Severity-adjusted evaluation of liver transplantation on health outcomes in urea cycle disorders.

PURPOSE: Liver transplantation (LTx) is performed in individuals with urea cycle disorders when medical management (MM) insufficiently prevents the occurrence of hyperammonemic events. However, there is a paucity of systematic analyses on the effects of LTx on health-related outcome parameters compared to individuals with comparable severity who are medically managed. METHODS: We investigated the effects of LTx and MM on validated health-related outcome parameters, including the metabolic disease course, linear growth, and neurocognitive outcomes. Individuals were stratified into "severe" and "attenuated" categories based on the genotype-specific and validated in vitro enzyme activity. RESULTS: LTx enabled metabolic stability by prevention of further hyperammonemic events after transplantation and was associated with a more favorable growth outcome compared with individuals remaining under MM. However, neurocognitive outcome in individuals with LTx did not differ from the medically managed counterparts as reflected by the frequency of motor abnormality and cognitive standard deviation score at last observation. CONCLUSION: Whereas LTx enabled metabolic stability without further need of protein restriction or nitrogen-scavenging therapy and was associated with a more favorable growth outcome, LTx-as currently performed-was not associated with improved neurocognitive outcomes compared with long-term MM in the investigated urea cycle disorders.

ASS1 deficiency is associated with impaired neuronal differentiation in zebrafish larvae.

Citrullinemia type 1 (CTLN1) is a rare autosomal recessive urea cycle disorder caused by deficiency of the cytosolic enzyme argininosuccinate synthetase 1 (ASS1) due to pathogenic variants in the ASS1 gene located on chromosome 9q34.11. Even though hyperammenomia is considered the major pathomechanistic factor for neurological impairment and cognitive dysfunction, a relevant subset of individuals presents with a neurodegenerative course in the absence of hyperammonemic decompensations. Here we show, that ASS1 deficiency induced by antisense-mediated knockdown of the zebrafish ASS1 homologue is associated with defective neuronal differentiation ultimately causing neuronal cell loss and consecutively decreased brain size in zebrafish larvae in vivo. Whereas ASS1-deficient zebrafish larvae are characterized by markedly elevated concentrations of citrulline - the biochemical hallmark of CTLN1, accumulation of L-citrulline, hyperammonemia or therewith associated secondary metabolic alterations did not account for the observed phenotype. Intriguingly, coinjection of the human ASS1 mRNA not only normalized citrulline concentration but also reversed the morphological cerebral phenotype and restored brain size, confirming conserved functional properties of ASS1 across species. The results of the present study imply a novel, potentially non-enzymatic (moonlighting) function of the ASS1 protein in neurodevelopment.

Impact of supplementation with L-citrulline/arginine after liver transplantation in individuals with Urea Cycle Disorders.

OBJECTIVE: Liver transplantation (LTx) is an intervention when medical management is not sufficiently preventing individuals with urea cycle disorders (UCDs) from the occurrence of hyperammonemic events. Supplementation with L-citrulline/arginine is regularly performed prior to LTx to support ureagenesis and is often continued after the intervention. However, systematic studies assessing the impact of long-term L-citrulline/arginine supplementation in individuals who have undergone LTx is lacking to date. METHODS: Using longitudinal data collected systematically, a comparative analysis was carried out by studying the effects of long-term L-citrulline/arginine supplementation vs. no supplementation on health-related outcome parameters (i.e., anthropometric, neurological, and cognitive outcomes) in individuals with UCDs who have undergone LTx. Altogether, 52 individuals with male ornithine transcarbamylase deficiency, citrullinemia type 1 and argininosuccinic aciduria and a pre-transplant "severe" disease course who have undergone LTx were investigated by using recently established and validated genotype-specific in vitro enzyme activities. RESULTS: Long-term supplementation of individuals with L-citrulline/arginine who have undergone LTx (n = 16) does neither appear to alter anthropometric nor neurocognitive endpoints when compared to their severity-adjusted counterparts that were not supplemented (n = 36) after LTx with mean observation periods between four to five years. Moreover, supplementation with L-citrulline/arginine was not associated with an increase of disease-specific plasma arithmetic mean values for the respective amino acids when compared to the non-supplemented control cohort. CONCLUSION: Although supplementation with L-citrulline/arginine is often continued after LTx, this pilot study does neither identify altered long-term anthropometric or neurocognitive health-related outcomes nor does it find an adequate biochemical response as reflected by the unaltered plasma arithmetic mean values for L-citrulline or L-arginine. Further prospective analyses in larger samples and even longer observation periods will provide more insight into the usefulness of long-term supplementation with L-citrulline/arginine for individuals with UCDs who have undergone LTx.

