Hypomagnesaemia with varying degrees of extrarenal symptoms as a consequence of heterozygous CNNM2 variants.

Variants in the CNNM2 gene are causative for hypomagnesaemia, seizures and intellectual disability, although the phenotypes can be variable. This study aims to understand the genotype-phenotype relationship in affected individuals with CNNM2 variants by phenotypic, functional and structural analysis of new as well as previously reported variants. This results in the identification of seven variants that significantly affect CNNM2-mediated Mg2+ transport. Pathogenicity of these variants is further supported by structural modelling, which predicts CNNM2 structure to be affected by all of them. Strikingly, seizures and intellectual disability are absent in 4 out of 7 cases, indicating these phenotypes are caused either by specific CNNM2 variant only or by additional risk factors. Moreover, in line with sporadic observations from previous reports, CNNM2 variants might be associated with disturbances in parathyroid hormone and Ca2+ homeostasis.

Description of clinical and genetic features of 122 patients included in the Spanish Pompe registry.

Pompe disease is a rare genetic disorder with an estimated prevalence of 1:60.000. The two main phenotypes are Infantile Onset Pompe Disease (IOPD) and Late Onset Pompe Disease (LOPD). There is no published data from Spain regarding the existing number of cases, regional distribution, clinical features or, access and response to the treatment. We created a registry to collect all these data from patients with Pompe in Spain. Here, we report the data of the 122 patients registered including nine IOPD and 113 LOPD patients. There was a high variability in how the diagnosis was obtained and how the follow-up was performed among different centres. Seven IOPD patients were still alive being all treated with enzymatic replacement therapy (ERT) at last visit. Ninety four of the 113 LOPD patients had muscle weakness of which 81 were receiving ERT. We observed a progressive decline in the results of muscle function tests during follow-up. Overall, the Spanish Pompe Registry is a valuable resource for understanding the demographics, patient's journey and clinical characteristics of patients in Spain. Our data supports the development of agreed guidelines to ensure that the care provided to the patients is standardized across the country.

Vitamin C and folate status in hereditary fructose intolerance.

BACKGROUND: Hereditary fructose intolerance (HFI) is a rare inborn error of fructose metabolism caused by the deficiency of aldolase B. Since treatment consists of a fructose-, sucrose- and sorbitol-restrictive diet for life, patients are at risk of presenting vitamin deficiencies. Although there is no published data on the status of these vitamins in HFI patients, supplementation with vitamin C and folic acid is common. Therefore, the aim of this study was to assess vitamin C and folate status and supplementation practices in a nationwide cohort of HFI patients. METHODS: Vitamin C and folic acid dietary intake, supplementation and circulating levels were assessed in 32 HFI patients and 32 age- and sex-matched healthy controls. RESULTS: Most of the HFI participants presented vitamin C (96.7%) and folate (90%) dietary intake below the recommended population reference intake. Up to 69% received vitamin C and 50% folic acid supplementation. Among HFI patients, 15.6% presented vitamin C and 3.1% folate deficiency. The amount of vitamin C supplementation and plasma levels correlated positively (R = 0.443; p = 0.011). Interestingly, a higher percentage of non-supplemented HFI patients were vitamin C deficient when compared to supplemented HFI patients (30% vs. 9.1%; p = 0.01) and to healthy controls (30% vs. 3.1%; p < 0.001). CONCLUSIONS: Our results provide evidence for the first time supporting vitamin C supplementation in HFI. There is great heterogeneity in vitamin supplementation practices and, despite follow-up at specialised centres, vitamin C deficiency is common. Further research is warranted to establish optimal doses of vitamin C and the need for folic acid supplementation in HFI.

Understanding gut-liver axis nitrogen metabolism in Fatty Liver Disease.

The homeostasis of the most important nitrogen-containing intermediates, ammonia and glutamine, is a tightly regulated process in which the gut-liver axis plays a central role. Several studies revealed that nitrogen metabolism is altered in Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD), a consensus-driven novel nomenclature for Non-Alcoholic Fatty Liver Disease (NAFLD), the most common chronic liver disease worldwide. Both increased ammonia production by gut microbiota and decreased ammonia hepatic removal due to impaired hepatic urea cycle activity or disrupted glutamine synthetase activity may contribute to hepatic ammonia accumulation underlying steatosis, which can eventually progress to hyperammonemia in more advanced stages of steatohepatitis and overt liver fibrosis. Furthermore, our group recently showed that augmented hepatic ammoniagenesis via increased glutaminase activity and overexpression of the high activity glutaminase 1 isoenzyme occurs in Fatty Liver Disease. Overall, the improved knowledge of disrupted nitrogen metabolism and metabolic miscommunication between the gut and the liver suggests that the reestablishment of altered gut-liver axis nitrogenous balance is an appealing and attractive therapeutic approach to tackle Fatty Liver Disease, a growing and unmet health problem.

