Characterization of clonal immunoglobulin heavy (IGH) V-D-J gene rearrangements and the complementarity-determining region in South Indian patients with precursor B-cell acute lymphoblastic leukemia

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.

Genotyping of human parvovirus B19 in clinical samples from Brazil and Paraguay using heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing

Heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing were utilised to genotype human parvovirus B19 samples from Brazil and Paraguay. Ninety-seven serum samples were collected from individuals presenting with abortion or erythema infectiosum, arthropathies, severe anaemia and transient aplastic crisis; two additional skin samples were collected by biopsy. After the procedure, all clinical samples were classified as genotype 1.

[DFNB59 gene mutations and its association with deafness in schoolchildren in kohgilooyeh and Boyerahmad province]

Hearing loss is a common disease affecting millions of people worldwide. Hearing loss can be caused due to genetic or environmental factors or even both. The genetic of hearing defect is highly heterogeneous and more than 100 genes are predicted to cause this disorder in humans. A newly identified gene [DFNB59] has been shown to cause deafness in some populations. Here we report mutation analysis for DFNB59 gene in 88 genetic non-syndromic hearing loss subjects. In this descriptive-lab based study which was conducted at the Cellular and Molecular Research Center of Shahrekord University of Medical Sciences, DNA was extracted from the peripheral blood samples using standard phenol chloroform procedure. Mutation analysis for DFNB59 gene was performed using PCR-SSCP/HA protocol. The suspected DFNB59 which was detected as shifted bands on PAGE were then confirmed by direct sequencing strategy. Two DFNB59 polymorphisms including c.793C>G and c.793C>T were detected in 8 and 1 deaf subjects respectively. We conclude that there is no association between DFNB59 mutations and deafness in the studied patients in the region

High genetic diversity in human immunodeficiency virus: type 1 in Jamaica / Elevada diversidad genética del virus de la inmunodeficiencia humana: tipo 1 en Jamaica

The subtypes of the human immunodeficiency virus - type 1 (HIV-1) strains from 54 HIV-1 - infected persons including 44 strains which were typed previously by heteroduplex mobility assay (HMA) were determined by DNA sequencing and phylogenetic analysis. Of 54 HIV- infected persons, 92.5% were infected with HIV-1 subtype B and 7.5% with other HIV-1 subtypes including subtypes D (3.7%), A (1.9%) and J (1.9%). In the phylogenetic analysis, the subtype A virus found in the sample clustered with subtype A reference strains and a circulating recombinant form (CRF) reference strain which originates in Central Africa and is circulating in Cuba indicating a close relationship between these viruses. There was 86% concordance between HMA and DNA sequencing in assigning subtype B viruses. For the non-B subtype viruses, there was less concordance between the two methods (67%). The results confirm the predominance of HIV-1 subtype B strains and the high genetic diversity of HIV-1 strains in circulation in Jamaica. The efficacies and some limitations of the HMA as a method of HIV-1 subtyping also were noted. It is important that the HIV/AIDS epidemic in Jamaica be monitored meticulously for possible expansions in non-B subtypes and the emergence of inter-subtype recombinant forms. We recommend that the more expensive DNA sequencing and phylogenetic analysis, including HIV-1 genotyping for antiretroviral drug resistance testing, be used as an adjunct to the more cost-effective HMA to track the HIV/AIDS epidemic in Jamaica.

Los subtipos de cepas de virus de la inmunodeficiencia humana-tipo-1 de 54 personas infectadas con el VIH-1, que incluyeron 44 cepas previamente clasificadas según su tipo mediante ensayo de movilidad de heterodúplex (HMA), fueron determinados mediante secuenciación de ADN y análisis filogenético. De 54 personas infectados con VIH, 92.5% estaban infectadas con VIH-1 subtipo B y 7.5% con otros subtipos de VIH-1 incluidos los subtipos D (3.7%), A (1.9%), J (1.9%). En el análisis filogenético, el virus de subtipo A hallado en la muestra, se agrupa con las cepas de referencias del subtipo A y una cepa de referencia de forma recombinante circulante (CRF), que tienesu origen en África Central y está circulando en Cuba, lo que indica una estrecha relación entre estos virus. Hubo un 86% de concordancia entre el HMA y la secuenciación del DNA en la asignación de virus de subtipo B. Para los virus de subtipo no B, hubo menos concordancia entre los dos métodos (67%). Los resultados confirman el predominio de las cepas del subtipo B del VIH-1, y la alta diversidad genética de las cepas del VIH-1 en circulación en Jamaica. También se señalaron las eficacias y algunas limitaciones del HMA como método de clasificación del VIH-1 en subtipos. Es importante monitorear meticulosamente la epidemia de VIH/SIDA en Jamaica, a fin de detectar posibles expansiones de subtipos no B y la aparición de formas recombinantes inter-subtipos. Recomendamos que por ser ambos métodos más costosos, tanto la secuenciación de ADN como el análisis filogenético - incluyendo el genotipado del VIH-1 para probar la resistencia antiretroviral del medicamento - sean usados como complementos del HMA, el cual es más costo-efectivo, para seguir de cerca el rastro de la epidemia VIH/SIDA en Jamaica.

