Roshan Mascarenhas, Vijaya Pai H, Lavanya G Rao, Shama Prasada K, Santhiya S T, Graw Jochen, Gopinath P M, Satyamoorthy K
Department of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal, Karnataka state, India.
Mol Vis. 2010 May 22;16:887-96.
Identification of causal mutation in the crystallin, connexin, and paired box gene 6 (PAX6) genes associated with childhood cataract in patients from India.
In this study, forty eight members from seventeen families and 148 sporadic cases of childhood cataract were evaluated. Clinical and ophthalmologic examinations were performed on available affected and unaffected family members. Samples of genomic DNA were PCR amplified to screen for mutations in the candidate genes viz., alpha-A crystallin (CRYAA), beta- B2 crystallin (CRYBB2), gamma-A crystallin (CRYGA), gamma-B crystallin (CRYGB), gamma-C crystallin (CRYGC), gamma-D crystallin (CRYGD), gap junction alpha-3 (GJA3), gap junction alpha-8 (GJA8), and PAX6 based on polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) analysis. Samples showing any band mobility shift were subjected to bidirectional sequencing to confirm the variation. Co-segregation of the observed change with the disease phenotype was further tested by restriction fragment length polymorphism (RFLP) for the appropriate restriction site.
DNA sequencing analysis of CRYAA, CRYBB2, CRYGA-D, GJA3, GJA8, and PAX6 of the affected members of a family (C-35) showed a novel heterozygous missense mutation C>A at position 229 in CRYGD in three affected members of family C-35 with anterior polar coronary cataract. This variation C229A substitution created a novel restriction site for AluI and resulted in a substitution of highly conserved arginine at position 77 by serine (R77S). AluI restriction site analysis confirmed the transversion mutation. Analysis of the available unaffected members of the family (C-35) and 100 unrelated control subjects (200 chromosomes) of the same ethnic background did not show R77S variation. Data generated using ProtScale and PyMOL programs revealed that the mutation altered the stability and solvent-accessibility of the CRYGD protein.
We describe here a family having anterior polar coronary cataract that co-segregates with the novel allele R77S of CRYGD in all the affected members. The same was found to be absent in the ethnically matched controls (n=100) studied. Interestingly the residue Arg has been frequently implicated in four missense (R15C, R15S, R37S, and R59H) and in one truncation mutation (R140X) of CRYGD. In two of the reported mutations Arg residues have been replaced with Serine. This finding further expands the mutation spectrum of CRYGD in association with childhood cataract and demonstrates a possible mechanism of cataractogenesis. Screening of other familial (n=48) and sporadic (n=148) cases of childhood cataract, did not reveal any previously reported or novel mutation in the candidate genes screened.
鉴定与印度儿童白内障患者相关的晶状体蛋白、连接蛋白和配对盒基因6（PAX6）基因中的致病突变。
在本研究中，对来自17个家庭的48名成员和148例儿童白内障散发病例进行了评估。对现有的患病和未患病家庭成员进行了临床和眼科检查。基于聚合酶链反应和单链构象多态性（PCR-SSCP）分析，对基因组DNA样本进行PCR扩增，以筛选候选基因即α-A晶状体蛋白（CRYAA）、β-B2晶状体蛋白（CRYBB2）、γ-A晶状体蛋白（CRYGA）、γ-B晶状体蛋白（CRYGB）、γ-C晶状体蛋白（CRYGC）、γ-D晶状体蛋白（CRYGD）、缝隙连接α-3（GJA3）、缝隙连接α-8（GJA8）和PAX6中的突变。显示任何条带迁移率变化的样本进行双向测序以确认变异。通过针对适当限制性位点的限制性片段长度多态性（RFLP）进一步测试观察到的变化与疾病表型的共分离。
对一个家族（C-35）的患病成员的CRYAA、CRYBB2、CRYGA-D、GJA3、GJA8和PAX6进行DNA测序分析，发现在患有前极性冠状白内障的家族C-35的三名患病成员中，CRYGD基因第229位存在一个新的杂合错义突变C>A。这种C229A替换产生了一个新的AluI限制性位点，并导致第77位高度保守的精氨酸被丝氨酸取代（R77S）。AluI限制性位点分析证实了这种颠换突变。对该家族（C-35）现有的未患病成员和100名相同种族背景的无关对照受试者（200条染色体）进行分析，未发现R77S变异。使用ProtScale和PyMOL程序生成的数据表明，该突变改变了CRYGD蛋白的稳定性和溶剂可及性。
我们在此描述了一个患有前极性冠状白内障的家族，在所有患病成员中该疾病与CRYGD的新等位基因R77S共分离。在所研究的种族匹配对照（n = 100）中未发现相同情况。有趣的是，CRYGD的四个错义突变（R15C、R15S、R37S和R59H）和一个截短突变（R140X）中经常涉及精氨酸残基。在两个已报道的突变中，精氨酸残基被丝氨酸取代。这一发现进一步扩展了与儿童白内障相关的CRYGD的突变谱，并证明了白内障发生的一种可能机制。对其他家族性（n = 48）和散发性（n = 148）儿童白内障病例的筛查，未在筛查的候选基因中发现任何先前报道的或新的突变。
