Fangtian Dong

Comprehensive Molecular Diagnosis of a Large Chinese Leber Congenital Amaurosis Cohort

PURPOSE Leber congenital amaurosis (LCA) is an inherited retinal disease that causes early-onset severe visual impairment. To evaluate the mutation spectrum in the Chinese population, we performed a mutation screen in 145 Chinese LCA families. METHODS First, we performed direct Sanger sequencing of 7 LCA disease genes in 81 LCA families. Next, we developed a capture panel that enriches the entire coding exons and splicing sites of 163 known retinal disease genes and other candidate retinal disease genes. The capture panel allowed us to quickly identify disease-causing mutations in a large number of genes at a relatively low cost. Thus, this method was applied to the 53 LCA families that were unsolved by direct Sanger sequencing of 7 LCA disease genes and an additional 64 LCA families. Systematic next-generation sequencing (NGS) data analysis, Sanger sequencing validation, and segregation analysis were used to identify pathogenic mutations. RESULTS Homozygous or compound heterozygous mutations were identified in 107 families, heterozygous autosomal dominant mutations were identified in 3 families and an X-linked mutation was found in 1 family, for a combined solving rate of 76.6%. In total, 136 novel pathogenic mutations were found in this study. In combination with two previous studies carried out in Chinese LCA patients, we concluded that the mutation spectrum in the Chinese population is distinct compared to that in the European population. After revisiting, we also refined the clinical diagnosis of 10 families based on their molecular diagnosis. CONCLUSIONS Our results highlight the importance of a molecular diagnosis as an integral part of the clinical diagnostic process.

Transthyretin Ala36Pro mutation in a Chinese pedigree of familial transthyretin amyloidosis with elevated vitreous and serum vascular endothelial growth factor

The familial transthyretin (TTR) amyloidosis (FTA) demonstrates variable penetrance of clinical features associated with mutations in the plasma thyroid hormone-binding protein TTR gene. The purpose of this study was to assess the ocular features, to analyze vitreous and serum vascular endothelial growth factor (VEGF) levels, and to identify the genetic defect in a Chinese family with TTR FTA. The pedigree of interest was a three-generation family with eleven members. The primary ocular signs were vitreous opacities, beginning from the third or fourth decade, accompanied by retinal vasculitis, hemorrhages, and widespread pinpoint deposits in the peripheral retina. Two patients underwent vitrectomy with marked improvement of visual acuity postoperatively. Vitreous and serum samples for VEGF were analyzed with an enzyme-linked immunosorbent assay (ELISA). Forty-eight healthy adult volunteers were enrolled as a control group for the analysis of serum VEGF. Eight subjects who underwent vitrectomy for a macular epiretinal membrane or macular hole were enrolled as control for the analysis of vitreous VEGF. Both serum and vitreous VEGF levels of patients were raised compared to that of controls. Venous blood was collected from family members and the genomic DNA was extracted. All exons and exon-intron boundaries of the TTR gene were sequenced. A previously-described pathogenic transversion in exon 2 (c.G106C, p.Ala36Pro) was identified. Within this family eight individuals were confirmed as affected. In conclusion, a Chinese family with TTR Ala36Pro associated FTA is characterized by early ocular involvement. Widespread pinpoint lesions indicate RPE lesions caused by TTR deposition. FTA is associated with increased VEGF levels, both in serum and vitreous.

De novo Mutations in the Cone-rod Homeobox Gene Associated with Leber Congenital Amaurosis in Chinese Patients

Abstract Background: The cone-rod homeobox (CRX) gene plays an important role in photoreceptor development. Recently, mutant alleles of the CRX gene have been associated with autosomal dominant Leber congenital amaurosis (LCA) and cone-rod dystrophy. The purpose of this study was to analyze the CRX mutations in a cohort of Chinese patients with LCA or early-onset severe retinal dystrophy (EOSRD) and to provide the clinical features of these patients. Methods: Patients with LCA or EOSRD were enrolled from 2003 to 2012. Detailed ocular examinations including optical coherence tomography (OCT) and standardized electrophysiology were performed. Genomic DNA was isolated with standard methods of genetic diagnosis. All three exons of CRX were amplified with PCR and screened for mutations through direct DNA sequencing. A total of 200 unrelated healthy Chinese subjects were screened to exclude nonpathogenic polymorphisms. Offspring-parent relationship was tested to confirm de novo mutation. Results: A total of 109 probands from 109 unrelated families were selected for mutation screening of the CRX gene. Two individuals with LCA were confirmed to carry de novo CRX mutations c.421delT (p.Ser141Pro fsX46) and c.571delT (p.Tyr191Met fsX3), respectively. The daughter of Case 1 also carried the same CRX mutation (c.421delT) and had LCA symptoms. Pigmentary retinopathy in the peripheral retina and macular atrophy were observed in the two probands. Macular atrophy without normal lamination structure was the retina phenotype under OCT. Conclusions: Two de novo mutations in CRX were found in Chinese patients with LCA. The CRX mutation might create a dominantly inherited trait.

