Establishment and application of an analytical method for PINK1 gene exon copy number / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a method for analyzing the PTEN-induced kinase 1 gene (PINK1) exon copy number and apply it to the analysis of PINK1 gene exon copy number variation (CNV) in patients with autosomal recessive early-onset Parkinsonism (AREP).</p><p><b>METHODS</b>Real-time PCR was used to analyze the exon copy number in 22 probands with AREP from unrelated Chinese Han families and 30 healthy controls.</p><p><b>RESULTS</b>Copy numbers of exons 1-8 of the PINK1 gene were analyzed, and satisfactory reaction conditions and primers for exons of the PINK1 gene were obtained. No exon CNV in the PINK1 gene was detected in this group.</p><p><b>CONCLUSION</b>An analytical method for PINK1 gene exon copy number was established. The exon CNV in the PINK1 gene was rare in Chinese patients with AREP.</p>

Polynucleotide repeat expansion of nine spinocerebellar ataxia subtypes and dentatorubral-pallidoluysian atrophy in healthy Chinese Han population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assist the establishment of platform and provide the reference standard for mutation detection in spinocerebellar ataxia (SCA) subtypes 1, 2, 3, 6, 7, 8, 10, 12, 17 and dentatorubral-pallidoluysian atrophy (DRPLA) in Chinese Han population.</p><p><b>METHODS</b>The nucleotide repeat numbers of the 9 SCA subtypes and DRPLA were detected using fluorescence-PCR and capillary gel electrophoresis technique in 300 healthy Chinese Han individuals.</p><p><b>RESULTS</b>Among the 300 healthy controls, the range of the CAG trinucleotide repeat number was 17 to 35 in SCA1, 14-28 in SCA2, 13-41 in SCA3/MJD, 4-16 in SCA6, 5-17 in SCA7, 5-21 in SCA12, 23-41 in SCA17, and 12-33 in DRPLA; and the range of CTA/CTG trinucleotide repeat number on SCA8 locus was 12-43 and the range of ATTCT pentanucleotide repeat number on SCA10 locus was 9-32. Of which, the 12 CTA/CTG repeats of SCA8, 9 ATTCT repeats of SCA10, 23 CAG repeats of SCA17 were the shortest normal repeat number, while the 41 CAG repeats of SCA3/MJD, 32 CAG repeats of SCA10 were the largest normal number that have not been reported.</p><p><b>CONCLUSION</b>The normal ranges of polynucleotide repeats of different subtypes of SCA vary with geographical areas and ethnicities. It might be associated with the genetic and ethnic backgrounds. This is the first normal reference standard of polynucleotide repeat number of these ten SCA subtypes in Chinese Han.</p>

Detection of the CAG trinucleotide repeats of MJD1 gene by recombinant DNA technology / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a stable, accurate and intuitive method for detecting the CAG trinucleotide repeats of MJD1 gene.</p><p><b>METHODS</b>The CAG trinucleotide polymorphism of the MJD1 gene was analyzed by recombinant DNA technology and DNA sequencing in 35 spinocerebellar ataxia 3/Machado-Joseph disease (SCA3/MJD) patients from Mainland China.</p><p><b>RESULTS</b>The range of the CAG repeat of the 35 patients was 65-81 (mean = 72.96 +/- 4.24). The CAG repeats contained two CAAs and one AAG variations in the CAG motif in all the patients and majority of the healthy controls. There was a CGG/GGG polymorphism at the 3' end of the CAG repeat. The GGG allele was consistently associated with smaller CAG repeats in healthy controls. On the other hand, the CGG allele consistently existed in the patients.</p><p><b>CONCLUSION</b>Recombinant DNA technology can stably, accurately and intuitively detect the CAG trinucleotide repeat of the MJD1 gene. It should be used as a major technique to diagnose the SCA3/MJD and analyze the polymorphism of CAG sequence.</p>

Mutation analysis of GCH1 gene in Chinese patients with dopa responsive dystonia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect mutations of guanosine triphosphate cyclohydrolase I (GCH1) gene in Chinese patients with dopa responsive dystonia (DRD).</p><p><b>METHODS</b>Six sporadic patients with DRD were examined. GCH1 gene mutations were detected using polymerase chain reaction (PCR), DNA sequence analysis and restriction enzyme digestion analysis. One hundred normal people were detected using PCR and restriction enzyme digestion analysis.</p><p><b>RESULTS</b>A new point mutation, 151(G-->A) in exon one was found in a patient. It lead to substitution of a methionine for isoleucine at amino acid 1(M1I). This mutation was not found in normal control people.</p><p><b>CONCLUSION</b>The authors report a new heterozygotic point mutation 151(G-->A) in GCH1 gene. There are GCH1 gene mutations in Chinese sporadic patients with DRD.</p>