Newborn screening for aromatic l-amino acid decarboxylase deficiency - Strategies, results, and implication for prevalence calculations.

BACKGROUND: Aromatic l-amino acid decarboxylase deficiency (AADCD) is a rare, autosomal-recessive neurometabolic disorder caused by variants in dopa decarboxylase (DDC) gene, resulting in a severe combined deficiency of serotonin, dopamine, norepinephrine, and epinephrine. Birth prevalence of AADCD varies by population. In pilot studies, 3-O-methyldopa (3-OMD) was shown to be a reliable biomarker for AADCD in high-throughput newborn screening (NBS) allowing an early diagnosis and access to gene therapy. To evaluate the usefulness of this method for routine NBS, 3-OMD screening results from the largest three German NBS centers were analyzed. METHODS: A prospective, multicenter (n = 3) NBS pilot study evaluated screening for AADCD by quantifying 3-OMD in dried blood spots (DBS) using tandem mass spectrometry (MS/MS). RESULTS: In total, 766,660 neonates were screened from January 2021 until June 2023 with 766,647 with unremarkable AADCD NBS (766,443 by 1st-tier analysis and 204 by 2nd-tier analysis) and 13 with positive NBS result recalled for confirmatory diagnostics (recall-rate about 1:59,000). Molecular genetic analysis confirmed AADCD (c.79C > T p.[Arg27Cys] in Exon 2 und c.215 A > C p.[His72Pro] in Exon 3) in one infant. Another individual was highly suspected with AADCD but died before confirmation (overall positive predictive value 0.15). False-positive results were caused by maternal L-Dopa use (n = 2) and prematurity (30th and 36th week of gestation, n = 2). However, in 63% (n = 7) the underlying etiology for false positive results remained unexplained. Estimated birth prevalence (95% confidence interval) was 1:766,660 (95% CI 1:775,194; 1:769,231) to 1:383,330 (95% CI 1:384,615; 1:383,142). The identified child remained asymptomatic until last follow up at the age of 9 months. CONCLUSIONS: The proposed screening strategy with 3-OMD detection in DBS is feasible and effective to identify individuals with AADCD. The estimated birth prevalence supports earlier estimations and confirms AADCD as a very rare disorder. Pre-symptomatic identification by NBS allows a disease severity adapted drug support to diminish clinical complications until individuals are old enough for the application of the gene therapy.

Impact of genetic and non-genetic factors on phenotypic diversity in NBAS-associated disease.

Biallelic pathogenic variants in neuroblastoma-amplified sequence (NBAS) cause a pleiotropic multisystem disorder. Three clinical subgroups have been defined correlating with the localisation of pathogenic variants in the NBAS gene: variants affecting the C-terminal region of NBAS result in SOPH syndrome (short stature, optic atrophy, Pelger-Huët anomaly), variants affecting the Sec 39 domain are associated with infantile liver failure syndrome type 2 (ILFS2) and variants affecting the ß-propeller domain give rise to a combined phenotype. However, there is still unexplained phenotypic diversity across the three subgroups, challenging the current concept of genotype-phenotype correlations in NBAS-associated disease. Therefore, besides examining the genetic influence, we aim to elucidate the potential impact of pre-symptomatic diagnosis, emergency management and other modifying variables on the clinical phenotype. We investigated genotype-phenotype correlations in individuals sharing the same genotypes (n = 30 individuals), and in those sharing the same missense variants with a loss-of-function variant in trans (n = 38 individuals). Effects of a pre-symptomatic diagnosis and emergency management on the severity of acute liver failure (ALF) episodes also were analysed, comparing liver function tests (ALAT, ASAT, INR) and mortality. A strong genotype-phenotype correlation was demonstrated in individuals sharing the same genotype; this was especially true for the ILFS2 subgroup. Genotype-phenotype correlation in patients sharing only one missense variant was still high, though at a lower level. Pre-symptomatic diagnosis in combination with an emergency management protocol leads to a trend of reduced severity of ALF. High genetic impact on clinical phenotype in NBAS-associated disease facilitates monitoring and management of affected patients sharing the same genotype. Pre-symptomatic diagnosis and an emergency management protocol do not prevent ALF but may reduce its clinical severity.

