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Citrin deficiency (CD) is a recessive, liver disease caused by sequence variants in the SLC25A13 gene encoding a mitochondrial aspartate-glutamate transporter. CD manifests as different age-dependent phenotypes and affects crucial hepatic metabolic pathways including malate-aspartate-shuttle, glycolysis, gluconeogenesis, de novo lipogenesis and the tricarboxylic acid and urea cycles. Although the exact pathophysiology of CD remains unclear, impaired use of glucose and fatty acids as energy sources due to NADH shuttle defects and PPARα downregulation, respectively, indicates evident energy deficit in CD hepatocytes. The present review summarizes current trends on available and potential treatments for CD. Baseline recommendation for CD patients is dietary management, often already present as a self-selected food preference, that includes protein and fat-rich food, and avoidance of excess carbohydrates. At present, liver transplantation remains the sole curative option for severe CD cases. Our extensive literature review indicated medium-chain triglycerides (MCT) as the most widely used CD treatment in all age groups. MCT can effectively improve symptoms across disease phenotypes by rapidly supplying energy to the liver, restoring redox balance and inducing lipogenesis. In contrast, sodium pyruvate restored glycolysis and displayed initial preclinical promise, with however limited efficacy in adult CD patients. Ursodeoxycholic acid, nitrogen scavengers and L-arginine treatments effectively address specific pathophysiological aspects such as cholestasis and hyperammonemia and are commonly administered in combination with other drugs. Finally, future possibilities including restoring redox balance, amino acid supplementation, enhancing bioenergetics, improving ureagenesis and mRNA/DNA-based gene therapy are also discussed., (© 2024 The Author(s). Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Citrin deficiency (CD) is a complex metabolic condition due to defects in SLC25A13 encoding citrin, an aspartate/glutamate carrier located in the mitochondrial inner membrane. The condition was first described in Japan and other East Asian countries in patients who were thought to suffer from classical citrullinemia type 1, and was therefore classified as a urea cycle disorder. With an improved understanding of its molecular basis, it became apparent that a defect of citrin is primarily affecting the malate-aspartate shuttle with however multiple secondary effects on many central metabolic pathways including glycolysis, gluconeogenesis, de novo lipogenesis and ureagenesis. In the meantime, it became also clear that CD must be considered as a global disease with patients identified in many parts of the world and affected by SLC25A13 genotypes different from those known in East Asian populations. The present short review summarizes the (hi)story of this complex metabolic condition and tries to explain the relevance of including CD as a differential diagnosis in neonates and infants with cholestasis and in (not only adult) patients with hyperammonemia of unknown origin with subsequent impact on the emergency management., (© 2024 The Author(s). Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The pathway of ammonia disposal in the mammalian organism has been described in 1932 as a metabolic cycle present in the liver in different compartments. In 1958, the first human disorder affecting this pathway was described as a genetic condition leading to cognitive impairment and constant abnormalities of amino acid metabolism. Since then, defects in all enzymes and transporters of the urea cycle have been described, referring to them as primary urea cycle disorders causing primary hyperammonemia. In addition, there is a still increasing list of conditions that impact on the function of the urea cycle by various mechanisms, hereby leading to secondary hyperammonemia. Despite great advances in understanding the molecular background and the biochemical specificities of both primary and secondary hyperammonemias, there remain many open questions: we do not fully understand the pathophysiology in many of the conditions; we do not always understand the highly variable clinical course of affected patients; we clearly appreciate the need for novel and improved diagnostic and therapeutic approaches. This study does look back to the beginning of the urea cycle (hi)story, briefly describes the journey through past decades, hereby illustrating advancements and knowledge gaps, and gives examples for the extremely broad perspective imminent to some of the defects of ureagenesis and allied conditions., (© 2024 The Author(s). Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

