OBJECTIVEUsing heteroduplex mobility assay (HMA) to subtype human immunodeficiency virus type 1 (HIV-1) for the purpose of understanding HIV-1 subtype epidemic in Fujian province.

METHODSDNA fragments of HIV-1 env gene were amplified from peripheral blood mononuclear cell (PBMC) cocultures of HIV-1 infected individuals by nested polymerase chain reaction (PCR). Heteroduplexs were formed through hybridizing PCR products from the samples and reference plasmid. According to the mobility of heteroduplexs in polyacrylamide gel electrophoresis, HIV-1 subtype from that sample was characterized and further confirmed by nucleotide sequencing analysis.

RESULTSThirteen of 15 (86.67%) samples were successfully subtyped by HMA, except 2 failures. Subtype E and B took up 80% (12/15) and 6.67% (1/15) respectively. Results indicated a high concordance between HMA and nucleotide sequencing analysis and concordance rate was 86.67% (13/15).

CONCLUSIONSSubtype E appeared to be the major epidemic strain of HIV-1 in Fujian. HMA showed the characteristics of fastness, easiness, economic and with high specificity, and can be used in the surviellance for the epidemic strain of HIV-1.

Genotype ; HIV-1 ; classification ; genetics ; Heteroduplex Analysis ; methods ; Polymerase Chain Reaction

OBJECTIVETo establish a simple, sensitive, specific and less-costly method for detecting genotypes of TT virus (TTV).

METHODSTTV DNA was tested by nested polymerase chain reaction (nPCR) in sera from 180 patients with different types of viral hepatitis and 96 normal individuals in Beijing. TTV genotypes were determined in 40 sera collected from TTV DNA positive patients by heteroduplex mobility assay (HMA) and through sequencing.

RESULTSThe positive rates of TTV DNA in viral hepatitis patients and normal individuals were 22.2% (40/180) and 19.8% (19/96), respectively (chi(2) = 0.220, P = 0.639). TTV DNA positive rates of patients with hepatitis A, B, C, E and non-A to E were 20.0% (6/30), 16.7% (5/30), 23.3% (7/30), 36.7% (11/30) and 18.3% (11/60), respectively. Of 40 TTV DNA positive patients, 20 (50.0%) were TTV G1, 7 (17.5%) TTV G2, 10 (25.0%) coinfected with different genotypes of TTV, and 3 untyped by HMA. Twenty G1 and 7 G2 detected by HMA were confirmed by sequence analysis. Of 10 patients coinfected with different genotypes of TTV, 5 were G1 and G2, 2 G1 and G3, 1 G1 and G4, 1 G1 and G3, and 1 with G1, G2 and G3 coinfections.

CONCLUSIONHMA was recognized as simple, sensitive, specific and less-costly, thus could be used for genotyping of TTV.

DNA, Viral ; analysis ; Genotype ; Hepatitis, Viral, Human ; virology ; Heteroduplex Analysis ; methods ; Humans ; Phylogeny ; Torque teno virus ; classification ; genetics

