Rare missense variants in IKZF5 are associated with thrombocytopenia. (A) BeviMed was applied gene by gene to infer associations between the genotypes of filtered rare variants and a case/control grouping defined by isolated thrombocytopenia. The posterior probabilities for genetic association inferred by BeviMed exceeding 0.4 are shown. The dots representing posterior probabilities for genes previously known to be implicated in hereditary thrombocytopenia are highlighted in blue. Genes not previously associated with thrombocytopenia are in gray. (B) Evolutionary conservation scores with respect to 66 protein sequences obtained using multiple sequence alignment within ConSurf,³¹  and the corresponding 95% confidence intervals, for amino acids (AAs) 1 through 419 of IKZF5, normalized to have a mean of 0 and standard deviation of 1. The filled/empty points indicate presence/absence of missense variants in GnomAD altering the AAs. Only 3 of the 141 456 individuals in GnomAD harbor a missense variant affecting a residue in the N-terminal Znfs with a normalized conservation score <−1 (AAs 102, 111, and 123). The blue boxes indicate the locations of the Znfs. The variants in cases with thrombocytopenia affecting the 5 conserved AAs have been followed up in cosegregation studies, while S200G, which affects a non-conserved AA, has not been followed up. (C) Sex-stratified histograms of PLT obtained using a Sysmex hematology analyzer from 48 345 blood donors from the Efficiency and Safety of Varying the Frequency of Whole Blood Donation study³²  after adjustment for technical artifacts. The red arrows superimposed on the histograms indicate the sex of and values for cases carrying 1 of the 5 missense rare variants. The green arrows indicate the sex of and values for relatives homozygous for the corresponding WT allele. Individual C II.3, marked with an asterisk, had thrombocytopenia as a child (with an unknown PLT) and was subsequently splenectomized, likely explaining why his PLT increased to 253 × 10⁹/L as an adult. (D) The 5 pedigrees recalled for cosegregation. Male (square) and female (circle) individuals are shown in black or white depending on whether they are affected or unaffected, respectively, whereas individuals with an unknown PLT are shown in gray. Genotyping results obtained by WGS ^(G), WES ^(E), or Sanger sequencing ^(S) are shown in terms of their predicted amino acid substitutions underneath the genotyped individuals. Crl, control; WT, homozygous for the reference allele. C III.4 carries the G134E variant but has a PLT of 184.