[Study on interferon gamma gene polymorphism in shaul sheep]

Interferon gamma [IFN-gamma] is a cytokine produced by activated T lymphocytes and natural killer cells. It plays an important role in innate and adaptive immunity. IFN-g gene is polymorphic and its haplotype has been associated with resistance to nematode infestations which are the most important parasites of domestic sheep worldwide. To evaluate IFN-g polymorphism in Iranian Shaul sheep population, 136 blood samples were collected. Genomic DNA were extracted and amplified by PCR using specific primers for exon 3 of IFN-g gene. After gene amplification, SSCP and Heteroduplex mobility analysis carried out on polyacrylamid gel. Five unique SSCP patterns were detected in this population which 3 of them represent 3 alleles. Although heteroduplex mobility analysis demonstrated that all samples were homozygous, SSCP could reveal heterozygosity at frequency of 40. 4% in the studied population. Data obtained from the present study have revealed that IFN-g gene was a highly polymorphic in Iranian Shaul ecotype. This will be useful to study host-pathogen interaction and to evaluate association between resistance or susceptibility to disease with IFN-g in this ecotype

Divergent strains of human immunodeficiency virus type 1 circulating in India, subtyped by heteroduplex mobility assay.

Genomic variations in HIV-1 represent a major problem in understanding disease progression, studying drug resistance and developing effective vaccines. Heteroduplex Mobility Assay (HMA) was used for analyzing HIV-1 subtypes resulting from genetic similarity or divergence of C2 -V3 -V5 region of envelope gene between HIV-1 strains obtained from clinical samples in a tertiary care center at Pune. DNA from the PBMCs of infected individuals was amplified by nested PCR. Heteroduplexes were then formed by denaturing DNA from the unknowns with DNA from the reference strains. The results were analyzed by polyacrylamide gel electrophoresis. Out of 177 samples analyzed, 170 were of subtype C (96%). Four samples were found to be of subtype B (2.2%); in three samples, no definitive assignment of subtype was possible by HMA and these perhaps could be circulating recombinant forms (CRFs) of HIV-1. These findings may have significant implications toward development of a candidate vaccine for India.

Diagnóstico de linfomas cutáneos mediante detección de clonalidad por PCR-heterodúplex / Diagnosis of cutaneous lymphomas by clonicity detection through PCR-heteroduplex technique

La detección de clonalidad en los síndromes linfoproliferativos mediante el estudio del reordenamiento de los genes de las inmuglobulinas y del receptor de células T, es utilizada para esclarecer si una proliferación o infiltrado de linfocitos es maligno o no. Este tipo de estudio es de particular utilidad en presencia de lesiones cutáneas cuyo origen linfoide o dermatológico resulta difícil de definir. Mediante la técnica de PCR-heterodúplex se estudiaron los genes de la cadena pesada de las inmunoglobulinas y de la cadena gamma del receptor de las células T, en 10 pacientes que presentaban manifestaciones dermatológicas atribuibles a algún tipo de linfoma cutáneo. Se observó reordenamiento clonal en 7 pacientes, lo cual permitió confirmar el diagnóstico de micosis fungoide y otros tipos de linfomas cutáneos. En 3 pacientes que no mostraron reordenamiento clonal, no fue posible confirmar por esta técnica un proceso linfoide de carácter maligno. Se demostró la utilidad del estudio cuando en presencia de una afección en la piel, es difícil diferenciar un proceso dermatológico de un síndrome linfoproliferativo con manifestaciones en piel.

