【作者】 金华；

【导师】 陈子江； 龚瑶琴；

【作者基本信息】 山东大学 ， 妇产科学， 2012， 博士

【摘要】 并多指(synpolydactyly, SPD, MIM18600, syndactyly type Ⅱ)是一种以肢端发育异常为主要特征的遗传性疾病,多呈常染色体显性遗传。其典型临床表现为3/4手指和4/5脚趾受累,两指(趾)间由蹼相连,不能分离,并指的蹼中可合并有骨性多指,且常伴有第5趾外端的多趾,第5指的侧弯、屈曲和短指,第2-5趾可有不同程度的并趾,中节趾骨发育不全等。受累肢体可为1-4个,受累程度可由单纯的皮肤相连到完全性骨性融合,甚至累及掌骨或跖骨。目前已经发现有SPD1、SPD2和SPD3三种SPD,致病基因分别定位于2q31,22q13.31和14q11.2-q12,其中,SPD1的致病基因为位于染色体2q31的HOXD13。HOXD13是HOX家族的成员之一,编码转录因子,在肢体发育中具有重要作用。典型的SPD由位于HOXD13基因N末端的15个聚丙氨酸残基的延伸所致。基因型和表型的相关性分析表明,聚丙氨酸区域短的延伸(如7个)与较低的外显率和表现度有关,而较长的聚丙氨酸延伸(如10个或更多)则导致较高的外显率和更严重的表型。而且,HOXD13同源结构域内的缺失和错义突变也可导致非典型的SPD,如特定的足部表型、短指合并并指畸形或者D型和E型短指。为SPD的高危妊娠进行产前诊断可以避免SPD患儿出生。迄今为止,仅有几例SPD的产前诊断报道。本研究中,我们利用在山东省济南市采集到的SPD家系,对其临床特点及系谱特征进行了分析,对其致病原因进行了分子遗传学研究,确定了致病基因及致病突变,并且在此基础上为家系高危妊娠提供了影像学和分子遗传学产前诊断。第一部分一个常染色体显性遗传并多指家系的家系调查及临床分析在遗传咨询过程中,我们发现了一SPD患者,因面临生育前来咨询并申请产前诊断。为了明确该SPD家系患者的致病基因,我们首先对该SPD家系进行了家系调查及临床分析。家系调查发现,家系中共有成员41名,其中患者16例,受累患者均表现有典型的并多指(趾)畸形。家系中男女均有发病,5代中共有男性患者9名,女性患者7名。家系中连续5代均有患者,在连续世代中呈垂直分布,无隔代遗传现象。家系中患者均存在不同程度的并(多)指(趾),但患者间存在较大的表现度差异。通过系谱分析,该SPD家系的遗传方式为常染色体显性遗传。该SPD家系中受累患者均表现有典型的并多指(趾)畸形。先证者出生时即发现严重手足畸形,表现为3/4指完全并指,4/5趾并趾并伴有多趾。经过详细的临床调查,该SPD家系临床特点总结如下：①先天性发病；②严重程度不同,家系中患者有表现度差异；③手部畸形严重,影响手部功能的发挥；④主要表现为3/4指完全并指,4/5趾并趾并伴有多趾；⑤X线检查有并多指(趾)表现；⑥呈常染色体显性遗传。根据典型的临床特征及放射线检查和系谱分析结果,可以确诊此家系患者为Ⅱ型并指,SPD1型,A4亚型。第二部分并多指家系的致病基因分析获得家系成员的系谱特征及标本后,为确定该家系致病基因,我们首先将HOXD13基因作为候选基因,在候选基因的两侧选取紧密连锁的STR位点D2S1238和D2S1245进行连锁分析。PCR扩增产物经聚丙烯酰胺凝胶电泳分析后确定家系中各个体基因型,经基因型分析和单体型分析发现,该家系致病基因与HOXD13紧密连锁,提示HOXD13也是该家系的致病基因。随后,我们应用PCR扩增结合测序分析检测HOXD13基因突变。首先我们分析了最常见的Poly-A突变。对包含该位点的第1外显子进行PCR扩增后,回收纯化目的片段进行TA克隆,挑选单克隆扩增后进行测序分析。测序结果采用BLAST程序进行序列对比分析。发现先证者的HOXD13基因在cDNA的189-190bp之间插入27bp,该插入突变使聚丙氨酸重复序列的第14和第15个丙氨酸之间插入9个丙氨酸残基,导致poly-A链的延伸。为证实该突变在家系中与疾病表型共分离,我们采用PCR扩增结合变性聚丙烯酰胺凝胶电泳方法确定家系中各成员的基因型,发现所有患者都带有27bp插入突变,正常人无此突变,证实该突变为家系患者的致病突变。第三部分并多指家系的产前诊断SPD属于一类严重致残致畸的单基因遗传性疾病,开展基因和超声学产前诊断,防止患儿出生是预防该病发生的最佳应对策略和最有效方法。为此,根据咨询者要求,我们在家系分析和基因型分析基础上,联合应用超声产前诊断检查方法和分子诊断方法对该家系中的1例高危妊娠进行产前影像学和分子遗传学诊断。孕16周起,通过超声进行四肢动态监测,动态观察胎儿手足形态；并于孕18周经腹抽取SPD孕妇的羊水,提取羊水中胎儿细胞基因组DNA,通过D2S1238和D2S1245两个STR位点连锁分析及HOXD13基因突变检测完成了对该家系的产前基因诊断,为家系成员提供了准确的产前诊断结果。新生儿出生时行体格检查进行验证。本部分结果显示,超声产前诊断未发现胎儿肢体异常。胎儿DNA连锁分析结果表明胎儿从母亲得到的是与正常等位基因连锁的等位基因,表明胎儿未从其母亲获得致病基因,进一步的突变分析证实胎儿HOXD13也未携带在该家系患者中检测到的插入突变。胎儿发育至足月分娩,对新生儿常规体格检查,发现新生儿手足正常,无并多指(趾)现象,同时未发现新生儿其他异常。验证了超声和基因产前诊断结果。以上研究表明,联合使用超声和基因诊断方法可为患者提供准确的产前诊断。更多还原

【Abstract】 Synpolydactyly （SPD, MIM186000, or syndactyly type2） is an autosomal dominant inherited malformation of the distal limbs. It is characterized by soft-tissue syndactyly between fingers3and4and between toes4and5with partial or complete digit duplication within the syndactylous web. The fifth-finger clinodactyly, camptodactyly, or brachydactyly, variable syndactyly of the second to fifth toes, and middle phalanx hypoplasia are also found to be associatated with SPD. Incomplete