Clinical feature difference between juvenile amyotrophic lateral sclerosis with SPTLC1 and FUS mutations / 中华医学杂志(英文版)

BACKGROUND@#Juvenile amyotrophic lateral sclerosis (JALS) is an uncommon form of amyotrophic lateral sclerosis whose age at onset (AAO) is defined as prior to 25 years. FUS mutations are the most common cause of JALS. SPTLC1 was recently identified as a disease-causative gene for JALS, which has rarely been reported in Asian populations. Little is known regarding the difference in clinical features between JALS patients carrying FUS and SPTLC1 mutations. This study aimed to screen mutations in JALS patients and to compare the clinical features between JALS patients with FUS and SPTLC1 mutations.@*METHODS@#Sixteen JALS patients were enrolled, including three newly recruited patients between July 2015 and August 2018 from the Second Affiliated Hospital, Zhejiang University School of Medicine. Mutations were screened by whole-exome sequencing. In addition, clinical features such as AAO, onset site and disease duration were extracted and compared between JALS patients carrying FUS and SPTLC1 mutations through a literature review.@*RESULTS@#A novel and de novo SPTLC1 mutation (c.58G>A, p.A20T) was identified in a sporadic patient. Among 16 JALS patients, 7/16 carried FUS mutations and 5/16 carried respective SPTLC1 , SETX , NEFH , DCTN1 , and TARDBP mutations. Compared with FUS mutation patients, those with SPTLC1 mutations had an earlier AAO (7.9 ± 4.6 years vs. 18.1 ± 3.9 years, P  < 0.01), much longer disease duration (512.0 [416.7-607.3] months vs. 33.4 [21.6-45.1] months, P  < 0.01), and no onset of bulbar.@*CONCLUSION@#Our findings expand the genetic and phenotypic spectrum of JALS and help to better understand the genotype-phenotype correlation of JALS.

Three-Dimensional Heterogeneity of Cerebellar Interposed Nucleus-Recipient Zones in the Thalamic Nuclei / 神经科学通报·英文版

The cerebellum is conceptualized as a processor of complex movements and is also endowed with roles in cognitive and emotional behaviors. Although the axons of deep cerebellar nuclei are known to project to primary thalamic nuclei, macroscopic investigation of the characteristics of these projections, such as the spatial distribution of recipient zones, is lacking. Here, we studied the output of the cerebellar interposed nucleus (IpN) to the ventrolateral (VL) and centrolateral (CL) thalamic nuclei using electrophysiological recording in vivo and trans-synaptic viral tracing. We found that IpN stimulation induced mono-synaptic evoked potentials (EPs) in the VL but not the CL region. Furthermore, both the EPs induced by the IpN and the innervation of IpN projections displayed substantial heterogeneity across the VL region in three-dimensional space. These findings indicate that the recipient zones of IpN inputs vary between and within thalamic nuclei and may differentially control thalamo-cortical networks.

Three-Dimensional Heterogeneity of Cerebellar Interposed Nucleus-Recipient Zones in the Thalamic Nuclei / 神经科学通报·英文版

The cerebellum is conceptualized as a processor of complex movements and is also endowed with roles in cognitive and emotional behaviors. Although the axons of deep cerebellar nuclei are known to project to primary thalamic nuclei, macroscopic investigation of the characteristics of these projections, such as the spatial distribution of recipient zones, is lacking. Here, we studied the output of the cerebellar interposed nucleus (IpN) to the ventrolateral (VL) and centrolateral (CL) thalamic nuclei using electrophysiological recording in vivo and trans-synaptic viral tracing. We found that IpN stimulation induced mono-synaptic evoked potentials (EPs) in the VL but not the CL region. Furthermore, both the EPs induced by the IpN and the innervation of IpN projections displayed substantial heterogeneity across the VL region in three-dimensional space. These findings indicate that the recipient zones of IpN inputs vary between and within thalamic nuclei and may differentially control thalamo-cortical networks.

