Detection And Functional Study Of Pathogenic Mutations That Associated With Congenital Cataract

Objective:Congenital Cataract(CC)is the most common cause of visual impairment or total blind in childhood.It is defined as any opacity of crystalline lens presenting at birth or within the first year of life in children.Hereditary cataracts are estimated to account for one fourth of all clinical type of CC,with autosomal dominant pattern in most cases or recessive and X-linked pattern in a few.CC can be classified in different clinical pattern according to the site and the shape of the opacity in lens,including total cataract,nuclear cataract,perinuclear cataract,and pulverulent sutural cataract.Among all the cataract cases,nuclear cataract has a higher incidence,which possess of approximately 30%of all cataract cases.CC has a high genetic heterogeneity.Many genes have been reported to be related with the pathogenesis of CC.Nonsymptom cataract includes AGK,BEST1,BFSP1,BFSP2,CHMP4B,CRYAA,CRYAB,CRYBA1,CRYBA4,CRYBB1,CRYBB2,CRYBB3,CRYGC,CRYGD,CRYGS,EPHA2,FYCOl,GJA3,GJA8,HSF4,P3H2,LIM2,MAF,MIP,NHS,PITX3,VIM,SLC16A12,and TDRD7.Symptom cataract includes ABHD12,CNBP,CTDP1,EYA1,FTL,GALK1,GCNT2,GFER,GJA1,JAM3,OPA 3,PAX6,RAB3GAP2,SIL1,SIX6,SLC33A1.Protein encoded by these genes have different fuctions such as:crystalline gene CRYAA、CRYBA1、CRYGC and CRYGD,gap junction gene such as GJA8、GJA3,membrane protein gene such as MIP and LIM2,transcriptional factor gene such as HSF4,MAF,PITX3 and PAX6.More and more new genes will be discovered soon.In this study,we recruited 16 Han Chinese families with typical cataract for pathogenic gene mutation screening.82 genes and area involved in the cataract panel have been screened by gene capture sequencing technique on the DNA sample extracted from the proband of these families.Sequencing was carried out to verify the mutation after the screening.Methods:1.Subjects.16 Han Chinese kindred from different Provence of China with congenital cataract were collected by China medical university,faculty of genomics.The major mode of inheritance is autosomal dominant inheritance with a few of autosomal recessive inheritance.The study was approved by Ethical Review Board of CMU and all subjects provided informed and written consent.2.Pedigree investgation and sample collection.Investgation on clinical manifestions and family history had been carried out before drawing family trees.Full consent were given and peripheral blood were extracted and stored at-80℃.3.Genomic DNA extraction Genomic DNA was extracted by using QIAamp DNA Blood Mini Kit(Qiagen,Valencia,CA,USA)from peripheral blood leucocytes using strictly following the manufacturer’s instructions,and was quantified by using NanoDrop 2000.4.Gene capture sequencing.Congenital cataract panel was used to perform gene capture sequencing on 82 genes related to the pathogenesis of congenital cataract in the genomic DNA samples from all 16 congenital cataract families in order to identify the pathogenic gene mutation.1)to order the hybridization primer which contains 82 CC related genes(CC Panel),2)NimbleGen SeqCap EZ Exome Library establishment 3)template prepare,4)Ion Torrent sequencing,5)data analysis.5.Sequencing for verifying of the mutation.PCR primers amplifying the nearby sequence of the point mutation were designed.Sanger sequencing was carried out on the PCR product after PCR fragment purification in order to verify the mutation.Results:1.Gene capture sequencing.Capture sequencing for 82 disease related genes in the probands of 16 families were completed.25 suspicious variations were identified,including 21 missense mutations,3 frameshift,and 1 deletion mutation.2.Sanger sequencing.Sanger sequencing was carried out on PCR products involving the mutation area in the gene sequence of all family members.Mutations were verified.The variation was found in affected patients and absent in all normal person in these families.Conclusion:Large scale second generation sequencing had been carried out in 16 probands of congenital cataract pedigrees by using cataract panel containing 82 cataract related genes.12 gene variations found in MIP、COL11A1、TRPM3、CRYGD、CRYBA4、TRPM3、CRYBB1、RGS6、PAX6、CRYGC、EPHA2 genes were isolated and primarily proved to be the pathogenic gene mutation of congenital cataract.Objective:Congenital Cataract(CC)is defined as any opacity of crystalline lens presenting at birth or within the first year of life in children.CC is the most common cause of childhood visual impairment or total blind with an incidence of 0.05%in mainland of China.22-30%of all children with visual disability are caused by CC.Hereditary cataracts are estimated to account for one fourth of all clinical type of CC,with autosomal dominant pattern in most cases or recessive in a few.CC can be divided in different clinical type according to opacity pattern such as total cataract,nuclear type,perinuclear type,etc.Clinical manifestation mainly appears white opacity in lens,sometimes with calcification and drop of lens capsule membrane.Visual impairment is obvious in bilateral eyes.To date,at least 80 genes are found to be related to congenital cataracts which have high genetic heterogeneity:they are GJA8 on 1q21-25,EPHA2 on 1p34-36,CRYGA,CRYGB,CRYGC,CRYGD,CRYBA2 on 2q33-36,BFSP2 on 3q21-22,CRYGS on 3q26.3,CRYAB on 11q23-24,AQPO on 12q12-14,GJA3 on 13q11-13,MAF on 15q21-22,HSF4 on 16q22-23,CRYBA1 on 17q11-12,CRYAA on 21q22.3,CRYBB1,CRYBB2,CRYBB3,CRYBA4 on 22q11.2.EPHA2 