penetrance and variable expressivity both between and within affected families are common and involvement is often asymmetrical. From one to four limbs can be involved, and the severity of involvements ranges from partial skin syndactyly to complete reduplication of a digit, extending as far proximally as the metacarpals/tarsals.Three loci have been identified at chromosomes2q31,22q13.31, and14q11.2-q12, and have been designated as SPD1, SPD2,and SPD3, respectively. Of these, SPD1is caused by the mutations in the HOXD13gene on chromosome2q31. HOXD13is a member of the HOX family that encodes for a transcription factor with a crucial role in limb development. Typical SPD is caused by expansions of a15-residue poly-Alanine tract in the N-terminal region of HOXD13. Genotype-phenotype correlation suggests that short poly-Alanine repeats （i.e.,+7repeats） are associated with lower penetrance and expressivity, whereas long repeats （+10and more） are associated with higher penetrance and more severe disease. Furthermore, deletion and missense mutations within the HOXD13homeodomain have been also found to cause atypical forms of SPD, characterized by a distinctive foot phenotype, brachydactyly-polydactyly or brachydactyly types D and E. Detection of SPD by prenatal diagnosis may be especially relevant in pregnancies at risk for severe affected individuals. To date, only few cases of prenatal diagnosis of SPD have been reported. Here, we present the clinical findings, genetic counseling, and prenatal diagnosis in a Chinese family. The family contains9affected. Linkage analysis mapped the disease locus to chromosome2q31where HOXD13located. Mutation analysis identified a9-residue poly-Alanine tract expansion in the N-terminal of HOXD13. Mid-trimester ultrasound and aminocentesis excluded that the fetus affected with SPD. Part1Phenotype and pedigree analysis of a large Chinese family with synpolydactylyA28-year-old woman with SPD from a large SPD kindred was referred for genetic counseling and prenatal diagnosis. She was born with severe bilateral hand-and foot abnormalities. She had complete syndactyly between the third and fourth fingers, and syndactyly between toes4th and5th with an extra toe at her feet. After we got the approval of the Ethics Committee of Shandong University School of Medicine, and informed consents from all participants, we performed clinical examination of the family members and obtained blood samples for linkage analysis and mutation detection. Further investigation of this family revealed8other family members with hand-and/or foot abnormalities: bilateral3/4syndactyly was noted in individuals Ⅳ-12, Ⅳ-14, Ⅲ-11, Ⅲ-15, V-4, IV7, and IV-6, unilateral syndactyly in indivudual Ⅲ-10. In the feet, the most obvious malformation was variable degrees