γ-crystallin gene screen in a congenital nuclear cataract family

Objects To screen the γ-crystallin genes in a Chinese autosomal dominant congenital nu-clear cataract (ADCC) family. Methods Tile genes CRYGA,CRYGB,CRYGC,CRYGD were screened by direct sequencing in the ADCC family. Family analysis was used to analyze the correlation of the mutations and tile disease. Results Nine sequence variants were found: 198G→A and 196T→C (LI48P) in CRYGA,2218-2219 in C,2437C→T and 5391C→A (L111I) in CRYGB, 18782T→G in CRYGC,2861T→C, 517T→C and 570C→T in CRYGD. Two mutations lead to amino acid changes. However,no mutation could be identi-fled as co-segregating with tile disease. L148P in CRYGA and L111I in CRYGB were supposed to be nu-cleotide polymorphisms. Conclusion Nine sequence variations have been found in the ADCC Family. But they seem not to be responsible for the disease.     

γ-crystallin gene screen in a congenital nuclear cataract family

Objects To screen the γ-crystallin genes in a Chinese autosomal dominant congenital nu-clear cataract (ADCC) family. Methods Tile genes CRYGA,CRYGB,CRYGC,CRYGD were screened by direct sequencing in the ADCC family. Family analysis was used to analyze the correlation of the mutations and tile disease. Results Nine sequence variants were found: 198G→A and 196T→C (LI48P) in CRYGA,2218-2219 in C,2437C→T and 5391C→A (L111I) in CRYGB, 18782T→G in CRYGC,2861T→C, 517T→C and 570C→T in CRYGD. Two mutations lead to amino acid changes. However,no mutation could be identi-fled as co-segregating with tile disease. L148P in CRYGA and L111I in CRYGB were supposed to be nu-cleotide polymorphisms. Conclusion Nine sequence variations have been found in the ADCC Family. But they seem not to be responsible for the disease.     

γ-crystallin gene screen in a congenital nuclear cataract family

Objects To screen the γ-crystallin genes in a Chinese autosomal dominant congenital nu-clear cataract (ADCC) family. Methods Tile genes CRYGA,CRYGB,CRYGC,CRYGD were screened by direct sequencing in the ADCC family. Family analysis was used to analyze the correlation of the mutations and tile disease. Results Nine sequence variants were found: 198G→A and 196T→C (LI48P) in CRYGA,2218-2219 in C,2437C→T and 5391C→A (L111I) in CRYGB, 18782T→G in CRYGC,2861T→C, 517T→C and 570C→T in CRYGD. Two mutations lead to amino acid changes. However,no mutation could be identi-fled as co-segregating with tile disease. L148P in CRYGA and L111I in CRYGB were supposed to be nu-cleotide polymorphisms. Conclusion Nine sequence variations have been found in the ADCC Family. But they seem not to be responsible for the disease.     

γ-crystallin gene screen in a congenital nuclear cataract family

Objects To screen the γ-crystallin genes in a Chinese autosomal dominant congenital nu-clear cataract (ADCC) family. Methods Tile genes CRYGA,CRYGB,CRYGC,CRYGD were screened by direct sequencing in the ADCC family. Family analysis was used to analyze the correlation of the mutations and tile disease. Results Nine sequence variants were found: 198G→A and 196T→C (LI48P) in CRYGA,2218-2219 in C,2437C→T and 5391C→A (L111I) in CRYGB, 18782T→G in CRYGC,2861T→C, 517T→C and 570C→T in CRYGD. Two mutations lead to amino acid changes. However,no mutation could be identi-fled as co-segregating with tile disease. L148P in CRYGA and L111I in CRYGB were supposed to be nu-cleotide polymorphisms. Conclusion Nine sequence variations have been found in the ADCC Family. But they seem not to be responsible for the disease.     

