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1.
Horm Res Paediatr ; 91(4): 262-270, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31256164

RESUMO

BACKGROUND: The HIV drugs lopinavir and ritonavir have recently been reported to cause transient adrenal insufficiency in preterm newborns. We, therefore, considered HIV drugs as a cause of transiently elevated 17-hydroxyprogesterone (17OHP) levels in a neonatal screening test for congenital adrenal hyperplasia in a preterm girl exposed to zidovudine, efavirenz, tenofovir, and emtricitabine. OBJECTIVE: So far, HIV drugs have not been tested for their effect on steroidogenesis and the steroidogenic enzyme activity of CYP21A2 specifically in an in vitro system. METHODS: We tested the effect of efavirenz, tenofovir, emtricitabine, and zidovudine on steroidogenesis of human adrenal H295R cells. Cells were treated with the drugs at different concentrations including concentrations in therapeutic use. The effect on CYP21A2 activity was assessed by testing the conversion of radiolabeled 17OHP to 11-deoxycortisol. Cell viability was tested by an MTT assay. In addition, recombinant human CYP21A2 protein was used to assess direct drug effects on CYP21A2 activity. RESULTS: We observed significantly decreased CYP21A2 activity in both in vitro testing systems after treatment with efavirenz at therapeutic concentrations. Moreover, efavirenz affected cell viability. By contrast, the other test drugs did not affect steroidogenesis. Follow-up of our patient revealed elevated 17OHP and androgen levels during the first weeks of life, but values normalized spontaneously. Genetic testing for CYP21A2 mutations was negative. Thus, it remains unsettled whether the transient 17OHP elevation in this baby was due to a drug effect. CONCLUSION: The HIV drug efavirenz inhibits CYP21A2 activity in vitro through direct interaction with enzyme catalysis at therapeutic concentrations. This may have clinical implications for HIV treatment in children and adults. However, so far, clinical data are scarce, and further studies are needed to be able to draw clinical conclusions.

2.
Am J Med Genet A ; 179(7): 1371-1375, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31075182

RESUMO

Accurate glycosylation of proteins is essential for their function and their intracellular transport. Numerous diseases have been described, where either glycosylation or intracellular transport of proteins is impaired. Coat protein I (COPI) is involved in anterograde and retrograde transport of proteins between endoplasmic reticulum and Golgi, where glycosylation takes place, but no association of defective COPI proteins and glycosylation defects has been described so far. We identified a patient whose phenotype at a first glance was reminiscent of PGM1 deficiency, a disease that also affects N-glycosylation of proteins. More detailed analyses revealed a different disease with a glycosylation deficiency that was only detectable during episodes of acute illness of the patient. Trio-exome analysis revealed a de novo loss-of-function mutation in ARCN1, coding for the delta-COP subunit of COPI. We hypothesize that the capacity of flow through Golgi is reduced by this defect and at high protein synthesis rates, this bottleneck also manifests as transient glycosylation deficiency.

3.
J Inherit Metab Dis ; 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30843237

RESUMO

A liver-humanized mouse model for CPS1-deficiency was generated by the high-level repopulation of the mouse liver with CPS1-deficient human hepatocytes. When compared with mice that are highly repopulated with CPS1-proficient human hepatocytes, mice that are repopulated with CPS1-deficient human hepatocytes exhibited characteristic symptoms of human CPS1 deficiency including an 80% reduction in CPS1 metabolic activity, delayed clearance of an ammonium chloride infusion, elevated glutamine and glutamate levels, and impaired metabolism of [15 N]ammonium chloride into urea, with no other obvious phenotypic differences. Because most metabolic liver diseases result from mutations that alter critical pathways in hepatocytes, a model that incorporates actual disease-affected, mutant human hepatocytes is useful for the investigation of the molecular, biochemical, and phenotypic differences induced by that mutation. The model is also expected to be useful for investigations of modified RNA, gene, and cellular and small molecule therapies for CPS1-deficiency. Liver-humanized models for this and other monogenic liver diseases afford the ability to assess the therapy on actual disease-affected human hepatocytes, in vivo, for long periods of time and will provide data that are highly relevant for investigations of the safety and efficacy of gene-editing technologies directed to human hepatocytes and the translation of gene-editing technology to the clinic.

