Screening for, andVariants in a Cohort of Chinese Patients with Charcot-Marie-Tooth / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>SH3TC2, PMP2, and BSCL2 genes are related to autosomal recessive (AR) Charcot-Marie-Tooth (CMT) disease type 1, autosomal dominant (AD)-CMT1, and AD-CMT2, respectively. Pathogenic variants in these three genes were not well documented in Chinese CMT patients. Therefore, this study aims to detect SH3TC2, PMP2, and BSCL2 pathogenic variants in a cohort of 315 unrelated Chinese CMT families.</p><p><b>METHODS</b>A total of 315 probands from 315 unrelated Chinese CMT families were recruited from the Department of Neurology of Third Xiangya Hospital and Xiangya Hospital. We screened for SH3TC2 pathogenic variants in 84 AR or sporadic CMT probands, PMP2 pathogenic variants in 39 AD or sporadic CMT1 probands, and BSCL2 pathogenic variants in 50 AD or sporadic CMT2 probands, using polymerase chain reaction and Sanger sequencing. All these patients were out of 315 unrelated Chinese CMT families and genetically undiagnosed after exclusion of pathogenic variants of PMP22, MFN2, MPZ, GJB1, GDAP1, HSPB1, HSPB8, EGR2, NEFL, and RAB7. Candidate variants were analyzed based on the standards and guidelines of American College of Medical Genetics and Genomics (ACMG). Clinical features were reevaluated.</p><p><b>RESULTS</b>We identified three novel heterozygous variants such as p.L95V (c.283C>G), p.L1048P (c.3143T>C), and p.V1105M (c.3313G>A) of SH3TC2 gene and no pathogenic variants of PMP2 and BSCL2 genes. Although evaluation in silico and screening in the healthy control revealed that the three SH3TC2 variants were likely pathogenic, no second allele variants were discovered. According to the standards and guidelines of ACMG, the heterozygous SH3TC2 variants such as p.L95V, p.L1048P, and p.V1105M were considered to be of uncertain significance.</p><p><b>CONCLUSIONS</b>SH3TC2, PMP2, and BSCL2 pathogenic variants might be rare in Chinese CMT patients. Further studies to confirm our findings are needed.</p>

Classification and molecular diagnostic procedure for Chacort-Marie-Tooth disease / 中华医学遗传学杂志

Charcot-Marie-Tooth disease (CMT) is the most common form of hereditary neuropathy with significant clinical and genetic heterogeneity. So far 28 genes have been cloned. The main clinical manifestations of CMT include progressive distal muscle wasting and weakness, impaired distal sensation, and diminishing or loss of tendon reflex. Patients may be classified into demyelinating type (CMT1) and axonal type (CMT2) according to electrophysiological and pathological characteristics. Establishment of a standard diagnostic procedure based on clinical, electrophysiological and pathological findings will enable accurate diagnosis in most CMT patients and provide guidance for gene consulting and prognosis.

Cellular expression of (R127W)HSPB1 and its co-localization with neurofilament light chain / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To observe the cellular expression of (R127W) HSPB1 and its influence on neurofilament light chain (NFL) self-assembly and co-localization with NFL.</p><p><b>METHODS</b>Eukaryotic expression vectors pEGFPN1-(wt) HSPB1 and pEGFPN1- (R127W) HSPB1 were constructed. Hela cells were transiently transfected with pEGFPN1-(wt) HSPB1 or pEGFPN1- (R127W) HSPB1 and observed under a confocal microscope. Hela cells were also transiently co-transfected with Pcl-NFL and pEGFPN1-(wt)HSPB1, or pCL-NFL and pEGFPN1-(R127W)HSPB1. The self-assembly of NFL was observed and the co-localization study of HSPB1/ (R127W)HSPB1 with NFL was carried out in these two cell models by immunofluorescence technique.</p><p><b>RESULTS</b>The aggregates formed by EGFP-(R127W)HSPB1 predominantly located around the nucleus, and EGFP-(wt)HSPB1 showed diffusion pattern in Hela cells. When co expressed with EGFP-(wt)HSPB1, NFL formed homogeneous structure in cytosol. When co-expressed with EGFP-(R127W)HSPB1, however, NFL had amorphous staining pattern predominantly consisting of NFL aggregates, and NFL co-localized with (R127W)HSPB1 in these aggregates.</p><p><b>CONCLUSION</b>The R127W mutant of HSPB1 may have reduced capacity to serve as a chaperone to prevent aggregate formation, and fail to correctly organize the neurofilament network. Dysfunction of the axon cytoskeleton and axon transport may be the primary mechanism of R127W mutation of HSPB1 in the pathogenesis of Charcot-Marie-Tooth disease.</p>

