Abstract: Hyperhomocysteinemia has occasionally been reported in patients with phenylketonuria (PKU) and B-vitamin deficiency. In our study total homocysteine (tHcy) and B-vitamins were measured in treated PKU patients and healthy controls. In the patients, dietary parameters and genetic polymorphisms affecting the Hcy pathway were investigated to identify parameters modulating tHcy. A case control study including 37 PKU patients and 63 healthy controls was conducted. t-Tests for independent samples were used to test between groups. Multiple regressions with tHcy as dependent variable were calculated. Hardy-Weinberg expectations were tested against the observed distribution of genotypes applying the Chi-square goodness-of-fit method. THcy concentrations were not significantly different (p=0.059) while folate and cobalamin (Cbl) concentrations were significantly higher in PKU patients compared to controls. However, 29.7% of patients had tHcy concentrations >97th centile. THcy did not vary with age nor correlate with folate and Cbl concentrations probably due to high saturatory levels. The presence of genetic polymorphisms had no impact on tHcy. In conclusion, in PKU patients treated with amino acid mixtures enriched with B-vitamins, tHcy is not significantly higher than in healthy controls, but tHcy concentrations exceed the 97th centile in about one third of patients. Even higher B-vitamin saturation may be required to further decrease tHcy concentrations and factors generally influencing tHcy such as betaine are to be investigated in PKU patients in the future.
Abstract: Pompe disease is an autosomal recessive disorder of glycogen metabolism caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). It presents at any age, with variable rates of progression ranging from a rapidly progressive course, often fatal by one-year of age, to a more slowly, but nevertheless relentlessly progressive course, resulting in significant morbidity and premature mortality. In infants, early initiation of enzyme replacement therapy is needed to gain the maximum therapeutic benefit, underscoring the need for early diagnosis. Several new methods for measuring GAA activity have been developed. The Pompe Disease Diagnostic Working Group met to review data generated using the new methods, and to establish a consensus regarding the application of the methods for the laboratory diagnosis of Pompe disease. Skin fibroblasts and muscle biopsy have traditionally been the samples of choice for measuring GAA activity. However, new methods using blood samples are rapidly becoming adopted because of their speed and convenience. Measuring GAA activity in blood samples should be performed under acidic conditions (pH 3.8-4.0), using up to 2mM of the synthetic substrate 4-methylumbelliferyl-alpha-d-glucoside or glycogen (50mg/mL), in the presence of acarbose (3-9muM) to inhibit the isoenzyme maltase-glucoamylase. The activity of a reference enzyme should also be measured to confirm the quality of the sample. A second test should be done to support the diagnosis of Pompe disease until a program for external quality assurance and proficiency testing of the enzymatic diagnosis in blood is established.
Abstract: Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a rare X-linked recessive disease caused by deficiency of the lysosomal enzyme iduronate-2-sulphatase, leading to progressive accumulation of glycosaminoglycans in nearly all cell types, tissues and organs. Clinical manifestations include severe airway obstruction, skeletal deformities, cardiomyopathy and, in most patients, neurological decline. Death usually occurs in the second decade of life, although some patients with less severe disease have survived into their fifth or sixth decade. Until recently, there has been no effective therapy for MPS II, and care has been palliative. Enzyme replacement therapy (ERT) with recombinant human iduronate-2-sulphatase (idursulfase), however, has now been introduced. Weekly intravenous infusions of idursulfase have been shown to improve many of the signs and symptoms and overall wellbeing in patients with MPS II. This paper provides an overview of the clinical manifestations, diagnosis and symptomatic management of patients with MPS II and provides recommendations for the use of ERT. The issue of treating very young patients and those with CNS involvement is also discussed. ERT with idursulfase has the potential to benefit many patients with MPS II, especially if started early in the course of the disease.
Abstract: The analysis of urinary organic acids is crucial for the diagnosis of many inborn errors of metabolism. A vital part of the analytical process is the extraction procedure. The sensitivity and linearity of the analysis of 26 diagnostically important urinary metabolites with tetrahydrofuran (THF) and ethyl acetate (EtOAc) as extraction solvents were determined by gas chromatography-mass spectrometry. Good linearity (r (2) > 0.90) was observed for all of the compounds in the investigated concentration range (290-900 mumol/L) for both solvents. For less polar compounds, THF extraction yielded lower or similar sensitivities as compared with EtOAc (sensitivity ratio: 0.6-1.3). For more polar compounds, however, much higher sensitivities were observed when THF was used (sensitivity ratio: 1.8-17.2). Our results provide information concerning the use of THF for the sensitive quantitative analysis of polar urinary metabolites which are difficult to quantify using EtOAc.
Abstract: Childhood T-cell precursor acute lymphoblastic leukemia (TCP ALL) is an aggressive disease with a presumably short latency that differs in many biologic respects from B-cell precursor (BCP) ALL. We therefore addressed the issue of in utero origin of this particular type of leukemia by tracing oncogenic mutations and clone-specific molecular markers back to birth. These markers included various first- and second-hit genetic alterations (TCRD-LMO2 breakpoint regions, n = 2; TAL1 deletions, n = 3; Notch1 mutations, n = 1) and nononcogenic T-cell receptor rearrangements (n = 13) that were derived from leukemias of 16 children who were 1.5 to 11.2 years old at diagnosis of leukemia. Despite highly sensitive polymerase chain reaction (PCR) approaches (1 cell with a specific marker among 100,000 normal cells), we identified the leukemic clone in the neonatal blood spots in only 1 young child. These data suggest that in contrast to BCP ALL most TCP ALL cases are initiated after birth.
