Lack of mutations of exon 2 of the MEN1 gene in endocrine and nonendocrine sporadic tumors

In addition to the mutations that underlie most cases of the multiple endocrine neoplasia type 1 (MEN1) syndrome, somatic mutations of the MEN1 gene have also been described in sporadic tumors like gastrinomas, insulinomas and bronchial carcinoid neoplasm. We examined exon 2 of this gene, where most of the mutations have been described, in 148 endocrine and nonendocrine sporadic tumors. DNA was obtained by phenol/chloroform extraction and ethanol precipitation from 92 formalin-fixed, paraffin-embedded samples, and from 40 fresh tumor tissue samples. We used 5 pairs of primers to encompass the complete coding sequence of exon 2 of the MEN1 gene that was screened by the polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) technique in 78 sporadic thyroid cancers: 28 follicular adenomas, 35 papillary carcinomas, 14 follicular carcinomas, and 1 anaplastic thyroid carcinoma. We also examined 46 adrenal lesions (3 hyperplasias, 3 adenomas and 35 adrenocortical carcinomas, 2 pheochromocytomas, 2 ganglioneuroblastomas, and 1 lymphoma) and 24 breast cancers (6 noninvasive, 16 infiltrating ductal, and 2 invasive lobular tumors). The PCR product of 5 tumors suspected to present band shifts by SSCP was cloned. Direct sense and antisense sequencing did not identify mutations. These results suggest that the MEN1 gene is not important in breast, thyroid or adrenal sporadic tumorigenesis. Because the frequency of mutations varies significantly among tumor subgroups and allelic deletions are frequently observed at 11q13 in thyroid and adrenal cancers, another tumor suppressor gene residing in this region is likely to be involved in the tumorigenesis of these neoplasms.

Griscelli syndrome-type 2 in twin siblings: case report and update on RAB27A human mutations and gene structure

Griscelli syndrome (GS) is a rare autosomal recessive disorder caused by mutation in the MYO5A (GS1, Elejalde), RAB27A (GS2) or MLPH (GS3) genes. Typical features of all three subtypes of this disease include pigmentary dilution of the hair and skin and silvery-gray hair. Whereas the GS3 phenotype is restricted to the pigmentation dysfunction, GS1 patients also show primary neurological impairment and GS2 patients have severe immunological deficiencies that lead to recurrent infections and hemophagocytic syndrome. We report here the diagnosis of GS2 in 3-year-old twin siblings, with silvery-gray hair, immunodeficiency, hepatosplenomegaly and secondary severe neurological symptoms that culminated in multiple organ failure and death. Light microscopy examination of the hair showed large, irregular clumps of pigments characteristic of GS. A homozygous nonsense mutation, C-T transition (c.550C>T), in the coding region of the RAB27A gene, which leads to a premature stop codon and prediction of a truncated protein (R184X), was found. In patient mononuclear cells, RAB27A mRNA levels were the same as in cells from the parents, but no protein was detected. In addition to the case report, we also present an updated summary on the exon/intron organization of the human RAB27A gene, a literature review of GS2 cases, and a complete list of the human mutations currently reported in this gene. Finally, we propose a flow chart to guide the early diagnosis of the GS subtypes and Chédiak-Higashi syndrome.

Variants of transcription factor 7-like 2 (TCF7L2) gene and incident glucose intolerance in Japanese-Brazilians