The effect of HSPB8 gene mutation on cell viability in Charcot-Marie-Tooth disease type 2L / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the effect of Charcot-Marie-Tooth 2L disease causing gene K141N mutation in heat shock protein B8 gene (HSPB8) on cell viability.</p><p><b>METHODS</b>By using liposome transfection technique, (wt)HSPB8, (K141N)HSPB8 eukaryotic expression vector and green fluorescent protein (GFP) vector were transfected into SHSY-5Y cell, respectively. Twenty-four hours later, the cells were treated with 44 degree centigrade lethal heat shock for 40 minutes. The relative viability of SHSY-5Y cells in each group was tested by using tetrazole blue colorimetric method (methyl thiazolyl tetrazolium, MTT).</p><p><b>RESULTS</b>There were significant differences among the light absorption value of GFP, pEGFP-(wt)HSPB8 and pEGFP-(K141N)HSPB8 transfected groups after heat shock (P<0.05), indicating that the relative viability of cells overexpressed with (wt)HSPB8 and (K141N)HSPB8 was different from that of control cells. The viability of cells overexpressing (wt)HSPB8 was highest, followed by cells overexpressed with (K141N)HSPB8. The viability of cells tranfected with GFP only was the lowest.</p><p><b>CONCLUSION</b>HSPB8 may play an important role in the protection of cells under lethal heat shock treatment, and the K141N mutation can impair the protective effect.</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>

Cloning to rule out 10 candidate genes located in chromosome 12q24 for Charcot-Marie-Tooth disease type 2L / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To clone the disease-causing genes possibly existing in 6.8 cM distance between microsatellite markers D12S1720 and D12S1611 in chromosome 12q24 for Charcot-Marie-Tooth disease type 2L (CMT2L).</p><p><b>METHODS</b>Ten positional and functional candidate genes were chosen among all known genes in this locus region by bioinformatics inqury. Mutation detection was performed by sequencing the exons and intron-exon junctions of the candidate genes.</p><p><b>RESULTS</b>Eleven sequence variations, that included 5 heterozygous and 6 homozygous variations, were detected in the exons and flanking areas of the 10 candidate genes. All the variations showed no co-segregation with disease phenotype.</p><p><b>CONCLUSION</b>Ten candidate genes(TAOK3, RAB35, RPLP0, PXN, RNF10, RHOF, VPS33A, RSN, DENR, RNP24) were ruled out as the disease-causing gene for CMT2L. Ten single nucleotide polymorphisms (SNP) were reported for the first time.</p>

