Jos Jansen

30 Chapter 2 This method provides additional glycan information, such as loss of galactose. Here, we report the identification of pathogenic mutations in coiled-coil domain containing 115 ( CCDC115 [GenBank: NM_032357.3], UCSC Genome Browser [GRCh37/hg19], chr2:131,095,506–131,099,956) in five unrelated families with abnormal N- and mucin type O-glycosylation, suggestive of a Golgi homeostasis defect. Materials and methods Participating Individuals Blood and, if obtained, fibroblasts of participating individuals were sent to the Radboud University Medical Center, Translational Metabolic Laboratory, for CDG diagnostics, based on clinical suspicion for an inborn error of metabolism. All participating affected individuals or their legal representatives gave informed consent for exome sequencing. Tissue and samples were obtained in accordance with the Declaration of Helsinki. Exome Sequencing and Interpretation Next- generation sequencing and analysis were performed as described earlier.(15) The SureSelect Human All Exon 50 Mb Kit (v.4, Agilent) was used for exome enrichment, covering ~21,000 genes. The exome library was sequenced on a 5500xl SOLiD sequencer (Life Technologies). Color space reads were iteratively mapped to the hg19 reference genome with the SOLiD LifeScope software v.2.1. We used our in-house annotation pipeline for annotation of called variants and indels. (16) Variants were excluded based on a frequency of > 0.2% in our inhouse database of > 1,300 exomes. Also, synonymous variants, deep intronic variants, and variants in UTRs were excluded. Quality criteria were applied and included variants called more than five times and with variation of more than 20% for heterozygous variants and 80% for homozygous variants. Bioinformatics Amino acid sequences of human CCDC115 and homologs of other species were aligned and visualized with Jalview v.2.8 (see Web Resources). The following accession numbers were used for the alignment in Figure 1B: GenBank: NP_115733.2 (H. sapiens); GenBank: NP_081435.1 (M. musculus); GenBank: NP_001013313. 1 (D. rerio); GenBank: NP_649550.1 (D. melanogaster); GenBank: NP_011927.1 (S. cerevisiae S288c); GenBank: NP_173500.1 (A. thaliana).