Correlation between protein C -1641A/-1654C haplotype and coagulation disorder in sepsis / 中华急诊医学杂志

Objective:To investigate the correlation between protein C -1641A/-1654C haplotype and coagulation disorder in Chinese Han septic patients.Methods:The genotypes of protein C gene -1641A>G (rs1799809) and -1654C>T (RS1799808) in septic patients were detected by direct sequencing, and their haplotypes were analyzed and divided into two groups according to the haplotype, -1641A/-1654C (AC) carriers and non-AC haplotype carriers. At the same time, unpaired t test or Mann-Whitney U test was used to compare the differences in coagulation/fibrinolytic parameters, including partial activated thrombin time, prothrombin time, internationally standardized ratio of prothrombin time, thrombin time, fibrinogen and D-dimer levels, as well as APC levels between the two groups. Results:A total of 174 septic patients were included in this study, including 60 AC haplotype carriers and 114 non-AC haplotype carriers. Compared with non-AC haplotype carriers, AC haplotype carriers had significantly lower platelet counts, significantly longer partial activated thrombin time, and significantly decreased activated protein C levels. Other coagulation/fibrinolytic parameters including prothrombin time, internationally standardized ratio of prothrombin time, thrombin time, fibrinogen and D-dimer were not significantly different between the two groups.Conclusions:In this study, the protein C-1641A/-1654C haplotype was found to lead to decreased circulating activated protein C levels decreased platelet counts, and prolonged partial activated thrombin time in septic patients. These results suggest that the protein C-1641A/-1654C haplotype may directly affect the APC level and consequently influence the coagulation disorder of sepsis.

Application of a new GC rich region sequence-specific polymerase chain reaction (SS-PCR) method for detection of C729T SNP / 西安交通大学学报(医学版)

Objective: To develop a sequence-specific polymerase chain reaction (SS-PCR) method for detection of single nucleotide polymorphism (SNP) in GC rich region and analyze the C729T SNP in the exon 3 of estrogen receptor α gene of endometrial cancer (EC). Methods: We selected 22 EC and 42 controls. A new GC rich region SS-PCR was developed. The two internal specific primers, identical to the two single strands of double strand DNA, were designed, and the 3' end of the primer coincided with the SNP locus. The PCR extension was controlled by the 3' end primer and the SNP was determined according to the bands of the extension product. The specificity of the extension reaction was increased by introducing a mismatched base at the third position upstream in the 3' end of the SNP specific primer. This method was used to confirm C729T genotypes in the exon 3 SNP, followed by direct sequencing. Results: The results showed that the SS-PCR was appropriate for genotyping C729T SNP of exon 3 in human ERα gene. Conclusion: A GC rich region SS-PCR method developed can be successfully applied in C729T SNP determination in the exon 3 of estrogen receptor α gene of endometrial cancer.

Identification for rare B(A) blood group / 临床检验杂志

Objective To investigate the serological and molecular identification of 2 rare B( A) blood groups. Methods The ABO blood groups of 2 samples from blood donors were detected by routine serological method. The genotype features was identified by PCR-sequence specific primer (PCR-SSP) and direct sequence analysis. Results The serological results for the 2 blood donors showed the characteristics of B(A) phenotype. The sample 1 was genotyped as BO2 subtype by PCR-SSP and direct sequencing showed B alleles in exon 7, presented nt640 A＞G mutation which was confirmed to be B(A)04/O02 genotype.The sample 2 was genotyped as BO1 sub-type by PCR-SSP and direct sequencing showed B alleles presented nt700 C＞G mutation in exon 7 which was confirmed to be B(A)02/O01 genotype. Conclusion The phenotype of the two samples should be B ( A ) and the genotypes should be rare B(A)04/O02 and B(A)02/O01.

First report of c. 1499G>C mutation in a 6-month-child with cystic fibrosis.

So far, more than 1800 mutations identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In this case report, we presented first report of c. 1499G>C mutation in a 6‑month‑old girl with cystic fibrosis (CF) diagnosis. A 6‑month‑old girl with weakness and meconium Ileus referred to the pediatric clinic in Ilam, in the west of Iran. Patient’s skin was dark and suffered from bronchiectasis. The sweat test was performed, and the concentration of chloride and sodium in patient’s sweat was 130-135 mmol/L and 125-128 mmol/L, respectively. The exon 10 mutation analysis of a CF patient was performed. CFTR mutation analysis revealed the identification of 2 mutations in patient, the mutations were p.F508del (ΔF508) and c. 1499G>C (cd500), respectively. The mutation c. 1499G>C (cd500) were found for the first time in the world. Assessing this mutation in future study and genetic investigation is recommended.