Novel CNGA3 mutations in Chinese patients with achromatopsia

Objective To study the clinical features and to identify the pathogenic mutations in Chinese patients with achromatopsia (ACHM). Design Fifteen patients from 10 unrelated families were included in this study. Detailed ocular examinations were performed for the affected subjects, including best-corrected visual acuity (BCVA), colour vision, slit lamp, fundus, electroretinography, perimetry, and spectral domain optical coherent topography (SD-OCT). Peripheral blood samples were obtained from all of the patients and their family members for genomic DNA extraction. All exons of CNGA3, CNGB3, GNAT2, PDE6C and PDE6H were amplified by a PCR and screened for mutation by direct Sanger sequencing. The sequences were analysed using the Blat tool and then compared with the gene transcript. A segregation test was conducted in the patients’ parents if they were available. The variants were compared with the database of the 1000 Genomes Project to exclude polymorphism. Results Nystagmus, photophobia, and impaired colour discrimination were observed in all patients. The BCVA of the affected subjects ranged from 0.05–0.2. Severely depressed and non-recordable cone electroretinograms were observed. Noticeable structural changes including disruption or loss of the macular inner/outer segments (IS/OS) junction of the photoreceptors were observed with SD-OCT. CNGA3 mutations were identified in 13 patients from eight families. Sequencing revealed seven novel missense mutations, three novel deletion mutations, and four previously reported mutations among those patients. Conclusions CNGA3 mutation is the most frequent cause of ACHM in this cohort of patients. Ten novel mutations were identified in CNGA3. Genetic characterisation of patients with ACHM is important for genetic counselling and future gene therapies. This study reports the comprehensive clinical and genetic features of Chinese patients with ACHM.

Thirty-two years follow-up of X-linked juvenile retinoschisis in a Chinese patient with RS1 mutation

Background: A Chinese family with X-linked juvenile retinoschisis (XLRS) was identified. The purpose of this study was to identify genetic defects in this family and to investigate the visual function during the progression of the disease in the proband from childhood to adulthood. Methods: The family history was collected and the proband underwent regular ophthalmologic examinations. Venous blood was collected from family members and genomic DNA was extracted. All exons and exon-intron boundaries of the RS1gene were sequenced for gene mutation in this family. Results: The pedigree of interest was a three-generation family with 43 family members, including three affected individuals. The proband clinically diagnosed with XLRS was investigated at 7 years of age with a follow-up of 32 years. Best corrected visual acuity was stable and complications such as vitreous hemorrhage, retinal detachment, and subcapsular cataract arose during this follow-up period. The R213Q mutation in exon6 of RS1 was identified in all affected individuals. Conclusions: Clinical follow-up of an XLRS patient with a typical juvenile retinoschisis phenotype revealed no significant decline in visual acuity during this time period. Various complications such as vitreous hemorrhage and cataract may occur during the progression of the disease which may lead to severe vision loss.

Clinical and genetic features of eight Chinese autosomal-dominant optic atrophy pedigrees with six novel OPA1 pathogenic variants

ABSTRACT Background: Autosomal-dominant optic atrophy (ADOA) is one of the most common types of inherited optic atrophy. We identify OPA1 pathogenic variants and assess the clinical features of a cohort of Chinese ADOA patients Materials and Methods: Detailed clinical evaluations were performed and genomic DNA was extracted from peripheral blood for all the participants. Sanger sequencing was used to analyze all exons and exon/intron junctions of OPA1 for eight pedigrees. Target exome capture plus next-generation sequencing (NGS) were applied for one atypical family with photophobia. Reverse transcription polymerase chain reaction was carried out to further characterize the mRNA change of selected splicing alteration. Results: All 17 patients had impaired vision and optic-disk pallor; however, the clinical severity varied markedly. Two patients complicated with hearing loss. Six novel and two reported pathogenic variants in OPA1 (GenBank Accession No. NM_130837.2) were identified including four nonsynonymous variants (c.2400T > G, c.1468T > C, c.1567A > G and c.1466T > C), two splicing variants (c.2984-1_2986delGAGA and c.2983 + 5G > A), one small deletion (c.2960_2968delGCGTTCAAC), and one small insertion (c.3009_3010insA). RNA analysis revealed the splicing variant c.2984-1_2986delGAGA caused small deletion of mRNA (r.2983_2988del). Conclusions: ADOA patients presented variable clinical manifestations. Novel OPA1 pathogenic variants are the main genetic defect for Chinese ADOA cases. NGS may be a useful molecular testing tool for atypical ADOA.