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OBJECTIVE: Ketogenic diet is the first line therapy for neurological symptoms associated with pyruvate dehydrogenase deficiency (PDHD) and intractable seizures in a number of disorders, including GLUT1 deficiency syndrome (GLUT1-DS). Because high-fat diet raises serious compliance issues, we investigated if oral L,D-3-hydroxybutyrate administration could be as effective as ketogenic diet in PDHD and GLUT1-DS. METHODS: We designed a partial or total progressive substitution of KD with L,D-3-hydroxybutyrate in three GLUT1-DS and two PDHD patients. RESULTS: In GLUT1-DS patients, we observed clinical deterioration including increased frequency of seizures and myoclonus. In parallel, ketone bodies in CSF decreased after introducing 3-hydroxybutyrate. By contrast, two patients with PDHD showed clinical improvement as dystonic crises and fatigability decreased under basal metabolic conditions. In one of the two PDHD children, 3-hydroxybutyrate has largely replaced the ketogenic diet, with the latter that is mostly resumed only during febrile illness. Positive direct effects on energy metabolism in PDHD patients were suggested by negative correlation between ketonemia and lactatemia (r 2 = 0.59). Moreover, in cultured PDHc-deficient fibroblasts, the increase of CO2 production after 14C-labeled 3-hydroxybutyrate supplementation was consistent with improved Krebs cycle activity. However, except in one patient, ketonemia tended to be lower with 3-hydroxybutyrate administration compared to ketogenic diet. CONCLUSION: 3-hydroxybutyrate may be an adjuvant treatment to ketogenic diet in PDHD but not in GLUT1-DS under basal metabolic conditions. Nevertheless, ketogenic diet is still necessary in PDHD patients during febrile illness.
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BACKGROUND: Type I congenital disorders of glycosylation (CDG-I) are mostly complex multisystemic diseases associated with hypoglycosylated serum glycoproteins. A subgroup harbour mutations in genes necessary for the biosynthesis of the dolichol-linked oligosaccharide (DLO) precursor that is essential for protein N-glycosylation. Here, our objective was to identify the molecular origins of disease in such a CDG-Ix patient presenting with axial hypotonia, peripheral hypertonia, enlarged liver, micropenis, cryptorchidism and sensorineural deafness associated with hypo glycosylated serum glycoproteins. RESULTS: Targeted sequencing of DNA revealed a splice site mutation in intron 5 and a non-sense mutation in exon 4 of the dehydrodolichol diphosphate synthase gene (DHDDS). Skin biopsy fibroblasts derived from the patient revealed ~20 % residual DHDDS mRNA, ~35 % residual DHDDS activity, reduced dolichol-phosphate, truncated DLO and N-glycans, and an increased ratio of [2-(3)H]mannose labeled glycoprotein to [2-(3)H]mannose labeled DLO. Predicted truncated DHDDS transcripts did not complement rer2-deficient yeast. SiRNA-mediated down-regulation of DHDDS in human hepatocellular carcinoma HepG2 cells largely mirrored the biochemical phenotype of cells from the patient. The patient also harboured the homozygous ALG6(F304S) variant, which does not cause CDG but has been reported to be more frequent in PMM2-CDG patients with severe/fatal disease than in those with moderate presentations. WES did not reveal other strong candidate causal genes. CONCLUSIONS: We describe a patient presenting with severe multisystem disease associated with DHDDS deficiency. As retinitis pigmentosa is the only clinical sign in previously reported cases, this report broadens the spectrum of phenotypes associated with this condition.