DNA methylation and congenital heart disease pathogenesis / 国际儿科学杂志

The roles that DNA methylation play in the congenital heart disease have recently begun to come to light.The abonormal DNA meghylation pattern on key regulation gne during heart development,such as T-box,CHD7,LINE1,methylenetrahydrofolate reductase,changed the expression of those gene,and increased the incidence of the congenital heart disease.The DNA methylation could be determined by sequencing,methylationsensitive PCR and combined bisulfate restriction anylysis.To study the DNA methylation will not only reveal the mechanism of congenital heart disease,but also provide a new strategy for clinical diagnosis and treatment.

A Case of Mycobacterium marinum Infection Diagnosed by PCR Amplification and Direct Sequencing / 대한피부과학회지

Mycobacterium marinum is an atypical mycobacterium (ATM) and is an uncommon cause of skin and soft tissue infections associated with contact with contaminated water. Diagnosis is often delayed when only a conventional identification method is used. PCR amplification and direct sequencing is recently available method for rapid identification of ATM. We report a case of M. marinum infection identified by PCR and sequencing. A 56-year-old female was referred for multiple erythematous nodules on both forearms which appeared two months ago. Skin biopsy showed suppurative granulomatous inflammation, and AFB culture showed nontuberculous Mycobacteria. PCR and sequencing were performed, and the obtained sequences were compared to the database using BLAST. The sequences of 16S rRNA and rpoB could not differentiate between M. marinum and M. ulcerans, showing 100% homology to both. Identification was possible using the sequences of the tuf and hsp65 genes, showing both 100% homology to M. marinum, while 99.8%, 99.7% to M. ulcerans. The patient was treated with clarithromycin, rifampicin, and ethambutol for 6 months.

Six Years' Experience Performing ABO Genotyping by PCR-direct Sequencing / 대한수혈학회지

BACKGROUND: ABO genotyping is essential for resolving ABO grouping discrepancy and for determinating ABO subgroups. Most clinical samples, including suspected inherited subgroups and acquired variant phenotypes, can be determined by PCR-sequencing of exons 6 and 7 in the ABO gene. Here, we describe our six years' experience performing ABO genotyping by PCR-direct sequencing. METHODS: We conducted a retrospective investigation of serological and genotypical data from 205 samples (158 patients and 47 of their family members) of patients who were referred to the Molecular Genetics Laboratory at Chonnam National University Hwasun Hospital for ABO genotyping between January 2007 and July 2012. ABO genotyping was performed on all samples with PCR-direct sequencing of exons 6 and 7 in the ABO gene; the standard serologic tests were also performed. RESULTS: The frequency of phenotypes consistent with their genotypes was 70.8% (112/158 cases) and the A2B3 phenotype with the cis-AB01 allele was the most common (31.0%, 49 cases) among them. The frequency of phenotypes inconsistent with their genotypes was 29.1% (46/158 cases) and the A1B3 phenotype was the most frequently recovered case (5.1%, 8 cases). Family study showed differential phenotype expression depending on the co-inherited ABO allele in five families with the B306, cis-AB01, Ael02, Aw14, or B305 allele and also showed a typical inheritance of a chimera with A102/B101/O04. CONCLUSION: We propose that ABO genotyping using PCR-direct sequencing is useful for the resolution of ABO discrepancies and for the investigation of ABO subgroups based on six years' experience. In addition, family study for analysis of phenotypic patterns of ABO subgroups is also crucial to ABO genotyping.

Six Years' Experience Performing ABO Genotyping by PCR-direct Sequencing / 대한수혈학회지

BACKGROUND: ABO genotyping is essential for resolving ABO grouping discrepancy and for determinating ABO subgroups. Most clinical samples, including suspected inherited subgroups and acquired variant phenotypes, can be determined by PCR-sequencing of exons 6 and 7 in the ABO gene. Here, we describe our six years' experience performing ABO genotyping by PCR-direct sequencing. METHODS: We conducted a retrospective investigation of serological and genotypical data from 205 samples (158 patients and 47 of their family members) of patients who were referred to the Molecular Genetics Laboratory at Chonnam National University Hwasun Hospital for ABO genotyping between January 2007 and July 2012. ABO genotyping was performed on all samples with PCR-direct sequencing of exons 6 and 7 in the ABO gene; the standard serologic tests were also performed. RESULTS: The frequency of phenotypes consistent with their genotypes was 70.8% (112/158 cases) and the A2B3 phenotype with the cis-AB01 allele was the most common (31.0%, 49 cases) among them. The frequency of phenotypes inconsistent with their genotypes was 29.1% (46/158 cases) and the A1B3 phenotype was the most frequently recovered case (5.1%, 8 cases). Family study showed differential phenotype expression depending on the co-inherited ABO allele in five families with the B306, cis-AB01, Ael02, Aw14, or B305 allele and also showed a typical inheritance of a chimera with A102/B101/O04. CONCLUSION: We propose that ABO genotyping using PCR-direct sequencing is useful for the resolution of ABO discrepancies and for the investigation of ABO subgroups based on six years' experience. In addition, family study for analysis of phenotypic patterns of ABO subgroups is also crucial to ABO genotyping.

