RESUMO
INTRODUCTION: Mutations in the glucokinase gene (GCK) produce a subtype of Maturity onset diabetes in the young (MODY), named MODY 2. To date over than 190 different mutations have been identified, distributed over the coding regions and the exon-intron boundaries of the gene. The aim of this work was to study the nature and frequency of mutations in the GCK gene, in a MODY clinically characterized Argentinean population. MATERIAL AND METHODS: Seventy unrelated individuals were selected based on MODY clinical features. The study methodology consisted in PCR amplification of the coding regions of the GCK gene, SSCP electrophoresis analysis of the amplified fragments and direct sequencing of the fragments with abnormal electrophoresis pattern. RESULTS: We identified a total of six patients with mutations in the GCK gene. This included two novel mutations: g.1831C>A, g.3792T>A, one already reported by our group, g.168fsdelC (same mutation in two non-related patients) and two already reported: p.Gln138Pro and p.Gly261Glu. With that data, we could establish the prevalence of MODY 2 among the patients in study reaching to 8.6%. DISCUSSION: The main contribution of this study is to inform about two novel mutations not described to date and to make an approach to the establishment of the prevalence of MODY 2 in the population under study. These findings contribute to confirm the allelic heterogeneity of GCK gene mutations and may provide an insight into the structure-function relationship of the GCK.
Assuntos
Diabetes Mellitus Tipo 2/genética , Testes Genéticos , Glucoquinase/genética , Adulto , Argentina , Glicemia/genética , Análise Mutacional de DNA , Humanos , Mutação , Linhagem , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
La diabetes autoinmune es una enfermedad multifactorial causada por factores genéticos predisponentes y ambientales desencadenantes. Se manifiesta en la edad infantojuvenil (diabetes tipo 1, DMID) y en la edad adulta (diabetes autoinmune latente del adulto, LADA). La predisposición genética es de tipo poligénico, se ha establecido asociación con alelos polimórficos del gen DQB del sistema HLA, VNTR del gen de insulina y polimorfismos en el gen CTLA4. En el presente trabajo se analizaron las frecuencias de los alelos polimórficos del gen HLA DQB1 en 63 pacientes LADA, 70 pacientes DMID y 79 individuos normales. La tipificación de los alelos del gen DQB1 se llevó a cabo mediante el Kit SSPTM DQ Olerup. Se observó una mayor frecuencia del genotipo *0201-*0302 y *0201-*0201 en ambas poblaciones diabéticas con respecto a normales (p<0.05). La presencia del genotipo *0201-*0302 fue mayor en DMID que en LADA (p<0.05). Por otra parte, el análisis del alelo protector *0602 muestra una alta prevalencia en individuos normales con respecto a la población diabética. El alelo de susceptibilidad más frecuente en pacientes LADA y DMID de nuestro país fue el *0201. En conclusión, LADA presenta susceptibilidad genética dada por alelos del gen HLA DQB1 pero en forma menos determinante que en diabetes tipo 1. A su vez, el hallazgo del aumento en la frecuencia del alelo *0201, tanto en frecuencias alélicas como genotípicas permite caracterizar nuestra población de pacientes tanto LADA como DMID a diferencia de otras poblaciones en las que el alelo más frecuente es el *0302. (AU)
Assuntos
Adulto , Humanos , RESEARCH SUPPORT, NON-U.S. GOVT , Antígenos HLA-DQ/genética , Genótipo , Doenças Autoimunes/genética , Diabetes Mellitus Tipo 1/genética , Polimorfismo Genético/genética , Frequência do Gene/genética , Estudos de Casos e Controles , Razão de Chances , Idade de Início , ArgentinaRESUMO
La diabetes autoinmune es una enfermedad multifactorial causada por factores genéticos predisponentes y ambientales desencadenantes. Se manifiesta en la edad infantojuvenil (diabetes tipo 1, DMID) y en la edad adulta (diabetes autoinmune latente del adulto, LADA). La predisposición genética es de tipo poligénico, se ha establecido asociación con alelos polimórficos del gen DQB del sistema HLA, VNTR del gen de insulina y polimorfismos en el gen CTLA4. En el presente trabajo se analizaron las frecuencias de los alelos polimórficos del gen HLA DQB1 en 63 pacientes LADA, 70 pacientes DMID y 79 individuos normales. La tipificación de los alelos del gen DQB1 se llevó a cabo mediante el Kit SSPTM DQ Olerup. Se observó una mayor frecuencia del genotipo *0201-*0302 y *0201-*0201 en ambas poblaciones diabéticas con respecto a normales (p<0.05). La presencia del genotipo *0201-*0302 fue mayor en DMID que en LADA (p<0.05). Por otra parte, el análisis del alelo protector *0602 muestra una alta prevalencia en individuos normales con respecto a la población diabética. El alelo de susceptibilidad más frecuente en pacientes LADA y DMID de nuestro país fue el *0201. En conclusión, LADA presenta susceptibilidad genética dada por alelos del gen HLA DQB1 pero en forma menos determinante que en diabetes tipo 1. A su vez, el hallazgo del aumento en la frecuencia del alelo *0201, tanto en frecuencias alélicas como genotípicas permite caracterizar nuestra población de pacientes tanto LADA como DMID a diferencia de otras poblaciones en las que el alelo más frecuente es el *0302.
