Study of Mutation in Tyrosine Protein Kinase of Insulin Receptor Gene in Patients with Polycystic Ovarian Syndrome

Objective To explore the molecular mechanism of insulin resistance in the patients with polycystic ovarian syndrome (PCOS ) Methods Polymerase chain reaction, silver staining-single strand conformation polymorphism(PCR-SSCP ) and DNA direct sequencing were used to detect the mutation of insulin receptor (INSR) gene in exon 17～21 with the abdominal wall adipose tissue from 31 patients with PCOS (PCOS Group) and 30 patients with pure hysteromyoma in reproductive lift (Control Group).Results Twenty-two variant SSCP patterns in exon 17 of INSR gene were detected.Direct sequence analysis of exon 17showed that homozygous nonsense mutation was two alleles single nucleotide polymorphism (SNP) at the codon 1058 ( CAC→CAT). Exons 18～21 were not detected with any significantly mutation. The INSR gene His^1058C→T substitution collecting rate and insulin resistance were significantly higher in the PCOS group than in the control group (P=0. 0293, P＜0. 05, P＜0. 01).Conclusion It is suggested that the SNP in codon 1058 of the INSR gene might be related with the insulin resistance in PCOS patients, which has hereditary tendency.And the missense mutation,nonsense mutation and frameshi ft mutation at exons 18～21 in tyrosine protein kinase region of INSR gene for PCOS patients were not frequently observed.     

Cholesteryl ester transfer protein levels and gene deficiency in Chinese patie nts with cardio-cerebrovascular diseases

Objective To detect cholesteryl ester transfer protein (CETP) levels, frequencies of CETP D442G and Ⅰ14A mutations and characteristics of abnormal lipids in patients wit h cardio-cerebro vascular diseases. Methods Ninety-four myocardial infarction (MI) patients,110 stroke patients and 335 hea lthy controls were selected.The CETP concentration was determined using ELISA .The CETP activity was measured using a substrate of ［14］C-radiol abeled discoidal bilayer particles.The CETP gene mutations were detected by PC R-RFLP. Results The CETP concentrations in the MI and stroke group, were higher than those in th e controls.The gene mutation frequencies of D442G in the MI, stroke and contro l group were 3.5%, 3.6% and 5%, respectively, and the frequencies of Ⅰ14A wer e 1.05%, 0.91% and 1%, respectively.One case of D442G homozygote was detected in the healthy group.The frequency of two CETP gene mutations showed no signi ficant difference among the patients and controls.The CETP concentration and a ctivity in subjects with CETP mutations were one-third of those in the control group.The level of HDL-C, apo-A1 increased in the mutation subjects, while t he TG level decreased. Conclusions The CETP level increased significantly in patients with cardio-cerebrovascular diseases.The carriers of CETP deficiency had CETP and lipid abnormalities.     

Novel Mutations of PRSS1 Gene in the Patients with Pancreatic Cancer among Han Population

Purpose: The aim of this study was to elaborate some novel mutations of PRSS1 gene in the patients with pancreatic cancer. Patients and Methods: There were 156 patients with pancreatic cancer and 220 unrelated individuals were studied as controls. The mutations of PRSS1 gene were analyzed by direct sequencing. K-ras Mutation Detection Kit was used to find the general k-ras gene disorder in the pancreatic cancer tissue. Then collected and analyzed the clinical data at the same time. Results: There were two patients carried novel mutations which is IVS 3 +157 G>C of PRSS1 gene from peripheral blood specimens and pancreatic cancer tissue. What’s more, we were surprised to find a novel complicated mutation of exon 3 in PRSS1 gene (c.409 A>G and c.416 C>T) in another young patient. The c complicated mutation made No.135 and No.137 amino acid transfer from Thr to Ala and Thr to Met respectively. All of the mutations weren’t found in the normal controls and no mutations of k-ras gene detected in the three patients. Conclusion: These observations imdicate that mutations of PRSS1 gene may be an important factor of pancreatic cancer.     

