleton & G.R. Dixon
Clatterbridge Cancer Research Trust, J K Douglas Cancer Research Laboratory, Clatterbridge Hospital, & Broadgreen Hospital, Liverpool, UK.
Br. J. Cancer (1994). 70, 297-303
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2033485/53 expression in normal and dysplastic bronchial epithelium and in lung carcinomas 

Bronchial epithelial dysplasia is thought to be a premalignant stage in the evolution of lung cancers. Using the CM-1 polyclonal antibody, we have examined the expression of the p53 protein in a larger series of bronchial dysplasias (n = 60) than hitherto investigated. The p53 protein was detected in 14% of mild, 25% of moderate and 59% of severe dysplasias; increased p53 expression correlated with the severity of dysplasia. p53-positive dysplasias had greater PCNA indices than p53-negative dysplasias. p53 expression in dysplastic tissues was compared with that in two groups of histologically normal epithelium: 14 bronchial biopsies from non-cancer patients of which all but one were negative and 32 bronchial margins from resected carcinomas, of which 17 showed infrequent solitary cells with p53-positive nuclei in predominantly basal locations scattered throughout the epithelium. These results for resection margins were confirmed by use of a second antibody, DO-1. Sixty-nine per cent of the corresponding carcinomas were p53 positive, but in 15 cases the p53 reactivity differed from resection margins. No correlation between p53 expression and any of the clinicopathological characteristics of these tumours was found. This study supports the observation that abnormal p53 expression may be an early but not obligatory event in malignant transformation in lung.

It is now widely agreed that all lung cancers are derived from a common pluripotent stem cell capable of expressing a variety of phenotypes. Although the sequence of events in the histogenesis of lung cancer is unknown, bronchial epithelial dysplasia is thought to be a premalignant stage in the evolution of lung carcinomas. Multistep genetic changes, which include

  • activation of cellular proto-oncogenes and
  • inactivation of tumour-suppressor genes, are
    • associated with the development of human cancers and are
    • thought to accompany the morphological changes that precede malignancy.

Currently the most commonly identified genetic change in human cancers is mutation in the p53 gene, located at position 13 on the short arm of chromosome 17. This gene is

  • a tumour suppressor gene and
  • encodes a 53 kDa nuclear phosphoprotein
  • capable of binding to DNA and
  • acting as a transcriptional factor.

The wild-type p53 protein inhibits cell proliferation, and

  • loss of this activity leads to neoplastic transformation.

This protein has a

  • short cellular half-life and
  • is usually present in normal cells, under normal physiological conditions, in extremely small amounts,
  • making it undetectable by standard immunohistochemical techniques.

Many mutations of the p53 gene, principally in exons 5-8,

  • lead to a functional inactivation of the -gene and
  • a protein product unable to regulate transcription, ultimately
  • resulting in deregulation of cell growth.

Mutant p53 has

  • an extended cellular half-life
  • enabling immunohistochemical detection of
  • the accumulated mutant protein in cell nuclei.

Although not all mutations lead to protein accumulation, in many studies a correlation between the p53 protein detected immunocytochemically and p53 gene mutations has been found.
Investigation of p53 overexpression in premalignant tissues has led to the observations that

  • alterations in the p53 gene
  • arise as late events in the evolution of some cancers,
    • e.g. in gastric carcinomas, prostatic carcinomas or melanomas, whereas in others,
    • e.g. oral , gall bladder and oesophageal, malignancies, abnormal p53 expression is an early event.

In attempts to define the type and temporal sequence of somatic genetic changes that precede the onset of invasive lung cancer, recent studies have reported mutations and allelic deletions in the p53 gene in preinvasive bronchial lesions. Immunodetectable p53 has been found in a few cases of bronchial dysplasia et al., and Nuorva et al. (1993) have reported that p53 overexpression correlated with the severity of dysplasia in 17 cases of dysplastic epithelium from cancer bearing patients. Thus lesions in the p53 gene have been reported as possible early events in the development of lung cancers.

p53 score and grade of dysplasia

The system of p53 scoring used in these experiments permitted a semiquantitative comparison of the degree of p53 expression in the various tissues examined. With increasing severity of dysplasia there was not only an increase in the percentage of cases demonstrating p53 staining but also an increase in the staining intensity of positive cells and an increase in the proportions of these positive cells. Thus, higher grades of dysplasia were associated with higher p53 scores; the Spearman rank correlation coefficient for the whole table is 0.47 (P = <0.0001), and considering  just the dysplasia cases it is 0.37 (P = 0.002). Comparison of p53 expression between the various grades of dysplasia by use of p53 score  results in more significant P-values by the Mann-Whitney test than obtained with the Fisher-Irwin tests.

