Differentiation and tumorigenicity of neural stem cells from human cord blood mesenchymal stem cells

BACKGROUND： Mesenchymal stem cells （MSCs） are capable of differentiating into a variety of tissues and exhibit low immunogenicity. OBJECTIVE： To investigate isolation and in vitro cultivation methods of human cord blood MSCs, to observe expression of neural stem cell （NSC） marker mRNA under induction, and to detect tumorigenicity in animals. DESIGN, TIME AND SETTING： A cell biological, in vitro trial and a randomized, controlled, in vivo experiment were performed at the Department of Neurology, Daping Hospital at the Third Military Medical University of Chinese PLA from August 2006 to May 2008. MATERIALS： Umbilical cord blood was collected from full-term-delivery fetus at the Department of Gynecology and Obstetrics of Daping Hospital, China. Eighteen BALB/C nu/nu nude mice were randomly assigned to three groups： back subcutaneous, cervical subcutaneous, and control, with 6 mice in each group. METHODS： Monocytes were isolated from heparinized human cord blood samples by density gradient centrifugation and then adherent cultivated in vitro to obtain MSC clones. After the cord blood MSCs were cultured for 7 days with nerve growth factor and retinoic acid to induce differentiation into NSCs, the cells （adjusted density of 1 × 10^7/mL） were prepared into cell suspension. In the back subcutaneous and cervical subcutaneous groups, nude mice were hypodermically injected with a 0.5-mL cell suspension into the back and cervical regions, respectively. In the control group, nude mice received a subcutaneous injection of 0.5 mL physiological saline into the back or cervical regions, respectively. MAIN OUTCOME MEASURES： Cellular morphology was observed by inverted microscopy, cultured cord blood MSCs were examined by flow cytometry, expression of nestin and musashi-1 mRNA was detected by reverse-transcriptase polymerase chain reaction prior to and after induction, and tumorigenicity following cord blood MSC transplantation was assayed by hematoxylin-eosin staining. RESULTS： Following adherent cultivation, the majority of cord blood monocytes became rhombic and strongly expressed CD29, but not CD34, CD1 la, or CD11 b. These results supported previously known characteristics of cord blood MSCs. Following differentiation induction, nestin and musashi-1 were expressed on the surface of NSCs, exhibiting strongest expression at 48 hours, and subsequently reducing expression. Cultured cord blood MSCs were not tumorigenic in the nude mice. Cellular morphology displayed no malignant changes between the control and subcutaneous groups. CONCLUSION： MSCs can be isolated from human cord blood, efficiently expanded under culture conditions, differentiated into NSCs following induction, and display no tumorigenicity in nude mice.

Do we need to screen every patient in intensive care unit for diabetes in community with high prevalence of diabetes?

Diabetes mellitus (DM) is marked as global health care challenge with almost 10% of the United States population being diagnosed with DM. A sizeable percentage of patients are oblivious of their disease, in spite of easily accessibility knowledge about its early signs and symptoms and rapid diagnostic modalities. Critically ill patients with undiagnosed DM are likely to have an increased mortality as compared to intensive care unit (ICU) patients with diagnosed DM. DM may have adverse effect on ICU patients causing organ failure and complications. Early Screening of patients at the risk of developing disease may prevent long term complications. Early screening and management may be beneficial as controlled DM patients have similar morbidity as non DM patients in ICU. An intense glycaemic and blood pressure control improves retinopathy and albuminuria, but may not affect the macrovascular outcomes.

Logistic regression analysis on risk factors for vascular dementia following cerebral infarction in 403 patients from Chongqing City Hospital and family follow-up studies