Assuntos
Adulto , Humanos , Doenças Autoimunes/genética , Diabetes Mellitus Tipo 1/genética , Genótipo , Frequência do Gene/genética , Antígenos HLA-DQ/genética , Polimorfismo Genético/genética , Idade de Início , Argentina , Estudos de Casos e Controles , Razão de ChancesRESUMO
La diabetes tipo MODY es una forma de diabetes tipo 2 que se presenta en pacientes menores de 25 años y con una forma de herencia autosómica dominante. Hasta el presente, las alteraciones genéticas descriptas en este tipo de pacientes determinan hiposecreción de insulina. De acuerdo a estas alteraciones,la diabetes tipo MODY se clasifica en MODY 1, que presenta mutaciones en el gen del factor nuclear hepático 4 alfa; MODY 2, con mutaciones en el gen de la enzima glucoquinasa; MODY 3, con mutaciones en el factor nuclear hepático 1a; y MODY 4, con mutaciones en el gen del factor promotor de insulina 1 y el gen del factor nuclear hepático 1ß...(AU)
Assuntos
Humanos , Diabetes Mellitus Tipo 2 , Biologia MolecularRESUMO
La diabetes tipo MODY es una forma de diabetes tipo 2 que se presenta en pacientes menores de 25 años y con una forma de herencia autosómica dominante. Hasta el presente, las alteraciones genéticas descriptas en este tipo de pacientes determinan hiposecreción de insulina. De acuerdo a estas alteraciones,la diabetes tipo MODY se clasifica en MODY 1, que presenta mutaciones en el gen del factor nuclear hepático 4 alfa; MODY 2, con mutaciones en el gen de la enzima glucoquinasa; MODY 3, con mutaciones en el factor nuclear hepático 1a; y MODY 4, con mutaciones en el gen del factor promotor de insulina 1 y el gen del factor nuclear hepático 1ß...
Assuntos
Humanos , Diabetes Mellitus Tipo 2 , Biologia MolecularRESUMO
We have previously reported a Brazilian family with congenital goiter, hypothyroidism, and marked impairment of thyroglobulin (Tg) synthesis. Analysis of the Tg mRNA in the goiter of one of the siblings revealed a cytosine to thymine transition creating a stop codon at position 1510. This point mutation is removed from the majority of Tg mRNA transcripts by the preferential generation in the goiter of a 171 nt deleted Tg mRNA by alternative splicing. The nonsense mutation destroys a TaqI site at this position in the mutant Tg gene. Using polymerase chain reaction (PCR) amplification and TaqI digestion we found that two siblings affected with goiter and hypothyroidism, as well as the father and three siblings with normal thyroid function, are all heterozygous for the nonsense mutation. This implies that an additional mutation must be present in the affected individuals, generating a compound heterozygote genotype. A new polymorphism within the thyroglobulin gene represented by three alleles has been detected. This was documented by the TaqI restriction enzyme and phTgM3 probe hybridization that showed a three allelic polymorphism with fragment sizes of 16.5 kb (allele A), 14.5 kb (allele B) and 11.0 kb (allele C). Segregation analysis of these alleles in the family indicated that the two affected siblings were homozygous for the allele C. In contrast the unaffected father and three other siblings, who carried the nonsense mutation, were heterozygous for alleles B and C. Analysis of the Tg genotypes implies that two additional mutations of the Tg gene must segregate in this family to account for the observed phenotypes.
