New mutations, hotspots, and founder effects in Brazilian patients with steroid 5alpha-reductase deficiency type 2.

Mutations of the steroid 5alpha-reductase type 2 (SRD5A2) gene in 46,XY subjects cause masculinization defects of varying degrees, due to reduced or impaired enzymatic activity. In this study, sequence abnormalities of the SRD5A2 gene were assessed by polymerase chain reaction with specific primers and automated sequencing analysis in DNA samples from 20 patients with suspected steroid 5alpha-reductase type 2 deficiency from 18 Brazilian families. Eleven subjects presented SRD5A2 homozygous single-base mutations (two first cousins and four unrelated patients with G183S, two with R246W, one with del642T, one with G196S, and one with 217_218insC plus the A49T variant in heterozygosis), whereas four were compound heterozygotes (one with Q126R/IVS3+1G>A, one with Q126R/del418T, and two brothers with Q126R/G158R). Three patients were heterozygous for A207D, G196S, and R266W substitutions. The V89L polymorphism was found in heterozygosis in one of them (with A207D) and in one case with an otherwise normal gene sequence. The A49T variant was also detected in heterozygosis in the second case without other sequencing abnormalities. Four patients harbor yet non-described SRD5A2 gene mutations: a single nucleotide deletion (del642T), a G158R amino acid substitution, a splice junction mutation (IVS3+1G>A), and the insertion of a cytosine (217_218insC) occurring at a CCCC motif. This is the first report of a single-nucleotide insertion in the coding sequence of the SRD5A2 gene. In addition to these new mutations, this investigation reveals the prevalence of G183S substitution among a subset of African-Brazilian patients and presents evidences of the recurrence of already known mutations.

Large-scale transcriptome analyses reveal new genetic marker candidates of head, neck, and thyroid cancer.

A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.

Genes envolvidos na determinação e diferenciação do sexo / Genes involved in sex determination and differentiation

O sexo cromossômico é estabelecido na fertilização pela presença de um cromossomo X ou Y. O desenvolvimento dos sexos masculino e feminino passa, num primeiro momento, pela especialização das gônadas em testículos ou ovários; os demais processos decorrem de efeitos secundários provocados pelos hormônios por elas produzidos. As etapas de determinação e diferenciação das gônadas em testículos ou em ovários e a diferenciação dos genitais externos masculinos ou femininos envolvem a expressão específica de uma cascata de genes. Esses genes, seus respectivos padrões de expressão, bem como seus envolvimentos na manifestação de patologias ligadas ao desenvolvimento gonadal e dos genitais externos serão abordados nesta revisão.

Deficiência de 5alfa-redutase tipo 2: experiências de Campinas (SP) e Salvador (BA) / 5alpha-reductase type 2 deficiency: experiences from Campinas (SP) and Salvador (BA)

OBJETIVO: Apresentar a experiência relativa a pacientes com deficiência da enzima 5alfa-redutase tipo 2 provenientes de três serviços distintos no Brasil. CASUíSTICA E MÉTODOS: Foram incluídos 25 pacientes com sinais clínicos e hormonais de deficiência da 5alfa-redutase 2 pertencentes a 23 famílias, 15 oriundas da Bahia, 7 de São Paulo e 1 de Minas Gerais. Foram avaliados dados clínicos, hormonais e moleculares. A análise molecular dos 5 éxons do gene SRD5A2 foi feita por meio da técnica de PCR, seguida de seqüenciamento automático ou manual. RESULTADOS: Em 10 famílias havia mutações no gene SRD5A2 em homozigose (5 com G183S, 2 com R246W, 1 com G196S, 1 com del642T, 1 com 217_218insC) e em 3 em heterozigose composta (1 com Q126R/IVS3+1G>A, 1 com Q126R/del418T e 1 com Q126R/G158R); em 3 casos os afetados eram heterozigotos, apresentando apenas uma mutação deletéria (1 com G196S, 1 com A207D e 1 com R246W). Em 7 casos não foram detectadas anormalidades ao seqüenciamento. Observou-se maior freqüência da G183S em pacientes miscigenados (Afro-Euro-Brasileiros) oriundos da Bahia. Os achados clínicos e hormonais não diferiram entre os casos com e sem mutação, à exceção da freqüência de consangüinidade e da maior gravidade da ambigüidade genital nos primeiros. CONCLUSÕES: Os resultados encontrados salientam a importância da investigação molecular para o diagnóstico dessa doença, ressaltando o achado de uma mutação bastante freqüente em nosso meio (G183S), especialmente em pacientes miscigenados oriundos da Bahia, e a descrição de mutações que até o momento só foram relatadas em pacientes brasileiros.