Resource use and costs of transitioning from paediatric to adult care for patients with chronic endocrine disease.

OBJECTIVE: Structured transition of adolescents and young adults with a chronic endocrine disease from paediatric to adult care is important. Until now, no data on time and resources required for the necessary components of the transition process and the associated costs are available. DESIGN, PATIENTS AND MEASUREMENTS: In a prospective cohort study of 147 patients with chronic endocrinopathies, for the key elements of a structured transition pathway including (i) assessment of patients' disease-related knowledge and needs, (ii) required education and counselling sessions, (iii) compiling an epicrisis and a transfer appointment of the patient together with the current paediatric and the future adult endocrinologist resource consumption and costs were determined. RESULTS: One hundred and forty-three of 147 enroled patients (97.3%) completed the transition pathway and were transferred to adult care. The mean time from the decision to start the transition process to the final transfer consultation was 399 ± 159 days. Transfer consultations were performed in 143 patients, including 128 patients jointly with the future adult endocrinologist. Most consultations were performed by a multidisciplinary team consisting of a paediatric and adult endocrinologist, psychologist, nurse, and a social worker acting also as a case manager with a median of three team members and lasted 87.6 ± 23.7 min. The mean cumulative costs per patient of all key elements were 519 ± 206 Euros. In addition, costs for case management through the transition process were 104.8 ± 28.0 Euros. CONCLUSIONS: Using chronic endocrine diseases as an example, it shows how to calculate the time and cost of a structured transition pathway from paediatric to adult care, which can serve as a starting point for sustainable funding for other chronic rare diseases.

MRI in LARS1 deficiency-Spectrum, patterns, and correlation with acute neurological deterioration.

Leucine aminoacyl tRNA-synthetase 1 (LARS1)-deficiency (infantile liver failure syndrome type 1 (ILFS1)) has a multisystemic phenotype including fever-associated acute liver failure (ALF), chronic neurologic abnormalities, and encephalopathic episodes. In order to better characterize encephalopathic episodes and MRI changes, 35 cranial MRIs from 13 individuals with LARS1 deficiency were systematically assessed and neurological phenotype was analyzed. All individuals had developmental delay and 10/13 had seizures. Encephalopathic episodes in 8/13 were typically associated with infections, presented with seizures and reduced consciousness, mostly accompanied by hepatic dysfunction, and recovery in 17/19 episodes. Encephalopathy without hepatic dysfunction occurred in one individual after liver transplantation. On MRI, 5/7 individuals with MRI during acute encephalopathy had deep gray matter and brainstem changes. Supratentorial cortex involvement (6/13) and cerebellar watershed injury (4/13) occurred with seizures and/or encephalopathy. Abnormal brainstem contour on sagittal images (8/13), atrophy (8/13), and myelination delay (8/13) were not clearly associated with encephalopathy. The pattern of deep gray matter and brainstem changes are apparently characteristic of encephalopathy in LARS1-deficiency, differing from patterns of hepatic encephalopathy or metabolic stroke in organic acidurias and mitochondrial diseases. While the pathomechanism remains unclear, fever and energy deficit during infections might be causative; thus, sufficient glucose and protein intake along with pro-active fever management is suggested. As severe episodes were observed during influenza infections, we strongly recommend seasonal vaccination.

Outcomes after newborn screening for propionic and methylmalonic acidemia and homocystinurias.

