发作性运动诱发性运动障碍PRRT2基因型—表型研究及应用全外显子测序技术鉴定Gordon Holmes综合征致病基因

发布时间：2018-03-08 02:00

  本文选题：发作性运动诱发性运动障碍　切入点：PKD基因突变　出处：《郑州大学》2014年博士论文　论文类型：学位论文

【摘要】：背景：发作性运动诱发性运动障碍(Paroxysmal Kinesigenit Dyskinesia，PKD)又称发作性运动诱发性舞蹈手足徐动症，是以在静止情况下，起始随意动作如起立、转身、迈步、举手等诱发的短暂的运动异常为临床特征的神经系统发作性疾病。其诊断要点为：运动诱发；发作持续时间短（1分钟）；发作不伴有意识丧失或疼痛；排除其他器质性疾病；神经系统检查正常。最近研究显示，PKD是一种遗传因素为主要发病因素的神经系统疾病，目前认为，有家族史的病人约占PKD发病总数的60%，通常为常染色体显性遗传，其他病人则为散发。长期以来PKD的致病基因不明，最新的分子遗传学研究发现：PKD的主要致病基因是PRRT2基因。目前，关于PKD人群中PRRT2基因突变检测的相关研究较少，PRRT2基因相关基因型与表型的关系尚不明确，为了明确本组PKD患者中PRRT2基因突变频率，及明确携带PRRT2基因突变对PKD病人表型的影响，我们开展了下列研究。 目的: 1.明确本研究纳入PKD患者的PRRT2基因突变频率及相关PRRT2基因突变位点信息； 2.探讨本组PKD患者中PRRT2基因相关基因型与表型的关系。 方法: 1.连续纳入2011年12月至2013年1月在郑州大学第一附属医院神经内科就诊的运动诱发性发作性运动障碍病人,所有病人均完成相关检查，并有两名神经内科专科医师详细进行体格检查，所有纳入病人均同意登记临床资料、抽血及定期随访。 2.所有患者行PRRT2外显子区域基因检测，明确本组PKD患者中PRRT2基因突变频率和突变位点，并在正常对照中检测发现的PRRT2基因突变是否存在。 3.对本研究纳入患者随访半年，观察其对抗癫痫药物卡马西平治疗的反应性，并比较PRRT2突变阳性患者和不携带PRRT基因突变PKD患者的表型差异。 结果: 1.本研究共纳入5个家系共11例家族性PKD患者和19例无家族史的散发PKD患者，其中男性患者21例，女性患者9例，平均发病年龄14.33±3.92岁。 2.在本组患者中，我们发现在5个PKD家系中的4个家系，共9名患者中PRRT2基因的c.649dupC (p.P217fsX7)突变是这些家系的致病突变，在19例散发PKD患者中同样发现PRRT2基因的c.649dupC (p.P217fsX7)突变导致了6例散发PKD患者致病，在1例散发患者中发现了一个PRRT2基因的同义突变c.1011C/T（p.Gly337Gly），在其余患者中未发现PRRT2基因突变。 3.在纳入PRRT2基因型-表型关系研究的27例PKD患者中，携带PRRT2基因突变的PKD病人发病年龄更早(平均发病年龄:11.62vs.17.21,P=0.01)；发病症状多呈对称性，但在PRRT2基因突变阴性组中全部为单侧起病（P＝0.001)；PRRT2基因突变组病人每日发作次数更多((P＝0.038)。在发作先兆方面，两组患者无明显差异。(P＝0.585)。6个月随访结束后，我们发现：卡马西平治疗对PRRT2基因突变阳性组患者全部有效（13/13,100%），但对PRRT2基因突变阴性组患者仅部分病人有效（5/14，35.7%）。 结论: 1. PRRT2基因突变是绝大多数家族性PKD患者及部分散发型PKD患者的致病原因，c.649dupC (p.P217fsX7)热点突变占到所有突变的93.75%，报道了一种可能致病的PRRT2基因c.1011C/T（p.Gly337Gly）同义突变。 2. PRRT2基因突变阳性的PKD患者发病年龄较早，发作更为频繁且发作时症状对称性更高。本研究提示PRRT2基因突变阳性对抗癫痫药物卡马西平的反应性具有预测作用，PRRT2基因检测可能对PKD患者的临床治疗具有潜在指导意义。 背景：遗传性共济失调（Hereditary ataxia, HA）是一组以慢性进行性小脑萎缩及小脑性共济失调为主要特征的神经系统遗传变性病，戈登-福尔摩斯氏综合征（Gordon Holmes Syndrome）是遗传性共济失调类疾病的一种特殊亚型，遗传方式为常染色隐性遗传，临床表现为青春期起病的，逐渐进展的步态不稳，，眼震等共济失调症状；病人同时合并性腺功能障碍，男性病人表现为外生殖器发育不良，性腺幼稚，不育，女性病人表现为青春期后仍无月经来潮及第二性征发育不全，临床上尽管在对Gordon Holmes综合征已有近100年时间的认识，但世界上仅有20余个该疾病家系被报道，其致病基因长期以来未被鉴定和克隆。2013年，其第一、二种致病基因RNF216和OTUD4被报道。 目的: 我们在前期工作中，收集到中国地区的首个Gordon Holmes综合征家系，本研究拟在前期收集本家系的基础上，通过外显子测序技术，探寻Gordon Holmes综合征的致病基因。 方法: 1收集本研究家系内所有成员的临床资料，并有两名神经内科专科医师详细进行体格检查，所有家系成员均同意登记临床资料、抽血及定期随访。 2应用全外显子组捕获+高通量测序技术对本家系内2例患者及家系内1位正常对照者进行全外显子组测序，对外显子组测序结果进行后期数据分析，寻找候选致病基因位点。 3对外显子组测序发现的候选基因致病位点进行Sanger测序验证，并在相关病人及正常对照中进行验证。 结果: 1本研究中的Gordon Holmes综合征家系内两名女性病人表现为青春期出现的共济失调伴小脑萎缩，性腺发育障碍，同时合并轻度的认知功能障碍。病人父母及一个弟弟表现正常，符合常染色体隐性遗传规律。 2本研究中的三个全外显子组测序样本每个样本均产生了8Gb左右的数据量，平均测序深度达到了90%以上，目标序列覆盖度超过99%，通过对SNP及Indel的注释，在本研究中的三个样本中，每个样本均发现了大约90000个SNP和2000个Indel。通过（1）数据库过滤，（2）隐性遗传模型分析，（3）IBD分析（，4）SNP致病性预测，我们发现STUB1基因的c.737C-T（p.T246M）突变是本家系最可能的致病位点。 3在正常人群、其他共济失调及Gordon Holmes综合征患者中进行验证，未发现STUB1基因突变，提示Gordon Holmes综合征的致病基因具有异质性。 结论:STUB1基因的c.737C-T（p.T246M）突变是本家系的最可能的致病位点，结合后续开展的体外功能研究及转基因动物研究结果，提示：STUB1基因是GordonHolmes综合征的第三种致病基因
[Abstract]:Background: paroxysmal kinesigenic dyskinesia (Paroxysmal Kinesigenit, Dyskinesia, PKD) also known as paroxysmal kinesigenic choreoathetosis, which is in the stationary case, initial random actions such as stand up, turn around, move, nervous system movement induced by different short hands often clinical features of paroxysmal diseases. The main points of diagnosis: exercise induced; short duration episodes (1 minutes); the attack was not accompanied by loss of consciousness or pain; exclusion of other organic disease; neurological examination is normal. Recent studies show that PKD is a disease