Mônica Barbosa de Melo

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Increased risk for acute myeloid leukaemia in individuals with glutathione S-transferase mu 1 (GSTM1) and theta 1 (GSTT1) gene defects

Abstract: Objectives: Glutathione S‐transferases (GST) modulate the effects of exposure to various cytotoxic and genotoxic agents, including those associated with increased risks of the myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML) and aplastic anemia (AA). Both the GST mu 1 (GSTM1) and GST theta 1 (GSTT1) genes have a null variant allele in which the entire gene is absent. In this study, we tested whether null genotypes for the GSTM1 and GSTT1 genes altered the risks for MDS, AML and AA. Methods: Genomic DNA from 49 MDS, 38 AML and 37 AA patients and 276 controls was analysed using the polymerase chain reaction (PCR). Results: The frequencies of GSTM1 (73.6%) and GSTT1 (34.2%) null genotypes were significantly higher in AML patients than in the controls (36.9 and 18.1%, respectively). A higher frequency of the combined null genotype for both genes was also observed in patients with AML (26.3% compared with 5.0% in the controls). In contrast, no differences in the frequencies of the null genotypes were found among MDS patients, AA patients and the controls. Conclusion: Our observation of a 4.7‐fold (95% CI: 2.1–11.0) and 2.3‐fold (95% CI: 1.0–5.2) increased risk associated with the GSTM1 and GSTT1 null genotypes, respectively, and a 6.6‐fold (95% CI: 2.4–7.9) increased risk associated with the combined null genotype presents preliminary evidence that the inherited absence of this carcinogen detoxification pathway may be an important determinant of AML.

N-ras Gene Point Mutations in Brazilian Acute Myelogenous Leukemia Patients Correlate with a Poor Prognosis

The frequency of ras gene mutations varies from 11 to 27% in AML populations from the United States and Europe but it seems that there is no study regarding the frequency of mutated N-ras gene in patients with AML in South America. In order to study the frequency of N-ras gene mutations (exons 1 and 2) in Brazilian patients with AML and to evaluate the possible correlation between the presence of the mutation and clinical features, 40 patients were analyzed. N-ras mutations were identified in DNA samples from eight of 40 AML patients (20%). No significant correlation was found between N-ras mutation and age, sex, race, response to therapy, FAB subtype or occupational exposure. However, the overall survival and AML-free survival were significantly shorter in patients with N-ras mutations than in those without these abnormalities.

Novel mutation in the MYOC gene in primary open angle glaucoma patients

Editor—Glaucoma is the worlds leading cause of irreversible blindness1 and is characterised by progressive optic disc cupping with corresponding visual field loss. Both intraocular pressure (IOP) and positive family history are risk factors for the development of the disease.2 Juvenile open angle glaucoma (JOAG) is a subtype of open angle glaucoma characterised by an early onset (10 to 35 years of age) and autosomal dominant inheritance with high penetrance,3 a characteristic which has led several authors to investigate affected families in an attempt to identify a gene or genes associated with this condition.4-10 With the use of genetic linkage analysis in families with JOAG, a genetic locus (GLC1A) was recognised on chromosome 1q21-q31.4 The gene associated with GLC1A has been identified and it codifies a 57 kDa protein named trabecular meshwork induced glucocorticoid response protein (TIGR),10also known as myocilin (MYOC).11 The MYOC gene is composed of three exons of 604, 126, and 785 bp, respectively.12 During screening for mutations in the MYOC gene in 25 unrelated Brazilian patients with JOAG, an unreported mutation (Cys433Arg) was detected, present in seven of them. Patients were followed at the Glaucoma Service of the State University of Campinas, Brazil. They underwent an ocular examination, including gonioscopy by Posner lens, applanation tonometry, slit lamp biomicroscopy, optic nerve evaluation, and automated perimetry (Humphrey 630, program 30-2). JOAG was defined as the presence of characteristic bilateral optic nerve damage and visual field loss in the presence of an open angle in subjects younger than 36 years of age. Each patient included in this study came from different families according to interview data. The study was approved by the Ethics Committee of the State University of Campinas. At the time of the ocular examination, the mean age …

Keeping an eye on myocilin: a complex molecule associated with primary open-angle glaucoma susceptibility.