Hibridização reversa e sequenciamento na genotipagem do vírus da hepatite C / Reverse hybridization and sequencing for genotyping the hepatitis C virus

INTRODUÇÃO: Os métodos de genotipagem do vírus da hepatite C têm sido muito discutidos. O objetivo deste trabalho foi comparar as metodologias de hibridização reversa e sequenciamento direto para a genotipagem do vírus da hepatite C. MÉTODOS: Noventa e uma amostras de plasma de pacientes assistidos na Faculdade de Medicina de Botucatu da Universidade Estadual Paulista foram utilizadas. A genotipagem por hibridização reversa foi realizada utilizando o kit comercial INNO-LiPA® v.1.0. O sequenciamento direto foi efetuado em sequenciador automático utilizando protocolos in house. RESULTADOS: A genotipagem por sequenciamento direto mostrou-se eficiente na resolução dos resultados inconclusivos pelo kit comercial. O kit mostrou resultados errôneos em relação à subtipagem viral. Além disso, a genotipagem por sequenciamento direto revelou um erro do kit com relação à determinação genotípica questionando a eficiência do método também para a identificação do genótipo viral. CONCLUSÕES: A genotipagem realizada por meio de sequenciamento direto permite uma maior acurácia na classificação viral quando comparada à hibridização reversa.

INTRODUCTION: The methods for genotyping the hepatitis C virus have been much discussed. The aim of this study was to compare the methodologies of reverse hybridization and direct sequencing for genotyping the hepatitis C virus. METHODS: Ninety-one plasma samples from patients attended at the Botucatu Medical School, São Paulo State University, were used. Genotyping by reverse hybridization was performed using the INNO-LiPA® v.1.0 commercial kit. Direct sequencing was performed in an automated sequencer using in-house protocols. RESULTS: Genotyping by direct sequencing was shown to be efficient for resolving cases that had remained inconclusive after using the commercial kit. The kit showed erroneous results in relation to virus subtyping. Moreover, direct sequencing revealed an error of the kit regarding the genotypic determination, thereby raising doubts about the efficiency of reverse hybridization for identifying the virus genotype. CONCLUSIONS: Genotyping by direct sequencing allowed greater accuracy of virus classification than did reverse hybridization.

Evaluation of the Genotypes of the Lewis Blood Group in a Korean Population Using Direct Sequencing / 대한혈액학회지

BACKGROUND: The FUT2 and FUT3 genes determine the Lewis phenotype of red blood cells (RBCs). Recently, the Lewis genes, the secretor genes, and several mutations that cause Lewis negative and nonsecretor phenotypes have been identified. The purpose of this study was to analyze the gene frequency of FUT2 and FUT3 in a Korean population by comparing the use of the direct sequencing method to the use of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for mutation detection in the FUT2 and FUT3 genes. METHODS: RBCs and peripheral blood leukocytes were obtained from 225 apparently healthy volunteers to determine the phenotype and genotype of the FUT2 and FUT3 genes. Lewis phenotypes were determined on K3EDTA-stablized fresh blood samples using the column agglutination method. Lewis blood group genotyping was performed by use of the direct sequencing method. For the detection of T59G, C357T, and A385T mutations, the PCR-RFLP method was performed. RESULTS: The frequencies of the Lewis blood group phenotype were 12.4% for Le(a+b-), 70.7% for Le(a-b+), 11.1% for Le(a-b-) and 5.8% for Le(a+b+), respectively. Direct Sequencing of the FUT2 gene identified 92.2% C357T, 56.9% A385T, 0.4% G244A mutations and 1.8% del396. Direct Sequencing of the FUT3 gene identified 46.9% T59G, 30.4% G508A, 1.1% T202C, 1.1% C314T, 0.7% A1029G, 3.0% T1067A and 13.3% G1242A mutations. The PCR-RFLP method results were discordant in nine cases (1 case for C357T, 4 cases for A385T and 2 cases for T59G) as compared to the direct sequencing method results. CONCLUSION: We have determined the frequencies of FUT2 and FUT3 gene mutations in a Korean population. The use of the direct sequencing method was more accurate than the use of the PCR-RFLP method for the determination of the genotype of the FUT2 and FUT3 genes.

