[Neuropathic pain in Fabry's disease: heterogeneous remission in three years of enzyme replacement therapy]. / Dolor neuropático en la enfermedad de Fabry: remisión heterogénea en tres años de tratamiento de reemplazo enzimático.

INTRODUCTION: Fabry's disease is associated with acute neuropathic pain (NP). When six males with classic Fabry's disease ended three years of enzyme replacement therapy (ERT), studies were conducted to analyse the progression of the NP. PATIENTS AND METHODS: All of them received 1 mg/kg of agalsidase beta every 14 days. NP in hands and feet was evaluated at 0, 6, 12, 24 and 36 months, two modalities being considered: a) very intense pain or Fabry crises (FC), which forced the patient to rest, take analgesics and apply cold locally; b) acroparesthesias (AP), lower intensity itching, tingling and burning sensations that did not prevent them from continuing with their activities of daily living. The intensity of the FC (IFC) and the AP (IAP) were recorded using the Visual Analogue Scale, and the frequency with which both (FrFC and FrAP, respectively) appeared was measured in days. The one-tailed Wilcoxon test, binomial distribution and bootstrap method were used to carry out the analysis. RESULTS: After six months of ERT the IFC, IAP and FrCF remained the same, although the FrAP had become worse. As shown by the NP indexes that relate IFC/FrFC and IAP/FrAP, progress was therefore favourable. At one year, IFC continued, but FrFC and IAP were lower. At two years, the four NP measurements improved in five patients, although the FC and AP indexes did not vary in one patient. At three years, all the NP variables improved in the six patients. The slope representing NP as a function of time in all the bootstrap analyses was found to be p < 0.001. CONCLUSIONS: NP responded in a favourable, significant and heterogeneous manner and therefore justifies the early indication of ERT.

Hurler-like phenotype: enzymatic diagnosis in dried blood spots on filter paper.

BACKGROUND: Clinical differentiation among mucopolysaccharidosis, oligosaccharidosis, and mucolipidosis II and III is difficult. We describe methods for the assay of 8 lysosomal enzymes in dried blood spots on filter paper that allow screening for 12 lysosomal storage diseases that present with a Hurler-like phenotype. METHODS: To test tubes containing 3-mm blood spots, we added elution liquid and fluorescent or radioactive substrate solution. After incubation at 37 degrees C, the reaction was terminated by the addition of a stop buffer. The amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. Sample stability was studied during storage for 21 days and during shipment of samples. We measured enzyme activities in 85 healthy controls (35 newborn, 50 adult), 57 patients suffering from 11 lysosomal storage diseases, and 46 obligate carriers. RESULTS: Intra- and interassay CVs were <9% and <15%, respectively. Mean activity losses during transportation or storage for up to 21 days at 4 degrees C were < or =27%. Enzyme activities in all patients were outside the ranges of values seen for carriers and controls. CONCLUSIONS: The described methodology distinguishes between patients and controls with samples that are sufficiently stable to be mailed to the testing laboratory.

Culture of porcine hepatocytes: the dogma of exogenous matrix revisited.

The use of exogenous matrices has been described as an essential component in securing the viability and functionality of hepatocytes in vitro whether cultured for extracorporeal devices or cell transplantation. Here we report on the in vitro culture of porcine hepatocytes in polystyrene tissue-culture flasks without exogenous matrices showing adequate attachment and viability. Cell proliferation was evidenced by uptake of 5-bromo-2'-deoxyuridine, with peaks at Days 2 (19.7 +/- 8.5%), 15 (20.8 +/- 3.3%), and 35 (21.4 +/- 0.3%). Detoxification capacity was assessed by determination of monoethylglycinexylidide, a product of lidocaine metabolism (highest value 156.5 +/- 10.1 ng/ml at Day 4), and by diazepam clearance (maximum clearance 66.2% at Day 6). Diazepam metabolite levels were highest at Day 4 both for temazepam and oxazepam (6.5 +/- 0.1 and 0.10 +/- 0.01, respectively). These results suggest that the need for an exogenous matrix to achieve sustained proliferative activity and differentiated hepatocyte function should not necessarily be considered a sine qua non condition.

Multiple acyl-CoA-dehydrogenase deficiency (MADD): use of acylcarnitines and fatty acids to monitor the response to dietary treatment.

