Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1. (C) 2007 Elsevier B.V. All fights reserved.

Mutational analysis of the GAP-related domain of the neurofibromatosis type 1 gene in Brazilian NF1 patients

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular analysis of the PROP1 and HESX1 genes in patients with septo-optic dysplasia and/or pituitary hormone deficiency

OBJETIVO: O presente estudo teve como objetivo avaliar os genes PROP1 e HESX1 em um grupo de pacientes com displasia septo-óptica (DSO) e deficiência hormonal hipofisária (combinada - DHHC; ou deficiência isolada de GH - DGH). Onze pacientes com apresentação clínica e bioquímica consistente com DHHC, DGH ou DSO foram avaliados. SUBJECTS and METHODS: em todos os pacientes, o gene HESX1 foi analisado pelo sequenciamento direto e, nos casos de DHHC, o gene PROP1 foi também sequenciado. RESULTADOS: Um polimorfismo no gene HESX1 (1772 A > G; N125S) foi identificado em um paciente com DSO. Foram encontrados três pacientes portadores da variação alélica 27 T > C; A9A e 59 A > G; N20S no éxon 1 do gene PROP1. Mutações no gene PROP1 e HESX1 não foram identificadas nesses pacientes com DGH, DHHC e DSO esporádicos. CONCLUSÃO: Alterações genéticas em um ou diversos outros genes ou mecanismos não genéticos devem estar implicados nesse processo patogênico.

Molecular analysis of the PROP1 and HESX1 genes in patients with septo-optic dysplasia and/ or pituitary hormone deficiency

Objective: The present study aimed at evaluating the PROP1 and HESX1 genes in a group of patients with septo-optic dysplasia (SOD) and pituitary hormone deficiency (combined – CPHD; isolated GH deficiency – GHD). Eleven patients with a clinical and biochemical presentation consistent with CPHD, GHD or SOD were evaluated. Subjects and methods: In all patients, the HESX1 gene was analyzed by direct sequence analysis and in cases of CPHD the PROP1 gene was also sequenced. Results: A polymorphism (1772 A > G; N125S) was identified in a patient with SOD. We found three patients carrying the allelic variants 27 T > C; A9A and 59 A > G; N20S in exon 1 of the PROP1 gene. Mutations in the PROP1 and HESX1 genes were not identified in these patients with sporadic GHD, CPHD and SOD. Conclusion: Genetic alterations in one or several other genes, or non-genetic mechanisms, must be implicated in the pathogenic process.

Sonic hedgehog (SHH) mutation in patients within the spectrum of holoprosencephaly

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gastric adenocarcinoma and Helicobacter pylori: Correlation with p53 mutation and p27 immunoexpression

Introduction: Helicobacter pylori infection is an established risk factor for gastric cancer development, but the exact underlying mechanism still remains obscure. The inactivation of tumor suppressor genes such as p53 and p27(KIP1) is a hypothesized mechanism, although there is no consensus regarding the influence of H. pylori cagA(+) in the development of these genetic alterations. Goals: To verify the relationship among H. pylori infection, p53 mutations and p27(Kip1) Protein (p27) expression in gastric adenocarcinomas (GA) seventy-four tissues were assayed by PCR for H. pylori and cagA presence. Mutational analysis of p53 gene was performed by single-strand conformation polymorphism (SSCP). Seventy tissues were analyzed by an immunohistochemical method for p27 expression. Results: From the samples examined, 95% (70/74) were H. pylori positive, 63% cagA(+). Altered p53 electrophoretic mobility was found in 72% of cases and significantly more frequent in the presence of cagA. Considerable reduction in p27 expression (19%) was found with a tendency for association between cagA(+) and p27(-), although the results were not statistically significant. Concomitant alterations of both suppressor genes were detected in 60% of cases. In the cases cagA(+), 66.7% of them had these concomitant alterations. Conclusions: The data suggest that H. pylori cagA(+) contributes to p53 alteration and indicate that concomitant gene inactivation, with reduced p27 expression, may be a mechanism in which H. pylori can promote the development and progression of gastric cancer. (C) 2010 Elsevier Ltd. All rights reserved.

The frequency of 844ins68 mutation in the cystathionine β-synthase gene is not increased in patients with venous thrombosis

Background and Objectives. A frequent mutation in the cystathionine β- synthase (CBS) gene (844ins68, a 68-bp insertion in the coding region of exon 8) was recently discovered. In the present study we investigated this mutation as a candidate risk factor for venous thrombosis. Design and Methods. The prevalence of the 844ins68 CBS mutation was determined in 101 patients with objectively diagnosed deep venous thrombosis and in 101 healthy controls matched for age, sex and race. PCR amplification of a DNA fragment containing exon 8 of the CBS gene was employed to determine the genotypes. Additionally, Bsrl restriction enzyme digestion of the PCR products was performed in all samples from carriers of the insertion, to test for concurrent presence of a second mutation (T833C) in the CBS gene. Results. The insertion was found in 21 out of 101 patients (20.8%; allele frequency 0.109) and in 20 out of 101 controls (19.8%; allele frequency 0.114), yielding a relative risk for venous thrombosis related to the 844ins68 CBS mutation close to 1.0. In addition, the T833C CBS mutation was detected in all alleles carrying the 844ins68 CBS insertion, confirming the co- inheritance of the two mutations. Interpretation and Conclusions. Our findings do not support the hypothesis that the 844ins68 mutation in the CBS gene is a genetic risk factor for venous thrombosis.