The current German newborn screening (NBS) panel includes 13 inherited metabolic diseases (IMDs). In addition, a NBS pilot study in Southwest Germany identifies individuals with propionic acidemia (PA), methylmalonic acidemia (MMA), combined and isolated remethylation disorders (e.g., cobalamin [cbl] C and methylenetetrahydrofolate reductase [MTHFR] deficiency), cystathionine ß-synthase (CBS) deficiency, and neonatal cbl deficiency through one multiple-tier algorithm. The long-term health benefits of screened individuals are evaluated in a multicenter observational study. Twenty seven screened individuals with IMDs (PA [N = 13], MMA [N = 6], cblC deficiency [N = 5], MTHFR deficiency [N = 2] and CBS deficiency [N = 1]), and 42 with neonatal cbl deficiency were followed for a median of 3.6 years. Seventeen screened IMD patients (63%) experienced at least one metabolic decompensation, 14 of them neonatally and six even before the NBS report (PA, cbl-nonresponsive MMA). Three PA patients died despite NBS and immediate treatment. Fifteen individuals (79%) with PA or MMA and all with cblC deficiency developed permanent, mostly neurological symptoms, while individuals with MTHFR, CBS, and neonatal cbl deficiency had a favorable clinical outcome. Utilizing a combined multiple-tier algorithm, we demonstrate that NBS and specialized metabolic care result in substantial benefits for individuals with MTHFR deficiency, CBS deficiency, neonatal cbl deficiency, and to some extent, cbl-responsive MMA and cblC deficiency. However, its advantage is less evident for individuals with PA and cbl-nonresponsive MMA. SYNOPSIS: Early detection through newborn screening and subsequent specialized metabolic care improve clinical outcomes and survival in individuals with MTHFR deficiency and cystathionine-ß-synthase deficiency, and to some extent in cobalamin-responsive methylmalonic acidemia (MMA) and cblC deficiency while the benefit for individuals with propionic acidemia and cobalamin-nonresponsive MMA is less evident due to the high (neonatal) decompensation rate, mortality, and long-term complications.

The Genetic Landscape and Epidemiology of Phenylketonuria.

Phenylketonuria (PKU), caused by variants in the phenylalanine hydroxylase (PAH) gene, is the most common autosomal-recessive Mendelian phenotype of amino acid metabolism. We estimated that globally 0.45 million individuals have PKU, with global prevalence 1:23,930 live births (range 1:4,500 [Italy]-1:125,000 [Japan]). Comparing genotypes and metabolic phenotypes from 16,092 affected subjects revealed differences in disease severity in 51 countries from 17 world regions, with the global phenotype distribution of 62% classic PKU, 22% mild PKU, and 16% mild hyperphenylalaninemia. A gradient in genotype and phenotype distribution exists across Europe, from classic PKU in the east to mild PKU in the southwest and mild hyperphenylalaninemia in the south. The c.1241A>G (p.Tyr414Cys)-associated genotype can be traced from Northern to Western Europe, from Sweden via Norway, to Denmark, to the Netherlands. The frequency of classic PKU increases from Europe (56%) via Middle East (71%) to Australia (80%). Of 758 PAH variants, c.1222C>T (p.Arg408Trp) (22.2%), c.1066-11G>A (IVS10-11G>A) (6.4%), and c.782G>A (p.Arg261Gln) (5.5%) were most common and responsible for two prevalent genotypes: p.[Arg408Trp];[Arg408Trp] (11.4%) and c.[1066-11G>A];[1066-11G>A] (2.6%). Most genotypes (73%) were compound heterozygous, 27% were homozygous, and 55% of 3,659 different genotypes occurred in only a single individual. PAH variants were scored using an allelic phenotype value and correlated with pre-treatment blood phenylalanine concentrations (n = 6,115) and tetrahydrobiopterin loading test results (n = 4,381), enabling prediction of both a genotype-based phenotype (88%) and tetrahydrobiopterin responsiveness (83%). This study shows that large genotype databases enable accurate phenotype prediction, allowing appropriate targeting of therapies to optimize clinical outcome.

Prospective Newborn Screening for SCID in Germany: A First Analysis by the Pediatric Immunology Working Group (API).

BACKGR OUND: T-cell receptor excision circle (TREC)-based newborn screening (NBS) for severe combined immunodeficiencies (SCID) was introduced in Germany in August 2019. METHODS: Children with abnormal TREC-NBS were referred to a newly established network of Combined Immunodeficiency (CID) Clinics and Centers. The Working Group for Pediatric Immunology (API) and German Society for Newborn Screening (DGNS) performed 6-monthly surveys to assess the TREC-NBS process after 2.5 years. RESULTS: Among 1.9 million screened newborns, 88 patients with congenital T-cell lymphocytopenia were identified (25 SCID, 17 leaky SCID/Omenn syndrome (OS)/idiopathic T-cell lymphocytopenia, and 46 syndromic disorders). A genetic diagnosis was established in 88%. Twenty-six patients underwent hematopoietic stem cell transplantation (HSCT), 23/26 within 4 months of life. Of these, 25/26 (96%) were alive at last follow-up. Two patients presented with in utero onset OS and died after birth. Five patients with syndromic disorders underwent thymus transplantation. Eight syndromic patients deceased, all from non-immunological complications. TREC-NBS missed one patient, who later presented clinically, and one tracking failure occurred after an inconclusive screening result. CONCLUSION: The German TREC-NBS represents the largest European SCID screening at this point. The incidence of SCID/leaky SCID/OS in Germany is approximately 1:54,000, very similar to previous observations from North American and European regions and countries where TREC-NBS was implemented. The newly founded API-CID network facilitates tracking and treatment of identified patients. Short-term HSCT outcome was excellent, but NBS and transplant registries will remain essential to evaluate the long-term outcome and to compare results across the rising numbers of TREC-NBS programs across Europe.

