Carrier Testing for Severe Childhood Recessive Diseases by Next-Generation Sequencing

Abstract

Of 7028 disorders with suspected Mendelian inheritance, 1139 are recessive and have an established molecular basis. Although individually uncommon, Mendelian diseases collectively account for ~20% of infant mortality and ~10% of pediatric hospitalizations. Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis. However, extension of preconception screening to most severe disease genes has hitherto been impractical. Here, we report a preconception carrier screen for 448 severe recessive childhood diseases. Rather than costly, complete sequencing of the human genome, 7717 regions from 437 target genes were enriched by hybrid capture or microdroplet polymerase chain reaction, sequenced by next-generation sequencing (NGS) to a depth of up to 2.7 gigabases, and assessed with stringent bioinformatic filters. At a resultant 160× average target coverage, 93% of nucleotides had at least 20× coverage, and mutation detection/genotyping had ~95% sensitivity and ~100% specificity for substitution, insertion/deletion, splicing, and gross deletion mutations and single-nucleotide polymorphisms. In 104 unrelated DNA samples, the average genomic carrier burden for severe pediatric recessive mutations was 2.8 and ranged from 0 to 7. The distribution of mutations among sequenced samples appeared random. Twenty-seven percent of mutations cited in the literature were found to be common polymorphisms or misannotated, underscoring the need for better mutation databases as part of a comprehensive carrier testing strategy. Given the magnitude of carrier burden and the lower cost of testing compared to treating these conditions, carrier screening by NGS made available to the general population may be an economical way to reduce the incidence of and ameliorate suffering associated with severe recessive childhood disorders.

Targeted next-generation sequencing as a comprehensive test for patients with and female carriers of DMD/BMD: a multi-population diagnostic study

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Navigating the current landscape of clinical genetic testing for inherited retinal dystrophies

“…42,[49][50][51][52][53][54][55] Many variants that have previously been reported as pathogenic in the medical literature (and thus are represented in databases used for molecular DNA analysis) are now known to have allele frequencies that make them very common occurrences in the general population, or they have been seen in the homozygous state in healthy controls. [56][57][58] Large polymorphic genes, such as the ABCA4 gene associated with Stargardt disease, arRP, autosomal-recessive CRD, and cone dystrophy, are likely to contain more variants that require scrutiny because some variants may have been previously erroneously asserted as being pathogenic. 59 When the appropriate level of evidence is not present to classify a variant as known/likely benign or deleterious, the laboratory must classify the variant as a "variant of unknown significance. "…”

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“…In 2011, an NGS study demonstrated the clinical and analytic validity of exome sequencing targeting a preselected region known to be associated with severe childhood autosomal recessive diseases, like Tay-Sachs disease and cystic fibrosis (Bell et al, 2011). Yin et al (2013) introduced a method of massively parallel sequencing for aneuploidy of blastocysts, reporting a 68.4% euploidy rate.…”