Penetrance estimation of PRRT2 variants in paroxysmal kinesigenic dyskinesia and infantile convulsions / 医学前沿

Proline-rich transmembrane protein 2 (PRRT2) is the leading cause of paroxysmal kinesigenic dyskinesia (PKD), benign familial infantile epilepsy (BFIE), and infantile convulsions with choreoathetosis (ICCA). Reduced penetrance of PRRT2 has been observed in previous studies, whereas the exact penetrance has not been evaluated well. The objective of this study was to estimate the penetrance of PRRT2 and determine its influencing factors. We screened 222 PKD index patients and their available relatives, identified 39 families with pathogenic or likely pathogenic (P/LP) PRRT2 variants via Sanger sequencing, and obtained 184 PKD/BFIE/ICCA families with P/LP PRRT2 variants from the literature. Penetrance was estimated as the proportion of affected variant carriers. PRRT2 penetrance estimate was 77.6% (95% confidence interval (CI) 74.5%-80.7%) in relatives and 74.5% (95% CI 70.2%-78.8%) in obligate carriers. In addition, we first observed that penetrance was higher in truncated than in non-truncated variants (75.8% versus 50.0%, P = 0.01), higher in Asian than in Caucasian carriers (81.5% versus 68.5%, P = 0.004), and exhibited no difference in gender or parental transmission. Our results are meaningful for genetic counseling, implying that approximately three-quarters of PRRT2 variant carriers will develop PRRT2-related disorders, with patients from Asia or carrying truncated variants at a higher risk.

Primary age-related tauopathy in a Chinese cohort / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Primary age-related tauopathy (PART) is characterized by the presence of tau neurofibrillary tangles (NFTs) which are typically observed in Alzheimer's disease (AD) brains, with few or without β-amyloid (Aβ) plaques. The diagnosis of PART can be categorized into "definite" or "possible" depending on the amount of Aβ plaques. Definite PART is diagnosed when NFTs are observed and the Braak stage is ≤IV, with Thal Aβ Phase 0 (Crary et al., 2014). According to the neuropathological diagnostic criteria, we reported that PART was frequently observed in the Chinese population according to our findings from specimens in our brain bank, with 47% of brain bank subjects meeting the criteria for PART. There is no consensus on the nature of PART. It remains to be elucidated whether PART is an early form of AD or a novel tauopathy (Duyckaerts et al., 2015; Jellinger et al., 2015).

Effect of Apolipoprotein E Genotypes on Huntington's Disease Phenotypes in a Han Chinese Population / 神经科学通报·英文版

Huntington's disease (HD) is an autosomal dominant degenerative disease that mainly encompasses movement, cognition, and behavioral symptoms. The apolipoprotein E (APOE) gene is thought to be associated with many neurodegenerative diseases. Here, we enrolled a cohort of 223 unrelated Han Chinese patients with HD and 1241 unrelated healthy controls in Southeastern China and analyzed the correlation between APOE genotypes and HD phenotypes. The results showed that the frequency of the E4 allele (7.1%) in HD patients was statistically less than that in controls (12.0%) (P =0.004). In addition, we divided patients into motor-onset and non-motor-onset groups, and analyzed the relationship with APOE genotypes. The results, however, were negative. Furthermore, the age at onset (AAO), defined as the age at the onset of motor symptoms, was compared in each APOE genotype subgroup and multivariate regression analysis was used to exclude the interference of CAG repeat length on AAO, but no association was found between APOE genotypes and AAO. Finally, we analyzed adult-onset HD to exclude the interference caused by juvenile HD (n = 13), and the results were negative. Therefore, our study suggests that APOE may not be a genetic modifier for HD, especially for adult-onset HD among Chinese of Han ethnicity. To the best of our knowledge, this is the first study of the correlation between APOE genotypes and HD phenotypes in a Han Chinese population.

Variant of Associated with Increasing Risk in Chinese Patients with Relapsing-remitting Multiple Sclerosis / 中华医学杂志(英文版)

