Jos Jansen

3 71 TMEM199 Deficiency is characterized by elevated liverenzymes, hypercholesterolemia and abnormal glycosylation For individual F2-II2, standard filtering of the whole exome sequencing data indicated a heterozygous variant in the splice acceptor site of TMEM199 exon 4 (c.376-1G > A) (Figure 1). The raw exome-sequencing data revealed an uncalled heterozygous missense variant in exon 1 (c.40G > C [p.Ala14Pro]). Sequencing of parental DNA showed heterozygosity for the c.40G > C mutation in the father and heterozygosity for the c.376-1G > A mutation in the mother. A healthy sister did not show any of these mutations (Figure 2A). To further study the effect of the splice site mutation, we extracted mRNA from skin fibroblasts, synthesized cDNA, and sequenced it by Sanger sequencing. The resulting cDNA sequence showed the c.40G > C variant in homozygous form (Figure 2B), suggesting nonsense mediated decay of mRNA transcribed from the allele with the splice site mutation. Splice site prediction software (Alamut Visual v.2.5.1, Interactive Biosoftware) predicted the mutation to induce a stop codon 14 positions downstream by skipping one nucleotide (Figure 2B). Sanger sequencing of TMEM199 in a cohort of individuals with genetically unsolved Golgi glycosylation disorders revealed an additional individual in family F3 with a homozygous missense mutation in TMEM199 exon 1 (c.92G > C [p.Arg31Pro]). This variant was heterozygous in both parents (Figures 1 and 2A). Western blot analysis of TMEM199 was performed in fibroblasts of affected individuals from families F1 and F2, and reduced protein levels were observed (Figure 2C, left). Specificity was demonstrated by detection of TMEM199-V5 with an anti-TMEM199 antibody after overexpression of a TMEM199-V5 construct in HeLa cells, showing a slightly higher molecular weight than endogenous TMEM199 (Figure 2C, right). TMEM199 (UCSC Genome Browser [GRCh37/hg19], chr17:26,684,687– 26,689,089) contains six exons and encodes a protein of 208 amino acids, which includes a conserved Vma12 domain (Pfam: PF11712) at the C terminus. The Vma12 domain is widespread among eukaryotes, including Arabidopsis thaliana, and includes two transmembrane helices as predicted by the CCTOP server (see Figure 1 and Web Resources). Interestingly, three of four mutations alter residues in the N terminus, which seems to be conserved only in higher eukaryotes. The missense mutations were variably predicted to be pathogenic by different prediction programs (Table 1). The Exome Aggregation Consortium (ExAC, see Web Resources) database showed an absence of all variants except the c.92G > C variant, which had a very low frequency (5.002e-05).