Dietary sulfur amino acid restriction in humans with overweight and obesity: Evidence of an altered plasma and urine sulfurome, and a novel metabolic signature that correlates with loss of fat mass and adipose tissue gene expression.

BACKGROUND: In animals, dietary sulfur amino acid restriction (SAAR) improves metabolic health, possibly mediated by altering sulfur amino acid metabolism and enhanced anti-obesogenic processes in adipose tissue. AIM: To assess the effects of SAAR over time on the plasma and urine SAA-related metabolites (sulfurome) in humans with overweight and obesity, and explore whether such changes were associated with body weight, body fat and adipose tissue gene expression. METHODS: Fifty-nine subjects were randomly allocated to SAAR (∼2 g SAA, n = 31) or a control diet (∼5.6 g SAA, n = 28) consisting of plant-based whole-foods and supplemented with capsules to titrate contents of SAA. Sulfurome metabolites in plasma and urine at baseline, 4 and 8 weeks were measured using HPLC and LC-MS/MS. mRNA-sequencing of subcutaneous white adipose tissue (scWAT) was performed to assess changes in gene expression. Data were analyzed with mixed model regression. Principal component analyses (PCA) were performed on the sulfurome data to identify potential signatures characterizing the response to SAAR. RESULTS: SAAR led to marked decrease of the main urinary excretion product sulfate (p < 0.001) and plasma and/or 24-h urine concentrations of cystathionine, sulfite, thiosulfate, H2S, hypotaurine and taurine. PCA revealed a distinct metabolic signature related to decreased transsulfuration and H2S catabolism that predicted greater weight loss and android fat mass loss in SAAR vs. controls (all pinteraction < 0.05). This signature correlated positively with scWAT expression of genes in the tricarboxylic acid cycle, electron transport and ß-oxidation (FDR = 0.02). CONCLUSION: SAAR leads to distinct alterations of the plasma and urine sulfurome in humans, and predicted increased loss of weight and android fat mass, and adipose tissue lipolytic gene expression in scWAT. Our data suggest that SAA are linked to obesogenic processes and that SAAR may be useful for obesity and related disorders. TRIAL IDENTIFIER: https://clinicaltrials.gov/study/NCT04701346.

Dietary sulfur amino acid restriction in humans with overweight and obesity: a translational randomized controlled trial.

BACKGROUND: Dietary sulfur amino acid restriction (SAAR) improves metabolic health in animals. In this study, we investigated the effect of dietary SAAR on body weight, body composition, resting metabolic rate, gene expression profiles in white adipose tissue (WAT), and an extensive blood biomarker profile in humans with overweight or obesity. METHODS: N = 59 participants with overweight or obesity (73% women) were randomized stratified by sex to an 8-week plant-based dietary intervention low (~ 2 g/day, SAAR) or high (~ 5.6 g/day, control group) in sulfur amino acids. The diets were provided in full to the participants, and both investigators and participants were blinded to the intervention. Outcome analyses were performed using linear mixed model regression adjusted for baseline values of the outcome and sex. RESULTS: SAAR led to a ~ 20% greater weight loss compared to controls (ß 95% CI - 1.14 (- 2.04, - 0.25) kg, p = 0.013). Despite greater weight loss, resting metabolic rate remained similar between groups. Furthermore, SAAR decreased serum leptin, and increased ketone bodies compared to controls. In WAT, 20 genes were upregulated whereas 24 genes were downregulated (FDR < 5%) in the SAAR group compared to controls. Generally applicable gene set enrichment analyses revealed that processes associated with ribosomes were upregulated, whereas processes related to structural components were downregulated. CONCLUSION: Our study shows that SAAR leads to greater weight loss, decreased leptin and increased ketone bodies compared to controls. Further research on SAAR is needed to investigate the therapeutic potential for metabolic conditions in humans. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT04701346, registered Jan 8th 2021, https://www. CLINICALTRIALS: gov/study/NCT04701346.

The Associations of Habitual Intake of Sulfur Amino Acids, Proteins and Diet Quality with Plasma Sulfur Amino Acid Concentrations: The Maastricht Study.

