【作者】 孙艺；

【导师】 王荣光； 袁慧军；

【作者基本信息】 中国人民解放军军医进修学院 ， 耳鼻咽喉科学， 2010， 博士

【摘要】 耳聋是一类严重影响人类生活质量的常见疾病,在所有耳聋患者中遗传性耳聋约占60%,主要表现为单基因遗传病,偶为双基因复合突变致病。由环境因素(如医疗因素、环境暴露、创伤、药物等)或基因与环境共同作用所致。本研究应用单核苷酸多态和微卫星标记作为遗传标记,分别在两个常染色体显性遗传非综合征型低频感音神经性耳聋大家系中定位及筛查出致病基因,在低—中频感音神经性耳聋散发患者中进行了WFS1基因第5、8外显子的突变筛查,并对一罕见的Pfeiffer综合征患者进行了致病基因筛查。主要研究发现概述如下：1.常染色体显性遗传非综合征型耳聋家系及低—中频感音神经性耳聋散发病例致病基因研究HB-S037家系为一个5代相传的耳聋大家系,耳聋患者的听力学表型为迟发性、渐进性、以低频听力下降为主的感音神经性耳聋。选取SNP作为遗传标记,应用Affymetrix 5.0 SNP芯片进行全基因组扫描及连锁分析,将HB-S037家系的致病基因初步定位于第11号染色体11q13.4-14.1之间(最大LOD值=4.346),选取初步定位区域内及附近的12个微卫星标记进行精细定位及单倍型分析,将致病基因定位于微卫星标记D11S987和D11S4172之间的区域(最大LOD值=4.18),与已知的DFNA11位点重叠。对定位区域内候选基因MY07A的49个外显子直接测序发现,在MY07A第17外显子有一个新的突变位点c.2011G>A,该位点突变与此家系疾病表型共分离,并引起编码第671位的甘氨酸替换为丝氨酸(G671S),该位点在多物种之间保守,100个听力正常人未发现此突变。通过蛋白质分子三维结构计算机模拟分析：该突变位于myosin蛋白头部换能区域。换能区域第727位苯丙氨酸(F727)与中转环第477位酪氨酸(Y477)和第482位异亮氨酸(I482)相互作用构成了保守的“疏水口袋”,该突变导致编码第671位的无侧链的甘氨酸替换为带有侧链的丝氨酸,并通过其侧链影响保守的“疏水口袋”结构,对临近的中转环第477位酪氨酸产生了立体阻碍作用,由此导致myosin蛋白结构、功能的变化。下一步的功能研究有利于阐明DFNA11的发病机制。BJ-L046家系是一个5代相传的耳聋大家系,耳聋患者的听力学表型为迟发性、渐进性、以低频听力下降为主的感音神经性耳聋。选取SNP作为遗传标记,利用Affymetrix 6.0 SNP基因芯片进行全基因组扫描连锁分析,将BJ-L046家系的致病基因定位于第4号染色体4p9.85-12.35之间的区域(最大LOD值=2.11),与已知的DFNA6/14/38位点重叠。对定位区域内候选基因WFS1的8个外显子直接测序,发现第8外显子有一个新的突变位点,即c.2086 C>T的杂合突变,该位点突变与此家系疾病表型共分离,并引起编码第696位的组氨酸替换为酪氨酸(H696Y),该突变位点在多物种之间保守,100个听力正常人中未发现此突变。本研究还选取了解放军总医院聋病分子诊断中心2002年～2010年收集的37名散发低—中频感音神经性耳聋患者DNA样品,进行WFS1基因第5和第8外显子测序突变筛查。发现1例患者WFS1第8外显子的新突变,即c.2108G>A的杂合突变,该突变引起编码第703位的精氨酸替换为组氨酸(R703H),该突变位点在多物种之间保守,100个听力正常人中未发现此突变。证实在低--中频感音神经性耳聋散发患者中进行WFS1基因热点突变筛查的必要性。2. Pfeiffer综合征致病基因的筛查及分子机制研究Pfeiffer综合征是一种罕见的常染色体显性遗传病,发病率为1／100,000,根据临床表现的严重程度不同而分为3型。本研究的患者表现为颅缝早闭、尖头畸形、斜形头、眼球突出、面中部发育不良、肘关节强直、传导性耳聋。根据临床表型诊断为Pfeiffer综合征Ⅰ型,对其可能的致病基因FGFR1、FGFR2、FGFR3的所有外显子直接测序,鉴定了FGFR2的第8外显子c.1021 A>C的杂合突变,此突变位点将编码第341位的苏氨酸替换为脯氨酸。对其父母的突变筛查显示该突变为生殖细胞嵌合体或新生突变。蛋白质分子三维结构计算机模拟分析,提示编码第342位的半胱氨酸与编码第278位的半胱氨酸之间形成二硫键,341位点毗邻342位点,推测该突变引起的氨基酸构象的变化导致编码第342位的半胱氨酸与编码第278位的半胱氨酸之间二硫键遭到破坏,由此产生了自由的半胱氨酸残基形成分子间的二硫键,受体不依赖配体二聚化并被激活,导致FGFR2突变后功能增强。本研究结果为该家庭下一步遗传咨询和产前诊断的需求提供了资料和依据。更多还原