BACKGROUND： Studies have demonstrated that the risk factors of vascular dementia following stroke are greatly different in region, race and other aspects. OBJECTIVE： To analyze the conditions and incidental risk factors of vascular dementia in patients with acute cerebral infarction from Chongqing City. DESIGN： Case analysis. SETTING： Department of Neurology, Daping Hospital, Third Military Medical University of Chinese PLA. PARTICIPANTS： Altogether 546 inpatients with acute ischemic stroke admitted to Department of Neurology, Daping Hospital, Third Military Medical University of Chinese PLA between May 1999 and December 2002 were involved in this study. The involved patients, including 295 males and 251 females, aged 55 - 94 years, dwelled in Chongqing over 5 years. They were admitted to hospital within 48 hours of attack of acute ischemic stroke, and survived for over 3 months. Informed consents were obtained from all the involved subjects. METHODS： ① Following the same standard, cognitive and social function evaluations were conducted by one physician on admission and 3 months after admission, Unified questionnaire, consisting of general characteristics, vascular risk factors, stroke characteristics, neurological physical sign, and other 28 factors of involved subjects, was used in all the patients. According to the investigation results, the patients were assigned into 2 groups： dementia group and non-dementia group. ②Ischemic stroke was diagnosed according to acute ischemic brain disorder 〉 24 hours and CT or MRI imageology.③ Neurophysiological examination was conducted in all the patients at 7 to l0 days after stroke （score was two SD less than or equaled to normal level was considered as abnormal）. ④Diagnosis and statistics of dementia were carried out with Mini-Mental State Examination and The Diagnostic and Statistical Manual of Mental Disorders-Ⅳ （published by American Psychiatric Association） on admission and 3 months after admission. Neurologic deficit scoring was carried out with the National Institutes of Health Stroke Scale. ⑤ Chi-square test was used for categorical variable, and t test for quantitative variable between dementia group and non-dementia group. Dementia-related factors were performed multiple-factor Logistic regression model analysis. MAIN OUTCOME MEASURES： Incidence of dementia and dementia-related risk factors of patients. RESULTS： Altogether 546 patients with stroke were involved in this study, 403 of them participated in the final analysis, and 143 dropped out. A total of 342 were followed-up in the hospital and 61 at home. At 3 months after cerebral infarction, vascular dementia occurred in 87 （21.6%） of 403 patients. The main risk factors were age （OR 1.179; 95%CI 1.130 - 1.230）, low education level （OR 1.806; 95%CI 1.024 - 3.186）, daily alcohol drinking （OR 3.447; 95%（1/ 1.591 - 7.468）, stroke history （OR 2.531; 95%CI 1.419 - 4.512）, atrial fibrilation（OR 3.475; 95%CI 1.712 - 7.057）, dysphonia （OR 5.873; 95%6/2.620 - 13.163） and left carotid artery infarction （OR 1.975; 95%（1/1.152 - 3.388）. CONCLUSION： The incidence of vascular dementia is determined by synthetic action of multiple risk factors. Dysphonia is the most important influencing factor.

Alzheimer’s disease early diagnostic and staging biomarkers revealed by large-scale cerebrospinal fluid and serum proteomic profiling

Amyloid-b,tau pathology,and biomarkers of neurodegeneration make up the core diagnostic biomarkers of Alzheimer disease(AD).However,these proteins represent only a fraction of the complex biological processes underlying AD,and individuals with other brain diseases in which AD pathology is a comorbidity also test positive for these diagnostic biomarkers.More ADspecific early diagnostic and disease staging biomarkers are needed.In this study,we performed tandem mass tag proteomic analysis of paired cerebrospinal fluid(CSF)and serum samples in a discovery cohort comprising 98 participants.Candidate biomarkers were validated by parallel reaction monitoring–based targeted proteomic assays in an independent multicenter cohort comprising 288 participants.We quantified 3,238 CSF and 1,702 serum proteins in the discovery cohort,identifying 171 and 860 CSF proteins and 37 and 323 serum proteins as potential early diagnostic and staging biomarkers,respectively.In the validation cohort,58 and 21 CSF proteins,as well as 12 and 18 serum proteins,were verified as early diagnostic and staging biomarkers,respectively.Separate 19-protein CSF and an 8-protein serum biomarker panels were built by machine learning to accurately classify mild cognitive impairment(MCI)due to AD from normal cognition with areas under the curve of 0.984 and 0.881,respectively.The 19-protein CSF biomarker panel also effectively discriminated patients with MCI due to AD from patients with other neurodegenerative diseases.Moreover,we identified 21 CSF and 18 serum stage-associated proteins re-flecting AD stages.Our findings provide a foundation for developing bloodbased tests for AD screening and staging in clinical practice.

Mutation Analysis of MR-1, SLC2A 1, and CLCN1 in 28 PRRT2-negative Paroxysmal Kinesigenic Dyskinesia Patients

Background： 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. Methods： 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-l, 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. Results： 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 SLC2A 1 c.363G〉A mutation was novel. Conclusions： 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.

Genotype-phenotype correlations of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis(ALS)is a devastating neurodegenerative disease characterized by progressive neuronal loss and degeneration of upper motor neuron(UMN)and lower motor neuron(LMN).The clinical presentations of ALS are heterogeneous and there is no single test or procedure to establish the diagnosis of ALS.Most cases are diagnosed based on symptoms,physical signs,progression,EMG,and tests to exclude the overlapping conditions.Familial ALS represents about 5~10% of ALS cases,whereas the vast majority of patients are sporadic.To date,more than 20 causative genes have been identified in hereditary ALS.Detecting the pathogenic mutations or risk variants for each ALS individual is challenging.However,ALS patients carrying some specific mutations or variant may exhibit subtly distinct clinical features.Unraveling the respective genotype-phenotype correlation has important implications for the genetic explanations.In this review,we will delineate the clinical features of ALS,outline the major ALS-related genes,and summarize the possible genotype-phenotype correlations of ALS.