Abstract: BACKGROUND: Dried blood filter cards, collected for newborn screening, are often stored for long periods of time. They may be suitable for the retrospective diagnosis of inborn errors of metabolism, but no data are currently available on the long-term stability of amino acids and acylcarnitine species. METHODS: We analyzed amino acids and acylcarnitines by tandem mass spectrometry in 660 anonymous, randomly selected filter cards from 1989 through 2004. We assessed long-term stability of metabolites by linear regression and estimated annual decrease of concentration for each metabolite. RESULTS: Concentrations of free carnitine increased by 7.6% per year during the first 5 years of storage and decreased by 1.4% per year thereafter. Alanine, arginine, leucine, methionine, and phenylalanine decreased by 6.5%, 3.3%, 3.1%, 7.3%, and 5.7% per year, respectively. Acetylcarnitine, propionylcarnitine, citrulline, glycine, and ornithine decreased by 18.5%, 27.4%, 8.1%, 14.7%, and 16.3% per year during the first 5 years, respectively; thereafter the decline was more gradual. Tyrosine decreased by 1.7% per year during the first 5 years and 7.9% per year thereafter. We could not analyze medium- and long-chain acylcarnitine species because of low physiological concentrations. CONCLUSIONS: Estimation of the annual decrease of metabolites may allow for the retrospective diagnosis of inborn errors of metabolism in filter cards that have been stored for long periods of time.
Abstract: This study aimed to estimate the number of infants who died of unrecognized congenital adrenal hyperplasia (CAH) in Austria and the Czech Republic within the past 13 years, before the introduction of adequate neonatal screening. The study was based on retrospective analysis of neonatal screening cards of 242 infants who died suddenly between 7 days and 12 months of age and whose cause of death could not be identified. 17-hydroxyprogesterone (17-OHP) was measured by fluoroimmunoassay and positive samples were subsequently genotyped. Three infants out of 242 may have had unrecognized CAH due to CYP21 (steroid 21-hydroxylase) gene defect. Their newborn 17-OHP levels and CYP21 genotypes were 706 nmol/l and del/conv//del/conv, 53 nmol/l and I2//I2, and 811 nmol/l and I2//Gln318stop, respectively. CAH due to CYP21 defect can lead to sudden unexpected death without prior symptoms typical for the condition. Hence, newborn screening would have prevented these deaths had it been available. In addition, we have shown that the I2 point mutation that is expected to lead to simple virilizing form may lead to a fatal outcome.
Abstract: Mutations in the gene for 4-hydroxyphenylpyruvic acid dioxygenase (HPD) cause either autosomal recessive tyrosinemia type III or autosomal dominant hawkinsinuria. We report a 6-month-old Indian infant who is compound heterozygous for both alleles and who has hawkinsinuria but not tyrosinemia type III based on biochemical investigations. The HPD gene was directly sequenced in the proband and both parents. The mechanistic model of the enzymatic function was built using the known structure of rat HPD. We identified a novel hawkinsinuria mutation, Asn241Ser, and a known tyrosinemia type III mutation, Ile335Met, in trans configuration. The structural analysis of the active site revealed that the IIe335Met mutation is situated in the close vicinity of one of the two highly conserved Phe rings which stack with the phenol ring of the substrate. The Asn241Ser mutation is situated further away from the 4-hydroxyphenylpyruvate binding pocket. Assuming that Asn241Ser causes hawkinsinuria, we propose positioning the dioxygen molecule in the HPD-catalyzed reaction as a novel role for the Asn residue. The IIe335Met allele is equivalent to a null mutation while the Asn241Ser allele results in a partially active enzyme with an uncoupled turnover causing hawkinsinuria.
Abstract: The authors studied methionine and creatine metabolism in females with Rett syndrome. Plasma metabolites (including methionine, homocysteine, guanidinoacetate) and urine creatine/creatinine ratios in 29 females with Rett syndrome were within the age-appropriate range. Although the authors have not been able to identify any abnormalities, it can be speculated that patients with Rett syndrome may benefit from dietary intervention to increase the supply of labile methyl groups to affected tissues.
Abstract: Guanidinoacetate methyltransferase deficiency typically presents with muscular hypotonia, global developmental delay, extrapyramidal signs, and seizures during infancy and childhood. The authors report a 5-year-old child with guanidinoacetate methyltransferase deficiency who presented with severe speech delay, emphasizing the importance of an early screening for disorders of creatine synthesis and transport in every infant or child with isolated speech delay of unknown cause.
Abstract: By January 2007 seven European countries had expanded, and more are considering the expansion of their newborn screening programmes by inclusion of ESI tandem mass spectrometry. We present an overview of the current status of expanded newborn screening programmes in Europe. While the first pilot programmes were initiated in 1998 in Germany, most countries started within the last 3 years. The number of disorders screened for by MS/MS ranges from two disorders (phenylketonuria and medium-chain acyl-CoA dehydrogenase deficiency) in some countries to 20 in others. The number of live births investigated per screening centre varies from 18,000 to 77,000. Few programmes have reported the number of positively identified cases and technical data, although many participate in quality assurance and proficiency test schemes. Given the relatively common genetic background of most European populations and similar health care systems, the reasons for the differences observed appear arbitrary and contrary to the optimal benefit of this important preventive health measure. Harmonization of disease screening panels, spectrum of metabolites analysed, sizes of screening laboratories, analytical procedures, follow-up management and proficiency and quality testing is urgently warranted on the European level. This will hopefully occur before screening by novel applications of tandem mass spectrometry for additional groups of disorders including lysosomal storage disorders and X-linked adrenoleukodystrophy are implemented.