<p><b>Background</b>Multiple sclerosis (MS) is a common central nervous system autoimmune disorder. Increasing number of genome-wide association study (GWAS) analyses hint that MS is strongly associated with genetics. Unfortunately, almost all the GWAS analyses were Caucasian population based. Numbers of risk loci might not be replicated in Chinese MS patients. Hence, we performed a MassArray Assay to genotype the previously reported variants located in the transcription regulation genes in order to elucidate their role in the Chinese MS patients.</p><p><b>Methods</b>One hundred and forty-two relapsing-remitting MS (RRMS) patients and 301 healthy controls were consecutively collected from September 2, 2008, to June 7, 2013, as stage 1 subjects. Eight reported transcription regulation-related single-nucleotide polymorphisms (SNPs) were genotyped using the Sequenom MassArray system. In stage 2, another 44 RRMS patients and 200 healthy controls were consecutively collected and Sanger sequenced from April 7, 2015, to June 29, 2017, for the validation of positive results in stage 1. Differences in allele and genotype frequencies between patients and healthy controls, odds ratios, and 95% confidence intervals were calculated with the Chi-square test or Fisher's exact test. Hardy-Weinberg equilibrium was tested also using the Chi-square test.</p><p><b>Results</b>In stage 1 analysis, we confirmed only one previously reported risk variant, rs11129295 in EOMES gene. We found that the frequency of T/T genotype was much higher in MS group (χ = 10.251, P = 0.005) and the T allele of rs11129295 increased the risk of MS (χ = 10.022, P = 0.002). In stage 2 and combined analyses, the T allele of rs11129295 still increased the risk of MS (χ = 4.586, P = 0.030 and χ = 16.378, P = 5.19 × 10, respectively).</p><p><b>Conclusions</b>This study enhances the knowledge that the variant of EOMES is associated with increasing risk in Chinese RRMS patients and provides a potential therapeutic target in RRMS.</p>

A Novel Missense Mutation in Peripheral Myelin Protein-22 Causes Charcot-Marie-Tooth Disease / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. A great number of causative genes have been described in CMT, and among them, the heterozygous duplication of peripheral myelin protein-22 (PMP22) is the major cause. Although the missense mutation in PMP22 is rarely reported, it has been demonstrated to be associated with CMT. This study described a novel missense mutation of PMP22 in a Chinese family with CMT phenotype.</p><p><b>METHODS</b>Targeted next-generation sequencing (NGS) was used to screen the causative genes in a family featured with an autosomal dominant demyelinating form of CMT. The potential variants identified by targeted NGS were verified by Sanger sequencing and classified according to the American College of Medical Genetics and Genomics standards and guidelines. Further cell transfection studies were performed to characterize the function of the novel variant.</p><p><b>RESULTS</b>Using targeted NGS, a novel heterozygous missense variant in PMP22 (c.320G>A, p.G107D) was identified. In vitro cell functional studies revealed that mutant PMP22 protein carrying p.G107D mutation lost the ability to reach the plasma membrane, was mainly retained in the endoplasmic reticulum, and induced cell apoptosis.</p><p><b>CONCLUSIONS</b>This study supported the notion that missense mutations in PMP22 give rise to a CMT phenotype, possibly through a toxic gain-of-function mechanism.</p>

AMutation in Dystrophin Causing Muscular Dystrophy in a Female Patient / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular diseases resulting from dystrophin (DMD) gene mutations. It has been known that the carrier of DMD mutations may also have symptoms of the disease. While de novo mutation is quite common in BMD/DMD patients, it is rarely reported in the female carriers.</p><p><b>METHODS</b>Two sporadic Chinese patients with progressive muscular dystrophy and their familial members were recruited. The targeted next-generation sequencing (NGS) and the multiplex ligation-dependent probe analysis (MLPA) were performed in the proband. Blood tests, electrocardiography, echocardiography, and electromyography were also evaluated.</p><p><b>RESULTS</b>Two novel mutations of DMD gene were identified, c.7318C>T (p.Q2440*) in the male proband and c.4983dupA (p.A1662Sfs*24) in the female carrier. The MLPA analysis did not detect any large rearrangements. The haplotype analysis indicated that the two mutations were derived from de novo mutagenesis.</p><p><b>CONCLUSIONS</b>We identified two novel de novo mutations of DMD gene in two Chinese pedigrees, one of which caused a female patient with muscular dystrophy. The mutational analysis is important for DMD patients and carriers in the absence of a family history. The NGS can help detect the mutations in MLPA-negative patients.</p>