China aims for national chain of anti-smoking clinics

Background Familial pulmonary arterial hypertension （FPAH） is an autosomal dominant disorder characterized by plexiform lesions of endothelial cells in pulmonary arterioles which leads to elevated pulmonary arterial pressure, right-sided heart failure and death. Heterozygous mutations in the bone morphogenetic protein type II receptor gene （BMPR2） have been found to underlie a majority of FPAH cases. More than 140 distinct mutations have been identified in FPAH cases and in idiopathic pulmonary arterial hypertension （IPAH） cases, but only one mutation has been reported in Chinese patients. Methods A three-generation pedigree of FPAH and another 10 patients with IPAH were collected. In the family, two of the 9 surviving and one deceased family member were diagnosed as FPAH. The entire protein-coding region and intron/exon boundaries of the BMPR2 gene were amplified by PCR using DNA samples from affected individuals. Direct sequencing of PCR products was performed on both the sense and antisense strands. To confirm the segregation of the mutation within the family and exclude the presence of the mutation in normal subjects, the relevant exon was amplified by PCR, followed by mutation-specific RPLP analysis. Results In the Chinese pedigree with FPAH an A-to-T transition at position 1157 in exon 9 of the BMPR2 gene was identified which resulted in a Glu386Val mutation. We confirmed the segregation of the mutation within the family and excluded the presence of the mutation in a panel of 200 chromosomes from normal subjects. No mutation was detected in BMPR2 in the other 10 patients with IPAH. Conclusions This amino acid substitution occurs at a glutamic acid that is highly conserved in all type Ⅱ TGF-β receptors. The nearly invariant Glu forms an ion pair with an invariant Arg at position 491 thereby helping to stabilize the large lobe. Substitution of Arg at position 491 is the most frequently observed missense mutation in FPAH, but until now no mutations at position 386 have been found in FPAH. The pr     

Identification of a mutation hotspot in exon 8 of W ilson disease gene by cycle sequencing

Objective To screen for mutation hotspot of Wilson disease (WD) gene in Chinese population.Methods Cycle sequencing was used to detect mutation in exon 8 of WD gene in 30 patients with Wilson disease. Results The same missense mutation, Arg779Leu, was identified in 14 WD patients, four of whom were homozygous and the other heterozygous for this mutation.The frequency of this mutation in Chinese patients was 30%.Conclusion The codon 779 (CCG→CTG) of exon 8 of WD gene was one of mutation hotspots in Chinese.     

Point Mutation in the Parkin Gene on Patients with Parkinson''''s Disease

Summary;To investigate the distribution of possible novel mutations from parkin gene in variant sub-set of patients with Parkinson’s disease(PD)in China and explore whether parkin gene plays an im-portant role in the pathogenesis of PD,70 patients were divided into early-onset group and late-onsetgroup; 70 healthy subjects were included as controls.Genomic DNA from 70 normal controls andfrom those of PD patients were extracted from peripheral blood leukocytes by using standard proce-dures.Mutations of parkin gene(exon 1—12)in all the subjects were screened by PCR-single strandconformation polymorphism(SSCP),and further sequencing was performed in tue samples with ab-normal SSCP results,in order to confirm the mutation and its location.A new missense mutationGly284Arg in a patient and 3 abnormal bands in SSCP electrophoresis from samples of another 3 pa-tients were found.All the DNA variants were sourced from the samples of the patients with early-on-set PD.It was concluded that Parkin point mutation a     

A novel CFTR mutation found in a Chinese patient with cystic fibrosis

Background Cystic fibrosis (CF) is rare in Chinese. We investigated the mutations in the gene of cystic fibrosis transmembrane conductance regulator ( CFTR ) in a Chinese CF patient and reviewed the clinical features, gene mutations in Chinese CF cases. Methods Blood samples were collected from a previously reported CF girl and her parents. The 24 coding exons of CFTR of the proband were amplified and sequenced. Results A Chinese girl of 16 years old was diagnosed as CF at the age of 14. She had recurrent productive cough with bronchiectasis in bilateral upper lobes, parasinusitis and otitis media, but without pancreatic involvement. Her sweat chloride was (108.9 ±3.3) mmol/L. A heterozygous novel missense mutation of 699 C → A which results in the amino acid change of N189K was identified in exon 5. In addition, a heterozygous 3821-3823 delT mutation in exon 19 was found in CFTR . The mutation 699C → A was inherited from her father, and the 3821-3823delT mutation was from her mother. Twenty patients with CF in Chinese reported from 1974 to 2004 were also reviewed. DelF508 mutation was not found in the nine cases whose CFTR mutations were analyzed. Conclusions The CF proband carries two heterozygous mutations (699C → A and 3821-3823delT) in CFTR . 699C → A mutation is a novel mutation which is not reported previously. Review of reported Chinese cases suggests that the genotype of Chinese CF may be different from those of white cases. More studies are needed to understand the spectra of CFTR and clinical CF features in Chinese.     

