identified in the ACMG 56 recommended gene
list (49) and additional GHS 20 genes for returnable secondary findings. We cross-referenced these
variants with the ClinVar dataset [updated
December 2015] restricting to those with a Pathogenic classification and a minor allele frequency
of less than 1% in the DiscovEHR population.
We also cross-referenced the variants with the
Human Gene Mutation Database [HGMD 2015.4]
restricting to DM-Disease causing mutations of
High-confidence variants only with a MAF <1%.
We compiled the list of returnable variants according to the published guidelines for the genes
where Expected Pathogenic (EP), comprising non-reported putative loss-of-function (pLoF), and/or
Known Pathogenic (KP) variants are recommended for clinically actionable return of results.
For a subset of sequenced samples, we applied
an orthogonal workflow for variant discovery,
adjudication of asserted pathogenicity, and development of clinical reports. We used a custom pipeline to annotate and prioritize clinically
important variants. This pipeline incorporates
standard annotations, including variant effect,
protein functional predictions, conservation, and
allele frequencies, along with public and private
databases of variants. Variants within the genes
of interest were filtered to extract all known
alleles of reported clinical relevance and rare,
novel and likely disruptive variants (e.g. frameshift, nonsense, splicing, etc). After potentially
important variants were identified, they were
subjected to manual assessment.
Manual assessments were performed as previously described (72), and were consistent with
the process developed through a national effort
to establish standardized clinical classification
criteria (50). Briefly, variants were assessed using
a comprehensive evidence review, which included published case-control, genetic and functional data as well as population frequency and
predictive assessment data. Classifications were
subsequently assigned according to a 5-tier system (pathogenic, likely pathogenic, uncertain
significance, likely benign, benign). For autosomal dominant disorders, reported variants were
limited to those classified as likely pathogenic or
pathogenic for the diseases of interest. For autosomal recessive and X-linked disorders, (i.e.
MUTYH-associated polyposis), likely pathogenic
or pathogenic variants were only reported when
present in a homozygous, compound heterozygous, or hemizygous state. All reported variants
were confirmed via Sanger sequencing.
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