Jos Jansen

2 31 CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation Mutation Analyses In silico analysis was done with Alamut v.2.4.6 (Interactive Biosoftware) and the effects of mutations were predicted with SIFT, PolyPhen, and MutationTaster (see Web Resources). The Exome Aggregation Consortium (ExAC) database (see Web Resources) was used for allele frequency. Primers (Biolegio and Sigma- Aldrich) flanked with universal M13 tags were constructed with the help of the UCSC Genome Browser, Primer3, and SNPCheck3 (see Web Resources).(17,18) For a list of primers used, see Table S1. Sanger sequencing was performed on DNA isolated from peripheral blood or cultured fibroblasts, according to standard protocols. DNA was amplified with a T100 ThermoCycler (Bio-Rad). An ABI 3730 DNA Analyzer (Life Technologies) was used for sequencing. Data analysis was done with Sequencher 4.8 (Gene Codes). Multiplex ligation- dependent probe amplification (MLPA) was performed as described previously. (19) In short, combinations of two adjacently annealing oligonucleotide probes were hybridized and ligated. After ligation, the common ends of the probes served as a template for PCR amplification with one primer pair, and due to the fluorescent labeling of the primer, the resulting products could be separated according to size via capillary electrophoresis on an ABI3130 Genetic Analyzer (Applied Biosystems). Fragment data were analyzed in GeneScan (Applied Biosystems). Peak heights of samples from affected individuals were compared with those from control individual probes, and ratios were calculated for all fragments (originating from CCDC115 , PTPN18 , and SMPD4 exons) via an Excel spreadsheet. Thresholds for deletions and duplications were set at 0.75 and 1.25 respectively, and all samples were tested at least twice. All reagents for the MLPA reaction and subsequent PCR amplification were purchased from MRC- Holland, with exception of the CCDC115 , PTPN18 , SMPD4 , and control primers (Biolegio). Primer sequences are described in Table S1. Cell Culture Skin fibroblasts from participants and healthy control individuals were cultured at 37.0°C under 5.0% CO2 in culture medium E199, supplemented with 10% fetal calf serum, and 1% penicillin/streptomycin. All cultures were tested for mycoplasma infection prior to cultivation.