MYOC encodes a secretary glycoprotein of 504 amino acids named myocilin. MYOC is the first gene to be linked to juvenile open-angle glaucoma (JOAG) and some forms of adult-onset primary open-angle glaucoma (POAG). The gene was identified as an up-regulated molecule in cultured trabecular meshwork (TM) cells after treatment with dexamethasone and was originally referred to as trabecular meshwork-inducible glucocorticoid response (TIGR). Elevated intraocular pressure (IOP), due to decreased aqueous outflow, is the strongest known risk factor for POAG. Increasing evidence showed that the modulation of the wild-type (wt) myocilin protein expression is not causative of glaucoma while some misfolded and self-assembly aggregates of mutated myocilin may be associated with POAG in related or unrelated populations. The etiology of the disease remains unclear. Consequently, a better understanding of the molecular mechanisms underlyingPOAG is required to obtain early diagnosis, avoid potential disease progression, and develop new therapeutic strategies. In the present study, we review and discuss the most relevant studies regarding structural characterizations, expressions, molecular interactions, putative functions of MYOC gene and/or its corresponding protein in POAG etiology.

Mutations in the p53 Gene in Acute Myeloid Leukemia Patients Correlate with Poor Prognosis

Inactivation of tumor suppressor genes, whose products exert an inhibitory influence on cell cycle progression, can lead to neoplastic transformation. In acute myeloid leukemia (AML), the frequency of p53 gene mutations ranges from 4 to 15% in populations from USA and Europe. In an attempt to investigate the frequency of point mutations in the p53 gene in AML Brazilian patients, DNA samples of 35 patients were studied using PCR-SSCP techniques, screening exons 4–10. Mutations were identified in bone marrow DNA in 5 of the 35 AML patients (14.3%), a frequency similar to those reported for Northern American and European populations. The overall survival of patients with mutations in the p53 gene was significantly shorter than for patients without mutations.

CYP1B1 Gene Analysis in Primary Congenital Glaucoma Brazilian Patients Novel Mutations and Association With Poor Prognosis

PurposeTo determine the spectrum of CYP1B1 gene mutations in Brazilian patients with primary congenital glaucoma, and to correlate the presence of alterations in the CYP1B1 gene sequence with clinical aspects of the disease. Materials and MethodsThirty nonrelated patients with primary congenital glaucoma were studied. Molecular analysis consisted of the codifying region sequencing (exons 2 and 3) and intron/exon boundaries. ResultsCYP1B1 gene mutations were present in 9 (30%) of the 30 patients. The structural changes in the CYP1B1 gene previously described in the literature and observed in our study were Q19X, P437L, A443G, g.4340delG, g.7901_79013delGAGTGCAGGCAGA, g.8182delG, and g.8214_8215delG. Three new mutations were observed: 4635delT, 4523delC, and L378Q, in addition to 3793T→C, R48G, A119S, L432V, D449D, and N453S polymorphisms. Patients carrying CYP1B1 gene mutations needed more surgical procedures to control intraocular pressure, either when both eyes were evaluated (P=0.003) or when the worst eye of the patient was analyzed (P=0.011). In relation to the number of affected eyes, all patients with mutations (n=9/9) developed bilateral glaucoma, whereas 11/21 patients without mutations in the CYP1B1 gene had bilateral glaucoma (P=0.013). ConclusionsIn this group of primary congenital glaucoma patients, a 30% mutation frequency in the CYP1B1 gene was observed. The presence of mutations was associated with a more severe form of the disease, requiring more surgeries for intraocular pressure control and with a higher rate of bilateral cases.