γ-crystallin gene screen in a congenital nuclear cataract family

Objects To screen the γ-crystallin genes in a Chinese autosomal dominant congenital nu-clear cataract (ADCC) family. Methods Tile genes CRYGA,CRYGB,CRYGC,CRYGD were screened by direct sequencing in the ADCC family. Family analysis was used to analyze the correlation of the mutations and tile disease. Results Nine sequence variants were found: 198G→A and 196T→C (LI48P) in CRYGA,2218-2219 in C,2437C→T and 5391C→A (L111I) in CRYGB, 18782T→G in CRYGC,2861T→C, 517T→C and 570C→T in CRYGD. Two mutations lead to amino acid changes. However,no mutation could be identi-fled as co-segregating with tile disease. L148P in CRYGA and L111I in CRYGB were supposed to be nu-cleotide polymorphisms. Conclusion Nine sequence variations have been found in the ADCC Family. But they seem not to be responsible for the disease.     

γ-crystallin gene screen in a congenital nuclear cataract family

Objects To screen the γ-crystallin genes in a Chinese autosomal dominant congenital nu-clear cataract (ADCC) family. Methods Tile genes CRYGA,CRYGB,CRYGC,CRYGD were screened by direct sequencing in the ADCC family. Family analysis was used to analyze the correlation of the mutations and tile disease. Results Nine sequence variants were found: 198G→A and 196T→C (LI48P) in CRYGA,2218-2219 in C,2437C→T and 5391C→A (L111I) in CRYGB, 18782T→G in CRYGC,2861T→C, 517T→C and 570C→T in CRYGD. Two mutations lead to amino acid changes. However,no mutation could be identi-fled as co-segregating with tile disease. L148P in CRYGA and L111I in CRYGB were supposed to be nu-cleotide polymorphisms. Conclusion Nine sequence variations have been found in the ADCC Family. But they seem not to be responsible for the disease.     

γ-crystallin gene screen in a congenital nuclear cataract family

Objects To screen the γ-crystallin genes in a Chinese autosomal dominant congenital nu-clear cataract (ADCC) family. Methods Tile genes CRYGA,CRYGB,CRYGC,CRYGD were screened by direct sequencing in the ADCC family. Family analysis was used to analyze the correlation of the mutations and tile disease. Results Nine sequence variants were found: 198G→A and 196T→C (LI48P) in CRYGA,2218-2219 in C,2437C→T and 5391C→A (L111I) in CRYGB, 18782T→G in CRYGC,2861T→C, 517T→C and 570C→T in CRYGD. Two mutations lead to amino acid changes. However,no mutation could be identi-fled as co-segregating with tile disease. L148P in CRYGA and L111I in CRYGB were supposed to be nu-cleotide polymorphisms. Conclusion Nine sequence variations have been found in the ADCC Family. But they seem not to be responsible for the disease.     

γ-crystallin gene screen in a congenital nuclear cataract family

Objects To screen the γ-crystallin genes in a Chinese autosomal dominant congenital nu-clear cataract (ADCC) family. Methods Tile genes CRYGA,CRYGB,CRYGC,CRYGD were screened by direct sequencing in the ADCC family. Family analysis was used to analyze the correlation of the mutations and tile disease. Results Nine sequence variants were found: 198G→A and 196T→C (LI48P) in CRYGA,2218-2219 in C,2437C→T and 5391C→A (L111I) in CRYGB, 18782T→G in CRYGC,2861T→C, 517T→C and 570C→T in CRYGD. Two mutations lead to amino acid changes. However,no mutation could be identi-fled as co-segregating with tile disease. L148P in CRYGA and L111I in CRYGB were supposed to be nu-cleotide polymorphisms. Conclusion Nine sequence variations have been found in the ADCC Family. But they seem not to be responsible for the disease.     

γ-crystallin gene screen in a congenital nuclear cataract family

Objects To screen the γ-crystallin genes in a Chinese autosomal dominant congenital nu-clear cataract (ADCC) family. Methods Tile genes CRYGA,CRYGB,CRYGC,CRYGD were screened by direct sequencing in the ADCC family. Family analysis was used to analyze the correlation of the mutations and tile disease. Results Nine sequence variants were found: 198G→A and 196T→C (LI48P) in CRYGA,2218-2219 in C,2437C→T and 5391C→A (L111I) in CRYGB, 18782T→G in CRYGC,2861T→C, 517T→C and 570C→T in CRYGD. Two mutations lead to amino acid changes. However,no mutation could be identi-fled as co-segregating with tile disease. L148P in CRYGA and L111I in CRYGB were supposed to be nu-cleotide polymorphisms. Conclusion Nine sequence variations have been found in the ADCC Family. But they seem not to be responsible for the disease.     