Mutation Analysis of MR-1, SLC2A1, and CLCN1 in 28 PRRT2-negative Paroxysmal Kinesigenic Dyskinesia Patients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Paroxysmal kinesigenic dyskinesia (PKD) is the most common subtype of paroxysmal dyskinesias and is caused by mutations in PRRT2 gene. The majority of familial PKD was identified to harbor PRRT2 mutations. However, over two-third of sporadic PKD patients did not carry anyPRRT2 mutation, suggesting an existence of additional genetic mutations or possible misdiagnosis due to clinical overlap.</p><p><b>METHODS</b>A cohort of 28 Chinese patients clinically diagnosed with sporadic PKD and excluded PRRT2 mutations were recruited. Clinical features were evaluated, and all subjects were screened for MR-1, SLC2A1, and CLCN1 genes, which are the causative genes of paroxysmal nonkinesigenic dyskinesia (PNKD), paroxysmal exertion-induced dyskinesia, and myotonia congenita (MC), respectively. In addition, 200 genetically matched healthy individuals were recruited as controls.</p><p><b>RESULTS</b>A total of 16 genetic variants including 4 in MR-1 gene, 8 in SLC2A1 gene, and 4 in CLCN1 gene were detected. Among them, SLC2A1 c.363G>A mutation was detected in one case, and CLCN1 c.1205C>T mutation was detected in other two cases. Neither of them was found in 200 controls as well as 1000 Genomes database and ExAC database. Both mutations were predicted to be pathogenic by SIFT and PolyPhen2. The SLC2A1 c.363G>A mutation was novel.</p><p><b>CONCLUSIONS</b>The phenotypic overlap may lead to the difficulty in distinguishing PKD from PNKD and MC. For those PRRT2- negative PKD cases, screening of SLC2A1 and CLCN1 genes are useful in confirming the diagnosis.</p>

Variants of Interferon Regulatory Factor 5 are Associated with Neither Neuromyelitis Optica Nor Multiple Sclerosis in the Southeastern Han Chinese Population / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Neuromyelitis optica (NMO) and multiple sclerosis (MS) are demyelinating disorders of the central nervous system. Interferon regulatory factor 5 (IRF5) is a common susceptibility gene to different autoimmune disorders. However, the association of IRF5 variants with NMO and MS patients has not been well studied. Therefore, we aimed to evaluate whether IRF5 variants were associated with NMO and MS in the Southeastern Han Chinese population.</p><p><b>METHODS</b>Four single nucleotide polymorphisms (SNPs) were selected and genotyped by matrix-assisted laser desorption/ionization time of flight mass spectrometry in 111 NMO patients, 145 MS patients and 300 controls from Southeastern China.</p><p><b>RESULTS</b>None of these 4 SNPs was associated with NMO or MS patients.</p><p><b>CONCLUSIONS</b>Our preliminary study indicates that genetic variants in IRF5 may affect neither NMO nor MS in the Southeastern Han Chinese population. Further studies with a large sample size and diverse ancestry populations are needed to clarify this issue.</p>

Huntington Disease in Asia / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>The objective was to review the major differences of Huntington disease (HD) in Asian population from those in the Caucasian population.</p><p><b>DATA SOURCES</b>Data cited in this review were obtained from PubMed database and China National Knowledge Infrastructure (CNKI) from 1994 to 2014. All the papers were written in English or Chinese languages, with the terms of Asia/Asian, HD, genotype, epidemiology, phenotype, and treatment used for the literature search.</p><p><b>STUDY SELECTION</b>From the PubMed database, we included the articles and reviews which contained the HD patients' data from Asian countries. From the CNKI, we excluded the papers which were not original research. Due to the language's restrictions, those data published in other languages were not included.</p><p><b>RESULTS</b>In total, 50 papers were cited in this review, authors of which were from the mainland of China, Japan, India, Thailand, Taiwan (China), Korea, and western countries.</p><p><b>CONCLUSIONS</b>The lower epidemiology in Asians can be partly explained by the less cytosine-adenine-guanine repeats, different haplotypes, and CCG polymorphisms. For the physicians, atypical clinical profiles such as the initial symptom of ataxia, movement abnormalities of Parkinsonism, dystonia, or tics need to be paid more attention to and suggest gene testing if necessary. Moreover, some pathogenesis studies may help progress some new advanced treatments. The clinicians in Asian especially in China should promote the usage of genetic testing and put more effects in rehabilitation, palliative care, and offer comfort of patients and their families. The unified HD rating scale also needs to be popularized in Asia to assist in evaluating the progression of HD.</p>