The clonicity detection in the lymphoproliferative syndromes by studying the rearrangement of the immunoglobulin genes and of the T-receptor cells is used to make clear if a proliferation or infiltrate of lymphocytes is malignant or not. This type of study is particularly useful in the presence of cutaneous lesions whose lymphoid or dermatological origin is difficult to define. By the PCR-heteroduplex technique, the genes of the immunoglobulin heavy chain and of the T-cell receptor chain were studied in 10 patients that presented dermatological manifestations attributable to some kind of cutaneous lymphoma. Clonal rearrangement was observed in 7 patients, which allowed to confirm the diagnosis of mycosis fungoides and other types of cutaneous lymphomas. It was not possible to confirm a lymphoid process of malignant character by this technique in 3 patients who did not show clonal rearrangement. The usefulness of the study was proved when in the presence of a skin affection, it was difficult to differentiate a dermatological process from a proliferative syndrome with cutaneous manifestations.

Analysis of Kell blood group system using polymerase chain reaction-restriction fragment-single strand conformation polymorphism combined with heteroduplex in Chinese / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the polymorphism of Kell blood group system in Chinese and to find a suitable method for large scale screening.</p><p><b>METHODS</b>An 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.</p><p><b>RESULTS</b>Two 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.</p><p><b>CONCLUSION</b>PCR-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.</p>

Evolutionary study of hepatitis C virus envelope genes during primary infection / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hepatitis 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.</p><p><b>METHODS</b>HCV 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>HCV persistence may be associated with positive selection pressures on HVR1, rather than functional constraints in the envelope region.</p>

Human immunodeficiency virus type- 1 subtypes of infected patients in Espírito Santo, Brazil

Genetic variability of human immunodeficiency virus type - 1(HIV-1) is a potential threat for both diagnosis and treatment of HIV/AIDS, as well as the development of effective vaccines. Up to now, HIV subtypes circulating among HIV-positive patients in the state of Espírito Santo were not known. In the present study, blood samples from 100 therapy-naïve HIV-1 infected patients were collected and the HIV subtype was determined through the Heteroduplex Mobility Assay (HMA). Ninety-seven out of 100 studied samples were subtyped by HMA, 73 samples (75.2 percent) were from subtype B, 9 (9.3 percent) from subtype F, 3 (3.1 percent) from subtype C, 6 (6.2 percent) Benv/Fgag, and another 6 (6.2 percent) Fenv/Bgag, what suggests that recombinant viruses were present in the studied samples. Twenty-eight percent of the subtype B samples were represented by the Brazilian B" subtype, which were identified by RFLP with Fok I. Data presented here demonstrate that the epidemiological characteristics of the HIV epidemic in the state of Espírito Santo are similar to those from the other Southeastern states and helped to better understand the genetic polymorphism of HIV in Brazil.

PCR-heterodúplex por agrupamiento: implementación de un método de identificación de portadores de la mutación más común causal de fibrosis quística en Colombia / PCR-heteroduplex by grouping: rapid screening carrier method for cystic fibrosis F508del mutation in Colombia

Introducción: La fibrosis quística (FQ) es una enfermedad autosómica recesiva frecuente, con una incidencia de 1 en 2,500 recién nacidos. La causan más de 1,300 mutaciones distintas en el gen regulador de la conductancia transmembranal de la fibrosis quística (CFTR). Sin embargo, la mutación F508del es la más común en la mayoría de las poblaciones. Objetivos: Desarrollo de una técnica rápida, de bajo costo y confiable que permita filtrar con rapidez a los portadores o afectados por esta mutación que mediante el asesoramiento genético, contribuya a disminuir la aparición de nuevos casos y a un diagnóstico temprano de los enfermos y así lograr un descenso en la morbilidad y la mortalidad asociadas con la fibrosis quística en Colombia. Metodología: En el presente estudio se aplicó la técnica PCR-heterodúplex por agrupamientos, gracias al análisis de 400 muestras de sangre en papel filtro obtenidas de individuos asintomáticos para la FQ. Resultados: En las pruebas de validación de la técnica PCR-heterodúplex por agrupamiento se obtuvo una eficiencia, reproducibilidad y especificidad de 100% y una sensibilidad de 92%. Conclusiones: Se demostró la sensibilidad y reproducibilidad de la técnica PCR Directa-heterodúplex por agrupamientos de hasta 10 muestras, que se pueden emplear en programas para filtrar heterocigotos y afectados de F508del.