先天性核性白内障家族的γ晶状体蛋白基因筛查

目的 在一例国人常染色体显性遗传的先天性核性白内障家族进行γ晶状体蛋白基因筛查,探讨基因与该家族发病的相关性.方法 运用直接测序的方法,在一例国人常染色体显性遗传的先天性核性白内障家族进行CRYGA、CRYGB、CRYGC、CRYGD的基因筛查,确定基因变异与该家族的关系.结果 在该家族中发现了9种,核苷酸序列改变,它们分别是:CRYGA的1986→A、196T→C(L48P)、CRYGB的2218→2219insC、2437C→T、5391C→A(L111I)、CRYGC的18782T→G和CRYGD的286T→C、517→、570C→T,其中两种导致了氨基酸的改变.但这些基因突变并没有在该家族中和疾病共分离.结论 初步排除γ晶状体蛋白基因与该先天性核性白内障家族有关.该家族白内障的发生可能由其他位置的基因突变引起.     

A New Arg54Gly Transthyretin Gene Mutation Associated with Vitreous Amyloidosis in Chinese

 Purpose:To analyse the hereditary features of a Chinese pedigree with familial vitreous amyloidosis in Liaoning Province, China, and to investigate the correlation between the clinical appearance of the disease and transthyretin（TTR）gene mutation, including the locus and type of TTR gene mutation． Methods:Five patients (10 eyes) from one Chinese family were diagnosed with vitreous amyloidosis between July 1996 and April 2009. Family members were followed up subsequently, and peripheral venous blood was obtained from 13 subjects (including 2 patients, and 11 controls without clinical signs of disease). DNA samples were extracted and 4 exons of the TTR gene were amplified by polymerase chain reaction (PCR). The gene fragments were subjected to sequencing analysis. The results were analyzed with DNAMAN Windows 5.2.2.0 and Chromas sequence chart analysis software, TTR gene exons were compared between affected patients and normal controls． Results:Family pedigree analysis revealed that patients were distributed in three generations. Male and female subjects had equal prevalence, and only one parent of affected patients had signs of disease. TTR gene exon sequencing showed that the sequence of patients was identical to that of normal individuals. No TTR gene mutations were noted in 10 un-affected family members. However, a TTR Gly-54 point mutation in the 2nd exon was detected in two patients and 1 unaffected family member (one of the patients' daughters)．Vitreous samples in 4 cases (7 eyes) showed positive Congo red staining, suggesting that these family members suffered from familial vitreous amyloidosis. Conclusion:This pedigree affected with familial vitreous amyloidosis was characterized by autosomal dominant inheritance; a TTR Gly-54 point mutation in the 2nd exon is presumed to be the cause. This Gly-54 point mutation of the TTR gene is a novel mutation in vitreous amyloidosis.     

Complete mitochondrial DNA sequence analysis in two southern Chinese pedigrees with Leber hereditary optic neuropathy revealed secondary mutations along with the primary mutation