Clinical,Neuroimaging,and Pathological Analyses of 13 Chinese Leigh Syndrome Patients with Mitochondrial DNA Mutations

Background: Leigh syndrome(LS) is a rare disease caused by mitochondrial defects and has high phenotypic and genotypic heterogeneity.We analyzed the clinical symptoms, neuroimaging, muscular histopathology, and genotypes of 13 Chinese LS patients with mitochondrial DNA(mtDNA) mutations.Methods: Mutations in mtDNA were identified by targeted sequencing. The brain imaging features on magnetic resonance imaging(MRI)were analyzed. The levels of lactate in fasting blood and cerebrospinal fluid(CSF) were routinely tested. The levels of urinary organic acids, plasma amino acids, and acylcarnitines were examined with gas chromatography–mass spectrometry and tandem mass spectrometry.The histopathological traits of skeletal muscles were analyzed under microscope.Results: Among 13 patients, mutations of MT?NDs(n = 8) and MT?ATP6(n = 4) genes were most common. Strabismus(8/13), muscle weakness(8/13), and ataxia(5/13) were also common, especially for the patients with late?onset age after 2 years old. However, respiratory distress was common in patients with early?onset age before 2 years old. The most frequently affected brain area in these patients was the brain stem(12/13), particularly the dorsal part of midbrain, followed by basal ganglia(6/13), thalamus(6/13), cerebellum(5/13),and supratentorial white matter(2/13). Besides, the elevated lactate levels in CSF(6/6) were more common than those in serum(7/13).However, the analysis of abnormal plasma amino acid and urinary organic acid showed limited results(0/3 and 1/4, respectively). Muscular histopathology showed mitochondrial myopathy in the three late?onset patients but not in the early?onset ones.Conclusions: Noninvasive genetic screening is recommended for mtDNA mutations in MT?NDs and MT?ATP6 genes in patients with ophthalmoplegia, muscle weakness, ataxia, and respiratory disorder. Furthermore, the lactate detection in CSF and the brain MRI scanning are suggested as the diagnosis methods for LS patients with mtDNA mutations.

Penetrance estimation of PRRT2 variants in paroxysmal kinesigenic dyskinesia and infantile convulsions

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.

Variants of Interleukin-7/Interleukin-7 Receptor Alpha are Associated with Both Neuromyelitis Optica and Multiple Sclerosis Among Chinese Han Population in Southeastern China

Background： Neuromyelitis optica （NMO） and multiple sclerosis （MS） are autoimmune demyelinating diseases of the central nerve system, lnterleukin-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 （rsl520333, rs1545298, rs4739140, rs6993386, rs7816065, and rs2887502） and one variant of IL-7RA （rs6897932） with NMO and MS among Chinese Han population in southeastern China. Methods： 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%. Results： 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. Conclusions： The study suggested that among Chinese Hart 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.

Three-Dimensional Heterogeneity of Cerebellar Interposed Nucleus-Recipient Zones in the Thalamic Nuclei

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.

Effect of Apolipoprotein E Genotypes on Huntington’s Disease Phenotypes in a Han Chinese Population

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 and1241 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 EOMES Associated with Increasing Risk in Chinese Patients with Relapsing-remitting Multiple Sclerosis

Background： 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 pertbrmed 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. Methods： 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. Results： In stage 1 analysis, we confirmed only one previously reported risk variant, rsl 1129295 in EOMES gene. We found that the frequency ofT/T genotype was much higher in MS group （χ2 = 10.251, P = 0.005） and the T allele ofrsl 1129295 increased the risk of MS （χ2 = 10.022, P = 0.002）. In stage 2 and combined analyses, the T allele of rsl 1129295 still increased the risk of MS （χ2 = 4.586, P- 0.030 and×2 = 16.378, P = 5.19×10 ^-5, respectively）. Conclusions： 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.

Genetic spectrum and clinical features in a cohort of Chinese patients with autosomal recessive cerebellar ataxias

Background:Although many causative genes have been uncovered in recent years,genetic diagnosis is still missing for approximately 50%of autosomal recessive cerebellar ataxia(ARCA)patients.Few studies have been performed to determine the genetic spectrum and clinical profile of ARCA patients in the Chinese population.Methods:Fifty-four Chinese index patients with unexplained autosomal recessive or sporadic ataxia were investigated by whole-exome sequencing(WES)and copy number variation(CNV)calling with ExomeDepth.Likely causal CNV predictions were validated by CNVseq.Results:Thirty-eight mutations including 29 novel ones were identified in 25 out of the 54 patients,providing a 46.3%positive molecular diagnostic rate.Ten different genes were involved,of which four most common genes were SACS,SYNE1,ADCK3 and SETX,which accounted for 76.0%(19/25)of the positive cases.The de novo microdeletion in SACS was reported for the first time in China and the uniparental disomy of ADCK3 was reported for the first time worldwide.Clinical features of the patients carrying SACS,SYNE1 and ADCK3 mutations were summarized.Conclusions:Our results expand the genetic spectrum and clinical profiles of ARCA patients,demonstrate the high efficiency and reliability of WES combined with CNV analysis in the diagnosis of suspected ARCA,and emphasize the importance of complete bioinformatics analysis of WES data for accurate diagnosis.