of the nervous system, as the main risk factors of a genetic factor is believed to have a family history of the PKD patients accounted for about 60% of the total incidence, usually autosomal dominant, other patients are sporadic. For a long time the pathogenic gene of PKD is unknown, molecular genetics, the new study found: mainly caused by PKD The disease gene is PRRT2 gene. At present, about PRRT2 in the population of PKD gene mutation detection research, the relationship between PRRT2 gene genotype and phenotype is not clear, in order to determine the PRRT2 of the patients with PKD gene mutation frequency, and clearly carrying PRRT2 gene mutation effect on the phenotype of PKD disease people, we carried out the following study.
Objective:
1. the PRRT2 gene mutation frequency and the related PRRT2 gene mutation sites of the PKD patients were included in this study.
2. to investigate the relationship between genotype and phenotype of PRRT2 gene in PKD patients.
Method:
1. consecutive December 2011 to January 2013 in the First Affiliated Hospital of Zhengzhou University Department of Neurology of motor evoked seizures in patients with dyskinesia, all patients completed related examination, and two neurological physicians with physical examination, all patients agreed to register the clinical data, blood and regular follow-up.
2. in all patients, the PRRT2 exon region gene detection was performed to identify the mutation frequency and mutation site of PRRT2 gene in PKD patients, and whether the PRRT2 gene mutation detected in normal controls existed.
3. the patients were included in the study for half a year to observe the reaction of C Masi Bing against epilepsy drugs. The phenotypic difference between PRRT2 mutation positive patients and PKD patients without PRRT gene mutation was observed.
Result:
1., a total of 5 families, 11 family PKD patients and 19 sporadic PKD patients without family history were included in the study, including 21 male patients and 9 female patients, with an average age of 14.33 + 3.92 years.
2. in this group of patients, we found 4 families in 5 PKD families, a total of 9 patients in the PRRT2 gene of c.649dupC (p.P217fsX7) mutations in these families in 19 sporadic mutation, PRRT2 gene c.649dupC was also found in PKD patients (p.P217fsX7) mutation in 6 cases patients with sporadic PKD disease, in 1 sporadic patients found a synonymous mutation in PRRT2 gene c.1011C/T (p.Gly337Gly), PRRT2 gene mutation was not found in the remaining patients.
3. in PRRT2 genotype - phenotype of 27 PKD patients, PKD patients with a younger age of PRRT2 gene mutation (average age: 11.62vs.17.21, P=0.01); the incidence of symptoms was symmetrical, but mutations in PRRT2 negative group were all unilateral onset (P = 0.001); PRRT2 gene mutation patients daily episodes more ((P = 0.038). At the onset of aura, no significant difference between the two groups. (P = 0.585) after.6 months follow-up, we found that C Masi Bing therapy on PRRT2 gene mutation positive group were all effective (13/13100%), but the PRRT2 gene mutation only some patients effective negative group (5/14,35.7%).
Conclusion:
1. PRRT2 gene mutation is the cause of most familial PKD patients and some sporadic PKD patients. C.649dupC (p.P217fsX7) hotspot mutation accounts for 93.75% of all mutations. A possible pathogenic PRRT2 gene c.1011C/T (p.Gly337Gly) synonymous mutation is reported.