Integrative Approach to Predict Severity in Nonketotic Hyperglycinemia.

OBJECTIVE: Glycine encephalopathy, also known as nonketotic hyperglycinemia (NKH), is an inherited neurometabolic disorder with variable clinical course and severity, ranging from infantile epileptic encephalopathy to psychiatric disorders. A precise phenotypic characterization and an evaluation of predictive approaches are needed. METHODS: Longitudinal clinical and biochemical data of 25 individuals with NKH from the patient registry of the International Working Group on Neurotransmitter Related Disorders were studied with in silico analyses, pathogenicity scores, and molecular modeling of GLDC and AMT variants. RESULTS: Symptom onset (p < 0.01) and diagnosis occur earlier in life in severe NKH (p < 0.01). Presenting symptoms affect the age at diagnosis. Psychiatric problems occur predominantly in attenuated NKH. Onset age ≥ 3 months (66% specificity, 100% sensitivity, area under the curve [AUC] = 0.87) and cerebrospinal fluid (CSF)/plasma glycine ratio ≤ 0.09 (57% specificity, 100% sensitivity, AUC = 0.88) are sensitive indicators for attenuated NKH, whereas CSF glycine concentration ≥ 116.5µmol/l (100% specificity, 93% sensitivity, AUC = 0.97) and CSF/plasma glycine ratio ≥ 0.15 (100% specificity, 64% sensitivity, AUC = 0.88) are specific for severe forms. A ratio threshold of 0.128 discriminates the overlapping range. We present 10 new GLDC variants. Two mild variants resulted in attenuated, whereas 2 severe variants or 1 mild and 1 severe variant led to severe phenotype. Based on clinical, biochemical, and genetic parameters, we propose a severity prediction model. INTERPRETATION: This study widens the phenotypic spectrum of attenuated NKH and expands the number of pathogenic variants. The multiparametric approach provides a promising tool to predict disease severity, helping to improve clinical management strategies. ANN NEUROL 2022;92:292-303.

The challenge of understanding and predicting phenotypic diversity in urea cycle disorders.

The Urea Cycle Disorders Consortium (UCDC) and the European registry and network for Intoxication type Metabolic Diseases (E-IMD) are the worldwide largest databases for individuals with urea cycle disorders (UCDs) comprising longitudinal data from more than 1100 individuals with an overall long-term follow-up of approximately 25 years. However, heterogeneity of the clinical phenotype as well as different diagnostic and therapeutic strategies hamper our understanding on the predictors of phenotypic diversity and the impact of disease-immanent and interventional variables (e.g., diagnostic and therapeutic interventions) on the long-term outcome. A new strategy using combined and comparative data analyses helped overcome this challenge. This review presents the mechanisms and relevant principles that are necessary for the identification of meaningful clinical associations by combining data from different data sources, and serves as a blueprint for future analyses of rare disease registries.

Kidney urinary biomarkers in patients with branched-chain amino acid and cobalamin metabolism defects.

