*Contributed equally
Retinitis pigmentosa (RP) belongs to a family of retinal disorders that is characterized by the progressive degeneration of rod and cone photoreceptors. The aim of the present study was to screen for possible disease-causing genetic variants in a non-consanguineous Chinese family with non-syndromic autosomal recessive RP. Whole-exome sequencing (WES) was performed in samples from the affected individual (the proband) and those from the two children of the proband. A novel compound heterozygous variant of c.C958T (p.R320X) and c.G1355A (p.R452H) in the
Retinitis pigmentosa (RP; OMIM no. 268000) is a group of highly heterogeneous but related retinal disorders that cause progressive vision loss (
One of the reasons for the heterogeneity of RP is the >80 disease-causing genes that have been identified (
In the present study, whole-exome sequencing (WES) was applied to screen for potential disease-causing variants in a non-consanguineous Chinese family with autosomal recessive RP. A novel compound heterozygous variant in
A Chinese family with RP, including six members with an affected individual and five unaffected individuals, was recruited from the Sichuan Provincial People's Hospital (Chengdu, China). The affected individual (sex, female) was 47 years old when diagnosed. Additionally, 400 unrelated healthy subjects, including 218 males and 182 females, were recruited from the Sichuan Provincial People's Hospital (Chengdu, China). The median age of the healthy controls was 42 (age range, 20-60). The study was performed in accordance with the tenets of the Declaration of Helsinki (
All participants underwent ophthalmological examinations. Fundus photography was performed for all members of the affected individual's family. The clinical data were assessed by ophthalmologists at Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital.
Peripheral blood samples were collected in EDTA tubes from all six members of the family and unrelated normal controls. Genomic DNA was extracted using a blood DNA extraction kit according to the manufacturer manual (Tiangen Biotech, Co., Ltd.) DNA samples were stored at -20˚C until required.
The DNA from individuals II-1 (the proband), III-1 and III-2 were analyzed by WES with a mean read depth of 100x. The samples were prepared following the Illumina standard procedure (Illumina, Inc.). Briefly, ~3 µg genomic DNA was randomly sheared into small fragments of 150-220 bp using a sonicator (Covaris). The sheared fragments were purified with reagents supplied with the AMPure XP system (Beckman Coulter, Inc.). Adapters (Agilent Technologies, Inc.) were ligated with the polished ends and the libraries were amplified by PCR. The amplified libraries were hybridized with biotin-labeled probes in the liquid phase. The DNA fragments bound to the probes, namely the captured library, were purified. Then, the library was sequenced on a Illumina HiSeq4000 platform (Illumina, Inc.) and paired-end 150 bp reads were obtained.
The mutations in
A Chinese family consisting of three generations of individuals with RP, but no history of consanguineous marriage was examined in the present study (
After WES analysis on the proband (II-1), and individuals III-1 and III-2, 29,734 variants in the coding regions and splice junctions were obtained, including 14,298 nonsynonymous single-nucleotide polymorphisms (SNPs), 14,927 synonymous SNPs, 509 SNPs of miscellaneous types and 744 indels. To screen for the disease-causative variant in the family with RP, common variants present in high frequencies in dbSNP138, 1000 Genomes Project, Exome Aggregation Consortium, OMIM, HGMD and other east Asian population databases were filtered out. Subsequently, variants located in introns, 5'untranslated regions (UTRs) and 3'UTRs, in addition to synonymous SNPs and non-frameshift indels were also filtered out since they typically do not influence gene function. A particular focus was placed on possible functional SNPs/indels in the homozygous or compound heterozygous variants, including frameshift indels, non-synonymous variants and splicing junction variants, which are more likely to be pathogenic. These SNP/indels were filtered further using the criterion that the candidate variants must be inherited in an autosomal recessive inheritance manner. Genes affected by these filtered variants were then compared with the reported genes that have been previously associated with retinal diseases using the Retnet database (
Direct Sanger sequencing confirmed the compound heterozygous variant in the proband. The proband's father (I-1) and daughter (III-1) possessed a heterozygous variant of c.G1355A (p.R452H), his mother (I-2) and son (III-2) were heterozygous carriers of c.C958T (p.R320X) (
The c.C958T replacement caused the substitution of arginine with a stop codon (p.R320X), whilst the c.G1355A replacement caused the substitution of histidine with arginine (p.R452H). Amino acid sequence alignment of the CYP4V2 protein among different species revealed that these two variants are located in two highly conserved regions (
In the present study, a novel compound heterozygous
Mutations in the
In the present pedigree, the proband (II-1) was found to be carrying the compound heterozygous variant of c.C958T and c.G1355A, whereas other family members were found to either carry none of the variants of interest or one heterozygous variant of c.C958T and c.G1355A. Furthermore, this compound heterozygous variant could not be found in 400 unrelated healthy Chinese control individuals or in any of the public databases probed, including HGMD (
CYP4V2 is also known as BCD or CYP4AH1, and belongs to a subfamily within the cytochrome P450 superfamily.
Structurally,
This compound variant of
In summary, a novel compound heterozygous mutation of c.C958T and c.G1355A in the
Not applicable.
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
TZ, TW, FZ and SD performed the experiments and analyzed the data. BG and HZ analyzed the data and supervised the project. TZ and HZ wrote the manuscript. All authors read and approved the final manuscript. TZ, BG and HZ confirmed the authenticity of all the raw data.
This study was approved by the Institutional Review Boards of Sichuan Provincial People's Hospital (Chengdu, China). Written informed consent was obtained from all participants or parents of children prior to their inclusion in the present study.
Not applicable.
The authors declare no competing interests.
Pedigree of the Chinese family with retinitis pigmentosa. The arrow points to the proband. Females and males are indicated by circles and squares, respectively. Empty and filled symbols represent normal and affected individuals, respectively. M1 and M2 represent the genetic variants of c.C958T (p.R320X) and c.G1355A (p.R452H) in the
Fundus photographs. Fundus images of unaffected individuals (A) I-1, (B) I-2 and (D) III-1. (C) Fundus images of the affected individual (II-1) in the family. OD, right eye; OS, left eye.
Identification of the mutations in the
Bioinformatics analysis of the effect of p.R320X and p.R452H in CYP4V2.
CYP4V2 Mutation | Mutation Taster | CADD, RawScore /Phred | PROVEAN | PolyPhen-2 | SIFT |
---|---|---|---|---|---|
p.R320X | Disease causing | 8.551470/43, Deleterious | -13.84, Deleterious | N/A | N/A |
p.R452H | Disease causing | 4.218231/29.6, Deleterious | -4.79, Deleterious | 0.999, Probably damaging | Damaging |
CYP4V2, Cytochrome P450 family 4 subfamily V member 2; CADD, Combined Annotation-Dependent Depletion; PROVEAN, Protein Variation Effect Analyzer; SIFT, Sorting Intolerant from Tolerant.