These data were combined through CATCH,37 and after analysis of the results, three variants remained for further evaluation (Table S1) in genes PIGG (c.928C>T [p.Gln310∗] [GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_001127178.2","term_id":"573459749","term_text":"NM_001127178.2"}}NM_001127178.2]), COQ5 (MIM: 616359; c.319G>A [p.Gly107Arg] [GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_032314.3","term_id":"116063535","term_text":"NM_032314.3"}}NM_032314.3]), and RGS12 (MIM: 602512; c.188A>T [p.Gln63Leu] [GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_002926.3","term_id":"108773791","term_text":"NM_002926.3"}}NM_002926.3]). All variants were confirmed by Sanger sequencing, and family segregation confirmed that the affected individuals, as well as the unaffected siblings, are heterozygous for the variants (Figure 3A). Variants in PIGG and COQ5 are not present in publically available databases, whereas the variant in RGS12 has a cumulative frequency of 4.95e−5 in the Exome Aggregation Consortium (ExAC) Browser.43 The variant in PIGG is nonsense, whereas the other two are missense.Open in a separate windowFigure 3Identified Variants(A) Sequencing chromatograms of the identified variant in family EG. The parents and unaffected siblings are heterozygous, whereas the affected individuals are homozygous for the c.928C>T mutation (noted by the black arrow).(B) Sequencing chromatograms of the identified variant in family JP. The father has the heterozygous c.2005C>T variant, but the mother does not (noted by the black arrow).(C) XHMM analysis using WES data of individual JP01. A microdeletion involving PIGG (chr4: 60,226–2,452,836) is indicated.(D) qPCR analysis of exons 7 and 12 of the PIGG genome from individual JP01 and her parents shows a heterozygous PIGG deletion in the affected individual, but not in her mother, suggesting that the deletion occurred de novo on the maternal chromosome.(E) Sequencing chromatograms of the identified variant in family PK are noted by the blue transparent boxes. The parents are heterozygous, and the affected individuals are homozygous for the c.2261+1G>C mutation.PIGG was considered the most prominent candidate because the variant identified causes a clear loss of function. A recent inquiry of the ExAC Browser verified that no homozygous loss-of-function variants have been reported and that the most common loss-of-function variant, c.1515G>A (p.Trp505∗) (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_001127178.2","term_id":"573459749","term_text":"NM_001127178.2"}}NM_001127178.2), has a minor allele frequency (MAF) of 0.0012 in the European population. This translates to a frequency of less than 1 in 600,000 individuals, compatible with a rare autosomal-recessive disorder.