There is a clinical need for early detection of chronic kidney disease (CKD) in patients with organic acidurias. We measured kidney markers in a longitudinal study over 5 years in 40 patients with methylmalonic aciduria (Mut0 ), propionic aciduria (PA), cobalamin A (CblA), and cobalamin C (CblC) deficiencies. Neutrophil gelatinase-associated lipocalin (NGAL), calprotectin (CLP), kidney injury molecule-1 (KIM-1), dickkopf-3 (DKK-3), albumin and beta-2-microglobulin (B2MG) in urine, as well as cystatin C (CysC) in serum were quantified. In Mut0 patients, mean concentrations of B2MG, KIM-1, and DKK-3 were elevated compared with healthy controls, all markers indicative of proximal tubule damage. In PA patients, mean B2MG, albumin, and CLP were elevated, indicating signs of proximal tubule and glomerulus damage and inflammation. In CblC patients, mean B2MG, NGAL, and CLP were increased, and considered as markers for proximal and distal tubule damage and inflammation. B2MG, was elevated in all three diseases, and correlated with DKK-3 in Mut0 /CblA and with eGFR(CysC) and KIM-1 in PA patients, respectively. None of the markers were elevated in CblA patients. Significant deterioration of kidney function, as determined by steady increase in CysC concentrations was noted in seven patients within the observation period. None of the investigated biomarker profiles showed a clear increase or added value for early detection. In conclusion, we identified disease-specific biomarker profiles for inflammation, tubular, and proximal damage in the urine of Mut0 , PA, and CblC patients. Whether these biomarkers can be used for early detection of CKD requires further investigation, as significant kidney function deterioration was observed in only a few patients.

Long-term anthropometric development of individuals with inherited metabolic diseases identified by newborn screening.

Newborn screening (NBS) for inherited metabolic diseases (IMDs) substantially shortens a patient's journey. It enables the early start of metabolic treatment which might prevent potentially lethal neonatal disease manifestations, while promoting favorable development and long-term clinical outcomes. This study aims to assess growth in screened individuals with IMDs under different dietary regimes. Anthropometric data (3585 prospective measures) of 350 screened individuals with IMDs born between 1999 and 2018 and participating in a German prospective multicenter observational study were evaluated. Overall, birth measures were within the reference ranges, suggesting unaffected prenatal growth, except for phenylketonuria (weight) and glutaric aciduria Type 1 (head circumference). After birth, longitudinal analysis of anthropometric measures revealed a loss of height standard deviation score (SDS; -0.5 SDS; p < 0.0001), head circumference SDS (-0.2 SDS; p = 0.0028), but not for weight SDS (0.1 SDS; p = 0.5097) until the age of 18 years, while BMI SDS increased (0.4 SDS; p < 0.0001). The significant interaction with age and diet groups was pronounced for the linear growth in individuals receiving diets being low in protein, long-chain triglycerides, and galactose (p < 0.001). Identification by NBS and subsequent early (dietary) treatment cannot completely protect against alterations in growths. Disease-specific (e.g., metabolic impairments, neurotoxins) and dietary-specific (e.g., diets reduced in protein) factors may have an amplified impact on longitudinal growth. Therefore, alongside other important follow-ups, the continuous observation of the anthropometric development of screened individuals with IMDs needs special attention to early identify and support individuals at risk.

Isovaleric aciduria identified by newborn screening: Strategies to predict disease severity and stratify treatment.

Newborn screening (NBS) allows early identification of individuals with rare disease, such as isovaleric aciduria (IVA). Reliable early prediction of disease severity of positively screened individuals with IVA is needed to guide therapeutic decision, prevent life-threatening neonatal disease manifestation in classic IVA and over-medicalization in attenuated IVA that may remain asymptomatic. We analyzed 84 individuals (median age at last study visit 8.5 years) with confirmed IVA identified by NBS between 1998 and 2018 who participated in the national, observational, multicenter study. Screening results, additional metabolic parameters, genotypes, and clinical phenotypic data were included. Individuals with metabolic decompensation showed a higher median isovalerylcarnitine (C5) concentration in the first NBS sample (10.6 vs. 2.7 µmol/L; p < 0.0001) and initial urinary isovalerylglycine concentration (1750 vs. 180 mmol/mol creatinine; p = 0.0003) than those who remained asymptomatic. C5 was in trend inversely correlated with full IQ (R = -0.255; slope = -0.869; p = 0.0870) and was lower for the "attenuated" variants compared to classic genotypes [median (IQR; range): 2.6 µmol/L (2.1-4.0; 0.7-6.4) versus 10.3 µmol/L (7.4-13.1; 4.3-21.7); N = 73]. In-silico prediction scores (M-CAP, MetaSVM, and MetaLR) correlated highly with isovalerylglycine and ratios of C5 to free carnitine and acetylcarnitine, but not sufficiently with clinical endpoints. The results of the first NBS sample and biochemical confirmatory testing are reliable early predictors of the clinical course of IVA, facilitating case definition (attenuated versus classic IVA). Prediction of attenuated IVA is supported by the genotype. On this basis, a reasonable algorithm has been established for neonates with a positive NBS result for IVA, with the aim of providing the necessary treatment immediately, but whenever possible, adjusting the treatment to the individual severity of the disease.

