Pedigree recruitment and genetic screening
The participating family of Pakistani descent was recruited from the Jammu and Kashmir for this study. The study was approved by the institutional ethical review boards of Mirpur University of Science and technology (MUST) and by the ethic committee of the Charité (EA1/212/08). Written informed consents were obtained from all individuals or custodians for the participation in the study. The behavior and phenotype of all affected individuals was analyzed with special attention to neurological, morphological, ophthalmological, dermatological and skeletal symptoms.
Genomic DNA from one affected and one not affected family member was extracted from the peripheral blood by using the QIAamp DNA blood mini kit (Qiagen, Frankfurt, Germany) according to the manufacturer’s instructions. The whole exome sequencing procedures were performed on a HiSeq4000 deep sequencer (Illumina, CA, USA) with a 150 bp paired-end protocol after enrichment in the coding regions by the SureSelect Human All Exon V5 kits (Agilent, CA, USA). The average sequencing coverage reached more than 150X, with at least 20X of more than 99% of the coding regions. The generated raw sequences were processed by the MERAP package for alignment, quality control, calling single nucleotide variant (SNV), insertion and deletion (Indel), structural variation (SV), and copy number variation (CNV), plus the variants annotation and prioritization (Hu et al., 2014). PCR amplification was performed by using the Q5 High-Fidelity Polymerase (5X, NEB, MA, USA) with the following specific primers targeted to the selected candidate variant: Forward: 5’-GTTTCCAGTGCCACCAAACC-3’, Reverse: 5’-AGCAAGAGCCTTTCCATTCCT-3’. The PCR products were Sanger sequenced to determine the carrier status of the specific variant.