Esmeralda Martins

Spectrum of MMACHC mutations in Italian and Portuguese patients with combined methylmalonic aciduria and homocystinuria, cblC type.

Methylmalonic aciduria (MMA) and homocystinuria, cblC type (MIM 277400) is the most frequent inborn error of vitamin B(12). The recent identification of the disease gene, MMACHC, has permitted preliminary genotype-phenotype correlations. We studied 24 Italian and 17 Portuguese patients with cblC defect to illustrate the spectrum of mutations in a southern European population and discuss the impact that mutation identification has on routine diagnostic procedures. Since the metabolic defect raises the serum levels of homocysteine, we also tested if variants in MTHFR-playing a key role in homocysteine remethylation pathway-could act as genetic modifier in cblC defect. We found that the c.271dupA (accounting for 55% of the MMACH alleles in our cohort) followed by c.394C>T (16%) and c.331C>T (9%) were the most frequent mutations. In our study we also identified a novel mutation (c.544T>C). On the other hand, the MTHFR genotype did not appear to influence age at onset, the clinical phenotype and outcome of patients with cblC defect. This study shows that mutation screening for the most common MMACH mutations occurring in early-onset forms (c.271dupA and c.331C>T) seems to have a high diagnostic yield in a southern European population with cblC defect. Although the identification of the gene defect per se does not predict completely time and severity of disease appearance, our data corroborate the importance of a molecular testing to offer accurate prenatal diagnosis to couples at high risk of having affected children.

Clinical, biochemical and molecular characterization of Cystinuria in a cohort of 12 patients

Barbosa M, Lopes A, Mota C, Martins E, Oliveira J, Alves S, De Bonis P, do Céu Mota M, Dias C, Rodrigues‐Santos P, Fortuna AM, Quelhas D, Lacerda L, Bisceglia L, Cardoso ML. Clinical, biochemical and molecular characterization of Cystinuria in a cohort of 12 patients.

Molecular and structural analyses of maple syrup urine disease and identification of a founder mutation in a Portuguese Gypsy community

Maple syrup urine disease (MSUD) is an autosomal recessive disorder, caused by the defective function of the branched-chain alpha-ketoacid dehydrogenase complex (BCKD). BCKD is a mitochondrial complex, encoded by four nuclear genes (BCKDHA, BCKDHB, DBT and DLD), involved in the metabolism of branched-chain amino acids (BCAAs). Since the MSUD mutational spectrum has not been previously assessed in Portugal, in this study we present the molecular characterization of 30 MSUD Portuguese patients. Seventeen putative mutations have been identified (six in BCKDHA, five in BCKDHB and six in DBT); seven of them are here described for the first time. The most common mutation identified was a C deletion in BCKDHA gene (c.117delC; p.R40GfsX23), already reported in the Spanish population. Interestingly, it was found in all patients of a Gypsy community from South of the country, so a founder effect is probably responsible for the high incidence of the disease in this community. Structural models of MSUD missense mutations have been performed to understand their pathogenic effect, in order to elucidate and often to predict the severity of a mutation clinical consequence.

Identification of a novel R21X mutation in the liver-type arginase gene (ARG1) in four Portuguese patients with argininemia

Argininemia is a rare autossomal recessive disorder caused by deficiency in the cytosolic liver‐type arginase enzyme (L‐arginine urea‐hydrolase; E.C. 3.5.3.1). In order to investigate the molecular basis for argininemia in four unrelated Portuguese patients (two from northern Portugal and two from Madeira Island) we performed a DNA sequence analysis of all the exons and exon/intron boundaries of the liver‐type arginase gene (ARG1). All patients were found to be homozygous for a newly identified C ‐>T transition in codon 21 (exon 2) substituting arginine for a premature stop codon (R21X: CGA to TGA) and generating a NlaIII restriction site. Restriction digestion following PCR amplification of ARG1 exon 2 confirmed the presence of the mutation. Hum Mutat 14:355–356, 1999.

Maple Syrup Disease Presenting as Paroxysmal Dystonia

An apparently normal 22-month-old boy was evaluated because of weekly paroxysmal episodes consisting of curvature of the body (alternating sides). During attacks, which lasted 5 minutes to 8 hours, the patient had extreme irritability. We hypothesized based on the clinical picture that the paroxysmal events might correspond to partial epileptic seizures, hemiplegic migraine, paroxysmal torticollis, or Sandifer’s syndrome. Blood count, hepatic and renal function, ionogram, and glycemia were normal. Electroencephalogram showed left temporal slow waves. Brain computed tomography scan was normal. The patient was put on sodium valproate (30mg/kg/ day PO). Two months later, the patient had similar weekly attacks. These episodes were videotaped, and we were able to diagnose a paroxysmal dystonia accompanied by irritability. Physical and neurological examination was normal between the attacks. Mental age showed a delay of 10 months of global development (Griffiths scale). Brain magnetic resonance imaging (MRI) showed symmetrical hypersignal in T2-weighted images in the brainstem, globus pallidus, thalami, and dentate nuclei (see Fig). Serum and urinary amino acid analysis in the interictal periods disclosed abnormally high leucine (489 mol/L; normal, 109), isoleucine (278 mol/L; normal, 94), valine (500 mol/L; normal, 246), and alloisoleucine (88 mol/L; normal, 3.4) levels. Urinary organic acid chromatography was normal. Serum leucine level was 300 mol/L after 5 days’ treatment with a vegetarian diet and milk substitute. Supplementation with high doses of thiamine did not improve the metabolic picture. Ten months after maple syrup urine disease (MSUD) was diagnosed, the patient had only two mild attacks of dystonia accompanied by metabolic decompensation episodes precipitated by fever. In addition, his psychomotor development improved, particularly verbal capacities. Brain MRI has not shown significant differences, however. Most episodes of paroxysmal dyskinesia in children are idiopathic. Secondary metabolic causes include nonketotic hyperglycinemia, hypoglycemia, hypocalcemia, thyrotoxicosis, kernicterus, cystinuria, and pyruvate carboxylase deficiency. We have presented a patient with episodes of nonkinesigenic paroxysmal dystonia accompanied by marked irritability. This makes this case different from typical idiopathic dystonias. Left temporal electroencephalogram abnormalities suggested a diagnosis of epilepsy, but it was quite improbable given the alternating location of the dystonic attacks and the failure to respond to antiepileptic therapy. Brain MRI pointed to a symptomatic cause (metabolic, toxic, or anoxic). The abnormal levels of branched-chain amino acids in the interictal periods with persistently high alloisoleucine levels have helped us diagnose chronic intermediate MSDU. Nonspecific developmental delay, progressive psychomotor retardation, dementia, and seizures can be observed during the course of this disease. Although age at onset, delayed psychomotor development, and typical brain imaging make this case similar to others, our patient atypically presented with paroxysmal dystonia. MSUD should be considered in the differential diagnosis of secondary causes of childhoodonset paroxysmal dyskinesia.