Abstract: OBJECTIVES: Sixty three healthy subjects were measured to assess dependence of brain metabolites on age using short- and long echo time spectroscopy in different brain regions. MATERIAL AND METHODS: Younger and elderly humans were measured with long echo time (TE=135ms) 3D-MR-spectroscopic imaging (MRSI) (10 subjects) and with ultra-short echo (TE=11ms) time 2D-MRSI (7 subjects). In addition, results from single voxel (1)H-spectroscopy (TE=20ms) of two cohorts of 46 healthy subjects were retrospectively correlated with age. RESULTS: 3D-MR SI revealed reduced NAA/Cr in the older group in the frontal lobe (-22%; p<0.01), parietal lobe (-28%; p<0.01) and semiovale (-9%; p<0.01) compared to the younger group. Cho/Cr was elevated in the semiovale (+35%; p<0.01) and in the n. lentiformis (+42%; p<0.01) in the older group. NAA/Cho was reduced in all regions measured, except the thalamus, in the older group compared to the younger group (from -21 to -49%; p<0.01). 2D-MRSI revealed decreased total NAA (-3.1% per decade; p<0.01) and NAA/Cr (-3.8% per decade; p<0.01), increased total Cho (+3.6% per decade; p<0.01) and Cho/Cr (+4.6% per decade; p<0.01) and increased total myo-Inositol (mI, +4.7% per decade; p<0.01) and mI/Cr (+5.4% per decade; p<0.01) and decreased NAA/Cho (-8% per decade; p<0.01) in semiovale WM. Results from single voxel spectroscopy revealed a significantly negative correlation of NAA/Cho in frontal (-13% per decade; p<0.01) and in temporal lobe (-7.4% per decade; p<0.01) as well as increased total Cr (10% per decade; p<0.01) in frontal lobe. Other results from single voxel measurements were not significant, but trends were comparable to that from multivoxel spectroscopy. CONCLUSION: Age-related changes measured with long echo time and short echo time 1H-MRS were comparable and cannot, therefore, be caused by different T2 relaxation times in young and old subjects, as suggested previously.
Abstract: BACKGROUND: Detection of amino acids (AA), acylcarnitines (AC), and guanidinoacetate (GAA) in dried blood spots by tandem mass spectrometry has made it possible to detect different inborn errors of metabolism in neonatal screening programs. Despite its proven sensitivity many issues related to sample preparation remain unsolved. Hematocrit has a profound effect on blood viscosity, and may thereby influence flux and diffusion properties of the blood. As newborn infants show a considerable interindividual variability of hematocrit levels, we investigated its effect on levels of AA and AC in dried blood spots. METHODS: Blood samples with defined hematocrit levels (20%, 30%, 40%, 50%, 60%) were produced by diluting blood cells with plasma from a single donor. Forty dried blood spots were made for each hematocrit level and a central as well as a peripheral 3 mm disk was punched and analysed for AA, AC, and GAA, respectively. RESULTS: Levels of most AA and GAA increased significantly with increasing hematocrit (p<0.001), while the effect of hematocrit on some AA was less pronounced. Total AC, free carnitine, some long, medium and short chain AC correlated positively with hematocrit levels (p<0.001). In samples with low hematocrit, levels of most AA and free carnitine were higher in the peripheral than in the central disk (p<0.0001). CONCLUSIONS: Both hematocrit and position of the disk within the dried blood spot have a significant and sometimes additive effect on levels of AA, AC and GAA in dried blood spots. Theoretically, diagnoses may be missed depending on hematocrit and position of the disk.
Abstract: AIMS: To investigate glucose and leucine kinetics in association with metabolic and endocrine investigations in children with ketotic hypoglycaemia (KH) in order to elucidate the underlying pathophysiology. METHODS: Prospective interventional study using stable isotope tracer in nine children (mean age 4.23 years, range 0.9-9.8 years; seven males) with KH and 11 controls (mean age 4.57 years, range 0.16-12.3 years; four males). RESULTS: Plasma insulin levels were significantly lower in KH compared to subjects in the non-KH group. Plasma ketone body levels were significantly higher in KH than in non-KH. Basal metabolic rate was significantly higher in subjects with KH (45.48+/-7.41 v 31.81+/-6.72 kcal/kg/day) but the respiratory quotients were similar in both groups (KH v non-KH, 0.84+/-0.05 v 0.8+/-0.04. Leucine oxidation rates were significantly lower in children with KH (12.25+/-6.25 v 31.96+/-8.59 micromol/kg/h). Hepatic glucose production rates were also significantly lower in KH (3.84+/-0.46 v 6.6+/-0.59 mg/kg/min). CONCLUSIONS: KH is caused by a failure to sustain hepatic glucose production rather than by increased glucose oxidation rates. Energy demand is significantly increased, whereas leucine oxidation is reduced.
Abstract: PURPOSE: Human mannose-binding lectin, encoded by the MBL2 gene, is an important component of innate immunity and an important regulator of inflammatory processes. MBL2 gene polymorphisms are associated with an increased risk of neonatal infections and some data suggest a relation between the maternal MBL2 genotype and the risk of premature delivery. In this study, we evaluated whether there is an association between the fetal MBL2 genotype and prematurity. METHODS: A microarray-based on-chip PCR method was used to simultaneously detect five common MBL2 polymorphisms (codon 52, 54, 57; promoter -550, -221) in 204 DNA samples isolated from archival blood cards. MBL2 genotypes of infants born before the 36th week of pregnancy (N = 102) were compared to a control group of infants born at term after the 37th week (N = 102). RESULTS: The frequency of the codon 52 polymorphism was significantly higher in the pre-term group compared to the term group (10.8% versus 4.9%, P = 0.04), while the frequency of the codon 54 polymorphism was equal in both groups (11.3% versus 11.8%). Interestingly, carriers of genotypes (O/O) likely conferring deficient MBL plasma levels were more common in the group of premature birth (9.8% versus 2.9%, P = 0.05), while the promoter -550 C/C genotype was underrepresented in the pre-term birth group (24.5% versus 39.2%, P = 0.03). CONCLUSION: Our data add to the knowledge about genetic predisposition to prematurity and suggest that the fetal MBL2 genotype might be an additional genetic factor contributing to the risk of premature delivery.
Abstract: The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.
Abstract: Methylation is an important aspect of many fundamental biological processes including creatine biosynthesis. We studied five patients with an inborn error of cobalamin metabolism to characterize the relation between homocysteine and creatine metabolism. Plasma guanidinoacetate concentrations were increased, 14.9 +/- 4.8 micromol/L (p < 0.0001), whereas plasma creatine concentrations were in the low reference range, 43.8 +/- 20.7 micromol/L (p = not significant). Individuals with combined methylmalonic aciduria and homocystinuria have a functional impairment of the creatine synthetic pathway probably secondary to a relative depletion of labile methyl groups. The neurotoxic effects of guanidinoacetate may be partly responsible for the observed neurological phenotype.