N-acetylglutamate synthase (NAGS) makes acetylglutamate, the essential activator of the first, regulatory enzyme of the urea cycle, carbamoyl phosphate synthetase 1 (CPS1). NAGS deficiency (NAGSD) and CPS1 deficiency (CPS1D) present identical phenotypes. However, they must be distinguished, because NAGSD is cured by substitutive therapy with the N-acetyl-L-glutamate analogue N-carbamyl-L-glutamate, while curative therapy of CPS1D requires liver transplantation. Since their differentiation is done genetically, it is important to ascertain the disease-causing potential of CPS1 and NAGS genetic variants. With this goal, we previously carried out site-directed mutagenesis studies with pure recombinant human CPS1. We could not do the same with human NAGS (HuNAGS) because of enzyme instability, leading to our prior utilization of a bacterial NAGS as an imperfect surrogate of HuNAGS. We now use genuine HuNAGS, stabilized as a chimera of its conserved domain (cHuNAGS) with the maltose binding protein (MBP), and produced in Escherichia coli. MBP-cHuNAGS linker cleavage allowed assessment of the enzymatic properties and thermal stability of cHuNAGS, either wild-type or hosting each one of 23 nonsynonymous single-base changes found in NAGSD patients. For all but one change, disease causation was accounted by the enzymatic alterations identified, including, depending on the variant, loss of arginine activation, increased K m Glutamate , active site inactivation, decreased thermal stability, and protein misfolding. Our present approach outperforms experimental in vitro use of bacterial NAGS or in silico utilization of prediction servers (including AlphaMissense), illustrating with HuNAGS the value for UCDs of using recombinant enzymes for assessing disease-causation and molecular pathogenesis, and for therapeutic guidance., (© 2024 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Citrin deficiency is an autosomal recessive disorder caused by a defect of citrin resulting from mutations in SLC25A13. The clinical manifestation is very variable and comprises three types: neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD: OMIM 605814), post-NICCD including failure to thrive and dyslipidemia caused by citrin deficiency, and adult-onset type II citrullinemia (CTLN2: OMIM 603471). Frequently, NICCD can run with a mild clinical course and manifestations may resolve in the post-NICCD. However, a subset of patients may develop CTLN2 when they become more than 18 years old, and this condition is potentially life-threatening. Since a combination of diet with low-carbohydrate and high-fat content supplemented with medium-chain triglycerides is expected to ameliorate most manifestations and to prevent the progression to CTLN2, early detection and intervention are important and may improve long-term outcome in patients. Moreover, infusion of high sugar solution and/or glycerol may be life-threatening in patients with citrin deficiency, particularly CTLN2. The disease is highly prevalent in East Asian countries but is more and more recognized as a global entity. Since newborn screening for citrin deficiency has only been introduced in a few countries, the diagnosis still mainly relies on clinical suspicion followed by genetic testing or selective metabolic screening. This paper aims at describing (1) the different stages of the disease focusing on clinical aspects; (2) the current published clinical situation in East Asia, Europe, and North America; (3) current efforts in increasing awareness by establishing management guidelines and patient registries, hereby illustrating the ongoing development of a global network for this rare disease., (© 2024 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The group of rare metabolic defects termed urea cycle disorders (UCDs) occur within the ammonia elimination pathway and lead to significant neurocognitive sequelae for patients surviving decompensation episodes. Besides orthotopic liver transplantation, curative options are lacking for UCDs, with dietary management being the gold clinical standard. Novel therapeutic approaches are essential for UCDs; however, such effort presupposes preclinical testing in cellular models that effectively capture disease manifestation. Several cellular and animal models exist and aim to recapitulate the broad phenotypic spectrum of UCDs; however, the majority of those lack extensive molecular and biochemical characterization. The development of cellular models is emerging since animal models are extremely time and cost consuming, and subject to ethical considerations, including the 3R principle that endorses animal welfare over unchecked preclinical testing. The aim of this study was to compare the extent of expression and functionality of the urea cycle in two commercial hepatoma-derived cell lines, induced pluripotent stem cell hepatocytes (iPSC-Heps), primary human hepatocytes (PHHs) and human liver cell preparations. Using immunoblotting, immunocytochemistry, and stable isotope tracing of the urea cycle metabolites, we identified that the hepatoma-derived, 2-week differentiated HepaRG cells are urea cycle proficient and behave as cellular alternatives to PHHs. Furthermore, HepaRG cells were superior to iPSC-Heps, which are known to exhibit batch-to-batch variabilities in terms of hepatic maturity and enzyme expression. Finally, HepG2 cells lack the urea cycle enzymes ornithine transcarbamylase and arginase 1, the transporter ORNT1, which limits their suitability as model for the study of UCDs., (© 2023 SSIEM.)

Citrin deficiency is an autosomal recessive disorder caused by a defect of citrin resulting from mutations in the SLC25A13 gene. Intrahepatic cholestasis and various metabolic abnormalities, including hypoglycemia, galactosemia, citrullinemia, and hyperammonemia may be present in neonates or infants in the "neonatal intrahepatic cholestasis caused by citrin deficiency" (NICCD) form of the disease. Because at present, newborn screening (NBS) for citrin deficiency using citrulline levels in dried blood spots (DBS) can only detect some of the patients, we tried to develop a new evaluation system to more reliably detect newborns with citrin deficiency utilizing parameters already in place in present NBS methods. To achieve this goal, we re-analyzed NBS profiles of amino acids and acylcarnitines in 96 NICCD patients, who were diagnosed through selective screening or positive family history. Hereby, we identified the combined evaluation of arginine (Arg), citrulline (Cit), isoleucine+leucine (Ile + Leu), tyrosine (Tyr), free carnitine (C0) / glutarylcarnitine (C5-DC) ratio in DBS as potentially sensitive to diagnose citrin deficiency in pre-symptomatic newborns. In particular, a scoring system using threshold levels for Arg (≥9 μmol/L), Cit (≥ 39 μmol/L), Ile + Leu (≥ 99 μmol/L), Tyr (≥ 96 μmol/L) and C0/C5-DC ratio (≥327) was significantly effective to detect newborns who later developed NICCD, and could thus be implemented in existing NBS programs at no extra analytical costs whenever citrin deficiency is considered to become a novel target disease., (© 2023 SSIEM.)

The SARS-CoV-2 pandemic challenges healthcare systems worldwide. Within inherited metabolic disorders (IMDs) the vulnerable subgroup of intoxication-type IMDs such as organic acidurias (OA) and urea cycle disorders (UCD) show risk for infection-induced morbidity and mortality. This study (observation period February 2020 to December 2021) evaluates impact on medical health care as well as disease course and outcome of SARS-CoV-2 infections in patients with intoxication-type IMDs managed by participants of the European Registry and Network for intoxication type metabolic diseases Consortium (E-IMD). Survey's respondents managing 792 patients (n = 479 pediatric; n = 313 adult) with intoxication-type IMDs (n = 454 OA; n = 338 UCD) in 14 countries reported on 59 (OA: n = 36; UCD: n = 23), SARS-CoV-2 infections (7.4%). Medical services were increasingly requested (95%), mostly alleviated by remote technologies (86%). Problems with medical supply were scarce (5%). Regular follow-up visits were reduced in 41% (range 10%-50%). Most infected individuals (49/59; 83%) showed mild clinical symptoms, while 10 patients (17%; n = 6 OA including four transplanted MMA patients; n = 4 UCD) were hospitalized (metabolic decompensation in 30%). ICU treatment was not reported. Hospitalization rate did not differ for diagnosis or age group (p = 0.778). Survival rate was 100%. Full recovery was reported for 100% in outpatient care and 90% of hospitalized individuals. SARS-CoV-2 impacts health care of individuals with intoxication-type IMDs worldwide. Most infected individuals, however, showed mild symptoms and did not require hospitalization. SARS-CoV-2-induced metabolic decompensations were usually mild without increased risk for ICU treatment. Overall prognosis of infected individuals is very promising and IMD-specific or COVID-19-related complications have not been observed., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Citrin deficiency is an autosomal recessive disorder caused by mutations in the SLC25A13 gene. The disease can present with age-dependent clinical manifestations: neonatal intrahepatic cholestasis by citrin deficiency (NICCD), failure to thrive, and dyslipidemia by citrin deficiency (FTTDCD), and adult-onset type II citrullinemia (CTLN2). As a nationwide study to investigate the clinical manifestations, medical therapy, and long-term outcome in Japanese patients with citrin deficiency, we collected clinical data of 222 patients diagnosed and/or treated at various different institutions between January 2000 and December 2019. In the entire cohort, 218 patients were alive while 4 patients (1 FTTDCD and 3 CTLN2) had died. All patients <20 years were alive. Patients with citrin deficiency had an increased risk for low weight and length at birth, and CTLN2 patients had an increased risk for growth impairment during adolescence. Liver transplantation has been performed in only 4 patients (1 NICCD, 3 CTLN2) with a good response thereafter. This study reports the diagnosis and clinical course in a large cohort of patients with citrin deficiency and suggests that early intervention including a low carbohydrate diet and MCT supplementation can be associated with improved clinical course and long-term outcome., (© 2022 SSIEM.)

