Mutations in RIT1 cause Noonan syndrome - additional functional evidence and expanding the clinical phenotype.

RASopathies are a clinically heterogeneous group of conditions caused by mutations in 1 of 16 proteins in the RAS-mitogen activated protein kinase (RAS-MAPK) pathway. Recently, mutations in RIT1 were identified as a novel cause for Noonan syndrome. Here we provide additional functional evidence for a causal role of RIT1 mutations and expand the associated phenotypic spectrum. We identified two de novo missense variants p.Met90Ile and p.Ala57Gly. Both variants resulted in increased MEK-ERK signaling compared to wild-type, underscoring gain-of-function as the primary functional mechanism. Introduction of p.Met90Ile and p.Ala57Gly into zebrafish embryos reproduced not only aspects of the human phenotype but also revealed abnormalities of eye development, emphasizing the importance of RIT1 for spatial and temporal organization of the growing organism. In addition, we observed severe lymphedema of the lower extremity and genitalia in one patient. We provide additional evidence for a causal relationship between pathogenic mutations in RIT1, increased RAS-MAPK/MEK-ERK signaling and the clinical phenotype. The mutant RIT1 protein may possess reduced GTPase activity or a diminished ability to interact with cellular GTPase activating proteins; however the precise mechanism remains unknown. The phenotypic spectrum is likely to expand and includes lymphedema of the lower extremities in addition to nuchal hygroma.

Update on transcobalamin deficiency: clinical presentation, treatment and outcome.

Transcobalamin (TC) transports cobalamin from blood into cells. TC deficiency is a rare autosomal recessive disorder usually presenting in early infancy with failure to thrive, weakness, diarrhoea, pallor, anemia, and pancytopenia or agammaglobulinemia. It can sometimes resemble neonatal leukemia or severe combined immunodeficiency disease. Diagnosis of TC deficiency is suspected based on megaloblastic anemia, elevation of total plasma homocysteine, and blood or urine methylmalonic acid. It is confirmed by studying the synthesis of TC in cultured fibroblasts, or by molecular analysis of the TCN2 gene. TC deficiency is treatable with supplemental cobalamin, but the optimal type, route and frequency of cobalamin administration and long term patient outcomes are unknown. Here we present a series of 30 patients with TC deficiency, including an update on multiple previously published patients, in order to evaluate the different treatment strategies and provide information about long term outcome. Based on the data presented, current practice appears to favour treatment of individuals with TC deficiency by intramuscular injections of hydroxy- or cyanocobalamin. In most cases presented, at least weekly injections (1 mg IM) were necessary to ensure optimal treatment. Most centres adjusted the treatment regimen based on monitoring CBC, total plasma homocysteine, plasma and urine methylmalonic acid, as well as, clinical status. Finally, continuing IM treatment into adulthood appears to be beneficial.

Expert recommendations for the laboratory diagnosis of MPS VI.

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine 4-sulfatase (arylsulfatase B, ASB). This enzyme is required for the degradation of dermatan sulfate. In its absence, dermatan sulfate accumulates in cells and is excreted in large quantities in urine. Specific therapeutic intervention is available; however, accurate and timely diagnosis is crucial for maximal benefit. To better understand the current practices for diagnosis and to establish diagnostic guidelines, an international MPS VI laboratory diagnostics scientific summit was held in February of 2011 in Miami, Florida. The various steps in the diagnosis of MPS VI were discussed including urinary glycosaminoglycan (uGAG) analysis, enzyme activity analysis, and molecular analysis. The following conclusions were reached. Dilute urine samples pose a significant problem for uGAG analysis and MPS VI patients can be missed by quantitative uGAG testing alone as dermatan sulfate may not always be excreted in large quantities. Enzyme activity analysis is universally acknowledged as a key component of diagnosis; however, several caveats must be considered and the appropriate use of reference enzymes is essential. Molecular analysis supports enzyme activity test results and is essential for carrier testing, subsequent genetic counseling, and prenatal testing. Overall the expert panel recommends caution in the use of uGAG screening alone to rule out or confirm the diagnosis of MPS VI and acknowledges enzyme activity analysis as a critical component of diagnosis. Measurement of another sulfatase enzyme to exclude multiple sulfatase deficiency was recommended prior to the initiation of therapy. When feasible, the use of molecular testing as part of the diagnosis is encouraged. A diagnostic algorithm for MPS VI is provided.