Abstract: Elevated plasma homocysteine levels may be an independent risk factor for premature vascular disease. Early detection and population screening are warranted to recognise hyperhomocysteinemia and initiate homocysteine lowering therapy. Current methods for homocysteine analysis are time consuming, labor intensive and/or expensive. We developed a sensitive and fast method for homocysteine analysis based on tandem mass spectrometry that avoids the need for derivatization and preanalytical chromatography.
Abstract: AIM: Fatty acid beta-oxidation defects comprise a heterogeneous group of disorders that may precipitate acute life threatening metabolic crises particularly during catabolic episodes. Several studies have demonstrated a possible association between fatty acid beta-oxidation defects, including long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and severe pregnancy complications. However, the precise percentage of women with haemolysis, elevated liver enzymes, low platelets (HELLP) syndrome associated with foetal fatty acid beta-oxidation defects is not known. METHODS: We carried out a multicentre retrospective study on 88 infants, born to women with HELLP syndrome. Acylcarnitine profiles from blood dried on filter paper cards were analysed by tandem mass spectrometry for the diagnosis of fatty acid beta-oxidation defects. In addition, we screened for the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation using a standard restriction fragment length polymorphism polymerase chain reaction method. RESULTS: None of the infants studied carried the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation. There was no evidence of fatty acid beta-oxidation defects, including long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, as expected by unremarkable acylcarnitine profiles, while three infants with fatty acid beta-oxidation defects were diagnosed in the control group. CONCLUSIONS: Neither foetal long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, including heterozygosity for the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation, nor fatty acid beta-oxidation defects in general are a major risk factor for HELLP syndrome in Austria.
Abstract: Mucopolysaccharidosis type II (Hunter disease, iduronate-2-sulfatase deficiency) was diagnosed in a 4-year-old female by demonstrating low iduronate-2-sulfatase activity both in leukocytes and fibroblasts and by the presence of a novel, complex rearrangement of the iduronate-2-sulfatase gene in heterozygous form. Mucopolysaccharidosis type II is inherited in an X-linked recessive manner and consequently females are rare. The disease phenotype in this case is due to complete unilateral inactivation of the nonmutant paternal X chromosome of the patient. The case presented here underscores the fact that a diagnosis of mucopolysaccharidosis type II should be suspected in any female who presents with the relevant clinical symptoms.
Abstract: Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive error of creatine synthesis characterized by cerebral creatine deficiency, accumulation of guanidinoacetate, mental retardation, epilepsy, and extrapyramidal symptoms. To date, 14 mutations of the GAMT gene in 27 patients have been reported. Mutation analysis was done using direct sequencing of PCR products and denaturing gradient gel electrophoresis in combination with direct sequencing. In contrast, we evaluated the efficiency of a newly developed DHPLC method to detect mutations in the GAMT gene by analysing DNA from 14 GAMT patients with known mutations. PCR amplification of both patient and control DNA was followed by formation of homoduplices and heteroduplices, and their detection by DHPLC. DHPLC identified all mutations tested and is the preferred choice of analytical method.
Abstract: Neurologic disease in glutaryl-CoA dehydrogenase (GCDH) deficiency usually presents with acute encephalopathic crises before 2 years of age. The authors report two previously asymptomatic patients with macrocephaly presenting with progressive neurologic deterioration and a severe leukoencephalopathy during adolescence or adulthood.
Abstract: OBJECTIVE: To evaluate prenatal treatment with hydroxycobalamin (OH-Cbl) in a pregnancy at risk for a severe form of the cobalamin C defect and postnatal treatment of the affected child. STUDY DESIGN: Observational study with non-randomized intervention. RESULTS: In contrast to reported pregnancies with affected fetuses in which maternal methylmalonic aciduria was found in the last trimester of pregnancy, there was no maternal methylmalonic aciduria in our case, given prenatal treatment with intramuscular OH-Cbl. We did not find that the concentration of odd long-chain fatty acids in cord blood erythrocytes reflects fetal methylmalonic academia. After birth, the infant was treated with intramuscular OH-Cbl and oral carnitine. Oral folate and betaine were added as adjunct therapy to decrease plasma total homocysteine. Because of inadequate metabolic control, a diet reduced in natural protein was introduced. The child had normal developmental milestones but had nystagmus, hyperpigmented retinopathy, and discrete truncal muscular hypotonia. CONCLUSIONS: Despite prenatal and postnatal treatment, adequate metabolic control, absence of metabolic crises, and normal developmental milestones, this patient with the cobalamin C defect had characteristic symptoms of the disease.
Abstract: BACKGROUND: Recurrent and persistent hypoketotic, hypofattyacidaemic hypoglycaemia in infancy and childhood is most frequently due to hyperinsulinism of infancy. This biochemical profile can also be due to non-islet cell tumour hypoglycaemia or circulating insulin-receptor autoantibodies. Hyperinsulinaemic hypoglycaemia is also seen in children with the Beckwith-Wiedemann syndrome, where it is usually transient. METHODS/RESULTS: We report a novel case of child with hemihypertrophy and severe persistent hypoketotic, hypofattyacidaemic hypoinsulinaemic hypoglycaemia. No 'big' pro-IGF2 forms or circulating insulin-receptor antibodies were found. Glucose and protein isotope turnover studies showed marked suppression of hepatic glucose production during fasting. There was no evidence for constitutive autophosphorylation of the insulin or IGF-1 receptor, and no evidence for up-regulation of IGF-1 receptor. CONCLUSION: The precise pathophysiology of this novel case is still unclear.
Abstract: Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive error of creatine synthesis characterized by cerebral creatine deficiency, accumulation of guanidinoacetate, mental retardation, epilepsy and extrapyramidal signs. So far, six mutations have been identified in seven patients. We investigated seven new patients by screening the promoter, 3'UTR, and six exons and exon/intron boundaries using direct sequencing and denaturing gradient gel electrophoresis. The clinical and biochemical phenotype was characterized by scoring the degree of main clinical manifestations and by determination of urinary guanidinoacetate concentrations and of GAMT activity in fibroblasts / lymphoblasts, respectively. We identified 7 novel mutations, including c.64dupG (exon 1; 4/14 alleles); c.59G>C (exon 1; 3/14 alleles); c.491delG (exon 5; 2/14 alleles); c.160G>C (exon 1; 2/14 alleles); and c.152A>C (exon 1; 1/14 alleles); c.526dupG (exon 5; 1/14 alleles); c.521G>A (exon 5; 1/14 alleles), and two polymorphisms c.626C>T (exon 6) and c.459+71G>A (intron 4). Frameshift and missense mutations in exon 1 were prevalent in the 4 patients with the severe phenotype, however a clear genotype-phenotype correlation has not been established in the limited number of patients characterized so far.