BACKGROUND: Recently a non-Jewish German family with writer's cramp was reported to have DYT1 mutation, expanding the phenotypic spectrum of DYT1. Although functional brain surgery has been tried for generalized dystonia, surgical outcome in focal dystonia patients with DYT1 mutation has not yet been reported. We investigated the clinical features and response to thalamotomy in familial writer's cramp with DYT1 mutation. METHODS: Family members were examined and clinically affected cases were video-taped. For the detection of DYT1 mutation, PCR-RFLP(restriction fragment length polymorphism) and heteroduplex analyses were performed as screening tests. Additional DNA sequencing was performed for the proband case to confirm the GAG deletion. RESULTS: Among this clinically homogeneous non-Jewish Korean family, five members in three generations were affected. Age of onset ranged from 7 to 20 years. Writing difficulty was the initial and the main disabling problem for all the affected individuals. All had the bilateral writer's cramp in succession. Dystonia remained focal and task-specific for the entire period after onset. Unilateral thalamotomy was performed in three patients, with remarkable improvement. Surgical benefit remained unchanged during the follow-up period of 6-8 years. DYT1 mutation cosegregated with the affected members. CONCLUSIONS: This study adds another evidence that DYT1 phenotype can present with purely focal and task-specific dystonia in all the affected members of a family. Marked and sustained improvement following thalamotomy in three of our patients suggests that stereotaxic thalamotomy is beneficial in familial focal dystonia with DYT1 mutation. (J Korean Neurol Assoc 19(2):110~115, 2001)
Age of Onset ; Brain ; Dystonia ; Dystonic Disorders* ; Family Characteristics ; Follow-Up Studies ; Heteroduplex Analysis ; Humans ; Mass Screening ; Phenotype ; Sequence Analysis, DNA ; Writing

BACKGROUND: Recently a non-Jewish German family with writer's cramp was reported to have DYT1 mutation, expanding the phenotypic spectrum of DYT1. Although functional brain surgery has been tried for generalized dystonia, surgical outcome in focal dystonia patients with DYT1 mutation has not yet been reported. We investigated the clinical features and response to thalamotomy in familial writer's cramp with DYT1 mutation. METHODS: Family members were examined and clinically affected cases were video-taped. For the detection of DYT1 mutation, PCR-RFLP(restriction fragment length polymorphism) and heteroduplex analyses were performed as screening tests. Additional DNA sequencing was performed for the proband case to confirm the GAG deletion. RESULTS: Among this clinically homogeneous non-Jewish Korean family, five members in three generations were affected. Age of onset ranged from 7 to 20 years. Writing difficulty was the initial and the main disabling problem for all the affected individuals. All had the bilateral writer's cramp in succession. Dystonia remained focal and task-specific for the entire period after onset. Unilateral thalamotomy was performed in three patients, with remarkable improvement. Surgical benefit remained unchanged during the follow-up period of 6-8 years. DYT1 mutation cosegregated with the affected members. CONCLUSIONS: This study adds another evidence that DYT1 phenotype can present with purely focal and task-specific dystonia in all the affected members of a family. Marked and sustained improvement following thalamotomy in three of our patients suggests that stereotaxic thalamotomy is beneficial in familial focal dystonia with DYT1 mutation. (J Korean Neurol Assoc 19(2):110~115, 2001)
Age of Onset ; Brain ; Dystonia ; Dystonic Disorders* ; Family Characteristics ; Follow-Up Studies ; Heteroduplex Analysis ; Humans ; Mass Screening ; Phenotype ; Sequence Analysis, DNA ; Writing

OBJECTIVETo study the prevalence of P53 protein expression and p53 gene mutation in laryngeal carcinoma.

METHODSUsing immunohistochemistry P53 expression was detected in 31 patients with laryngeal carcinoma. In 11 P53 negative patients,microdissection-PCR-HA technique was used to determine mutation in p53 exon 5, 6, 7, 8.

RESULTSAmong the 31 patients tested with immunostaining, the overall average positive rate was 64.5%. Positive rates for T3 and T4 tumors were 86.7% vs 43.8% in T1 and T2 tumors.The positive rate was 91.7% in those with cervical node metastasis compared with 47.4% in those without lymph node metastasis. The positive P53 immunostaining was more frequently found in poor differentiated carcinoma (87.5%) and moderate-differentiated carcinoma (66.7%),than in well differentiated carcinoma (45.5%). The abnormal exon 5 or 7 of p53 gene were detected in 2 out of 11 cases, in which P53 was negative.

CONCLUSIONP53 gene mutation is related with TNM grading and cervical lymph node metastasis in laryngeal carcinoma. P53 mutation tents to be correlated to pathologic grading.