Mutation detection of parkin gene by denaturing high performance liquid chromatography / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect parkin gene mutation of early-onset parkinsonism (EOP) by denaturing high performance liquid chromatography (DHPLC).</p><p><b>METHODS</b>The blood cell genomic DNA of 82 EOP patients was isolated. Exons of parkin gene were amplified by PCR. The PCR products were detected by DHPLC. The sample with abnormal peak shape was sequenced.</p><p><b>RESULTS</b>Three point mutations were identified in 82 EOP patients compared with 100 healthy controls. Mutations in intron include IVS1-39 G --> T and IVS9 +18 C --> T. The T1422C mutation was in coding region and resulted in 441 Cys --> Arg.</p><p><b>CONCLUSION</b>Three heterozygous mutations are found in sporadic EOP patients and genetic diagnosis of parkin gene by DHPLC is applicable in EOP patients.</p>

The wild-type alpha-synuclein over-expression to induce the protein aberrant aggregation of alpha-synuclein in HEK293 cells in vitro / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate over-expression of wild-type alpha-synuclein inducing the aberrant aggregation of alpha-synuclein in HEK293 cell in vitro.</p><p><b>METHODS</b>The cDNA encoding the human alpha-synuclein without the stop code was cloned into PGEM T-easy vector. Using enzyme map and DNA sequencing analyzed and determined the recombinant plasmid, and then sub-clone the alpha-synuclein cDNA fragment into pEGFP-N1 vector. The recombinant plasmids alpha-synuclein-pEGFP were transfected into HEK293 cells by lipofectamin 2000. The aberrant aggregation of alpha-synuclein was measured by EGFP fluorescence, anti-alpha-synuclein immunocytochemistry. The inclusions in the cultured cells were identified with HE staining.</p><p><b>RESULTS</b>The restriction enzyme map suggested that eukaryotic expression vector for human wild-type alpha-synuclein gene was constructed successfully. By EGFP fluorescence, anti-alpha-synuclein immunocytochemistry, it could be observed that the alpha-synuclein protein could aggregate in cytoplasm and the Lewy body-like inclusions found in cytoplasm of cultured cells.</p><p><b>CONCLUSION</b>The over-expression of wild-type alpha-synuclein can induce protein aberrant aggregation and Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro.</p>

Genotype and phenotype analyses of three families with autosomal recessive juvenile parkinsonism / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the gene mutations and the clinical features of Chinese patients with autosomal recessive juvenile parkinsonism(AR-JP).</p><p><b>METHODS</b>the polymerase chain reaction (PCR), DNA sequence analysis, and restriction enzyme digestion analysis were applied to check parkin gene mutations of 15 index patients from 15 families with AR-JP.</p><p><b>RESULTS</b>Three families were detected to have parkin mutations. Two of them had heterozygous deletion mutations (202-203 del AG in exon 2, 1069-1074 del GTGTCC in exon 9) and another of them carried a heterozygous missense mutation [1422(T-->C) in exon 12]. Two of the mutations [1069-1074delGTGTCC and 1422(T-->C)] were not reported previously. There were six patients in the three families. Mean age at onset was 25.2+/-5.7 years, ranging from 18 to 31 years. The symptoms were under slow progression, diurnal fluctuation with sleep benefit, and hyperreflexia were relatively prominent. Response to levodopa was satisfactory.</p><p><b>CONCLUSION</b>There are parkin mutations happened in Chinese patients with AR-JP. Patients with parkin mutations have distinct clinical features besides the common clinical features of Parkinson's disease.</p>

Clinical and mutational analysis of KCNQ3 gene in a Chinese family with benign familial neonatal convulsions / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the clinical and genetic characteristics of a Chinese family with benign familial convulsions (BFNC).</p><p><b>METHODS</b>The clinical data of this family was analyzed. The blood samples were collected from 13 members of this family. By four microsatellite markers which are located in the gene loci of both K+ channel KCNQ2 and KCNQ3, the linkage analysis was performed in the family. With DNA direct sequencing and restriction endonuclease cutting analysis, the mutation analysis of KCNQ3 gene was made for the proband, other 12 family members and 76 unrelated normal individuals.</p><p><b>RESULTS</b>There were 7 patients with BFNC observed in the three generation of family. The BFNC seizures of all patients disappeared during one month and no recurrence of seizures was found. The linkage analysis suggested the disease gene linked to KCNQ3 gene locus in the family. The mutation 988(C to T) of KCNQ3 gene was found in the proband by DNA-direct sequencing. Cosegregation of this mutation with BFNC was confirmed by restriction endonuclease cutting analysis.</p><p><b>CONCLUSION</b>Chinese patients with BFNC can be caused by KCNQ3 gene mutation.</p>