gene locates on chromosome 1p36,3964bp in length and contains 17 exons,976 AAs are encoded.EPHA2 protein is tyrosin kinase receptor,loss of function of SAM domain and ephrinA5 can cause CC.Previous study on an Australia family showed that congenital total cataract is related with mutations of EPHA2 gene.PCR sequencing of EPHA2 gene of the pedigree revealed a point mutation in intron 16(c.2826-9G>A),which caused a novel splicing site and a wide range frame-shift followed by a delayed stop codon.In this study,we established congenital cataract mouse model in C57BL/6J by constructing transgenic vector composed of cDNA of mutant EPHA2 gene,in order to confirm whether the point mutation(c.2826-9G>A)is the pathogenic mutation of the pedigree we studied before.We also observed the shape change of crystalline epithelial cells and fibrous cells and the expression of relative protein in Eph-ephrin signal pathway.Eukaryotes expression vectors were constructed and transfected to HEK-293T cell line to prove the results founded in the morphological study in animal model and discuss the pathogenic mechanisms,which could cause congenital cataract.Methods:1.The establishment of EPHA2 splicing mutation transgenic mouse modelTransgenic gene composed of mutant EPHA2 cDNA was use to proceed male pronucleus injection in fertilized ovum of mouse after linearization by restriction enzyme KpnI.Then treated fertilized ova were transferred into uteruses of female mouse for surrogacy.2 pairs of primers used for genotyping were designed in different functional sites of transgenic gene vector.Genomic DNA was extracted from tail tissue of founder mice 3 weeks after birth.PCR was carried out for genotyping by using 2 pairs of primers designed from different site.Transgenic mouse was verified to be intergration success by 2 PCR positive results.Transgenic mice with intergration were hybridized with wild type of opposite sex.The offspring were genotyped by the same pairs of primers 3weeks after birth.The hybridization was carried out till F3 mice were born.2.Phenotype analysis(1)sex ratio:The number of gender of mutant and negative siblings was recorded and analysed by using Graphpad prism to evaluate the affection of transgene to sex ratio.(2)General phenotype:The length,weight and hair were observed and measured in transgenic mice and the siblings at 8 weeks after birth.Photos were taken by digital camera.(3)Phenotype of eyes:split light and digital camera was used to observe and record the lens opacity of transgenic mice.Clinical classification was defined according to the results.The sibling mice were used as control.3.mRNA expression analysis of transgene EPHA2 cDNA sequence in mice lensTotal RNA was extracted by Trizol method from the lens of transgenic positive and the siblings who is transgenic negative.Double strand cDNA sequcnce was obtained by using reverse transcription kit.RT-PCR was then carried out by using the primers designed between EPHA2 cDNA sequence and poly(A)sequence to confirm the specific expression of transgene in transgenic mice lens.4.HE stain for morphological analysis of mouse lens.Bilateral eyeballs of transgenic mouse and controls were excised and fixed into fixation buffer after cervical vertebra malposition.Routine dehydration and clearing by using gradient ethanol solution and xylene was carried out.The eyeballs were embedded with paraffin and were incised at 5μm thickness.HE stain was carried out to exam the morphological difference between mutant mouse and wild type.Results:1.The establishment of EPHA2 splicing mutation transgenic mouse model75 founder mice were born totally after transgene injection and fertilized ova implantation to female C57BL/6 J mice.3 mice were verified as the positive founder.After crossed with wild C57BL/6J mice with opposite gender,3 generations(in total)were born.Mice were determined as transgenic positive after PCR genotyping with a positive rate of 38%.2.Phenotype analysis(1)Gender ratio:The ratio of male to female is 1:1.19,which has no significant difference by biological statistics.(2)General phenotype:transgenic positive and the control mice 8 weeks after birth were taken to be measured and assessed in length,weight and hair.No significant difference was found.(3)Phenotype of eyes:all transgenic positive mice presented opacity in lens by slit lamp diagnosis.The clinical classification is total CC,i.e.the crystalline lens is all nepheloid without clear part.Congenital microphthalmia was found in some cases.Transgenic mice model with EPHA2 gene splicing mutation was established successfully.3.mRNA expression analysis of transgene EPHA2 cDNA sequence in mice lensPositive result was obtained in the RT-PCR amplification by using cDNA obtained from lens of transgenic mice.The result showed that there is a specific expression of transgene sequence in transgenic mice lens.4.HE stain of transgenic mouse lens slice:Wild type C57BL/6J mouse presented a clear and complete construction of lens.The arrangement of lens epithelial cells and lens fiber cells is dense and regular.Bubble-like structure appeared around the lens epithelial cells in mutant mouse.The lens tissue structure was incomplete,which indicated a more fragile tendency.Conclusion:EPHA2 splicing mutation transgenic mouse model has been established successfully.The point mutation of intron 16 of EPHA2 gene(c.2826-9G>A)has been proved as the pathogenic mutation of the congenital cataract pedigree in animal model.