4.
Eur J Nutr ; 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30843107

RESUMO

PURPOSE: Despite longstanding voluntary salt iodisation in Switzerland, data suggest inadequate iodine intake in vulnerable population groups. In response, the salt iodine concentration was increased from 20 to 25 mg/kg and we assessed the impact on iodine status. METHODS: We conducted a cross-sectional national study in school-age children (n = 731), women of reproductive age (n = 353) and pregnant women (n = 363). We measured urinary iodine concentration (UIC) and urinary sodium concentration (UNaC) in spot urine samples. The current median UIC was compared with national data from 1999, 2004 and 2009. We measured TSH, total T4 and thyroglobulin (Tg) on dried blood spot samples collected in women. RESULTS: The median UIC (bootstrapped 95% CI) was 137 µg/L (131, 143 µg/L) in school children, 88 µg/L (72, 103 µg/L) in women of reproductive age and 140 µg/L (124, 159 µg/L) in pregnant women. Compared to 2009, the median UIC increased modestly in school children (P < 0.001), but did not significantly change in pregnant women (P = 0.417). Estimated sodium intake exceeded the recommendations in all population groups. The prevalence of thyroid disorders in women was low, but Tg was elevated in 13% of the pregnant women. CONCLUSION: Iodine intake is overall adequate in Swiss school-age children, but only borderline sufficient in pregnant and non-pregnant women, despite high salt intakes and satisfactory household coverage with iodized salt. Our findings suggest increasing the concentration of iodine in salt may not improve iodine intakes in women if iodised salt is not widely used in processed foods. REGISTRATION: This trial was registered at clinicaltrials.gov as NCT02312466.

5.
J Inherit Metab Dis ; 42(1): 128-139, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30740731

RESUMO

PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.

6.
J Inherit Metab Dis ; 2019 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-30714172

RESUMO

The most common ureagenesis defect is X-linked ornithine transcarbamylase (OTC) deficiency which is a main target for novel therapeutic interventions. The spf ash mouse model carries a variant (c.386G>A, p.Arg129His) that is also found in patients. Male spf ash mice have a mild biochemical phenotype with low OTC activity (5%-10% of wild-type), resulting in elevated urinary orotic acid but no hyperammonemia. We recently established a dried blood spot method for in vivo quantification of ureagenesis by Gas chromatography-mass spectrometry (GC-MS) using stable isotopes. Here, we applied this assay to wild-type and spf ash mice to assess ureagenesis at different ages. Unexpectedly, we found an age-dependency with a higher capacity for ammonia detoxification in young mice after weaning. A parallel pattern was observed for carbamoylphosphate synthetase 1 and OTC enzyme expression and activities, which may act as pacemaker of this ammonia detoxification pathway. Moreover, high ureagenesis in younger mice was accompanied by elevated periportal expression of hepatic glutamine synthetase, another main enzyme required for ammonia detoxification. These observations led us to perform a more extensive analysis of the spf ash mouse in comparison to the wild-type, including characterization of the corresponding metabolites, enzyme activities in the liver and plasma and the gut microbiota. In conclusion, the comprehensive enzymatic and metabolic analysis of ureagenesis performed in the presented depth was only possible in animals. Our findings suggest such analyses being essential when using the mouse as a model and revealed age-dependent activity of ammonia detoxification.

7.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1106-1107: 64-70, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30641270

RESUMO

BACKGROUND: Congenital disorders of glycosylation (CDG) are a growing group of inherited diseases causing manifold symptoms. Routine diagnostic procedures are high performance liquid chromatography (HPLC) or isoelectric focusing (IEF) of serum transferrin. METHODS: We introduce a modified method to screen for glycosylation abnormalities from dried blood spot (DBS) samples based on isoelectric focusing. In PGM1-CDG, glycosylation analysis and enzyme activity measurement were performed from a single DBS sample. Furthermore, we present the possibility to use capillary blood samples for quantification of transferrin isoforms. RESULTS: IEF from DBS samples is possible and results are identical to the ones obtained in serum samples. Gel analysis using the ImageJ software allows quantification of IEF results. Storage at -20 °C ensures stable samples for more than six months. Capillary blood samples are equally suitable for glycosylation analysis and show no inferiority to serum samples. CONCLUSION: In view of a growing number of treatable CDG subtypes, the proposed methods allow reliable diagnosis and therapy control of CDG while being easily applicable. Capillary blood samples can be taken at home and sent in for follow-up. DBS are widely used in new-born screening programs and have the potential to broaden the knowledge of glycosylation abnormalities in early infancy. By its possible application in the context of alcohol abuse, the proposed method bears the potential for widespread use in a non-metabolic context.