Abstract: Nuclear magnetic resonance (NMR) spectroscopy is a safe, noninvasive method that is the preferred technique for in vivo analysis of specific chemical compounds in localized brain regions. Besides quantification of compounds, NMR spectroscopy allows the detailed analysis of neurotransmitter, glucose and lactate metabolism following peripheral infusions of stable isotopically labelled precursors. The latter has been successfully applied to patients with different neurological disease states not including glutaryl-CoA dehydrogenase (GCDH) deficiency. In contrast, single patients with GCDH deficiency who were neurologically unremarkable have been studied with conflicting results. One patient was shown to have an increase in intracerebral creatine and phosphocreatine concentrations, while the second studied had unremarkable levels. In a 15-year-old patient, we were able to demonstrate elevated levels of intracerebral lactate and elevated choline/N -acetylaspartate ratios, indicating potentially increased myelin turnover and reduced neuronal integrity in periventricular white matter. Interestingly, spectra in basal ganglia were within normal limits. Systematic studies to address well-defined questions in GCDH deficiency are urgently needed. In particular, analysis of in vivo neurotransmitter metabolism following administration of isotopically labelled precursors in patients with GCDH deficiency, both when metabolically stable and when unstable, may help to advance our understanding of the pathophysiology of GCDH deficiency.
Abstract: Creatine deficiency syndromes are a newly described group of inborn errors of creatine synthesis (arginine:glycine amidinotransferase (AGAT) deficiency and guanidinoaceteate methyltransferase (GAMT) deficiency) and creatine transport (creatine transporter (CRTR) deficiency). The common clinical denominator of creatine deficiency syndromes is mental retardation and epilepsy, suggesting the main involvement of cerebral grey matter (grey matter disease). Patients with GAMT deficiency exhibit a more complex clinical phenotype with dystonic hyperkinetic movement disorder and epilepsy that in some cases is unresponsive to pharmacological treatment. The common biochemical denominator of creatine deficiency syndromes is cerebral creatine deficiency which is demonstrated by in vivo proton magnetic resonance spectroscopy. Measurement of guanidinoacetate in body fluids may discriminate GAMT (high concentration), AGAT (low concentration) and CRTR (normal concentration). Further biochemical characteristics include changes in creatine and creatinine concentrations in body fluids. GAMT and AGAT deficiency are treatable by oral creatine supplementation, while patients with CRTR deficiency do not respond to this type of treatment. Further recognition of patients will be of major importance for the estimation of the frequency, for the understanding of phenotypic variations and for treatment strategies.
Abstract: In a male patient with hereditary tyrosinaemia type I (HTI), NTBC [2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexandion] treatment and a diet low in phenylalanine and tyrosine were started at the age of 4 wk. At the recommended average dosage (1 mg kg(-1)), liver failure improved transiently. After 4 mo of treatment, with increased body weight, the dose had decreased to 0.7 mg kg(-1), and diffuse cirrhotic changes in liver parenchyma and multiple nodules were visualized by ultrasonography. Multiple nodules in the liver parenchyma were differentiated from hepatocellular carcinoma by magnetic resonance imaging (MRI) using mangafodipir trisodium as a paramagnetic liver-specific contrast agent. Augmentation of NTBC dosage resulted in a decrease in serum alpha-fetoprotein levels and in significant regression of liver nodules on MRI. CONCLUSION: In HTI patients with a poor response to NTBC treatment and/or development of cirrhotic changes of liver parenchyma, augmentation of the recommended NTBC dosage may result in significant improvement of symptoms.
Abstract: To explore the pathogenesis of cystathionine beta-synthase (CBS) deficiency and to test the efficacy of pharmacological therapy we examined a panel of metabolites in nine homocystinuric patients under treated and/or untreated conditions. Off pharmacological treatment, the biochemical phenotype was characterized by accumulation of plasma total homocysteine (median 135 micromol/L) and blood S -adenosylhomocysteine (median 246 nmol/L), and by normal levels of guanidinoacetate and creatine. In addition, enhanced remethylation was demonstrated by low serine level (median 81 micromol/L), and by increased concentration of methionine (median 76 micromol/L) and N -methylglycine (median 6.8 micromol/L). Despite the substantially blocked transsulphuration, which was evidenced by undetectable cystathionine and severely decreased total cysteine levels (median 102 micromol/L), blood glutathione was surprisingly not depleted (median 1155 micromol/L). In 5 patients in whom pharmacological treatment was withdrawn, the differences of median plasma total homocysteine levels (125 micromol/L after withdrawal versus 33 micromol/L under treatment conditions), total cysteine levels (139 versus 211 micromol/L) and plasma serine levels (53 versus 103 micromol/L) on and off treatment demonstrated the efficacy of long-term pyridoxine/betaine administration ( p <0.05). The treatment also decreased blood S -adenosylhomocysteine level (133 versus 59 nmol/L) with a borderline significance. In summary,our study shows that conventional treatment of CBS deficiency by diet and pyridoxine/betaine normalizes many but not all metabolic abnormalities associated with CBS deficiency. We propose that the finding of low plasma serine concentration in untreated CBS-deficient patients merits further exploration since supplementation with serine might be a novel and safe component of treatment of homocystinuria.
