Genetic Mapping And Mutation Analysis In Two Kinds Of Ophthalmic Genetic Diseases

ObjectiveCongenital cataract is the most common cause of treatable childhood visual impairment and half of them are estimated to have genetic cause.Inherited cataract is a lens disease with remarkable phenotypic and genetic heterogeneity.It shows considerable inter and intra familial clinical variation but high penetrance,and it most often presents as an autosomal dominant Mendelian trait.To date,at least 39 mapped loci including 19 causative genes associated with isolated congenital cataracts were reported.These genes can be grouped as crystallins(CRYAA,CRYAB,CRYBA1, CRYBA4,CRYBB1,CRYBB2,CRYBB3,CRYGC,CRYGD,CRYGS),membrane transport proteins(GJA3,GJA8,MIP),transcription factors(HSF4,PITX3,MAF), cytoskeletal proteins(BFSP2) and chromatin modifying protein 4B(CHMP4B).The identification of genes causing inherited cataract will improve our understanding of the mechanisms underlying cataractogenesis and will also provide further insights into development and physiology of normal lens.In this study,we have collected five Chinese families with congenital bilateral cataract and tried to identify the genetic defect leading to the cataract in these families.Norrie disease is a rare X-inked recessive condition characterized by congenital blindness in males due to degenerative and proliferative processes in the neuroretina. The NDP gene is the Norrie disease causative gene.We have collected a four generation Norrie disease family and tried to find the pathogenic mutation in this family.In addition,we planed to perform prenatal diagnosis for a fetus with high risk in this family.Materials and MethodsProbands and family members from 5 Chinese families with congenital cataracts underwent detailed ophthalmic examination.After informd consent was obtained, peripheral blood samples(5 -10ml per individual) were collected from family members (affected and unaffected).Genomic DNA was extracted from peripheral blood lymphocytes using standard phenol/chloroform-proteinase K method.25 of the known ADCC loci was chosen to be screened by linkage analysis using 62 microsatellite markers at chromosome 1p36,1p22.3,1q21-25,2p12,2p24-pter,2q33-36,3p22-24.2,3q21-25,3q27.3,10q25,11q22.1-23.21,12q13-14,13q12.11,14q22-23,15q21-22,16q23.1,16q22.1,17p13.3,17q11-12,19q13.33,20p11.23-p12.1,20p12.1,20q11.22,21q22.3,22q11.2-12.1.These markers were selected and their PCR primers were designed according to the genomic sequences on the UCSC Genome Browser on Human Mar.2006 assembly.PCR were performed using standard conditions.The PCR products were separated by electrophoresis on 8% denaturing polyacrylamide gel and allele fragments were detected by routine silver staining.Two-point linkage LOD scores were calculated using the MLINK program in the LINKAGE software package(version 5.2).Haplotypes were constructed for each family according to the allele information.To detect pathogenic mutations in the gene CRYBB1,CRYBB2,CRYBB3,CRYBA4,HSF4,PTCHD2,KAZRIN,CASP9,SPEN,RSC1A1,PRMD2,PAX6, PCR primers were designed to amplify all coding exons and their flanking intronic sequences.PCR products were examined on 2%agarose gels to confirm the amplification and purified using the TaKaRa Agarose Gel DNA Purification Kit.The purified PCR fragments were submitted for direct automatic sequencing.In family 4,to confirm the mutation in PTCHD2 gene(INV20+5G＞A),the exon 20 of PTCHD2 gene and its flanking intronic sequences were amplified from their genomic DNA of all family members and 268 unrelated normal controls,and the resulted PCR fragments were analyzed by ApalI digestion and 2%agarose gels electrophoresis.The mutation INV20 + 5G＞A will introduce a ApalI restriction site.In order to determine whether this change(INV20 + 5G＞A) affects mRNA splicing, primers were designed to amplify genomic region spanning exon 19 to 21 and the PCR products from a patient were cloned into pcDNA3.1 vector to construct a PTCHD2 minigene expressing vector.COS-7 cell and HeLa cell were cultured and transfected with the vector;then the total RNA was extracted;reverse transcription- PCR was carried out using primer located in exon 19 and exon 21 of PTCHD2 gene respectively.To confirm the c.356G＞A(p.Arg119His) mutation of HSF4 gene in family 5,a mismatch primer was designed to introduce a NsbI restriction site into the normal allele by nested PCR,and the resulted PCR fragments of 188bp were analyzed by NsbI digestion and polyacrylamide gel electrophoresis.In the Chinese family with Norrie disease we collected,the clinical diagnoses were made according to familial history,clinical signs and B ultrasonic examination.After informed consents were obtained,peripheral blood samples were collected from all available family members.Genomic DNA was extracted from peripheral blood lymphocytes using the standard SDS-proteinase K-phenol/chloroform method.The three exons and all intron-exon boundaries of the NDP gene were amplified by PCR using three pairs of primers from the proband and his mother,and subjected to automatic DNA sequence.The causative mutation was confirmed by restriction enzyme analysis and PAGE analysis in all family members.For the fetus pregnant in a mother carring the causative mutation,Genomic DNA was extracted from chorionic