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.