AIM: To investigate mitochondrial factors associated with Leber hereditary optic neuropathy (LHON) through complete sequencing and analysis of the mitochondrial genome of Chinese patients with this disease. METHODS: Two unrelated southern Chinese families with LHON and 10 matched healthy controls were recruited, and their entire mitochondrial DNA (mtDNA) was amplified and sequenced with the universal M13 primer. Then DNA sequence analysis and variation identification were perfomed by DNAssist and Chromas 2 software and compared with authoritative databases such as Mitomap. RESULTS: Mutational analysis of mtDNA in these two Chinese pedigrees revealed one common LHON-associated mutation, G11778A (Arg→His), in the MT-ND4 gene. In addition, there were two secondary mutations in Pedigree 1: C3497T (Ala→Val), and C3571T (Leu→Phe) in the MT-ND1 gene, which have not been reported; and two secondary mutations occurred in Pedigree 2: A10398G (Thr→Ala) in the MT-ND3 gene, and T14502C (Ile→Val) in the MT-ND6 gene. Three polymorphisms, A73G, G94A and A263G in the mtDNA control region, were also found. CONCLUSION: Our study confirmed that the known MT-ND4*G11778A mutation is the most significant cause of LHON. The C3497T and C3571T mutations in Pedigree 1 were also both at hot-spots of MT-ND1; they may affect the respiratory chain in coordination with the primary mutation G11778A. In Pedigree 2, the two secondary mutations A10398G of MT-ND3 and T14502C of MT-ND6 may influence mitochondrial respiratory complex I, leading to the mitochondrial respiratory chain dysfunction which results in optic atrophy together with G11778A. Therefore, not only the common primary LHON mutation is responsible for the visual atrophy, but other secondary mtDNA mutations should also be considered when giving genetic counseling.     

Reliability of Chinese web-based ocular surface disease index questionnaire in dry eye patients: a randomized, crossover study

AIM: To investigate the causal gene mutation and clinical characteristics in two Chinese families with autosomal dominant congenital coralliform cataracts. METHODS: Two unrelated Chinese pedigrees with congenital cataract were investigated. Routine ophthalmic examinations were performed on all patients and non-affected family members. Peripheral blood samples were collected, and the genomic DNAs were extracted. The coding regions of probands’ DNAs were analyzed with cataract gene panel. The identified gene was amplified by polymerase chain reaction and sequenced in other members of two families to verify if the mutated gene was co-segregated with the disease. RESULTS: Congenital coralliform cataract was inherited in an autosomal dominant mode in both pedigrees. For each family, more than half of the family members were affected. All patients presented with severe visual impairment after birth as a result of bilateral symmetric coralliform lens opacification. An exact same defect in the same gene, heterozygous mutation of c.70C>A (p. P24T) in exon 2 of γD-crystallin CRYGD gene, was identified in both probands from each family. Sanger sequencing analysis demonstrated that the mutated CRYGD was co-segregated in these two families. CONCLUSION: A known substitution c.70C>A (p. P24T) in CRYGD gene appears to be the disease-causing gene in these two Chinese families. It is known that mutated CRYGD caused most of the congenital coralliform cataracts, suggesting that the CRYGD gene are associated with phenotype of congenital coralliform cataract.     

A mutated CRYGD associated with congenital coralliform cataracts in two Chinese pedigrees

AIM: To investigate the causal gene mutation and clinical characteristics for two Chinese families with autosomal dominant congenital coralliform cataract. METHODS: Two Chinese pedigrees with congenital cataract were investigated. Routine ophthalmic examinations were performed on all patients and non-affected family members. Peripheral blood samples were collected, and the genomic DNAs were extracted. The coding regions of proband’s DNAs were analyzed with cataract gene panel. The identified mutation was amplified by polymerase chain reaction, and automated sequencing was performed in other members of two families to verify whether the mutated gene was co-segregated with the disease. RESULTS: Congenital coralliform cataract was inherited in an autosomal dominant mode in both pedigrees. For each family, more than half of the family members were affected. All patients presented with severe visual impairment after birth as a result of bilateral symmetric coralliform lens opacification. An exact the same defect in the same gene, a heterozygous mutation of c.70C>A (p. P24T) in exon 2 of γD-crystallin gene, was detected in both probands from each family. Sanger sequencing analysis demonstrated that the mutated CRYGD was co-segregated in these two families. CONCLUSION: A c.70C>A (p. P24T) variant in CRYGD gene was reconfirmed to be the causal gene in two Chinese pedigrees. It is known that mutated CRYGD caused most of the congenital coralliform cataracts, suggesting that the CRYGD gene is associated with coralliform congenital cataract.     

