2012 has been a very exciting year for our laboratory with the launching of our first series of next generation sequencing (NGS)-based tests.  While the power of this technology is now well established, it has been very satisfying to experience it first hand and to see how it is changing what we can do in the diagnostic laboratory.  With the applications of NGS unraveling all around us at a dizzying pace it has been difficult not to get caught up in it all.  We have spent a fair amount of time upfront setting up a robust, efficient and accurate workflow and infrastructure for data analysis, which has paid off and made transition of this technology (with its several-fold increase in data generation) to clinical use relatively painless.  Thanks go to two talented individuals in the laboratory, a previous fellow and currently our new assistant director, Scott Topper, and our very own bioinformatician, Viswateja Nelakuditi. 

Our strategy has been to start with the development of NGS panels for neurodevelopmental disorders, building on the existing strengths and expertise of our laboratory.  To date, we have developed comprehensive gene panels for primary microcephaly, early infantile epileptic encephalopathy, lissencephaly and other brain abnormalities, Seckel syndrome and intellectual disability.  We seem to be observing an increase in the number of patient samples with positive findings, compared to our previous Sanger sequence-based tests, when a more limited number of genes could be analyzed.  For example, with our previous Sanger sequencing-based tier 2 primary microcephaly panel (not including the ASPM gene that accounts for approximately 40% of primary microcephaly patients performed as a tier 1 test), approximately 4% of samples tested were positive; with our more comprehensive NGS-based primary microcephaly test approximately 11% of samples tested have been positive, not including ASPM positive results.  While such an observation is not surprising and indeed to be expected it is nonetheless very satisfying to experience this for ourselves. 

Significant genetic heterogeneity exists for the vast majority of genetic disorders.  Even for rare orphan genetic conditions that are thought to be more ‘simple’ with regards to their genetic basis, more and more genes are being identified that underlie these conditions.  With NGS, as increased numbers of genes get analyzed in more and more patients with particular disease phenotypes, we will be able to gather data to more accurately determine the percent contribution of specific genes to disease.  For many genes in which disease association is limited to small sample size (often single families and specific ethnic groups) limited research studies have been performed.  The inclusion of such ‘low frequency’ genes in NGS panels will result in the study of larger numbers of patients for these genes and give a more accurate representation of the contribution of these genes to disease.  We are already seeing this happening.  Through our NGS-based microcephaly testing, we recently identified a Caucasian patient from a non-consanguineous family, with compound heterozygosity for mutations in the CDK5RAP2 gene, the first such patient described.  To date only a handful of patients with CDK5RAP2 mutations have been described and all have been from consanguineous families primarily of Pakistani origin.  This suggests that CDK5RAP2 mutations are not restricted to certain ethnic groups and may account for more microcephaly patients worldwide.  We expect more and more of such observations to occur with an increasing number of genes.

We are in the process of developing several more NGS-based gene panels for additional disease conditions and look forward to launching them in the coming year.  For each of our panels we are working with research groups and clinicians with expertise in the genetics of the different disorders to put together as meaningful and comprehensive a gene panel as possible.  As gene panels get larger to cover broader phenotypes, the question of whether whole exome sequencing may be more worthwhile arises.  The primary advantage of focused gene panels is complete sequence coverage of all the genes on the panel and therefore increased assay sensitivity compared to whole exome sequencing where sequence coverage of the genes of interest will remain incomplete.  However with whole exome sequencing there is the ability to analyze a broader range of genes, indeed all our genes, and for patients where the phenotype may not be so clear or where all previous testing has been negative this may be a better approach.  We therefore feel that there is utility for both comprehensive gene panel testing and whole exome sequencing for clinical purposes, the former in instances where a patient’s phenotype is more defined and the latter when this is less clear. 

Multiple efforts are ongoing at the national level, through the American College of Medical Genetics, National Institutes of Health, College of American Pathologists and others, to help with standardized, safer and more responsible implementation of NGS, including whole exome and whole genome sequencing, for clinical purposes.  There remain many challenges to clinical implementation of whole exome and/or whole genome sequencing and the development of guidelines will be helpful, as these technologies become the norm for clinical laboratories.  Like many other laboratories, we are in the process of evaluating whole exome sequencing for clinical testing purposes and it will be exciting to see how this unfolds.  2013 promises to be another exciting year for us as we generate new findings from our NGS tests, launch new NGS tests and implement clinical exome sequencing.