PCNA indices

For 39 cases of bronchial dysplasia, PCNA indices had been determined previously (Pendleton et al., 1993). p53-positive dysplasias had significantly greater PCNA indices than p53-negative dysplasias (Table IVa), indicating abnormal growth in these p53-positive biopsies

Bronchial carcinomas and resection margins

In previous studies, a series of bronchial carcinomas (Burnett et al., 1993) and their corresponding resection margins which contained histologically normal epithelium (Pendleton et al., 1993) had been collected prospectively following surgery. Using the CM-1 antibody, p53-positive nuclei were seen in 22/32 (69%) of the tumours and in histologically normal epithelium in 17/32 (53%) of the resection margins (Table V).  p53-positive cells in resection margins were predominantly basal, solitary and scattered throughout the epithelium (Figure 2a), and were less frequent than in tumour tissues (Figure 2b) or many samples of dysplastic epithelium. Many nuclei were weakly stained, but some showed a staining intensity similar to p53-positive tumour cells. Compared with dysplasias and tumour tissues the p53 scores of resection margins were low (Tables III and V), with only one case with a score of 3 and no higher scores. To confirm these results, sections of resection margins were stained with the monoclonal antibody DO-1; all of the cases positive for the CM-1 antibody were also DO-1 positive, but two cases (numbers 16 and 21) which were negative for CM-1 were clearly positive for DO-1.


During the course of the preparation of this manuscript, it has been reported that

  • the p53 protein accumulates frequently in early bronchial neoplasia.

This study differs only in that biopsies, not resected tumours, were examined and all tissues were derived from a single treatment centre.  The results of all published studies (p53 expression has so far been investigated in a combined total of 23 mild, 31 moderate and 77 severe dysplasias) yields

  • 19% of the mild,
  • 28% of the moderate and
  • 63% of the severe dysplasias
    • found to be p53 positive.

In other similar studies investigating the expression of the p53 protein in premalignant lesions of lung and other tissues,

  • results were analysed by assessment of p53 positivity.

In this study, analysis was either

    1. by comparison of p53-positive and -negative groups or
    2. by use of a p53 scoring system similar to that described by Vojtesk et al. (1993).

The advantage of this scoring system is that it allows comparison of the degree of p53 expression between tissue groups.
The p53-positive group, equivalent to the positive group in other similar studies,

  • had a p53 score of two or more.

Cells with the p53 score for intensity of 1 were clearly p53 positive and were

  • found in tumours as well as in dysplasias and normal tissues.

Unlike other studies of preinvasive lung lesions,

  • PCNA indices for many of the dysplasias in this series had been determined.

The greater PCNA indices of the p53-positive group

  • indicates that p53-positive dysplasias contain higher proportions of cells in the proliferative phase of the cell cycle;

this suggests that p53-positive dysplasias

  • may have abnormalities in their growth control mechanisms.

It is possible that alterations in the p53 gene confer a growth advantage on these cells, leading to

  • expansion of p53-positive cells as severity of dysplasia increases.

A close relation between p53 overexpression and PCNA indices has also been observed in

  • pancreatic duct cell carcinomas,
  • hepatocellular carcinomas, and
  • gastric cancers.

In this study, p53 expression in dysplastic tissues was

  • compared with two groups of histologically normal epithelium.

All but one of the first group, taken from patients who did not have cancer at the time of biopsy, were negative. Comparison of p53 expression in this group with that in

  • dysplastic bronchial biopsies
    • showed a highly significant difference between these groups.

The second group of histologically normal epithelium analysed,

  • from the resection margins of bronchial carcinomas,
    • showed p53-positive cells in a high proportion of cases,
      • indicating differences in the normal bronchial epithelium of cancer and non-cancer patients.

p53 positivity in the normal mucosa of resection margins did not result in a measurable increase in proliferation, as indicated by PCNA indices. This may suggest that the mechanism whereby the p53 protein is elevated in normal mucosa differs from that in dysplasia. Whatever the mechanism to account for these p53-positive cells in normal bronchial mucosa, it seems that their presence, even if not associated with mutation in the p53 gene, indicates abnormalities that are not reflected in the histological appearance of these cells.