Deficiency of the biotinidase (BTD) enzyme is an inborn error of biotin metabolism caused by biallelic pathogenic variants in the BTD gene. There are two forms, partial and profound BTD deficiency, which both can be successfully treated with pharmacological doses of biotin, justifying the inclusion of this disorder in the newborn screening in numerous countries. We investigated the BTD deficiency cohort (N = 87) in our metabolic center, as it was detected upon newborn screening since 2005, and aimed to better understand the long-term course of BTD enzyme activity and how it may relate to the patients' genetic background. We observed that individuals with partial BTD deficiency display an elevation of BTD enzyme activity with increasing age in 48% of cases-a recovery which allowed adjustment or stop of biotin supplementation in 20% of all individuals. In addition, we were able to recruit 56 patients (64%) for genetic testing, revealing 19 different variants (2 novel), and constituting 22 different genotypes. Genotype-phenotype correlations revealed that the most abundant allele in our cohort p.(Asp444His) was also the most common variant in patients displaying recovery of BTD enzyme activity. Based on our results, we recommend to retest all patients with partial BTD deficiency at the age of 5 years, as this may result in an impact on therapy. Moreover, genetic testing of BTD deficient individuals can allow prediction of the severity of BTD deficiency and of the likelihood of BTD enzyme activity recovery with age., (© 2022 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Urea cycle disorders (UCDs) are inherited metabolic diseases causing hyperammonemia by defects in urea cycle enzymes or transporters. Liver transplantation (LT) currently is the only curative treatment option until novel therapies become available. We performed a nationwide questionnaire-based study between January 2000 and March 2018 to investigate the effect of LT in patients with UCDs in Japan. A total of 231 patients with UCDs were enrolled in this study. Of them, a total of 78 patients with UCDs (30 male and 16 female ornithine transcarbamylase deficiency (OTCD), 21 carbamoyl phosphate synthetase 1 deficiency (CPSD), 10 argininosuccinate synthetase deficiency (ASSD) and 1 arginase 1 deficiency (ARGD)) had undergone LT. Concerning the maximum blood ammonia levels at the onset time in the transplanted male OTCD (N = 28), female OTCD (N = 15), CPSD (N = 21) and ASSD (N = 10), those were median 634 (IQR: 277-1172), 268 (211-352), 806 (535-1382), and 628 (425-957) μmol/L, respectively. The maximum blood ammonia levels in female OTCD were thus significantly lower than in the other UCDs (all P < .01). LT was effective for long-term survival, prevented recurrent hyperammonemia attack, and lowered baseline blood ammonia levels in patients with UCDs. LT had limited effect for ameliorating neurodevelopmental outcome in patients with severe disease because hyperammonemia at the onset time already had a significant impact on the brain. Patients with ASSD may be more likely to survive without cognitive impairment by receiving early LT despite severe neonatal hyperammonemia ≥ 360 μmol/L. In patients with neonatal onset OTCD or CPSD, there may be additional factors with adverse effects on the brain that are not improved by LT., (© 2021 SSIEM.)

Urea cycle disorders (UCDs) are inherited metabolic disorders with impaired nitrogen detoxification caused by defects in urea cycle enzymes. They often manifest with hyperammonemic attacks resulting in significant morbidity or death. We performed a nationwide questionnaire-based study between January 2000 and March 2018 to document all UCDs in Japan, including diagnoses, treatments, and outcomes. A total of 229 patients with UCDs were enrolled in this study: 73 males and 53 females with ornithine transcarbamylase deficiency (OTCD), 33 patients with carbamoylphosphate synthetase 1 deficiency, 48 with argininosuccinate synthetase deficiency, 14 with argininosuccinate lyase deficiency, and 8 with arginase deficiency. Survival rates at 20 years of age of male and female patients with late-onset OTCD were 100% and 97.7%, respectively. Blood ammonia levels and time of onset had a significant impact on the neurodevelopmental outcome (P < .001 and P = .028, respectively). Hemodialysis and liver transplantation did not prevent poor neurodevelopmental outcomes. While treatment including medication, hemodialysis, and liver transplantation may aid in decreasing blood ammonia and/or preventing severe hyperammonemia, a blood ammonia level ≥ 360 μmol/L was found to be a significant indicator for a poor neurodevelopmental outcome. In conclusion, although current therapy for UCDs has advanced and helped saving lives, patients with blood ammonia levels ≥ 360 μmol/L at onset often have impaired neurodevelopmental outcomes. Novel neuroprotective measures should therefore be developed to achieve better neurodevelopmental outcomes in these patients., (© 2021 SSIEM.)