Variants of Interleukin-7/Interleukin-7 Receptor Alpha are Associated with Both Neuromyelitis Optica and Multiple Sclerosis Among Chinese Han Population in Southeastern China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Neuromyelitis optica (NMO) and multiple sclerosis (MS) are autoimmune demyelinating diseases of the central nerve system. Interleukin-7 (IL-7) and interleukin-7 receptor alpha (IL-7Rα) were proved to be important in the pathogenesis of both diseases because of the roles they played in the differentiations of autoimmune lymphocytes. The variants of both genes had been identified to be associated with MS susceptibility in Caucasian, Japanese and Korean populations. However, the association of these variants with NMO and MS has not been well studied in Chinese Southeastern Han population. Here, we aimed to evaluate the association of six IL-7 variants (rs1520333, rs1545298, rs4739140, rs6993386, rs7816065, and rs2887502) and one variant of IL-7RA (rs6897932) with NMO and MS among Chinese Han population in southeastern China.</p><p><b>METHODS</b>Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MassARRAY system) and Sanger sequencing were used to determine the variants of IL-7 and IL-7RA in 167 NMO patients, 159 MS patients and 479 healthy controls among Chinese Han population in southeastern China. Samples were excluded if the genotyping success rate <90%.</p><p><b>RESULTS</b>Statistical differences were observed in the genotypes of IL-7 rs1520333 in MS patients and IL-7RA rs6897932 in NMO patients, compared with healthy controls (P = 0.035 and 0.034, respectively). There was a statistically significant difference in the genotypes of IL-7 rs2887502 between MS and NMO patients (P = 0.014). And there were statistically significant differences in the rs6897932 genotypes (P = 0.004) and alleles (P = 0.042) between NMO-IgG positive patients and healthy controls.</p><p><b>CONCLUSIONS</b>The study suggested that among Chinese Han population in southeastern China, the variant of IL-7RA (rs6897932) was associated with NMO especially NMO-IgG positive patients while the variant of IL-7 (rs1520333) with MS patients. And the genotypic differences of IL-7 rs2887502 between MS and NMO indicated the different genetic backgrounds of these two diseases.</p>

Wilson’s disease in Asia

Wilson’s disease is an autosomal recessive disorder of copper metabolism. The resultant accumulation of copper primarily damages the liver and brain, resulting in hepatic, neurological and psychiatric symptoms. There have been many recent studies advancing the understanding of Wilson’s disease in Asia. There are indications that the incidence of Wilson’s disease in parts of Asia may be relatively high. Many genetic studies have identifi ed various hot spots in theATP7B gene in a variety of the Asian populations. Screening of these hotspot mutations may thus be useful in confi rming the diagnosis. Despite the advances in treatment, lack of familiarity by the health care profession resulting in late diagnosis, and poor access to treatment particularly among those from the developing economies remain areas of major concern.

Analysis of alleles 4qA and 4qB of the chromosome 4q subtelomere in Chinese Han population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze two alleles (4qA and 4qB) distal to D4Z4 of the 4q subtelomere in Chinese population, and to elucidate the interrelationship between these variants of 4qter and facioscapulohumeral muscular dystrophy (FSHD).</p><p><b>METHODS</b>Eighty unrelated healthy individuals from a random Chinese Han population were investigated. The genomic DNA was extracted from peripheral blood lymphocytes according to the specific procedure designed to minimize DNA shearing, then digested with EcoRI, HindIII or double digested with EcoRI and BlnI. The cleaved DNA was separated by pulsed field gel electrophoresis (PFGE) and Southern blotting with the probes p13E-11, 4qA and 4qB. The sizes of 4q35 EcoRI/4qA and EcoRI/4qB arrays were obtained by "curve fitting", and the frequencies of alleles and genotypes were calculated. Data were analyzed using a commercially available statistical package (Version 13.0 SPSS).</p><p><b>RESULTS</b>In normal individuals, frequencies of 4qA and 4qB alleles (46.9% and 53.1%) were observed of no significant difference (chi(2) = 1.250, P>0.05). The frequency of 4qA/4qB heterozygote was much higher than that of homozygote (P<0.05). The means of EcoRI/4qA and EcoRI/ 4qB arrays (115.8+/-11.9 kb and 98.3+/-8.6 kb) were of significant difference (t=23.04, P<0.001). 8.8% (7/80) of the individuals displayed a translocation repeat array configuration. 4qB-type EcoRI arrays smaller than 35 kb were found in two individuals.</p><p><b>CONCLUSION</b>The structural polymorphism of 4qA/4qB alleles within 4q35 and 10q26 is confirmed using PFGE in normal Chinese Han population. Although both alleles are almost equally common, shorten 4qB-type EcoRI fragment is not pathogenic. The frequency of 4qA/4qB heterozygote is significantly higher than that of homozygote.</p>

