Targeting de novo loss of function variants in constrained disease genes improves diagnostic rates in the 100,000 Genomes Project

Whole genome sequencing was first offered clinically in the UK through the 100,000 Genomes Project (100KGP); however, data analysis was time and resource intensive with 3 million variants found per patient. Consequently, analysis was restricted to predefined gene panels associated with the patients phenotype. However, panels rely on clearly characterised phenotypes and risk missing diagnostic variants outside of the panel(s) applied. We propose a complementary method to rapidly identify diagnostic variants, including those missed by 100KGP methods. The Loss-of-function Observed/Expected Upper-bound Fraction (LOEUF) score quantifies gene constraint, with low scores correlated with haploinsufficiency. We applied DeNovoLOEUF, a filtering strategy to sequencing data from 13,949 rare disease trios in the 100KGP, by filtering for rare, de novo, single nucleotide loss-of-function variants in OMIM disease genes with a LOEUF score <0.2. We conducted our analysis prospectively in 2019 and compared our findings with the corresponding diagnostic reports as returned in 2019 and again in 2021. 324/336 (96%) of the variants identified through DeNovoLOEUF were classified as diagnostic or partially diagnostic. We identified 39 diagnoses that were "missed" by 100KGP standard analyses, which are now being returned to patients. We have demonstrated a highly specific and rapid method with a 96% positive predictive value that has good concordance with standard analysis, low false positive rate, and can identify additional diagnoses. Globally, as more patients are being offered genome sequencing, we anticipate that DeNovoLOEUF will rapidly identify new diagnoses and facilitate iterative analyses when new disease genes are discovered.