Collaborative evaluation study on 18 candidate diseases for newborn screening in 1.77 million samples.

Analytical and therapeutic innovations led to a continuous but variable extension of newborn screening (NBS) programmes worldwide. Every extension requires a careful evaluation of feasibility, diagnostic (process) quality and possible health benefits to balance benefits and limitations. The aim of this study was to evaluate the suitability of 18 candidate diseases for inclusion in NBS programmes. Utilising tandem mass spectrometry as well as establishing specific diagnostic pathways with second-tier analyses, three German NBS centres designed and conducted an evaluation study for 18 candidate diseases, all of them inherited metabolic diseases. In total, 1 777 264 NBS samples were analysed. Overall, 441 positive NBS results were reported resulting in 68 confirmed diagnoses, 373 false-positive cases and an estimated cumulative prevalence of approximately 1 in 26 000 newborns. The positive predictive value ranged from 0.07 (carnitine transporter defect) to 0.67 (HMG-CoA lyase deficiency). Three individuals were missed and 14 individuals (21%) developed symptoms before the positive NBS results were reported. The majority of tested candidate diseases were found to be suitable for inclusion in NBS programmes, while multiple acyl-CoA dehydrogenase deficiency, isolated methylmalonic acidurias, propionic acidemia and malonyl-CoA decarboxylase deficiency showed some and carnitine transporter defect significant limitations. Evaluation studies are an important tool to assess the potential benefits and limitations of expanding NBS programmes to new diseases.

Levodopa-refractory hyperprolactinemia and pituitary findings in inherited disorders of biogenic amine metabolism.

Elevated serum prolactin concentrations occur in inherited disorders of biogenic amine metabolism because dopamine deficiency leads to insufficient inhibition of prolactin secretion. This work from the International Working Group on Neurotransmitter Related Disorders (iNTD) presents the results of the first standardized study on levodopa-refractory hyperprolactinemia (LRHP; >1000 mU/L) and pituitary magnetic resonance imaging (MRI) abnormalities in patients with inherited disorders of biogenic amine metabolism. Twenty-six individuals had LRHP or abnormal pituitary findings on MRI. Tetrahydrobiopterin deficiencies were the most common diagnoses (n = 22). The median age at diagnosis of LRHP was 16 years (range: 2.5-30, 1st-3rd quartiles: 12.25-17 years). Twelve individuals (nine females) had symptoms attributed to hyperprolactinemia: menstruation-related abnormalities (n = 7), pubertal delay or arrest (n = 5), galactorrhea (n = 3), and decreased sexual functions (n = 2). MRI of the pituitary gland was obtained in 21 individuals; six had heterogeneity/hyperplasia of the gland, five had adenoma, and 10 had normal findings. Eleven individuals were treated with the dopamine agonist cabergoline, ameliorating the hyperprolactinemia-related symptoms in all those assessed. Routine monitoring of these symptoms together with prolactin concentrations, especially after the first decade of life, should be taken into consideration during follow-up evaluations. The potential of slow-release levodopa formulations and low-dose dopamine agonists as part of first-line therapy in the prevention and treatment of hyperprolactinemia should be investigated further in animal studies and human trials. This work adds hyperprolactinemia-related findings to the current knowledge of the phenotypic spectrum of inherited disorders of biogenic amine metabolism.

Recommendations for diagnosing and managing individuals with glutaric aciduria type 1: Third revision.