The onset age of patients with PKD positive early 2. mutations in the PRRT2 gene, more frequent episodes and symptoms of higher symmetry. This study suggests a role in predicting the response of the PRRT2 mutation of antiepileptic drug C Masi Bing, detection of PRRT2 gene may be of clinical patients with PKD treatment has potential significance.
Background: hereditary ataxia (Hereditary ataxia HA) is a chronic progressive cerebellar atrophy and cerebellar ataxia as genetic neural degeneration disease system is the main feature of the Gordon - Holmes syndrome (Gordon Holmes Syndrome) is a kind of disease of hereditary ataxia is a special subtype, genetic mode autosomal recessive, clinical manifestations of adolescent onset, progressive ataxia, nystagmus and ataxia; patients with gonadal dysfunction, male patient presented with genital hypoplasia, gonadal immature, infertility, female patients showed no menstruation after puberty and sex characteristics hypoplasia, clinical although in the understanding of Gordon Holmes syndrome in nearly 100 years, but only 20 more than the world in the family have been reported. The pathogenic gene has not been identified And cloned.2013, first, second of its pathogenic genes, RNF216 and OTUD4, were reported.
Objective:
In our previous work, collected the first Gordon Holmes Chinese area syndrome family, this study based on the pedigree collected earlier on by exome sequencing technology, to explore the pathogenic gene Gordon Holmes syndrome.
Method:
1 the clinical data of all the members in the family were collected, and two neurologes were examined in detail. All the family members agreed to register clinical data, blood sampling and regular follow-up.
2 Application of whole exome capture and high-throughput sequencing of the family in 2 patients and families in 1 normal control subjects by whole exome sequencing, exon sequencing results of group data analysis, identify genetic loci of candidate pathogenicity.
3 the pathogenicity site of the candidate gene was confirmed by Sanger sequencing and verified in the related patients and normal controls.
Result:
1 in this study Gordon Holmes syndrome family in two female patients presenting to the onset of puberty ataxia with cerebellar atrophy, gonadal dysgenesis, and mild cognitive impairment patients. Parents and a brother showed normal, consistent with autosomal recessive inheritance.
2 in this study three whole exome sequencing samples each have about 8Gb the amount of data, the average sequencing depth reached more than 90%, the target sequence coverage of more than 99%, according to the SNP and Indel notes, three samples in this study in each sample were found. About 90000 SNP and 2000 Indel. (1) through the filtering database (2), analysis of the recessive genetic model, (3) IBD (4, SNP) prediction analysis of pathogenicity, we found that STUB1 c.737C-T gene (p.T246M) mutation is pathogenic sites most likely family.
3 in the normal population, other ataxia and Gordon Holmes syndrome patients were verified, and no STUB1 gene mutation was found, suggesting that the Gordon Holmes syndrome gene is heterogeneous.
Conclusion: STUB1 gene c.737C-T (p.T246M) is the most likely pathogenic mutation pedigree, combined with the results, to carry out follow-up and in vitro functional study of transgenic animal study showed that the STUB1 gene is third kinds of pathogenic gene of GordonHolmes syndrome

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R741


本文编号：1581891