Clinical analysis of dopa-responsive dystonia and mutation analysis of the GCH I gene / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the clinical characteristics and GCH I gene mutations in patients with dopa-responsive dystonia (DRD).</p><p><b>METHODS</b>The clinical features of 3 families with 6 affected members and 8 sporadic cases were analyzed to determine the clinical characteristics, and 2 families with 4 affected members and 2 sporadic cases were screened for mutations of the GCH I gene.</p><p><b>RESULTS</b>Age at onset was (10 +/- 3) years. Onset occurred earlier in female (9 +/- 4) years than in male (12 +/- 1) years. The initial symptom was a gait disorder, dystonia or tremor in most patients and nine patients (64%) presented with diurnal fluctuation. Thirteen patients (93%) were cured and one was improved after administration of low doses of levodopa for 3 months and no long-term side effects of levodopa had occurred. Two independent mutations were found in three patients. Gln161Pro, a new missense mutation, was found in a sporadic case, leading to a relatively severe phenotype. The two patients with mild phenotype in one family were found to have Lys224Arg mutation, as previously described.</p><p><b>CONCLUSIONS</b>DRD patients have diverse phenotypes and diurnal fluctuation is an important feature. They have dramatic and sustained response to levodopa. There may be a correlation between genotype and phenotype. The detection of GCH I mutations is helpful in early diagnosis of non-typical cases.</p>

Rapid diagnosis of spinal muscular atrophy using denaturing high-performance liquid chromatography / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To introduce the application of denaturing high-performance liquid chromatography (DHPLC) in the diagnosis of childhood type spinal muscular atrophy (SMA).</p><p><b>METHODS</b>Exon 7 and flanking area of survival motor neuron (SMN) gene were amplified by PCR in 1 standard sample, 25 normal individuals and 25 patients with SMA. The PCR products were then directly loaded onto the DHPLC system after denaturing and annealing. Different DNA segments were separated by changing the concentration of buffer A relative to that of buffer B.</p><p><b>RESULTS</b>Different DNA segments were separable on the DHPLC chromatogram. Three peaks including SMN1/SMN2 heteroduplex peak, SMN2 homoduplex peak and SMN1 homoduplex peak were detected in 23 out of 25 normal individuals. Only SMN1 homoduplex peak was detected in 2 normal individuals and the standard sample, indicating the deletion of SMN2 On the contrary, only the SMN2 homoduplex peak was detected in 22 out of 25 patients with SMA, indicating deletion of SMN1. The three peaks as those of normal individuals were detected in the other 3 patients, indicating no SMN1 or SMN2 deletion.</p><p><b>CONCLUSION</b>As a new technology for diagnosing SMA, DHPLC is sensitive, accurate, rapid and convenient.</p>

Quantitative studies on SMN1 gene and carrier testing of spinal muscular atrophy / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To construct a method for detecting the copy number of survival of motor neuron 1 gene (SMN1) with single copy difference based on real-time fluorescence quantitative PCR, and to make practical use of the method for acquiring the data on SMN1 copy number in Chinese as well as for screening the carriers of spinal muscular atrophy (SMA) from healthy individuals and SMA families.</p><p><b>METHODS</b>Exon 7 and flanking area of SMN1 gene were amplified by real-time fluorescence quantitative PCR in 264 healthy individuals, in 1 standard sample having 2 SMN1 but having no SMN2, and in 88 parents of SMA patients. The samples for detecting were diluted to 30 ng/microL and the standard sample was diluted to 15 ng/microL, 30 ng/microL, 45 ng/microL, 60 ng/microL; the unknown samples and 4 standard samples with different concentrations were amplified at the same time, a standard curve could be drawn out according to the results of the 4 standard samples, then the copy number of samples could be calculated.</p><p><b>RESULTS</b>Of 88 parents' samples, 84 samples each had 1 copy of SMN1, and the rest 4 each had 2 copies of SMN1. Of 264 healthy individuals' samples, 5 samples each had only 1 copy of SMN1 (an indicator of definite gene carriers), 232 samples each had 2 copies of SMN1, 25 samples each had 3 copies of SMN1, and 2 samples each had 4 copies of SMN1. Of the samples of 32 members of SMA families, 2 samples each had only 1 copy of SMN1 indicating definite gene carriers, 25 samples each had 2 copies of SMN1, and 5 samples each had 3 copies of SMN1.</p><p><b>CONCLUSION</b>SMN1 copy number could be detected precisely by real-time fluorescence quantitative PCR; the screening of gene carriers could provide essential data for genetic counseling.</p>