Assuntos
Defeitos Congênitos da Glicosilação , Transferrina/análise , Coleta de Amostras Sanguíneas , Cromatografia Líquida de Alta Pressão , Defeitos Congênitos da Glicosilação/sangue , Defeitos Congênitos da Glicosilação/diagnóstico , Glicosilação , Humanos , Focalização Isoelétrica
8.
Thyroid ; 29(2): 268-277, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30648484

RESUMO

BACKGROUND: In areas with incomplete salt iodization coverage, infants and children aged 6-24 months weaning from breast milk and receiving complementary foods are at risk of iodine deficiency. However, few data exist on the risk of excessive iodine intake in this age group. Thyroglobulin (Tg) is a sensitive marker of iodine intake in school-age children and adults and may be used to estimate the optimal iodine intake range in infancy. The aim of this study was to assess the association of low and high iodine intakes with Tg and thyroid function in weaning infants. METHODS: This multicenter cross-sectional study recruited infants aged 6-24 months (n = 1543; Mage = 12.2 ± 4.6 months) receiving breast milk with complementary foods, from seven countries in areas with previously documented deficient, sufficient, or excessive iodine intake in schoolchildren or pregnant women. Urinary iodine concentration (UIC) and Tg, total thyroxine, and thyrotropin were measured using dried blood spot testing. RESULTS: Median UIC ranged from 48 µg/L (interquartile range 31-79 µg/L) to 552 µg/L (interquartile range 272-987 µg/L) across the study sites. Median Tg using dried blood spot testing was high (>50 µg/L) at estimated habitual iodine intakes <50 µg/day and >230 µg/day. Prevalence of overt thyroid disorders was low (<3%). Yet, subclinical hyperthyroidism was observed in the countries with the lowest iodine intake. CONCLUSIONS: Tg is a sensitive biomarker of iodine intake in 6- to 24-month-old infants and follows a U-shaped relationship with iodine intake, suggesting a relatively narrow optimal intake range. Infants with low iodine intake may be at increased risk of subclinical thyroid dysfunction. In population monitoring of iodine deficiency or excess, assessment of iodine status using UIC and Tg may be valuable in this young age group.

9.
JAMA Neurol ; 76(3): 342-350, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30575854

RESUMO

Importance: The identification and understanding of the monogenic causes of neurodevelopmental disorders are of high importance for personalized treatment and genetic counseling. Objective: To identify and characterize novel genes for a specific neurodevelopmental disorder characterized by refractory seizures, respiratory failure, brain abnormalities, and death in the neonatal period; describe the outcome of glutaminase deficiency in humans; and understand the underlying pathological mechanisms. Design, Setting, and Participants: We performed exome sequencing of cases of neurodevelopmental disorders without a clear genetic diagnosis, followed by genetic and bioinformatic evaluation of candidate variants and genes. Establishing pathogenicity of the variants was achieved by measuring metabolites in dried blood spots by a hydrophilic interaction liquid chromatography method coupled with tandem mass spectrometry. The participants are 2 families with a total of 4 children who each had lethal, therapy-refractory early neonatal seizures with status epilepticus and suppression bursts, respiratory insufficiency, simplified gyral structures, diffuse volume loss of the brain, and cerebral edema. Data analysis occurred from October 2017 to June 2018. Main Outcomes and Measures: Early neonatal epileptic encephalopathy with glutaminase deficiency and lethal outcome. Results: A total of 4 infants from 2 unrelated families, each of whom died less than 40 days after birth, were included. We identified a homozygous frameshift variant p.(Asp232Glufs*2) in GLS in the first family, as well as compound heterozygous variants p.(Gln81*) and p.(Arg272Lys) in GLS in the second family. The GLS gene encodes glutaminase (Enzyme Commission 3.5.1.2), which plays a major role in the conversion of glutamine into glutamate, the main excitatory neurotransmitter of the central nervous system. All 3 variants probably lead to a loss of function and thus glutaminase deficiency. Indeed, glutamine was increased in affected children (available z scores, 3.2 and 11.7). We theorize that the potential reduction of glutamate and the excess of glutamine were a probable cause of the described physiological and structural abnormalities of the central nervous system. Conclusions and Relevance: We identified a novel autosomal recessive neurometabolic disorder of loss of function of glutaminase that leads to lethal early neonatal encephalopathy. This inborn error of metabolism underlines the importance of GLS for appropriate glutamine homeostasis and respiratory regulation, signal transduction, and survival.

