Introdução: A homocistinúria clássica é um erro inato do metabolismo causado pela atividade deficiente da cistationina β-sintase (CβS). É caracterizada pela elevação sérica de homocisteína e metionina e redução dos níveis de cisteína. As manifestações clínicas clássicas envolvem os sistemas ocular, vascular, nervoso central e ósseo. Entretanto, observações recentes sugerem que alterações na composição corporal, especialmente a redução de tecido adiposo, também pode ser uma manifestação frequente e clinicamente relevante nesta doença. Além disso, a gravidade do fenótipo clínico parece ser influenciada por outros genes além do CβS. O objetivo deste estudo foi investigar alterações na composição corporal e detectar mutações comuns nos genes CβS, MTHFR e F5 em pacientes com homocistinúria clássica; relacionando dados clínicos, bioquímicos e genéticos. Métodos: Realizada avaliação da composição corporal em 8 pacientes através de duas técnicas: antropometria (aferição das dobras cutâneas triciptal, biciptal, subescapular e suprailíaca) e bioimpedância elétrica (BIA). A partir dos resultados obtidos, calculou-se o percentual de gordura pelas fórmulas de Siri (antropometria) e Kushner (BIA). O índice de massa corporal (IMC) também foi calculado. Os níveis de metionina, homocisteína e cisteína foram mensurados por HPLC. A avaliação genotípica foi realizada em 11 pacientes através de PCR-RFLP. Pesquisadas as mutações p.I278T, p.T191M, p.G307S e c.844ins68 no gene CβS; c.677TC>T e c.1298A>C no gene MTHFR; e p.R506Q no gene F5 (Fator V de Leiden). Resultados: Cinco pacientes apresentaram baixo percentual de gordura por pelo menos um dos métodos; o restante apresentou percentual médio. De acordo com o IMC, apenas dois pacientes foram classificados em baixo peso. O percentual de gordura total não se relacionou com os achados bioquímicos ou ósseos, mas medida isolada da dobra cutânea triciptal correlacionou-se positivamente com os níveis de cisteína (p=0,03) Além disso, os níveis de homocisteína e metionina correlacionaram-se negativamente com o IMC, enquanto que a cisteína teve associação positiva (p<0.05). Houve tendência do escore T de fêmur associar-se negativamente com homocisteína e positivamente com cisteína (p=0,09). Entre as mutações do gene CβS, a p.1278T foi a única encontrada (n= 3/9 famílias); entretanto, em 2/3 famílias estava em cis com a c.844ins68. Assim, em apenas um caso tinha caráter patogênico. Não foi possível estabelecer associação entre mutações em MTHFR e F5 e a gravidade do fenótipo. Conclusões: Os resultados sugerem que deficiência de CβS gera alterações significativas na composição corporal – observou-se redução do percentual de gordura independentemente do IMC. Este pode ser um mecanismo patogênico importante envolvido na etiologia da 2 osteoporose na homocistinúria clássica, mas estudos adicionais são necessários para demonstrar esta relação. Os achados sugerem que as mutações patogênicas no gene CBS sejam raras ou privadas nesta amostra, e não corroboram a ação dos genes MTHFR e F5 como modificadores da doença. / Introduction: Classical homocystinuria is an inborn error of metabolism caused by the deficient activity of cystathionine β-synthase (CβS). It is characterized by plasma increase in homocysteine and methionine and a decrease in cysteine levels. Its classical clinical manifestations involve the following systems: ocular, vascular, central nervous, and skeletal. However, recent observations have suggested that changes in the body composition, especially a reduction of the adipose tissue, may also be a frequent manifestation of the disease and clinically relevant to it. Moreover, the severity of its clinical phenotype may be influenced by other genes besides CβS. The objective of the present study was to investigate alterations in the body composition and to detect common mutations in CβS, MTHFR and F5 genes in patients with classical homocystinuria and to correlate them to the clinical, biochemical, and genetic data. Methods: Body composition was assessed in 8 patients using the techniques of anthropometry (measurement of the following skinfolds: triciptal, biciptal, subscapular, and suprailiac) and electrical bioimpedance (BIA). Based on the results obtained, the percentage of body fat was calculated using the formulas of Siri (anthropometry) and Kushner (BIA). The body mass index (BMI) was also calculated. The levels of methionine, homocysteine, and cysteine were measured by HPLC. The genotypic evaluation was done in 11 patients by PCR-RFLP. The following mutations were investigated: p.I278T, p.T191M, p.G307S, and c.844ins68 in the CβS gene; c.677TC>T and c.1298A>C in the MTHFR gene; and p.R506Q in the F5 gene (Factor V Leiden). Results: Five patients presented low percentage of body fat by at least one of the methods; the other patients presented a medium percentage. According to the BMI, only 2 patients were classified as being underweight. The percentage of total body fat was not related to either bone or biochemical findings, but the isolate measurement of the triciptal skinfold was positively correlated to the levels of cysteine (p=0.03). Also, the levels of homocysteine and methionine were negatively correlated to the BMI, while cysteine had a positive association (p<0.05). The T score of the femur tended to associate negatively with homocysteine and positively with cysteine (p=0.09). The genetic evaluation revealed only one pathogenic mutation in CβS (p.I278T). An association between mutations in MTHFR and F5 and phenotype severity could not be established. Conclusions: The present results suggest that the deficiency of CβS causes significant alterations in the body composition; a reduction of the percentage of body fat was observed independently from the BMI. This may be an important pathogenic mechanism involved in the etiology of osteoporosis in classical homocystinuria. The present findings suggest that in this sample the 4 pathogenic mutations in the CBS gene are rare or private and do not corroborate with the action of the MTHFR and F5 genes as disease modifiers. Further studies are needed.