The Prevalence of Maturity Onset Diabetes of the Young(MODY) 3 in Children with Type 2 Diabetes Mellitus / 소아과

PURPOSE: Maturity-onset diabetes of the young(MODY) is a subtype of type 2 diabetes defined by autosomal dominant mode of inheritance, onset of diabetes usually before the age of 25 yrs, and a primary defect in the function of the beta cells of the pancreas. MODY3 is known as the most common form and is caused by mutations in hepatocyte nuclear factor(HNF)-1alpha. We examined the prevalence of MODY3 in children with type 2 diabetes mellitus(DM). METHODS: Children with type 2 DM(N=17) and their family members with type 2 DM(N=5) were enrolled. Inclusion criteria for the children were fasting C-peptide and postprandial C-peptide more than 1.0 ng/mL and 1.5 ng/mL respectively, familial type 2 DM in at least two generations, and body mass index(BMI)(kg/m(2)) less than 95th percentile. Genomic DNA was extracted from blood samples. We analyzed HNF-1alpha for mutation by DNA microarray method and direct sequencing. RESULTS: We found one case with a mutation of the promoter region of HNF-1alpha(5'-ctaGGCTAGTGGGGTTTTGCGGGGGCAGTGGGTGCAAGG-3') in one child's family member among 22 children and adult subjects with type 2 DM. CONCLUSION: Although we found a mutation of HNF-1alpha in an adult family member with type 2 DM, we did not find this mutation in a child with type 2 DM. The further investigation of MODY in children, including other types, is required.

Characterization of Bruton's Tyrosine Kinase Genetic Mutations in One Korean X-linked Agammaglobulinemia Family

PURPOSE: X-linked agammaglobulinemia(XLA) is an immunodeficiency caused by abnormalities in Bruton's tyrosine kinase(Btk), and is characterized by a deficiency of peripheral blood B cells. We studied the cytoplasmic expression of Btk protein and analyzed the Btk gene in peripheral blood mononuclear cells from two siblings and one cousin with XLA, as well as additional family members. METHODS: Btk protein expression was analyzed by flow cytometry. Isolation of the coding sequence of the Btk gene was performed by amplification using the reverse transcription-polymerase chain reaction(RT-PCR) technique. Sequence alterations were screened by the single-stranded conformation polymorphism(SSCP) method and characterized by standard sequencing protocols. RESULTS: Cytoplasmic expression of Btk protein in monocytes was not detected in three patients with XLA. In addition, Btk protein analysis clearly showed cellular mosaicism in monocytes from four obligate carriers, findings further supported by SSCP. A single base pair mutation(T to C) in Btk-exon three, which encodes the PH domain, was identified in four XLA patients. A diagnostic sequencing analysis was established to detect heterozygotic pattern in 4 carrier females. Furthermore, we found significant clinical heterogeneity in individuals with the same gene mutation. CONCLUSION: The implicating genetic alteration provided valuable clues to the pathogenesis of XLA in Korea and the flow cytometric analysis was suggested as a useful tool for rapid detection of XLA patients and carriers. The present study has identified a genetic mutation in the Btk coding region and demonstrated heterogeneity in clinical manifestations among patients with the same mutation. A flow cytometric analysis was found to be informative in establishing a deficiency of Btk protein in both patients and carriers and is recommended as a frontline procedure in the molecular diagnosis and work-up of XLA.

Characterization of Mutations in Bruton's Tyrosine Kinase(Btk)Gene from Unrelated 3 X-linked Agammaglobulinemia(XLA) Families in Korea

PURPOSE: X-linked agammaglobulinemia(XLA) is an immunodeficiency caused by abnormalities in Bruton's tyrosine kinase(Btk), and is characterized by a deficiency of peripheral blood B cells. We studied cytoplasmic expression of Btk protein and analyzed the Btk gene in peripheral blood mononuclear cells(PBMC) from three XLA families in Korea. METHODS: Heparinized venous blood samples were collected from four XLA patients and additional family members in three unrelated XLA families. Mononuclear cells were separated from their blood and the intracellular Btk protein was characterized by a flow cytometry. The mutation analysis was performed using direct sequencing. RESULTS: Cytoplasmic expression of Btk protein in monocytes was not detected in the patients with XLA. We observed a novel deletion and two point mutations within introns(intron 1 and intron 18) resulting in alternative splicings. In XLA family 2, a 980 bp deletion(from intron 9+191 T to intron 10-215 C) including exon 10 was found in patient P2. He was the only sporadic case in this study, because his mother and brother showed a normal Btk expression by flow cytometry. CONCLUSION: These identified genetic alterations support the molecular heterogeneity of Btk gene in XLA disease. Additionally, by means of flow cytometric analysis, we diagnosed three hypogammaglobulinemia patients as XLA. Advancements in diagnostic methods has facilitated a prompt and definite diagnosis of this disease.