Since 1972, 18 patients (10 females/8 males) have been detected by newborn bloodspot screening (NBS) with neonatal-onset maple syrup urine disease (MSUD) in Ireland. Patients were stratified into three clusters according to clinical outcome at the time of data collection, including developmental, clinical, and IQ data. A fourth cluster comprised of two early childhood deaths; a third patient died as an adult. We present neuroimaging and electroencephalography together with clinical and biochemical data. Incidence of MSUD (1972-2018) was 1 in 147 975. Overall good clinical outcomes were achieved with 15/18 patients alive and with essentially normal functioning (with only the lowest performing cluster lying beyond a single SD on their full scale intelligence quotient). Molecular genetic analysis revealed genotypes hitherto not reported, including a possible digenic inheritance state for the BCKDHA and DBT genes in one family. Treatment has been based on early implementation of emergency treatment, diet, close monitoring, and even dialysis in the setting of acute metabolic decompensation. A plasma leucine ≥400 μmol/L (outside therapeutic range) was more frequently observed in infancy or during adolescence, possibly due to infections, hormonal changes, or noncompliance. Children require careful management during metabolic decompensations in early childhood, and this represented a key risk period in our cohort. A high level of metabolic control can be achieved through diet with early implementation of a "sick day" regime and, in some cases, dialysis as a rescue therapy. The Irish cohort, despite largely classical phenotypes, achieved good outcomes in the NBS era, underlining the importance of early diagnosis and skilled multidisciplinary team management., (© 2020 SSIEM.)

The enzyme carbamoyl phosphate synthetase 1 (CPS1; EC 6.3.4.16) forms carbamoyl phosphate from bicarbonate, ammonia, and adenosine triphosphate (ATP) and is activated allosterically by N-acetylglutamate. The neonatal presentation of bi-allelic mutations of CPS1 results in hyperammonemia with reduced citrulline and is reported as the most challenging nitrogen metabolism disorder to treat. As therapeutic interventions are limited, patients often develop neurological injury or die from hyperammonemia. Survivors remain vulnerable to nitrogen overload, being at risk for repetitive neurological injury. With transgenic technology, our lab developed a constitutive Cps1 mutant mouse and reports its characterization herein. Within 24 hours of birth, all Cps1 -/- mice developed hyperammonemia and expired. No CPS1 protein by Western blot or immunostaining was detected in livers nor was Cps1 mRNA present. CPS1 enzymatic activity was markedly decreased in knockout livers and reduced in Cps1 +/- mice. Plasma analysis found markedly reduced citrulline and arginine and markedly increased glutamine and alanine, both intermolecular carriers of nitrogen, along with elevated ammonia, taurine, and lysine. Derangements in multiple other amino acids were also detected. While hepatic amino acids also demonstrated markedly reduced citrulline, arginine, while decreased, was not statistically significant; alanine and lysine were markedly increased while glutamine was trending towards significance. In conclusion we have determined that this constitutive neonatal mouse model of CPS1 deficiency replicates the neonatal human phenotype and demonstrates the key biochemical features of the disorder. These mice will be integral for addressing the challenges of developing new therapeutic approaches for this, at present, poorly treated disorder., (© 2019 SSIEM.)

The first patients affected by argininosuccinic aciduria (ASA) were reported 60 years ago. The clinical presentation was initially described as similar to other urea cycle defects, but increasing evidence has shown overtime an atypical systemic phenotype with a paradoxical observation, that is, a higher rate of neurological complications contrasting with a lower rate of hyperammonaemic episodes. The disappointing long-term clinical outcomes of many of the patients have challenged the current standard of care and therapeutic strategy, which aims to normalize plasma ammonia and arginine levels. Interrogations have raised about the benefit of newborn screening or liver transplantation on the neurological phenotype. Over the last decade, novel discoveries enabled by the generation of new transgenic argininosuccinate lyase (ASL)-deficient mouse models have been achieved, such as, a better understanding of ASL and its close interaction with nitric oxide metabolism, ASL physiological role outside the liver, and the pathophysiological role of oxidative/nitrosative stress or excessive arginine treatment. Here, we present a collaborative review, which highlights these recent discoveries and novel emerging concepts about ASL role in human physiology, ASA clinical phenotype and geographic prevalence, limits of current standard of care and newborn screening, pathophysiology of the disease, and emerging novel therapies. We propose recommendations for monitoring of ASA patients. Ongoing research aims to better understand the underlying pathogenic mechanisms of the systemic disease to design novel therapies., (© 2019 SSIEM.)

The urea cycle disorder (UCD) argininosuccinate lyase (ASL) deficiency, caused by a defective ASL enzyme, exhibits a wide range of phenotypes, from life-threatening neonatal hyperammonemia to asymptomatic patients, with only the biochemical marker argininosuccinic acid (ASA) elevated in body fluids. Remarkably, even without ever suffering from hyperammonemia, patients often develop severe cognitive impairment and seizures. The goal of this study was to understand the effect on the known toxic metabolite ASA and the assumed toxic metabolite guanidinosuccinic acid (GSA) on developing brain cells, and to evaluate the potential role of creatine (Cr) supplementation, as it was described protective for brain cells exposed to ammonia. We used an in vitro model, in which we exposed three-dimensional (3D) organotypic rat brain cell cultures in aggregates to different combinations of the metabolites of interest at two time points (representing two different developmental stages). After harvest and cryopreservation of the cell cultures, the samples were analyzed mainly by metabolite analysis, immunohistochemistry, and western blotting. ASA and GSA were found toxic for astrocytes and neurons. This toxicity could be reverted in vitro by Cr. As well, an antiapoptotic effect of ASA was revealed, which could contribute to the neurotoxicity in ASL deficiency. Further studies in human ASL deficiency will be required to understand the biochemical situation in the brain of affected patients, and to investigate the impact of high or low arginine doses on brain Cr availability. In addition, clinical trials to evaluate the beneficial effect of Cr supplementation in ASL deficiency would be valuable., (© 2019 SSIEM.)

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., (© 2019 SSIEM.)