Abstract: Familial complex chromosomal rearrangements (CCRs) are rare and tend to involve fewer breakpoints and fewer chromosomes than CCRs that are de novo in origin. We report on a CCR identified in a child with congenital heart disease and dysmorphic features. Initially, the child's karyotype was thought to involve a straightforward three-way translocation between chromosomes 3, 8, and 16. However, after analyzing the mother's chromosomes, the mother was found to have a more complex rearrangement that resulted in a recombinant chromosome in the child. The mother's karyotype included an inverted chromosome 2 and multiple translocations involving chromosomes 3, 5, 8, and 16. No evidence of deletion or duplication that could account for the clinical findings in the child was identified.
Abstract: Combined methylmalonicaciduria and homocystinuria is a disorder of intracellular cobalamin metabolism that remains a challenge to the physician unfamiliar with the diagnosis. We have followed six patients with combined methylmalonicaciduria and homocystinuria (four males, two females, age 4.2-24 years) for a median of 6.5 years (range 4-9 years). Age at diagnosis was between 18 days and 14 months in early-onset cases (n = 4) and 15 and 19 years in late-onset cases. Predominant clinical features include microcephaly, hydrocephalus, seizures, and white-matter changes on magnetic resonance imaging in early-onset cases. The white-matter changes may be secondary to impaired methylation owing to a lack of readily available methyl groups. Spastic quadriparesis and diplegia are long-term sequelae in late-onset cases. Management consists of hydroxycobalamin intramuscular injections, oral folate, betaine, and carnitine supplementation. Dietary protein restriction may be necessary when metabolic control remains difficult. The implementation of an emergency regimen should alleviate episodes of metabolic decompensation and reduce the rate of hospital admissions.
Abstract: A 20-month-old girl with developmental delay, dysmorphic features, horizontal supranuclear gaze palsy, retrocollis, and episodes of laryngospasm was diagnosed with variant neuronopathic Gaucher disease. The diagnosis was made enzymatically. Mutation analysis showed compound heterozygosity for D409H and a previously unreported mutation C16S. The presence of dysmorphic features, laryngospasm, absent cardiac findings, and the severe clinical phenotype distinguishes our case from other cases of variant neuronopathic Gaucher disease. We therefore propose to extend the spectrum of variant Gaucher disease.
Abstract: OBJECTIVE: Uncooked cornstarch (UCCS) is used widely for the treatment of patients with glycogen storage disease type I (GSD-I). Previous studies suggested that glucose absorption may be impaired in GSD-I. In order to measure utilization of UCCS in young adults with GSD-Ia and healthy controls, we used a C-breath test based on the natural enrichment of C in UCCS. DESIGN: Open, not randomized, prospective interventional study. METHODS: Following 1 g/kg UCCS, we studied eight subjects with GSD-Ia (7 males, 1 female; mean age 28.3 years, range 16-42 years) and 15 healthy controls (10 males, 5 females; mean age 23.5 years, range 19-36 years). Breath samples for analysis of CO enrichment were collected at baseline and at 30-min intervals for 6 h or until hypoglycaemia occurred. Indirect calorimetry was used to measure respiratory gas exchange. Intermediate metabolites, lipids and glucose were measured in plasma. Breath H concentrations were measured as an indicator of malabsorption. RESULTS: Cumulative utilization over 6 h was significantly higher in controls (18.35 +/- 6.2% of total carbohydrate intake) than in subjects with GSD-Ia (11.5 +/- 4.7%) (P < 0.02). However, utilization of UCCS was virtually identical up to 2.5 h. Two subjects with GSD-Ia fulfilled the criteria for malabsorption. CONCLUSIONS: Starch digestion and absorption are not impaired in GSD-Ia. However, overall utilization of UCCS appears to be lower in GSD-Ia, which is most likely secondary to perturbed intermediary metabolism. There are important implications for treatment of this disorder. Ways to improve the efficacy of UCCS in GSD-I are needed.
Abstract: BACKGROUND: Autosomal dominant familial hypercholesterolemia (FH) attributable to mutations in the LDL receptor (LDLR) gene is one of the most common genetic disorders associated with significant morbidity and mortality. Definitive diagnosis would help to initiate appropriate treatment to prevent premature cardiovascular disease. Currently, clinical diagnosis of FH is imprecise, and molecular diagnosis is labor-intensive and expensive because of the size of the LDLR gene and number of coding exons. METHODS: We used PCR to amplify all exons, including exon/intron boundaries, and the promoter of the LDLR gene. Nine individuals from five families with typical findings for a clinical diagnosis of heterozygous FH, 2 heterozygous FH cell lines, and 50 control individuals were screened for mutations by denaturing HPLC (DHPLC) followed by direct sequencing of aberrantly migrating fragments. RESULTS: Mutations that were previously reported to be disease causing were identified in eight of nine individuals with FH and both cell lines (V502M, C146X, E207X, C660X, C646Y, and delG197), but none were found in controls. The one individual with FH in whom no mutation was found had a previously unreported change in the 5'-untranslated region of unknown significance. In addition, we identified several previously reported polymorphism both in controls and individuals with FH. CONCLUSIONS: DHPLC can be used to detect mutations causing FH. On the basis of our current experience with DHPLC, this method combined with confirmatory DNA sequencing is likely to be sensitive and efficient.
Abstract: We report a male with late infantile glycogen storage disease type II (Pompe's disease) who presented at 12 months of age with muscular hypotonia and developmental delay. Oral supplementation with L-alanine has been administered for 5 years. Progression of skeletal myopathy was slow, and cardiomyopathy resolved almost completely. L-alanine may be a valuable supplement for infants with glycogen storage disease type II.
Abstract: We report a patient with mitochondrial DNA depletion, partial complex II and IV deficiencies, and 3-methylglutaconic aciduria. Complex II deficiency has not been previously observed in mitochondrial DNA depletion syndromes. The observation of 3-methylglutaconic and 3-methylglutaric acidurias may be a useful indicator of a defect in respiratory chain function caused by mitochondrial DNA depletion.