Assuntos
Alquil e Aril Transferases/metabolismo , Defeitos Congênitos da Glicosilação/enzimologia , Cromatografia em Camada Fina , Defeitos Congênitos da Glicosilação/sangue , Defeitos Congênitos da Glicosilação/metabolismo , Dolicóis/análogos & derivados , Dolicóis/metabolismo , Éxons/genética , Glicoproteínas/sangue , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Células Hep G2 , Humanos , Recém-Nascido , Masculino , Mutação , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , RNA Interferente Pequeno/genética , Pele/metabolismoRESUMO
The facilitative glucose transporter-1 (GLUT1) deficiency or de Vivo syndrome is a rare neuropediatric disorder characterized by drug-resistant epilepsy, acquired microcephaly, delayed psychomotor development, intermittent ataxia, and other paroxysmal neurological disorders due to the presence of dominant mutations in the SLC2A1 gene. Alternating hemiplegia of childhood (AHC) is another rare neuropediatric disorder characterized by episodes of hemiplegia developing during the first 1.5 years of life. Before the recent finding of the gene ATP1A3 as the major cause of AHC, a heterozygous missense mutation in the SLC2A1 gene encoding GLUT1 was described in one child with atypical AHC, suggesting some clinical overlap between AHC and GLUT1 deficiency syndrome (GLUT1DS1). Half of patients with symptoms evocative of GLUT1DS1 with hypoglycorrhachia and up to 25 % of patients with AHC remain molecularly undiagnosed. We investigated whether mutations in SLC2A3 encoding GLUT3, another glucose transporter predominant in the neuronal cell, may account the case of a cohort of 75 SLC2A1 negative GLUTDS1-like patients and seven patients with AHC who were negative for ATP1A3 and SLC2A1 mutations. Automated Sanger sequencing and qPCR analyses failed to detect any mutation of SLC2A3 in the patients analyzed, excluding this gene as frequently mutated in patients with GLUT1DS1 like or AHC.
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Dystroglycanopathies are a heterogeneous group of muscular dystrophies with autosomal recessive inheritance characterized by abnormal glycosylation of alpha-dystroglycan. The most severe phenotypes are Walker-Warburg Syndrome (WWS) and muscle-eye-brain disease (MEB) presenting with lissencephaly type II (LIS II) and in which muscular dystrophy is associated with mental retardation and eye abnormalities. To date, six distinct genes, POMT1, POMT2, POMGNT1, FKTN, FKRP, LARGE and recently in one case DPM3, have been shown to be involved in dystroglycanopathies. Genomic sequencing alone is still frequently used for diagnosis purpose, not allowing detection of intragenic rearrangements at the heterozygous state contrarily to RNA analysis, quantitative PCR and CGH array analysis. These latter methods enabled us to identify four new intragenic rearrangements in the LARGE gene in three fetuses with WWS, born to two unrelated families: deletion of exons 9-10 and duplication of introns 1-4 for the first family and deletion of exons 4 and 7 for the second one; and a deletion of the last six exons of the POMGNT1 gene in two unrelated MEB patients. Genomic dosage studies using emerging tools such as CGH array should be included in routine molecular analysis of dystroglycanopathies, not only for the screening of the LARGE gene in which this kind of mutation seems to be more frequent than point mutations, but also for the other involved genes, especially in severe clinical cases.
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Duplicação Gênica/genética , N-Acetilglucosaminiltransferases/genética , Deleção de Sequência/genética , Síndrome de Walker-Warburg/genética , Criança , Consanguinidade , Análise Mutacional de DNA , Éxons/genética , Feto , Humanos , Masculino , Repetições de Microssatélites/genética , Fenótipo , Análise de Sequência de RNA , Síndrome de Walker-Warburg/fisiopatologiaRESUMO
BACKGROUND: In type I congenital disorders of glycosylation (CDG I), proteins necessary for the biosynthesis of the lipid-linked oligosaccharide (LLO) required for protein N-glycosylation are defective. A deficiency in guanosine diphosphate-mannose: GlcNAc(2)-PP-dolichol mannosyltransferase-1 (MT-1) causes CDG Ik (OMIM 608540), and only five patients, with severe multisystemic clinical presentations, have been described with this disease. Objective To characterise genetic, biochemical and clinical data in five new CDG Ik cases and compare these findings with those of the five previously described patients. Methods LLO biosynthesis was examined in skin biopsy fibroblasts, mannosyltransferases were assayed in microsomes prepared from these cells, and ALG1-encoding MT-1 was sequenced at the DNA and complementary DNA levels. Clinical data for the five new patients were collated. RESULTS: Cells from five patients with non-typed CDG I revealed accumulations of GlcNAc(2)-PP-dolichol, the second intermediate in the biosynthesis of LLO. Assay of MT-1, -2 and -3, the first three mannosyltransferases required for extension of this intermediate, demonstrated only MT-1 to be deficient. DNA sequencing of ALG1 revealed nine different mutations, seven of which have not been previously reported. Clinical presentations are severe, with dysmorphias, CNS involvement and ocular disturbances being prevalent. CONCLUSIONS: 5 patients with CDG Ik are described, and their identification reveals that in France, this disease and CDG Ib (mannose phosphate isomerase deficiency: OMIM 602579) are the most frequently diagnosed CDG I after CDG Ia (phosphomannomutase 2 deficiency: OMIM 601785) and substantiate previous observations indicating that this disease presents at the severe end of the CDG I clinical spectrum.