synpolydactyly between toes4and5with an extra toe. Moreover, most of affected feet had additional metatarsal between fourth and fifth metatarsals. This is not commonly observed in other families. Compared with the previously reported patients, the patients in this family had more severe phenotypes at their feet. The disease phenotype in this kindred clearly followed a pattern of autosomal dominant inheritance, though with variable expressivity.Part2Linkage analysis and mutation detectionBecause mutations were identified in the HOXD13gene in several unrelated Chinese families with SPD, we conducted a linkage analysis with previously used markers flanking the HOXD13gene. Blood samples were obtained from16family members （9affected,7unaffected）, and leukocyte genomic DNA was extracted via standard techniques. Using two markers （D2S1238, and D2S1245） spanning the HOXD13gene, we showed that SPD was linked to chromosome2q31. These results suggested that the HOXD13gene was a candidate for the SPD phenotype in this family. Therefore, we carried out the mutation detection by directly sequencing HOXD13gene. We identified a heterozygous27-bp expansion in the imperfect GCN triplet-repeat of exon1, c.184₂10dup. This mutation resulted in an addition of9alanine residues between the14th and15th alanine of the normally15-amino-acid-long poly-Alanine tract. This expansion represents a perfect duplication of the sixth to fourteenth triplet of the wild-type sequence. PCR amplification of the corresponding products comprising part of exon1revealed that this mutation cosegregated with the disease phenotype in the family, confirming full penetrance.Part3Prenatal ultrasonographic and molecular diagnosisPrenatal diagnosis was requested by the proband. Previous studies have shown that reliable observation of all fingers is possibly made by ultrasound examination between13and19weeks of gestation. To verify the fetal hand development, ultrasound examinations were conducted at16-21weeks, and demonstrated fetal biometry consistent with dates and normal amniotic fluid volume. All fingers and toes appeared normal. No other anomalies were observed. As mentioned above, enoumous phenotypic variation exists in SPD patients, and such clinical variability makes a prenatal diagnosis based on sonography alone difficult. At the proband’s request, her aminotic fluid sample was obtained by ultrasound-guided amniocentesis at18weeks of gestation. Linkage analysis and mutation detection confirmed that the fetus did not inherite the mutant allele from his affected mother. Pregnancy progressed uneventfully. At term a female infant was born. Normal limb development was confirmed.To conclude, accurate prenatal diagnosis of SPD can be performed by combing the ultrasound diagnosis and molecular analysis.更多还原