Abstract: Amniotic Band Sequence (ABS) is a disruption sequence that results in a variable group of abnormalities secondary to the disruption process and subsequent deformations. The incidence of ABS ranges from 1:1,200 to 1:15,000 live-born, and is even higher in still-born [Froster and Baird, 1993: Am J Med Genet 46:497-500]. The pathophysiology of ABS remains controversial, but a close look to critical periods of embryogenesis and/or organogenesis has helped in understanding pathogenetic mechanisms leading to the ABS disruption. The abnormalities are typically limited to external structures; however, associated internal malformations as seen in the case reported here may occur [Hunter and Carpenter, 1986: Am J Med Genet 24:691-700]. The prognosis depends on the severity of the abnormalities and the involvement of internal organs [Froster and Baird; 1993: Am J Med Genet 46:497-500; Levy, 1998: Ped Rev 19:249].
Abstract: A diagnosis of severe infantile, autosomal recessive osteopetrosis (I-ARO) was made in a 3-month-old female based on characteristic radiological and histological findings. The finding of multiple fractures at presentation in this infant is highly unusual. Deficiency of carbonic-anhydrase type II was excluded.
Abstract: Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine metabolism characterized by low plasma creatine concentrations in combination with elevated guanidinoacetate (GAA) concentrations. Although rare, GAMT deficiency has been identified in children with seizures, extrapyramidal movements, developmental delay, myopathies and behavioral abnormalities. Treatment with creatine monohydrate has been proven to be effective. We describe an isotope dilution electrospray tandem mass spectrometry (ES-MS/MS) assay for the simultaneous determination of plasma GAA and creatine using multiple reaction monitoring (MRM), d(3)-creatine as the internal standard and derivatization of GAA and creatine as butyl-esters. We analysed plasma of 16 healthy adults and 20 healthy children as well as three affected children. Plasma GAA concentrations were 5.02+/-1.84 micromol/l (mean+/-S.D.) in adults, 3.91+/-0.76 micromol/l in children age 5-10 years and 11.57, 15.16, 14.36 micromol/l in children with GAMT deficiency. Plasma creatine concentrations were 34.7+/-15.25 micromol/l in adults, 58.96+/-22.30 micromol/l in children and 5.37, 8.15, 403.5 micromol/l in two untreated children and one treated child with GAMT deficiency, respectively. GAA can also be reliably measured from filter cards, which is sufficient to make the correct diagnosis while creatine is consistently falsely elevated probably secondary to liberation of red cell creatine. In nine healthy newborn infants, GAA concentrations from filter cards were 4.83+/-1.43 and 5.04+/-1.84 micromol/l in 16 healthy adults. We conclude that isotope dilution ES-MS/MS is ideal for rapid high-throughput diagnosis of GAMT deficiency both from plasma and filter paper cards. Using this technique neonatal screening is feasible for this treatable inborn error of creatine metabolism.
Abstract: Inborn errors of metabolism often present with a variety of psychiatric symptoms. With improved diagnosis and treatment options, many patients have increased lifespans; consequently, issues of long-term quality of life are coming to the forefront. Mental health concerns are among these issues. To demonstrate the connection between the course of metabolic disease and its psychiatric manifestations, four different inborn errors of metabolism are reviewed: phenylketonuria, Wilson disease, acute intermittent porphyria, and metachromatic leukodystrophy.
Abstract: We report on a 22-month-old girl with minor facial anomalies, global developmental delay, growth retardation, seizures, and leukoencephalopathy. Initial clinical assessment suggested the diagnosis of Williams syndrome. Results of fluorescence in situ hybridization testing for elastin were normal. However, chromosome analysis showed a 46,XX,del(9)(p13.1p21.1) karyotype in peripheral lymphocytes. Parental chromosomes were normal, indicating a de novo deletion. This patient's manifestations are compared with those of two other cases with overlapping deletions of the proximal short arm of chromosome 9.
Abstract: BACKGROUND: CO2 production is most commonly measured by using indirect calorimetry to quantify elimination of CO(2) in breath (VCO2). An alternative is to measure the rate at which CO2 appears in the body pool (RaCO2) by infusing a (13)C labelled bicarbonate tracer. VCO2 and RaCO2 generally differ but are related by c, a factor that adjusts for the incomplete recovery of infused tracer in the breath. The literature relating to human studies cites a wide range of values for c but the only neonatal study to determine c empirically estimated a mean value of 0.77. AIM: To estimate fractional recovery rate, c, in very low birthweight babies, and assess the feasibility of using the isotopic technique to measure CO2 production during mechanical ventilation. METHOD: Eleven spontaneously breathing, continuously fed, very low birthweight infants (median birth weight 1060 g, median gestational age 29 weeks) were studied. RESULTS: Mean (SD) VCO2 was 9.0 (2.0) ml/min (standard temperature and pressure dry, STPD) and mean (SD) RaCO2 was 9.6 (2.1) ml/min (STPD). The mean (SD) value of c was estimated as 0.95 (0.13). The 95% confidence intervals of the mean were 0.87-1.03. CONCLUSIONS: The results emphasise the importance of measuring c for a given study population rather than assuming a value based on adult studies. The close approximation of RaCO2 and VCO2 in this group of babies implies that the labelled bicarbonate infusion technique could be used to measure simply CO2 production during mechanical ventilation.
Abstract: OBJECTIVES: During intercurrent illness children with methylmalonic acidemia were found to have increased resting energy expenditure (REE). We measured REE in children with disorders of propionate metabolism (methylmalonic and propionic acidemia) when they were well and compared the values with those predicted by the Schofield equation. STUDY DESIGN: Prospective study in tertiary care facility. REE was measured with open-circuit indirect calorimetry under standardized conditions. Predicted REE values were calculated with the Schofield equation. Fourteen subjects with propionic acidemia (n = 3) and methylmalonic acidemia (n = 11) were studied. RESULTS: The median REE was 690 kcal/d (range 186 to 1687 kcal/d), which is significantly reduced, representing 80% +/- 18% of that predicted by the Schofield height and weight equation (P <.01). REE was significantly lower in female compared with male patients for unknown reasons. There were no differences with age or neurologic state. REE was not further reduced in those with chronic renal failure. CONCLUSION: REE in patients with disorders of propionate metabolism is reduced when they are well.