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Defeitos Congênitos da Glicosilação/genética , Manosiltransferases/genética , Mutação , Sequência de Bases , Células Cultivadas , Pré-Escolar , Defeitos Congênitos da Glicosilação/classificação , Análise Mutacional de DNA , Éxons/genética , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Lactente , Lipopolissacarídeos/biossíntese , Masculino , Manosiltransferases/deficiência , Manosiltransferases/metabolismoRESUMO
Alternating hemiplegia of childhood (AHC) is a rare neuropediatric disorder classically characterized by episodes of hemiplegia developing in the first months of life, various non-epileptic paroxysmal events and global neurological impairment. If the etiology is unresolved, the disorder is highly suspected to be monogenic with DE NOVO autosomal dominant mutations. A missense mutation in the SLC2A1 gene encoding the facilitative glucose transporter-1 (GLUT1) was recently described in a child fulfilling the existing criteria for the diagnosis of AHC, with the exception of age at onset, thus suggesting a clinical overlap between AHC and GLUT1 deficiency syndrome due to SLC2A1 mutations. We have studied a cohort of 23 patients to investigate whether patients with classical AHC harbor SLC2A1 mutations. Automated Sanger sequencing and MLPA analyses failed to detect any SLC2A1 mutations in the 23 patients analyzed, thus excluding mutations of this gene as a frequent cause of classical AHC.
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Transportador de Glucose Tipo 1/genética , Mutação , Adolescente , Adulto , Idade de Início , Alelos , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Genótipo , Hemiplegia/genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The congenital disorders of glycosylation (CDG) are a recently described group of inherited multisystem disorders characterized by defects predominantly of N- and O-glycosylation of proteins. Cardiomyopathy in CDG has previously been described in several subtypes; it is usually associated with high morbidity and mortality and the majority of cases present in the first 2 years of life. This is the first case with presentation in late childhood and the article reviews current literature. An 11-year-old female with a background of learning difficulties presented in cardiac failure secondary to severe dilated cardiomyopathy. Prior to the diagnosis of CDG, her condition deteriorated; she required mechanical support (Excor Berlin Heart) and was listed for cardiac transplant. Investigations included screening for glycosylation disorders, and isoelectric focusing of transferrin revealed an abnormal type 1 pattern. Analysis of phosphomannomutase and phosphomannose isomerase showed normal enzyme activity, excluding PMM2 (CDG Ia) and MPI (CDG Ib). Lipid-linked oligosaccharide and mutational studies have not yet defined the defect. Despite aggressive therapy there were persistent difficulties achieving adequate anticoagulation and she developed multiple life-threatening thrombotic complications. She was removed from the transplant list and died from overwhelming sepsis 5 weeks following admission. This case emphasizes the need to screen all children with an undiagnosed cardiomyopathy for CDG, regardless of age, and where possible to exclude CDG before the use of cardiac bridging devices. It highlights the many practical and ethical challenges that may be encountered where clinical knowledge and experience are still evolving.