Glutaric aciduria type 1 is a rare inherited neurometabolic disorder of lysine metabolism caused by pathogenic gene variations in GCDH (cytogenic location: 19p13.13), resulting in deficiency of mitochondrial glutaryl-CoA dehydrogenase (GCDH) and, consequently, accumulation of glutaric acid, 3-hydroxyglutaric acid, glutaconic acid and glutarylcarnitine detectable by gas chromatography/mass spectrometry (organic acids) and tandem mass spectrometry (acylcarnitines). Depending on residual GCDH activity, biochemical high and low excreting phenotypes have been defined. Most untreated individuals present with acute onset of striatal damage before age 3 (to 6) years, precipitated by infectious diseases, fever or surgery, resulting in irreversible, mostly dystonic movement disorder with limited life expectancy. In some patients, striatal damage develops insidiously. In recent years, the clinical phenotype has been extended by the finding of extrastriatal abnormalities and cognitive dysfunction, preferably in the high excreter group, as well as chronic kidney failure. Newborn screening is the prerequisite for pre-symptomatic start of metabolic treatment with low lysine diet, carnitine supplementation and intensified emergency treatment during catabolic episodes, which, in combination, have substantially improved neurologic outcome. In contrast, start of treatment after onset of symptoms cannot reverse existing motor dysfunction caused by striatal damage. Dietary treatment can be relaxed after the vulnerable period for striatal damage, that is, age 6 years. However, impact of dietary relaxation on long-term outcomes is still unclear. This third revision of evidence-based recommendations aims to re-evaluate previous recommendations (Boy et al., J Inherit Metab Dis, 2017;40(1):75-101; Kolker et al., J Inherit Metab Dis 2011;34(3):677-694; Kolker et al., J Inherit Metab Dis, 2007;30(1):5-22) and to implement new research findings on the evolving phenotypic diversity as well as the impact of non-interventional variables and treatment quality on clinical outcomes.

High Throughput Newborn Screening for Sickle Cell Disease - Application of Two-Tiered Testing with a qPCR-Based Primary screen.

BACKGROUND: Sickle cell disease (SCD) is a group of hemoglobinopathies with a common point mutation causing the production of sickle cell hemoglobin (HbS). In high-throughput newborn screening (NBS) for SCD, a two-step procedure is suitable, in which qPCR first pre-selects relevant samples that are differentiated by a second method. METHODS: Three NBS centers using qPCR-based primary screening for SCD performed a laboratory comparison. Methods using tandem MS or HPLC were used for differentiation. RESULTS: In a benchmarking test, 450 dried blood samples were analyzed. Samples containing HbS were detected as reliably by qPCR as by methods established for hemoglobinopathy testing. In a two-step screening approach, the 2nd-tier-analyses have to distinguish the carrier status from pathological variants. In nine months of regular screening, a total of 353,219 samples were analyzed using two-stage NBS procedures. The 1st-tier screening by qPCR reduced the number of samples for subsequent differentiation by>99.5%. Cases with carrier status or other variants were identified as inconspicuous while 78 cases with SCD were revealed. The derived incidence of 1:4,773, is in good agreement with previously published incidences. CONCLUSION: In high-throughput NBS for SCD, qPCR is suitable to focus 2nd-tier analyses on samples containing HbS, while being unaffected by factors such as prematurity or transfusions. The substantial reduction of samples numbers positively impacts resource conservation, sustainability, and cost-effectiveness. No false negative cases came to attention.

RINT1 Bi-allelic Variations Cause Infantile-Onset Recurrent Acute Liver Failure and Skeletal Abnormalities.

Pediatric acute liver failure (ALF) is life threatening with genetic, immunologic, and environmental etiologies. Approximately half of all cases remain unexplained. Recurrent ALF (RALF) in infants describes repeated episodes of severe liver injury with recovery of hepatic function between crises. We describe bi-allelic RINT1 alterations as the cause of a multisystem disorder including RALF and skeletal abnormalities. Three unrelated individuals with RALF onset ≤3 years of age have splice alterations at the same position (c.1333+1G>A or G>T) in trans with a missense (p.Ala368Thr or p.Leu370Pro) or in-frame deletion (p.Val618_Lys619del) in RINT1. ALF episodes are concomitant with fever/infection and not all individuals have complete normalization of liver function testing between episodes. Liver biopsies revealed nonspecific liver damage including fibrosis, steatosis, or mild increases in Kupffer cells. Skeletal imaging revealed abnormalities affecting the vertebrae and pelvis. Dermal fibroblasts showed splice-variant mediated skipping of exon 9 leading to an out-of-frame product and nonsense-mediated transcript decay. Fibroblasts also revealed decreased RINT1 protein, abnormal Golgi morphology, and impaired autophagic flux compared to control. RINT1 interacts with NBAS, recently implicated in RALF, and UVRAG, to facilitate Golgi-to-ER retrograde vesicle transport. During nutrient depletion or infection, Golgi-to-ER transport is suppressed and autophagy is promoted through UVRAG regulation by mTOR. Aberrant autophagy has been associated with the development of similar skeletal abnormalities and also with liver disease, suggesting that disruption of these RINT1 functions may explain the liver and skeletal findings. Clarifying the pathomechanism underlying this gene-disease relationship may inform therapeutic opportunities.