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Multiplexed Reference Materials as Controls for Cardiomyopathy Diagnostic Next-Generation Sequencing

Posted by Dale Yuzuki on Dec 13, 2016 12:00:00 AM

Hypertrophic Cardiomyopathy (HCM) is a disease where the heart muscle is enlarged and a significant cause of sudden cardiac death, and is frequently asymptomatic.  HCM is commonly caused by a mutation in one of nine heart muscle genes that comprise the sarcomere, and occurs at a prevalence of about 1 in 500 in the general population. HCM is the leading cause of cardiac death in young athletes in the United States.

Clinical genetic testing for mutations in the HCM-related genes has been ongoing for over a decade;  the GeneTest.org database reveals 105 laboratories offering some version of genetic testing.  While knowledge of prevalent pathogenic variants are available, the majority of variants remain private (that is, unpublished and not widely available). The move to NGS-based gene panels for HCM testing has lead to new challenges for test development, validation and routine quality control due to the inherent scarcity of samples, the cost of including numerous single mutations from these individual samples, and the lack of these materials for laboratories without a long history of testing.

This paper uses expert-designed multiplexed controls that spike biosynthetic fragments, containing  10 common pathogenic or technically challenging variants from the top HCM genes, into human genomic DNA. The multiplexed variants are spiked into genomic DNA GM24385, a highly characterized reference material from The Genome-in-a-Bottle Consortium.  GM24385 reference material enables laboratories to validate SNPs throughout other areas of the genome and establishes foundation material for inter-laboratory comparisons and peer review. This panel was expertly designed by a group at Laboratory of Molecular Medicine, Harvard Partners Healthcare at the Massachusetts General Hospital (Boston, MA). The allelic fraction was adjusted as a range (45%, 50% and 55%) to represent ideal and realistic fractional percentages for heterozygous germline variants, and the results were compared across NGS-based test data from patient samples with the identical causative variants.

Alternative approaches  for quality control, such as in silico-mutated FASTQ files or CRISPR-Cas9 mutated cell lines, which have their utility, are also discussed; however this article makes a strong case for biosynthetic DNA for its ease of engineering, wide multiplexing ability and blending into precise allelic ratios.

The laboratory that performed this work at Partners HealthCare Personalized Medicine also point out the consistent results across both SNVs and indels between the biosynthetic approach and patient-derived DNA, calling it ‘essentially indistinguishable’. The group notes that with a second lot of material (provided by SeraCare Life Sciences), there was favorable lot-to-lot reproducibility.

As far as future direction, a higher degree of multiplexing is being considered (the 10 mutations chosen for this pilot study cover about 30% of all pathogenic HCM mutations), as well as the inclusion of a larger number of technically challenging indels (one 25-bp indel reported lower-than-expected allele ratio due to a known problem of NGS analysis pipelines for accurate mapping and calling of large indels).

Do you perform routine NGS-based analysis of Hypertrophic Cardiomyopathy? Could a reference material like this meet an important unmet need in your laboratory?  Click here to find out more. 


Download Now: Multiplexed Reference Materials as Controls for Diagnostic Next-Generation Sequencing



  1. Kudalkar EM, Almontashiri NA, Huang C, Anekella B, Bowser M, Hynes E, Garlick R, Funke BH. Multiplexed Reference Materials as Controls for Diagnostic Next-Generation Sequencing: A Pilot Investigating Applications for Hypertrophic Cardiomyopathy. J Mol Diagn. 2016 Nov;18(6):882-889. doi:10.1016/j.jmoldx.2016.07.005. 



Topics: clinical genomics, NGS