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Cardiomiopatias/etiologia , Defeitos Congênitos da Glicosilação/complicações , Transtornos da Coagulação Sanguínea/etiologia , Cardiomiopatias/diagnóstico , Cardiomiopatias/terapia , Criança , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/metabolismo , Evolução Fatal , Feminino , Coração Auxiliar , HumanosAssuntos
Defeitos Congênitos da Glicosilação/diagnóstico , Cardiopatias Congênitas/diagnóstico , Defeitos Congênitos da Glicosilação/complicações , Defeitos Congênitos da Glicosilação/genética , Análise Mutacional de DNA , Diagnóstico Diferencial , Evolução Fatal , Feminino , Cardiopatias Congênitas/etiologia , Humanos , Masculino , Mutação , Fosfotransferases (Fosfomutases)/genéticaRESUMO
Fukuyama congenital muscular dystrophy (FCMD) is frequent in Japan, due to a founder mutation of the fukutin gene (FKTN). Outside Japan, FKTN mutations have only been reported in a few patients with a wide spectrum of phenotypes from Walker-Warburg syndrome to limb-girdle muscular dystrophy (LGMD2M). We studied four new Caucasian patients from three unrelated families. All showed raised serum CK initially isolated in one case and muscular dystrophy. Immunohistochemical studies and haplotype analysis led us to search for mutations in FKTN. Two patients (two sisters) presented with congenital muscular dystrophy, mental retardation, and posterior fossa malformation including cysts, and brain atrophy at Brain MRI. The other two patients had normal intelligence and brain MRI. Sequencing of the FKTN gene identified three previously described mutations and two novel missense mutations. Outside Japan, fukutinopathies are associated with a large spectrum of phenotypes from isolated hyperCKaemia to severe CMD, showing a clear overlap with that of FKRP.
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Predisposição Genética para Doença/genética , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Distrofias Musculares/genética , Mutação/genética , Malformações do Sistema Nervoso/genética , Adulto , Atrofia/genética , Atrofia/patologia , Atrofia/fisiopatologia , Encéfalo/anormalidades , Encéfalo/patologia , Encéfalo/fisiopatologia , Criança , Creatina Quinase/análise , Creatina Quinase/sangue , Análise Mutacional de DNA , Evolução Fatal , Feminino , Marcadores Genéticos/genética , Predisposição Genética para Doença/etnologia , Genótipo , Humanos , Deficiência Intelectual/patologia , Deficiência Intelectual/fisiopatologia , Masculino , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia , Malformações do Sistema Nervoso/patologia , Malformações do Sistema Nervoso/fisiopatologia , Fenótipo , Síndrome , População Branca , Adulto JovemRESUMO
Type II lissencephaly (type II LIS) is a group of autosomal recessive congenital muscular dystrophies (CMD) associated with defects in alpha-DG O-glycosylation, which comprises Walker-Warburg syndrome, Fukuyama cerebral and muscular dystrophy, or muscle-eye-brain disease. The most severe forms of these diseases often have a fetal presentation and lead to a pregnancy termination. We report here the first molecular study on fetal type II LIS in a series of 47 fetuses from 41 unrelated families. Sequencing of the different genes known to be involved in alpha-DG O-glycosylation allowed the molecular diagnosis in 22 families: involvement of POMT1 was demonstrated in 32% of cases, whereas POMGNT1 and POMT2 were incriminated in 15% and in 7% of cases, respectively. We found 30 different mutations in these three genes, 25 were described herein for the first time, 15 in POMT1, and five in POMT2 and POMGNT1. Despite sequencing of FKRP, FCMD, and LARGE, no definitive molecular diagnosis could be made for the other half of our cases. Preliminary results concerning genotype-phenotype correlations show that the choice of the first gene sequenced should depend on the clinical severity of the type II LIS; POMT1 and POMT2 for severest clinical picture and POMGNT1 for milder disease. The other genes, FKRP, FCMD, and LARGE, seem not to be implicated in the fetal form of CMD.
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Regulação da Expressão Gênica , Distrofias Musculares/embriologia , Distrofias Musculares/genética , Alelos , Distroglicanas/metabolismo , Feminino , Genótipo , Idade Gestacional , Humanos , Masculino , Manosiltransferases/genética , Repetições de Microssatélites , Modelos Genéticos , Mutação , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Walker Warburg syndrome (WWS) is the most severe of a group of multiple congenital disorders known as lissencephaly type II ( LIS Type II) associated with congenital muscular dystrophy and eye abnormalities. The POMT1 gene is the most frequently affected found in 20% of patients with WWS. We describe five fetuses with WWS in three non-related families carrying a same mutation in the POMT1 gene. All fetuses presented with tetra ventricular hydrocephaly, and arachnoidal neuroglial ectopia and cortical dysplasia characteristic of LIS type II. We performed sequencing of the POMT1 gene on fetal DNA. The five fetuses were found to share an insertion of an inversed Alu repeated DNA element within exon 3 of the POMT1 gene, all at the heterozygous state except one at the homozygous state. This mutation was associated with a common transition c.2203 C > T (p.Arg735Cys) in exon 20 on the same allele and similar intragenic haplotype, suggesting that the three families could be related or indicating a possible founder effect in France. Insertions of Alu sequences, which are rarely found in coding regions, have occasionally been reported to cause other genetic diseases. However, this is the first report of a retrotransposon insertion in the POMT1 gene associated with WWS.