Relative frequency of known causes of multiple mtDNA deletions: Two novel POLG mutations

Diseases affecting mtDNA stability, termed nuclear-mitochondrial intergenomic communication disorders, are caused by a primary nuclear gene defect resulting in multiple mtDNA deletions. The aim of this study was to estimate the frequency of known etiologies and the spectrum of mutations in a cohort of 21 patients harboring multiple mtDNA deletions in skeletal muscle. We showed that 10 cases (48%) display mutations in POLG, including eight previously reported variants and two novel mutations (namely, p.Trp585X and p.Arg1081Gln). The novel mutations affect evolutionary conserved residues and were absent in a large set of control chromosomes. These findings expand the array of mutations associated with multiple rearranged mtDNA attributed to mutations in POLG. The relatively high diagnostic yield (about one in two cases) supports the notion that it is recommended to test POLG routinely in diagnostic laboratories whenever multiple mtDNA deletions are present, regardless of the age of onset of patients and their clinical phenotype.

Short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency: the clinical relevance of an early diagnosis and report of four new cases

Short-chain 3-hydroxyacyl-CoA dehydrogenase (HADH, SCHAD) deficiency (OMIM #231530) represents a recently described disorder of mitochondrial fatty acid beta-oxidation, with less than ten cases described worldwide. The main clinical presentation of this metabolic disease is different from other inherited defects of fatty acid β−oxidation as the hypoglycemia is associated with hyperinsulinism. We present the clinical, biochemical and molecular findings of four new Caucasian patients with HADH deficiency. These new cases contribute to a more comprehensive description of the phenotype, diagnostic biomarkers and treatment options for this poorly defined disease.

Incidence of maple syrup urine disease in Portugal

Maple syrup urine disease is an autosomal recessive disorder of branched-chain amino acids metabolism with a worldwide frequency of 1/185,000 live newborns. In Portugal, the incidence of the disease has not been assessed. Based on the review of the cases diagnosed by tandem mass spectrometry an incidence of 1/86,800 live newborns was estimated in Portugal, indicating that the disease is more frequent in this country than reported in most populations.

Lethal dilated cardiomyopathy due to long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

logical abnormalities including impaired function of lymphocytes, the presence of lupus erythematosus cells and thrombocytopenia. Immunological dysfunctions such as impaired functions of T and B cells, hypofunction of suppressor T cells, and abnormal proliferation of B cells are thought to be due to amino acid imbalance in tissue and cells (Nagata et al 1987). Congenital thymic hypoplasia detected at autopsy is rather interesting and has not been reported in LPI before. The coincidence of this abnormality and LPI is not excluded. In conclusion, these patients must be investigated for further immunological abnormalities including structural thymic disorders.

A frequent splicing mutation and novel missense mutations color the updated mutational spectrum of classic galactosemia in Portugal

Classic galactosemia is an autosomal recessive disorder caused by deficient galactose-1-phosphate uridylyltransferase (GALT) activity. Patients develop symptoms in the neonatal period, which can be ameliorated by dietary restriction of galactose. Many patients develop long-term complications, with a broad range of clinical symptoms whose pathophysiology is poorly understood. The high allelic heterogeneity of GALT gene that characterizes this disorder is thought to play a determinant role in biochemical and clinical phenotypes. We aimed to characterize the mutational spectrum of GALT deficiency in Portugal and to assess potential genotype-phenotype correlations. Direct sequencing of the GALT gene and in silico analyses were employed to evaluate the impact of uncharacterized mutations upon GALT functionality. Molecular characterization of 42 galactosemic Portuguese patients revealed a mutational spectrum comprising 14 nucleotide substitutions: ten missense, two nonsense and two putative splicing mutations. Sixteen different genotypic combinations were detected, half of the patients being p.Q188R homozygotes. Notably, the second most frequent variation is a splicing mutation. In silico predictions complemented by a close-up on the mutations in the protein structure suggest that uncharacterized missense mutations have cumulative point effects on protein stability, oligomeric state, or substrate binding. One splicing mutation is predicted to cause an alternative splicing event. This study reinforces the difficulty in establishing a genotype-phenotype correlation in classic galactosemia, a monogenic disease whose complex pathogenesis and clinical features emphasize the need to expand the knowledge on this “cloudy” disorder.