Features of a Chinese family with cerebral cavernous malformation induced by a novel CCM1 gene mutation

Background Familial cerebral cavernous malformations （CCMs）, characterized by hemorrhagic stroke, recurrent headache and epilepsy, are congenital vascular anomalies of the central nervous system. Familial CCMs is an autosomal dominant inherited disorder and three CCM genes have been identified. We report a Chinese family with CCMs and intend to explore clinical, pathological, magnetic resonance imaging （MRI） features and pathogenic gene mutation of this family. Methods Totally 25 family members underwent brain MRI examination and clinical check. Two patients with surgical indications had surgical treatment and the specimens were subjected to histopathological and microstructural examination. In addition, polymerase chain reaction （PCR） and direct sequencing were performed with genomic DNA extracted from 25 family members＇ blood samples for mutation detection. Results Brain MRI identified abnormal results in seven family members. All of them had multiple intracranial lesions and four cases had skin cavernous hemangioma. T2-weighted sequence showed that the lesions were typically characterized by an area of mixed signal intensity. Gradient-echo （GRE） sequence was more sensitive to find micro- cavernous hemangiomas. There was a wide range in the clinical manifestations as well as the age of onset in the family. The youngest patient was an 8-year-old boy with least intracranial lesions. Histopathological and microstructural examination showed that CCMs were typically discrete multi-sublobes of berry-like lesions, with hemorrhage in various stages of illness evolution. They were formed by abnormally enlarged sinusoids and the thin basement membranes. A novel T deletion mutation in exon 14 of CCM1 gene was identified by mutation detection in the seven patients. But unaffected members and healthy controls did not carry this mutation. Conclusions The clinical manifestations were heterogenic within this family. We identified a novel mutation （c.1396delT） was the disease-causing mutation for this family and extended the mutational spectrum of CCMs.     

Relationship between serum S-100 protein level and ischemic damage degree in patients with acute cerebral infarction

Objective： To investigate the time course of serum S-100 concentrations of patients with acute cerebral infarction,and their relation with the clinical data and the prognosis. Methods： Serum S-100 levels were serially determined in 36 patients with acute cerebral infarction within 12 h, at 24 h and day 2, 3, 4, 5, 7 and 10 after acute cerebral infarction and in 20 age- and sex-matched control subjects. An S-100 content assay was performed using a two-site radioimmunoassay technique. The clinical status was assessed using NIH Stroke Scale. The functional deficit at 4 weeks after acute cerebral infarction was scored using the modified Rankin scale. A cranial computed tomography was performed initially. Results： Elevated concentrations of S-100 （〉0.2μg/L） were observed in 29 of 36 patients with acute cerebral infarction,but none of the control subjects. The S-100 peak levels were at day 2 and 3 after acute cerebral infarction and were significantly high in those patients with severe neurological deficit at admission, with extensive infarction or with space-occupying effect of ischemic edema as compared with the rest of the populations. Conclusion： Serum S-100 level assay can be used as a peripheral marker of ischemic brain damage, and may be helpful for evaluation of therapeutic effects in acute ischemic stroke.     