Common variants of the transcription factor 7-like 2 (TCF7L2) gene have been found to be associated with type 2 diabetes in different ethnic groups. The Japanese-Brazilian population has one of the highest prevalence rates of diabetes. Therefore, the aim of the present study was to assess whether two single-nucleotide polymorphisms (SNPs) of TCF7L2, rs7903146 and rs12255372, could predict the development of glucose intolerance in Japanese-Brazilians. In a population-based 7-year prospective study, we genotyped 222 individuals (72 males and 150 females, aged 56.2 ± 10.5 years) with normal glucose tolerance at baseline. In the study population, we found that the minor allele frequency was 0.05 for SNP rs7903146 and 0.03 for SNP rs12255372. No significant allele or genotype association with glucose intolerance incidence was found for either SNP. Haplotypes were constructed with these two SNPs and three haplotypes were defined: CG (frequency: 0.94), TT (frequency = 0.027) and TG (frequency = 0.026). None of the haplotypes provided evidence for association with the incidence of glucose intolerance. Despite no associations between incidence of glucose intolerance and SNPs of the TCF7L2 gene in Japanese-Brazilians, we found that carriers of the CT genotype for rs7903146 had significantly lower insulin levels 2 h after a 75-g glucose load than carriers of the CC genotype. In conclusion, in Japanese-Brazilians, a population with a high prevalence of type 2 diabetes, common TCF7L2 variants did not make major contributions to the incidence of glucose tolerance abnormalities.

Association of rs12255372 in theTCF7L2 gene with type 2 diabetes mellitus: a meta-analysis

Our objective was to evaluate the association of rs12255372 in theTCF7L2 gene with type 2 diabetes mellitus (T2DM) in the world population. We carried out a survey of the literature about the effect of rs12255372 on genetic susceptibility to T2DM by consulting PubMed, the Cochrane Library, and Embase from 2006 to 2012, and then performed a meta-analysis of all the studies in order to evaluate the association between rs12255372 and T2DM. A total of 33 articles including 42 studies (with 34,076 cases and 36,192 controls) were confirmed to be eligible and were included in the final meta-analysis: 6 studies conducted on Europeans, 14 on Caucasians, 17 on Asians, 2 on Africans, and 3 on Americans. Overall, the effect size was as follows: for the variant allele T (OR = 1.387, 95%CI = 1.351-1.424), for the TT genotype (OR = 1.933, 95%CI = 1.815-2.057), for the GT genotype (OR = 1.363, 95%CI = 1.315-1.413), for the dominant model (OR = 1.425, 95%CI = 1.344-1.510), and for the recessive model (OR = 1.659, 95%CI = 1.563-1.761). In summary, by pooling all available qualified data from genetic studies on rs12255372 and T2DM, we have confirmed that rs12255372 is significantly associated with susceptibility to T2DM in the global population.

Genetic analysis of the ELOVL6 gene polymorphism associated with type 2 diabetes mellitus

Recent animal studies have indicated that overexpression of the elongation of long-chain fatty acids family member 6 (Elovl6) gene can cause insulin resistance and β-cell dysfunction. These are the major factors involved in the development of type 2 diabetes mellitus (T2DM). To identify the relationship between single nucleotide polymorphisms (SNP) ofELOVL6 and T2DM pathogenesis, we conducted a case-control study of 610 Han Chinese individuals (328 newly diagnosed T2DM and 282 healthy subjects). Insulin resistance and islet first-phase secretion function were evaluated by assessment of insulin resistance in a homeostasis model (HOMA-IR) and an arginine stimulation test. Three SNPs of the ELOVL6 gene were genotyped with polymerase chain reaction-restriction fragment length polymorphism, with DNA sequencing used to confirm the results. Only genotypes TT and CT of the ELOVL6 SNP rs12504538 were detected in the samples. Genotype CC was not observed. The T2DM group had a higher frequency of the C allele and the CT genotype than the control group. Subjects with the CT genotype had higher HOMA-IR values than those with the TT genotype. In addition, no statistical significance was observed between the genotype and allele frequencies of the control and T2DM groups for SNPs rs17041272 and rs6824447. The study indicated that the ELOVL6 gene polymorphism rs12504538 is associated with an increased risk of T2DM, because it causes an increase in insulin resistance.

