Ubaldo Caruso

Organization of the mevalonate kinase ( MVK ) gene and identification of novel mutations causing mevalonic aciduria and hyperimmunoglobulinaemia D and periodic fever syndrome

Mevalonic aciduria (MA) and hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) are two autosomal recessive inherited disorders both caused by a deficient activity of the enzyme mevalonate kinase (MK) resulting from mutations in the encoding MVK gene. Thus far, disease-causing mutations only could be detected by analysis of MVK cDNA. We now describe the genomic organization of the human MVK gene. It is 22 kb long and contains 11 exons of 46 to 837 bp and 10 introns of 379 bp to 4.2 kb. Three intron-exon boundaries were confirmed from natural splice variants, indicating the occurrence of exon skipping. Sequence analysis of 27 HIDS and MA patients confirmed all previously reported genotypes based on cDNA analysis and identified six novel nucleotide substitutions resulting in missense or nonsense mutations, providing new insights in the genotype/phenotype relation between HIDS and MA.

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.

Characteristic Acylcarnitine Profiles in Inherited Defects of Peroxisome Biogenesis: A Novel Tool for Screening Diagnosis Using Tandem Mass Spectrometry

Patients with inherited defects of peroxisomal metabolism, a class of diseases with marked clinical and genetic heterogeneity, show a characteristic phenotype in most cases with severe neurologic impairment, craniofacial abnormalities, and hepatic and kidney dysfunction. For the differential diagnosis of clinically suspected cases, a complex biochemical and genetic approach is required. Analysis of plasma very-long-chain fatty acids is a reliable screening method to detect most but not all peroxisomal disorders. To study the potential presence of abnormal acylcarnitine species in plasma and blood, we screened by tandem mass spectrometry a series of patients affected by a peroxisome biogenesis disorder (PBD) and compared the results with those obtained in patients with isolated peroxisomal defects (e.g. D-bifunctional protein deficiency, X-linked adrenoleukodystrophy) and mitochondrial long-chain fatty acid oxidation defects. The most relevant finding observed in plasma of patients with PBD was a significant increase of long-chain dicarboxylic C16- and C18-carnitine, i.e. hexadecanedioyl- and octadecanedioyl-carnitine, with high dicarboxylycarnitine/monocarboxylylcarnitine ratio. Elevation of very long-chain acylcarnitines C24- and C26-, i.e. lignoceroyl- and cerotoyl-carnitine, was detected in some PBDs and in D-bifunctional protein deficiency. Similar abnormalities were also found in neonatal screening blood spots. Detection of these compounds alone, in the absence of other shorter-chain acylcarnitines, is highly specific and characteristic of PBD, as confirmed by the differing profiles observed in patients with adrenoleukodystrophy and mitochondrial long-chain fatty acid oxidation defects. Our study adds a novel method to the diagnosis of PBD, which may also be of benefit for future neonatal mass screening programs based on acylcarnitine profiling.

An electrogenic amino acid transporter in the apical membrane of cultured human bronchial epithelial cells

We performed Ussing chamber experiments on cultured human bronchial epithelial cells to look for the presence of electrogenic dibasic amino acid transport. Apical but not basolaterall-arginine (10-1,000 μM) increased the short-circuit current. Maximal effect and EC50 were ∼3.5 μA/cm2 and 80 μM, respectively, in cells from normal subjects and cystic fibrosis patients. The involvement of nitric oxide was ruled out because a nitric oxide synthase inhibitor ( N G-nitro-l-arginine methyl ester) did not decrease the arginine-dependent current. Apicall-lysine,l-alanine, andl-proline, but not aspartic acid, were also effective in increasing the short-circuit current, with EC50 values ranging from 26 to 971 μM. Experiments performed with radiolabeled arginine demonstrated the presence of an Na+-dependent concentrative transporter on the apical membrane of bronchial cells. This transporter could be important in vivo to maintain a low amino acid concentration in the fluid covering the airway surface.We performed Ussing chamber experiments on cultured human bronchial epithelial cells to look for the presence of electrogenic dibasic amino acid transport. Apical but not basolateral L-arginine (10-1, 000 microM) increased the short-circuit current. Maximal effect and EC50 were approximately 3.5 microA/cm2 and 80 microM, respectively, in cells from normal subjects and cystic fibrosis patients. The involvement of nitric oxide was ruled out because a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester) did not decrease the arginine-dependent current. Apical L-lysine, L-alanine, and L-proline, but not aspartic acid, were also effective in increasing the short-circuit current, with EC50 values ranging from 26 to 971 microM. Experiments performed with radiolabeled arginine demonstrated the presence of an Na+-dependent concentrative transporter on the apical membrane of bronchial cells. This transporter could be important in vivo to maintain a low amino acid concentration in the fluid covering the airway surface.

Early-onset cobalamin C/D deficiency: Epilepsy and electroencephalographic features

Summary:  Purpose: To describe epilepsy and EEG findings in the early‐onset cobalamin (Cbl) C/D deficiency, an inborn error of intracellular Cbl metabolism characterized by high plasma levels of methylmalonic acid, homocystine, and homocysteine.

