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 investigate the polymorphism of Kell blood group system in Chinese and to find a suitable method for large scale screening.

METHODSAn analysis method of polymerase chain reaction-restriction fragment-single strand conformation polymorphism (PCR-RF-SSCP) combined with heteroduplex was established to detect abnormal sample in KEL exon 7-9 area, then sequencing was used to find out the mutation site.

RESULTSTwo mutations were found from 500 samples: 966G > A mutation in exon 9 and C > A mutation in 67th site of intron 7, both with no amino acid change. The mutation rate was 4/1000. No mutation was found as missed in using PCR-RF-SSCP combined with heteroduplex.

CONCLUSIONPCR-RF-SSCP combined with heteroduplex is confirmed as an effective, economical and simple method, it is quite suitable for large scale population screening study with unclear gene background and unavailable positive controls. Since there is special polymorphism for Kell blood group system in Chinese, further study is needed.

Asian Continental Ancestry Group ; genetics ; China ; Heteroduplex Analysis ; methods ; Humans ; Kell Blood-Group System ; genetics ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single-Stranded Conformational

BACKGROUNDHepatitis C virus (HCV) envelope genes encoding glycoproteins E1 and E2 exhibits a high degree of variability that gives rise to differing phenotypic traits; including alterations in receptor-binding affinity and immune recognition and escape. This study aims to elucidate the relationship of the evolutionary patterns for HCV envelope glycoproteins to viral persistence.

METHODSHCV quasispecies were characterized in specimens collected every two to six months from a cohort of acutely HCV-infected subjects. We evaluated two individuals who spontaneously cleared viremia and three individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2; including hypervariable region 1 (HVR1). To detect representative variants for sequencing thirty-three cloned cDNAs representing each specimen were assessed by a method that combined analysis of a single-stranded conformational polymorphism (SSCP) method and heteroduplex analysis (HDA). For each patient, the rates of both synonymous and nonsynonymous substitutions for the E1, HVR1 and E2 regions outside HVR1 were evaluated. The amino acid sequences and predicted antigenic profiles were analyzed.

RESULTSThe genetic diversity within HVR1 was consistently higher than that in the E1 and E2 regions outside HVR1 in individuals with persistent viremia, but did not change markedly over time in those with clearance of viremia. For individuals with persistent viremia, the rate of nonsynonymous substitutions within the HVR1 region predominated and gradually increased, compared to that in the E1 and E2 regions outside HVR1. By contrast, the rates of both nonsynonymous and synonymous substitutions for the E1 and E2 regions, including HVR1, were consistently lower in individuals with clearance of viremia. HVR1 had a higher antigenic variable and lower positive charge in subjects with persistent viremia. All cysteine residues and N-linked glycosylation sites, some of which were known to play a major role in protein folding and others play a role in HCV entry, were 100% conserved among the sequenced cloned cDNAs from the two outcome groups.

CONCLUSIONHCV persistence may be associated with positive selection pressures on HVR1, rather than functional constraints in the envelope region.

Acute Disease ; Adult ; Amino Acid Sequence ; Evolution, Molecular ; Female ; Hepacivirus ; genetics ; Hepatitis C ; virology ; Heteroduplex Analysis ; Humans ; Male ; Molecular Sequence Data ; Polymorphism, Single-Stranded Conformational ; Viral Envelope Proteins ; genetics

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

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

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

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

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

BACKGROUND: Recently, the molecular pathologic investigation for clonality in lymphomas has been introduced and has gained a role in the diagnosis of lymphomas. In fact, the clonality test using TCRGR phenomenon has been done by Southern blot analysis (SBA) and polymerase chain reaction (PCR) for molecular pathologic diagnosis of T cell lymphomas. However, it is difficult to perform SBA with paraffin embedded specimens or with samples of small skin biopsies. OBJECTIVE: We investigated the efficacy of PCR amplification of TCR gene in paraffin em-bedded cutaneous T cell lymphomas. METHODS: Iii this study, the clonality was assessed by polymerase chain reaction (PCR) analysis of T cell receptor gamma (TCR) gene from the DNA extracts obtained from paraffin em-bedded tissues (PET) of malignant T cells, B cell lymphomas, and benign cutaneous T cell proliferative disorders. Heteroduple-x-analyses were also performed to rule out the false positives. RESULTS: Among the total of 62 cases analyzed, monoclonality was observed in 4 out of 10 mycosis fungoides, 7 out of 9 cutaneous T cell lymphomas excluding mycosis fungoides, 1 out of 3 angiocentric lymphomas, 2 out of 2 lymphomatosis papulosis, 1 out of 7 large plaque parapsoriasis, and 1 out of 2 T cell lymphomas in other organs. No monoclonality was observed in 9 inflammatory cutaneous diseases, 5 small plaque parapsoriasis, 4 cutaneous B cell lymphomas, and 11 B cell lymphomas in lymph nodes. CONCLUSION: The results suggest that the PCR method and heteroduplex analysis used in this study were not only practical but also efficacious for the diagnosis of cutaneous T cell lymphomas using tissues embedded in paraffins.
Biopsy ; Blotting, Southern ; Diagnosis ; DNA ; Gene Rearrangement* ; Genes, T-Cell Receptor ; Heteroduplex Analysis* ; Lymph Nodes ; Lymphoma ; Lymphoma, B-Cell ; Lymphoma, T-Cell ; Lymphoma, T-Cell, Cutaneous ; Mycosis Fungoides ; Paraffin ; Parapsoriasis ; Polymerase Chain Reaction* ; Receptors, Antigen, T-Cell* ; Skin ; T-Lymphocytes

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