Folate deficiency, a prevalent vitamin deficiency in America, can stem from environmental and/or genetic causes. The most common inborn error of folate metabolism is deficiency of methylenetetrahydrofolate reductase (MTHFR), which catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Severe MTHFR deficiency results in hyperhomocysteinemia and homocystinuria; patients present with developmental delay, and various neurological and vascular disorders. This thesis describes three mutations identified in the MTHFR locus in patients with severe deficiency: 1025T&rarr;C (M&rarr;T), 1027T&rarr;G (W&rarr;G), and 1768G&rarr;A (E&rarr;K). Genotype-phenotype correlations are described, along with biochemical characterization of three mutations (983A&rarr;G (N&rarr;S), 1025T&rarr;C, 1027T&rarr;G). All three mutations exert their effect by decreasing Vmax without changing the enzyme's affinity for its substrate, 5-methyltetrahydrofolate. The 983A&rarr;G variant also conferred decreased affinity for FAD, a cofactor. / The more common and mild deficiency observed in the general healthy population is probably due in part to insufficient dietary intake of folate. Folate deficiency has been associated with increased risk for colon cancer. In a pilot study presented here, the impact of altered folate intake on tumor multiplicity in the Min mouse, a model for multiple intestinal neoplasia, was assessed. Folate deficient diets did not produce a consistent change in tumor numbers. However, a linear correlation between S-adenosylmethionine and S-adenosylhomocysteine content of preneoplastic tissue and tumor multiplicity was identified. / This thesis contributes to our understanding of the impact of genetic- and/or dietary-induced folate deficiency on cellular and organismal functions.

Introdução: A homocistinúria clássica é um erro inato do metabolismo causado pela atividade deficiente da cistationina β-sintase (CβS). É caracterizada pela elevação sérica de homocisteína e metionina e redução dos níveis de cisteína. As manifestações clínicas clássicas envolvem os sistemas ocular, vascular, nervoso central e ósseo. Entretanto, observações recentes sugerem que alterações na composição corporal, especialmente a redução de tecido adiposo, também pode ser uma manifestação frequente e clinicamente relevante nesta doença. Além disso, a gravidade do fenótipo clínico parece ser influenciada por outros genes além do CβS. O objetivo deste estudo foi investigar alterações na composição corporal e detectar mutações comuns nos genes CβS, MTHFR e F5 em pacientes com homocistinúria clássica; relacionando dados clínicos, bioquímicos e genéticos. Métodos: Realizada avaliação da composição corporal em 8 pacientes através de duas técnicas: antropometria (aferição das dobras cutâneas triciptal, biciptal, subescapular e suprailíaca) e bioimpedância elétrica (BIA). A partir dos resultados obtidos, calculou-se o percentual de gordura pelas fórmulas de Siri (antropometria) e Kushner (BIA). O índice de massa corporal (IMC) também foi calculado. Os níveis de metionina, homocisteína e cisteína foram mensurados por HPLC. A avaliação genotípica foi realizada em 11 pacientes através de PCR-RFLP. Pesquisadas as mutações p.I278T, p.T191M, p.G307S e c.844ins68 no gene CβS; c.677TC>T e c.1298A>C no gene MTHFR; e p.R506Q no gene F5 (Fator V de Leiden). Resultados: Cinco pacientes apresentaram baixo percentual de gordura por pelo menos um dos métodos; o restante apresentou percentual médio. De acordo com o IMC, apenas dois pacientes foram classificados em baixo peso. O percentual de gordura total não se relacionou com os achados bioquímicos ou ósseos, mas medida isolada da dobra cutânea triciptal correlacionou-se positivamente com os níveis de cisteína (p=0,03) Além disso, os níveis de homocisteína e metionina correlacionaram-se negativamente com o IMC, enquanto que a cisteína teve associação positiva (p<0.05). Houve tendência do escore T de fêmur