【Abstract】 Hearing impairment is a common sensory disorder in human. It is caused by both environmental factors(medical factor,environment exposure, injury,medicine) and genetic factors. About 60% of deafness cases is attributed to genetic defects.In this study, by means of SNP typing and microsatellite marker (STR) mapping,we successfully mapped and identified causative genes in two Chinese families with autosomal-dominant nonsyndromic low-frequency hearing loss.We conducted the screening of two exons of WFS1 gene in sporadic cases with low and middle frequency hearing loss. We have also identified the causative gene for one patient with Pfeiffer syndrome.Part 1:Identifacation of causative genes in two families with autosomal-dominant nonsyndromic low-frequency hearing loss family and in 37 sporadic casesIn family HB-S037 we have mapped the disease locus on on chromosome 11q13.4-q14.1 region between D11S1314 and D11S4166 (two-point lod-score of 4.18) by applying the Affymetrix 5.0 SNP Genechip and linkage analysis.By direct sequencing of candidate genes in the critical interval, we identified a novel heterozygous missense mutation c.2011G>A in exonl7 of MYO7A,resulting in amino acid change of G671S, which was faithfully cosegregated with hearing loss in the family HB-S037. This mutation was absent in 100 unrelated control DNA samples of Chinese origin.In famalily BJ-L046 we have mapped the disease locus on chromosome 4p 12.0-12.40 by applying the Affymetrix 6.0 SNP Genechip and linkage analysis.By direct sequencing of candidate genes in mapping region,we have identified a novel heterozygous missense mutation c.2086 C>T in exon8 of WFS1, resulting in amino acid change of H696Y, which was faithfully cosegregated with hearing loss in this family. The mutation was absent in 100 unrelated control DNA samples of Chinese origin. To determine the genetic loading of WFS1 mutation in sporadic cases with low and middle frequency hearing loss,we have screened the WFS1 mutations.in exon 5 and 8 of 37 individuals with non-syndrome low and middle frequency hearing impairment. Single nucleotide variations were present in 14 out of 37 patients, but only heterozygous missense c.2108G> A identified in one patient, resulting in amino acid change of R703H,while the other changes are single nucleotide polymorphisms (SNPs). In addition, the mutation was absent in 100 unrelated control DNA samples of Chinese origin. This study suggests that the necessity of WFS1 screening in non-syndromic low frequency sporadic cases.Part 2:Mutation screening in one patient with Pfeiffer syndromePfeiffer syndrome is a rare autosomal dominantly inherited disorder that associates craniosynostosis,broad and deviated thumbs and big toes,and partial syndactyly on hands and feet. Hydrocephaly may be found occasionally, along with severe ocular proptosis, ankylosed elbows, abnormal viscera, and slow development. Based on the severity of the phenotype, Pfeiffer syndrome is divided into three clinical subtypes. Pfeiffer syndrome affects about 1 in 100,000 individuals. The disorder can be caused by mutations in the fibroblast growthfactor receptor genes FGFR1,FGFR2 and FGFR3. Molecular genetic testing is important to confirm the diagnosis. In this study, we have indentified the causative gene of a family diagnosed as Pfeiffer syndrome I subtype. By direct sequencing of candidate genes (FGFR1, FGFR2, FGFR3), we identified a heterozygous missense mutation c.1021 A>C in exon8 of FGFR2, resulting in amino acid change of T341P. The mutation was absent in his parents. We used molecular modeling to construct three-dimensional representation of the Ig-3 domain of FGFR2 based on the crystallographic coordinates of telokin, a myosin light chain kinase homolog, an approach that has been used previously. Cys342 mutation is the hotspot mutaton of FGFR2 T341 lie close to the disulfide-bonded cysteines, the T341P mutation would alter theβ-strand containing Cys342, which would be expected to disrupt its bonding with Cys278. From this analysis, it is apparent that the noncysteine craniosynostosis mutations function through disruption of the Ig3 disulfide bond, creating free cysteine residues that can form intermolecular disulfide bonds resulting in receptor dimerization and activation. We presumed the mutaton probably resulting from cytochimera or fresh mutation. Mutation screening can provide solid information for genetic counseling in this family.更多还原