A novel, homozygous nonsense variant of the CDHR1 gene in a Chinese family causes autosomal recessive retinal dystrophy by NGS‐based genetic diagnosis

Abstract

Retinal dystrophy is an inherited, heterogeneous, chronic and progressive disorder of visual functions. The mutations of patients with autosomal recessive retinal retinopathy cone‐and‐rod dysfunction and macular dystrophy have not been well described in the Chinese population. In this study, a three‐generation Chinese retinal dystrophy family was recruited. Ophthalmic examinations were performed. Targeted next generation sequencing (TGS) was used to identify causative genes, and Sanger sequencing was conducted to verify candidate mutations and co‐segregation. Reverse transcription (RT)‐PCR was applied to investigate the spatial and temporal expression patterns of cdhr1 gene in mouse. A novel, homozygous, deleterious and nonsense variant (c.T1641A; p.Y547*) in the CDHR1 gene was identified in the family with autosomal recessive retinal dystrophy, which was co‐segregated with the clinical phenotypes in this family. RT‐PCR analysis revealed that cdhr1 is ubiquitously expressed in eye, particularly very high expression in retina; high expression in lens, sclera, and cornea; and high expression in brain. In conclusion, our study is the first to indicate that the novel homozygous variant c.T1641A (p.Y547*) in the CHDR1 gene might be the disease‐causing mutation for retinal dystrophy in our patient, extending its mutation spectrums. These findings further the understanding of the molecular pathogenesis of this disease and provide new insights for diagnosis as well as new implications for genetic counselling.

A Novel Variant of the FZD4 Gene in a Chinese Family Causes Autosomal Dominant Familial Exudative Vitreoretinopathy

“…Only very rare variants were considered, with a dominant frequency cutoff of 0.05% [5]. Sequencing depth 4, estimated copy number 2, SNP quality 20, and a distance between two SNPs of>5 were used as the filtration criteria for candidate SNPs, according to previously reported studies [29,30]. Sequence variants should not be annotated in any of the above public databases.…”

“…Total RNA was extracted from the mice at the indicated ages and the indicated tissues using an RNA kit according to our previously reported standard protocols [30]. RNA quality was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and 1.5% agarose gel electrophoresis.…”

“…RNA quality was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and 1.5% agarose gel electrophoresis. cDNA synthesis was performed using random oligomer primers and a reverse transcriptase kit [30]. The RT-primer sequences, product size, and PCR conditions (annealing temperature) are listed in Table 1.…”

“…For mutation verification and co-segregation analysis, polymerase chain reaction (PCR) amplification and direct Sanger sequencing of the variant were applied to gDNA of all available individuals [29,30]. A locus-specific primer pair (M362-FZD4) was designed using the online Primer3 program (http://primer3.…”

“…The RT-primer sequences, product size, and PCR conditions (annealing temperature) are listed in Table 1. Semiquantitative RT-PCR was performed using the primer pair RT-fzd4 for the mouse Fzd4 gene; the ß-actin gene of mouse was used as an internal control and amplified using the primer pair RT-b-actin-mL and RT-b-actinmR [30] (Table 1). Each RT-PCR assay was done in three independent tests for verification.…”