BACKGROUND: Plasma sulfur amino acids (SAAs), i.e., methionine, total cysteine (tCys), total homocysteine (tHcy), cystathionine, total glutathione (tGSH), and taurine, are potential risk factors for obesity and cardiometabolic disorders. However, except for plasma tHcy, little is known about how dietary intake modifies plasma SAA concentrations. OBJECTIVE: To investigate whether the intake of SAAs and proteins or diet quality is associated with plasma SAAs. METHODS: Data from a cross-sectional subset of The Maastricht Study (n = 1145, 50.5% men, 61 interquartile range: [55, 66] y, 22.5% with prediabetes and 34.3% with type 2 diabetes) were investigated. Dietary intake was assessed using a validated food frequency questionnaire. The intake of SAAs (total, methionine, and cysteine) and proteins (total, animal, and plant) was estimated from the Dutch and Danish food composition tables. Diet quality was assessed using the Dutch Healthy Diet Index, the Mediterranean Diet Score, and the Dietary Approaches to Stop Hypertension score. Fasting plasma SAAs were measured by liquid chromatography (LC) tandem mass spectrometry (MS) (LC/MS-MS). Associations were investigated with multiple linear regressions with tertiles of dietary intake measures (main exposures) and z-standardized plasma SAAs (outcomes). RESULTS: Intake of total SAAs and total proteins was positively associated with plasma tCys and cystathionine. Associations were stronger in women and in those with normal body weight. Higher intake of cysteine and plant proteins was associated with lower plasma tHcy and higher cystathionine. Higher methionine intake was associated with lower plasma tGSH, whereas cysteine intake was positively associated with tGSH. Higher intake of methionine and animal proteins was associated with higher plasma taurine. Better diet quality was consistently related to lower plasma tHcy concentrations, but it was not associated with the other SAAs. CONCLUSION: Targeted dietary modifications might be effective in modifying plasma concentrations of tCys, tHcy, and cystathionine, which have been associated with obesity and cardiometabolic disorders.

Newborn Screening in a Pandemic-Lessons Learned.

The COVID-19 pandemic affected many essential aspects of public health, including newborn screening programs (NBS). Centers reported missing cases of inherited metabolic disease as a consequence of decreased diagnostic process quality during the pandemic. A number of problems emerged at the start of the pandemic, but from the beginning, solutions began to be proposed and implemented. Contingency plans were arranged, and these are reviewed and described in this article. Staff shortage emerged as an important issue, and as a result, new work schedules had to be implemented. The importance of personal protective equipment and social distancing also helped avoid disruption. Staff became stressed, and this needed to be addressed. The timeframe for collecting bloodspot samples was adapted in some cases, requiring reference ranges to be modified. A shortage of essential supplies and protective equipment was evident, and laboratories described sharing resources in some situations. The courier system had to be adapted to make timely and safe transport possible. Telemedicine became an essential tool to enable communication with patients, parents, and medical staff. Despite these difficulties, with adaptations and modifications, some centers evaluated candidate conditions, continued developments, or began new NBS. The pandemic can be regarded as a stress test of the NBS under real-world conditions, highlighting critical aspects of this multidisciplinary system and the need for establishing local, national, and global strategies to improve its robustness and reliability in times of shortage and overloaded national healthcare systems.

Recent therapeutic approaches to cystathionine beta-synthase-deficient homocystinuria.

Cystathionine beta-synthase (CBS)-deficient homocystinuria (HCU) is the most common inborn error of sulfur amino acid metabolism. The pyridoxine non-responsive form of the disease manifests itself by massively increasing plasma and tissue concentrations of homocysteine, a toxic intermediate of methionine metabolism that is thought to be the major cause of clinical complications including skeletal deformities, connective tissue defects, thromboembolism and cognitive impairment. The current standard of care involves significant dietary interventions that, despite being effective, often adversely affect quality of life of HCU patients, leading to poor adherence to therapy and inadequate biochemical control with clinical complications. In recent years, the unmet need for better therapeutic options has resulted in development of novel enzyme and gene therapies and exploration of pharmacological approaches to rescue CBS folding defects caused by missense pathogenic mutations. Here, we review scientific evidence and current state of affairs in development of recent approaches to treat HCU.

Elevated homocysteine levels: What inborn errors of metabolism might we be missing?