Importance of Timely Treatment Initiation in Infantile-Onset Pompe Disease, a Single-Centre Experience.

Classic infantile Pompe disease (IPD) is a rare lysosomal storage disorder characterized by severe hypertrophic cardiomyopathy and profound muscle weakness. Without treatment, death occurs within the first 2 years of life. Although enzyme replacement therapy (ERT) with alglucosidase alfa has improved survival, treatment outcome is not good in many cases and is largely dependent on age at initiation. The objective of the study was (a) to analyse the different stages in the diagnosis and specific treatment initiation procedure in IPD patients, and (b) to compare clinical and biochemical outcomes depending on age at ERT initiation (<1 month of age vs. <3 months of age). Here, we show satisfactory clinical and biochemical outcomes in two IPD patients after early treatment initiation before 3 months of life with immunomodulatory therapy in the ERT-naïve setting, with a high ERT dose from the beginning. Despite the overall good evolution, the patient who initiated treatment <1 month of life presented even better outcomes than the patient who started treatment <3 months of life, with an earlier normalization of hypertrophic cardiomyopathy, along with CK normalization, highlighting the importance of early treatment initiation in this progressive disease before irreversible muscle damage has occurred.

Urine Phenylacetylglutamine Determination in Patients with Hyperphenylalaninemia.

Phenylketonuria (PKU), an autosomal-recessive inborn error of phenylalanine (Phe) metabolism is the most prevalent disorder of amino acid metabolism. Currently, clinical follow-up relies on frequent monitoring of Phe levels in blood. We hypothesize that the urine level of phenylacetylglutamine (PAG), a phenyl-group marker, could be used as a non-invasive biomarker. In this cross-sectional study, a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS) method was used for urinary PAG quantification in 35 participants with hyperphenylalaninemia (HPA) and 33 age- and sex-matched healthy controls. We have found that (a) PKU patients present higher urine PAG levels than healthy control subjects, and that (b) there is a significant correlation between urine PAG and circulating Phe levels in patients with HPA. In addition, we show a significant strong correlation between Phe levels from venous blood samples and from capillary finger-prick dried blood spot (DBS) samples collected at the same time in patients with HPA. Further research in order to assess the potential role of urine PAG as a non-invasive biomarker in PKU is warranted.

Transferrin Isoforms, Old but New Biomarkers in Hereditary Fructose Intolerance.

Hereditary Fructose Intolerance (HFI) is an autosomal recessive inborn error of metabolism characterised by the deficiency of the hepatic enzyme aldolase B. Its treatment consists in adopting a fructose-, sucrose-, and sorbitol (FSS)-restrictive diet for life. Untreated HFI patients present an abnormal transferrin (Tf) glycosylation pattern due to the inhibition of mannose-6-phosphate isomerase by fructose-1-phosphate. Hence, elevated serum carbohydrate-deficient Tf (CDT) may allow the prompt detection of HFI. The CDT values improve when an FSS-restrictive diet is followed; however, previous data on CDT and fructose intake correlation are inconsistent. Therefore, we examined the complete serum sialoTf profile and correlated it with FSS dietary intake and with hepatic parameters in a cohort of paediatric and adult fructosemic patients. To do so, the profiles of serum sialoTf from genetically diagnosed HFI patients on an FSS-restricted diet (n = 37) and their age-, sex- and body mass index-paired controls (n = 32) were analysed by capillary zone electrophoresis. We found that in HFI patients, asialoTf correlated with dietary intake of sucrose (R = 0.575, p < 0.001) and FSS (R = 0.475, p = 0.008), and that pentasialoTf+hexasialoTf negatively correlated with dietary intake of fructose (R = -0.386, p = 0.024) and FSS (R = -0.400, p = 0.019). In addition, the tetrasialoTf/disialoTf ratio truthfully differentiated treated HFI patients from healthy controls, with an area under the ROC curve (AUROC) of 0.97, 92% sensitivity, 94% specificity and 93% accuracy.

MMADHC premature termination codons in the pathogenesis of cobalamin D disorder: Potential of translational readthrough reconstitution.

