Detection of miRNA gene sequence variations in multiple myeloma and its significance / 白血病·淋巴瘤

Objective To investigate sequence variations of 12 miRNA genes in multiple myeloma(MM) in order to find whether sequence variations in miRNA genes are associated with tumorigenesis and discuss the clinical significance of MM associated with miRNA genes mutations. Methods The miRNA gene mutations in 20 cases of MM, 4 MM-derived cell lines and 20 controls were detected by the methods of polymerase chain reaction single stranded conformation polymorphism (PCR-SSCP) and silver staining technique. Both clinical features and laboratory results were analyzed simultaneously. Results The electrophoretic patterns showed a total of three variations in miR-19a, miR-19b and miRNA-335,which were observed in 3 MM cells (15 %, 3/20). We also found variations of miRNA-335 in MM-derived cell lines KM-3and RPMI8226. However, no sequence alteration in the miRNA genes was observed in our set of controls. One of the three MM patients died, and two of them were detected mutations at the terminal stage of the disease.Conclusion A relative high frequency of miRNA gene mutation was found in MM and MM derived cell lines, which suggests possibility of a main mechanism underlying tumorigenesis. And, detecting miRNA gene mutations in MM might be benefit to evaluate the progression and prognosis of disease.

Gene diagnosis of X-linked spondyloepiphyseal dysplasia tarda / 中华检验医学杂志

ObjectiveTo investigate the molecular pathogenesis of a pedigree of X-linked spondyloepiphyseal dysplasia atarda (SEDL) and to establish methods of gene diagnosis. Methods Clinical diagnosis was made based on height measurement, radiological examination and pedigree analysis. Peripheral blood samples of relevant family members were collected. After genomic DNA extraction, single strand conformation polymorphism (SSCP) followed with DNA sequencing was used to detect SEDL gene exons 36. Microsatellite marker DXS16 was selected for linkage analysis. Results The abnormal electrophoretic bands were detected in exon 4 of probands by PCR-SSCP. A c. 218C ＞ T mutation in exon 4 of SEDL gene was found in three probands, which resulted in a change in amino acid sequence S37L. The heterozygous exon 4 mutation was identified in three carriers, but not in healthy individuals, and no mutations were detect in exon 3, 5 and 6 of probands. Three unmarried young females (Ⅲ10, Ⅳ6 and Ⅳ7) were found to harbor the mutation by DNA sequencing analysis. ConclusionsA c. 218C ＞ T missense mutation in exon 4 of SEDL gene is the cause of molecular pathogenesis of the pedigree. SSCP and DNA sequencing can be used for prenatal gene diagnosis.

SSCP screening of mutation in exon 13 of low density lipoprotein receptor gene in Chinese familial hypercholesterolemia patients / 中华检验医学杂志

Objective To investigate the application of polymerase chain reaction and single strand conformation polymorphism analysis(PCR-SSCP)to the screening of gene mutation of exon 13 of the LDLR gene in familial hypercholesterolemia(FH).Methods Peripheral blood DNA of 16 clinically diagnosed FH patients was extracted and the exon 13 coding region of the LDLR gene was amplified by PCR.PCR products were separated by optimized SSCP electrophoresis and visualized by silver staining.DNA fragments with abnormal mobility were sequenced to determine the nature and position of mutations.Results The SSCP electrophoresis conditions were optimized as 8%polyaerylamide(degree of cross linking 49:1)gel without glycerin at a electrophoresis temperature of 10℃ or 8%polyacrylamide gel with 5%glycerin at room temperature,gel thickness of＜0.4 mm,and a voltage of 5 V/cm.DNA fragments were well resolved with the conditions and sequencing of the abnormal bands resuhed in detections of missense mutations of A606T,D601N,Y601D and G636V together with a synonymous mutation of 1959C→T in 4 patients and a sole synonymous mutation of 1959C→T in other 4 patients.Conclusion PCR-SSCP is an effective method for the screening of exon13 mutations of LDLR gene in FH patients.

