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NGS Cardiovascular Diagnostics: Long-QT Genes Sequenced – A Potential Replacement for Molecular Pathology

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cardiovascular Pharmacogenomics, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Genomic Testing: Methodology for Diagnosis, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Population Health Management, Genetics & Pharmaceutical, Technology Transfer: Biotech and Pharmaceutical, tagged , , , , , , , , , on October 1, 2012| 3 Comments »

 

Reporter: Aviva Lev-Ari, PhD, RN

J Cardiovasc Transl Res. 2012 Sep 7. [Epub ahead of print]

Next Generation Diagnostics in Inherited Arrhythmia Syndromes : A Comparison of Two Approaches.

Ware JS, John S, Roberts AM, Buchan R, Gong S, Peters NS, Robinson DO, Lucassen A, Behr ER, Cook SA.

Source

MRC Clinical Sciences Centre, Imperial College London, London, UK, j.ware@imperial.ac.uk.

Abstract

Next-generation sequencing (NGS) provides an unprecedented opportunity to assess genetic variation underlying human disease. Here, we compared two NGS approaches for diagnostic sequencing in inherited arrhythmia syndromes. We compared PCR-based target enrichment and long-read sequencing (PCR-LR) with in-solution hybridization-based enrichment and short-read sequencing (Hyb-SR). The PCR-LR assay comprehensively assessed five long-QT genes routinely sequenced in diagnostic laboratories and “hot spots” in RYR2. The Hyb-SR assay targeted 49 genes, including those in the PCR-LR assay. The sensitivity for detection of control variants did not differ between approaches. In both assays, the major limitation was upstream target capture, particular in regions of extreme GC content. These initial experiences with NGS cardiovascular diagnostics achieved up to 89 % sensitivity at a fraction of current costs. In the next iteration of these assays we anticipate sensitivity above 97 % for all LQT genes. NGS assays will soon replace conventional sequencing for LQT diagnostics and molecular pathology.

PMID: 22956155 [PubMed]
Source: 
http://www.ncbi.nlm.nih.gov/pubmed/22956155

Researchers in the UK have compared a PCR-based and a capture hybridization-based assay for sequencing panels of inherited cardiovascular disease genes and have found both to be suitable for diagnostics in principle, though their sensitivity needs to be optimized.

According to James Ware, a clinical lecturer at Imperial College London, the purpose of the study, published online this month in the Journal of Cardiovascular Translational Research, was to evaluate different approaches for sequencing cardiovascular disease genes, both for molecular diagnosis and for large-scale resequencing research studies.

His group, in the National Institute for Health Research Royal Brompton Cardiovascular Biomedical Research Unit, is interested in a range of inherited heart disease types, including cardiomyopathies and inherited arrhythmia syndromes such as long QT syndrome.

For their study, they compared two next-gen sequencing assays: a PCR-based approach that uses Fluidigm’s Access Array to amplify 96 amplicons in five LQT genes and one other gene, followed by sequencing on the 454 GS Junior; and an in-solution hybridization approach that uses Agilent’s SureSelect to target 49 inherited arrhythmia genes and sequences them on Life Technologies’ SOLiD 4.

The study focused on the sensitivity of the assays, or how well they were able to capture their intended targets, rather than their specificity, or their ability to avoid false positives.

Ware said that at the time of the study, PCR and in-solution capture were the two main target selection methods available. The researchers are still using both approaches but are now employing “a wide range of sequencers” from various providers for both types of assays, including Illumina instruments and Life Tech’s Ion Torrent.

For their comparison, they analyzed 48 samples, of which they sequenced 33 with both approaches and 15 using either one or the other.

The samples included 19 known variants in three disease genes, of which the hybridization-SOLiD method detected 17 and the PCR-454 method 14. Undetected variants were generally in areas that were not well covered, either due to a failure in enrichment, sequencing, or because the alignment was not unique. One variant that was missed by both approaches fell in a very GC-rich region.

Consumables costs for both assays were considerably lower than with Sanger sequencing: While sequencing five genes by Sanger costs more than $700 in consumables, the five-gene PCR/454 assay cost about $55 and the 49-gene hybridization/SOLiD assay cost about $200, according to the study.

Turnaround time is the shortest for Sanger sequencing, which, according to the study, can be done in one day for five genes and 17 samples, not including sample prep. The PCR/454 assay takes about two days for target enrichment and sequencing 48 samples, and the hybridization/SOLiD assay takes about two weeks for sequencing alone, they wrote.

Overall, Ware said, both sequencing approaches performed “reasonably well” and are significantly cheaper than Sanger sequencing. He said that in the UK, molecular diagnosis for inherited cardiovascular disease has traditionally been performed by Sanger, at a cost of approximately £500 to £1,000 ($800 to $1,600) for several genes involved in a clinical condition. However, for cost reasons, not all relevant genes are usually sequenced.

Target selection was the performance-limiting step for both approaches, a result the researchers expected. “It sounds obvious, but not all genes are equally easy to target,” Ware said. For example, in the hybridization assay, the overall target coverage was about 98 percent, but for some genes, it was only 80 percent or 90 percent. The two most important genes in long QT syndrome, KCNQ1 and KCNH2, “proved to be the hardest to sequence.”

Thus, for diagnostic use of NGS gene panels, “it’s important to know not just how the system performs overall but really how it’s performing for the specific genes you’re interested in,” he said.

To use either approach in diagnostics, the target selection step would need to be optimized. Ware’s team has already improved both assays and is now trying them in a number of fully Sanger-sequenced samples to study both sensitivity and specificity.

Longer term, the sensitivity of next-gen sequencing could approach that of Sanger sequencing, he said. And even if it does not reach 100 percent, because NGS approaches can target so many more genes, “maybe you can afford a very slight tradeoff in the per-gene sensitivity if the overall diagnostic sensitivity of the panel goes up,” he said. “At the moment, because we don’t have that much experience in sequencing the less-common genes, we don’t exactly know where that tradeoff lies.” In addition, any gaps could be filled by Sanger sequencing, while the test would probably still be cost effective.

Each approach also has some features that make it more suitable for certain applications. The PCR-based method has a fast turnaround and an “extremely user-friendly workflow,” Ware said, but it can only accommodate a small number of genes at the moment. His team also found it to be easier to optimize and improve. Thus, in the short term, PCR and sequencing “is probably closer to providing a diagnostic solution,” he said, especially for conditions where only a few genes are causative.

The hybridization-based approach, on the other hand, has much greater capacity, and there are advantages in “having a single assay that covers everything,” he said. It might also be possible to detect copy number variants using this approach, but not the more limited PCR method, he added.

Ware and his colleagues are currently using the hybridization approach to study a large panel of genes in 2,000 well-phenotyped volunteers, both healthy individuals and heart disease patients.

They have also started to use the hybridization method to sequence the TTN gene, truncating mutations in which were recently found to be a common cause of dilated cardiomyopathy. They are running the TTN test routinely for patients consented for research diagnostic testing that is not available anywhere else. Because this gene is so large, it is “completely impractical to be sequenced by conventional Sanger,” Ware said.

Julia Karow tracks trends in next-generation sequencing for research and clinical applications for GenomeWeb’s In Sequenceand Clinical Sequencing News. E-mail her here or follow her GenomeWeb Twitter accounts at @InSequence and@ClinSeqNews.

 

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