Mutations in the MMADHC gene cause cobalamin D disorder (cblD), an autosomal recessive inborn disease with defects in intracellular cobalamin (cbl, vitamin B12) metabolism. CblD patients present methylmalonic aciduria (MMA), homocystinuria (HC), or combined MMA/HC, and usually suffer developmental delay and cognitive deficits. The most frequent MMADHC genetic alterations associated with disease generate MMADHC truncated proteins, in many cases due to mutations that create premature termination codons (PTC). In this study, we have performed a comprehensive and global characterization of MMADHC protein variants generated by all annotated MMADHC PTC mutations in cblD patients, and analyzed the potential of inducible translational PTC readthrough to reconstitute MMADHC biosynthesis. MMADHC protein truncation caused by disease-associated PTC differentially affected the alternative usage of translation initiation sites, protein abundance, and subcellular localization of MMADHC. Aminoglycoside compounds induced translational PTC readthrough of MMADHC truncated variants, allowing the biosynthesis of full-length MMADHC in a PTC-specific manner. Our results suggest that translational PTC readthrough-based interventions could complement current therapies for cblD patients carrying specific MMADHC PTC mutations.

Leigh syndrome associated with TRMU gene mutations.

tRNA 5-methylaminomethyl-2-thiouridylate methyltransferase (TRMU) deficiency causes an early onset potentially reversible acute liver failure, so far reported in less than 30 patients. We describe two new unrelated patients with an acute liver failure and a neuroimaging compatible with Leigh syndrome (LS) due to TRMU deficiency, a combination not previously reported. Our report enlarges the phenotypical spectrum of TRMU disease.

Brain Circuit Alterations and Cognitive Disability in Late-Onset Cobalamin D Disorder.

Neuroimaging studies describing brain circuits' alterations in cobalamin (vitamin B12)-deficient patients are limited and have not been carried out in patients with inborn errors of cobalamin metabolism. The objective of this study was to assess brain functionality and brain circuit alterations in a patient with an ultra-rare inborn error of cobalamin metabolism, methylmalonic aciduria, and homocystinuria due to cobalamin D disease, as compared with his twin sister as a healthy control (HC). We acquired magnetic resonance imaging (including structural, functional, and diffusion images) to calculate brain circuit abnormalities and combined these results with the scores after a comprehensive neuropsychological evaluation. As compared with HC, the patient had severe patterns of damage, such as a 254% increment of ventricular volume, pronounced subcortical and cortical atrophies (mainly at striatum, cingulate cortex, and precuneus), and connectivity alterations at fronto-striato-thalamic circuit, cerebellum, and corpus callosum. In agreement with brain circuit alterations, cognitive deficits existed in attention, executive function, inhibitory control, and mental flexibility. This is the first study that provides the clinical, genetic, neuroanatomical, neuropsychological, and psychosocial characterization of a patient with the cobalamin D disorder, showing functional alterations in central nervous system motor tracts, thalamus, cerebellum, and basal ganglia, that, as far as we know, have not been reported yet in vitamin B12-related disorders.

Non-alcoholic fatty liver in hereditary fructose intolerance.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is characterized by fat accumulation affecting >5% of the liver volume that is not explained by alcohol abuse. It is known that fructose gives rise to NAFLD and it has been recently described that the ingestion of fructose in low amounts in aldolase B deficient mice is associated with the development of fatty liver. Therefore, it is reasonable that patients with HFI (Hereditary Fructose Intolerance) present fatty liver at diagnosis, but its prevalence in patients treated and with adequate follow-up is not well documented in the literature. The aim of this study is to analyze the association between HFI and NAFLD in treated patients. METHODS: A cross-sectional observational study was conducted. The population comprised 16 genetically diagnosed HFI patients aged from 3 years to 48 and in dietary treatment of fructose, sorbitol and sacarose exclusion at least for two years. Blood samples were obtained for analytical studies and anthropometric measurements of each patient were performed. RESULTS: Patients presented a Body Mass Index (BMI) of 17.9 ± 2.9 kg/m2. The HOMA index and Quick index were in normal range for our population. The S-adenosyl-methionine (SAM)/S-adenosyl-l-homocysteine (SAH) ratio was increased in the patients in whom this analysis was performed. By imaging techniques it was observed that 9 of the 16 patients presented fatty liver (7 by hepatic MRI). Of these 9 patients, only 3 presented hepatomegaly. 7 of 9 patients affected by the c.448G > C mutation had fatty infiltration, of which three of them presented in addition hepatomegaly. CONCLUSIONS: There is a high prevalence of fatty liver in HFI patients and it is not related to obesity and insulin resistance. The diagnosis of fatty liver in HFI patients and, above all, the identification of new therapeutic approaches, can positively impact the quality of life of these patients.

Quantification of urinary derivatives of Phenylbutyric and Benzoic acids by LC-MS/MS as treatment compliance biomarkers in Urea Cycle disorders.

