Mutation analysis in 54 propionic acidemia patients.

Deficiency of propionyl CoA carboxylase (PCC), a dodecamer of alpha and beta subunits, causes inherited propionic acidemia. We have studied, at the molecular level, PCC in 54 patients from 48 families comprised of 96 independent alleles. These patients of various ethnic backgrounds came from research centers and hospitals in Germany, Austria and Switzerland. The thorough clinical characterization of these patients was described in the accompanying paper (Grünert et al. 2012). In all 54 patients, many of whom originated from consanguineous families, the entire PCCB gene was examined by genomic DNA sequencing and in 39 individuals the PCCA gene was also studied. In three patients we found mutations in both PCC genes. In addition, in many patients RT-PCR analysis of lymphoblast RNA, lymphoblast enzyme assays, and expression of new mutations in E.coli were carried out. Eight new and eight previously detected mutations were identified in the PCCA gene while 15 new and 13 previously detected mutations were found in the PCCB gene. One missense mutation, p.V288I in the PCCB gene, when expressed in E.coli, yielded 134% of control activity and was consequently classified as a polymorphism in the coding region. Numerous new intronic polymorphisms in both PCC genes were identified. This study adds a considerable amount of new molecular data to the studies of this disease.

Propionic acidemia: neonatal versus selective metabolic screening.

BACKGROUND: Whereas propionic acidemia (PA) is a target disease of newborn screening (NBS) in many countries, it is not in others. Data on the benefit of NBS for PA are sparse. STUDY DESIGN: Twenty PA patients diagnosed through NBS were compared to 35 patients diagnosed by selective metabolic screening (SMS) prompted by clinical findings, family history, or routine laboratory test results. Clinical and biochemical data of patients from 16 metabolic centers in Germany, Austria, and Switzerland were evaluated retrospectively. Additionally, assessment of the intelligent quotient (IQ) was performed. In a second step, the number of PA patients who have died within the past 20 years was estimated based on information provided by the participating metabolic centers. RESULTS: Patients diagnosed through NBS had neither a milder clinical course regarding the number of metabolic crises nor a better neurological outcome. Among NBS patients, 63% were already symptomatic at the time of diagnosis, and <10% of all patients remained asymptomatic. Among all PA patients, 76% were found to be at least mildly mentally retarded, with an IQ <69. IQ was negatively correlated with the number of metabolic decompensations, but not simply with the patients' age. Physical development was also impaired in the majority of patients. Mortality rates tended to be lower in NBS patients compared with patients diagnosed by SMS. CONCLUSION: Early diagnosis of PA through NBS seems to be associated with a lower mortality rate. However, no significant benefit could be shown for surviving patients with regard to their clinical course, including the number of metabolic crises, physical and neurocognitive development, and long-term complications.

Dihydropyrimidine dehydrogenase deficiency caused by a novel genomic deletion c.505_513del of DPYD.

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disorder of the pyrimidine degradation pathway. In a patient presenting with convulsions, psychomotor retardation and Reye like syndrome, strongly elevated levels of uracil and thymine were detected in urine. No DPD activity could be detected in peripheral blood mononuclear cells. Analysis of the gene encoding DPD (DPYD) showed that the patient was homozygous for a novel c.505_513del (p.169_171del) mutation in exon 6 of DPYD.

Rapid quantification of conjugated and unconjugated bile acids and C27 precursors in dried blood spots and small volumes of serum.

The aim of the study was to develop a method for fast and reliable diagnosis of peroxisomal diseases and to facilitate differential diagnosis of cholestatic hepatopathy. For the quantification of bile acids and their conjugates as well as C(27) precursors di- and trihydroxycholestanoic acid (DHCA, THCA), in small pediatric blood samples we combined HPLC separation on a reverse-phase C18 column with ESI-MS/MS analysis in the negative ion mode. Analysis was done with good precision (CV 3,7%-11.1%) and sufficient sensitivity (LOQ: 11-91 nmol/L) without derivatization. Complete analysis of 17 free and conjugated bile acids from dried blood spots and 10 microL serum samples, respectively, was performed within 12 min. Measurement of conjugated primary bile acids plus DHCA and THCA as well as ursodeoxycholic acid was done in 4.5 min. In blood spots of healthy newborns, conjugated primary bile acids were found in the range of 0.01 to 2.01 micromol/L. Concentrations of C(27) precursors were below the detection limit in normal controls. DHCA and THCA were specifically elevated in cases of peroxysomal defects and one Zellweger patient.