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., (© 2019 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

In 2012, we published guidelines summarizing and evaluating late 2011 evidence for diagnosis and therapy of urea cycle disorders (UCDs). With 1:35 000 estimated incidence, UCDs cause hyperammonemia of neonatal (~50%) or late onset that can lead to intellectual disability or death, even while effective therapies do exist. In the 7 years that have elapsed since the first guideline was published, abundant novel information has accumulated, experience on newborn screening for some UCDs has widened, a novel hyperammonemia-causing genetic disorder has been reported, glycerol phenylbutyrate has been introduced as a treatment, and novel promising therapeutic avenues (including gene therapy) have been opened. Several factors including the impact of the first edition of these guidelines (frequently read and quoted) may have increased awareness among health professionals and patient families. However, under-recognition and delayed diagnosis of UCDs still appear widespread. It was therefore necessary to revise the original guidelines to ensure an up-to-date frame of reference for professionals and patients as well as for awareness campaigns. This was accomplished by keeping the original spirit of providing a trans-European consensus based on robust evidence (scored with GRADE methodology), involving professionals on UCDs from nine countries in preparing this consensus. We believe this revised guideline, which has been reviewed by several societies that are involved in the management of UCDs, will have a positive impact on the outcomes of patients by establishing common standards, and spreading and harmonizing good practices. It may also promote the identification of knowledge voids to be filled by future research., (© 2019 SSIEM.)

Classical homocystinuria (HCU) is the most common inborn error of metabolism in Qatar, with an incidence of 1:1800, and is caused by the Qatari founder p.R336C mutation in the CBS gene. This study describes the natural history and clinical manifestations of HCU in the Qatari population. A single center study was performed between 2016 and 2017 in 126 Qatari patients, from 82 families. Detailed clinical and biochemical data were collected, and Stanford-Binet intelligence, quality of life and adherence to treatment assessments were conducted prospectively. Patients were assigned to one of three groups, according to the mode of diagnosis: (a) late diagnosis group (LDG), (b) family screening group (FSG), and (c) newborn screening group (NSG). Of the 126 patients, 69 (55%) were in the LDG, 44 (35%) in the NSG, and 13 (10%) in the FSG. The leading factors for diagnosis in the LDG were ocular manifestations (49%), neurological manifestations (45%), thromboembolic events (4%), and hyperactivity and behavioral changes (1%). Both FSG and NSG groups were asymptomatic at time of diagnosis. NSG had significantly higher intelligence quotient, quality of life, and adherence values compared with the LDG. The LDG and FSG had significantly higher methionine levels than the NSG. The LDG also had significantly higher total homocysteine levels than the NSG and FSG. Regression analysis confirmed these results even when adjusting for age at diagnosis, current age, or adherence. These findings increase the understanding of the natural history of HCU and highlight the importance of early diagnosis and treatment. SYNOPSIS: A study in 126 Qatari patients with HCU, including biochemical, clinical, and other key assessments, reveals that patients with a late clinical diagnosis have a poorer outcome, hereby highlighting the importance of early diagnosis and treatment., (© 2019 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Classical homocystinuria is a recessive inborn error of metabolism caused by mutations in the cystathionine beta-synthase (CBS) gene. The highest incidence of CBS deficiency in the world is found in the country of Qatar due to the combination of high rates of consanguinity and the presence of a founder mutation, c.1006C>T (p.R336C). This mutation does not respond to pyridoxine and is considered severe. Here we describe the creation of a mouse that is null for the mouse Cbs gene and expresses human p.R336C CBS from a zinc-inducible transgene (Tg-R336C Cbs -/- ). Zinc-treated Tg-R336C Cbs -/- mice have extreme elevation in both serum total homocysteine (tHcy) and liver tHcy compared with control transgenic mice. Both the steady-state protein levels and CBS enzyme activity levels in liver lysates from Tg-R336C Cbs -/- mice are significantly reduced compared to that found in Tg-hCBS Cbs -/- mice expressing wild-type human CBS. Treatment of Tg-R336C Cbs -/- mice with the proteasome inhibitor bortezomib results in stabilization of liver CBS protein and an increase in activity to levels found in corresponding Tg-hCBS Cbs -/- wild type mice. Surprisingly, serum tHcy did not fully correct even though liver enzyme activity was as high as control animals. This discrepancy is explained by in vitro enzymatic studies of mouse liver extracts showing that p.R336C causes reduced binding affinity for the substrate serine by almost 7-fold and significantly increased dependence on pyridoxal phosphate in the reaction buffer. These studies demonstrate that the p.R336C alteration effects both protein stability and substrate/cofactor binding., (© 2019 SSIEM.)

Neonatal onset hyperammonemia in patients with urea cycle disorders (UCDs) is still associated with high morbidity and mortality. Current protocols consistently recommend emergency medical and dietary management. In case of increasing or persistent hyperammonemia, with continuous or progressive neurological signs, dialysis is performed, mostly as ultima ratio. It is presently unknown whether the currently defined ammonia threshold (e.g., at 500 μmol/L) to start dialysis is useful to improve clinical outcome. A systematic review of clinical and biochemical data from published neonatal onset UCD patients was performed to identify factors determining clinical outcome and to investigate in which clinical and biochemical setting dialysis was most effective. A total of 202 patients (118 proximal and 84 distal UCDs) described in 90 case reports or case series were included according to predefined inclusion/exclusion criteria. Median age at onset was three days and mean ammonia that triggered start of dialysis was 1199 μmol/L. Seventy-one percent of all patients received any form of dialysis. Total mortality was 25% and only 20% of all patients had a "normal" outcome. In general, patients with higher ammonia levels were more likely to receive dialysis, but this had for most patients no influence on outcome. In conclusion, in severe neonatal onset hyperammonemia, the current practice of dialysis, which effectively clears ammonia, had no impact on outcome. It may be essential for improving outcome to initiate all available treatment options, including dialysis, as early as possible.

