小热休克蛋白22基因在腓骨肌萎缩症中的突变分析

目的 探讨小热休克蛋白22（small heat-shock protein 22，HSP22）基因在中国人腓骨肌萎缩症（Charcot-Marie-Tooth disease，CMT）中的突变特点。方法 应用聚合酶链反应和DNA直接测序方法，对1个发现HSP22基因423（G→T）突变的CMT2L家系外的114个CMT家系先证者进行了且HSP22基因的突变分析。结果 114个先证者中有2例患者在且HSP22基因的第3外显子发生了582（C→T）碱基改变，由于编码的氨基酸未改变，均为色氨酸（Thr），为一种同义突变。结论 HSP22基因突变在中国人的腓骨肌萎缩症患者中少见，突变率为0.87％（1／115）。     

伴声带麻痹的X连锁腓骨肌萎缩症GJB1基因新突变

目的 报告1个临床可疑的伴声带麻痹的X连锁显性遗传腓骨肌萎缩症(X-1inked Charcot-Marie-Tooth disease,CMTX)家系,并探讨与连接蛋白(gap jurction protein beta 1,GJB1)基因突变的关系.方法 对1个具有声音嘶哑、吞咽困难、致死性呼吸衰竭的临床可疑的CMTX家系进行临床、纤维喉镜、头部MRI及电生理检查,对先证者应用实时PCR排除PMP22基因大片段重复突变后,应用PCR直接测序法对先证者及其家系内其他4例患者、5名有血缘关系的正常家系成员以及50名无血缘关系的正常人进行GJB1基因突变检测.结果 该家系3代共8例患者,先证者临床表现为进行性双下肢胫前肌、腓骨肌严重萎缩、无力;该家系中共4例患者出现声音嘶哑,吞咽困难,经纤维喉镜检查证实为声带麻痹,其中2例双侧声带完全麻痹最终因吞咽困难,呼吸衰竭而死亡;先证者的头部MRI检查正常,神经电生理检查揭示周围神经运动和感觉神经传导速度明显减慢或动作电位消失;分子遗传学结果显示该家系GJB1基因第2外显子存在c.186 C＞G错义突变,该突变与疾病表型共分离.结论 c.186 C＞G突变为GJB1基因新突变,目前国内外尚无报道;因声带麻痹而呼吸衰竭是CMT罕见的严重的临床症状;CMTX具有明显的临床和遗传异质性.     

神经丝轻链基因在腓骨肌萎缩症中的突变分析

目的：探讨神经丝轻链基因（neurofilament-light gene,NF-L）在中国人腓骨肌萎缩症（Charcot-Marie-Tooth disease,CMT）中的突变特点。方法：应用聚合酶链反应－单链构象多态性技术结合DNA序列分析方法，对32个来自全国5省汉族的CMT家系先证者进行了NF－L基因的突变分析。结果：32例先证者中只有1例患者出现异常条带，经DNA测序证实该患者在NF－L基因的外显子3发生了1329C→T碱基改变，由于编码的氨基酸未改变，均为酪氨酸（Tyr），为一种同义突变。结论：NF－L基因突变可能在中国人的腓骨肌萎缩症患者中少见。     

伴有脑干听觉诱发电位异常的腓骨肌萎缩症发现连接蛋白32基因新突变

目的：报告一个脑干听觉诱发电位有异常改变的腓骨肌萎缩症（Charcot-Marie-Tooth disease,CMT）家系，并探讨与连接蛋白32(connexin32，Cx32)基因突变的关系。方法：对整个家系进行临床检查，对先证者进行肌电图及脑干听觉诱发电位检查，并应用聚合酶链式反应-单链构象多态（polymerase chain reaction -single strand conformation polymouphism，PCR-SSCP）技术结合DNA序列分析方法检测了先证者、家系内8人及家系外50名无血缘关系的正常人。结果：先证者肌电图检查示神经传导速度明显减慢，脑干听觉诱发电位示中枢传导延长，该家系中先证者及另3人均出现异常SSCP条带，经测序证实为392T→C(Leu131Pro)突变。结论：Leu131Pro是未报道过的突变，腓骨肌萎缩症患者可以出现脑干听觉诿发电位异常。     