Atypical features in two adult patients with Cockayne syndrome and analysis of genotype-phenotype correlation

To the Editor:Cockayne syndrome(CS;Mendelian Inheritance in Man#133540,216400)is a rare autosomal recessive neurodegenerative disorder described by Edward Cockayne in 1936.[1]The prevalence of CS is 2.7 per million live births,[2]and the disease is probably underdiagnosed.The major clinical features are progressive growth failure and microcephaly as well as other characteristics such as a“cachectic dwarfism”appearance with sunken eyes,cutaneous photosensitivity,mental retardation,demyelinating peripheral neuropathy,pigmentary retinopathy,cataracts,deafness,dental anomalies,and premature death.[1,3]There is considerable variation in the severity of the disorder,leading to classification into three main types.

Variants of Interferon Regulatory Factor 5 are Associated with Neither Neuromyelitis Optica Nor Multiple Sclerosis in the Southeastern Han Chinese Population

Background： 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 Southeastenl Han Chinese population. Methods： Four single nucleotide polymorphisnls （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. Results： None of these 4 SNPs was associated with NMO or MS patients. Conclusions： Our preliminary study indicates that genetic variants in IRI~ 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 isstie.

Recommendations for the diagnosis and treatment of paroxysmal kinesigemc dyskinesia: an expert consensus in China

Paroxysmal dyskinesias are a group of neurological diseases characterized by intermittent episodes of involuntary movements with different causes.Paroxysmal kinesigenic dyskinesia(PKD)is the most common type of paroxysmal dyskinesia and can be divided into primary and secondary types based on the etiology.Clinically,PKD is characterized by recurrent and transient attacks of involuntary movements precipitated by a sudden voluntary action.The major cause of primary PKD is genetic abnormalities,and the inheritance pattern of PKD is mainly autosomal-dominant with incomplete penetrance.The proline-rich transmembrane protein 2(PRRT2)was the first identified causative gene of PKD,accounting for the majority of PKD cases worldwide.An increasing number of studies has revealed the clinical and genetic characteristics,as well as the underlying mechanisms of PKD.By seeking the views of domestic experts,we propose an expert consensus regarding the diagnosis and treatment of PKD to help establish standardized clinical evaluation and therapies for PKD.In this consensus,we review the clinical manifestations,etiology,clinical diagnostic criteria and therapeutic recommendations for PKD,and results of genetic analyses in PKD patients performed in domestic hospitals.

Wilson's Disease in China

Wilson＇s disease（WD） is an autosomal recessive disorder of copper metabolism. Its incidence is higher in China than in western countries. ATP7 B is the causative gene and encodes a P-type ATPase, which participates in the synthesis of holoceruloplasmin and copper excretion. Disease-causing variants of ATP7 B disrupt the normal structure or function of the enzyme and cause copper deposition in multiple organs,leading to diverse clinical manifestations. Given the variety of presentations, misdiagnosis is not rare. Genetic diagnosis plays an important role and has gradually become a routine test in China. The first Chinese spectrum of disease-causing mutations of ATP7 B has been established. As a remediable hereditary disorder, most WD patients have a good prognosis with an early diagnosis and chelation treatment. However, clinical trials are relatively few in China, and most treatments are based on the experience of experts and evidences from other countries. It is necessary to study and develop appropriate regimens specific for Chinese WD patients.

A De novo Mutation in Dystrophin Causing Muscular Dystrophy in a Female Patient

Background： 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. Methods： 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. Results： 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. Conclusions： 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.

Amyotrophic Lateral Sclerosis： Precise Diagnosis and Individualized Treatment

INTRODUCTION Amyotrophic lateral sclerosis （ALS） is a progressive neurodegenerative disease characterized by selective death of upper motor neurons （UMNs） and lower motor neurons （LMNs）, typically may die from respiratory failure within 2-5 years of symptorn onset）H About 10% orALS patients are familial whereas the remaining patients are sporadic. ALS is highly heterogeneous in genetic and clinical phenotype, with lack of definitive diagnostic tools, making it extremely difficult to make early diagnosis.

A Novel Missense Mutation in Peripheral Myelin Protein-22 Causes Charcot-Marie-Tooth Disease

Background：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.Methods：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.Results：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.G 107D mutation lost the ability to reach the plasma membrane,was mainly retained in the endoplasmic reticulum,and induced cell apoptosis.Conclusions：This study supported the notion that missense mutations in PMP22 give rise to a CMT phenotype,possibly through a toxic gain-of-function mechanism.