Free asymmetric dimethylarginine (ADMA) is low in children and adolescents with classical phenylketonuria (PKU).

INTRODUCTION: Free asymmetric dimethylarginine (ADMA) is a competitive inhibitor of the nitric oxide synthases (NOS). Suppression of nitric oxide (NO) synthesis increases the risk of atherosclerosis. Nevertheless, in the condition of oxidative stress, NOS blockade by ADMA may exert protective effects. Protein metabolism is altered in patients with phenylketonuria (PKU) on dietary treatment and as shown recently, oxidative stress is high in PKU. Since free ADMA concentrations are determined by both protein metabolism and oxidative stress we hypothesized, that free ADMA levels may be elevated in PKU patients. DESIGN: Sixteen patientswith PKU on dietary treatment (mean age 10.1 ± 5.2 yrs), and 91 healthy children (mean age 11.6 ± 3.7 yrs) participated in a cross sectional study. RESULTS: ADMA, total homocysteine (tHcy) and blood glucose were lower and the L-arginine/ADMA ratio was higher in PKU patients compared to controls. No significant correlation was present between phenylalanine (Phe) concentrations, protein intake, and lipid profile, history of cardiovascular disease or ADMA. DISCUSSION: In contrast to our hypothesis, ADMAwas lower and the L-arginine/ADMA ratio was higher in PKU patients. Therefore, in PKU patients, the regulating function of ADMA on NO synthesis is altered and may thus contribute to oxidative stress.

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.

Danon disease: case report and detection of new mutation.

Danon disease is an X-linked disorder resulting from mutations in the lysosome-associated membrane protein-2 (LAMP2) gene. We report a male patient with skeletal myopathy, mental retardation, and massive hypertrophic obstructive cardiomyopathy necessitating heart transplantation. Immunohistochemistry of skeletal muscle and leukocytes, western blot analysis of leukocytes and cardiac muscle, flow cytometry, and DNA sequencing were performed. Muscle biopsy revealed autophagic vacuolar myopathy and lack of immunohistochemically detectable LAMP-2. Diagnosis of Danon disease was confirmed by western blot analysis of myocardial tissue and peripheral blood sample of the patient showing deficiency of LAMP-2 in myocardium and leukocytes. Moreover, absence of LAMP-2 in lymphocytes, monocytes and granulocytes was shown by flow cytometric analysis. Genetic analysis of the LAMP2 gene revealed a novel 1-bp deletion at position 179 (c.179delC) at the 3' end of exon 2, resulting in a frameshift with a premature stop codon.

Prediction of outcome in isolated methylmalonic acidurias: combined use of clinical and biochemical parameters.

Objectives Isolated methylmalonic acidurias (MMAurias) are caused by deficiency of methylmalonyl-CoA mutase or by defects in the synthesis of its cofactor 5'-deoxyadenosylcobalamin. The aim of this study was to evaluate which parameters best predicted the long-term outcome. Methods Standardized questionnaires were sent to 20 European metabolic centres asking for age at diagnosis, birth decade, diagnostic work-up, cobalamin responsiveness, enzymatic subgroup (mut(0), mut(-), cblA, cblB) and different aspects of long-term outcome. Results 273 patients were included. Neonatal onset of the disease was associated with increased mortality rate, high frequency of developmental delay, and severe handicap. Cobalamin non-responsive patients with neonatal onset born in the 1970s and 1980s had a particularly poor outcome. A more favourable outcome was found in patients with late onset of symptoms, especially when cobalamin responsive or classified as mut(-). Prevention of neonatal crises in pre-symptomatically diagnosed newborns was identified as a protective factor concerning handicap. Chronic renal failure manifested earlier in mut(0) patients than in other enzymatic subgroups. Conclusion Outcome in MMAurias is best predicted by the enzymatic subgroup, cobalamin responsiveness, age at onset and birth decade. The prognosis is still unfavourable in patients with neonatal metabolic crises and non-responsiveness to cobalamin, in particular mut(0) patients.