中国人腓骨肌萎缩症小热休克蛋白27基因突变分析

目的 探讨中国人腓骨肌萎缩症（Clmrcot-Marie-Tooth disease，CMT）小热休克蛋白27基因（small heat-shock protein 27，HSP27）的突变特点。方法 应用聚合酶链反应结合DNA序列分析方法，对114个CMT家系的先证者进行HSP27基因突变研究，并进一步对基因突变家系进行单体型分析。结果 在4个常染色体显性遗传CM32家系中发现一个HSP27基因错义突变C379T，单体型分析提示这4个家系很可能具有共同祖先。结论 中国人CMT患者存在HSP27基因突变，但突变率较低（0．90％）。HSP27基因C397T突变除引起远端型遗传性运动神经病外尚可导致CMT2，进一步证实同一CMT疾病基因的同一突变可引起不同的表现型。     

Duchenne肌营养不良症患者及携带者的基因缺失和重复突变检测

目的利用多重连接依赖探针PCR扩增技术检测Duchenne肌营养不良症（Duchenne muscular dystrophy，DMD）患者及其可能的女性携带者的dystrophin基因的缺失、重复突变。方法利用多重连接依赖探针PCR扩增对32例DMD患者及其27个可能的女性携带者的dystrophin基因缺失、重复进行检测。结果32个先证者中，共检测出了24例DMI）患者具有一个或多个外显子的缺失，l例DMD患者具有重复突变，l例患者为第19外显子的无义突变（R768X），6例没有检测出缺失、重复突变的先证者可能是点突变所致。17个先证者的18位女性亲属具有和先证者相同的缺失、重复突变。结论多重连接依赖探针PCR扩增技术可用于检测DMD基因的缺失、重复突变，可以检测DMD基因女性携带者的基因杂合情况，在检测DMD基因缺失和重复方面，具有一定的应用价值。     

中国人遗传性痉挛性截瘫spastin基因突变研究

目的 探讨中国人遗传性痉挛性截瘫（hereditary spastic paraplegia,HSP)spastin基因的突变特点，为该病的基因诊断提供依据。方法 应用聚合酶链反应—单链构象多态性(PCR—single strand conformation polymorphism，PCR—SSCP)结合DNA序列分析方法，对22个常染色体显性遗传HSP家系的先证者和9例散发性HSP患者的spastin基因进行研究，对发现异常SSCP条带的家系内成员进行突变研究。结果 在22例常染色体显性遗传HSP家系的先证者和9例散发性HSP患者中发现异常SSCP条带6例，进行DNA序列分析，共发现3种spastin基因突变，为外显子8的T1258A和A1293G，外显子14的1667delACT或1668delCTA或1669delTAC，均未见报道，突变位点均位于spastin基因功能区域，其中两个家系存在同一种突变(T1258A)，各突变家系内患者存在同样的异常SSCP条带。结论 中国人遗传性痉挛性截瘫患者存在spastin基因突变，该基因在中国人常染色体显性遗传的遗传性痉挛性截瘫家系中的突变率较低(18．2％)，点突变是主要的突变形式，外显子8可能是中国人spastin基因的突变热点。     

PINK1基因外显子拷贝数分析方法的建立及应用

目的 建立PINK1(PTEN-induced kinase 1)基因外显子拷贝数分析方法 ,并应用该方法 分析常染色体隐性遗传性早发型帕金森综合征(autosomal recessive early onset Parkinsonism,AREP)PINK1基因拷贝数变异(copy-number variation,CNV)的特点.方法 应用实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)技术分析方法 ,对30个正常对照和22个中国AREP汉族家系先证者的PINK1基因进行外显子拷贝数分析.结果 应用实时荧光定量PCR技术完成了PINK1基因第1～8外显子拷贝数分析,获得了扩增效率和特异性均满意的反应条件及各外显子引物.所检测的AREP先证者未发现PINK1基因的外显子拷贝数变异.结论 建立了PINK1基因外显子拷贝数分析方法 .中国人AREP患者PINK1基因外显子拷贝数变异可能罕见.     