Effects of 5-aza-2242;deoxycytidine on RECK gene expression and tumor invasion in salivary adenoid cystic carcinoma

Reversion-inducing cysteine-rich protein with kazal motifs (RECK), a novel tumor suppressor gene that negatively regulates matrix metalloproteinases (MMPs), is expressed in various normal human tissues but downregulated in several types of human tumors. The molecular mechanism for this downregulation and its biological significance in salivary adenoid cystic carcinoma (SACC) are unclear. In the present study, we investigated the effects of a DNA methyltransferase (DNMT) inhibitor, 5-aza-2242;deoxycytidine (5-aza-dC), on the methylation status of the RECK gene and tumor invasion in SACC cell lines. Methylation-specific PCR (MSP), Western blot analysis, and quantitative real-time PCR were used to investigate the methylation status of the RECK gene and expression of RECK mRNA and protein in SACC cell lines. The invasive ability of SACC cells was examined by the Transwell migration assay. Promoter methylation was only found in the ACC-M cell line. Treatment of ACC-M cells with 5-aza-dC partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression of mRNA and protein, and 5-aza-dC significantly suppressed ACC-M cell invasive ability. Our findings showed that 5-aza-dC inhibited cancer cell invasion through the reversal of RECKgene hypermethylation, which might be a promising chemotherapy approach in SACC treatment.

Triagem familiar para o gene HBB*S e detecção de novos casos de traço falciforme em Pernambuco

OBJETIVO: Estimar o incremento no número adicional de afetados com base na prevalência de síndromes falciformes em familiares de casos-índice. MÉTODOS: Estudo transversal em familiares de amostra aleatória dos casos-índice identificados por programa de triagem neonatal em Pernambuco, no período de 2001 a 2005. O modelo de triagem familiar ampliado incluiu 463 membros familiares de 21 casos-índice. Os familiares foram categorizados como: núcleo reduzido (NR -pai, mãe e irmãos); de primeiro grau (N1 - avós, tios e primos de primeiro grau); de segundo grau (N2 - filhos dos primos de primeiro grau); ampliado (NA - NR+N1+N2) e ampliado de primeiro grau (NA1 -NR+N1). A confirmação da presença de HBB*S e detecção de hemoglobinas anormais foram realizadas por meio da High Performance Liquid Chromathgraphy. A associação entre a presença de HBB*S e variáveis foi testada pelo cálculo da razão de prevalência e respectivos IC 95% e a diferença entre médias verificadas pelo teste t de Student, ao nível de significância de 5%. RESULTADOS: A anemia falciforme era desconhecida por 81% dos familiares; o gene HBB*S esteve presente em 114 familiares. Observou-se que 53,3% da população estudada estava na faixa considerada reprodutiva e 80% das pessoas portadoras do gene HBB*S já tinham gerado filhos. A freqüência foi maior no núcleo NR (69%), mas também elevada no N1 (22,8%). O NA1 resultou na detecção de 69 portadores adicionais (aumento de 172%). CONCLUSÕES: Os resultados indicam que a triagem familiar para identificação de portadores de síndrome falciforme deve ser estendida para os familiares até o primeiro grau.

Molecular characterization of Dengue viruses type 1 and 2 isolated from a concurrent human infection

In 2001, an autochthonous case of dual viremia, resulting from naturally acquired dengue virus DEN-1 and DEN-2 infections was detected during the dengue outbreak that occurred in Barretos, a city with about 105,000 inhabitants in the North region of São Paulo State. Serotype identification was based on virus isolation to C6/36 mosquito cells culture and immunofluorescence assays using type-specific monoclonal antibodies. The double infection was also confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Comparative analysis of the 240-nucleotide sequences of E/NS1 gene junction region between the genome of DEN-1 and DEN-2 isolates of the corresponding reference Nauru and PR 159S1 strains, respectively, showed some nucleotide differences, mainly silent mutations in the third codon position. Results of maximum likelihood phylogenetic analysis of E/NS1 gene sequences indicated that both genotypes of DEN-1 and DEN-2 viruses recovered from double infection in Barretos belonged to genotypes I and III, respectively.

Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

Digoxigenin-labeled probe for rabies virus nucleoprotein gene detection

A digoxigenin-labeled probe was produced from the Pasteur virus strain for the detection of the rabies virus N gene. The probe hybridization was performed from amplified N gene obtained by reverse transcription polymerase chain reaction and the results by RT-PCR and hybridization showed 100% agreement. The hybridization, when carried out in products amplified by RT-PCR, increases the sensitivity of this technique even more and confers specificity to the diagnosis. The technique described in this work will be useful in rabies diagnosis laboratories, once the cost is compatible with traditional rabies diagnostic techniques.

Nested-PCR do gene que codifica o antígeno b aplicada ao diagnóstico da tuberculose pulmonar

A reação em cadeia da polimerase usada para amplificação de uma seqüência interna de um fragmento previamente amplificado (nested-PCR) foi investigada como uma alternativa complementar a pesquisa de bacilos álcool ácido resistentes e a cultura do Mycobacterium tuberculosis em meio de Lowenstein-Jensen. Foram investigadas 144 amostras de escarro de pacientes suspeitos de tuberculose encaminhados ao Laboratório de Tuberculose do Instituto Evandro Chagas em Belém, no período de junho de 2002 a dezembro de 2003. Das 144 amostras, 121 foram caracterizadas como tuberculose, 119 foram positivas na cultura, 95 na baciloscopia e 128 na nested-PCR. A sensibilidade da nested-PCR foi 96% (116/121), enquanto a especificidade foi 48% (11/23). A nested-PCR poderá ser uma ferramenta complementar para o diagnóstico da tuberculose, pois apresenta sensibilidade equivalente à cultura, no entanto, necessita de maiores avaliações visando minimizar o número de resultados falso-positivos.

Distribution of QPY and RAH haplotypes of granzyme B gene in distinct Brazilian populations

INTRODUCTION: The cytolysis mediated by granules is one of the most important effector functions of cytotoxic T lymphocytes and natural killer cells. Recently, three single nucleotide polymorphisms (SNPs) were identified at exons 2, 3, and 5 of the granzyme B gene, resulting in a haplotype in which three amino acids of mature protein Q48P88Y245 are changed to R48A88H245, which leads to loss of cytotoxic activity of the protein. In this study, we evaluated the frequency of these polymorphisms in Brazilian populations. METHODS: We evaluated the frequency of these polymorphisms in Brazilian ethnic groups (white, Afro-Brazilian, and Asian) by sequencing these regions. RESULTS: The allelic and genotypic frequencies of SNP 2364A/G at exon 2 in Afro-Brazilian individuals (42.3% and 17.3%) were significantly higher when compared with those in whites and Asians (p < 0.0001 and p = 0.0007, respectively). The polymorphisms 2933C/G and 4243C/T also were more frequent in Afro-Brazilians but without any significant difference regarding the other groups. The Afro-Brazilian group presented greater diversity of haplotypes, and the RAH haplotype seemed to be more frequent in this group (25%), followed by the whites (20.7%) and by the Asians (11.9%), similar to the frequency presented in the literature. CONCLUSIONS: There is a higher frequency of polymorphisms in Afro-Brazilians, and the RAH haplotype was more frequent in these individuals. We believe that further studies should aim to investigate the correlation of this haplotype with diseases related to immunity mediated by cytotoxic lymphocytes, and if this correlation is confirmed, novel treatment strategies might be elaborated.