A Study on Mutations of p53 Tumor Suppressor Gene in Oral Tumors

Nowadays, there are a lot of evidence that mutation of the p53 tumor suppressor gene is one of the most common genetic abnormalities in neoplastic progression. In this study, we analyzed 20 specimens of oral tumors (squamous cell carcinoma 14 cases, ameloblastoma 3 cases, adenoid cystic carcinoma 2 cases, malignant schwannoma 1 case) using polymerase chain reaction and direct sequencing which used an automated DNA sequencer and software for detection of mutations. Polymerase chain reactions were performed with 4 sets of primers encompassing exon 5, 6, 7, 8, and direct sequencing method was employed. The results were as followings. 1. We detected 10 piont mutations out of 20 specimens (50%). 2. The genetic alterations included 7 mis-sense mutations resulting in single amino acid subtitutions, 2 silent mutations, 1 non-sense mutations encoding a stop codon. 3. Mutations were mostly in exon 7(7 out of 10 mutations, 70%) and involved codons 225, 234, 235, 236, 238, 247. 4. Therse were 4 cases of T-->A transversion, 2 cases of C-->A transversion, A-->G transition, 1 case of C-->G, T-->G transversion respectively. 5. We could find out point mutations more conveniently using PCR-Automated Direct Sequencing method.

Detection of Mutations to Zidovudine in the pol Gene of Human Immunodeficiency Virus-1 by Direct Sequencing

No abstract available.

A molecular model of a point mutation (Val297Met) in the serine protease domain of protein C

A heterozygous GTG to ATG (Val297Met) mutation was detected in a patient with inherited protein C deficiency and deep vein thrombosis. Cosegregation of the mutation with protein C deficiency was observed through a family pedigree study. Molecular models of the serine protease domains of wild type and mutant protein C were constructed by standard comparative method. Val 297 was found to be located in the hydrophobic core of the protein. Although the substitution of Met for Val does not greatly alter the hydrophobicity of the protein, it introduces a bulkier side chain, which yields steric hindrance between this residue and adjacent residues, such as Met364, Tyr393, Ile321, Ile323, and Val378. It seems that the Met can not fit into the tight packing into which it is trapped, thereby probably inducing misfolding and/or greater instability of the protein. Such misfolding and/or instability thereby eventually disturbs the catalytic triad, in consistent with the observed type I deficiency state.

Analysis of mutations of ras and p53 gene in multiple myeloma / 대한내과학회지

BACKGROUND: Multiple myeloma is a malignant prolif eration of plasma cells producing monoclonal immunog lobulins. The pathogenesis of this disease is still unkno wn. Karyotypic complexity and stepwise disease progre ssion in multiple myeloma suggest that the development of multiple myeloma is a multistep process in genetic events, such as oncogene activation or tumor suppressor gene inactivation. Alterations of ras oncogene or p53 tumor sup pressor gene are involved in various type of human cancers. The aim of this study was to determine the frequency of ras and p53 gene mutations in multiple myeloma, and to analyze its association with clinical parameter and clinical outcome. METHODS: Mutations of N-, K-ras exon 1 & 2 and p53 exon 5-8 were observed in 33 patients with multiple myeloma. Genomic DNA was isolated from mononuclear cells separated from bone marrow samples. Extracted DNAs were screened for mutations by single-strand con formation polymorphism analysis of PCR products (PCR SSCP). DNA fragments displaying an altered electrophore tic mobility were further studied by direct sequencing to confirm and characterize the nature of the mutations. RESULTS: No mutation was found at N-, K-ras exon 1, K-ras exon 2 or p53 exon 5-8. Only one patient has N-ras exon 2 mutation(1/33 patients, 3%). By direct sequencing of PCR products, I confirmed and detected a CAA-->AAA transversion(glutamine-->lysine). The patient was a 61-year-old male in progressive state. M protein was IgG/kappa type. Bone marrow aspirate revealed a 67% plasma cell infiltration. CONCLUSION: Although the number of patients is small, these data revealed low frequency of N-ras, K-ras and p53 gene mutation in multiple myeloma. Ras and p53 gene mutations may have limited role in pathogenesis of mulitple myeloma and may associate with tumor progres sion rather than initiation.