[Genes involved in sex determination and differentiation]. / Genes envolvidos na determinação e diferenciação do sexo.

Chromosomal sex is established at fertilization by the presence of an X or Y chromosome. The first step of male and female development is gonadal specialization in testes or ovaries; all other processes that follow result from secondary effects produced by testis and ovary hormones. Gonadal determination and differentiation and the development of external genitalia involve time- and tissue-specific expression of genes forming a gene cascade. Those genes, their expression profile and their role in the pathological manifestations related to gonadal and external genitalia development will be discussed in this review.

[5alpha-reductase type 2 deficiency: experiences from Campinas (SP) and Salvador (BA)]. / Deficiência de 5alfa-redutase tipo 2: experiências de Campinas (SP) e Salvador (BA).

OBJECTIVE: To report the experience regarding patients with steroid 5alpha-reductase type 2 deficiency from three different clinical services in Brazil. CASUISTIC AND METHODS: Twenty five patients with clinical and hormonal features of 5alpha-reductase deficiency from 23 families (15 from Bahia, 7 from São Paulo and 1 from Minas Gerais) were included in this study. Clinical, hormonal and molecular data were evaluated. The molecular analysis of the five exons of the SRD5A2 gene was done by automatic or manual sequencing of PCR products. RESULTS: In ten families, SRD5A2 mutations were found in homozygosis (5 with G183S, 2 with R246W, 1 with G196S, 1 with del642T, 1 with 217_218insC), in three in compound heterozygosis (1 with Q126R/IVS3+1G>A, 1 with Q126R/del418T, 1 with Q126R/G158R) while other three were heterozygous, with only one deleterious mutation (1 with G196S, 1 with A207D, and 1 with R246W). In seven cases, no sequencing abnormalities were detected. The G183S substitution was the most frequently found among miscegenated patients (Afro-Euro-Brazilians) from Bahia. Hormonal and clinical findings did not differ between patients with or without mutations, exception made to a higher frequency of consanguinity and greater severity of genital ambiguity in the first group. CONCLUSION: Our results reinforce the importance of molecular investigation for the diagnosis of this disease and point out to the finding of a very frequent mutation (G183S) in our series, especially in patients with mixed ethnic background from Bahia, and the description of mutations that have only been reported in Brazilian patients so far.

Diagnosis of 5alpha-reductase type 2 deficiency: contribution of anti-Müllerian hormone evaluation.

AIM: To evaluate anti-Müllerian hormone (AMH) levels in patients with clinical and molecular diagnosis of 5alpha-reductase 2 deficiency. PATIENTS AND METHODS: Data from 14 patients whose age ranged from 21 days to 29 years were analyzed according to age and pubertal stage. Sexual ambiguity was rated as Prader III in 11 patients. LH, FSH, testosterone (T), dihydrotestosterone (DHT) and AMH serum levels were measured in all but two patients, who had been previously submitted to gonadectomy; T and DHT were also measured in 20 age-matched controls. RESULTS: Gonadotropin levels were normal in all but one patient who retained gonads (six of whom had reached puberty) and T/DHT ratio was elevated in all patients when compared to controls. All prepubertal patients had AMH levels < -1 SD for age, while most pubertal patients had AMH levels compatible with pubertal stage. CONCLUSIONS: Prepubertal patients with 5alpha-reductase 2 deficiency have AMH values in the lower part of the normal range. These data indicate that T does not need to be converted to DHT to inhibit AMH secretion by Sertoli cells.

Molecular analysis of KAL-1, GnRH-R, NELF and EBF2 genes in a series of Kallmann syndrome and normosmic hypogonadotropic hypogonadism patients.