Association between SLC25A12 and SCN2A2 gene polymorphisms and autism / 吉林大学学报(医学版)

Objective To investigate the association SLC25A12 and SCN2A2 gene single nucleotide polymorphisms(SNPs) and susceptibility to autism among 105 Japanese family trios consisting of fathers,mothers,and affected offsprings with autism.Methods Genomic DNA was isolated from the whole blood samples.The PCR-single stranded conformational polymorphism(SSCP) technique was used to test genotype of SNPs(rs3770448,rs3769955) at SLC25A12 and SCN2A2 genes.Results The distributions of genotypic and allelic frequencies of rs3770448 and rs3769955 were not deviated from the Hardy-Weinberg equilibrium.The results of transmission disequilibrium test(TDT) indicated that the allelic frequency transmitted from the heterozygote parents didn′t deviate 50%.Conclusion The polymorphism of rs3770448 in the SLC25A12 and rs3769955 in the SCN2A2 locus may not be associated with autism.But the association of the other SNPs at the SLC25A12 and SCN2A2 locus with the illness can not be ruled out.

Gene Mutations in a Chinese Family with Hailey-Hailey Disease / 中华皮肤科杂志

Objective To analyse gene mutation in members of a Chinese family with Hailey-Hailey disease(HHD)and study the relationship between the genotype and clinical features of the disease.Meth-ods Genomic DNA of leucocytes were obtained from members of the Chinese family with HHD including4patients and6normal persons.Ten exons of ATP2C1gene were amplified by polymerase chain reaction(PCR)and the products were analysed by single-strand conformation polymorphism(SSCP)and direct DNA sequencing.Results A novel mutation was identified in this family.The sequence of"TGTAGCCAT"(2068→2076)was substituded by"AGATGGAACA",which caused a frame shift of open reading frame and premature termination codon(PTC)in gene ATP2C1.There was no relationship between the genotypes and the phenotypes.Conclusion Gene mutation of ATP2C1gene at exon21is the cause for HHD in this fami-ly.

No Association of Factor XIII Val34Leu Polymorphism with Primary Intracerebral Hemorrhage and Healthy Controls in Korean Population

The polymorphism in the factor XIII A-subunit gene (FXIII Val34Leu) has been recognized as a risk factor for primary intracerebral hemorrhage (PICH). In addition, FXIII Val34Leu has a significant ethnic heterogeneity. FXIII Val34Leu was detected in 41.7-54.8% of the Westerners, but in 2.5% of the Asians. We aimed to evaluate the prevalence of FXIII Val34Leu in patients with PICH and in healthy controls among Koreans. We recruited 58 in-patients with PICH, defined by brain computed tomography or magnetic resonance imaging, and 48 controls matched for age, sex, and risk factors for cerebrovascular diseases. Genomic DNA was extracted from blood. A 183-bp fragment of exon 2/intron B of the factor XIII Asubunit gene was amplified by polymerase chain reaction (PCR). The factor XIII genotype was determined through a single-stranded conformational polymorphism. Fifty-eight patients and 48 controls showed the same band patterns on SSCP. In addition, we directly sequenced six random-selected DNA segments using DNA auto-sequencer. In conclusion, the results of this study suggest that FXIII Val34Leu be absent or rare both in patients with PICH and in healthy controls among Koreans.

embB mutations in Mycobacterium tuberculosis ethambutol-resistant isolates / 中华检验医学杂志

Objectives To understand the mutations of embB genes in M. tuberculosis isolates, and to evaluate their clinical value. Method 102 clinical isolates were identified for their mycobacterial species, and then analyzed their embB genes with PCR SSCP, PCR RFLP, and PCR direct sequencing. Results Mycobacterium tuberculosis strain H 37 R v was used as a control. 102 clinical isolates all had the same 16S rDNA SSCP profiles as M. tuberculosis . Forty one drug sensitive isolates had normal embB SSCP and RFLP profiles. Of 61 ethambutol resistant isolates, 23 (37.7%) displayed abnormal embB SSCP profiles. Eight isolates had abnormal RFLP profiles. All embB mutations situated at codon 306, whose EMB MICs were more than 20 ?g/ml. Eight isolates had ATG to ATA or ATT mutations at codon 306. Thirty isolates had ATG to GTG or CTG mutations at codon 306, whose EMB MICs were more than 30 ?g/ml. Conclusions Ethabutol resistances in some M. tuberculosis isolates were due to mutations on embB genes. PCR SSCP and PCR RFLP method might become a simple and rapid diagnostic test for genotypes of M. tuberculosis ethabutol resistance.

