摘要目的 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.

abstractsObjective 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.