Adult ; Aged ; Female ; Genes, p53 ; Heteroduplex Analysis ; Humans ; Immunohistochemistry ; Laryngeal Neoplasms ; chemistry ; genetics ; Male ; Middle Aged ; Mutation ; Tumor Suppressor Protein p53 ; analysis

OBJECTIVES: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is most often caused by a deficiency in steroid 21-hydroxylase (21-OH), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21-OH deficiency, genotyping of the 21-OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. This study was aimed to analyze the complete characterization of the CYP21 gene coding region in a Korean CAH patient and to conform the PCR-based single strand conformation polymorphism (SSCP) and heteroduplex analysis as a diagnostic tool. METHODS: We used a highly sensitive, non-radioactive method allowing PCR-based single strand conformation polymorphism (SSCP) analysis. This method was applied to the characterization of all the exons and intron-exon junctions of the CYP21 gene in one patients affected by the salt wasting form and 4 normal controls. RESULTS: In all samples showing SSCP abnormal band patterns, sequence analysis showed the presence of sequence variants. In particular, one mutation (I172N) which is already known to cause the disease and 3 silent mutations were detected. CONCLUSION: PCR-based single strand conformation polymorphism (SSCP) and heteroduplex analysis should be useful for the clinical application as a diagnostic tool for the detection of 21-hydroxylase gene mutations.
Adrenal Hyperplasia, Congenital* ; Alleles ; Clinical Coding ; Diagnosis* ; Exons ; Gene Conversion ; Heteroduplex Analysis ; Humans ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis ; Steroid 21-Hydroxylase* ; Virilism

OBJECTIVETo confirm that PCR products with heterozygous mutations contain not only wide-type and mutant homoduplexes, but also two types of heteroduplexes.

METHODSAn insertion-deletion mutation in the exon 1 of KRT9 gene (497delAinsGGCT), which caused Chinese epidermolytic palmoplantar keratoderma (EPPK) was investigated by polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and denaturing high-performance liquid chromatography(DHPLC).

RESULTSTwo heteroduplexes and two homoduplexes in the PCR product from the heterozygous mutation of the exon 1 of KRT9 (497delAinsGGCT) were detected.

CONCLUSIONPCR products from KRT9 gene with heterozygous mutations contain two types of heteroduplexes. It is without the need to perform heating and cooling PCR products obtained from heterozygous mutations in advance before the mutation screening steps such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), conformation-sensitive gel electrophoresis (CSGE), DHPLC and heteroduplex analysis (HA), etc.

Base Pair Mismatch ; Chromatography, High Pressure Liquid ; DNA Mutational Analysis ; Electrophoresis, Polyacrylamide Gel ; Heteroduplex Analysis ; Heterozygote ; Humans ; Keratin-9 ; Keratins ; genetics ; Mutation ; Nucleic Acid Heteroduplexes ; Polymerase Chain Reaction

BACKGROUND: The clonality of lymphoid infiltrates determined by polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) or T cell receptor (TCR) genes is not only useful in confirming the diagnosis of malignant lymphoma but also in establishing the lineage of a clonal lymphoid proliferation. We analyzed the efficiency of PCR analyses for IgH and TCRgenes that have been routinely applied for the diagnosis of malignant lymphoma in our laboratory. METHODS: Paraffin sections of 200 cases were analyzed by seminested PCR. Primers were FRIIIA-LJH/VLJH consensus primer for IgH gene and V-J consensus primer for TCR gene. The cases showing negative results by PCR for TCR gene were further analyzed by multiplex V family primers with heteroduplex analysis. RESULTS: PCR approach for IgH gene allowed detection of clonality in 100% of cases with false positive rate of 0.3% and false negative rate of 0%. The combination of PCR for TCR consensus primers with multiplex V family primers allowed detection of clonality in 91% of cases with false positive rate of 0.6% and false negative rate of 10.3%. CONCLUSIONS:Combined analysis of IgH and TCR gene rearragnements by the PCR technique followed by heteroduplex analysis can be a useful diagnostic adjunct to determine the clonality of various lymphoproliferative diseases with high sensitivity. But clinical, morphological and immunophenotypical correlation should be considered to reach the final diagnosis due to a few false positive cases.
Consensus ; Diagnosis ; Gene Rearrangement* ; Genes, T-Cell Receptor ; Heteroduplex Analysis ; Humans ; Immunoglobulin Heavy Chains* ; Immunoglobulins* ; Lymphoma ; Paraffin ; Polymerase Chain Reaction ; Receptors, Antigen, T-Cell* ; T-Lymphocytes*