Preparation of polyclonal antibody against survival motor neuron protein and study on the expression of survival motor neuron protein in the skeletal muscular of patients with spinal muscular atrophy / 中华神经科杂志

Objective To prepare the survival motor neuron(SMN)polyclonal antibody and explore the localization of SMN protein in transfected cells and its expression in skeletal muscles of patients with spinal muscular atrophy(SMA).Methods A prokaryotic expressional plasmid named pET-28? (+)/SMN was constructed and SMN-His fusion protein was induced.The fusion protein was used to immunize New Zealadd rabbits to prepare SMN polyclonal antibody.A eukaryotic expressional plasmid named pcDNA3.1/myc-HisB-SMN was constructed and used to transfect CHO cells.Skeletal muscles were collected from 3 patients with bone fracture who were regarded as normal controls, and 3 SMA patients of type Ⅰ, 3 of type Ⅱ and 3 of type Ⅲ who were ascertained by genetic analysis.Western-blotting and immunofluorescence stain were applied to study the expression of SMN in transfected CHO cells and skeletal muscles of normal individuals and SMA patients.Results Correct pET-28a(+)/SMN prokaryotic expressive plasmid was constructed and SMN-His fusion protein was obtained from E coli BL21 transformed with pET-28a(+)/SMN.Then, rabbit anti-human full-length SMN polyclonal antibody of high specificity and sensitivity was obtained from rabbits immunized by SMN-His fusion protein.SMN proteins were shown diffusedly locating in the cytoplasm and nucleus of CHO cells transfected with pcDNA3.1/myc-HisB-SMN plasmid and mainly accumulating around the nucleus.The results of Western-blotting were as follows:the average ratio of SMN band density to glyceraldehyde phosphate dehydrogenase(GAPDH)band density (SMN/GAPDH)is 0.619 in skeletal muscles from normal controls, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type Ⅲ and Ⅱ were 0.347 and 0.340 respectively, which were lower than that of normal controls.However, the average values of SMN/GAPDH in skeletal muscle from SMA patients of type I was only 0.079, which was quite lower than that of normal controls.Conclusions The rabbit anti-human full-length SMN polyclonal antibody is of high specificity and sensitivity, which makes the basis for the research of SMN function and SMA pathogenesis.There may be a correlation between the SMN level in skeletal muscle and the severity of disease.

Optimization of short tandem repeats and their application in prenatal diagnosis of spinal muscular atrophy / 中华神经科杂志

Objective To optimize the short tandem repeats(STR)which link closely to survival motor neuron(SMN)and have redundant polymorphism information contents,and to use these STR in the prenatal diagnosis of spinal muscular atrophy(SMA).Methods Eleven STR loci(D5S435,D5F153, DSF151,D5S637,D5S1413,D5S125,D5S464,D5S1556,DSF149,D5S351,MAP1B-5')were amplified by PCR.Then the PCR products were detected by polyacrylamide gel electrophoresis(PAGE)and analyzed by silver staining.STR loci were evaluated and optimized by their PIC values.PCR-PAGE and gene scan were combined to make genetic link analysis for SMA families based on the optimized STR.Results Three STR loci(D5S435,DSF149 and D5S351)were selected with 8,19 and 18 polymorphic fragments detected respectively in 100 normal individuals.Their PIC values were 0.84,0.91 and 0.92 respectively.Four carriers and 2 normal individuals were detected from 6 SMA families with linkage analysis by using the 3 STR.Conclusion This genetic diagnosis system based on the 3 STR loci can provide rapid prenatal diagnosis for SMA families,can eliminate maternal blood contamination,and also can discriminate carriers from normal individuals in the fetuses,which makes the prenatal diagnosis system of SMA perfect.