Molecular and Cytogenetic Analyses on Brazilian Youths with Pervasive Developmental Disorders

The Pervasive Developmental Disorders (PDDs) constitute a group of behavioral and neurobiological impairment conditions whose main features are delayed communicative and cognitive development. Genetic factors are reportedly associated with PDDs and particular genetic abnormalities are frequently found in specific diagnostic subgroups such as the autism spectrum disorders. This study evaluated cytogenetic and molecular parameters in 30 youths with autism or other PDDs. The fragile X syndrome was the most common genetic abnormality detected, presented by 1 patient with autism and 1 patient with PPD not-otherwise specified (PPD-NOS). One girl with PDD-NOS was found to have tetrasomy for the 15q11-q13 region, and one patient with autism exhibited in 2/100 metaphases an inv(7)(p15q36), thus suggesting a mosaicism 46,XX/46,XX,inv(7)(p15q36) or representing a coincidental finding. The high frequency of chromosomopathies support the hypothesis that PDDs may develop as a consequence to chromosomal abnormalities and justify the cytogenetic and molecular assessment in all patients with PDDs for establishment of diagnosis.

Utilization of different methodologies for the characterization of Hb Hasharon heterozygotes

Hb Hasharon has an electrophoretic mobility similar to that of Hb S in cellulose acetate and a mobility between Hb S and C at acid pH. In high-performance liquid chromatography, Hb Hasharon shows a distinct chromatographic profile and retention time. The origin of this variant is a mutation in codon 47 (GAC → CAC) of the α2-globin gene, resulting in the replacement of asparagine by histidine during the translation process. Ten blood samples from individuals suspected of being Hb Hasharon carriers were analyzed. In addition to classic laboratory tests and high-performance liquid chromatography, molecular analysis by polymerase chain reaction with restriction fragment length polymorphism designed in the laboratory was performed to confirm this mutation. The study of these cases showed that a combination of classical and molecular methodologies is necessary in the diagnosis of hemoglobinopathies for a correct hemoglobin mutant identification. The accurate identification of hemoglobin variants is essential for genetic counseling and choice of therapy. ©FUNPEC-RP.

Clinical and molecular phenotype of Aicardi-Goutières syndrome

Aicardi-Goutières syndrome (AGS) is a genetic encephalopathy whose clinical features mimic those of acquired in utero viral infection. AGS exhibits locus heterogeneity, with mutations identified in genes encoding the 3′→5′ exonuclease TREX1 and the three subunits of the RNASEH2 endonuclease complex. To define the molecular spectrum of AGS, we performed mutation screening in patients, from 127 pedigrees, with a clinical diagnosis of the disease. Biallelic mutations in TREX1, RNASEH2A, RNASEH2B, and RNASEH2C were observed in 31, 3, 47, and 18 families, respectively. In five families, we identified an RNASEH2A or RNASEH2B mutation on one allele only. In one child, the disease occurred because of a de novo heterozygous TREX1 mutation. In 22 families, no mutations were found. Null mutations were common in TREX1, although a specific missense mutation was observed frequently in patients from northern Europe. Almost all mutations in RNASEH2A, RNASEH2B, and RNASEH2C were missense. We identified an RNASEH2C founder mutation in 13 Pakistani families. We also collected clinical data from 123 mutation-positive patients. Two clinical presentations could be delineated: an early-onset neonatal form, highly reminiscent of congenital infection seen particularly with TREX1 mutations, and a later-onset presentation, sometimes occurring after several months of normal development and occasionally associated with remarkably preserved neurological function, most frequently due to RNASEH2B mutations. Mortality was correlated with genotype; 34.3% of patients with TREX1, RNASEH2A, and RNASEH2C mutations versus 8.0% RNASEH2B mutation-positive patients were known to have died (P = .001). Our analysis defines the phenotypic spectrum of AGS and suggests a coherent mutation-screening strategy in this heterogeneous disorder. Additionally, our data indicate that at least one further AGS-causing gene remains to be identified. © 2007 by The American Society of Human Genetics. All rights reserved.

Serum ferritin and transferrin saturation levels in β0 and β + thalassemia patients

There have been few studies on the mutations that cause heterozygous beta-thalassemia and how they affect the iron profile. One hundred and thirty-eight individuals were analyzed, 90 thalasemic β0 and 48 thalasemic β+, identified by classical and molecular methods. Mutations in the hemochromatosis (HFE) gene, detected using PCR-RFLP, were found in 30.4% of these beta-thalassemic patients; heterozygosity for H63D (20.3%) was the most frequent. Ferritin levels and transferrin saturation were similar in beta-thalassemics with and without mutations in the HFE gene. Ferritin concentrations were significantly higher in men and in individuals over 40 years of age. Transferrin saturation also was significantly higher in men, but only in those without HFE gene mutations. There was no significant difference in the iron profile among the β0 and β+ thalassemics, with and without HFE gene mutations. The frequency of ferritin values above 200 ng/mL in women and 300 ng/mL in men was also similar in β0 and β+ thalassemics (P > 0.72). Our conclusion is that ferritin levels are variable in the beta-thalassemia, trait regardless of the type of beta-globin mutation. Furthermore, HFE gene polymorphisms do not change the iron profile in these individuals. ©FUNPEC-RP www.funpecrp.com.br.

MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25. years, and lack of auto-antibodies. It accounts for 2-5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil. © 2012 Elsevier B.V.