PURPOSE: Salts of phenylacetic acid (PAA) and phenylbutyric acid (PBA) have been used for nitrogen elimination as a treatment for hyperammonaemia caused by urea cycle disorders (UCD). A new analytical method for PBA measurement in urine which helps to evaluate the drug adherence has been implemented. METHODS: Urine specimens from UCD patients receiving PBA were analysed by tandem mass spectrometry to measure urine phenylacetylglutamine (PAGln). Some clinical and biochemical data for each patient were collected. RESULTS: Our study included 87 samples from 40 UCD patients. The PAGln levels did not correlate with height, weight or age. However, the PAGln values showed correlation with PBA dose (r = 0.383, P = 0.015). Plasma glutamine and ammonia levels presented a positive correlation (r = 0.537, P < 0.001). The stability for PAGln in urine was determined at different storage temperatures. CONCLUSIONS: We have developed a simple method for the determination of PAGln in urine, which acts as useful biomarker of effective drug delivery. PAGln in urine is stable at room temperature at least for 15 days, and for several months when frozen at -20 °C. This procedure is useful for the optimization and monitorization of the drug dose allowing the use of spot urine samples.

International best practice for the evaluation of responsiveness to sapropterin dihydrochloride in patients with phenylketonuria.

Phenylketonuria (PKU) is an inherited metabolic disease caused by phenylalanine hydroxylase (PAH) deficiency. As the resulting high blood phenylalanine (Phe) concentration can have detrimental effects on brain development and function, international guidelines recommend lifelong control of blood Phe concentration with dietary and/or medical therapy. Sapropterin dihydrochloride is a synthetic preparation of tetrahydrobiopterin (6R-BH4), the naturally occurring cofactor of PAH. It acts as a pharmacological chaperone, reducing blood Phe concentration and increasing dietary Phe tolerance in BH4-responsive patients with PAH deficiency. Protocols to establish responsiveness to sapropterin dihydrochloride vary widely. Two meetings were held with an international panel of clinical experts in PKU management to develop recommendations for sapropterin dihydrochloride response testing. At the first meeting, regional differences and similarities in testing practices were discussed based on guidelines, a literature review, outcomes of a global physician survey, and case reports. Statements developed based on the discussions were sent to all participants for consensus (>70% of participants) evaluation using a 7-level rating system, and further discussed during the second meeting. The experts recommend sapropterin dihydrochloride response testing in patients with untreated blood Phe concentrations of 360-2000 µmol/L, except in those with two null mutations. For neonates, a 24-h sapropterin dihydrochloride loading test is recommended; responsiveness is defined as a decrease in blood Phe ≥30%. For older infants, children, adolescents, and adults, a test duration of ≥48 h or a 4-week trial is recommended. The main endpoint for a 48-h to 7-day trial is a decrease in blood Phe, while improved Phe tolerance is the endpoint to be assessed during a longer trial. Longer trials may not be feasible in some locations due to lack of reimbursement for hospitalization, while a 4-week trial may not be possible due to limited access to sapropterin dihydrochloride or public health regulation. A 48-h response test should be considered in pregnant patients who cannot achieve blood Phe ≤360 µmol/L with a Phe-restricted diet. Durability of response and clinical benefits of sapropterin dihydrochloride should be assessed over the long term. Harmonization of protocols is expected to improve identification of responders and comparability of test results worldwide.

Asymmetric dimethylarginine as a potential biomarker for management and follow-up of phenylketonuria.

Phenylketonuria's (PKU) treatment based on low-protein diet may affect other metabolic pathways, such as that of asymmetric dimethylarginine (ADMA). The aim of this study was to evaluate the reliability of ADMA as a biomarker of adequate metabolic control and possible nutritional risk in a long-term PKU patient population. One hundred and six dietary-treated PKU patients from four hospitals in Spain were enrolled in this cross-sectional study. Their lipid profile, total homocysteine, ADMA, and symmetric dimethylarginine (SDMA) concentrations were analyzed and compared with a control group. Sensitivity, specificity, and likelihood ratios of the proposed biomarker were calculated. PKU patients had statistically significant lower plasmatic ADMA, SDMA, and arginine concentrations as compared with the control group (p < 0.001). Significant correlations were found between ADMA, phenylalanine, and total homocysteine levels. The ADMA/creatinine ratio correlated with phenylalanine levels as metabolic control and nutritional risk in PKU patients. Its reliability as a management biomarker was studied with positive results. The ADMA/creatinine ratio might serve as an independent biomarker in the management of PKU patients, different from blood phenylalanine levels. It could be of particular usefulness to detect those who are following an unbalanced diet that could have long-term negative effects.Conclusion: In this study, we have evaluated the reliability of ADMA as a potential biomarker of adequate metabolic control and possible nutritional risk in a long-term PKU patient population. What is Known: • Although PKU individuals have lower values of ADMA even with blood Phe levels in the recommended range, little attention is payed to other metabolic pathways. What is New: • ADMA could be used as new biomarker for PKU management and follow-up of the diet, after evaluating their reliability in a long-term PKU patient population.