villi.Molecular examinations in two aspects were carried out for precise prenatal diagnosis:two-point linkage analysis using microsatillite markers and Restriction analysis.ResultsThe pattern of phenotype transmission in all 5 families with congenital cataract suggested a mode of autosomal dominant inheritance.Of the 5 families,2 were diagnosed as nuclear cataract,2 as posterior polar cataract,and 1 as polymorphic cataract.Microsatellite polymorphic markers were successfully genotyped by polyacrylamide gel electrophoresis and silver staining.Two-point linkage analysis did not provide evidence for linkage to markers from chromosome regions 1p22.3,1q21-25,2p12,2p24-pter,2q33-36,3p22-24.2,3q21-25,3q27.3,10q25,11q22.1-23.21,12q13-14,13q12.11,14q22-23,15q21-22,16q23.1,17p13.3,17q11-12,19q13.33,20p11.23-p12.1,20p12.1,20q11.22,21q22.3 in each of the 5 families.However,we obtained a maximum lod score of 2.71 atθ=0 with marker D22S689 at chromosome 22q11.2-12.1 in family 3,indicative of linkage.Haplotypes were constructed for the markers analyzed on chromosome 22 and cosegregation was observed in all affected individuals with all markers.It suggested possible involvement of the CRYB gene cluster in the cataractogenesis in the family 3.A maximum LOD score of 3.36 was obtained with marker D1S1151 atθ=0 in family 4 that was the evidence of the linkage.Haplotypes were constructed for the markers analyzed on chromosome 1p36.IndividualⅢ8 was recombinant at marker 1pTTTC andⅢ6 was recombinant at marker 1pCAAA and cosegregation was observed in all affected individuals between marker 1pTTTC and 1pCAAA.The cataract locus was thus mapped to the 7.3cM region bound by 1pTTTC and 1pCAAA,which overlaps with the smallest interval reported so far.A maximum LOD score of 2.63 was obtained with marker D16S3085 atθ=0 in family 5,and haplotype analysis suggested possible involvement of the HSF4 gene in the cataractogenesis in the family 5.Mutation profiling of the candidate genes at the cataract loci was done in the 3 families which had been linked to these loci.In family 3,mutation analysis of CRYBB1,CRYBB2,CRYBB3 and CRYBA4 showed no disease associated changes. While in family 4,DNA sequencing of the 7 candidate genes(PTCHD2,KAZRIN, CASP9,SPEN,RSC1A1,PRMD2,PAX6) revealed a G＞T substitution in intron 20 of PTCHD2(INV20 + 5G＞A).Restriction enzyme analysis showed the same substitution in all affected family members,but not in the 268 normal individuals.RT-PCR analysis of PTCHD2 transcripts showed that the INV20 + 5G＞A substitution does not affect mRNA splicing.Sequencing of HSF4 in family 5 revealed a c.356G＞A change in exon 3 which leads to the substitution of Arg by His at the 119th coden.Restriction enzyme analysis showed cosegregation of the same substitution with all affected family members,but not with the 100 normal individuals.In the family suffering from Norrie disease,sequencing of the affected male proband and his mother revealed a missense mutation in NDP(c.220C＞T,p.Arg74Cys) in both of them,while the proband is a hemizygote for this mutation and his mother is a heterogenous carrier.Restriction enzyme analysis showed 4 female family members (Ⅲ3,Ⅳ3,Ⅲ5 andⅡ5) with normal phenotype also carrying the same mutation. Linkage analysis using microsatillite markers and restriction analysis showed the fetus is a female mutation-carrier. ConclusionsHere we report five Chinese families with congenital cataract,which were inherited in autosomal dominance pattern in these families.Both the phenotypic and the genetic heterogeneity were obvious in these families,since there are 2 families suffering from nuclear cataract,2 families showing posterior polar cataract,and the rest one manifesting the polymorphic cataract;and they were mapped at least 3 different genetic loci.Linkage analysis of family 1 and 2 has excluded all known candidate genes and most of known candidate loci,which indicated that other loci may be responsible for congenital cataract in these two families.In family 3,linkage analysis suggested the phenotype possibly link to chromosome 22q11.2-12.1 and definitely not link with all other loci;however,no disease-associated mutations were detected in all four candidate genes,which indicate the possibility of unknown pathogenic mechanisms including novel genetic locus.In family 4,the linkage analysis proved a definite link between the disease phenotype and the chromosome 1p36 region;our haplotype analysis has narrowed the disease interval into a 2.4 Mb region,which is smaller than the known 7.3 Mb smallest interval in this locus;and sequencing showed no disease-causing mutation in PTCHD2, KAZRIN,CASP9,SPEN,RSC1A1,PRMD2 and PAX6 genes.The cataract phenotype in family 5 was suggested to link to chromosome 16q22.1 and sequencing of HSF4 gene in patients revealed a novel pathogenic mutation, c.356G＞A,which leads to the substitution of Arg by His at the highly conserved 119th coden.In the Chinese family suffering from Norrie disease,we identified a pathogenic mutation in NDP gene,c.220C＞T,the same causative mutation in a foreign Norrie disease patient.This is the first report of mutation detection in a Chinese Norrie disease family.In addition,we performed prenatal diagnosis for a high-risk fetus and found it is a female fetus carring the causative mutation,c.220C＞T.