Impact of the IL-18 gene polymorphism in response to antiviral therapy in chronic HCV genotype 4 patients

^a Introduction Interleukin (IL)-18 is a well-known major proinflammatory cytokine with broad biological effects. The major immunomodulatory functions of IL-18 include enhancing T cell and natural killer cell cytotoxicity. Serum levels of this cytokine were shown to increase in chronic hepatitis C patients compared to non-infected healthy people. An association between IL-18 gene promoter polymorphisms and pegylated interferon (PEG-IFN) and ribavirin treatment outcomes has been reported for individuals with chronic hepatitis C virus genotype 1 (HCV-1). In this study, HCV genotype 4 (HCV-4) patients were assessed for IL-18 gene polymorphisms and treatment outcomes or severity of liver disease because data concerning the impact of IL-18 gene polymorphisms on patients with HCV-4 infections are limited. Methods This study included 123 chronic HCV-4 Egyptian patients and 123 apparently healthy volunteer blood donors who served as a control group. HCV genotyping was performed using the line probe assay. IL-18 genotyping was performed using the TaqMan Real-Time PCR method in all 246 patient and control samples. Results In our study, all patients had HCV-4. IL-18 gene single nucleotide polymorphism (SNP) (-607C/A) genotype distributions and allele frequencies did not differ between HCV patients and normal healthy subjects or between patient groups when compared according to the therapeutic response. Moreover, the presence of an IL-18 SNP was not associated with histological disease severity. We conclude that the presence of the IL-18 SNP rs1946518 does not affect the outcome of chronic HCV-4 treatment in Egyptian patients. Conclusions The IL-18 SNP rs1946518 does not affect response to treatment in chronic HCV-4 patients.

Toll-like receptor 3 gene polymorphisms are not associated with the risk of hepatitis B and hepatitis C virus infection

INTRODUCTION: The present study investigated the prevalence of two single-nucleotide polymorphisms (SNPs) in the Toll-like receptor 3 (TLR3) gene in patients infected with hepatitis B virus (HBV) and hepatitis C virus (HCV). METHODS: Samples collected from HCV (n = 74) and HBV (n = 35) carriers were subjected to quantitative real-time PCR (qPCR) to detect the presence of the SNPs rs5743305 and rs3775291 in TLR3 and to measure the following biomarkers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and prothrombin time (PT). A healthy control group was investigated and consisted of 299 HCV- and HBV-seronegative individuals. RESULTS: No significant differences in allele, genotype and haplotype frequencies were observed between the investigated groups, and no association was observed between the polymorphisms and histopathological results. Nevertheless, genotypes TA/AA (rs5743305) and GG (rs3775291) appear to be associated with higher levels of ALT (p<0.01), AST (p<0.05) and PT (p<0.05). In addition, genotypes TT (rs5743305; p<0.05) and GG (rs3775291; p<0.05) were associated with higher GGT levels. CONCLUSIONS: This genetic analysis revealed the absence of an association between the polymorphisms investigated and susceptibility to HBV and HCV infection; however, these polymorphisms might be associated with a greater degree of biliary damage during the course of HCV infection.

Use of nonradioactive labeling to detect large gene rearrangements in 21-hydroxylase deficiency

PURPOSE: To establish the Southern blotting technique using hybridization with a nonradioactive probe to detect large rearrangements of CYP21A2 in a Brazilian cohort with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH-21OH). METHOD: We studied 42 patients, 2 of them related, comprising 80 non-related alleles. DNA samples were obtained from peripheral blood, digested by restriction enzyme Taq I, submitted to Southern blotting and hybridized with biotin-labeled probes. RESULTS: This method was shown to be reliable with results similar to the radioactive-labeling method. We found CYP21A2 deletion (2.5%), large gene conversion (8.8%), CYP21AP deletion (3.8%), and CYP21A1P duplication (6.3%). These frequencies were similar to those found in our previous study in which a large number of cases were studied. Good hybridization patterns were achieved with a smaller amount of DNA (5 mug), and fragment signs were observed after 5 minutes to 1 hour of exposure. CONCLUSIONS: We established a non-radioactive (biotin) Southern blot/hybridization methodology for CYP21A2 large rearrangements with good results. Despite being more arduous, this technique is faster, requires a smaller amount of DNA, and most importantly, avoids problems with the use of radioactivity.