Aortic Customized Stent-Graft Treatment of Postnephrectomy Renal Pedicle Arteriovenous Fistula.

A postnephrectomy renal acquired arteriovenous fistula (AVF) is a rare clinical entity that may cause high-output heart failure. Most of the cases are identified time along after surgery. We present a case of a postnephrectomy renal AVF treated with aortic customized stent-graft.

A morphological method for ammonia detection in liver.

Hyperammonemia is a metabolic condition characterized by elevated levels of ammonia and a common event in acute liver injury/failure and chronic liver disease. Even though hepatic ammonia levels are potential predictive factors of patient outcome, easy and inexpensive methods aiming at the detection of liver ammonia accumulation in the clinical setting remain unavailable. Thus, herein we have developed a morphological method, based on the utilization of Nessler´s reagent, to accurately and precisely detect the accumulation of ammonia in biological tissue. We have validated our method against a commercially available kit in mouse tissue samples and, by using this modified method, we have confirmed the hepatic accumulation of ammonia in clinical and animal models of acute and chronic advanced liver injury as well as in the progression of fatty liver disease. Overall, we propose a morphological method for ammonia detection in liver that correlates well with the degree of liver disease severity and therefore can be potentially used to predict patient outcome.

An update on the use of benzoate, phenylacetate and phenylbutyrate ammonia scavengers for interrogating and modifying liver nitrogen metabolism and its implications in urea cycle disorders and liver disease.

INTRODUCTION: Ammonia-scavenging drugs, benzoate and phenylacetate (PA)/phenylbutyrate (PB), modulate hepatic nitrogen metabolism mainly by providing alternative pathways for nitrogen disposal. Areas covered: We review the major findings and potential novel applications of ammonia-scavenging drugs, focusing on urea cycle disorders and liver disease. Expert opinion: For over 40 years, ammonia-scavenging drugs have been used in the treatment of urea cycle disorders. Recently, the use of these compounds has been advocated in acute liver failure and cirrhosis for reducing hyperammonemic-induced hepatic encephalopathy. The efficacy and mechanisms underlying the antitumor effects of these ammonia-scavenging drugs in liver cancer are more controversial and are discussed in the review. Overall, as ammonia-scavenging drugs are usually safe and well tolerated among cancer patients, further studies should be instigated to explore the role of these drugs in liver cancer. Considering the relevance of glutamine metabolism to the progression and resolution of liver disease, we propose that ammonia-scavenging drugs might also be used to non-invasively probe liver glutamine metabolism in vivo. Finally, novel derivatives of classical ammonia-scavenging drugs with fewer and less severe adverse effects are currently being developed and used in clinical trials for the treatment of acute liver failure and cirrhosis.

Cholestane-3ß,5α,6ß-triol: high levels in Niemann-Pick type C, cerebrotendinous xanthomatosis, and lysosomal acid lipase deficiency.

Niemann-Pick type C (NPC) is a progressive neurodegenerative disease characterized by lysosomal/endosomal accumulation of unesterified cholesterol and glycolipids. Recent studies have shown that plasma cholestane-3ß,5α,6ß-triol (CT) and 7-ketocholesterol (7-KC) could be potential biomarkers for the diagnosis of NPC patients. We aimed to know the sensitivity and specificity of these biomarkers for the diagnosis of NPC compared with other diseases that can potentially lead to oxysterol alterations. We studied 107 controls and 122 patients including 16 with NPC, 3 with lysosomal acid lipase (LAL) deficiency, 8 with other lysosomal diseases, 5 with galactosemia, 11 with cerebrotendinous xanthomatosis (CTX), 3 with Smith-Lemli-Opitz, 14 with peroxisomal biogenesis disorders, 19 with unspecific hepatic diseases, 13 with familial hypercholesterolemia, and 30 with neurological involvement and no evidence of an inherited metabolic disease. CT and 7-KC were analyzed by HPLC-ESI-MS/MS as mono-dimethylglycine derivatives. Levels of 7-KC were high in most of the studied diseases, whereas those of CT were only high in NPC, LAL, and CTX patients. Consequently, although CT is a sensitive biomarker of NPC disease, including those cases with doubtful filipin staining, it is not specific. 7-KC is a very unspecific biomarker.