Elevated total plasma homocysteine (hyperhomocysteinemia) is a marker of cardiovascular, thrombotic, and neuropsychological disease. It has multiple causes, including the common nutritional vitamin B12 or folate deficiency. However, some rare but treatable, inborn errors of metabolism (IEM) characterized by hyperhomocysteinemia can be missed due to variable presentations and the lack of awareness. The aim of this study is to identify undiagnosed IEM in adults with significantly elevated homocysteine using key existing clinical data points, then IEM specific treatment can be offered to improve outcome. We conducted a retrospective study with data mining and chart review of patients with plasma total homocysteine >30 µmol/L over a two-year period. We offer biochemical and genetic testing to patients with significant hyperhomocysteinemia without a clear explanation to diagnose IEM. We identified 22 subjects with significant hyperhomocysteinemia but no clear explanation. Subsequently, we offered genetic testing to seven patients and diagnosed one patient with classic homocystinuria due to cystathionine beta-synthase deficiency. With treatment, she lowered her plasma homocysteine and improved her health. This study stresses the importance of a thorough investigation of hyperhomocysteinemia in adults to identify rare but treatable IEM. We propose a metabolic evaluation algorithm for elevated homocysteine levels.

Cysteine restriction-specific effects of sulfur amino acid restriction on lipid metabolism.

Decreasing the dietary intake of methionine exerts robust anti-adiposity effects in rodents but modest effects in humans. Since cysteine can be synthesized from methionine, animal diets are formulated by decreasing methionine and eliminating cysteine. Such diets exert both methionine restriction (MR) and cysteine restriction (CR), that is, sulfur amino acid restriction (SAAR). Contrarily, SAAR diets formulated for human consumption included cysteine, and thus might have exerted only MR. Epidemiological studies positively correlate body adiposity with plasma cysteine but not methionine, suggesting that CR, but not MR, is responsible for the anti-adiposity effects of SAAR. Whether this is true, and, if so, the underlying mechanisms are unknown. Using methionine- and cysteine-titrated diets, we demonstrate that the anti-adiposity effects of SAAR are due to CR. Data indicate that CR increases serinogenesis (serine biosynthesis from non-glucose substrates) by diverting substrates from glyceroneogenesis, which is essential for fatty acid reesterification and triglyceride synthesis. Molecular data suggest that CR depletes hepatic glutathione and induces Nrf2 and its downstream targets Phgdh (the serine biosynthetic enzyme) and Pepck-M. In mice, the magnitude of SAAR-induced changes in molecular markers depended on dietary fat concentration (60% fat >10% fat), sex (males > females), and age-at-onset (young > adult). Our findings are translationally relevant as we found negative and positive correlations of plasma serine and cysteine, respectively, with triglycerides and metabolic syndrome criteria in a cross-sectional epidemiological study. Controlled feeding of low-SAA, high-polyunsaturated fatty acid diets increased plasma serine in humans. Serinogenesis might be a target for treating hypertriglyceridemia.

Human ultrarare genetic disorders of sulfur metabolism demonstrate redundancies in H2S homeostasis.

Regulation of H2S homeostasis in humans is poorly understood. Therefore, we assessed the importance of individual enzymes in synthesis and catabolism of H2S by studying patients with respective genetic defects. We analyzed sulfur compounds (including bioavailable sulfide) in 37 untreated or insufficiently treated patients with seven ultrarare enzyme deficiencies and compared them to 63 controls. Surprisingly, we observed that patients with severe deficiency in cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE) - the enzymes primarily responsible for H2S synthesis - exhibited increased and normal levels of bioavailable sulfide, respectively. However, an approximately 21-fold increase of urinary homolanthionine in CBS deficiency strongly suggests that lacking CBS activity is compensated for by an increase in CSE-dependent H2S synthesis from accumulating homocysteine, which suggests a control of H2S homeostasis in vivo. In deficiency of sulfide:quinone oxidoreductase - the first enzyme in mitochondrial H2S oxidation - we found normal H2S concentrations in a symptomatic patient and his asymptomatic sibling, and elevated levels in an asymptomatic sibling, challenging the requirement for this enzyme in catabolizing H2S under physiological conditions. Patients with ethylmalonic encephalopathy and sulfite oxidase/molybdenum cofactor deficiencies exhibited massive accumulation of thiosulfate and sulfite with formation of large amounts of S-sulfocysteine and S-sulfohomocysteine, increased renal losses of sulfur compounds and concomitant strong reduction in plasma total cysteine. Our results demonstrate the value of a comprehensive assessment of sulfur compounds in severe disorders of homocysteine/cysteine metabolism and provide evidence for redundancy and compensatory mechanisms in the maintenance of H2S homeostasis.

Towards Achieving Equity and Innovation in Newborn Screening across Europe.