Background: Cystic fibrosis (CF) is the most frequent autosomal recessive disorder in the Caucasian population with an incidence of 1 in 2,500 newborns. More than 1,300 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that causes CF have been described. However, mutation F508del is the most common mutation in different populations around the world. Objective: To develop a fast, reliable and low-cost technique to screen carriers and affected individuals for the F508del mutation. This kind of analysis will have an impact on genetic counselling to decrease the incidence of new cases, in the early diagnosis and instauration of appropriate treatment to decrease morbidity and mortality associated to CF in Colombia. Methods: The reliability of the PCR-heteroduplex by grouping technique by analysis of 400 blood spot samples from asymptomatic CF patients was defined. Results: Using PCR-heteroduplex by grouping technique 100% efficiency, reproducibility and specificity and 92%sensitivity were found.Conclusions: The sensitivity and reproducibility of the PCR-heteroduplex by grouping technique up to pooling of 10 samples were demonstrated. This kind of analysis could be used in heterozygotes and affected screening programs.

HIV-1 genetic diversity in Argentina and early diagnosis of perinatal infection

HIV-1 diagnosis of perinatally exposed children is usually performed by molecular biology-based methods, allowing the direct detection of the virus. Thus, HIV-1 genomic variability within and across strains plays a major role in relation to the sensitivity of these tests, often leading to misdiagnosis. We describe the performance of an in-house multiplex nested PCR (nPCR) for early detection of HIV-1 infection in perinatally exposed children born in Argentina, where the percentage of diverse BF recombinants is as high as 80%. After evaluation of 1316 HIV-1 perinatally exposed children collected over a 7-year period, the specificity and sensitivity of the diagnostic nPCR was of 100% and 99.2% respectively, with only two false negative cases indicating a good performance of the diagnostic nPCR in the Argentine pediatric cohort. In search of unusual HIV-1 subtypes among 22 HIV-1 infected cases presenting partial or complete HIV-1 gene amplification failure, we performed phylogenetic and recombination analysis of a vpu-env fragment in addition to gag and env Heteroduplex Mobility Assay screening. The most unusual findings included two subtypes A and a novel BC recombinant, while the majority of the strains were a variety of different BF recombinants. These results indicate the presence of novel and heterogeneous genotypes in our country and the need of continuous viral surveillance not only for diagnostic test optimization but also for the eventual implementation of a successful vaccine.

Detection of genetic polymorphism among and within Echinococcus granulosus strains by heteroduplex analysis of a microsatellite from the U1 snRNA genes

Polymerase chain reaction of a pentanucleotide microsatellite in the U1 snRNA gene complex generated a multiple band pattern due to the priming of paralogous sequences. Denaturation and slow renaturation of polymerase chain reaction products allow the formation of heteroduplex DNA that can be detected by its differential mobility in polyacrylamide gel electrophoresis. Heteroduplex analysis was used to determine if the U1 snRNA microsatellite could be a useful genetic marker in Echinococcus granulosus. A U1 snRNA microsatellite fragment from E. granulosus was isolated and characterized by Southern blot and sequencing. Four E. granulosus strains were analyzed: sheep, Tasmanian sheep, cattle, and camel strains. The former two showed polymorphism and shared three of the six patterns found for sheep strain. The cattle strain displayed two patterns, and the camel strain was monomorphic. The electrophoretic profiles were used for statistical analysis in order to determine genetic distance and the relationship among strains. Heteroduplex analysis can be helpful in genotyping E. granulosus strains and is useful in detecting polymorphism within strains.

HIV-1 subtyping using gag/env heteroduplex mobility assay and peptide enzyme-linked immunosorbent assay.

Two HIV-1 subtypes have accounted for virtually all infections in Thailand: subtype B', found mainly in injection drug users (IDUs), and CRF01_AE (initially subtype E), found in over 90% of sexually infected persons and increasingly in IDUs in recent years. During 1997-1998, 227 blood samples were collected from HIV-1 infected individuals consisting of 92 mothers, 35 children and 100 IDUs. The blood samples were subtyped by heteroduplex mobility assay (HMA) and peptide enzyme-linked immunosorbent assay (PEIA). Using gag and env HMA, CRF01_AE and subtype B' accounted for 96-97% and 3-4% of both the mothers and the children, respectively. In the IDU group, 10% of the plasma samples could only be performed by gag HMA and gave the result as CRF01_AE. CRF01_AE and subtype B' using PEIA accounted for 67% and 33% of the IDUs. There was 100% concordance of the results between gag HMA and env HMA. Ninety-five percentages of concordant results were observed between HMA and PEIA. Of the 6/134 (5%) subjects with discordant results, nucleotide sequencing, used as a gold standard, confirmed the HMA result. In this study, HIV-1 was successfully genotyped by HMA and PEIA. However, a comparison of the subtyping results between HMA and PEIA revealed that HMA was slightly more accurate than PEIA.