The number of p53-positive tumours in the series (69% overall and 68% for non-small-cell lung cancers) agreed well

  • with the incidence of p53 positivity for lung cancers reported in some studies
  • but was higher than that found in others.

Although the number of tumours in this series was small no correlation in p53 overexpression was found with any of the clnical characteristics of these tumours. This contrasts with reports of a relationship between p53 overexpression and

  • poor prognosis and shortened survival,
  • tumour grade  or lymph node involvement and
  • a greater incidence in squamous cell carcinomas compared with other types of lung carcinoma .

This study supports the observation that abnormal p53 expression is an early but not obligatory event in the evolution of lung cancers. Immunodetection of p53 overexpression in bronchial epithelium

    • may be a useful tool in the identification of those early lesions which may progress to malignancy.
Age-standardised death rates from Trachea, bro...

Age-standardised death rates from Trachea, bronchus, lung cancers by country (per 100,000 inhabitants). (Photo credit: Wikipedia)


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Genome Sequencing of the Healthy

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


Key Issues in Genome Sequencing of Healthy Individuals
Eric Topol, MD, Genomic Medicine

I briefly review 3 important articles that recently appeared, each touching on important controversies in the use of whole genome sequencing


I briefly review 3 important articles that recently appeared, each touching on important controversies in the use of whole genome sequencing:
1. Should Healthy People Have Their Genomes Sequenced At This Time? Wall Street Journal, February 15, 2013.
2. A Genetic Code for Genius? Wall Street Journal, February 15, 2013.
3. Francke U, Djamco C, Kiefer AK, et al. Dealing with the unexpected: consumer responses to direct-access BRCA mutation testing. PeerJ. 2012;1:e8. DOI 10.7717/peerj.8
Welcome to another segment on genomic medicine. Today, I want to get into 3 different articles: 2 from the Wall Street (“Medical”) Journal and the other from a new open access journal, PeerJ. All of them are related to the issues of genome sequencing.
First, there was a debate about whether all healthy people should have their genomes sequenced. It was a debate between Atul Butte from Stanford and Robert Green from Harvard. In this debate, they made a number of really good points, and I have linked you to that article if you’re interested.
Basically, is it too early to get sequencing because we need millions of people to have whole genome sequencing who are healthy in order for that information to be truly informative. The price continues to drop. So even though the sequencing that is done today would still be valid if it’s done accurately, the problem we have, of course, is a lack of enough people who are phenotyped with a particular condition to extract all the best information that is truly informative from whole genome sequencing.
 it’s unlikely that even 2000 individuals with high IQ will be particularly informative but also, of course, what this could do from a bioethical standpoint. I’ll leave that to your imagination and thoughts as to where this could go – that is, trying to understand, even with limited power, the genomics of intelligence.
The third article, which is also very interesting, comes from this new journal called PeerJ. I’m on the editorial board of that journal, and I think it’s terrific to see open access, high-quality biomedical articles.
This one comes from the company 23andMe. From a very large number of individuals – now over 200,000 and rapidly approaching 1 million – who have had genome scans, a large number of women had information about the BRCA gene and whether they had a significant mutation. From these women who volunteered to participate in this study, we learned that they had no serious psychological repercussions from knowledge of this highly actionable BRCA pathogenic mutation.
This goes along with the previous study that we had done at Scripps led by my colleague Cinnamon Bloss in the New England Journal of Medicine, where, in thousands of individuals who had genome scans and had such data as ApoE4 status known to them for the first time, there were no significant negative psychological repercussions.

Should Healthy People Have Their Genomes Sequenced At This Time?

‘Patients in Waiting’

Injecting so much uncertain genetic information into the doctor-patient relationship could create legions of “patients in waiting” leading to unnecessary tests, harmful outcomes and lifelong anxiety. As private software companies compete to provide more genomic “findings” to a medical culture that is trained to search for diagnostic fire when they smell the smoke of disease risk, there are potential benefits. But there is also a real possibility that medical resources will be squandered and patients could be harmed.

Perhaps we all underestimated how complicated it would be to move genomic knowledge into the practice of medicine and public health. Now is the time to make sure we get this right through rigorous basic and clinical studies that define which mutations are dangerous, and distinguish useful from unnecessary interventions. Soon, genomic insights will give us early warnings about life-threatening illnesses that we may be able to prevent. Soon, standards will be available to guide doctors about which findings are meaningful and which are not.

Soon, there may be evidence to support the benefits of screening healthy individuals. But not today.