Polymorphism in insulin receptor gene exon 17 in women with polycystic ovary syndrome / 中华妇产科杂志

Objective To investigate insulin receptor (INSR) genotype exon 17 frequencies in women with polycystic ovary syndrome(PCOS) and to elucidate its role in the pathogenesis of PCOS. Methods The study involved 33 women with PCOS and 28 healthy control women who were genotyped for polymorphism of INSR gene exon 17 by single strand conformation polymorphism (SSCP) analysis. Body mass index (BMI), insulin sensitive index (ISI), the expression of INSR beta subunit, and serum concentration of luteinizing hormone(LH), total testosterone between the genotypes were compared. Results (1) The T -to- C mutation was observed in the INSR gene exon 17 (1008 bp). The frequency of the C/C genotype was significantly higher in patients (39%) than in the controls (11%) (P

Detection of H ras mutation in urine exfoliated cells complements cytology in 48 TCC patients / 中华检验医学杂志

Objective To determine the value of detection of H ras oncogene mutation in urine exfoliated cells as clinical indicator of tumor presence, recurrence and stage.Methods Point mutation at codon 12 of H ras gene was assayed by polymerase chain reaction followed by analysis of single strand conformation polymorphism in urine exfoliated cells from 48 patients with transitional cell carcinoma before operation and 28 patients with non urothelial cancer or normal individuals. The mutation was further confirmed by dideoxy mediated chain termination method of DNA sequencing. Cytology analysis was carried out simultaneously. Bladder tumor specimens were obtained from 48 patients during operation, and histologically elevated for tumor content and grading.Results 48%(23 of 48) of the patients were detected by their aberrant band in SSCP. All aberrant bands displayed a mutant H ras sequence, where 15% (7 of 48) of the patients displayed, apositive cytological analysis. Analysis of abnormalities with tumor stage revealed that the greater detection of high pathological stage (Ⅲ Ⅳ) compared with low stage (Ⅰ Ⅱ) was related to the recurrence of transitional cell carcinoma.Conclusion Our results suggest that the detection of H ras mutations may be of clinical value in the detection of TCC.

Polymorphism within the interleukin-10 receptor cDNA gene in patients with systemic lupus erythematosus / 中华皮肤科杂志

Objective To assess the association between polymorphism within the interleukin-10 receptor cDNA gene (IL-10R) and Chinese patients with systemic lupus erythematosus (SLE). Methods The IL-10R genotypes of 94 SLE patients and 80 healthy subjects were examined by reverse transcription-polymerase chain reaction-single strand conformation polymorphism method (RT-PCR-SSCP), RT-PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing. Results There were significant differences in the IL-10R2 genotype frequencies of these two groups. The IL-10R2 G520/G520 genotype increased the risk of developing SLE (OR = 0.515, 95% CI 0.414-0.579, P = 0.004) and individuals who had G520/A520 genotype also had a higher susceptibility to SLE (OR = 1.968, 95% CI 0.981-3.949, P = 0.055). There was no significant association between SLE and IL-10R1 genotypes. The risk of developing SLE was detected in the individuals who had the combination of IL-10R1 G241/G241 and IL-10R2 G520/G520 (OR = 0.515, 95% CI 0.444-0.597, P = 0.004). Conclusion The IL-10R2 genotypes of G520/G520 and G520/A520 as well as the combination of genotypes IL-10R1G241/G241 and IL-10R2 G520/G520 may increase the susceptibility to SLE in Chinese people.