10.
Br J Haematol ; 183(4): 648-660, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30334577

RESUMO

Sickle Cell Disease (SCD) is an increasing global health problem and presents significant challenges to European health care systems. Newborn screening (NBS) for SCD enables early initiation of preventive measures and has contributed to a reduction in childhood mortality from SCD. Policies and methodologies for NBS vary in different countries, and this might have consequences for the quality of care and clinical outcomes for SCD across Europe. A two-day Pan-European consensus conference was held in Berlin in April 2017 in order to appraise the current status of NBS for SCD and to develop consensus-based statements on indications and methodology for NBS for SCD in Europe. More than 50 SCD experts from 13 European countries participated in the conference. This paper aims to summarise the discussions and present consensus recommendations which can be used to support the development of NBS programmes in European countries where they do not yet exist, and to review existing programmes.

11.
Nat Med ; 24(10): 1519-1525, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30297904

RESUMO

CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah)enu2 mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU)1, using recently developed CRISPR-Cas-associated base editors2-4. These systems enable conversion of C∙G to T∙A base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an intein-split base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pahenu2 gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pahenu2 mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.

12.
Thyroid ; 28(9): 1198-1210, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30019625

RESUMO

BACKGROUND: Acute excess iodine intake can damage the thyroid, but the effects of chronic excess iodine intake are uncertain. Few data exist for pregnant and lactating women and infants exposed to excessive iodine intake. METHODS: This was a multicenter cross-sectional study. At study sites in rural Kenya and urban Tanzania previously reporting iodine excess in children, urinary iodine concentration (UIC), thyrotropin, total thyroxine, and thyroglobulin (Tg) were measured in school-age children (SAC), women of reproductive age, pregnant (PW) and lactating women, and breast-feeding and weaning infants. In a national study in Djibouti, UIC was measured in SAC and PW. At all sites, daily iodine intake was estimated based on UIC, and iodine concentration was measured in household salt and drinking water. RESULTS: The total sample size was 4636: 1390, 2048, and 1198 subjects from Kenya, Tanzania, and Djibouti, respectively. In Kenya and Tanzania: (i) median UIC was well above thresholds for adequate iodine nutrition in all groups and exceeded the threshold for excess iodine intake in SAC; (ii) iodine concentrations >40 mg of iodine/kg were found in approximately 55% of household salt samples; (iii) iodine concentrations ≥10 µg/L were detected in 9% of drinking water samples; (iv) Tg was elevated in all population groups, but the prevalence of thyroid disorders was negligible, except that 5-12% of women of reproductive age had subclinical hyperthyroidism and 10-15% of PW were hypothyroxinemic. In Djibouti: (i) the median UIC was 335 µg/L (interquartile range [IQR] = 216-493 µg/L) in SAC and 265 µg/L (IQR = 168-449 µg/L) in PW; (ii) only 1.6% of Djibouti salt samples (n = 1200) were adequately iodized (>15 mg/kg); (iii) the median iodine concentration in drinking water was 92 µg/L (IQR = 37-158 µg/L; n = 77). In all countries, UIC was not significantly correlated with salt or water iodine concentrations. CONCLUSIONS: Although iodine intake was excessive and Tg concentrations were elevated, there was little impact on thyroid function. Chronic excess iodine intake thus appears to be well tolerated by women, infants, and children. However, such high iodine intake is unnecessary and should be avoided. Careful evaluation of contributions from both iodized salt and groundwater iodine is recommended before any review of iodization policy is considered.

13.
Hum Mutat ; 39(8): 1029-1050, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29726057

RESUMO

The urea cycle disorder argininemia is caused by a defective arginase 1 (ARG1) enzyme resulting from mutations in the ARG1 gene. Patients generally develop hyperargininemia, spastic paraparesis, progressive neurological and intellectual impairment, and persistent growth retardation. Interestingly, in contrast to other urea cycle disorders, hyperammonemia is rare. We report here 66 mutations (12 of which are novel), including 30 missense mutations, seven nonsense, 10 splicing, 15 deletions, two duplications, one small insertion, and one translation initiation codon mutation. For the most common mutations (p.Thr134Ile, p.Gly235Arg and p.Arg21*), which cluster geographically in Brazil, China, or Turkey, a structural rationalization of their effect has been included. In order to gain more knowledge on the disease, we have collected clinical and biochemical information of 112 patients, including the patients' genetic background and ethnic origin. We have listed as well the missense variants with unknown relevance. For all missense variants (of both known and unknown relevance), the conservation, severity prediction, and ExAc scores have been included. Lastly, we review ARG1 regulation, animal models, diagnostic strategies, newborn screening, prenatal testing, and treatment options.