Association between fifteen risk factors and progressing ischemic stroke in the Han population of northeast China

Background The mortality and disability associated with progressing ischemic stroke are much higher than general ischemic stroke. This study was conducted to determine the risk factors for progressing ischemic stroke in the Han population of northeast China. Methods A total of 2511 patients with ischemic stroke within 24 hours admitted to Department of Neurology, First Affiliated Hospital of Harbin Medical University were studied, from November 2007 to May 2009. All of the patients were classified into the progressing or non-progressing group according to the scores of the Scandinavian Neurological Stroke Scale. Fifteen putative risk factors were evaluated. The influence of risk factors for progressing ischemic stroke was analyzed with the simple Logistic analysis, the multiple Logistic analysis, and the stepwise Logistic regression model. All the statistical analysis was performed by SAS 9.1. Results Totally 359 （14.3%） patients met the criteria for progressing ischemic stroke. The Logistic analysis showed that age, family stroke history, smoking history, hypertension on admission, a drop in blood pressure after admission to the hospital, high serum glucose on admission, and fever were related to progressing ischemic stroke in the Han population of northeast China. Conclusion People of the ischemic stroke with these factors are more likely to develop progressing ischemic stroke.     

DYT1 MUTATIONS AMONGST EARLY ONSET PRIMARY DYSTONIA PATIENTS IN CHINA

Objective To investigate the frequency of GAG deletion in the DYT1 gene among early onset primary dystonia patients in China. Methods Thirteen patients with early onset primary torsion dystonia were screened for mutation in exon 5 of the DYT1 gene using denaturing high-performance liquid chromatography （DHPLC） and DNA sequencing, and the results were confirmed with polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP）. Results The GAG deletion mutation which results in Glu302del in exon 5 of the DYT1 gene was found in 5 patients. The detecting results were consistent between with DHPLC and PCR-RFLP. We did not find any other mutations in the DYT1 gene. Conel~iotm The GAG deletion in the DYT1 gene is common amongst early onset primary torsion dystonia patients in Chin＆ The frequency of DYT1 mutation is not significantly different between European and Asian patients with early onset primary dystonia.     

Proteolipid protein 1 gene mutation in nine patients with Pelizaeus-Merzbacher disease

Background Pelizaeus-Merzbacher disease （PMD） is a rare X-linked recessive disorder with symptoms including nystagmus, impaired motor development, ataxia, and progressive spasticity. The proteolipidprotein 1 （PLP1） gene is the only pathogenic gene of PMD. Duplication of the PLP1 gene is the most frequent gene defect, accounting for 50%-70% of PMD cases, whereas point mutations in the coding sequence or the splice sites account for 10%-25% of PMD cases. This study aimed to identify PLP1 mutations in nine unrelated Chinese patients （P1-9） with PMD, and 14 subjects from the family of patient 2 were also described. Methods Genomic DNA was extracted from peripheral multiplex ligation-dependent probe amplification （MLPA）. AI amplified and analyzed using direct DNA sequencing. blood samples. Gene dosage was determined using the 7 exons and exon-intron boundaries of the PLP1 gene were Results Of these nine patients, there were four transitional, four classical, and one connatal PMD according to their clinical and radiological presentations. PLP1 duplications were identified in patients 1-7 with PMD. Their mothers were PLPI duplications carriers as well. Both duplication carriers and normal genotypes of PLP1 were identified in the family members of patient 2. A c.517C〉T （p. P173S） hemizygous missense mutation in exon 4 was found in patient 8 with PMD, and his mother was shown to be a heterozygote of this mutation. Conclusions We identified seven genomic duplications and one missense mutation （p. P173S） of the PLP1 gene in eight Chinese patients with PMD. This is the report about PLP1 mutations in PMD patients from the mainland of China.     

Clinical and laboratory observations on polysaccharide sulphate (PSS) in 282 cases of ischemic cerebrovascular disease.

Polysaccharide sulphate (PSS) is a new heparinoid drug. The therapeutic effect and laboratory findings in the treatment of 282 cases with ischemic cerebrovascular disease by PSS were analysed in this study. In treating patients with acute cerebral infarction, the effective rate (93.2%) and highly effective rate (62.9%) were both significantly higher in comparison with controls. Excellent results were also obtained in treating patients with cerebral infarction at late and sequela stage and patients with transient ischemic attack. Laboratory observations and animal experiments showed that PSS has anticoagulative and vasodilatory effects. It can also reduce blood viscosity and serum lipids. So PSS is an effective drug for the prevention and treatment of ischemic cerebrovascular diseases.