associar-se negativamente com homocisteína e positivamente com cisteína (p=0,09). Entre as mutações do gene CβS, a p.1278T foi a única encontrada (n= 3/9 famílias); entretanto, em 2/3 famílias estava em cis com a c.844ins68. Assim, em apenas um caso tinha caráter patogênico. Não foi possível estabelecer associação entre mutações em MTHFR e F5 e a gravidade do fenótipo. Conclusões: Os resultados sugerem que deficiência de CβS gera alterações significativas na composição corporal – observou-se redução do percentual de gordura independentemente do IMC. Este pode ser um mecanismo patogênico importante envolvido na etiologia da 2 osteoporose na homocistinúria clássica, mas estudos adicionais são necessários para demonstrar esta relação. Os achados sugerem que as mutações patogênicas no gene CBS sejam raras ou privadas nesta amostra, e não corroboram a ação dos genes MTHFR e F5 como modificadores da doença. / Introduction: Classical homocystinuria is an inborn error of metabolism caused by the deficient activity of cystathionine β-synthase (CβS). It is characterized by plasma increase in homocysteine and methionine and a decrease in cysteine levels. Its classical clinical manifestations involve the following systems: ocular, vascular, central nervous, and skeletal. However, recent observations have suggested that changes in the body composition, especially a reduction of the adipose tissue, may also be a frequent manifestation of the disease and clinically relevant to it. Moreover, the severity of its clinical phenotype may be influenced by other genes besides CβS. The objective of the present study was to investigate alterations in the body composition and to detect common mutations in CβS, MTHFR and F5 genes in patients with classical homocystinuria and to correlate them to the clinical, biochemical, and genetic data. Methods: Body composition was assessed in 8 patients using the techniques of anthropometry (measurement of the following skinfolds: triciptal, biciptal, subscapular, and suprailiac) and electrical bioimpedance (BIA). Based on the results obtained, the percentage of body fat was calculated using the formulas of Siri (anthropometry) and Kushner (BIA). The body mass index (BMI) was also calculated. The levels of methionine, homocysteine, and cysteine were measured by HPLC. The genotypic evaluation was done in 11 patients by PCR-RFLP. The following mutations were investigated: p.I278T, p.T191M, p.G307S, and c.844ins68 in the CβS gene; c.677TC>T and c.1298A>C in the MTHFR gene; and p.R506Q in the F5 gene (Factor V Leiden). Results: Five patients presented low percentage of body fat by at least one of the methods; the other patients presented a medium percentage. According to the BMI, only 2 patients were classified as being underweight. The percentage of total body fat was not related to either bone or biochemical findings, but the isolate measurement of the triciptal skinfold was positively correlated to the levels of cysteine (p=0.03). Also, the levels of homocysteine and methionine were negatively correlated to the BMI, while cysteine had a positive association (p<0.05). The T score of the femur tended to associate negatively with homocysteine and positively with cysteine (p=0.09). The genetic evaluation revealed only one pathogenic mutation in CβS (p.I278T). An association between mutations in MTHFR and F5 and phenotype severity could not be established. Conclusions: The present results suggest that the deficiency of CβS causes significant alterations in the body composition; a reduction of the percentage of body fat was observed independently from the BMI. This may be an important pathogenic mechanism involved in the etiology of osteoporosis in classical homocystinuria. The present findings suggest that in this sample the 4 pathogenic mutations in the CBS gene are rare or private and do not corroborate with the action of the MTHFR and F5 genes as disease modifiers. Further studies are needed.