14.
Hum Mol Genet ; 2018 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-29771303

RESUMO

Developmental eye defects often severely reduce vision. Despite extensive efforts, for a substantial fraction of these cases the molecular causes are unknown. Recessive eye disorders are frequent in consanguineous populations and such large families with multiple affected individuals provide an opportunity to identify recessive causative genes. We studied a Pakistani consanguineous family with three affected individuals with congenital vision loss and progressive eye degeneration. The family was analyzed by exome sequencing of one affected individual and genotyping of all family members. We have identified a non-synonymous homozygous variant (NM_001128918.2:c.1708C>G:p.Arg570Gly) in the MARK3 gene as the likely cause of the phenotype. Given that MARK3 is highly conserved in flies (I: 55%; S: 67%) we knocked down the MARK3 homologue, par-1, in the eye during development. This leads to a significant reduction in eye size, a severe loss of photoreceptors and loss of vision based on electroretinogram (ERG) recordings. Expression of the par-1 p.Arg792Gly mutation (equivalent to the MARK3 variant found in patients) in developing fly eyes also induces loss of eye tissue and reduces the ERG signals. The data in flies and human indicate that the MARK3 variant corresponds to a loss of function. We conclude that the identified mutation in MARK3 establishes a new gene-disease link, since it likely causes structural abnormalities during eye development and visual impairment in humans, and that the function of MARK3/par-1 is evolutionarily conserved in eye development.

15.
J Nutr ; 148(4): 587-598, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29659964

RESUMO

Background: Dietary iodine requirements are high during pregnancy, lactation, and infancy, making women and infants vulnerable to iodine deficiency. Universal salt iodization (USI) has been remarkably successful for preventing iodine deficiency in the general population, but it is uncertain if USI provides adequate iodine intakes during the first 1000 d. Objective: We set out to assess if USI provides sufficient dietary iodine to meet the iodine requirements and achieve adequate iodine nutrition in all vulnerable population groups. Methods: We conducted an international, cross-sectional, multicenter study in 3 study sites with mandatory USI legislation. We enrolled 5860 participants from 6 population groups (school-age children, nonpregnant nonlactating women of reproductive age, pregnant women, lactating women, 0-6-mo-old infants, and 7-24-mo-old infants) and assessed iodine status [urinary iodine concentration (UIC)] and thyroid function in Linfen, China (n = 2408), Tuguegarao, the Philippines (n = 2512), and Zagreb, Croatia (n = 940). We analyzed the iodine concentration in household salt, breast milk, drinking water, and cow's milk. Results: The salt iodine concentration was low (<15 mg/kg) in 2.7%, 33.6%, and 3.1%, adequate (15-40 mg/kg) in 96.3%, 48.4%, and 96.4%, and high (>40 mg/kg) in 1.0%, 18.0%, and 0.5% of household salt samples in Linfen (n = 402), Tuguegarao (n = 1003), and Zagreb (n = 195), respectively. The median UIC showed adequate iodine nutrition in all population groups, except for excessive iodine intake in school-age children in the Philippines and borderline low intake in pregnant women in Croatia. Conclusions: Salt iodization at ∼25 mg/kg that covers a high proportion of the total amount of salt consumed supplies sufficient dietary iodine to ensure adequate iodine nutrition in all population groups, although intakes may be borderline low during pregnancy. Large variations in salt iodine concentrations increase the risk for both low and high iodine intakes. Strict monitoring of the national salt iodization program is therefore essential for optimal iodine nutrition. This trial was registered at clinicaltrials.gov as NCT02196337.