PINK1基因外显子拷贝数分析方法的建立及应用

目的 建立PINK1(PTEN-induced kinase 1)基因外显子拷贝数分析方法 ,并应用该方法 分析常染色体隐性遗传性早发型帕金森综合征(autosomal recessive early onset Parkinsonism,AREP)PINK1基因拷贝数变异(copy-number variation,CNV)的特点.方法 应用实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)技术分析方法 ,对30个正常对照和22个中国AREP汉族家系先证者的PINK1基因进行外显子拷贝数分析.结果 应用实时荧光定量PCR技术完成了PINK1基因第1～8外显子拷贝数分析,获得了扩增效率和特异性均满意的反应条件及各外显子引物.所检测的AREP先证者未发现PINK1基因的外显子拷贝数变异.结论 建立了PINK1基因外显子拷贝数分析方法 .中国人AREP患者PINK1基因外显子拷贝数变异可能罕见.     

PINK1基因外显子拷贝数分析方法的建立及应用

目的 建立PINK1(PTEN-induced kinase 1)基因外显子拷贝数分析方法 ,并应用该方法 分析常染色体隐性遗传性早发型帕金森综合征(autosomal recessive early onset Parkinsonism,AREP)PINK1基因拷贝数变异(copy-number variation,CNV)的特点.方法 应用实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)技术分析方法 ,对30个正常对照和22个中国AREP汉族家系先证者的PINK1基因进行外显子拷贝数分析.结果 应用实时荧光定量PCR技术完成了PINK1基因第1～8外显子拷贝数分析,获得了扩增效率和特异性均满意的反应条件及各外显子引物.所检测的AREP先证者未发现PINK1基因的外显子拷贝数变异.结论 建立了PINK1基因外显子拷贝数分析方法 .中国人AREP患者PINK1基因外显子拷贝数变异可能罕见.     

Effectiveness of real-time quantitative PCR compare to repeat PCR for the diagnosis of Charcot-Marie-Tooth Type 1A and hereditary neuropathy with liability to pressure palsies

The majority of cases of Charcot-Marie-Tooth type 1A (CMT1A) and of hereditary neuropathy with a liability to pressure palsies (HNPP) are the result of heterozygosity for the duplication or deletion of peripheral myelin protein 22 gene (PMP22) on 17p11.2. Southern blots, pulsed-field gel electrophoresis (PFGE), fluorescence in situ hybridization (FISH) and polymorphic marker analysis are currently used diagnostic methods. But they are time-consuming, labor-intensive and have some significant limitations. We describe a rapid real- time quantitative PCR method for determining gene copy number for the identification of DNA duplication or deletion occurring in CMT1A or HNPP and compare the results obtained with REP-PCR. Six patients with CMT1A and 14 patients with HNPP [confirmed by Repeat (REP)-PCR], and 16 patients with suspicious CMT1A and 13 patients with suspicious HNPP [negative REP-PCR], and 15 normal controls were studied. We performed REP-PCR, which amplified a 3.6 Kb region (including a 1.7Kb recombination hotspot), using specific CMT1A-REP and real-time quantitative PCR on the LightCycler system. Using a comparative threshold cycle (Ct) method and beta -globin as a reference gene, the gene copy number of the PMP22 gene was quantified. The PMP22 duplication ratio ranged from 1.35 to 1.74, and the PMP22 deletion ratio from 0.41 to 0.53. The PMP22 ratio in normal controls ranged from 0.81 to 1.12. All 6 patients with CMT1A and 14 patients with HNPP confirmed by REP-PCR were positive by real-time quantitative PCR. Among the 16 suspicious CMT1A and 13 suspicious HNPP with negative REP-PCR, 2 and 4 samples, respectively, were positive by real-time quantitative PCR. Real-time quantitative PCR is a more sensitive and more accurate method than REP-PCR for the detection of PMP22 duplications or deletions, and it is also faster and easier than currently available methods. Therefore, we believe that the real-time quantitative method is useful for diagnosing CMT1A and HNPP.     