A Chinese family with Axenfeld-Rieger syndrome: report of the clinical and genetic findings

AIM: To describe a Chinese family affected by a severe form of Axenfeld-Rieger syndrome (ARS) and characterize the molecular defect in PITX2 in the family. METHODS: Patients presented with typical ARS from a Chinese family were investigated. We performed genome-wide linkage scan and exome sequencing to identify the pathogenic mutations. Candidate mutations were verified for co-segregation in the whole pedigree using Sanger sequencing. Real-time polymerase chain reaction (RT-PCR) and Western blotting were performed to verify the expression of the pathogenic gene. RESULTS: Genome-wide linkage and exome sequencing analyses showed PITX2 as the disease candidate gene. A>G substitution at position -11 of 3’ss of exon 5 (IVS5-11A>G) that co-segregated with the disease phenotype was discovered in the family. The PITX2 messenger ribonucleic acid and protein levels were about 50% lower in patients with ARS than in unaffected family members in the family. CONCLUSION: Our findings implicate the first intronic mutation of the PITX2 gene in the pathogenesis of a severe form of ARS in a Chinese family. This study highlights the importance of a systematic search for intronic mutation in ARS cases for which no mutations in the exons of PITX2 have been found.     

Exome sequencing analysis identifies novel homozygous mutation in ABCA4 in a Chinese family with Stargardt disease

AIM: To identify the disease-associated mutations in a Chinese Stargardt disease(STGD) family, extend the existing spectrum of disease-causing mutations and further define the genotype-phenotype correlations.METHODS: A Chinese STGD family and 200 normal controls were collected. Whole exome sequencing(WES) and bioinformatics analysis were performed to find the pathogenic gene mutation. Physico-chemical parameters of mutant and wildtype proteins were computed by Prot Param tool. Domains analysis was performed by SMART online software. HOPE online software was used to analyze the structural effects of mutation. Immunofluorescence, quantitative real-time polymerase chain reaction and Western blotting were used for expression analysis.RESULTS: Using WES, a novel homozygous mutation(NM_000350: c.G3190 C, p.G1064 R) in ABCA4 gene was identified. This mutation showed co-segregation with phenotype in this family. It was not found in the 200 unrelated health controls and absent from any databases. It was considered "Deleterious" as predicted by five function prediction softwares, and was highly conserved during evolution. ABCA4 was expressed highly in the human eye and mouse retina. The p.G1064 R was located in AAA domain, may force the local backbone into an incorrect conformation, disturb the local structure, and reduce the activity of ATPase resulting in the disease pathology. CONCLUSION: We define a novel pathogenic mutation(c.G3190 C of ABCA4) of STGD. This extends the existing spectrum of disease-causing mutations and further defines the genotype-phenotype correlations.     

Novel mutations in the BEST1 gene cause distinct retinopathies in two Chinese families

AIM:To describe the clinical heterogeneity of patients with novel mutations in BEST1.METHODS:All the members in the two Chinese families underwent detailed clinical evaluations including best-corrected visual acuity,slit-lamp examination,applanation tonometry,and dilated fundus examination.Fundus autofluorescence,fundus fluorescein angiography,spectral-domain optical coherence tomography,electrooculography,and electroretinogram were also performed.Genomic DNA was extracted from venous blood for all the participants.The targeted next-generation sequencing of inherited retinal disease-associated genes was conducted to identify the causative mutation.RESULTS:A novel BEST1 missense mutation c.41T>C(p.Leu14Ser) was identified in Family 1.It was co-segregated with the phenotype of best vitelliform macular dystrophy(BVMD) and bioinformatics analysis confirmed it was harmful.Another novel BEST1 frameshift mutation c.345₃46insGGCAAGGACG(p.Glu119Glyfs*116) and a novel USH2A missense mutation c.12560G>A,p.Arg4187 His were identified in family 2 with retinitis pigmentosa(RP),which might interact and lead to the phenotype of RP.CONCLUSION:Two novel mutations in the BEST1 gene in two unrelated families with distinct phenotypes and BEST1 mutation accompanied with USH2A mutation would result in RP,which could be enormously helpful in understanding the pathogenesis of the inherited retinal disease caused by a BEST1 mutation.     