Abstract: Myopathy in glycogen storage disease type II (GSD-II) is slowly progressive. Five subjects with the late-onset form of GSD-II (age range, 15 to 47 years) and seven healthy control subjects (age range, 28 to 55 years) were studied. Following alanine supplementation, resting energy expenditure decreased in patients with GSD-II (p < 0.05) compared to values seen in control subjects. Leucine flux decreased (p < 0.004), as did leucine oxidation, to levels lower than those observed in control subjects (p < 0.001). l-Alanine reduces protein turnover and catabolism in GSD-II.
Abstract: OBJECTIVE: To evaluate the effects of four weeks of fish oil supplementation on apolipoprotein B100 production and lipoprotein metabolism in normolipidaemic males. DESIGN AND SUBJECTS: Very low density lipoprotein (VLDL) apolipoprotein B100 (apoB100) kinetics in ten healthy, white males, aged 22-43 y (mean 32 y) were investigated using 13C-leucine technique and gas chromatography-mass spectrometry before and after fish oil supplementation. INTERVENTION: All subjects received 10 g (1.8 g EPA, 1.2 g DHA)/d of fish oil concentrate for four weeks. RESULTS: Fish oil supplementation resulted in a decrease of total plasma VLDL (mean +/- s.d. 1.11 +/- 0.41 vs 0.87 +/- 0.28 mmol/l, P < 0.05) and triacylglycerol concentrations (0.74 +/- 0.27) vs 0.48 +/- 0.21 mmol/l, P < 0.01). VLDL apoB100 pool size was decreased without alteration of the fractional synthetic rate but a significant decrease of apoB100 production (2.23 +/- 0.90 vs 1.54 +/- 0.52 mg/dl/h, P < 0.02). Following fish oil supplementation plasma concentrations of glucose and insulin as well as lipoprotein and hepatic lipase activities were unchanged. Fasting plasma concentrations of non-esterified fatty acid (NEFA) were decreased (0.45 +/- 0.12 vs 0.33 +/- 0.10 mmol/l, P < 0.05). CONCLUSIONS: Dietary supplementation with fish oil in healthy males results in decreased VLDL-triacylglycerol concentrations through a decrease in VLDL particle synthesis. The decrease in NEFA substrate supply also contributes.
Abstract: The assessment of energy expenditure is valuable for the management of children with various conditions such as obesity and failure to thrive. Total daily energy expenditure (TDEE) includes resting energy expenditure (REE), energy expenditure during physical activity, dietary thermogenesis and growth. TDEE can be assessed by using the double-labelled water technique, but it has complex pitfalls and potential sources of errors and is impractical for everyday use. As REE is a substantial part of TDEE (65%-70%) and computerised indirect calorimeters have become recently available, this non-invasive, relatively cheap and easy to use technique is valuable for the assessment of short-term changes in energy metabolism. This can be used to assess REE of children with inborn errors of metabolism, whilst well and during episodes of metabolic decompensation and therefore to accurately determine energy intake.
Abstract: Aspects of glucose metabolism have been investigated quantitatively employing stable isotopes for 20 year. Use of non-recycling [6,6-2H2] or [U-13C] glucose labels provides a value for total hepatic glucose production (glycogenolysis plus gluconeogenesis). Quantitation of gluconeogenesis with isotopic tracers has itself recently been revisited employing protocols and analytical options that purport to overcome the isotope exchanges (dilution) experienced at the level of oxaloacetate when the rate of incorporation of label into glucose from infused alanine, lactate or pyruvate is monitored. Labelled glucose has been employed to investigate metabolic disturbances of glycogen storage disease type I (GSD-I) and type III (GSD-III). Endogenous glucose production and glucose recycling have been studied in both these storage diseases employing a primed-continuous infusion of D-[U-13C] glucose and quantitation of isotope enrichments and isotopomer distribution observed in plasma glucose either by mass spectrometry or nuclear magnetic resonance spectroscopy. General aspects of glucose metabolism as investigated with stable isotopes are presented.
Abstract: Stable isotope studies have considerable potential to help understand the biochemistry and pathophysiology of inborn errors of metabolism but this has not yet been fully realised. It is to be hoped that this symposium will prove to be a valuable stimulus to this field of research.
Abstract: There is a paucity of data documenting the metabolic response to catabolic stress in childhood in general and about protein turnover in critically ill children in particular. Despite a high overall morbidity and mortality rate there is little information on which to base decisions to improve the management either by dietary therapy or by use of growth factors. Protein turnover is a key metabolic process that significantly alters during the catabolic state. Protein kinetics are easy to quantify using various stable isotope models, with some having advantages in the critically ill child. The 1-13C leucine technique is the most widely used and best validated model to date, requiring accurate estimation of CO2 production. There is also uncertainty about the bicarbonate kinetics and pool sizes in ventilated children whose respiratory function is severely impaired. The value of the 15N glycine (end product) technique is more limited because the time to achieve isotopic equilibrium is lengthy and considerable concerns about the validity of the model exist. The ring-D5 phenylalanine technique has the advantage of not requiring the measurement of CO2 production or 13C enrichment, but the model has not yet been validated in critically ill children. Despite it is of obvious value to measure protein turnover, few studies in critically ill children have been done.
Abstract: Late-onset acid maltase deficiency or glycogen storage disease type II (GSD II) is a rare disorder of intralysosomal glycogen metabolism, resulting in progressive myopathy that is secondary to increased muscle protein breakdown. Stable isotope studies in the postabsorptive state have confirmed that mean protein breakdown in GSD II is increased by 31% compared to control subjects. 6.86 versus 4.69 g/kg per day, that mean protein balance is reduced is GSD II -1.32 versus -1.06 g/kg per day. Indirect calorimetry has demonstrated an increase in mean resting energy expenditure in GSD II, 41.8 versus 31.2 kcal/kg per day. Compliance following the introduction of a high-protein diet is often poor due to the large quantities of protein necessary and to the high caloric intake with the consequent weight gain. Only 25% of all reported subjects with GSD II showed an improvement of muscle or respiratory function after a high-protein diet. Careful evaluation of the underlying pathophysiological changes in GSD II is necessary to develop more logical and therefore more beneficial forms of dietary treatment.
