Pedigree with 3 cases affected by a novel pathogenic DHFR gene mutation. (A) Pedigree of the index case in 1 branch and 2 additional cases diagnosed in another branch of the same family. Segregation studies showed full penetrance in the case of homozygosity, whereas heterozygous carriers had neither clinical symptoms nor any abnormality in the hematological and immunological parameters. Individuals with the year of birth indicated in the extended pedigree have been tested. Filled symbols indicate the 3 homozygous cases in the family (also marked by 1/1 as determined by WGS [whole-genome sequencing]). Carriership of the DHFR mutation is indicated by 0/1, as was confirmed by Sanger sequencing. (B) NGS demonstrating the homozygous mutation in DHFR (NM_000791.3; c.61G>A; p.Gly21Arg) at chromosome 5q14.1 [OMIM 126060]. (C) Absent activity of DHFR in EBV-transformed lymphoblasts from patient 3 vs control (measured in duplicate according to the exact methods as reported by Cario et al).²