The treatment of multiple acyl-CoA-dehydrogenase deficiency (MADD) includes a low-fat, low-protein, high-carbohydrate diet, avoiding long fasting periods. However, there is no useful biochemical marker to determine the response to different diets or fasting periods. The aims of this study are to report a patient with MADD, diagnosed through a newborn screening program using tandem mass spectrometry, to assess her response to different feedings, and to evaluate the usefulness of acylcarnitines and FFA to monitor the response to dietary changes. The patient was diagnosed at 6 d. Family history revealed three dead siblings. Five tests were performed, one with breast milk and the subsequent four after giving the patient a bottle of a low-fat, low-protein formula (F), F with glucose polymers (GP), F+GP plus uncooked corn starch (CS), or F+GP+CS preceded by amylase. The results showed that acylcarnitines, FFA, and total nonesterified fatty acids levels were greatly improved at 2 and 4 h on F+GP compared with breast milk. At 6 mo of age, the test with F+CS was repeated to assess the response to a longer fast. The results were similar at 2 and 4 h, but showed a marked increase of acylcarnitines, FFA, and total nonesterified fatty acids at 6 h. The increase of these metabolites could not be avoided by the use of F+GP+CS, but was prevented when amylase was used simultaneously. The patient is currently 3.9 y old and has normal growth and development. We conclude that diagnosis of MADD through a newborn screening program using tandem mass spectrometry is suitable; acylcarnitines and FFA are useful to monitor the response to treatment; and exogenous amylase allows the use of CS in small children with MADD. This therapeutic approach may be an alternative to the use of continuous overnight feedings used for young children with severe fatty acid oxidation defects. Early diagnosis and treatment may change the natural history of MADD.

Evaluation of urinary acylglycines by electrospray tandem mass spectrometry in mitochondrial energy metabolism defects and organic acidurias.

We analyzed the urinary acylglycine excretion in 26 patients with mitochondrial energy metabolism disorders and in 55 patients with organic acidurias by electrospray tandem mass spectrometry (ESI-MS/MS), monitoring precursor ions of m/z 90. Urinary concentrations of the different acylglycines were quantified using deuterated internal standards. Normal values for the most important acylglycines were established. In MCAD and MAD (neonatal form) deficiencies, typical excretion patterns of urinary acylglycines were found in all the samples. In isovaleric aciduria, propionic aciduria, and 3-methylcrotonylglycinuria typical glycine conjugates were always found. Methylmalonic aciduria (mutase deficiency), multiple carboxylase deficiency, and 3-hydroxy-3-methylglutaric aciduria revealed pathological acylglycine profiles, even if not specific for the disease. In all these diseases acylglycine excretion seems to be less influenced by the clinical status than organic acid excretion. This method is a useful diagnostic tool for these metabolic disorders, complementary to organic acids and acylcarnitine profiles.

An unexpected affected female patient in a classical Lesch-Nyhan family.

Lesch-Nyhan disease is a genetic disorder of purine metabolism caused by defective activity of the enzyme hypoxanthine-guanine phosphoribosyl transferase (HPRT), resulting from mutation in the corresponding gene on the long arm of the X chromosome (Xq26). The classical phenotype, which includes spasticity, involuntary movements, developmental disability, and self-injurious behavior, occurs exclusively in males, while heterozygous, carrier females are clinically normal. We analyzed an Argentine family in which there were male and female siblings with clinically identical classic features of Lesch-Nyhan disease. The mother and an older daughter were carriers and had normal phenotypes. We identified the HPRT mutation in the family. It is a C --> T transition at position 508 of the cDNA (c.508 C --> T) that changes the CGA codon for Arg(169) to the TGA stop codon (R169X). The female patient was karyotypically normal and heterozygous for the mutation. She inherited the HPRT mutation from her mother, but she also had unexpected nonrandom inactivation of the paternal X chromosome carrying the normal HPRT gene. This additional genetic alteration is the cause of the clinical expression of disease in this female patient.

Familial neonatal SIDS revealing carnitine-acylcarnitine translocase deficiency.

UNLABELLED: A patient with a severe phenotype of carnitine-acylcarnitine translocase deficiency (CATR)(McKusick 212138) is reported. Prior to birth, a defect in beta-oxidation was suspected because of neonatal death of six siblings. Dietary treatment during neonatal adaptation and the subsequent six months of life and a trial of carnitine supplementation are reported. The rapidity with which long chain fatty acid metabolites can accumulate and induce secondary carnitine deficiency within a few hours after birth in an infant with CATR is noteworthy. CONCLUSION: High rates of glucose suppressed neonatal lipolysis in this infant, but did not seem sufficient to avoid secondary carnitine deficiency as in severe forms of CATR. Therefore simultaneous use of insulin and glucose may be necessary to control neonatal lipolysis. Carnitine supplementation and the possible adverse effects of MCT systematically administrated, should be further assessed in patients with CATR.