We report the results of molecular analysis in a series of twelve Kallmann syndrome (KS) and five normosmic hypogonadotropic hypogonadism (nHH) Brazilian patients. Kallman syndrome 1 (KAL-1) gene analysis was performed in all patients and the gonadotrophin releasing hormone receptor (GnRH-R) gene was investigated in nHH patients using PCR analysis with exon-flanking primers followed by automated sequencing techniques. Two-point mutations at the KAL-1 locus were found in two KS patients. One case exhibited a novel C deletion (del1956C) in exon 12 leading to a premature stop codon at position 617. The second case, a C to T transition at exon 5, showed a stop codon at aminoacid 191 (Arg191X). Renal agenesis and bimanual synkinesis, which are frequently found in patients with the KAL-1 mutation, were observed in these cases. Among the KS patients, two previously reported cases had intragenic deletions of exons 5-10, while a third patient had a KAL-1 gene microdeletion detected by fluorescence in situ hybridization. For the nHH patients, no abnormalities were observed at the exonic and flanking sequences of the KAL-1 or GnRH-R genes. Nasal embryonic LHRH factor (NELF) and early B-cell factor 2 (EBF2) exons were evaluated in KAL-1/GnRH-R mutation-negative cases (seven KS and five nHH) by sequence analysis but no mutations were identified in the coding regions in these patients. In conclusion, this report includes the description of a novel point mutation of the KAL-1 gene and suggests that the KAL-1 mutations and deletions might be more prevalent in KS Brazilian patients than previously described in other series. NELF and EBF2 genes have been considered good candidates for HH and a large number of patients need to be studied to assess their contribution to reproductive function.

A transcript finishing initiative for closing gaps in the human transcriptome.

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms.

Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences.

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.

Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.

Similar interstitial deletions of the KAL-1 gene in two Brazilian families with X-linked Kallmann Syndrome

Mutations in the KAL-1 gene localized at Xp22.3 have been shown to be responsible for the X-linked Kallmann syndrome (KS), a disorder characterized by the association of hypogonadotropic hypogonadism and anosmia. In this paper, we describe the investigation of two families with X-linked KS, in which similar interstitial deletions ning exons 5 to 10 of the KAL-1 gene were identified. The presence of interspersed repetitive DNA sequences within the KAL-1 gene might have predisposed to this type of mutation.

[Chromosome instability induced by agrochemicals among farm workers in Passo Fundo, Rio Grande do Sul, Brazil]. / Instabilidade cromossômica induzida por agroquímicos em trabalhadores rurais na região de Passo Fundo, Rio Grande do Sul, Brasil.

A major share of the grain farming (wheat and soybeans) in the State of Rio Grande do Sul is in the Passo Fundo area. For crop pest control, large amounts of agrochemicals (fungicides, insecticides, and herbicides) are used. To evaluate the genotoxicity of these products, the micronucleus test was performed in farm workers directly exposed to these chemicals. Heparinized blood samples were drawn by venipuncture from 30 exposed workers and 30 non-exposed controls. Micronuclei frequency was evaluated by counting 1,000 binucleated cells per individual in both groups. Smoking habits, age, and duration of exposure showed no effect on the frequency of micronuclei in both groups. However, statistical analysis showed significantly higher mean numbers of binucleated cells with micronuclei in exposed individuals (14.3/1,000 cells) as compared to controls (7.1/1,000 cells), allowing the authors to conclude that the micronucleus test is an efficient biological assay for monitoring population exposure to mixtures of agrochemicals.

Instabilidade cromossômica induzida por agroquímicos em trabalhadores rurais na regiäo de Passo Fundo, Rio Grande do Sul, Brasil / Chromosome instability induced by agrochemicals among farm workers in Passo Fundo, Rio Grande do Sul, Brazil

A regiäo de Passo Fundo no Planalto Médio do Rio Grande do Sul, caracteriza-se pela produçäo de gräos (trigo, soja), nas quais grandes quantidades de agroquímicos (fungicidas, inseticidas e herbicidas) säo utilizadas. Para avaliar a atividade genotóxica desses produtos em seres humanos, utilizou-se a técnica de micronúcleos, através de amostras de sangue periférico de trinta trabalhadores expostos e de trinta indivíduos controles näo expostos. A freqüência de micronúcleos foi avaliada em 1.000 células binucleadas por indivíduo em ambos os grupos. Fatores como tabagismo, idade e tempo de exposiçäo näo exerceram qualquer efeito sobre a freqüência de micronúcleos em ambos os grupos. No entanto, a análise estatística revelou números significativamente mais elevados de micronúcleos em expostos (14,3/1.000 células) do que em näo expostos (7,1/1.000 células), indicando que o teste do micronúcleo é um ensaio biológico eficiente para monitorar populaçöes expostas a misturas de agroquímicos

Polymorphisms in the DNA repair gene XRCC1 and susceptibility to alcoholic liver cirrhosis in older Southeastern Brazilians.