Compliance to clinical guidelines determines outcome in glutaric aciduria type I in the era of newborn screening.

We report on a 4.5-year-old patient diagnosed with Glutaric aciduria type I (GAI), an autosomal recessive inborn error of lysine, hydroxylysine and tryptophan metabolism. Enzymatic assay in cultivated skin fibroblasts revealed complete absence of glutaryl-CoA dehydrogenase activity. All 11 Exons of the GCDH-Gen were sequenced and homozygosity for a yet undescribed mutation was identified. The patient was treated following the recently published guidelines for GA-I. Following this treatment regimen, the child developed normally without any manifest clinical crises. Our patient provides evidence that early commencement and strict adherence to treatment improves clinical outcome even in patients with complete absence of enzyme activity.

Peripheral neuropathy in a patient with D-2-hydroxyglutaric aciduria.

D-2-hydroxyglutaric aciduria (D-2-HGA; OMIM 600721) is a rare autosomal recessive neurometabolic disorder with a wide clinical spectrum. The severe phenotype is homogeneous and is characterized by early infantile-onset epileptic encephalopathy with hypotonia, delayed cerebral visual development, cardiomyopathy and facial dysmorphic features. The mild phenotype has a more variable clinical expression with hypotonia and developmental delay. We present peripheral neuropathy as an additional clinical and electrophysiological feature in a 16-year-old boy with a homozygous missense mutation in exon 3 of the D-2-hydroxyglutarate dehydrogenase gene (D2HGDH) at position c.458T>C. This mutation results in replacement of a methionine residue, which was highly conserved during evolution, by threonine (p.Met153Thr).

L-2-hydroxyglutaric aciduria and brain tumors in children with mutations in the L2HGDH gene: neuroimaging findings.

L-2-Hydroxyglutaric aciduria (L-2-HGA, MIM 236792) is a rare autosomal recessive neurodegenerative disorder characterized by psychomotor delay, cerebellar and extrapyramidal signs and subcortical leukoencephalopathy with basal ganglia and dentate nuclei involvement. Mutations in the gene L2HGDH ( C14ORF160/DURANIN/) have been identified as causative for L-2-HGA. A feature disproportionally associated with L-2-HGA is the development of malignant brain tumors. In our cohort of 40 patients with L-2-HGA, two developed medulloblastoma and glioblastoma multiforme during the course of the disease. Two missense mutations in two patients were identified in the L2HGDH gene in exon 3 (c.292C-->T) and in exon 7 (c.887T-->A). Both mutations were present in the homozygous state. Serial MR imaging findings as well as MR spectroscopy imaging is reported in a patient who developed glioblastoma multiforme.

The relation of cerebrospinal fluid and plasma glycine levels in propionic acidaemia, a 'ketotic hyperglycinaemia'.

The characteristic elevation of plasma glycine concentrations observed in propionic acidaemia (PA) and other 'ketotic hyperglycinaemias' has been attributed to secondary inhibition of the hepatic glycine cleavage system (GCS) by accumulating CoA derivatives of branched-chain amino acid metabolites. In nonketotic hyperglycinaemia (NKH), cerebrospinal fluid (CSF) and plasma glycine levels and their ratio are increased due to primary deficiency of central nervous system (CNS) as well as hepatic GCS. Whether the GCS in the CNS is also inhibited in PA is unclear, as there are scant data available on CSF glycine levels in this disorder. We studied the relation of CSF and plasma glycine levels in 6 paired samples from 4 PA patients, including one PA patient with bacterial meningitis who underwent ventriculoperitoneal shunting and multiple CSF analyses (n = 26). In contrast to the CSF glycine levels which were generally elevated in all four PA patients, the CSF/plasma glycine concentration ratios in paired samples were normal (0.016-0.029), with the exception of a single sample (0.132) with extremely high CSF protein concentration (2010 mg/L) during the course of meningitis indicating a disturbed blood-brain barrier. This finding of normal CSF/plasma glycine ratio in PA suggests that the observed elevations of CSF glycine levels are a reflection of the concurrent hyperglycinaemia resulting from secondary inhibition of hepatic GCS, but that brain GCS is not affected, in contrast to the situation in NKH. The neurological sequelae in PA are therefore unlikely to be related to disturbed glycine metabolism.

