Based On The Identification Of New HVA Genes In Two Lines And The Phenotypic Difference Analysis Of HVA Caused By Different Pathogenic Genes

Objective:(1)To screen new pathogenic genes of two familial hereditary vitreous amyloidosis(HVA)families and analyze their clinical and genetic characteristics.(2)To explore the relationship between Gly83 Arg mutation as well as upstream c.-743 A > T mutation of Transthyretin(TTR)and the phenotypic difference of vitreous opacity.Methods:Clinical data and genetic characteristics of members from two HVA families were collected,the peripheral venous blood of 6 patients and 2 carriers from two families were sampled for whole exome sequencing,and gene annotation was carried out for the results.The genetic characteristics of the families were analyzed by co-segregation method,and the suspected pathogenic genes were determined by relevant biological information software.Finally,the mutant genes were verified by Sanger sequencing.The data of preoperative B-ultrasonography were collected from 2 families who had undergone pars plana vitrectomy(PPV).The phenotypes of vitreous opacity were classified and the differences of gene mutations and phenotypes were compared.Results: 13273 amyloidosis-related genes were identified by Phenolyzers and 54 seed genes were identified by phenotype.The above seed genes were searched for 130188 SNV results and 21557 INDEL results,and 38 SNV and INDEL variants that were closely related to amyloidosis were obtained.These 38 sites were screened to a certain extent,namely non-coding and synonymous mutations were eliminated,and 10 suspected mutation sites related to diseases were obtained on this basis.The heterozygous mutations of TTR c.311 T > A and TTR c.307 G > C were found in pedigree 1 and pedigree 2 patients after removing the mutations of MAF ≥ 1% in the 1000-person genome database.According to the above results,Sanger sequencing and co-segregation were carried out in each family.In family 1,HVA patients Ⅱ 7(proband’s sister)and Ⅱ 9(proband)all carried TTR c.311 T > A heterozygous mutation,while phenotypic normal members Ⅲ 7,Ⅲ 8,Ⅲ10(proband’s niece)and Ⅳ 7(proband’s sister’s granddaughter)did not carry this mutation.HVA patients Ⅲ 2(proband),Ⅲ 4,Ⅲ 14,Ⅲ 17(sister of proband),Ⅲ 9,Ⅲ 15(brother of proband)in pedigree 2 all carried TTR c.307 G > C heterozygous mutation,while phenotypic normal members Ⅳ 17(daughter of sister Ⅲ 4 of proband),Ⅳ 32(son of brother Ⅲ 9 of proband)and Ⅳ 43(son of brother Ⅲ 15 of proband)did not carry TTR c.307 G > C heterozygous mutation,which was consistent with family co-segregation.It was confirmed that the pathogenic gene of HVA family 1 was TTR c.311 T > A,the pathogenic gene of HVA family 2 was TTR c.307 G > C,and MEFV c.442 G > C might be a new pathogenic gene of HVA.There were 3 loose type patients in family 1.Among the patients in family 2,8 were loose and 15 were compact.However,the presence or absence of TTR c.-743 A > T and/or Gly83 Arg mutations had no significant effect on the vitreous opacity phenotype of 6 patients according to B-ultrasound phenotype.Conclusion:(1)The disease-causing genes of family 1 and family 2 are TTR c.311T>A and TTR c.307G>C respectively;It is very likely that MEFV c.442G>C is the pathogenic genes of HVA,however,the association with vitreous amyloidosis needs to be further verified in the population.(2)There is no significant association between TTR c.-743A>T and patient phenotype differences.