Jos Jansen

2 41 CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation from the paternal allele. This complete deletion of CCDC115 is in agreement with the severe phenotype of individual F5-II1. Sanger sequencing of additional individuals with unsolved Golgi glycosylation defects identified missense mutations in CCDC115 in individuals from two unrelated families: individual F3-II2with the same homozygous c.92T > Cmutation and siblings F4-II1 and F4-II2 with a homozygous missense mutation, c.31G > T, leading to a p.Asp11Tyr substitution (Figure 1A and Table 1). The p.Asp11Tyr substitution was also predicted to be pathogenic by SIFT, PolyPhen-2, and MutationTaster (Table S3). The allele frequency in the ExAC database was 0. In total, we found two misseBse mutations and one deletion in eight individuals fromfive families. CCDC115 is located on the negative strand, contains five exons, and encodes coiled-coil domain containing 115 with 180 amino acids and two predicted coiled-coil domains (Figure 1B). Both CCDC115 missense mutations are located in the first predicted coiled-coil domain and affect highly conserved positions. CCDC115 is widespread among eukaryotes, including Arabidopsis thaliana, indicating its origin at the root of the eukaryotic tree. Glycosylation Studies Global defects inglycosylation canbedetectedby IEF of serumTf (N-glycosylation) and serum ApoC-III (mucin type O-glycosylation). Tf has two N-glycosylation sites, and the most abundant fraction corresponds with four sialic acids. ApoC- III has one mucin-type O-linked glycan that can host one or two sialic acids. An increase in fractions associated with hyposialylated Tf or ApoC-III is indicative of abnormal N- or O-glycosylation. All individuals showed a similarly abnormal type 2 N-glycosylation profile of Tf (see Figure 2A and Table S5 for quantifications). ApoC-III IEF was abnormal for all tested individuals (see Figure 2A and Table S6 for quantifications). We performed MALDI-LTQ mass spectrometry of total plasma N-glycans of individual F1-II1 and compared the spectrum with that of a healthy control individual. Most notably, the glycans with theoretical masses of 2,433 m/z and 2,229 m/z were increased, indicating loss of either one sialic acid (2,433 m/z) or one sialic acid plus one galactose (2,229 m/z) (Figure 2B). In accordance with total plasma N-glycan analysis, nanochip-C8 QTOF mass spectrometry of intact serum Tf (79,555 amu, peak 1) showed accumulation of