Assuntos
Bócio/genética , Hipotireoidismo/genética , Mutação/fisiologia , Tireoglobulina/genética , Adulto , Alelos , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Brasil , Códon sem Sentido/genética , Hipotireoidismo Congênito , DNA/análise , DNA/genética , Feminino , Frequência do Gene , Genoma Humano , Bócio/congênito , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da PolimeraseRESUMO
1. Hereditary goiter and the various degrees of thyroid hypofunction are the result of structural changes in the thyroglobulin (Tg) or thyroperoxidase (TPO) proteins, the inability to couple iodotyrosines or defective iodination, impairing or substantially altering the synthesis of T4 and T3. 2. The first mutations in the Tg and TPO genes responsible for human cases of dyshormonogenesis have been described. The mutation in two siblings with hereditary goiter and marked impairment of Tg synthesis was a cytosine to thymine transition creating a stop codon at position 1510. The point mutation is removed by the preferential accumulation of a 171-nt deleted Tg mRNA. In another subject, molecular studies revealed that exon 4 was missing from the major Tg transcript due to a cytosine to guanine transversion at position minus 3 in the acceptor splice site of intron 3. 3. Genomic DNA studies identified a duplication of a 4-base sequence in the eighth exon of the TPO gene. Interestingly, besides abolishing the enzymatic activity by disrupting the reading frame of the messenger RNA and introducing stop codons, the GGCC duplication also unmasks a cryptic acceptor splice site in exon 9. 4. In conclusion, the identification of different molecular defects provided evidence that hereditary goiter associated with abnormal Tg or TPO synthesis is caused by heterogeneous genetic alterations.
Assuntos
Bócio/genética , Peroxidase/genética , Tireoglobulina/genética , Sequência de Aminoácidos , Sequência de Bases , Regulação da Expressão Gênica , Bócio/enzimologia , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Tireoglobulina/biossínteseRESUMO
1. Hereditary goiter and the various degrees of thyroid hypofunction are the result of structural changes in the thyroglobulin (Tg) or thyroperoxidase (TPO) proteins, the inability to couple iodotyrosines or defective iodination, impairing or substantially altering the synthesis of T4 and T3. 2. The first nmutations in the Tg and TPO genes responsable for human cases of dys-hormonogenesis have been described. The mutation in two siblings with hereditary goiter and marked impairment of Tg synthesis was a cytosine to thymine transition creating a stop codon at postion 1510. The point mutation is removed by the preferential accumulation of a 171-nt deleted Tg mRNA. In another subject, molecular studies revealed that exon 4 was missing from the major Tg transcript due to a cytosine to guanine transversion at postion minus 3 in the acceptor splice site of intron 3. 3. Genomic DNA studies identified a duplication of a 4-base sequence in the eight exon of the TPO gene. Interestingly, besides abolishing the enzymatic activity by disrupting the reading frame of the messenger RNA and introducing stop codons, the GGCC duplication also unmasks a cryptic acceptor splice site in exon 9. 4. In conclusion, the identification of different molecular defects provied evidence that hereditary goiter associated with abnormal Tg or TPO synthesis is caused by heterogeneous genetic alterations
Assuntos
Humanos , Bócio/genética , Técnicas In Vitro , Peroxidase/genética , Tireoglobulina/genética , Distribuição por Idade , Sequência de Aminoácidos , Regulação da Expressão Gênica , Bócio/enzimologia , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Tireoglobulina/biossínteseRESUMO
Thyroid tissue total RNAs from multinodular goiter (G2) and from hereditary goiter with defective Tg synthesis (JNA) were hybridized with a 5'albumin cDNA probe (F-47), a 3' albumin cDNA probe (B-44) and a thyroglobulin cDNA probe (phTgM3). JNA refers to tissue obtained from a patient with virtual absence of Tg in thyroid tissue and the presence of increased concentration of an albumin-like labeled protein in the thyroid. No hybridization signal was detected in both G2 and JNA with albumin probes at Northern Blot studies. Those results were confirmed by dot-blot analysis of total RNA where no hybridization signal was detected in G2 and JNA. To confirm that thyroid tissues do not express thyroalbumin total RNA from JNA and normal control thyroid tissue (C) were amplified by PCR using albumin and Tg primers. An expected fragment of 592 bp was observed in a human liver sample with the albumin primers. However JNA and C samples showed absence of an amplification product of the same size. We concluded that thyroid cells do not contain the albumin transcript. Albumin is probably taken up from circulation and iodinated by the thyroid follicular cell with subsequent release of iodoalbumin into the circulation.