Although individual rare disorders are uncommon, it is estimated that, together, 6000+ known rare diseases affect more than 30 million people in Europe, and present a substantial public health burden. Together with the psychosocial burden on affected families, rare disorders frequently, if untreated, result in a low quality of life, disability and even premature death. Newborn screening (NBS) has the potential to detect a number of rare conditions in asymptomatic children, providing the possibility of early treatment and a significantly improved long-term outcome. Despite these clear benefits, the availability and conduct of NBS programmes varies considerably across Europe and, with the increasing potential of genomic testing, it is likely that these differences may become even more pronounced. To help improve the equity of provision of NBS and ensure that all children can be offered high-quality screening regardless of race, nationality and socio-economic status, a technical meeting, endorsed by the Slovenian Presidency of the Council of the European Union, was held in October 2021. In this article, we present experiences from individual EU countries, stakeholder initiatives and the meeting's final conclusions, which can help countries attempting to establish new NBS programmes or expand existing provision.

Influence of early identification and therapy on long-term outcomes in early-onset MTHFR deficiency.

MTHFR deficiency is a severe inborn error of metabolism leading to impairment of the remethylation of homocysteine to methionine. Neonatal and early-onset patients mostly exhibit a life-threatening acute neurologic deterioration. Furthermore, data on early-onset patients' long-term outcomes are scarce. The aims of this study were (1) to study and describe the clinical and laboratory parameters of early-onset MTHFR-deficient patients (i.e., ≤3 months of age) and (2) to identify predictive factors for severe neurodevelopmental outcomes in a cohort with early and late onset MTHFR-deficient patients. To this end, we conducted a retrospective, multicentric, international cohort study on 72 patients with MTHFR deficiency from 32 international metabolic centres. Characteristics of the 32 patients with early-onset MTHFR deficiency were described at time of diagnosis and at the last follow-up visit. Logistic regression analysis was used to identify predictive factors of severe neurodevelopmental outcome in a broader set of patients with early and non-early-onset MTHFR deficiency. The majority of early-onset MTHFR-deficient patients (n = 32) exhibited neurologic symptoms (76%) and feeding difficulties (70%) at time of diagnosis. At the last follow-up visit (median follow-up time of 8.1 years), 76% of treated early-onset patients (n = 29) exhibited a severe neurodevelopmental outcome. Among the whole study population of 64 patients, pre-symptomatic diagnosis was independently associated with a significantly better neurodevelopmental outcome (adjusted OR 0.004, [0.002-0.232]; p = 0.003). This study provides evidence for benefits of pre-symptomatic diagnosis and appropriate therapeutic management, highlighting the need for systematic newborn screening for MTHFR deficiency and pre-symptomatic treatment that may improve outcome.

Postauthorization safety study of betaine anhydrous.

Patient registries for rare diseases enable systematic data collection and can also be used to facilitate postauthorization safety studies (PASS) for orphan drugs. This study evaluates the PASS for betaine anhydrous (Cystadane), conducted as public private partnership (PPP) between the European network and registry for homocystinurias and methylation defects and the marketing authorization holder (MAH). Data were prospectively collected, 2013-2016, in a noninterventional, international, multicenter, registry study. Putative adverse and severe adverse events were reported to the MAH's pharmacovigilance. In total, 130 individuals with vitamin B6 nonresponsive (N = 54) and partially responsive (N = 7) cystathionine beta-synthase (CBS) deficiency, as well as 5,10-methylenetetrahydrofolate reductase (MTHFR; N = 21) deficiency and cobalamin C (N = 48) disease were included. Median (range) duration of treatment with betaine anhydrous was 6.8 (0-9.8) years. The prescribed betaine dose exceeded the recommended maximum (6 g/day) in 49% of individuals older than 10 years because of continued dose adaptation to weight; however, with disease-specific differences (minimum: 31% in B6 nonresponsive CBS deficiency, maximum: 67% in MTHFR deficiency). Despite dose escalation no new or potential risk was identified. Combined disease-specific treatment decreased mean ± SD total plasma homocysteine concentrations from 203 ± 116 to 81 ± 51 µmol/L (p < 0.0001), except in MTHFR deficiency. Recommendations for betaine anhydrous dosage were revised for individuals ≥ 10 years. PPPs between MAH and international scientific consortia can be considered a reliable model for implementing a PASS, reutilizing well-established structures and avoiding data duplication and fragmentation.

Knock-Out of Retrovirus Receptor Gene Tva in the Chicken Confers Resistance to Avian Leukosis Virus Subgroups A and K and Affects Cobalamin (Vitamin B12)-Dependent Level of Methylmalonic Acid.