Rapid detection of duplication/deletion of the PMP22 gene in patients with Charcot-Marie-Tooth disease Type 1A and hereditary neuropathy with liability to pressure palsy by real-time quantitative PCR using SYBR Green I dye

Mutations and altered gene dosage of the peripheral myelin protein (PMP22) gene in chromosome 17p11.2-12 are the main causes for hereditary neuropathies, accounting for approximately 70% of all cases. Patients with duplication of the PMP22 develop Charcot-Marie-Tooth disease type 1A (CMT1A) and deletion of one PMP22 allele leads to hereditary neuropathy with liability to pressure palsy (HNPP). Twenty patients with CMT1A, 17 patients with HNPP, and 18 normal family members and 28 normal controls were studied by real-time quantitative PCR using SYBR Green I on the ABI 7700 Sequence Detection System. The copy number of the PMP22 gene was determined by the comparative threshold cycle method and the albumin was used as a reference gene. The PMP22 duplication ratio ranged from 1.45 to 2.06 and the PMP22 deletion ratio ranged from 0.42 to 0.64. The PMP22 ratio in normal controls, including normal family members, ranged from 0.85 to 1.26. No overlap was found between patients with CMT1A or patients with HNPP and normal controls. This method is fast, highly sensitive, specific, and reproducible in detecting PMP22 duplication and deletion in CMT1A and HNPP patients, respectively.     

A new quantitative PCR multiplex assay for rapid analysis of chromosome 17p11.2-12 duplications and deletions leading to HMSN/HNPP

A 1.4-Mb tandem duplication, including the gene for peripheral myelin protein 22 (PMP22) in chromosome 17p11.2-12 is responsible for 70% of the cases of the demyelinating type 1 of Charcot-Marie-Tooth disease or hereditary motor and sensory neuropathy I (CMT1A/HMSN I). A reciprocal deletion of this CMT1A region causes the hereditary neuropathy with liability to pressure palsies (HNPP). The CMT1A duplication increases the PMP22 gene dosage from two to three, the HNPP deletion reduces the gene dosage from two to one. Currently, routine diagnosis of HMSN/HNPP patients is mainly performed with polymorphic markers in-between the repetitive elements flanking the CMT1A region. These show quantitative and/or qualitative changes in case of a CMT1A duplication and a homozygous allele pattern in case of HNPP deletion. In HNPP patients the deletion is usually confirmed by fluorescence in situ hybridisation (FISH). We now developed a reliable, single tube real-time quantitative PCR assay for rapid determination of PMP22 gene dosage directly. This method involves a multiplex reaction using FAM labelled Taqman-probe with TAMRA quencher derived from PMP22 exon 3 and a VIC labelled probe with non-fluorescent quencher from exon 12 of the albumin gene as internal reference. Copy number of the PMP22 gene was determined by the comparative threshold cycle method (deltadeltaCt). Each sample was run in quadruplicate and analysed at two different threshold levels. The level giving the smallest standard deviation was scored. We evaluated this method through the retrospective analysis of 252 HMSN patients with known genotype and could confirm the previous findings in 99% of cases. Two patients were wrongly diagnosed with microsatellite analysis while quantitative real-time PCR identified the correct genotype, as confirmed by FISH. Thus, this method shows superior sensitivity to microsatellite analysis and has the additional advantage of being a fast and uniform assay for quantitative analysis of both CMT1A and HNPP.     

Asian hereditary neuropathy patients with peripheral myelin protein-22 gene aneuploidy

Japanese hereditary neuropathy with liability to pressure palsy (HNPP) patients have a deletion of one peripheral myelin protein-22 (PMP22) gene region in distal chromosome band 17p11.2 as do Caucasian patients. Japanese and Asiatic Indian CMT1A patients have a PMP22 gene duplication that results in Charcot-Marie-Tooth disease type IA (CMT1A; HMSNIA) in patients of European and Middle Eastern ancestry. About 70% of Japanese CMT1 patients have a PMP22 duplication as do Caucasians, while Japanese CMT1B, CMT2 and Dejerine-Sottas patients do not have PMP22 gene region aneuploidy. Although HNPP and CMT1A genotypes are generated simultaneously by unequal recombination that results in PMP22 gene aneuploidy in each daughter cell, only 3 Japanese HNPP probands with PMP22 deletion from a large patient population were referred to a single center compared to 18 referred CMT1A probands with PMP22 duplication. This lower HNPP frequency more likely reflects lower HNPP reproductive fitness than patient ascertainment bias because disease severity and variation in severity is about the same in CMT1A and HNPP patients and because all patients of both types were referred regardless of disease severity. These results, along with an apparently high de novo CMT1A mutation rate, suggest that common ancestors of Japanese, Asian Indians, and Caucasians carried PMP22 geneflanking sequences that enhance unequal crossing over. © 1995 Wiley-Liss, Inc.     