The population of Southeastern Brazil has a very high mortality rate from liver cirrhosis, a disease that is considered an irreversible pre-malignant condition. This is largely due to the high prevalence of alcohol abuse in the region. Chronic alcohol consumption is associated with the production of free radical intermediates that can cause several DNA lesions. Reduced repair of these DNA lesions would, therefore, constitute a significant risk factor for liver cirrhosis and subsequent cancer. Recently, a number of polymorphisms in several DNA repair genes have been discovered, and it is possible that these polymorphisms may affect DNA repair capacity and thus modulate susceptibility to the disease. In this study, we tested the hypothesis that polymorphisms in the DNA repair gene XRCC1 are associated with increased risk of liver cirrhosis in Southeastern Brazilians. We conducted a pilot case-control study of 97 liver cirrhosis cases and 96 controls (matched for age, sex, and ethnicity) to investigate the role of two allelic variants coding for amino acid changes in the XRCC1 gene (the Arg194Trp and the Arg399Gln polymorphisms). Overall, we observed a 1.8-fold increase in the relative risk of liver cirrhosis associated with the 399Gln allele (either the heterozygous Arg/Gln or the homozygous Gln/Gln genotypes). The adjusted odds ratio (OR) was 1.82 (95% confidence limit (CL) 1.10-3.30). The relative risk appears to be highest among the Mestiso ethnic group (OR 2.60, 95% CL 0.92-7.34). There was a significant association between the 399Gln polymorphism and the risk of liver cirrhosis in older individuals over the age of 45 years (OR 2.70 (95% CL 1.14-6.48) compared to an OR of 1.24 (95% CL 0.55-2.78) for those under 45 years of age. No association was observed between the XRCC1 194Trp polymorphism and risk of liver cirrhosis. These preliminary results suggest that the XRCC1 399Gln polymorphism may be a significant risk modifier for alcoholic liver cirrhosis and justifies additional studies in that direction.

Cytogenetic analysis and detection of KAL-1 gene deletion with fluorescence in situ hybridization (FISH) in patients with Kalmann syndrome

Kallmann syndrome (KS) is a disease clinically characterized by the association of hypogonadotropic hypogonadism and anosmia or hyposmia, for which three modes of transmission have been described: X-linked, autosomal recessive and autosomal dominant. The KAL-1 gene, responsible for the X-linked form of the disease, has been isolated and its intron-exon organization determined. In this study, two families with X-linked KS and four sporadic male patients with hypogonadotropic hypogonadism and anosmia were cytogenetically investigated with high-resolution techniques and FISH. Chro-mosomal analysis did not reveal any rearrangements or deletions. Deletion of the KAL-1 gene was detected by FISH in only one sporadic patient with the typical features of KS and a high palate. Among the familial cases renal abnormalities and pes cavus deformity were observed.

Interphase cytogenetics using fluorescence in situ hybridization: an overview of its application to diffuse and solid tissue

Técnicas para marcaçäo de hibridaçäo in situ por fluorescência (FISH) têm sido amplamente utilizadas para análise citogenética em células interfásicas de tecidos sólidos e difusos. Na maioria dos casos, os estudos com FISH têm sido desenvolvidos em células naturalmente isoladas, como as células sangüíneas ou de medula óssea. Mais raramente, a análise citogenética tem sido desenvolvida em fluidos corporais como líquido amniótico, urina, esperma ou escarro. As amostras de tecidos sólidos, na maioria dos casos, säo submetidas à desagregaçäo mecânica ou química anteriormente à aplicaçäo de FISH. Contudo, FISH também tem sido aplicada a amostras de tecido com arquitetura conservada, tais como os "imprints" ou cortes histológicos finos, principalmente em estudos com tumores sólidos. Este artigo exemplifica vantagens e limitaçöes da técnica, ilustra o uso de FISH para análise citogenética em células interfásicas de diferentes tipos e destaca alguns resultados interessantes obtidos com a metodologia.

Bloom syndrome in a Brazilian child

Os autores descrevem o caso de um menino com quadro clínico característico da síndrome de Bloom e uma alta freqüência quadrirradiais, quebras cromossômicas e outras aberraçöes cromossômicas estruturais

Partial trisomy 10q in a child born to a woman with a reciprocal translocation t (7;10) (p22;q24): case report and review of the literature

Descreve-se uma dupl(10) (q24 - qter) em uma menina cuja mäe possui uma translocaçäo equilibrada t(7;10) (p22;q24)> Säo ainda revisados 46 casos de trissomia parcial 10q com respeito a caracteres clínicos e citogenéticos