Mutation analysis of Gaucher disease patients from Argentina: high prevalence of the RecNciI mutation.

Gaucher disease (GD) is caused by a deficiency of beta-glucocerebrosidase activity mainly due to mutations in the gene coding for the enzyme. More than 100 mutations have been identified to date and their frequencies have been established in several populations, including Ashkenazi Jews, among whom the disease is particularly prevalent. In order to study the molecular pathology of the disease in patients from Argentina, we conducted a systematic search for mutations in the glucocerebrosidase gene. Genomic DNA from 31 unrelated GD patients was screened for seven previously described mutations: N370S (1226A-->G), L444P (1448T-->C), D409H (1342G-->C), R463C (1504C-->T), 1263de155, RecNciI, and RecTL. This allowed the identification of 77.4% of the GD alleles: N370S and RecNciI were the most prevalent mutations found (46.8% and 21% respectively). Southern analysis demonstrated three distinct patterns for the RecNciI alleles. In order to identify the remaining alleles, the full coding region of the gene, all the splice sites, and part of the promoter region were analyzed by single-strand conformational polymorphism analysis (SSCP) after polymerase chain reaction amplification. This extensive screening allowed the identification of 13 different mutations, accounting for 93% of the total number of GD alleles. Three novel missense mutations, I161S (599T-->G), G265D (911G-->A), and F411I (1348T-->A), were detected. Twelve polymorphic sites within the glucocerebrosidase gene are in complete linkage disequilibrium and define two major haplotypes, "-" and "+". Mutation N370S was always associated with the "-" haplotype, as described in other populations. Interestingly, the RecNciI alleles with the same Southern-blot pattern were always associated with the same haplotype.

[Glutaric aciduria type 1: phenotypic variability. Report of 6 patients]. / Acidúria glutárica tipo 1. Variabilidade fenotípica. Estudo de seis pacientes.

We report six patients with glutaric aciduria type 1 in four families. The patients had marked clinical variability, even within families. Three of the patients studied were normal until the onset of neurologic abnormalities, that presented as an encephalitis-like illness in the first year of age. One patient had an early and important developmental delay, but never suffered an encephalopathic crisis. Two patients have intellectual preservation; one of them has a mild tremor and choreoathetosis since the first year of age, and the other had only two afebrile seizures in infancy and no other neurologic signs. Three patients are severely handicapped, with a severe dystonic-dyskinetic disorder and unable to even sit. All the six patients have macrocephaly and in all the computed tomography showed enlarged CSF spaces and sulcal separation over the frontal and temporal lobes. Urine organic acids study of all patients showed large quantities of glutaric acid.

Diagnosis of isovaleric acidaemia by tandem mass spectrometry: false positive result due to pivaloylcarnitine in a newborn screening programme.

Tandem mass spectrometric analysis of acylcarnitines and amino acids has been applied in newborn screening programmes for the detection of several inborn errors of metabolism. We report a false positive result for isovaleric acidaemia in a newborn screening programme using this method. The newborn screening sample showed a very prominent signal corresponding to the mass of isovalerylcarnitine. Repeat samples (age 6 days) of blood and urine showed similar results. However, urine organic acids were normal. Acylcarnitine analysis in blood, breast milk and urine of the mother also showed a prominent signal of the same mass. Gas chromatography-mass spectrometry of the methyl esters demonstrated that the signal in the patient's urine was due to the presence of pivaloylcarnitine, which is isomeric with isovalerylcarnitine. The patient's mother was receiving an antibiotic containing a derivative of pivalic acid to treat a urinary tract infection. Follow-up samples in the patient and the mother confirmed a decrease in the levels of pivaloylcarnitine, concomitant with the discontinuation of the treatment. We conclude that pivaloylcarnitine can give a false positive result for isovaleric acidaemia in newborns whose mothers are on treatment with pivoxilsulbactam-containing antibiotics.

Patient with an Xp21 contiguous gene deletion syndrome in association with agenesis of the corpus callosum.