Mutation analysis of DJ1 gene in patients with autosomal recessive early-onset Parkinsonism / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the mutation characteristics of DJ1 gene in Chinese patients with autosomal recessive early-onset Parkinsonism (AR-EP).</p><p><b>METHODS</b>Mutations of DJ1 gene were screened by polymerase chain reaction combined with DNA direct sequencing in index patients with AR-EP from 11 unrelated families.</p><p><b>RESULTS</b>No pathogenetic mutations in the DJ1 gene were detected in this group. Six intronic DJ1 polymorphisms (IVS1-15T-->C, IVS4+30T-->G, IVS4+45G-->A, IVS4+46G-->A, IVS5+31G-->A, g.168-185del) were found. Three of them (IVS1-15T-->C, IVS4+45G-->A, IVS4+46G-->A) were not reported previously.</p><p><b>CONCLUSION</b>DJ1 mutations were rare in Chinese patients with autosomal recessive early-onset Parkinsonism.</p>

Mutation analysis of small heat-shock protein 22 gene in Chinese patients with Charcot-Marie-Tooth disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the characteristics of the mutation of small heat-shock protein 22 (HSP22) gene in Chinese patients with Charcot-Marie-Tooth (CMT) disease.</p><p><b>METHODS</b>A CMT2L proband with 423(G--> T) mutation in HSP22 gene had been studied and reported by the present authors. In this study, mutation analysis of HSP22 gene was performed using polymerase chain reaction and DNA direct sequencing in 114 CMT probands.</p><p><b>RESULTS</b>In the 114 CMT probands, a 582(C--> T)(T194T)samesense mutation was found in two unrelated families.</p><p><b>CONCLUSION</b>The rate of HSP22 gene mutation in Chinese patients with CMT is as low as 0.87%(1/115).</p>

Clinical features of hereditary spastic paraplegia with thin corpus callosum: report of 5 Chinese cases / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hereditary spastic paraplegia is a clinically and genetically heterogeneous group of neurodegenerative disorders of the motor system, characterized by slowly progressive spasticity and weakness of the lower extremities. This study was conducted to investigate the clinical features of hereditary spastic paraplegia with thin corpus callosum (HSP-TCC).</p><p><b>METHODS</b>Clinical data from five patients and thirty-five previously published case reports of HSP-TCC were analyzed retrospectively.</p><p><b>RESULTS</b>Most patients were adolescents at the onset of the disease, presenting with spastic paraparesis of the lower limbs and mental impairment. Some patients also had other clinical features, including spasticity of the upper limbs, cerebellar ataxia, and sensory disturbances. Cranial MRIs of the five patients revealed an extremely thin corpus callosum, sometimes with widened cerebral sulci and ventricles, as well as with cerebellar and cerebral atrophy.</p><p><b>CONCLUSION</b>The main clinical features of HSP-TCC include slowly progressive spastic paraplegia, mental impairment during the second decade of life, and an extremely thin corpus callosum as shown on cranial MRIs.</p>

Spastin gene mutation in Chinese patients with hereditary spastic paraplegia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the mutation characteristics of spastin gene in Chinese patients with hereditary spastic paraplegia (HSP) and thus provide a basis for the gene diagnosis of HSP.</p><p><b>METHODS</b>Mutation of spastin gene was screened by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) combined with DNA direct sequencing in 31 unrelated affected HSP individuals in China, of whom 22 were from autosomal dominant families and 9 were sporadic HSP patients. Co-segregation analysis was carried out after the finding of abnormal SSCP bands.</p><p><b>RESULTS</b>Six cases were found to have abnormal SCP bands, and among them, two missense mutations (T1258A, A1293G in exon 8) and one deletion mutation (1667delACT or 1668delCTA or 1669delTAC in exon 14) were found and all of them were not reported previously. They were all co-segregated with the disease and were localized within the functional domain of spastin gene. Besides, T1258A was seen in two unrelated families.</p><p><b>CONCLUSION</b>The mutation rate (18.2%) in autosomal dominant HSP in Chinese patients is comparatively low. Point mutation is the major mutation type and exon 8 may be the mutation hot spot.</p>