Identification and characteristics of pathogenic variants in RASGRP2. (A) BeviMed inference analysis applied to rare, nonsynonymous RASGRP2 variants observed in all index cases from the NIHR BioResource. Cases were designated as having the indicator phenotype of CalDAG-GEFI deficiency if they had HPO terms indicating bleeding and reduced light transmission aggregation responses to ≥3 activating agonists. The indicator phenotype was present in 119 index cases and was absent in 5982 index cases. The posterior probability of the association model was 1 (prior was 0.1), and the posterior probability of recessive inheritance was 1 (prior was 0.5). The RASGRP2 exons are represented by gray blocks. The bar chart above shows the marginal posterior probabilities of pathogenicity for individual variants observed in all NIHR BioResource index cases, conditional on an association under a recessive mode of inheritance. The bar chart beneath indicates whether the variant was observed in an index case with (pink) or without (blue) the indicator phenotype and whether it was present as a heterozygous (het.) or homozygous (hom.) allele. Variants observed in index cases as compound heterozygous alleles are linked. (B) The characteristics of the 11 likely pathogenic RASGRP2 variants across the NIHR BioResource (NBR) and ThromboGenomics (TG) collections. Variants were annotated against the canonical transcript ENST00000354024 by using the Variant Effect Predictor (VEP).¹⁴  Population allelic frequencies are derived from the Exome Aggregation Consortium (ExAC).¹⁵  The likely pathogenicity of the variants is expressed as the Combined Annotation Dependent Depletion (CADD) score,¹⁷  and the percentage conservation as the proportion of CalDAG-GEFI orthologs in 9 species that have the same amino acid as human CalDAG-GEFI. (C) Localization and predicted consequence of the likely pathogenic RASGRP2 variants identified in the NIHR BioResource and ThromboGenomics collections (below the protein diagram) and the previously reported variants^9-11  (above the diagram). The protein domains indicated are the Ras exchange motif (REM), catalytic domain (CDC25), calcium-binding EF hands (EF) and diacylglycerol-binding domain (C1). The exploded view shows the predicted consequence of the 11:64507638 G>C variant identified as a homozygous allele in ThromboGenomics case K II.1. Bioinformatic analysis of the variant sequence predicts preferential use of an alternative exonic splice acceptor, resulting in codon deletion, frameshift, and a stop gain at codon 125.