16.
J Am Heart Assoc ; 7(5)2018 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-29478971

RESUMO

BACKGROUND: Uninephrectomy (UNX) is performed for various reasons, including kidney cancer or donation. Kidneys being the main site of l-arginine production in the body, we tested whether UNX mediated kidney mass reduction impacts l-arginine metabolism and thereby nitric oxide production and blood pressure regulation in mice. METHODS AND RESULTS: In a first series of experiments, we observed a significant increase in arterial blood pressure 8 days post-UNX in female and not in male mice. Further experimental series were performed in female mice, and the blood pressure increase was confirmed by telemetry. l-citrulline, that is used in the kidney to produce l-arginine, was elevated post-UNX as was also asymmetric dimethylarginine, an inhibitor of nitric oxide synthase that competes with l-arginine and is a marker for renal failure. Interestingly, the UNX-induced blood pressure increase was prevented by supplementation of the diet with 5% of the l-arginine precursor, l-citrulline. Because l-arginine is metabolized in the kidney and other peripheral tissues by arginase-2, we tested whether the lack of this metabolic pathway also compensates for decreased l-arginine production in the kidney and/or for local nitric oxide synthase inhibition and consecutive blood pressure increase. Indeed, upon uninephrectomy, arginase-2 knockout mice (Arg-2-/-) neither displayed an increase in asymmetric dimethylarginine and l-citrulline plasma levels nor a significant increase in blood pressure. CONCLUSIONS: UNX leads to a small increase in blood pressure that is prevented by l-citrulline supplementation or arginase deficiency, 2 measures that appear to compensate for the impact of kidney mass reduction on l-arginine metabolism.

17.
PLoS One ; 12(9): e0184897, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28915261

RESUMO

BACKGROUND AND AIMS: Increased propionylcarnitine levels in newborn screening are indicative for a group of potentially severe disorders including propionic acidemia (PA), methylmalonic acidemias and combined remethylation disorders (MMACBL). This alteration is relatively non-specific, resulting in the necessity of confirmation and differential diagnosis in subsequent tests. Thus, we aimed to develop a multiplex approach for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid from the same dried blood spot (DBS) as in primary screening (second-tier test). We also set out to validate the method using newborn and follow-up samples of patients with confirmed PA or MMACBL. METHODS: The assay was developed using liquid chromatography-tandem mass spectrometry and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients. RESULTS: Reliable determination of all three analytes in DBSs was achieved following simple and fast (<20 min) sample preparation without laborious derivatization or any additional pipetting steps. The method clearly distinguished the pathological and normal samples and differentiated between PA and MMACBL in all stored newborn specimens. Methylcitric acid was elevated in all PA samples; 3-hydroxypropionic acid was also high in most cases. Methylmalonic acid was increased in all MMACBL specimens; mostly together with methylcitric acid. CONCLUSIONS: A liquid chromatography-tandem mass spectrometry assay allowing simultaneous determination of the biomarkers 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in DBSs has been developed. The assay can use the same specimen as in primary screening (second-tier test) which may reduce the need for repeated blood sampling. The presented preliminary findings suggest that this method can reliably differentiate patients with PA and MMACBL in newborn screening. The validated assay is being evaluated prospectively in a pilot project for extension of the German newborn screening panel (?Newborn screening 2020"; Newborn Screening Center, University Hospital Heidelberg).


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/sangue , Citratos/sangue , Teste em Amostras de Sangue Seco/métodos , Ácido Láctico/análogos & derivados , Programas de Rastreamento/métodos , Ácido Metilmalônico/sangue , Acidemia Propiônica/sangue , Cromatografia Líquida/métodos , Feminino , Humanos , Recém-Nascido , Ácido Láctico/sangue , Masculino , Espectrometria de Massas/métodos
18.
Mol Genet Metab Rep ; 13: 33-40, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28794993

RESUMO

INTRODUCTION: Phosphoglucomutase 1 deficiency (PGM1 deficiency) has been identified as both, glycogenosis and congenital disorder of glycosylation (CDG). The phenotype includes hepatopathy, myopathy, oropharyngeal malformations, heart disease and growth retardation. Oral galactose supplementation at a dosage of 1 g per kg body weight per day is regarded as the therapy of choice. RESULTS: We report on a patient with a novel disease causing mutation, who was treated for 1.5 years with oral galactose supplementation. Initially, elevated transaminases were reduced and protein glycosylation of serum transferrin improved rapidly. Long-term surveillance however indicated limitations of galactose supplementation at the standard dose: 1 g per kg body weight per day did not achieve permanent correction of protein glycosylation. Even increased doses of up to 2.5 g per kg body weight did not result in complete normalization. Furthermore, we described for the first time heart rhythm abnormalities, i.e. long QT Syndrome associated with a glycosylation disorder. Mass spectrometry of IGFBP3, which was assumed to play a major role in growth retardation associated with PGM1 deficiency, revealed no glycosylation abnormalities. Growth rate did not improve under galactose supplementation. CONCLUSIONS: The results of our study indicate that the current standard dose of galactose might be too low to achieve normal glycosylation in all patients. In addition, growth retardation in PGM1 deficiency is complex and multifactorial. Furthermore, heart rhythm abnormalities must be considered when treating patients with PGM1 deficiency.