The so-called Xp21 contiguous deletion syndrome or complex glycerol kinase deficiency (GKD) usually presents with classical Duchenne muscular dystrophy (DMD) or a milder dystrophic myopathy, adrenal hypoplasia, and GKD. A number of syndromic and nonsyndromic cases of agenesis of the corpus callosum (ACC) also map to that location. To date, none of the cases of complex GKD have been associated with ACC. Here, we report on a patient with a complex phenotype as a result of the Xp21 contiguous deletion syndrome in association with ACC. Biochemical, cytogenetic, and molecular analyses were performed to detect and establish the size of the genomic deletion. It is at least 3 million base pairs in length; however, exact limits could not be determined in the present study. Nevertheless, we suggest the presence of a primary gene involved in the embryogenesis of the corpus callosum between Xp21.1 and Xp22.11.

The molecular basis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in compound heterozygous patients: is there correlation between genotype and phenotype?

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of mitochondrial beta-oxidation. It is potentially fatal, but shows a wide clinical spectrum. The aim of the present study was to investigate whether any correlation exists between MCAD genotype and disease phenotype. We determined the prevalence of the 14 known and seven previously unknown non-G985 mutations in 52 families with MCAD deficiency not caused by homozygosity for the prevalent G985 mutation. This showed that none of the non-G985 mutations are prevalent, and led to the identification of both disease-causing mutations in 14 families in whom both mutations had not previously been reported. We then evaluated the severity of the mutations identified in these 14 families. Using expression of mutant MCAD in Escherichia coli with or without co-overexpression of the molecular chaperonins GroESL we showed that five of the missense mutations affect the folding and/or stability of the protein, and that the residual enzyme activity of some of them could be modulated to a different extent depending on the amounts of available chaperonins. Thus, some of the missense mutations may result in relatively high levels of residual enzyme activity, whereas the mutations leading to premature stop codons will result in no residual enzyme activity. By correlating the observed types of mutations identified to the clinical/biochemical data in the 14 patients in whom we identified both disease-causing mutations, we show that a genotype/phenotype correlation in MCAD deficiency is not straightforward. Different mutations may contribute with different susceptibilities for disease precipitation, when the patient is subjected to metabolic stress, but other genetic and environmental factors may play an equally important role.

[Severe cardiac failure in Kearns-Sayre syndrome]. / Insuficiencia cardíaca grave en el síndrome de Kearns-Sayre.

Mitochondrial disorders are a group of diseases that can affect virtually all organ systems. A 19 year old man was seen in 1993 with neurologic abnormalities consisting of impaired function of muscles, diplopia, progressive loss of vision, impaired phonation and swallowing, during the last 10 years. Physical examination disclosed moderate wasting of the four limb muscles, mild motor weakness of neck muscles, symmetrical hyporeflexia, cerebellar dysfunction, severe external ophtalmoplegia and ptosis. Fundii oculi examination showed retinitis pigmentosa. The electromyogram demonstrated myopathic changes with normal nerve conduction velocities. The cerebrospinal fluid was normal, except for a mild increase in lactic acid. Histochemical study of a muscle biopsy specimen demonstrated ragged red fibers and increase of the subsarcolemal oxidative activity of mitochondriae. The diagnosis of Kearns-Sayre disease was confirmed and he was discharged advising physical therapy. On February 1995, he was again admitted, this time with right cardiac failure and worsening of all his previous symptoms and signs. He complained of myalgias and his muscle weakness was more striking on clinical examination. Echocardiography showed biventricular dilatation and left ventricular hypertrophy with preserved systolic function. A new muscle biopsy revealed an heteroplasmic deletion of 5 Kb with 80% of mutant mitochondrial DNA. In brief, we report a patient with the clinical phenotype of Kearns-Sayre syndrome who presented an acute congestive cardiac failure due to cardiomyopathy, an association which has seldom been, reported in the literature.

[Autoimmune hypoglycemia syndrome with specific anti-human insulin antibodies]. / Síndrome de hipoglucemia autoinmune con anticuerpos específicos anti-insulina humana.

A 33 year old woman with episodes of severe hypoglycemia is presented. The studies showed anti-insulin antibodies and variable C-peptide levels. Circulating insulin measured after acid-ethanol extraction, was of 1,600 uU/ml and shown to be human insulin after characterization by HLPC. Specific anti-human insulin antibodies were of high affinity (Ka1: 6.20 x 10(10) M-1; Ka2: 2.42 x 10(9) M-1). A small cross-reactive porcine and bovine antibody subpopulation was also detected (IgG, light k type chain). Plasmapheresis was undertaken when symptoms were spontaneously declining and turned antibody title negative. Prolonged follow-up showed no relapse of this syndrome.