Methods for a prompt and reliable laboratory diagnosis of Pompe disease: report from an international consensus meeting.

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 2 mM of the synthetic substrate 4-methylumbelliferyl-alpha-D-glucoside or glycogen (50 mg/mL), in the presence of acarbose (3-9 microM) 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.

Congenital disorders of glycosylation--a challenging group of IEMs.

Congenital disorders of glycosylation (CDG) are a rapidly growing group of inherited errors of metabolism (IEMs) due to an impairment of one or several glycosylation pathways. During recent years over 30 CDG subtypes have been identified at a molecular and biochemical level. The clinical manifestations in CDG are heterogeneous and may be highly variable within the same subtype and even among affected siblings. Novel insights into the extremely complex glycosylation pathways have necessitated several reclassifications of the group of CDG. Today CDG comprise not only the formerly known multisystem glycosylation defects but also some tissue-specific glycosylation defects, implicating a different diagnostic work-up depending on the underlying glycosylation defect. In 2007 the expanding group of CDG is an enormous challenge to all specialists working in the field of IEMs. This review gives a brief overview about the expanded group of CDG and summarizes the main implications for clinicians.

Comparison of tetrahydrofuran and ethyl acetate as extraction solvents for urinary organic acid analysis.

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.

Diagnostic work-up and management of patients with isolated methylmalonic acidurias in European metabolic centres.

The long-term outcome of patients with methylmalonic aciduria (MMA) is still uncertain due to a high frequency of complications such as chronic renal failure and metabolic stroke. The understanding of this disease is hampered by a huge variation in the management of these patients. The major aim of this study was to evaluate the current practice in different European metabolic centres. A standardized questionnaire was sent to 20 metabolic centres asking for standard procedures for confirmation of diagnosis, testing cobalamin responsiveness, dietary treatment, pharmacotherapy, and biochemical and clinical monitoring. Sixteen of 20 metabolic centres (80%) returned questionnaires on 183 patients: 89 of the patients were classified as mut(0), 36 as mut(-), 13 as cblA, 7 as cblB, and 38 as cblA/B. (1) Confirmation of diagnosis: All centres investigate enzyme activity by propionate fixation in fibroblasts; six centres also perform mutation analysis. (2) Cobalamin response: Ten centres follow standardized protocols showing large variations. A reliable exclusion of nonspecific effects has not yet been achieved by these protocols. (3) Long-term treatment: In cobalamin-responsive patients, most centres use hydroxocobalamin (1-14 mg/week i.m. or 5-20 mg/week orally), while two centres use cyanocobalamin. All cobalamin-nonresponsive patients and most cobalamin-responsive patients are supplemented with L: -carnitine (50-100 mg/kg per day). Fourteen centres use intestinal decontamination by antibiotic therapy. Most centres follow D-A-CH (n = 6) or Dewey (n = 4) recommendations for protein requirements. Fourteen centres regularly use precursor-free amino acid supplements. Standardized monitoring protocols are available in seven centres, again showing high variability.

Expanded newborn screening in Europe 2007.

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.

Severe speech delay as the presenting symptom of guanidinoacetate methyltransferase deficiency.

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.

The effects of l-alanine supplementation in late-onset glycogen storage disease type II.

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.

Atypical presentation of amniotic band sequence.

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].

Deletion (9) (p13.1 p21.1).

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.

Analysis of guanidinoacetate and creatine by isotope dilution electrospray tandem mass spectrometry.

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.

Effects of fish oil supplementation on apolipoprotein B100 production and lipoprotein metabolism in normolipidaemic males.

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.

Measurement of carbon dioxide production in very low birth weight babies.

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.