19.
J Clin Endocrinol Metab ; 102(1): 23-32, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27732337

RESUMO

Context: Thyroglobulin (Tg) could be a sensitive biomarker of iodine nutrition in pregnant women (PW). A dried blood spot (DBS) assay would simplify collection and transport in field studies. Objectives: Our aims were to (1) establish and test a reference range for DBS-Tg in PW; (2) determine whether co-measurement of Tg antibodies (Abs) is necessary to define population iodine status. Design, Setting, and Participants: Standardized cross-sectional studies of 3870 PW from 11 countries. For the DBS-Tg reference range, we included TgAb-negative PW (n = 599) from 3 countries with sufficient iodine intake. Main Outcome Measures: We measured the urinary iodine concentration and DBS thyroid-stimulating hormone, total thyroxin, Tg, and TgAb. Results: In the reference population, the median DBS-Tg was 9.2 µg/L (95% confidence interval, 8.7 to 9.8 µg/L) and was not significantly different among trimesters. The reference range was 0.3 to 43.5 µg/L. Over a range of iodine intake, the Tg concentrations were U-shaped. Within countries, the median DBS-Tg and the presence of elevated DBS-Tg did not differ significantly between all PW and PW who were TgAb-negative. Conclusions: A median DBS-Tg of ∼10 µg/L with <3% of values ≥44 µg/L indicated population iodine sufficiency. Concurrent measurement of TgAb did not appear necessary to assess the population iodine status.


Assuntos
Biomarcadores/sangue , Teste em Amostras de Sangue Seco/métodos , Teste em Amostras de Sangue Seco/normas , Iodo/deficiência , Trimestres da Gravidez/sangue , Tireoglobulina/sangue , Adulto , Autoanticorpos/sangue , Estudos Transversais , Feminino , Seguimentos , Humanos , Iodo/sangue , Gravidez , Prognóstico , Valores de Referência , Adulto Jovem
20.
Clin Chim Acta ; 464: 236-243, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27923571

RESUMO

BACKGROUND: Clinical management of inherited or acquired hyperammonemia depends mainly on the plasma ammonia level which is not a reliable indicator of urea cycle function as its concentrations largely fluctuate. The gold standard to assess ureagenesis in vivo is the use of stable isotopes. METHODS: Here we developed and validated a simplified in vivo method with [15N]ammonium chloride ([15N]H4Cl) as a tracer. Non-labeled and [15N]urea were quantified by GC-MS after extraction and silylation. RESULTS: Different matrices were evaluated for suitability of analysis. Ureagenesis was assessed in ornithine transcarbamylase (OTC)-deficient spfash mice with compromised urea cycle function during fasted and non-fasted feeding states, and after rAAV2/8-vector delivery expressing the murine OTC-cDNA in liver. Blood (5µL) was collected through tail vein puncture before and after [15N]H4Cl intraperitoneal injections over a two hour period. The tested matrices, blood, plasma and dried blood spots, can be used to quantify ureagenesis. Upon [15N]H4Cl challenge, urea production in spfash mice was reduced compared to wild-type and normalized following rAAV2/8-mediated gene therapeutic correction. The most significant difference in ureagenesis was at 30min after injection in untreated spfash mice under fasting conditions (19% of wild-type). Five consecutive injections over a period of five weeks had no effect on body weight or ureagenesis. CONCLUSION: This method is simple, robust and with no apparent risk, offering a sensitive, minimal-invasive, and fast measurement of ureagenesis capacity using dried blood spots. The stable isotope-based quantification of ureagenesis can be applied for the efficacy-testing of novel molecular therapies.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Ureia/sangue , Animais , Jejum/sangue , Isótopos , Masculino , Camundongos , Ornitina Carbamoiltransferase/metabolismo
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