The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva -/- chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/- siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken.

Impact of Newborn Screening and Early Dietary Management on Clinical Outcome of Patients with Long Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency and Medium Chain Acyl-CoA Dehydrogenase Deficiency-A Retrospective Nationwide Study.

Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD/MTPD) and medium chain acyl-CoA dehydrogenase deficiency (MCADD) were included in the expanded neonatal screening program (ENBS) in Czechia in 2009, allowing for the presymptomatic diagnosis and nutritional management of these patients. The aim of our study was to assess the nationwide impact of ENBS on clinical outcome. This retrospective study analysed acute events and chronic complications and their severity in pre-ENBS and post-ENBS cohorts. In total, 28 children (12 before, 16 after ENBS) were diagnosed with LCHADD/MTPD (incidence 0.8/100,000 before and 1.2/100,000 after ENBS). In the subgroup detected by ENBS, a significantly longer interval from birth to first acute encephalopathy was observed. In addition, improvement in neuropathy and cardiomyopathy (although statistically non-significant) was demonstrated in the post-ENBS subgroup. In the MCADD cohort, we included 69 patients (15 before, 54 after ENBS). The estimated incidence rose from 0.7/100,000 before to 4.3/100,000 after ENBS. We confirmed a significant decrease in the number of episodes of acute encephalopathy and lower proportion of intellectual disability after ENBS (p < 0.0001). The genotype-phenotype correlations suggest a new association between homozygosity for the c.1528C > G variant and more severe heart involvement in LCHADD patients.

Shifting landscapes of human MTHFR missense-variant effects.

Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.

Absence of MMACHC in peripheral retinal cells does not lead to an ocular phenotype in mice.

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B12 metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.

Sulfur amino acid restriction, energy metabolism and obesity: a study protocol of an 8-week randomized controlled dietary intervention with whole foods and amino acid supplements.

BACKGROUND: Dietary sulfur amino acid (SAA) restriction is an established animal model for increasing lifespan and improving metabolic health. Data from human studies are limited. In the study outlined in this protocol, we will evaluate if dietary SAA restriction can reduce body weight and improve resting energy expenditure (REE) and parameters related to metabolic health. METHOD/DESIGN: Men and women (calculated sample size = 60), aged 18-45 years, with body mass index of 27-35 kg/m2 will be included in a double-blind 8-week dietary intervention study. The participants will be randomized in a 1:1 manner to a diet with either low or high SAA. Both groups will receive an equal base diet consisting of low-SAA plant-based whole foods and an amino acid supplement free of SAA. Contrasting SAA contents will be achieved using capsules with or without methionine and cysteine (SAAhigh, total diet SAA ~ 50-60 mg/kg body weight/day; SAAlow, total diet SAA ~ 15-25 mg/kg body weight/day). The primary outcome is body weight change. Data and material collection will also include body composition (dual X-ray absorptiometry), resting energy expenditure (whole-room indirect calorimetry) and samples of blood, urine, feces and adipose tissue at baseline, at 4 weeks and at study completion. Measures will be taken to promote and monitor diet adherence. Data will be analyzed using linear mixed model regression to account for the repeated measures design and within-subject correlation. DISCUSSION: The strength of this study is the randomized double-blind design. A limitation is the restrictive nature of the diet which may lead to poor compliance. If this study reveals a beneficial effect of the SAAlow diet on body composition and metabolic health, it opens up for new strategies for prevention and treatment of overweight, obesity and its associated disorders. Trial registration ClinicalTrials.gov: NCT04701346, Registration date: January 8th, 2021.

Neonatal Screening in Europe Revisited: An ISNS Perspective on the Current State and Developments Since 2010.

Neonatal screening (NBS) was initiated in Europe during the 1960s with the screening for phenylketonuria. The panel of screened disorders ("conditions") then gradually expanded, with a boost in the late 1990s with the introduction of tandem mass spectrometry (MS/MS), making it possible to screen for 40-50 conditions using a single blood spot. The most recent additions to screening programmes (screening for cystic fibrosis, severe combined immunodeficiency and spinal muscular atrophy) were assisted by or realised through the introduction of molecular technologies. For this survey, we collected data from 51 European countries. We report the developments between 2010 and 2020 and highlight the achievements reached with the progress made in this period. We also identify areas where further progress can be made, mainly by exchanging knowledge and learning from experiences in neighbouring countries. Between 2010 and 2020, most NBS programmes in geographical Europe matured considerably, both in terms of methodology (modernised) and with regard to the panel of conditions screened (expanded). These developments indicate that more collaboration in Europe through European organisations is gaining momentum. We can only accomplish the timely detection of newborn infants potentially suffering from one of the many rare diseases and take appropriate action by working together.

