Molecular Genetics Study Of Congenital Cataract

Paper1Molecular Genetic Study of Isolated Congenital CataractCongenital cataract （CC） is defined as any opacity of the crystalline lens presenting at birth or within the first year of life. With an incidence of0.05%in the mainland of China, it is particularly serious as the major cause of childhood visual impairment. Hereditary cataracts, isolated or part of a syndrome, are estimated to acount for one third of congenital cataracts with remarkable phenotypic and genetic heterogeneity. It shows considerable inter and intra familial clinical variation with high penetrance, and most often presents as an autosomal dominant Mendelian trait. To date, at least42mapped loci including29causative genes associated with isolated congenital cataracts were reported. These genes can be grouped as crystallins （CRYAA, CRYAB, CRYBA1, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD and CRYGS）, membrane transport proteins （GJA3, GJA8, MIP and LIM2）, transcription factors （HSF4, PITX3, MAF, CHX10, EYA1and FOXE3）, cytoskeletal proteins （BFSP1and BFSP2）, chromatin modifying protein4B （CHMP4B） and the others （FTL, OCRL, GCNT2and GALK1）.Chapter1Mutation Identification and Functional Study of Congenital Cataract in Three Different FamiliesLoci for autosomal dominant posterior polar cataract-1（CTPP1; MIM#116600） and total CC were both been mapped to chromosome1p36in1997and2005, respectively, without any further determination of disease causing gene. In the previous study, a genetic linkage to the same region was confirmed by linkage analysis in a five-generation Chinese Han family （Family1） with isolated congenital posterior polar cataract. Taken together with the published data in the Australian family, our haplotype analysis in the Chinese family reduced the critical region to an interval between marker D1S436and D1S199, representing a genomic extent of4Mb containing52RefSeq genes.Firstly, we sequenced genomic DNA from an affected individual in Family1for six candidate genes, DNAJC16, EPHA2, FBLIM1, PAX7, RSC1A1and SPEN, and identified in EPHA2a missense mutation, C.2819C<T （p.T940I）. CDD （Conserved Domain Dtabase） search showed that the mutation replaces the threonine at residue940, one of the two amino acids in the C-terminal sterile alpha motif （SAM） domain of the EPHA2receptor that form the oligomerization interface. The C.2819C>T mutation creates an Mbo I restriction site in the mutant allele. By restriction analysis using Mbo I, this mutation was confirmed in all affected individuals but was not detected in unaffected family members or in other202unrelated Chinese controls. To investigate whether EPHA2is also mutated in other cataract families mapped to1p36, we sequenced DNA samples from the British family （Family2） with autosomal dominant posterior polar CC and the Australian family （Family3） with autosomal dominant total CC mentioned above. A deletion of2bp （c.2915₂916delTG） in exon17, the last exon of EPHA2, and a single base substitution （c.2826-9G>A） in intron16were found in Family2and Family3, respectively. The c.2915₂916delTG （p.V972GfsX39） frameshift mutation is predicted to produce a mutant EPHA2protein with a novel C-terminal polypeptide of39amino acid residues. Using a minigene assay, we confirmed that the c.2826-9G>A substitution creates a novel splice acceptor site causing an intronic sequence of7bp to be included in the processed transcript. This aberrant splicing is predicted to result in translation of a novel C-terminal polypeptide of71amino acid residues of which the last39are identical to that of the novel polypeptide produced by the c.2915₂916delTG frameshift mutation. In the following text, we quote the encoded proteins of mutant p.T940I, c.2915₂916delTG and c.2826-9G>A as T940I, C39and C71respectively.EPHA2is a member of Eph family and widely expresses in epithelial cells. Stimulated by its ligands, ephrinAs, EPHA2can modulate multiple development processes and tumorigenesis by regulating cell migration and apoptosis. To further investigate the pathologic role of EPHA2during the onset of cataract, we first determined the expression of EPHA2in lens. Using RT-PCR, we detected mRNA expression of EPHA2in a human lens epithelial cell line （SRA01/04） and human anterior lens capsule tissues. Furthermore, immunohistochemical findings on lens of BALB/C mice revealed that EPHA2was most abundantly expressed in lens epithelial cells and cortical fiber cells.To investigate the role of mutant EPHA2proteins in cell migration and apoptosis, SRA01/04cells were transfected with wild type and mutant EPHA2expression constructs respectively. Without any observation of significant difference, we consequently hypothesized that the "loss-of-function effect" may be the common pathogenic mechanism of the three distinct mutations of EPHA2. Furthermore, there’s an obviously lower level of mutant proteins compared to wild type after transfection, while pretreatment of proteasomal inhibitor （MG-132） increased the amount of them. These data suggest that the mutant proteins are susceptible to degradation, which represents the aforementioned "loss-of-function effect".It is well documented that ligand binding promotes rapid aggregation, autophosphorylation and consequent degredation of EPHA2. Meanwhile, the receptor aggregation is necessary for its phosphorylation. As is well known, SHIP2and LMW-PTP are the two molecules recruited to the C-terminal domains of EPHA2participating in the maintenance of its stability. With yeast-two-hybrid assay, we found out an impaired interaction between T940I and SHIP2, and increased interactions between C39and C71themselves, suggesting that those three mutant proteins may be degraded through different pathways. Besides, yeast-two-hybridization also shows increased interactions of C39and C71to LMW-PTP and the wild type EPHA2, implying that a "dominant negative effect" and a "gain-of-function effect" may also contribute to the onset of cataract. Chapter2Genome Mapping of Congenital Total Cataract in a Family of QuadrupletsWe performed linkage analysis in a two-generation Chinese Han family with congenital total cataracts. This is a family with quadruplets while three of them are suffering from congenital total cataracts accompanied by microphthalmia and microcornea. Firstly, we excluded the possibility of Congenital Rubella Syndrome （CRS）, which is a group of physical abnormalities （especially congenital cataract） that have developed in an infant as a result of maternal infection and subsequent fetal infection with rubella virus. In the following linkage analysis, we excluded40reported loci for human CCs and9candidate loci related to the development of cataract and microcornea, and finally identified a novel locus in chromosome6q15-qter cosegregating with the phenotype of the family. Fourteen candidate genes （GJAJ, GJA10, HSF2, COL10A1, SLC35F1, SLC22A16, SLC2A12, SAMD3, PRDM1, EPHA7, FYN, FOXO3, SNX and WASF1） from the previously defined interval were selected based on their expression in lens and their known or putative function. However, no segregating mutations were identified in any of them. Paper2Mutation Identification and prenatal diagnosis of a Chinese pedigree with Lowe’s syndromeLowe syndrome （Lowe Oculocerebrorenal syndrome, OCRL, MIM309000）, also named the oculocerebrorenal syndrome of Lowe, is a rare X-linked disorder characterized by mental retardation, congenital cataracts, and Fanconi syndrome of the proximal renal tubules, and the estimated incidence in general population is1/500000. OCRL, located in Xq25-q26, is the only known gene accounts for Lowe syndrome.We identifid the pathogenic mutation in OCRL and provided a prenatal diagnosis in a Chinese Han family with Lowe syndrome. By microsatellite linkage analysis, the OCRL locus could not be excluded, and the sequence analysis of the proband revealed a reported nonsence mutation, c.2032C>T, in the18th exon of OCRL gene. Restriction analysis showed that this mutation existed in the proband and three obligate carriers, but not in the other7family members or a fetus of a high risk pregnant woman in the family. These data suggested that the nonsence mutation in OCRL gene, c.2032C>T, was the pathogenic mutation of the family and the fetus didn’t carry the mutation.