BACKGROUND: This study characterized clonal IG heavy V-D-J (IGH) gene rearrangements in South Indian patients with precursor B-cell acute lymphoblastic leukemia (precursor B-ALL) and identified age-related predominance in VDJ rearrangements. METHODS: IGH rearrangements were studied in 50 precursor B-ALL cases (common ALL=37, pre-B ALL=10, pro-B ALL=3) by polymerase chain reaction (PCR) heteroduplex analysis. Twenty randomly selected clonal IGH rearrangement sequences were analyzed using the IMGT/V-QUEST tool. RESULTS: Clonal IGH rearrangements were detected in 41 (82%) precursor B-ALL cases. Among the IGHV1-IGHV7 subgroups, IGHV3 was used in 25 (50%) cases. Among the IGHD1-IGHD7 genes, IGHD2 and IGHD3 were used in 8 (40%) and 5 (25%) clones, respectively. Among the IGHJ1-IGHJ6 genes, IGHJ6 and IGHJ4 were used in 9 (45%) and 6 (30%) clones, respectively. In 6 out of 20 (30%) IGH rearranged sequences, CDR3 was in frame whereas 14 (70%) had rearranged sequences and CDR3 was out of frame. A somatic mutation in Vmut/Dmut/Jmut was detected in 14 of 20 IGH sequences. On average, Vmut/Dmut/Jmut were detected in 0.1 nt, 1.1 nt, and 0.2 nt, respectively. CONCLUSION: The IGHV3 gene was frequently used whereas lower frequencies of IGHV5 and IGHV6 and a higher frequency of IGHV4 were detected in children compared with young adults. The IGHD2 and IGHD3 genes were over-represented, and the IGHJ6 gene was predominantly used in precursor-B-ALL. However, the IGH gene rearrangements in precursor-B-ALL did not show any significant age-associated genotype pattern attributed to our population.
Child ; Clone Cells ; Complementarity Determining Regions* ; Gene Rearrangement* ; Genotype ; Heteroduplex Analysis ; Humans ; Immunoglobulins* ; Polymerase Chain Reaction ; Precursor Cell Lymphoblastic Leukemia-Lymphoma* ; Precursor Cells, B-Lymphoid* ; Young Adult

OBJECTIVETo study the relationship of Chinese familial Parkinson disease with alpha-synuclein gene.

METHODSPolymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and polymerase chain reaction-heteroduplex analysis(PCR-HA) were employed to detect the abnormal mobilization in the familial Parkinson disease and sporadic Parkinson disease patients, then it was verified by gene sequencing.

RESULTSNo mutation was found in alpha-synuclein gene exons 3 and 4 by PCR-SSCP together with PCR-HA. An inserted c and an inserted t were found in intron 4, position 23 and position 67 respectively.

CONCLUSION(1) Exons 3 and 4 of alpha-synuclein gene are not the mutational hot spots of Chinese familial Parkinson disease. (2) Two polymorphisms were found in intron 4 of alpha-synuclein gene. They are 23 ins c and 67 ins t.

Adult ; Aged ; Exons ; Female ; Heteroduplex Analysis ; Humans ; Male ; Middle Aged ; Mutation ; Nerve Tissue Proteins ; genetics ; Parkinson Disease ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Synucleins ; alpha-Synuclein