Informing Parents about Newborn Screening: A European Comparison Study.

Knowledge about newborn screening (NBS) is an important factor for parents to make an informed decision about participation. In Europe, countries inform parents differently about their NBS program, potentially including different knowledge aspects in their information. The aim of this study was to assess twenty-six European parental information products and to analyze their knowledge aspects through a content analysis. The analyzed aspects were compared to a list of eight knowledge aspects from scientific literature. The list includes aspects important for parents' decision-making, such as the purpose of screening. The study showed that most of the eight knowledge aspects are included in NBS information products of the majority of countries. However, there were differences between countries, for example in the amount of detail and phrasing of the information. Additional relevant knowledge aspects have also been identified and are recommended to optimize information products, such as the handling of residual bloodspot samples. This study only assessed knowledge aspects in information products meant for printing, but many countries also use other communication methods, and the impact on knowledge of the delivery of the information needs further study. Preferences of parents on alternative communication methods need to be considered and evaluated on their effectiveness.

U-IMD: the first Unified European registry for inherited metabolic diseases.

BACKGROUND: Following the broad application of new analytical methods, more and more pathophysiological processes in previously unknown diseases have been elucidated. The spectrum of clinical presentation of rare inherited metabolic diseases (IMDs) is broad and ranges from single organ involvement to multisystemic diseases. With the aim of overcoming the limited knowledge about the natural course, current diagnostic and therapeutic approaches, the project has established the first unified patient registry for IMDs that fully meets the requirements of the European Infrastructure for Rare Diseases (ERDRI). RESULTS: In collaboration with the European Reference Network for Rare Hereditary Metabolic Disorders (MetabERN), the Unified European registry for Inherited Metabolic Diseases (U-IMD) was established to collect patient data as an observational, non-interventional natural history study. Following the recommendations of the ERDRI the U-IMD registry uses common data elements to define the IMDs, report the clinical phenotype, describe the biochemical markers and to capture the drug treatment. Until today, more than 1100 IMD patients have been registered. CONCLUSION: The U-IMD registry is the first observational, non-interventional patient registry that encompasses all known IMDs. Full semantic interoperability for other registries has been achieved, as demonstrated by the use of a minimum common core data set for equivalent description of metabolic patients in U-IMD and in the patient registry of the European Rare Kidney Disease Reference Network (ERKNet). In conclusion, the U-IMD registry will contribute to a better understanding of the long-term course of IMDs and improved patients care by understanding the natural disease course and by enabling an optimization of diagnostic and therapeutic strategies.

Clinical and molecular characterization of adult patients with late-onset MTHFR deficiency.

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency usually presents as a severe neonatal disease. This study aimed to characterize natural history, biological and molecular data, and response to treatment of patients with late-onset MTHFR deficiency. The patients were identified through the European Network and Registry for Homocystinuria and Methylation Defects and the Adult group of the French Society for Inherited Metabolic Diseases; data were retrospectively colleted. To identify juvenile to adult-onset forms of the disease, we included patients with a diagnosis established after the age of 10 years. We included 14 patients (median age at diagnosis: 32 years; range: 11-54). At onset (median age: 20 years; range 9-38), they presented with walking difficulties (n = 8), cognitive decline (n = 3) and/or seizures (n = 3), sometimes associated with mild mental retardation (n = 6). During the disease course, symptoms were almost exclusively neurological with cognitive dysfunction (93%), gait disorders (86%), epilepsy (71%), psychiatric symptoms (57%), polyneuropathy (43%), and visual deficit (43%). Mean diagnostic delay was 14 years. Vascular events were observed in 28% and obesity in 36% of the patients. One patient remained asymptomatic at the age of 55 years. Upon treatment, median total homocysteine decreased (from 183 µmol/L, range 69-266, to 90 µmol/L, range 20-142) and symptoms improved (n = 9) or stabilized (n = 4). Missense pathogenic variants in the C-terminal regulatory domain of the protein were over-represented compared to early-onset cases. Residual MTHFR enzymatic activity in skin fibroblasts (n = 4) was rather high (17%-58%). This series of patients with late-onset MTHFR deficiency underlines the still unmet need of a prompt diagnosis of this treatable disease.