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Apr 1, 2014 (Vol. 34, No. 7)

Clinical Genomics Transcends Sequencing

  • To establish a link between a specific disease and a genetic abnormality, researchers must first obtain tissue samples from affected patients. Nucleic acids are then isolated and sequenced.

    Then, the data obtained can be used to identify susceptibility loci in families where sequence variants such as single nucleotide polymorphisms (SNPs) or copy number variants (CNVs) are co-inherited with the disease. The data must then be validated by comparison with sequences from unrelated individuals who have the same disease, as well as reference genomes from different populations, before a relationship can be established.

    Although sequence variants are essential for understanding the genetic basis of diseases, they represent marker sequences, not actual mutations. Only about 1.5% of the human genome contains sequences that code for proteins. Thus, identifying actual mutations that affect protein function resulting in a pathogenic phenotype requires the sequencing and analysis of many genomes from unrelated individuals.

    Both whole-genome sequencing (WGS) and exome sequencing (sequencing the expressed regions of the genome) make it possible to identify mutations in a relatively short time through next-generation sequencing (NGS) technologies. In addition, data from genome-wide expression, in vitro, and in vivo studies provide a framework for assessing the relevance of mutations and developing panels for targeted genetic diagnostics.

    The rapid evolution of NGS technology has made it possible to sequence genomes from individual patients in the clinic and to use this information to both identify the genetic causes of disease and also determine the best course of treatment, based on how patients with a given genotype respond to drugs or surgery.

  • NGS versus Classic Sequencing

    WGS, exome sequencing, and gene-panel-based resequencing are relatively new. Once DNA sequencing became possible in the 1970s, so did its potential as a clinical diagnostic tool. For example, automated sequencing enabled the first standardized diagnostic tests, such as targeted sequencing for BRCA1 mutations in breast cancer patients and their family members. Classic (Sanger) sequencing-based tests remain the diagnostic standard for a number of diseases and gene-testing panels today.

    Jan Jongbloed, Ph.D., a laboratory specialist at the University of Groningen in the Netherlands, has compared the efficacy of gene-panel-based resequencing methods with established Sanger sequencing methods for the identification of inherited cardiomyopathy mutations. He found that these methods provide sequencing results of comparable quality. He also noticed, however, that NGS methods offer certain advantages.

    According to Dr. Jongbloed, the primary advantage of NGS in the clinic is that more genes can be analyzed: “NGS methods provide greater depth than Sanger sequencing and allow us to analyze more genes in the clinic. Sanger depends on amplifying a region first using specific primers, an approach that has its disadvantages. NGS methods do not require this type of amplification.”

    “Also, with Sanger, we were only able to analyze three or four genes at the same time compared with 55 genes using NGS methods,” adds Dr. Jongbloed. “This also allowed us to identify new mutations where we were unable to do so before, such as in the titin gene.”

  • WGS versus Exome Sequencing

    Dr. Jongbloed’s clinic is one of eight in the Netherlands that provides comprehensive sequencing services including WGS, exome analysis, and gene-panel testing. About 75 patients are evaluated there each month; however, WGS is still relatively rare.

    “We do about one entire genome per month,” Dr. Jongbloed says. “While we can sequence a lot of genes at the same time, some regions are more difficult to sequence than others, such as those with high GC content, and repetitive elements in certain sequences make them difficult to map.” Another issue he and other laboratory specialists face is determining whether certain mutations are indeed pathogenic. “For this, the only solution is to find other affected family members with the same mutation, which can be difficult for a rare disease.”

    Dr. Jongbloed believes that WGS, exome sequencing, gene-panel-based resequencing, and Sanger all currently have their place in the clinic. “WGS is actually faster because we don’t need to enrich for the genes as we do for exome analysis. Also, WGS should be the method of choice for neonatal screening “where we don’t know what’s going on.”

    Regarding exome sequencing, he states, “It isn’t necessarily faster, but it is cheaper. For now, we should focus on exomes because it is easier to understand the consequences.” Sanger sequencing will always have its place in the clinic, he thinks: “It is well established and [Sanger] panels already exist for many diseases…we will always need to use it to validate NGS results and to look in other family members.”

    Dr. Jongbloed would like to see lower costs for NGS technologies and more collaboration in the field through a centralized facility. He believes that other applications, such as Ion Torrent (Thermo Fisher Scientific) sequencing, will help reduce the cost of NGS itself and ultimately replace Sanger.

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