Mutational analysis and genotype/phenotype correlation in Turkish Charcot-Marie-Tooth Type 1 and HNPP patients

The major Charcot- Marie-Tooth Type 1 (CMT1) locus, CMT1A, and Hereditary neuropathy with liability to pressure palsies (HNPP) cosegregate with a 1.5-Mb duplication and a 1.5-Mb deletion, respectively, in band 17p11.2. Point mutations in peripheral myelin gene 22 (PMP22), myelin protein zero (MPZ), and connexin 32 (Cx32) have been reported in CMT1, and in PMP22 in HNPP patients without deletion. We have screened 54 CMT1 patients, of variable clinical severity, and 25 HNPP patients from Turkey, with no duplication or deletion, for mutations in the PMP22 and Cx32 genes. A novel frameshift mutation affecting the second extracellular domain of PMP22 was found in an HNPP patient, while a point mutation in the second transmembrane domain of the protein was detected in a CMT1 patient. Two point mutations affecting different domains of Cx32 were identified in two CMTX patients. Another patient was found to carry a polymorphism in a non-conserved codon of the Cx32 gene. The clinical phenotypes of the patients correlate well with the effect of the mutation on the protein.     

Diagnosis of CMT1A duplications and HNPP deletions by interphase FISH: Implications for testing in the cytogenetics laboratory

Charcot-Marie-Tooth (CMT) disease type 1A is an inherited peripheral neuropathy characterized by slowly progressive distal muscle wasting and weakness, decreased nerve conduction velocities, and genetic linkage to 17p12. Most (>98%) CMT1A cases are caused by a DNA duplication of a 1.5-Mb region in 17p12 containing the PMP22 gene. The reciprocal product of the CMT1A duplication is a 1.5-Mb deletion which causes hereditary neuropathy with liability to pressure palsies (HNPP). The most informative current diagnostic testing requires pulsed-field gel electrophoresis to detect DNA rearrangement-specific junction fragments. We investigated the use of interphase FISH for the detection of duplications and deletions for these disorders in the clinical molecular cytogenetics laboratory. Established cell lines or blood specimens from 23 individuals with known molecular diagnoses and 10 controls were obtained and scored using a two-color FISH assay. At least 70% of CMT1A cells displayed three signals consistent with duplications. Using this minimum expected percentile to make a CMT1A duplication diagnosis, all patients with CMT1A showed a range of 71–92% of cells displaying at least three signals. Of the HNPP cases, 88% of cells displayed only one hybridization signal, consistent with deletions. The PMP22 locus from normal control individuals displayed a duplication pattern in ∼9% of cells, interpreted as replication of this locus. The percentage of cells showing replication was significantly lower than in those cells displaying true duplications. We conclude that FISH can be reliably used to diagnose CMT1A and HNPP in the clinical cytogenetics laboratory and to readily distinguish the DNA rearrangements associated with these disorders from individuals without duplication or deletion of the PMP22 locus. Am. J. Med. Genet. 69:325–331, 1997. © 1997 Wiley-Liss, Inc.     

Mutation analysis of the nerve specific promoter of the peripheral myelin protein 22 gene in CMT1 disease and HNPP.

We analysed the nerve specific promoter of the peripheral myelin protein 22 gene (PMP22) in a set of 15 unrelated patients with Charcot-Marie-Tooth type 1 disease (CMT1) and 16 unrelated patients with hereditary neuropathy with liability to pressure palsies (HNPP). In these patients no duplication/deletion nor a mutation in the coding region of the CMT1/ HNPP genes was detected. In one autosomal dominant CMT1 patient, we identified a base change in the non-coding exon 1A of PMP22 which, however, did not cosegregate with the disease in the family. This study indicates that mutations in the nerve specific PMP22 promoter and 5' untranslated exon will not be a common genetic cause of CMT1A and HNPP.     