Background: Patients with urea cycle disorders (UCDs) have an increased risk of neurological disease manifestation., Aims: Determining the effect of diagnostic and therapeutic interventions on the neurological outcome., Methods: Evaluation of baseline, regular follow-up and emergency visits of 456 UCD patients prospectively followed between 2011 and 2015 by the E-IMD patient registry., Results: About two-thirds of UCD patients remained asymptomatic until age 12 days [i.e. the median age at diagnosis of patients identified by newborn screening (NBS)] suggesting a potential benefit of NBS. In fact, NBS lowered the age at diagnosis in patients with late onset of symptoms (>28 days), and a trend towards improved long-term neurological outcome was found for patients with argininosuccinate synthetase and lyase deficiency as well as argininemia identified by NBS. Three to 17 different drug combinations were used for maintenance therapy, but superiority of any single drug or specific drug combination above other combinations was not demonstrated. Importantly, non-interventional variables of disease severity, such as age at disease onset and peak ammonium level of the initial hyperammonemic crisis (cut-off level: 500 μmol/L) best predicted the neurological outcome., Conclusions: Promising results of NBS for late onset UCD patients are reported and should be re-evaluated in a larger and more advanced age group. However, non-interventional variables affect the neurological outcome of UCD patients. Available evidence-based guideline recommendations are currently heterogeneously implemented into practice, leading to a high variability of drug combinations that hamper our understanding of optimised long-term and emergency treatment.

Background: Arginase 1 (ARG1) deficiency is a rare urea cycle disorder (UCD). This hypothesis-generating study explored clinical phenotypes, metabolic profiles, molecular genetics, and treatment approaches in a cohort of children and adults with ARG1 deficiency to add to our understanding of the underlying pathophysiology., Methods: Clinical data were retrieved retrospectively from physicians using a questionnaire survey. Plasma aminoacids, guanidinoacetate (GAA), parameters indicating oxidative stress and nitric oxide (NO) synthesis as well as asymmetric dimethylarginine (ADMA) were measured at a single study site., Results: Nineteen individuals with ARG1 deficiency and 19 matched controls were included in the study. In patients, paraparesis, cognitive impairment, and seizures were significantly associated suggesting a shared underlying pathophysiology. In patients plasma GAA exceeded normal ranges and plasma ADMA was significantly elevated. Compared to controls, nitrate was significantly higher, and the nitrite:nitrate ratio significantly lower in subjects with ARG1 deficiency suggesting an advantage for NO synthesis by inducible NO synthase (iNOS) over endothelial NOS (eNOS). Logistic regression revealed no significant impact of any of the biochemical parameters (including arginine, nitrates, ADMA, GAA, oxidative stress) or protein restriction on long-term outcome., Conclusion: Three main hypotheses which must be evaluated in a hypothesis driven confirmatory study are delineated from this study: 1) clinical manifestations in ARG1 deficiency are not correlated with arginine, protein intake, ADMA, nitrates or oxidative stress. 2) GAA is elevated and may be a marker or an active part of the pathophysiology of ARG1 deficiency. 3) Perturbations of NO metabolism merit future attention in ARG1 deficiency.

Background: Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn defect disturbing the remethylation of homocysteine to methionine (<200 reported cases). This retrospective study evaluates clinical, biochemical genetic and in vitro enzymatic data in a cohort of 33 patients., Methods: Clinical, biochemical and treatment data was obtained from physicians by using a questionnaire. MTHFR activity was measured in primary fibroblasts; genomic DNA was extracted from cultured fibroblasts., Results: Thirty-three patients (mean age at follow-up 11.4 years; four deceased; median age at first presentation 5 weeks; 17 females) were included. Patients with very low (<1.5%) mean control values of enzyme activity (n = 14) presented earlier and with a pattern of feeding problems, encephalopathy, muscular hypotonia, neurocognitive impairment, apnoea, hydrocephalus, microcephaly and epilepsy. Patients with higher (>1.7-34.8%) residual enzyme activity had mainly psychiatric symptoms, mental retardation, myelopathy, ataxia and spasticity. Treatment with various combinations of betaine, methionine, folate and cobalamin improved the biochemical and clinical phenotype. During the disease course, patients with very low enzyme activity showed a progression of feeding problems, neurological symptoms, mental retardation, and psychiatric disease while in patients with higher residual enzyme activity, myelopathy, ataxia and spasticity increased. All other symptoms remained stable or improved in both groups upon treatment as did brain imaging in some cases. No clear genotype-phenotype correlation was obvious., Discussion: MTHFR deficiency is a severe disease primarily affecting the central nervous system. Age at presentation and clinical pattern are correlated with residual enzyme activity. Treatment alleviates biochemical abnormalities and clinical symptoms partially.

Background: The clinical presentation of patients with organic acidurias (OAD) and urea cycle disorders (UCD) is variable; symptoms are often non-specific., Aims/methods: To improve the knowledge about OAD and UCD the E-IMD consortium established a web-based patient registry., Results: We registered 795 patients with OAD (n = 452) and UCD (n = 343), with ornithine transcarbamylase (OTC) deficiency (n = 196), glutaric aciduria type 1 (GA1; n = 150) and methylmalonic aciduria (MMA; n = 149) being the most frequent diseases. Overall, 548 patients (69 %) were symptomatic. The majority of them (n = 463) presented with acute metabolic crisis during (n = 220) or after the newborn period (n = 243) frequently demonstrating impaired consciousness, vomiting and/or muscular hypotonia. Neonatal onset of symptoms was most frequent in argininosuccinic synthetase and lyase deficiency and carbamylphosphate 1 synthetase deficiency, unexpectedly low in male OTC deficiency, and least frequently in GA1 and female OTC deficiency. For patients with MMA, propionic aciduria (PA) and OTC deficiency (male and female), hyperammonemia was more severe in metabolic crises during than after the newborn period, whereas metabolic acidosis tended to be more severe in MMA and PA patients with late onset of symptoms. Symptomatic patients without metabolic crises (n = 94) often presented with a movement disorder, mental retardation, epilepsy and psychiatric disorders (the latter in UCD only)., Conclusions: The initial presentation varies widely in OAD and UCD patients. This is a challenge for rapid diagnosis and early start of treatment. Patients with a sepsis-like neonatal crisis and those with late-onset of symptoms are both at risk of delayed or missed diagnosis.

Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

Background: The disease course and long-term outcome of patients with organic acidurias (OAD) and urea cycle disorders (UCD) are incompletely understood., Aims: To evaluate the complex clinical phenotype of OAD and UCD patients at different ages., Results: Acquired microcephaly and movement disorders were common in OAD and UCD highlighting that the brain is the major organ involved in these diseases. Cardiomyopathy [methylmalonic (MMA) and propionic aciduria (PA)], prolonged QTc interval (PA), optic nerve atrophy [MMA, isovaleric aciduria (IVA)], pancytopenia (PA), and macrocephaly [glutaric aciduria type 1 (GA1)] were exclusively found in OAD patients, whereas hepatic involvement was more frequent in UCD patients, in particular in argininosuccinate lyase (ASL) deficiency. Chronic renal failure was often found in MMA, with highest frequency in mut(0) patients. Unexpectedly, chronic renal failure was also observed in adolescent and adult patients with GA1 and ASL deficiency. It had a similar frequency in patients with or without a movement disorder suggesting different pathophysiology. Thirteen patients (classic OAD: 3, UCD: 10) died during the study interval, ten of them during the initial metabolic crisis in the newborn period. Male patients with late-onset ornithine transcarbamylase deficiency were presumably overrepresented in the study population., Conclusions: Neurologic impairment is common in OAD and UCD, whereas the involvement of other organs (heart, liver, kidneys, eyes) follows a disease-specific pattern. The identification of unexpected chronic renal failure in GA1 and ASL deficiency emphasizes the importance of a systematic follow-up in patients with rare diseases.

Loss of function of the urea cycle enzyme argininosuccinate lyase (ASL) is caused by mutations in the ASL gene leading to ASL deficiency (ASLD). ASLD has a broad clinical spectrum ranging from life-threatening severe neonatal to asymptomatic forms. Different levels of residual ASL activity probably contribute to the phenotypic variability but reliable expression systems allowing clinically useful conclusions are not yet available. In order to define the molecular characteristics underlying the phenotypic variability, we investigated all ASL mutations that were hitherto identified in patients with late onset or mild clinical and biochemical courses by ASL expression in human embryonic kidney 293 T cells. We found residual activities >3% of ASL wild type (WT) in nine of 11 ASL mutations. Six ASL mutations (p.Arg95Cys, p.Ile100Thr, p.Val178Met, p.Glu189Gly, p.Val335Leu, and p.Arg379Cys) with residual activities ≥16% of ASL WT showed no significant or less than twofold reduced Km values, but displayed thermal instability. Computational structural analysis supported the biochemical findings by revealing multiple effects including protein instability, disruption of ionic interactions and hydrogen bonds between residues in the monomeric form of the protein, and disruption of contacts between adjacent monomeric units in the ASL tetramer. These findings suggest that the clinical and biochemical course in variant forms of ASLD is associated with relevant residual levels of ASL activity as well as instability of mutant ASL proteins. Since about 30% of known ASLD genotypes are affected by mutations studied here, ASLD should be considered as a candidate for chaperone treatment to improve mutant protein stability.

Unlabelled: The cblC defect is the most common inborn error of vitamin B12 metabolism. Despite therapeutic measures, the long-term outcome is often unsatisfactory. This retrospective multicentre study evaluates clinical, biochemical and genetic findings in 88 cblC patients. The questionnaire designed for the study evaluates clinical and biochemical features at both initial presentation and during follow up. Also the development of severity scores allows investigation of individual disease load, statistical evaluation of parameters between the different age of presentation groups, as well as a search for correlations between clinical endpoints and potential modifying factors., Results: No major differences were found between neonatal and early onset patients so that these groups were combined as an infantile-onset group representing 88 % of all cases. Hypotonia, lethargy, feeding problems and developmental delay were predominant in this group, while late-onset patients frequently presented with psychiatric/behaviour problems and myelopathy. Plasma total homocysteine was higher and methionine lower in infantile-onset patients. Plasma methionine levels correlated with "overall impression" as judged by treating physicians. Physician's impression of patient's well-being correlated with assessed disease load. We confirmed the association between homozygosity for the c.271dupA mutation and infantile-onset but not between homozygosity for c.394C>T and late-onset. Patients were treated with parenteral hydroxocobalamin, betaine, folate/folinic acid and carnitine resulting in improvement of biochemical abnormalities, non-neurological signs and mortality. However the long-term neurological and ophthalmological outcome is not significantly influenced. In summary the survey points to the need for prospective studies in a large cohort using agreed treatment modalities and monitoring criteria.

Urea cycle disorders (UCDs) are inherited disorders of ammonia detoxification often regarded as mainly of relevance to pediatricians. Based on an increasing number of case studies it has become obvious that a significant number of UCD patients are affected by their disease in a non-classical way: presenting outside the newborn period, following a mild course, presenting with unusual clinical features, or asymptomatic patients with only biochemical signs of a UCD. These patients are surviving into adolescence and adulthood, rendering this group of diseases clinically relevant to adult physicians as well as pediatricians. In preparation for an international workshop we collected data on all patients with non-classical UCDs treated by the participants in 20 European metabolic centres. Information was collected on a cohort of 208 patients 50% of which were ≥ 16 years old. The largest subgroup (121 patients) had X-linked ornithine transcarbamylase deficiency (OTCD) of whom 83 were female and 29% of these were asymptomatic. In index patients, there was a mean delay from first symptoms to diagnosis of 1.6 years. Cognitive impairment was present in 36% of all patients including female OTCD patients (in 31%) and those 41 patients identified presymptomatically following positive newborn screening (in 12%). In conclusion, UCD patients with non-classical clinical presentations require the interest and care of adult physicians and have a high risk of neurological complications. To improve the outcome of UCDs, a greater awareness by health professionals of the importance of hyperammonemia and UCDs, and ultimately avoidance of the still long delay to correctly diagnose the patients, is crucial.

