Following our initial linkage study (14
), we performed whole-genome genotyping of the affected individuals and identified a shared homozygous segment that spans >2 Mb on chromosome 19p13 (Chr 19: 13 610 401–15 645 116 bp in hg Build 36.3). To facilitate the discovery of the causal mutation and gene, we included both obligate carrier parents in an exome sequencing study. We identified 71 variants (33 missense, 37 synonymous, 1 non-protein coding) in the critical region in the parents. Given the pattern of homozygosity in the critical region in Family G, we assumed that the NSMR in this family was due to a fully penetrant autosomal recessive mutation (14
). Therefore, we considered 18 of the 71 variants that were heterozygous in both parents as candidates for the NSMR mutation (Supplementary Material, Table S1
). Seventeen of these 18 variants were present in the dbSNP (v131) and were, therefore, further excluded as NSMR mutations (Supplementary Material, Table S2
). The remaining novel mutation occurred in exon 8 of the TECR
(trans-2,3-enoyl-CoA reductase) gene (Chr 19: 14 536 653 in hg Build 36.6), causing a Pro to Leu substitution at amino acid 182 in the TECR protein (Fig. A). Using Sanger sequencing, we confirmed that the mutation in the TECR
gene segregated with NSMR in Family G: the non-reference allele was homozygous in the five affected siblings, heterozygous in the parents and seven unaffected siblings and absent in one unaffected sibling (Fig. B).
Figure 2. (A) Schematic representation of the TECR primary transcript, exons and processed transcript. The location of the mutation affecting Pro182 in exon 8 of the TECR gene is indicated with a red line. (B) Results of sequencing studies. The nucleotide sequences (more ...)
Additional genotyping of this mutation in 1523 individuals that belong to the same religious community as Family G revealed no homozygotes other than those in the affected sibship. Overall, there were 109 carriers of the TECR Leu182 mutation (including those in Family G), yielding a carrier frequency of 7.1% and mutation frequency of 3.9% in this population. The observed genotype distributions fit with the Hardy–Weinberg expectations (P > 0.10). Despite the high carrier frequency of the TECR mutation (1 in 14), none of the other carriers other than the parents of Family G were married to each other. Ninety-seven of 109 carriers were also genotyped with the Affymetrix 500k or 6.0 SNP arrays. In those individuals, the mutation was present on the same haplotype that was homozygous in the affected individuals in Family G, indicating a single ancestral founder haplotype carrying the mutation in this population (Fig. ). The combined segregation and population data suggest that the Pro182Leu mutation in the TECR gene is the NSMR causal mutation in Family G.
Figure 3. Haplotype analysis of the Pro182Leu mutation; the arrowhead shows the position of the mutation. The x-axis shows the 141 SNPs that define the 2.2 Mb homozygous segment in the affected siblings; individuals representing each genotype are shown in each (more ...)
We next applied bioinformatic approaches to predict the potential effect of the Pro182Leu mutation. First, we used ClustalW algorithm (15
) to align the human TECR protein sequence with the amino acid sequences of the orthologous proteins in six other species. The proline at amino acid 182 was highly conserved, being present in all species as distantly related to humans as zebrafish and Xenopus laevis
(Fig. C). We then used two in silico
methods: Polymorphism Phenotyping (PolyPhen) (16
) and Sorting Intolerant from Tolerant (SIFT) (17
). The effect of Pro182Leu mutation was predicted to be ‘probably damaging’ by PolyPhen (PSIC score difference 2.97) and ‘not tolerated’ by SIFT with a substitution score of 0.01. Thus, two in silico
approaches predict that the substitution of a leucine for a highly conserved proline at amino acid 182 in the TECR protein is a deleterious/damaging mutation that very likely alters protein function and, therefore, downstream phenotypes.