Severe Epilepsy in X-Linked Creatine Transporter Defect (CRTR-D)

Disorders of creatine synthesis or its transporter resulting in neurological impairment with mental retardation and epilepsy have only been recognized in recent years. To date, the epileptic disorder observed in creatine transporter deficiency (CRTR‐D) has been described as a mild phenotype with infrequent seizures and favorable response to common antiepileptic drugs.

Severe lactic acidosis due to thiamine deficiency in a patient with B-cell leukemia/lymphoma on total parenteral nutrition during high-dose methotrexate therapy.

An 11-month-old girl with B-cell leukemia/lymphoma developed profound lethargy due to severe lactic acidosis during chemotherapy and total parenteral nutrition (TPN). Initial treatment with NaHCO3 was ineffective. Treatment with a vitamin cocktail (OH-cobalamin, pyridoxine, thiamine, riboflavine, biotin, carnitine) at pharmacologic doses rapidly improved the childs clinical and laboratory status. Lactic acidosis was caused by an impairment of pyruvate dehydrogenase complex, which was due to lack of its necessary cofactor thiamine in the TPN. This case report indicates that lactic acidosis may be a front-line diagnosis in patients on TPN with lethargy and outlines the need for monitoring thiamine supply in TPN.

X-linked creatine transporter deficiency: clinical description of a patient with a novel SLC6A8 gene mutation.

Creatine transporter deficiency is an X-linked disorder characterized by mental retardation and language delay. The authors report a patient affected by creatine transport deficiency caused by a novel mutation in the SLC6A8 gene. Impairment in social interaction represents a consistent clinical finding in the few cases described to date and may be a diagnostic clue for creatine transporter deficiency in males affected by mental retardation, seizures, and language impairment.

Minor facial anomalies in combined methylmalonic aciduria and homocystinuria due to a defect in cobalamin metabolism.

Methylmalonic acidaemia and homocystinuria (MMA/HC) is a very rare inborn error of cellular metabolism of cobalamin (Cbl), that results in functional defects of both methylmalonyl-CoA mutase and methionine synthase because of deficient synthesis of 5-deoxyadenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl). AdoCbl is the cofactor of methylmalonyl-CoA mutase, the enzyme catalysing the intramitochondrial isomerization of methylmalonyl-CoA to succinyl-CoA, while MeCbl is the cofactor of the cytosolic remethylation from homocysteine to methionine with recycling of the folate derivative tetrahydrofolate (THF). Seven different defects of the intracellular metabolism of Cbl have been described; of these seven, three forms involve the synthesis of both AdoCbl and MeCbl and are distinct genetically and biochemically, namely Cbl-C (the most frequent variant), Cbl-D and Cbl-F. Age of onset and clinical findings vary widely among the three complementation groups and among known patients with Cbl-C disease: the affected patients generally show feeding difficulties, failure to thrive, neurological dysfunction (developmental delay, hypotonia, seizures, extrapyramidal signs in childhood, dementia, psychosis), haematological changes, ocular abnormalities and renal involvement. The acute neonatal onset is severe and can lead to death (Fenton and Rosenblatt 1995; Rosenblatt et al 1997)). To date, minor facial anomalies have been reported only in two cases of the Cbl-F form, but not in the other variants of combined MMA and HC, whereas peculiar facial dysmorphism has been described in several different metabolic diseases such as peroxisomal disorders, carbohydrate glycoprotein deficiency syndrome, Smith-Lemli-Opitz syndrome, mevalonic aciduria, pyruvate dehydrogenase deficiency and respiratory chain deficiencies. We report here our personal observation of these clinical findings in seven patients with MMA and HC.

Intermittent-relapsing pyruvate dehydrogenase complex deficiency: a case with clinical, biochemical, and neuroradiological reversibility

Pyruvate dehydrogenase complex (PDHC) deficiency causes encephalomyopathies, of which there are four major categories: (1) neonatal encephalopathy with lactic acidosis; (2) an early infantile form, which (3) at times resembles Leigh syndrome; and (4) a later‐onset form. Long‐term clinical and radiological follow‐up is still incompletely elucidated. We report a 12‐year‐old male with intermittent‐relapsing PDHC deficiency who presented with three typical acute episodes of metabolic decompensation over 7 years. Neuroimaging showed reversible signal abnormalities in the basal ganglia, inferior olivary nuclei, periaqueductal grey matter, and dentate nuclei, with evidence of lactate on magnetic resonance spectroscopy. Molecular analysis of PDH1A revealed a novel hemizygous c.1045G>A mutation, predicting a p.A349T missense mutation. He was treated with thiamine supplementation and, while on this regimen, he experienced several intercurrent febrile episodes without neurological compromise. This case report stresses the importance of performing neuroimaging during acute clinical episodes because brain lesions in PDHC deficiency may be transient and reversible, and false‐negative results may mislead the diagnosis and delay the treatment.