Detection of RB Germline Mutations Using Exon-by-exon Heteroduplex Analysis Compared with SSCP

Purpose: To compare heteroduplex analysis with SSCP and to develop a simple and effective method for mutational screening of RB gene.Materials and Methods: Leukocyte DNA was prepared from 12 unrelated Japanese patients with hereditary retinoblstoma. PCR combined with simultaneous nonisotopic heteroduplex and SSCP analysis was used to screen leukocyte DNA for such mutations, exon-by-exon, without the use of restriction endonu-clease digestion. PCR was conducted using 28 pairs of primers flanking all 27 ex-ons and the promoter region of the RB gene, with PCR products ranging from 159bp to 326bp. Mutations were identified by sequencing.Results: Heterozygous germline mutations were detected in 8 of 12 Japanese patients. The mutations were identified by sequencing as follows; G→C/acceptor of exon 11, T insertion/codon 389, C→T/codon 455, 33bp insertion/codon 455 (C GA), G→T/codon 533, C→T/codon 579, C deletion/codon 674, and C→T/ codon 787.Conclusion: Our results suggest that small RB gene mutations are     

Compound heterozygous mutations of CRB1 gene in a Chinese family with Leber congenital amaurosis by whole exome sequencing北大核心CSCD

Objective: To investigate the disease-causing mutation in a family with Leber congenital amaurosis (LCA). Methods: A Chinese Han pedigree with LCA from Chaoshan area was recruited in Shantou International Eye Center in August 2011.The clinical features of the families were evaluated, including medical history, best corrected visual acuity, intraocular pressure and fundus photography. The peripheral blood sample of 5 ml was collected from each of the family members for the extraction of genomic DNA.DNA of the proband was investigated by whole exome sequencing (WES) and was filtered for function of variants and inheritance pattern. Then, Sanger sequencing was performed to confirm the WES result on all the participating subjects in the pedigree. Results: There were 11 families of 3 generations in this pedigree, and 2 female LCA patients were found (Ⅱ2 and Ⅱ4) who were sisters. The parents (Ⅰ-1 and Ⅰ-2) and children (Ⅲ-1, Ⅲ-2, Ⅲ-3 and Ⅲ-4) of the patients showed normal phenotype, suggesting an autosomal recessive pattern. The patients appeared severe visual impairment during early childhood. Ophthalmic examination showed diffuse pigmentation on the retina and attenuation of retinal artery in both patients.WES of proband revealed two compound heterozygous mutations (c.2234C>T, p.T745M; c. 3488G>T, p.C1163F) of the CRB1 gene. Sanger sequencing confirmed the mutations in both patients (Ⅱ-2 and Ⅲ-4), and the parents of the patients were found to carry one mutations respectively and the other subjects with normal phenotype had neither none or only one mutation. Conclusions: The compound heterozygous mutation of c. 2234C>T, p.T745M and c. 3488G>T, p.C1163F in CRB1 is responsible for LCA pathogenesis this Chinese Han pedigree. Copyright © 2018 by the Chinese Medical Association.     

Utilization of Gene Mapping and Candidate Gene Mutation Screening for Diagnosing Clinically Equivocal Conditions： A Norrie Disease Case Study

Prenatal diagnosis was requested for an undiagnosed eye disease showing X-linked inheritance in a family. No medical records existed for the affected family members. Mapping of the X chromosome and candidate gene mutation screening identified a c.C267A[p.F89L] mutation in NPD previously described as possibly causing Norrie disease. The detection of the c.C267A[p.F89L] variant in another unrelated family confirms the pathogenic nature of the mutation for the Norrie disease phenotype. Gene mapping, haplotype analysis, and candidate gene screening have been previously utilized in research applications but were applied here in a diagnostic setting due to the scarcity of available clinical information. The clinical diagnosis and mutation identification were critical for providing proper genetic counseling and prenatal diagnosis for this family.