Table 1. Performance values for genome sequenc...

Table 1. Performance values for genome sequencing technologies including Sanger methods and Massively Parallel Seqeuncing methods. Sinville, R. and Soper, S. A. High resolution DNA separations using microchip electrophoresis. J. Sep. Sci. 2007, 30, 1714 – 1728 Morozova,O. and Marra, M. A. Applications of next-generation sequencing technologies in functional genomics. Genomics. 92 (2008) 255–264 (Photo credit: Wikipedia)


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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

A number of novel genes have been identified in association with a variety of endocrine phenotypes over the last few years. However, although mutations in a number of genes have been described in association with disorders such as

  • hypogonadotropic hypogonadism,
  • congenital hypopituitarism,
  • disorders of sex development, and
  • congenital hyperinsulinism,

these account for a minority of patients with these conditions, suggesting that many more genes remain to be identified.

How will these novel genes be identified? Monogenic disorders can arise as a result of genomic microdeletions or microduplications, or due to single point mutations that lead to a functional change in the relevant protein. Such disorders may also result from altered expression of a gene, and hence altered dosage of the protein. Candidate genes may be identified by utilizing naturally occurring or transgenic mouse models, and this approach has been particularly informative in the elucidation of the genetic basis of a number of disorders.

Other approaches include the identification of chromosomal rearrangements using conventional karyotyping techniques, as well as novel assays such as array comparative genomic hybridization (CGH) and single nucleotide polymorphism oligonucleotide arrays (SNP arrays). These molecular methods usually result in the identification of gross abnormalities as well as submicroscopic deletions and duplications, and eventually to the discovery of single gene defects that are associated with a particular phenotype.

However, there is no doubt that the major advances in novel gene identification will be made as a result of the sequencing of the genome of affected individuals and comparison with control data that are already available. Chip techniques allow hybridization of DNA or RNA to hundreds of thousands of probes simultaneously. Microarrays are being used for mutational analysis of human disease genes.

Complete sequencing of genomes or sequencing of exons that encode proteins (exome sequencing) is now possible, and will lead to the elucidation of the etiology of a number of human diseases in the next few years. High-throughput, high-density sequencing using microarray technology potentially offers the option of obtaining rapid, accurate, and relatively inexpensive sequence of large portions of the genome. One such technique is oligo-hybridization sequencing, which relies on the differential hybridization of target DNA to an array of oligonucleotide probes. This technique is ideally suited to the analysis of DNA from patients with defined disorders, such as disorders of sex development and retinal disease, but suffers from a relatively high false positive rate and failure to detect insertions and deletions.

It is often difficult to perform studies in humans, and so the generation of animal models may be valuable in understanding the etiology and pathogenesis of disease. A number of naturally occurring mouse models have led to the identification of corresponding candidate genes in humans, with mutations subsequently detected in human patients. More frequently, genes of interest are often deleted and lead to the generation of disease models.

In general, mouse models correlate well with human disease; however species-specific defects need to be taken into account. Additionally, the transgenic models could be used to manipulate a condition, with the potential for new therapies. The advent of conditional transgenesis has led to an exponential increase in our understanding of how the mutation of a single gene impacts on a single organ. Using technology such as inducible gene expression systems, the effect of switching on or switching off a gene at a particular stage in development can be determined.

Advances in genomics will also have a major impact on therapeutics. Micro RNAs (miRNA) are small non-coding RNAs that regulate gene expression by targeting mRNAs of protein coding genes or non-coding RNA transcripts. Micro RNAs also have an important role in developmental and physiological processes and can act as tumor suppressors or oncogenes in the ontogenesis of cancers. The use of small interfering RNA (siRNA) offers promise of novel therapies in a range of conditions, such as cystic fibrosis and Type II autosomal dominant IGHD. Elucidation of the genetic basis of disease also allows more direct targeting of therapy. For instance, children with permanent neonatal-onset diabetes mellitus (PNDM) due to mutations in SUR1 or KIR6.2 were previously treated with insulin but have now been shown to respond well to sulfonylureas, thereby allowing the cessation of insulin therapy.

Finally, we are now entering the era of pharmacogenetics when the response of an individual to various therapeutic agents may be determined by their genotype. For example, a polymorphism in the GH receptor that results in deletion of exon 3 may be associated with an improved response to GH. Thus the elucidation of the genetic basis of many disorders will aid their management, and permit the tailoring of therapy in individual patients.

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