Jos Jansen

68 Chapter 3 DNA of individuals F1-II2, F1-II3 and F2-II2 was isolated from fibroblasts and subjected to whole-exome sequencing as described.(18) All individuals, or their legal representatives, participating in this study provided informed consent, and all biological materials were obtained in accordance with the Declaration of Helsinki. Our in-house bioinformatics pipeline was used to annotate called variants and indels.(19) A frequency of > 0.2% in an in-house database of over 1,300 exomes was set for exclusion of variants. We searched for non-synonymous variants located in exons and canonical splice sites. As quality criteria, we only included variants called more than five times and with a frequency of more than 20% for heterozygous variants and 80% for homozygous variants. According to a model of autosomal-recessive inheritance, no obvious candidate genes could be identified. For brothers F1-II2 and F1-II3 from family F1, previous homozygosity mapping and targeted chip-based gene sequencing of all genes in the largest homozygous intervals did not reveal a clear candidate gene either. Inspection of the raw exome-sequencing data of both brothers was carried out with the Integrative Genomics Viewer (IGV, v.2.3.14, Broad Institute). Zooming in on all TMEM199 exons (GenBank: NM_152464.2) revealed a missense mutation, c.20C > A (p.Ala7Glu), in exon 1 (Figure 1). This variant had a sequence depth of three reads in one sibling and no coverage for the other affected sibling. Sanger sequencing confirmed the missense mutation to be homozygous in DNA from both affected siblings and heterozygous in both parents. The mutation was absent from a healthy sister, thereby showing complete segregation of the mutation in the family, in agreement with autosomal-recessive inheritance (Figure 2A).