L-2-hydroxyglutaric aciduria: identification of ten novel mutations in the L2HGDH gene.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a metabolic disease with an autosomal recessive mode of inheritance. It was first reported in 1980. Patients with this disease have mutations in both alleles of the L2HDGH gene. The clinical presentation of individuals with L-2-HGA is somewhat variable, but affected individuals typically suffer from progressive neurodegeneration. Analysis of urinary organic acids reveals an increased signal of 2-hydroxyglutaric acid, mainly as the L-enantiomer. L-2-HGA is known to occur in individuals of various ethnic backgrounds, but up to now mutation analysis has been mainly focused on patients of Turkish and Portuguese origin. This led us to confirm the diagnosis on the DNA level and undertake the corresponding mutation analysis in individuals of diverse ethnicity previously diagnosed with L-2-HGA on the basis of urinary metabolites and clinical/neuroimaging data. In 24 individuals from 17 families with diverse ethnic and geographic origins, 13 different mutations were found, 10 of which have not been reported previously. At least eight of the patients were compound heterozygotes. The identification of two mutations (c.751C > T and c.905C > T in exon 7) in patients with different origins supports the view that they occurred independently in different families. In contrast, the mutation c.788C > T was detected in all six Venezuelan patients originating from the same Caribbean island of Margarita, but not in other patients, thus rendering a founder effect likely. None of the mutations was found in the control population, indicating that they are most probably causative. Mutation analysis may improve the quality of diagnosis and prenatal diagnosis of L-2-HGA.

Possible genotype-phenotype correlations in children with mild clinical course of Canavan disease.

Canavan disease is characterised as a rare, neurodegenerative disease that usually causes death in early childhood. It is an autosomal recessive disorder due to an aspartoacylase (ASPA) deficiency. The causative gene has been mapped to chromosome 17 pter-p13. Here we describe three affected children from two Greek families with an unusually mild course of Canavan disease. All children presented with muscular hypotonia and macrocephaly. Diagnosis was based on elevated N-acetylaspartate in urine, reduced aspartoacylase activity in fibroblasts, and marked white matter changes on cerebral imaging. All three affected individuals exhibited continuous psychomotor development without any regression. Genetic analyses revealed compound heterozygous mutations (Y288 C; F295 S) in two individuals. The Y288 C variant was previously described in a child with macrocephaly, mild developmental delay, increased signal intensity in the basal ganglia, partial cortical blindness and retinitis pigmentosa, and slightly elevated N-acetylaspartate in the urine. Demonstration of the same variant in two unusually mildly affected Canavan disease patients and absence of this variant in 154 control chromosomes suggest a possible pathogenic role in mild Canavan disease. In the third individual, two homozygous sequence variants were identified, which comprise the known G274R mutation and a novel K213E variant.

Propionic acidemia revisited: a workshop report.

Propionic acidemia (PA) is one of the most frequent organic acidurias, but information on the outcome of individuals with PA is rather limited. We present data of 49 patients with PA, which were gathered from 18 metabolic centers throughout Central Europe on the occasion of an international workshop. All patients were identified by selective metabolic screening, and 86% of them were classified as having early-onset PA owing to their presentation with clinical symptoms within the first 90 days of life. Mortality rate was one third, and details of symptoms and treatment of the surviving patients are discussed. The great variation of phenotypic expression of the disease and different therapeutic strategies (especially in regard to the degree of protein restriction) used at the various institutions involved in this study imply the need for a registry of PA patients and for a multicenter prospective treatment study.