     

Neuron-specific enolase in patients with acute ischemic stroke and related dementia.

Neuron-specific enolase (NSE) levels of cerebrospinal fluid (CSF) were measured in 39 patients with ischemic stroke and 15 controls. There was a significant increase of CSF NSE in acute ischemic stroke patients as compared with the controls. The altered CSF NSE levels correlated well with the infarct size in CT scan. The CSF NSE levels were higher in 6-multiinfarct dementia (MID) patients who were diagnosed after 6-month follow-up than those in 22 non-MID patients of this series. Our research supports the view that CSF NSE can be a useful biochemical marker for brain ischemia. The importance of CSF NSE in the study of dementia related to ischemic stroke is worth further studies.     

Polymorphism of human platelet alloantigen in Chinese patients with acute myocardial infarction and acute ischemic stroke

Objective To investigate the gene frequencies of 5 major human platelet alloantiqens (HPA 1-5) in Chinese population and to assess if polymorphism of HPA was associated with Chinese acute myocardial infarction (AMI) and acute ischemic stroke (AIS). Methods HPA 1-5 genotyping was performed by PCR using allele specific primers and restriction enzyme digestion based on PCR products in 95 AMI cases, 188 AIS cases and 270 normal controls. Gene frequency distribution was tested by Hardy-Weinberg equilibrium and comparison of HPA gene frequencies between the patient and control groups by χ(2) test. Results The gene frequencies of HPA 1-5 were the followings: HPA1a: 91%; 1b: 9%; HPA 2a: 94%; 2b: 6%; HPA 3a: 83%; 3b: 17%; HPA 4a: 98%; 4b: 2%; HPA5a: 97%; 5b: 3%. We found there were no significant differences in HPA 1-5 gene frequencies between AMI patients and normal controls. In AIS patient group HPA-2a allele frequency was significant higher than in controls, but this allele gene frequency in two groups (0.94 and 0.99) was very close and too many subjects in these two groups were overlapped. Otherwise no differences was found in other 4 HPA systems between cases and controls. Conclusion Polymorphism of HPA were not inherited risky factors and not associated with chinese arterial thrombotic diseases such as AMI and AIS.     

TUMOR NECROSIS FACTOR-ALPHA POLYMORPHISM AND SECRETION IN MYASTHENIA GRAVIS

Objective To analyze the relationship between tumor necrosis factor-alpha (TNFo) gene promoter -308 polymorphism and myasthenia gravis (MG) in Chinese and analyze secretion of TNFo in peripheral blood mononuclear cells (PBMC) in MG patients.Methods A biallelic polymorphism at position -308 in the promoter of TNFα gene was screened by PCR amplification and NcoI recognition site. One hundred and twenty-three MG cases and 115 healthy controls were included in this study. MG patients were classified to different groups according to clinical type, age at onset, and sex respectively. PBMC were isolated from 20 patients and 20 healthy controls, and then cultured in the presence or absence of phytohemagglutinin (PHA) and acetycholine receptors (AchR). The supernatants were harvested after incubation and stored until TNFαwas assayed by enzyme-linked immunosorbent assay.Results The frequency of TNFα-308 allele 2 (A) was found significantly increase in MG patients and showed a trend especially in late onset (≥ 40 years) and male patients (P ＜ 0.05). The allele A had no relationship with thymic pathogenesis in MG patients. But frequency of allele A was significantly higher in general type than in ocular type (P ＜ 0.05). MG patients had a higher inducible level of TNFα by PHA and AchR, and could be down regulated after treatment.Conclusion Polymorphism in TNFα gene promoter -308 is associated with onset of MG. The microsatellite allele TNFα2 confer risk for the development of MG in Chinese patients. MG patients have a higher inducible level of TNFα.     