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Anormalidades Múltiplas/enzimologia , Anormalidades Múltiplas/genética , Elementos Alu/genética , Substituição de Aminoácidos/genética , Sequência de Bases , Encéfalo/anormalidades , Encéfalo/embriologia , Encéfalo/enzimologia , Anormalidades do Olho/enzimologia , Anormalidades do Olho/genética , Feminino , França , Humanos , Manosiltransferases/genética , Dados de Sequência Molecular , Gravidez , SíndromeRESUMO
Congenital disorders of glycosylation are a group of inherited disorders, characterized by a central nervous system dysfunction and multiorgan failure associated with defective N-glycosylation. CDG-I comprises all defects in the assembly of the dolichol-linked glycan and its transfer to the protein, whereas CDG-II refers to defects in the processing of the protein-bound glycans. The diagnosis is done by the presence of hypoglycosylated glycoproteins in the serum and typing by enzymatic assay (available for CDG-Ia and Ib) and/or mutation detection. We give an overview of the latest results of molecular diagnosis from the French CDG I families. We report novel mutations and their functional study. In addition we looked for a founder effect for the most frequent mutations observed in the French population.
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Erros Inatos do Metabolismo dos Carboidratos/genética , Adulto , Alelos , Erros Inatos do Metabolismo dos Carboidratos/diagnóstico , Erros Inatos do Metabolismo dos Carboidratos/enzimologia , Criança , Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/enzimologia , Defeitos Congênitos da Glicosilação/genética , Feminino , França , Frequência do Gene , Pesquisa em Genética , Glicosídeo Hidrolases/deficiência , Glicosilação , Glicosiltransferases/deficiência , Haplótipos , Heterozigoto , Humanos , Masculino , Manose-6-Fosfato Isomerase/deficiência , MutaçãoAssuntos
Defeitos Congênitos da Glicosilação/genética , Predisposição Genética para Doença , Mutação de Sentido Incorreto , Fosfotransferases (Fosfomutases)/genética , Alelos , Defeitos Congênitos da Glicosilação/diagnóstico , Feminino , Triagem de Portadores Genéticos , Genótipo , Glicosilação , Humanos , Fosfotransferases (Fosfomutases)/deficiência , Gravidez , Diagnóstico Pré-NatalRESUMO
Congenital disorders of glycosylation (CDG) is a fast growing group of autosomal recessive inherited diseases caused by defects in glycosylation. The biosynthesis of the glycans is a pathways which occurs in the endoplasmic reticulum and Golgi complex thanks to highly specific enzymes: glycosidases and glycosyltransferases. The sequential addition of monosaccharides needs precursors which are nucleotide sugars or dolichyl sugars. CDG are divided into two groups: CDG I composed of defects in enzymes involved in the assembly of dolicholpyrophosphate oligosaccharide and in the transfer of oligosaccharide from dolicholpyrophosphate to an Asn residue on nascent proteins; CDG II composed of defects in the processing of protein-bound glycans with alterations in enzymes or in the transporters of monosaccharides. Clinical symptoms are poorly specific and multisystemic, biochemistry provides the diagnosis: Isoelectrofocalisation and western blot of serum transferrin and some other glycoproteins; Measurement of enzyme activities; Research of gene mutations. Today, thirteen CDG are identified, the most frequent is CDG Ia due to a defect in the phosphomannomutase activities and CDG Ib due to a defective phosphomannose isomerase, is the only CDG which is successfully treated with mannose.