Lack of association between optineurin gene variants T34T, E50K, M98K, 691_692insAG and R545Q and primary open angle glaucoma in Brazilian patients.

Purpose: To verify the frequencies of T34T, E50K, M98K, 691_692insAG, and R545Q variants in the optineurin (OPTN) gene in Brazilian subjects with primary open-angle glaucoma (POAG) and controls. Patients and Methods: Ninety-nine patients with POAG and 100 normal controls were enrolled in this study. The frequency of alterations in the OPTN gene was analyzed by direct sequencing and enzymatic digestion of PCR products. Results: None of the five alterations evaluated was significantly associated with POAG when compared to controls. However, the T34T silent change was present in greater frequency in POAG patients (37.37% vs. 23.00% in controls), while the R545Q change was more prevalent in controls (23.00% vs. 10.10% in POAG). The M98K and 691_692insAG presented with low frequencies in POAG patients (1.01% and 2.02%, respectively) and controls (2.00% and 2.00%, respectively). The E50K substitution was not observed. Conclusion: Our data show no association between the five evaluated variants and POAG in the Brazilian population.

Graves’ Disease in Brazilian Children and Adults: Lack of Genetic Association with CTLA-4 +49A>G Polymorphism

Background/Aim: In several populations, major histocompatibility complex and CTLA-4 (cytotoxic T lymphocyte antigen-4) gene polymorphisms are related to adult subjects with Graves’ disease (GD). Our aim was to study the association of +49A>G polymorphism of the CTLA-4 gene in Brazilian children and adults with GD and its correlation with clinical and laboratory markers of disease severity. Methods: CTLA-4 +49A>G polymorphism was established by polymerase chain reaction-restriction fragment length polymorphism analysis in 44 children and 72 adults with GD and compared to a stringent control group consisting of octogenarians with no history of thyroid disease; free T4 and T3 levels and T3/T4 ratio, antithyroid antibodies, and Graves’ ophthalmopathy were also evaluated according to genotype. Results: No significant difference was found in the frequency of CTLA-4 +49A>G polymorphism among children and adults with GD compared to controls and within groups. There was no significant correlation between the presence of G allele and Graves’ ophthalmopathy, gender, age at diagnosis, and biochemical markers of disease severity. Conclusion: The frequency of CTLA-4 +49A>G polymorphism is not different in children and adults with GD compared to the normal control population and does not seem to contribute independently to the severity of the clinical presentation of GD.

New mutation in the myocilin gene segregates with juvenile-onset open-angle glaucoma in a Brazilian family.

Mutations in the myocilin gene (MYOC) account for most cases of autosomal dominant juvenile-onset open-angle glaucoma (JOAG), an earlier and more severe form of POAG. We accessed seven members of a Brazilian JOAG family by clinical and molecular investigation. Four out of seven family members were diagnosed with JOAG. All of these patients presented high intraocular pressure and two of them were bilaterally blind. The disease onset varied from 20 to 30years old. There was a nine-year-old family member who had not yet manifested the disease, although he was also a carrier of the mutation. Ophthalmologic examination included: evaluation of the visual field and optic disc, intraocular pressure measurement, and gonioscopy. The three exons and intron/exon junctions of the MYOC gene were screened for mutations through direct sequencing of PCR-amplified DNA fragments. Mutation screening revealed an in-frame mutation in the third exon of the MYOC gene: an insertion of six nucleotides between the cDNA positions 1187 and 1188 (c.1187_1188insCCCAGA, p.D395_E396insDP). This mutation presented an autosomal dominant pattern of inheritance, segregating with the disease in four family members for three generations, and it was absent in 60 normal controls. We also performed a computational structure modeling of olfactomedin-like domain of myocilin protein and conducted in silico analysis to predict the structural changes in the myocilin protein due to the presence of the mutation. These findings may be important for future diagnosis of other presymptomatic family members, as well as for the increase of the panel of MYOC mutations and their effects on phenotype.