Relationship between tumor suppressor gene p53 and tumors of adipose tissue

Objective　To investigate the relationship between p53 gene and tumors of adipose tissue at the level of protein and gene. Methods　Immunohistochemical LSAB, PCR-SSCP and DNA sequencing were used in 82 cases. Results　p53 protein is expressed only in liposarcomas, in which the positive staining rate was 48.08% (25/52). In different subtypes of liposarcomas, the positive staining rate in well differentiated liposarcomas was 30.00% (9/30), which is much lower than that of the poorly differentiated liposarcomas (P<0.005). Abnormality in the single-stranded DNA pattern was determined in 2 samples (pleomophic liposarcomas) by PCR-SSCP analysis. Missense mutations in exon 8 codon 268 of p53 gene (AAC→ATC) were detected by DNA sequencing. Another heterozygotic cosense mutation may exist at exon 6 codon 221 of p53 gene (GAG→GAA). Conclusions　The data suggest that the p53 protein has a relationship with development, differentiation and malignancy of liposarcoma. Detecting the level of p53 protein expression may be valuable in evaluating the level of differentiation and malignancy of liposarcoma. There appear point mutation on exon 8,6 of p53 gene.     

Genetic characterization and protein stability analysis of a Chinese family with Von Hippel-Lindau disease

Background Von HippeI-Lindau disease （VHL）,a heritable autosomal dominant disease characterized by neoplasia in multiple organ systems,has rarely been reported in Asia.We genetically investigated a unique Chinese family with VHL disease and performed an analysis of the VHL protein stability.Methods Genomic deoxyribonucleic acid （DNA） extracted from peripheral blood was amplified by polymerase chain reaction （PCR） to three exons of the VHL gene in 9 members of the Chinese family with VHL disease.PCR products were directly sequenced.We estimated the effects of VHL gene mutation on the stability of pVHL,which is indicated by the free energy difference between the wild-type and the mutant protein （△△G）.Results The Chinese family was classified as VHL type 1.Three family members,including two patients and a carrier,had a T to G heterozygotic missense mutation at nucleotide 515 of the VHL gene exon 1.This missense mutation resulted in the transition from leucine to arginine in amino acid 101 of the VHL protein.There was low stability of the VHL protein （the △△G was 12.71 kcal/mol） caused by this missense mutation.Conclusions We first reported a family with this VHL gene mutation in Asia.This missense mutation is predicted to significantly reduce the stability of the VHL protein and contribute to the development of the renal cell carcinoma （RCC） phenotype displayed by this family.The genetic characterization and protein stability analysis of families with VHL disease are important for early diagnosis and prevention of the disease being passed on to their offspring.     

Exon Deletions of Parkin Gene in Patients with Parkinson Disease

Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 ( 1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.     

p21WAF1/CIP1 gene DNA sequencing and its expression in human osteosarcoma

Background Mutation and expression change of p21^WAF1/CIP1 may play a role in the growth of osteosarcoma. This study was to investigate the expression of the p21^WAF1/CIP1 gene in human osteosarcoma, p21^WAF1/CIP1 gene DNA sequence change and their relationships with the phenotype and clinical prognosis.Methods p21^WAF1/CIP1 gene in 10 normal people and the tumours of 45 osteosarcoma patients were examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with silver staining. The PCR product with an abnormal strand was sequenced directly. The p21^WAF1/CIP1 gene mRNA and P21 protein of 45 cases of osteosarcoma were investigated by using in situ hybridization and immunohistochemistrv, respectively.Results The occurrence of P21 protein in osteosarcoma was 17. 78% (8/45), and p21^WAF1/CIP1 mRNA expression in osteosarcoma was 42.22% (19/45). The p21^WAF1/CIP1 gene DNA sequencing of amplified production showed that in p21^WAF1/CIP1 gene exon 3 of 36 cases of human osteosarcoma,there were 17 cases (47.22%) with C→T at position 609; 10 normal blood samples‘ DNA sequence analysis yielded 8 cases (80.00%) with C→T at the same position.Conclusions Along with the increase of malignancy, the expression of p21^WAF1/CIP1 mRNA and P21 protein in osteosarcoma tends to decrease. It is uncommon for the p21^WAF1/CIP1 gene mutation to occur in human osteosarcoma. As a result, the possible existence of tumour subtypes of p21^WAF1/CIP1 gnemutation should be investigated. Our research leads to the location of p21^WAF1/CIP1 gene polymorphism of Chinese osteosarcoma patients, which can provide a basis for further research.