Study on aggregate formation mechanism of HSPB8 gene mutation resulting in CMT2L / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the possible mechanism of the intracellular aggregate formation of small heat shock protein HSPB8 (HSPB8)(K141N) mutation resulting in axonal Charcot-Marie-Tooth disease type 2L(CMT2L).</p><p><b>METHODS</b>The cell models which transiently expressed pEGFPN1-HSPB8 and pEGFPN1-(K141N)HSPB8 were established. The immunofluorescent co-location study of EGFP-(K141N)HSPB8 and HSPB1, EGFP-(K141N)HSPB8 and neurofilament light chain (NEFL) was carried out in the SHSY5Y cell models. The aggregate formation of EGFP-(K141N)HSPB8 in cell models was investigated and the possible mechanism of cellular aggregate formation was analyzed by t test and analysis of variance between group(ANOVA).</p><p><b>RESULTS</b>EGFP-(K141N)HSPB8 formed large aggregate which predominantly located around the nucleus in cell models. EGFP-(K141N)HSPB8 co-localized perfectly with HSPB1 and NEFL in the SHSY5Y cell models. The aggregate formation was different in different cell types, there were fewer aggregates formed in an sHSPs deficient milieu than in HEK293T cells.</p><p><b>CONCLUSION</b>(K141N)HSPB8 formed aggregates predominantly locate around the nucleus in cells. (K141N)HSPB8 co-localizes perfectly with HSPB1 and NEFL. The aggregate formation may be due to (K141N)HSPB8 conformational change leading to self aggregation and its abnormal interaction with other sHSPs such as HSPB1.</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, pathological and genetic studies in a Chinese Charcot-Marie-Tooth disease type 2 family / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To report a Chinese Charcot-Marie-Tooth disease type 2 (CMT2) family.</p><p><b>METHODS</b>All the members in the family were studied clinically,and 6 patients were studied electrophysiologically. Sural nerve biopsy was performed in the proband. PMP22 gene duplications were detected by highly polymorphic short tandem repeat. Point mutation analysis of PMP22, MPZ and NEFL gene was screened by PCR-SSCP combined with DNA direct sequencing. A genome-wide screening was carried out to the family.</p><p><b>RESULT</b>Except 2 who had weakness and atrophy in both proximal and distal muscles of the lower limbs, all patients presented muscle wasting and a predominating weakness of distal parts of the lower limbs, and mild to moderate sensory impairments. In 6 patients who were subjected to elctrophysiological examinations, median-nerve conduction velocity (NCV) of the median nerve was normal. Electromyograms (EMGs) revealed signs of denervation with large motor unit potentials, fibrillation potentials and positive sharp waves. Sural nerve biopsy of the proband confirmed the presence of axonal neuropathy with an important loss of large myelinating fibers and a large number of clusters with mostly thinly myelinated axons. PMP22, MPZ and NEFL gene mutations were not found. The results of genome-wide screening revealed a linkage of CMT2 to a locus at chromosome 12q24.</p><p><b>CONCLUSION</b>The results are consistent with the diagnosis of CMT2. This family represents a rare genetic type of CMT2 which can be designated as CMT2L.</p>

Mutation analysis of small heat shock protein 27 gene in Chinese patients with Charcot-Marie-Tooth disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the features of small heat shock protein 27 (HSP27) gene mutation in Chinese patients with Charcot-Marie-Tooth disease (CMT).</p><p><b>METHODS</b>DNA samples from 114 CMT probands were screened for mutations in HSP27 gene by polymerase chain reaction and direct sequencing, and haplotype analysis was further carried out on the mutation detected families.</p><p><b>RESULTS</b>One missense mutation C379T was detected in 4 autosomal dominant CMT2 families. Haplotype analysis indicated that the 4 families probably had a common ancestor.</p><p><b>CONCLUSION</b>To the authors' knowledge, this is the first report of HSP27 gene mutation in Chinese patients with CMT, but it may be not common(0.90%). The C379T mutation in HSP27 gene also causes CMT2 except for distal hereditary motor neuropathy, thus providing further evidence that even the same mutation in the same gene may lead to distinct phenotypes.</p>

Detection of duplications or deletions of the PMP22 gene using real-time quantitative PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect the duplication or deletion of peripheral myelin protein 22(PMP22) gene in Chinese patients with Charcot-Marie-Tooth disease(CMT) or hereditary neuropathy with liability to pressure palsies(HNPP) using real-time quantitative polymerase chain reaction.</p><p><b>METHODS</b>Duplications or deletions of PMP22 gene were detected in 113 CMT cases, 4 HNPP cases and 50 normal controls by using real-time quantitative PCR.</p><p><b>RESULTS</b>Thirty-six of 113 CMT cases had the PMP22 duplication, 4 HNPP cases had the PMP22 deletion. No duplication or deletion was found in 50 normal controls.</p><p><b>CONCLUSION</b>The PMP22 duplication rate in Chinese patients with CMT is 31.9%(36/113). PMP22 deletion is the common cause of HNPP.</p>

Mutation analysis of ganglioside-induced differentiation associated protein-1 gene in Chinese Charcot-Marie-Tooth disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the mutation feature of ganglioside-induced differentiation associated protein-1 (GDAP1) gene in Chinese Charcot-Marie-Tooth disease(CMT) patients.</p><p><b>METHODS</b>Mutation analysis was carried out by use of polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) combined with DNA direct sequencing of the six exons and their flanking regions of GDAP1 gene in twenty-three CMT patients, including 8 probands of autosomal recessive CMT families and 15 sporadic patients.</p><p><b>RESULTS</b>A compound heterozygous mutation A533G and A767G were unveiled in one autosomal recessive CMT kindred. The homozygous and heterozygous T507G were common SNPs in Chinese population.</p><p><b>CONCLUSION</b>A533G and A767G of GDAP1 gene were new mutations firstly reported.</p>