An 11-month-old male proband is the first child of the non-consanguineous parents from China. He presented with creamy white skin, yellow white hair, accompanied with nystagmus, astigmatism, and hypermetropia. His father was phenotypically normal, his mother presented with yellow hair. We obtained approval from the BGI-Shenzhen ethics committee (No. BGI-IRB 17168). Written informed consent was obtained from the patient’s parents for participation in the present study before collecting peripheral blood. The parents of the proband declined publication of the clinical images.
Genetic analysis
In order to identify the etiology, targeted NGS was carried out on the proband with the 54 inherited eye disease genes panel, which includes four prevalent nsOCA genes: TYR, OCA2, TYRP1, and SLC45A2. (Additional file 1: Table S1). The total DNA extraction was performed using lymphocyte of peripheral blood by the QIAamp DNA extraction kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions and recommendations. Genomic DNA was fragmented into 200 bp to 300 bp using an ultrasonoscope (Covaris S2, Massachusetts, USA). Then library construction was operated as previously published procedure [9]. The enriched library was sequenced using a HiSeq2500 Analyzers (Illumina, San Diego, CA, USA). The pipeline of bioinformatics analysis was performed to screen the mutations as a previous study [10]. The produced sequenceing paired-end reads (90 bp) were aligned to the reference human genome (GRCh37/hg19) by Burrows Wheeler Aligner (bio-bwa.sourceforge.net). Single-nucleotide variant (SNV) and insertion and deletion (indel) were detected by SOAPsnp software (sourceforge.net/projects/soapsnp/) and the SAMtools (samtools.sourceforge.net) respectively. All SNVs and indels were filtered in the dbSNP, HapMap, 1 K human genome database and in-house database of 100 Chinese controls. Calling copy number variation (CNV) was performed according to a previous paper [11,12,13]. The deletion was identified by comparing the intra- and inter-sample normalized sequencing depth of each exon. Exons with a depth ratio 0.5 were considered to have heterozygous deletion, in contrast to the given sample. The sequence variants interpretation was conducted based on the guideline of the American College of Medical Genetics (ACMG).
We looked for candidate pathogenic variants in the proband by targeted NGS. The captured targeted region was 238,836 bp, and the coverage of the targeted region was 98.9%. The average sequencing depth of the panel was 204.43-fold, with 95.61% of the targeted bases covered a minimum of 30-fold. Three heterozygous variants were detected in the proband. The first was a missense mutation (c.1865 T > C, p.Leu622Pro) in exon 18 of OCA2 gene, the second was a gross deletion with exons 17–21 encompassed introns in OCA2 gene, and the third was a previously reported mutaiton (c.4805G > A, p.Arg1602Gln) in exon 35 of MYO7A gene with Usher Syndrome (US) [14], which clinically characterized with deafness and gradual vision loss.
To further evaluate the pathogenesis of the variant, three prediction programs PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/), and Mutation Taster (http://www.mutationtaster.org/) were used to predict the probability of variant c.1865 T > C in OCA2 gene. All prediction tools showed the variant was probably damaging. Multiple amino acid sequence alignment of OCA2 amino acid sequences with CLUSTALW (https://www.genome.jp/tools-bin/clustalw) showed that the missense variant (c.1865 T > C) was in a highly conserved region among species (Fig. 1). The three-dimensional (3D) models of native and mutant proteins were generated to predict the structure of proteins using the I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). The result showed that the variant (c.1865 T > C) disturbed the formation of the α-helix, and formed a random coil structure (Fig. 2a, b), suggesting that it was a pathogenic mutation. Deletion of exons 17–21 affected the 3D structure of OCA2 protein, and leaded to a truncated and non-functional protein (Fig. 2c).
Confirmation of the novel mutation in the family
To ascertain three variants revealed by the targeted NGS, we conducted Sanger sequencing and quantitative PCR (qPCR) in the family with primers in Additional file 2: Table S2. The heterozygous variants c.1865 T > C(OCA2) and c.4805G > A (MYO7A) were both detected in the proband and his father (Fig. 3a, b). The quantity of exon 17–21 detected in the proband was consistent with his mother, almost half of his father and the control sample by qPCR (Fig. 4). The results demonstrated that the compound heterozygous variants in OCA2 gene of the proband were inherited from the parents respectively, and the proband was also a carrier of US.