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Erros Inatos do Metabolismo dos Carboidratos/genética , Glicosilação , Erros Inatos do Metabolismo dos Carboidratos/enzimologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Mutação/genéticaAssuntos
Anormalidades Múltiplas/genética , Defeitos Congênitos da Glicosilação/genética , Cirrose Hepática/patologia , Enteropatias Perdedoras de Proteínas/patologia , Anormalidades Múltiplas/patologia , Defeitos Congênitos da Glicosilação/enzimologia , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , Diarreia Infantil/patologia , Saúde da Família , Haplótipos , Humanos , Lactente , Manose-6-Fosfato Isomerase/deficiência , Manose-6-Fosfato Isomerase/genética , Mutação de Sentido Incorreto , Quebeque , SíndromeRESUMO
A T911C (F304S) substitution in the ALG6 gene involved in congenital disorder of glycosylation type Ic (OMIM 603147) has been described. However, whether the F304S substitution is a common polymorphism or a causal mutation remains unclear. We screened for the T911C substitution in the ALG6 gene in 54 unrelated healthy French individuals. We developed a restriction fragment length polymorphism assay with a mutagenic primer introducing a diagnostic DdeI restriction site. We found 23 heterozygotes (42.6%) and 3 homozygous individuals (5.5%). This result indicates that T911C is a common polymorphism with an allele frequency of 27% in a French population and not a causal mutation of congenital disorder of glycosylation type Ic.
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Defeitos Congênitos da Glicosilação/genética , Glucosiltransferases/genética , Proteínas de Membrana , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Alelos , Substituição de Aminoácidos , Frequência do Gene , Genótipo , Glucosiltransferases/química , Glicosilação , Heterozigoto , Homozigoto , HumanosRESUMO
The PMM2 gene, which is defective in CDG-Ia, was cloned three years ago [Matthijs et al., 1997b]. Several publications list PMM2 mutations [Matthijs et al., 1997b, 1998; Kjaergaard et al., 1998, 1999; Bjursell et al., 1998, 2000; Imtiaz et al., 2000] and a few mutations have appeared in case reports or abstracts [Crosby et al., 1999; Kondo et al., 1999; Krasnewich et al., 1999; Mizugishi et al., 1999; Vuillaumier-Barrot et al., 1999, 2000b]. However, the number of molecularly characterized cases is steadily increasing and many new mutations may never make it to the literature. Therefore, we decided to collate data from six research and diagnostic laboratories that have committed themselves to a systematic search for PMM2 mutations. In total we list 58 different mutations found in 249 patients from 23 countries. We have also collected demographic data and registered the number of deceased patients. The documentation of the genotype-phenotype correlation is certainly valuable, but is out of the scope of this molecular update. The list of mutations will also be available online (URL: http://www.kuleuven. ac.be/med/cdg) and investigators are invited to submit new data to this PMM2 mutation database.
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Defeitos Congênitos da Glicosilação/genética , Mutação de Sentido Incorreto , Fosfotransferases (Fosfomutases)/genética , Adolescente , Adulto , Sequência de Aminoácidos/genética , Criança , Defeitos Congênitos da Glicosilação/classificação , Defeitos Congênitos da Glicosilação/enzimologia , Defeitos Congênitos da Glicosilação/epidemiologia , Éxons/genética , Genótipo , Glicosilação , Humanos , Lactente , Recém-Nascido , Dados de Sequência Molecular , Fenótipo , Fosfotransferases (Fosfomutases)/metabolismo , Polimorfismo Genético/genéticaRESUMO
We screened 11 unrelated French patients with congenital disorders of glycosylation (CDG) Ia for PMM2 mutations. Twenty one missense mutations on the 22 chromosomes (95%) including four novel mutations were identified: C9Y (G26A) in exon 1, L32R (TA95GC) in exon 2, and T226S (C677G) and C241S (G722C) in exon 8. We studied the PMM activity of these four novel mutant proteins and of the R141H mutant protein in an E coli expression system. The T226S, C9Y, L32R, and C241S mutant proteins have decreased specific activity (23 to 41% of normal), are all more or less thermolabile, and R141H has no detectable activity. Our results indicate that the new mutations identified here are less severe than the inactive R141H mutant protein, conferring residual PMM activity compatible with life.