杜氏/贝氏肌营养不良症433个家系的基因突变分析

摘要

Objective Mutation analysis of unrelated families with Duchenne/Becker muscular dystrophy (DMD/BMD) was performed to investigate the characteristic of DMD gene mutation,especially the distribution pattern of point mutation of DMD gene in Chinese population.Methods A total of 433 unrelated DMD/BMD families were collected at the Center of Prenatal Diagnosis of the First Affiliated Hospital of Zhengzhou University from March 2010 to December 2014.The deletions or duplications in 79 exons of DMD gene were screened using multiplex ligation-dependent probe amplification (MLPA).Any single-exon deletion detected by MLPA was further validated by PCR amplification.In the 117 unrelated Chinese families in which large-scale deletions and duplications had been excluded by MLPA,the point mutation in 79 exons of DMD gene were tested in the propositus using next-generation sequencing (NGS),and further verified the point mutation using Sanger sequencing.Results In the 433 unrelated DMD/BMD families,316 families with DMD deletions/duplications were identified by MLPA.Out of 57 single-exon deletions detected by MLPA,3 were found as point mutations by PCR and Sanger sequencing,including 2 nonsense mutation (c.1729G ＞ T [p.Glu577X],c.3346A ＞ T [p.Lys1116X]) and 1 frame-shift mutation (c.8605_8606delGT [p.Val2869ThrfsX25]).Direct sequencing with Ion PGM and Sanger sequencing in 117 families with negative results in MLPA detected 92 different point mutations in 96 families,including 46 novel mutations,42 previously reported ones,and 4 possible polymorphisms (rs189143447,rs202008454,rs200213555,rs187617705).The 46 novel mutations consisted of 16 nonsense mutations (c.100A ＞ T [p.Lys34X],c.1201C ＞ T [p.Gln401X],c.1707C ＞ A [p.Cys569X],c.1831G ＞ T [p.Glu611X],c.1912C ＞ T [p.Gln638X],c.2213C ＞ G [p.Ser738X],c.3673_3673delA [p.Ile1225X],c.3774C ＞ A [p.Cys1258X],c.4858G ＞ T [p.Glu 1620X],c.5764A ＞ T [p.Lys1922X],c.6035T ＞ G [p.Leu2012X],c.6408G ＞ A [p.Trp2136X],c.7717C ＞ T [p.Gln2573X],c.7864G ＞ T [p.Glu2622X],c.8184_8185insT [p.Lys2729X],c.8215C ＞ T [p.Gln2739X]),5 missense mutations (c.139G ＞ A [p.Gly47Arg],c.238G ＞C [p.Ala80Pro],c.335G ＞ T [p.Trp112Leu],c.804A ＞ C [p.Leu268Phe],c.1149G ＞ T [p.Glu383Asp]),6 splice-site mutations (c.2293-3C ＞ A,c.2380 + 1G ＞ T,c.3277-1G ＞ C,c.4519-7A ＞G,c.5740-15G ＞ T,c.7661-1 G ＞ C),16 small deletions (c.688_688delA [p.Met230CysfsX14],c.1760_1791del32 [p.Thr587IlefsX37],c.2271 _ 2271delA [p.Asp774ThrfsX22],c.2281 _ 2285delGAAAA [p.Glu761SerfsX10],c.2527_2527delG [p.Glu843SerfsX3],c.3405_3405delC [p.Asn1135LysfsX18],c.4450_4450delC [p.His1484ThrfsX14],c.4770 _4770delA [p.Thr1590ThrfsX5],c.4937 _4937delA [p.Glu1646GlyfsX11],c.5253 _ 5256delATTA [p.Lys1751LysfsX2],c.5654 _ 5654delA [p.Gln1885ArgfsX6],c.7441_7441delG [p.Glu2481AsnfsX13],c.7860_7860delC [p.Ile2620IlefsX18],c.8668-8668delG/c.8668 + 1-8668 + 1delG,c.9009_9009delC [p.Thr3003ThrfsX18],c.9021 _9021delT [p.Ile3007IlefsX14]),and 3 small insertions (c.305_306insG [p.Gly102GlyfsX4],c.3116_3117insA [p.His1039GlufsX11],c.9197 _9198insATCTC [p.Ser3066SerfsX25]).And 87.4％ (83/95) of the pathologic point mutations disrupted the translational reading frame (46 nonsense mutations,24 frame-shift mutations,and 13 splice-site mutations).Conclusions Inexpensive and efficient genetic/prenatal diagnosis of DMD/BMD may be plausible by MLPA analysis,NGS,and Sanger sequencing.Most of the mutations identified in this study led to a predictable premature stop codon or splicing defects,resulting in defective function of dystrophin.%目的 对无亲缘关系的杜氏/贝氏肌营养不良症(DMD/BMD)家系进行DMD基因突变分析,探讨DMD基因突变特点,并主要阐明DMD基因点突变在中国汉族人群中的分布模式.方法 在2010年3月至2014年12月郑州大学第一附属医院收集的433个无亲缘关系的中国汉族DMD/BMD家系中,首先应用多重连接依赖性探针扩增(MLPA)技术对DMD/BMD家系进行DMD基因79个外显子进行缺失或重复的分析;应用PCR扩增技术针对MLPA单个外显子缺失的样本排除MLPA假阳性;对应用MLPA技术已排除DMD基因大片段缺失和重复的117个家系中的先证者,应用二代测序(NGS)技术对DMD基因79个外显子进行点突变分析,应用Sanger测序对点突变进一步验证.结果 在433个无亲缘关系的中国汉族DMD/BMD家系中,应用MLPA技术在316个家系中检测到DMD基因存在外显子大片段缺失和重复突变;其中MLPA检测的57单个外显子缺失样本,应用PCR扩增技术和Sanger测序发现3个新的点突变包括:2个无义突变c.1729G ＞T[p.Glu577X]、c.3346A＞T[p.Lys1 116X]和1个移码突变c.8605_8606delGT[p.Val2869ThrfsX25].应用NGS(Ion PGM高通量平台)和Sanger测序在117个MLPA阴性结果家系对DMD基因进行直接测序,在96个家系中检测到DMD基因92个不同点突变,包括46个新突变、42个已知致病突变和4个可疑多态位点(rs189143447、rs202008454、rs200213555、rs187617705).46个新突变包括16个无义突变(c.100A＞T[p.Lys34X]、c.1201C＞T[p.Gln401X]、c.1707C＞A[p.Cys569X]、c.1831G＞ T[p.Glu611X]、c.1912C＞ T[p.Gln638X]、c.2213C＞G[p.Ser738X]、c.3673_3673delA[p.Ile1225X]、c.3774C＞A[p.Cys1258X]、c.4858G ＞T[p.Glu1620X]、c.5764A ＞T [p.Lys1922X]、c.6035T＞G[p.Leu2012X]、c.6408G＞A[p.Trp2136X]、c.7717C ＞T [p.Gln2573X]、c.7864G ＞T[p.Glu2622X]、c.8184_8185insT[p.Lys2729X]、c.8215C＞T[p.Gln2739X]),5个错义突变(c.139G＞A[p.Gly47Arg]、c.238G＞C[p.Ala80Pro]、c.335G ＞T[p.Trp112Leu]、c.804A ＞C [p.Leu268Phe]、c.1149G ＞T [p.Glu383Asp]),6个剪切位点突变(c.2293-3C＞A、c.2380+ 1G＞T、c.3277-1G＞C、c.4519-7A＞G、c.5740-15G＞T、c.7661-1G＞C),16个缺失突变(c.688_688delA[p.Met230CysfsX14]、c.1760_1791del32[p.Thr587IlefsX37] 、c.2271_2271delA [p.Asp774ThrfsX22] 、c.2281_2285delGAAAA[p.Glu761SerfsX10]、c.2527_2527delG[p.Glu843SerfsX3]、c.3405_3405delC[p.Asn1135LysfsX18]、c.4450_4450delC[p.His1484ThrfsX14]、c.4770_4770delA[p.Thr1590ThrfsX5]、c.4937 _4937delA[p.Glu1646GlyfsX11] 、c.5253_5256delATTA [p.Lys1751LysfsX2] 、c.5654_5654delA [p.Gln1885ArgfsX6]、c.7441_7441delG[p.Glu2481AsnfsX13]、c.7860_7860delC[p.Ile2620IlefsX18]、c.8668-8668delG/c.8668+ 1-8668+1delG、c.9009_9009delC[p.Thr3003ThrfsX18]、c.9021_9021delT [p.Ile3007IlefsX14])和3个插入突变(c.305_306insG[p.Gly102GlyfsX4]、c.3116_3117insA [p.His1039GlufsX11]、c.9197_9198insATCTC[p.Ser3066SerfsX25]).87.4％ (83/95)点突变破坏了阅读框(46个无义突变、24个移码突变、13个剪切突变).结论 结合应用MLPA技术、NGS技术、Sanger测序可为患者提供经济高效的基因诊断服务.研究发现的点突变多数是可预测的提前终止密码子或导致剪接缺陷,导致肌营养不良蛋白功能缺陷.