CAJ | 학술논문

目的揭示一疑似糖原贮积症(GSD)Ⅲ型家系发病的分子遗传学机制,并对其致病的AGL基因的新突变做致病性鉴定.方法先证者于2012年初在福建医科大学附属二院诊断为疑似GSD患者,并转诊至中山大学中山医学院医学遗传学教研室.首先对先证者的AGL基因的所有编码区及各编码区与内含子交界部位的序列进行直接测序.在检出突变后,再对其父母的相应位点进行检测并确认患儿突变的来源.对所发现的杂合新突变再通过克隆测序予以确证,并对新突变的致病性进行一系列鉴定,包括对100名健康对照(近2年来人群中随机取样获得)进行变性高效液相色谱(DHPLC)筛检以统计突变的频率；在11个跨物种间对突变所在氨基酸序列的保守性进行分析；比对并分析突变蛋白和正常蛋白的高级结构的差异程度.结果先证者为“c.100C＞ T(p.R34X)”无义突变和“c.1176_1178 del TCA(p.392delHis)”缺失突变的复合杂合子.其中,p.R34X国外已报道为致病性突变,而c.1176_1178 del TCA为新发现的缺失突变.其父为p.R34X无义突变的携带者；其母为c.1176_1178 del TCA缺失突变的携带者.dbSNP数据库、HGMD数据库及近几年最新文献的检索确认c.1176_1178 delTCA为一新突变,并排除多态性变异的可能.11个跨物种的保守性分析结果表明:该突变位点所在氨基酸在进化上具有高度保守性.突变蛋白和正常蛋白预测的高级结构的比对结果显示:此缺失突变造成了AGL蛋白空间立体构象的显著改变.结论所发现的c.1176_1178 del TCA(p.392delHis)缺失突变是一新的致病性突变,它和“c.100C＞ T(p.R34X)”无义突变是先证者患GSDⅢa病的根本原因,这两种突变分别是由母亲和父亲各遗传一个而来.
목적 게시일의사당원저적증(GSD)Ⅲ형가계발병적분자유전학궤제,병대기치병적AGL기인적신돌변주치병성감정.방법 선증자우2012년초재복건의과대학부속이원진단위의사GSD환자,병전진지중산대학중산의학원의학유전학교연실.수선대선증자적AGL기인적소유편마구급각편마구여내함자교계부위적서렬진행직접측서.재검출돌변후,재대기부모적상응위점진행검측병학인환인돌변적래원.대소발현적잡합신돌변재통과극륭측서여이학증,병대신돌변적치병성진행일계렬감정,포괄대100명건강대조(근2년래인군중수궤취양획득)진행변성고효액상색보(DHPLC)사검이통계돌변적빈솔；재11개과물충간대돌변소재안기산서렬적보수성진행분석；비대병분석돌변단백화정상단백적고급결구적차이정도.결과 선증자위“c.100C＞ T(p.R34X)”무의돌변화“c.1176_1178 del TCA(p.392delHis)”결실돌변적복합잡합자.기중,p.R34X국외이보도위치병성돌변,이c.1176_1178 del TCA위신발현적결실돌변.기부위p.R34X무의돌변적휴대자；기모위c.1176_1178 del TCA결실돌변적휴대자.dbSNP수거고、HGMD수거고급근궤년최신문헌적검색학인c.1176_1178 delTCA위일신돌변,병배제다태성변이적가능.11개과물충적보수성분석결과표명:해돌변위점소재안기산재진화상구유고도보수성.돌변단백화정상단백예측적고급결구적비대결과현시:차결실돌변조성료AGL단백공간입체구상적현저개변.결론 소발현적c.1176_1178 del TCA(p.392delHis)결실돌변시일신적치병성돌변,타화“c.100C＞ T(p.R34X)”무의돌변시선증자환GSDⅢa병적근본원인,저량충돌변분별시유모친화부친각유전일개이래.
Objective To reveal the molecular genetic pathogenesis of the glycogen storage disease type Ⅲ (GSD Ⅲ) and to provide a prerequisite for prenatal gene diagnosis in future.Method All the coding regions as well as the border areas between exons and introns of the AGL gene and the parental relevant mutation sites were directly sequenced,so that to affirm the origin of the mutation.Then,detected novel heterozygous mutation was confirmed by cloning sequencing.Finally,definite diagnoses of the novel mutation were performed by a series of identification methods,including screening for the 100 normal controls by DHPLC in order to count the mutational frequency,analyze the conservative of the mutant amino acid sequence from 11 kinds of species and comprise the difference of the tertiary structure between the mutant protein and the normal one.Result The patient had compound heterozygous mutations,the c.100C ＞T(p.R34X) nonsense mutation and c.1176_1178 del TCA deletion mutation.The p.R34X has been reported abroad,but the 1176_1178 del TCA/p.His392fs mutation is a novel one.The proband's father is heterozygous with the p.R34X mutation while his mother carries the c.1176_1178 del TCA mutation.The result from searching the dbSNP database,HGMD database and papers published in recent years showed that the c.1176_1178 del TCA is a novel mutation,but not an SNP.Conservative analysis results in 11 species indicate that the amino acid of the mutation site is highly conserved in the stage of evolution.Comparison results between the mutant protein and the normal one demonstrate that the deletion mutation results in the obvious variation of the spatial conformation of AGL protein.Conclusion The "c.1176_ 1178 del TCA (p.392delHis)" mutation is a novel pathogenic mutation.This mutation and the c.100C ＞ T(p.R34X) is the cause that the proband suffer from the GSD Ⅲ a disease.These two mutations are inherited from mother and father respectively.The methods from this paper can be used for further prenatal gene diagnosis.