Isobutyryl-CoA dehydrogenase deficiency: isobutyrylglycinuria and ACAD8 gene mutations in two infants.

Isobutyryl-CoA dehydrogenase (IBD) is an enzyme involved in the catabolism of the branched-chain amino acid valine. We report a third and a fourth child with IBD deficiency who were both detected during newborn screening with tandem mass spectrometry and so far do not receive any treatment. The diagnosis was confirmed by biochemical and molecular studies. One of the children is homozygous for the mutation M128I in the ACAD8 gene, which is predicted to affect the substrate binding cavity. The other child is compound heterozygous for a frameshift mutation F33fsins and a missense mutation V203I. It is as yet uncertain whether IBD deficiency may cause significant morbidity in affected children and whether treatment is necessary. In view of the limited experience worldwide, careful monitoring of the children is recommended.

Trichothiodystrophy: quantification of cysteine in human hair and nails by application of sodium azide-dependent oxidation to cysteic acid.

The term "trichothiodystrophy" (TTD) covers several autosomal recessive diseases whose diagnostic hallmark is short, brittle hair low in sulfur and cystine because of impaired synthesis of high-sulfur matrix protein. Clinical symptoms associated with TTD represent a variable range of abnormalities in organs derived from ectoderm and neuroectoderm. Important laboratory tests of the hair for the diagnosis of TTD comprise polarizing microscopy ("tiger-tail" pattern), electron microscopy, and amino acids analysis of hydrolyzed hair with a special focus on cystine. However, only very few institutions determine the amino acid composition of human hair and nail clippings, which requires special sample preparation including hydrolysis. If no special precautions are taken, quantification of cysteine and cystine becomes inaccurate because of decomposition of these residues during hydrolysis. We therefore performed the sample work-up with azide-dependent oxidation which we have for the first time adapted for analysis of hair and nail clippings. With our control and parent data resembling published data on hair and nail samples, we obtained a decreased proportion of cysteine (half cystine, determined as cysteic acid) in materials obtained from a boy with TTD. Clearly, the method for the quantification of cysteine following sodium azide-dependent oxidation is a suitable and rather convenient approach to the quantification of cyst(e)ine and other amino acids in hair and nail proteins, and is a valuable contribution to the diagnosis of TTD.

Effects of all-trans-retinoic acid and all-trans-retinoyl glucuronide in two in vitro systems of distinct biological complexity.

In vitro systems are widely used to evaluate the embryotoxic potential of retinoids. The effective concentrations of these retinoids, however, are not consistent in the various in vitro systems used in evaluating embryotoxicity. This may be explained by the different level of complexity for each individual system, which may lead to different concentrations of the substances in the target tissues. To verify this hypothesis we have compared two in vitro systems of distinct biological complexity: the rat whole embryo culture system, and the mouse limb bud organ culture system. The lipid soluble, teratogenic retinoid all-trans-retinoic acid (ATRA), and all-trans-retinoyl-beta-D-glucuronide (ATRAG), an endogenous, water-soluble and biologically active retinoid with limited placental transfer, were compared with regard to their embryotoxic potential in vitro. In both in vitro systems, ATRAG showed a lower degree of embryotoxicity than ATRA. In the limb bud organ culture, ATRAG revealed only slightly less toxicity than ATRA, whereas the effective concentrations of the two compounds in the whole embryo culture system differed by almost two orders of magnitude. During incubation with ATRAG, ATRA is generated by hydrolysis and is found in culture media and exposed tissues. The presence of membrane barriers around the developing embryo in the whole embryo culture system possibly prevents the transfer of ATRAG to the embryo and, therefore, its exposure to the active hydrolysis product ATRA. From these results we conclude that analysis of retinoid concentrations in the culture media and in the exposed tissues is essential for the interpretation of results obtained from in vitro toxicity testing.