Introdução: A homocistinúria clássica é um erro inato do metabolismo causado pela atividade deficiente da cistationina β-sintase (CβS). É caracterizada pela elevação sérica de homocisteína e metionina e redução dos níveis de cisteína. As manifestações clínicas clássicas envolvem os sistemas ocular, vascular, nervoso central e ósseo. Entretanto, observações recentes sugerem que alterações na composição corporal, especialmente a redução de tecido adiposo, também pode ser uma manifestação frequente e clinicamente relevante nesta doença. Além disso, a gravidade do fenótipo clínico parece ser influenciada por outros genes além do CβS. O objetivo deste estudo foi investigar alterações na composição corporal e detectar mutações comuns nos genes CβS, MTHFR e F5 em pacientes com homocistinúria clássica; relacionando dados clínicos, bioquímicos e genéticos. Métodos: Realizada avaliação da composição corporal em 8 pacientes através de duas técnicas: antropometria (aferição das dobras cutâneas triciptal, biciptal, subescapular e suprailíaca) e bioimpedância elétrica (BIA). A partir dos resultados obtidos, calculou-se o percentual de gordura pelas fórmulas de Siri (antropometria) e Kushner (BIA). O índice de massa corporal (IMC) também foi calculado. Os níveis de metionina, homocisteína e cisteína foram mensurados por HPLC. A avaliação genotípica foi realizada em 11 pacientes através de PCR-RFLP. Pesquisadas as mutações p.I278T, p.T191M, p.G307S e c.844ins68 no gene CβS; c.677TC>T e c.1298A>C no gene MTHFR; e p.R506Q no gene F5 (Fator V de Leiden). Resultados: Cinco pacientes apresentaram baixo percentual de gordura por pelo menos um dos métodos; o restante apresentou percentual médio. De acordo com o IMC, apenas dois pacientes foram classificados em baixo peso. O percentual de gordura total não se relacionou com os achados bioquímicos ou ósseos, mas medida isolada da dobra cutânea triciptal correlacionou-se positivamente com os níveis de cisteína (p=0,03) Além disso, os níveis de homocisteína e metionina correlacionaram-se negativamente com o IMC, enquanto que a cisteína teve associação positiva (p<0.05). Houve tendência do escore T de fêmur associar-se negativamente com homocisteína e positivamente com cisteína (p=0,09). Entre as mutações do gene CβS, a p.1278T foi a única encontrada (n= 3/9 famílias); entretanto, em 2/3 famílias estava em cis com a c.844ins68. Assim, em apenas um caso tinha caráter patogênico. Não foi possível estabelecer associação entre mutações em MTHFR e F5 e a gravidade do fenótipo. Conclusões: Os resultados sugerem que deficiência de CβS gera alterações significativas na composição corporal – observou-se redução do percentual de gordura independentemente do IMC. Este pode ser um mecanismo patogênico importante envolvido na etiologia da 2 osteoporose na homocistinúria clássica, mas estudos adicionais são necessários para demonstrar esta relação. Os achados sugerem que as mutações patogênicas no gene CBS sejam raras ou privadas nesta amostra, e não corroboram a ação dos genes MTHFR e F5 como modificadores da doença. / Introduction: Classical homocystinuria is an inborn error of metabolism caused by the deficient activity of cystathionine β-synthase (CβS). It is characterized by plasma increase in homocysteine and methionine and a decrease in cysteine levels. Its classical clinical manifestations involve the following systems: ocular, vascular, central nervous, and skeletal. However, recent observations have suggested that changes in the body composition, especially a reduction of the adipose tissue, may also be a frequent manifestation of the disease and clinically relevant to it. Moreover, the severity of its clinical phenotype may be influenced by other genes besides CβS. The objective of the present study was to investigate alterations in the body composition and to detect common mutations in CβS, MTHFR and F5 genes in patients with classical homocystinuria and to correlate them to the clinical, biochemical, and genetic data. Methods: Body composition was assessed in 8 patients using the techniques of anthropometry (measurement of the following skinfolds: triciptal, biciptal, subscapular, and suprailiac) and electrical bioimpedance (BIA). Based on the results obtained, the percentage of body fat was calculated using the formulas of Siri (anthropometry) and Kushner (BIA). The body mass index (BMI) was also calculated. The levels of methionine, homocysteine, and cysteine were measured by HPLC. The genotypic evaluation was done in 11 patients by PCR-RFLP. The following mutations were investigated: p.I278T, p.T191M, p.G307S, and c.844ins68 in the CβS gene; c.677TC>T and c.1298A>C in the MTHFR gene; and p.R506Q in the F5 gene (Factor V Leiden). Results: Five patients presented low percentage of body fat by at least one of the methods; the other patients presented a medium percentage. According to the BMI, only 2 patients were classified as being underweight. The percentage of total body fat was not related to either bone or biochemical findings, but the isolate measurement of the triciptal skinfold was positively correlated to the levels of cysteine (p=0.03). Also, the levels of homocysteine and methionine were negatively correlated to the BMI, while cysteine had a positive association (p<0.05). The T score of the femur tended to associate negatively with homocysteine and positively with cysteine (p=0.09). The genetic evaluation revealed only one pathogenic mutation in CβS (p.I278T). An association between mutations in MTHFR and F5 and phenotype severity could not be established. Conclusions: The present results suggest that the deficiency of CβS causes significant alterations in the body composition; a reduction of the percentage of body fat was observed independently from the BMI. This may be an important pathogenic mechanism involved in the etiology of osteoporosis in classical homocystinuria. The present findings suggest that in this sample the 4 pathogenic mutations in the CBS gene are rare or private and do not corroborate with the action of the MTHFR and F5 genes as disease modifiers. Further studies are needed.

No description available.

Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...

Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...