A rapid and reliable detection system for the analysis of PMP22 gene dosage by MP/DHPLC assay

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are caused by a 1.5-Mb duplication and a deletion at chromosome 17p11.2–12 encompassing the peripheral myelin protein 22 gene (PMP22), respectively. We developed a rapid and reliable detection system for duplications/deletions of the PMP22 gene based on measurement of gene copy number. The method involves amplification of a test locus with unknown copy number and a reference locus of known copy number by multiplex PCR (MP), followed by denaturing high-performance liquid chromatography (DHPLC) or capillary electrophoresis detection to identify single copy changes. Thirty-two patients with CMT1A, 17 patients with HNPP, and 61 unaffected individuals were analyzed. Using the same competitive MP protocol, the measured PMP22 gene dosage revealed concordant results between DHPLC and capillary electrophoresis analysis. The results of the MP/DHPLC or the MP/capillary electrophoresis assay were all confirmed by PCR–restriction fragment length polymorphism analysis. We concluded that the MP/DHPLC assay is an efficient, accurate, and reliable technique for gene dosage determination of the PMP22 gene for CMT1A duplication and HNPP deletion. This technique further extends the application of DHPLC as an alternative method for the measurement of gene amplifications and heterozygous deletions in different genetic diseases.Chia-Yun Lin and Yi-Ning Su share the first authorship.     

Rapid detection of 17p11.2 rearrangements by FISH without cell culture (direct FISH,DFISH): a prospective study of 130 patients with inherited peripheral neuropathies

Charcot-Marie-Tooth (CMT) disease and hereditary neuropathy with pressure palsies (HNPP) are two frequent hereditary motor and sensory neuropathies. CMT is characterized by slowly progressive weakness and atrophy, primarily in peroneal and distal leg muscles. The most frequent form, CMT1A, is due, in most cases, to the duplication of a 1.5 Mb region on chromosome 17p11.2 containing the peripheral myelin protein 22 gene (PMP22). The phenotype seems to result from dosage of the PMP22 gene. This hypothesis is reinforced by the existence of HNPP, which is clinically characterized by various recurrent truncular palsies or sensory loss precipitated by minor trauma, which is caused by deletion of the same 1.5 Mb region in 17p11.2. In clinical practice, the detection of the duplication or the deletion in 17p11.2, which permits a positive diagnosis, is still performed by time consuming methods (Southern blot or various combinations of molecular tools). We developed a method for the rapid detection of 17p11.2 rearrangements, using "direct FISH" and PRINS analyses, which does not require cell culture. In a prospective study of 92 patients with CMT and 38 with suspected HNPP, we compared this new technique to classical strategies like Southern blot. The results demonstrate the high sensitivity and specificity of the new FISH technique for the diagnosis of CMT1A and HNPP. Moreover, because of its simplicity and rapidity, this technique provides a useful alternative to the molecular approaches that have been used to diagnose segmental aneusomies, especially in the case of duplications that often go undetected.     

Genetic basis of inherited peripheral neuropathies

Progress in the elucidation of the genetic basis for inherited peripheral neuropathies has been remarkable over the last years. In particular, the molecular mechanisms underlying the autosomal dominantly inherited disorders Charcot–Marie–Tooth disease type 1A (CMT1 A), Charcot–Marie–Tooth disease type 1B (CMT1B), and hereditary neuropathy with liability to pressure palsies (HNPP) have been determined. While mutation in the gene encoding the major myelin protein, P_o has been associated with CMT1B, CMT1A and HNPP have been shown to be associated with reciprocal recombination events leading either to a large submicroscopic duplication in CMT1 A, or the corresponding DNA deletion in HNPP. Available evidence is consistent with the hypothesis that one or more genes within the relevant rearranged segment of 1.5 Mb on chromosome 17 is sensitive to gene dosage providing a novel mechanism for inherited human disorders. It is likely that the gene encoding the peripheral myelin protein PMP22 is at least one of the genes involved since the PMP22 gene maps within the CMT1A duplication (or HNPP deletion), and point mutations within it have been shown to cause a CMT phenotype in humans and comparable neuropathies in rodents (trembler and trembler^J). The mechanism(s) by which gene dosage and point mutations affecting the same gene might lead to a similar phenotype are currently unknown but recent transgenic mouse experiments suggest that similar mechanisms may also underlie other genetic diseases. © 1994 Wiley-Liss, Inc.