Δ(1)-Pyrroline-5-carboxylate synthetase (P5CS) catalyzes the first two steps of ornithine/proline biosynthesis. P5CS deficiency has been reported in three families, with patients presenting with cutis/joint laxity, cataracts, and neurodevelopmental delay. Only one family exhibited metabolic changes consistent with P5CS deficiency (low proline/ornithine/citrulline/arginine; fasting hyperammonemia). Here we report a new P5CS-deficient patient presenting the complete clinical/metabolic phenotype and carrying p.G93R and p.T299I substitutions in the γ-glutamyl kinase (γGK) component of P5CS. The effects of these substitutions are (1) tested in mutagenesis/functional studies with E.coli γGK, (2) rationalized by structural modelling, and (3) reflected in decreased P5CS protein in patient fibroblasts (shown by immunofluorescence). Using optical/electron microscopy on skin biopsy, we show collagen/elastin fiber alterations that may contribute to connective tissue laxity and are compatible with our angio-MRI finding of kinky brain vessels in the patient. MR spectroscopy revealed decreased brain creatine, which normalized after sustained arginine supplementation, with improvement of neurodevelopmental and metabolic parameters, suggesting a pathogenic role of brain creatine decrease and the value of arginine therapy. Morphological and functional studies of fibroblast mitochondria show that P5CS deficiency is not associated with the mitochondrial alterations observed in Δ(1)-pyrroline-5-carboxylate reductase deficiency (another proline biosynthesis defect presenting cutis laxa and neurological alterations).

Background: The urea cycle defect argininosuccinate lyase (ASL) deficiency has a large spectrum of presentations from highly severe to asymptomatic. Enzyme activity assays in red blood cells or fibroblasts, although diagnostic of the deficiency, fail to discriminate between severe, mild or asymptomatic cases. Mutation/phenotype correlation studies are needed to characterize the effects of individual mutations on the activity of the enzyme., Methods: Bacterial in-vitro expression studies allowed the enzyme analysis of purified mutant ASL proteins p.I100T (c.299 T > C), p.V178M (c.532 G > A), p.E189G (c.566A > G), p.Q286R (c.857A > G), p.K315E (c.943A > G), p.R379C (c.1135 C > T) and p.R385C (c.1153 C > T) in comparison to the wildtype protein., Results: In the bacterial in-vitro expression system, ASL wild-type protein was successfully expressed. The known classical p.Q286R, the novel classical p.K315E and the known mutations p.I100T, p.E189G and p.R385C, which all have been linked to a mild phenotype, showed no significant residual activity. There was some enzyme activity detected with the p.V178M (5 % of wild-type) and p.R379C (10 % of wild-type) mutations in which K(m) values for argininosuccinic acid differed significantly from the wild-type ASL protein., Conclusion: The bacterially expressed enzymes proved that the mutations found in patients and studied here indeed are detrimental. However, as in the case of red cell ASL activity assays, some mutations found in genetically homozygous patients with mild presentations resulted in virtual loss of enzyme activity in the bacterial system, suggesting a more protective environment for the mutant enzyme in the liver than in the heterologous expression system and/or in the highly dilute assays utilized here.

Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the last step in proline synthesis. Deficiency of PYCR1, caused by a defect in PYCR1, was recently described in patients with cutis laxa, intrauterine growth retardation, developmental dysplasia of the hips and mental retardation. In this paper, we describe additional six patients (ages ranging from 4 months to 55 years) from four Iranian families with clinical manifestations of a wrinkly skin disorder. All patients have distinct facial features comprising triangular face, loss of adipose tissue and thin pointed nose. Additional features are short stature, wrinkling over dorsum of hand and feet, visible veins over the chest and hyperextensible joints. Three of the patients from a large consanguineous family do not have mental retardation, while the remaining three patients from three unrelated families have mental and developmental delay. Mutation analysis revealed the presence of disease-causing variants in PYCR1, including a novel deletion of the entire PYCR1 gene in one family, and in each of the other patients the homozygous missense mutations c.616G > A (p.Gly206Arg), c.89T > A (p.Ile30Lys) and c.572G > A (p.Gly191Glu) respectively, the latter two of which are novel. Light- and electron microscopy investigations of skin biopsies showed smaller and fragmented elastic fibres, abnormal morphology of the mitochondria and their cristae, and slightly abnormal collagen fibril diameters with irregular outline and variable size. In conclusion, this study adds information on the natural course of PYCR1 deficiency and sheds light on the pathophysiology of this disorder. However, the exact pathogenesis of this new disorder and the role of proline in the development of the clinical phenotype remain to be fully explained.

Blood sampling for newborn screening cannot be standardized as for example blood collection in adults after an overnight fast. Therefore the influence of postprandial changes and individual variation is valuable information for the assessment of sensitivity and specificity of newborn screening for certain disorders. We have analyzed 92 pairs of dried blood samples taken pre- and one hour postprandially, respectively. We have determined the mean increase in metabolite concentration and calculated its significance. Individual variation after an overnight fast in healthy adults (n = 3) was between 12 and 32% (SD). Postprandial increases of acylcarnitines were mostly not significant and not exceeding 10%. Postprandial increase of amino acids was highly significant for most proteinogenic amino acids, but not for all. With the collected data we were able to estimate that mainly decreased levels of methionine and, to a lesser extent, of free carnitine could be "masked" by postprandial increases of the respective metabolites, and could therefore lead to false negative results for the detection of disorders of cobalamin metabolism and carnitine transporter deficiency.

Glutamine synthetase (GS) is ubiquitously expressed in human tissues, being involved in ammonia detoxification and interorgan nitrogen flux. Inherited systemic deficiency of glutamine based on a defect of glutamine synthetase was recently described in two newborns with an early fatal course of disease. Glutamine was largely absent in their serum, urine and cerebrospinal fluid. Each of the patients had a homozygous mutation in the glutamine synthetase gene and enzymatic investigations confirmed that these mutations lead to a severely reduced glutamine synthetase activity. From the observation in the first patients with congenital glutamine synthetase deficiency, brain malformation can be expected as one of the leading signs. In addition, other organ systems are probably involved as observed in one of the index patients who suffered from severe enteropathy and necrolytic erythema of the skin. Deficiency of GS has to be added to the list of inherited metabolic disorders as a rare example of a defect in the biosynthesis of an amino acid.