Unbiased phenotype and genotype matching maximizes gene discovery and diagnostic yield.

PURPOSE: Widespread application of next-generation sequencing, combined with data exchange platforms, has provided molecular diagnoses for countless families. To maximize diagnostic yield, we implemented an unbiased semi-automated genematching algorithm based on genotype and phenotype matching. METHODS: Rare homozygous variants identified in 2 or more affected individuals, but not in healthy individuals, were extracted from our local database of ∼12,000 exomes. Phenotype similarity scores (PSS), based on human phenotype ontology terms, were assigned to each pair of individuals matched at the genotype level using HPOsim. RESULTS: 33,792 genotype-matched pairs were discovered, representing variants in 7567 unique genes. There was an enrichment of PSS ≥0.1 among pathogenic/likely pathogenic variant-level pairs (94.3% in pathogenic/likely pathogenic variant-level matches vs 34.75% in all matches). We highlighted founder or region-specific variants as an internal positive control and proceeded to identify candidate disease genes. Variant-level matches were particularly helpful in cases involving inframe indels and splice region variants beyond the canonical splice sites, which may otherwise have been disregarded, allowing for detection of candidate disease genes, such as KAT2A, RPAIN, and LAMP3. CONCLUSION: Semi-automated genotype matching combined with PSS is a powerful tool to resolve variants of uncertain significance and to identify candidate disease genes.

Exome sequencing for structurally normal fetuses-yields and ethical issues.

The yield of chromosomal microarray analysis (CMA) is well established in structurally normal fetuses (0.4-1.4%). We aimed to determine the incremental yield of exome sequencing (ES) in this population. From February 2017 to April 2022, 1,526 fetuses were subjected to ES; 482 of them were structurally normal (31.6%). Only pathogenic and likely pathogenic (P/LP) variants, per the American College of Medical Genetics and Genomics (ACMG) classification, were reported. Additionally, ACMG secondary findings relevant to childhood were reported. Four fetuses (4/482; 0.8%) had P/LP variants indicating a moderate to severe disease in ATP7B, NR2E3, SPRED1 and FGFR3, causing Wilson disease, Enhanced S-cone syndrome, Legius and Muenke syndromes, respectively. Two fetuses had secondary findings, in RET and DSP. Our data suggest that offering only CMA for structurally normal fetuses may provide false reassurance. Prenatal ES mandates restrictive analysis and careful management combined with pre and post-test genetic counseling.

Parental exome analysis identifies shared carrier status for a second recessive disorder in couples with an affected child.

Consanguinity, commonplace in many regions around the globe, is associated with an increased risk of autosomal recessive (AR) genetic disorders. Consequently, consanguineous couples undergoing preimplantation genetic diagnosis (PGD) for one Mendelian disorder may be at increased risk for a child with a second, unrelated AR genetic disorder. We examined the yield of exome analysis for carrier screening of additional AR disorders, beyond the primary diagnosis, amongst consanguineous vs. non-consanguineous populations. Parental samples from trio exomes of 102 consanguineous families and 105 non-consanguineous controls were evaluated for shared carrier status, after disregarding the primary molecular diagnosis. Results were sub-classified according to disease severity. Secondary shared carrier status for pathogenic and likely pathogenic variants leading to AR disorders of moderate to profound severity was identified in 10/102 (9.8%) consanguineous couples, as compared to 1/105 (0.95%) non-consanguineous couples (χ2 = 8.0565, p value < 0.005). Higher inbreeding coefficient values, calculated from individual exomes, correlated with secondary shared carrier status for diseases of moderate to profound severity (r = 0.17, p value < 0.0125). Our results indicate that consanguineous couples undergoing PGD are at increased risk for a second genetic disease of moderate to profound severity. This study represents an underestimate of the rate of secondary shared carrier status due to inability to detect deep intronic variants, no assessment of copy number variants, and false negative results stemming from stringent variant interpretation. False positive results may result from inaccuracies in public databases. Additional studies in consanguineous populations will determine whether exome-based carrier screening should be recommended to all couples undergoing PGD.