Morphological and molecular characterization of three species of Anastrepha Schiner and of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

Four species of Tephritoidea, three from genus Anastrepha: A. obliqua (Macquart), A. sororcula Zucchi and A. serpentina (Wiedemann), and one from genus Ceratitis, C. capitata (Wiedemann) were compared based on puparium morphology and application of the Heteroduplex Mobility Assay (HMA). Puparia were characterized for the first time using the spiracular posterior plate morphology. Application of HMA allowed the detection of variability in the D2 domain from 28S rRNA gene in all four species (confirmed by sequencing). This is a fast, simple, sensitive and inexpensive assay that was used for the first time in the analysis of two Tephritoidea genera. The detected variability suggests that the tecnique has great potential for rapid determination of infestations with two or more species in the same host fruit.

Foram comparadas com base na morfologia do pupário e na técnica de HMA (Heteroduplex Mobility Assay)) quatro espécies de Tephritoidea, três delas pertencentes ao gênero Anastrepha, A. obliqua (Macquart), A. sororcula Zucchi e A. serpentina (Wiedemann) e uma do gênero Ceratitis, C. capitata (Wiedemann). Os pupários foram caracterizados pela primeira vez com base na morfologia da placa espiracular posterior. A aplicação da técnica de HMA possibilitou a detecção de variabilidade no segmento D2 do gene 28S rRNA nas quatro espécies (confirmada por seqüenciamento). Esta é uma técnica rápida, simples, sensível e barata que é aplicada pela primeira vez na análise de dois gêneros de Tephritoidea. A variabilidade observada sugere grande potencial da técnica no caso da determinação da infestação por duas ou mais espécies num mesmo fruto hospedeiro.

PCR products with heterozygous mutations containing two types of heteroduplexes / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To confirm that PCR products with heterozygous mutations contain not only wide-type and mutant homoduplexes, but also two types of heteroduplexes.</p><p><b>METHODS</b>An 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).</p><p><b>RESULTS</b>Two heteroduplexes and two homoduplexes in the PCR product from the heterozygous mutation of the exon 1 of KRT9 (497delAinsGGCT) were detected.</p><p><b>CONCLUSION</b>PCR 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.</p>

Molecular epidemiology of human immunodeficiency virus-1 in the state of Ceará, Northeast, Brazil

We analyzed, by env and gag heteroduplex mobility assay, 149 human immunodeficiency virus (HIV-1) positive samples collected in Ceará during the year 2000. The prevalence of subtype B was 81.2 percent and the prevalence of subtype F and B/F recombinants were both 2.7 percent. Eight (5.4 percent) and 12 (8 percent) out of 149 samples showed indeterminate results in the env and gag analysis respectively. By FokI restriction fragment length polymorphism, 34 percent of the subtype B samples were identified as the typical Brazilian subtype B.In the present study, we identified HIV-1 subtype F and B/F in Ceará for the first time. Our results contribute to the understanding of HIV in Brazil, and may prove useful for the development of vaccine candidates

Alteration of P53 protein expression and p53 gene in laryngeal carcinoma / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To study the prevalence of P53 protein expression and p53 gene mutation in laryngeal carcinoma.</p><p><b>METHODS</b>Using 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.</p><p><b>RESULTS</b>Among 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.</p><p><b>CONCLUSION</b>P53 gene mutation is related with TNM grading and cervical lymph node metastasis in laryngeal carcinoma. P53 mutation tents to be correlated to pathologic grading.</p>

Study on exons 3 and 4 of alpha-synuclein gene in Chinese familial Parkinson disease patients / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the relationship of Chinese familial Parkinson disease with alpha-synuclein gene.</p><p><b>METHODS</b>Polymerase 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.</p><p><b>RESULTS</b>No 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.</p><p><b>CONCLUSION</b>(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.</p>

Rapid detection of genotypes of TT virus using a heteroduplex mobility assay / 中华流行病学杂志

<p><b>OBJECTIVE</b>To establish a simple, sensitive, specific and less-costly method for detecting genotypes of TT virus (TTV).</p><p><b>METHODS</b>TTV 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>HMA was recognized as simple, sensitive, specific and less-costly, thus could be used for genotyping of TTV.</p>