New Cases of Maleylacetoacetate Isomerase Deficiency with Detection by Newborn Screening and Natural History over 32 Years: Experience from a German Newborn Screening Center.

Newborn screening (NBS) for hepatorenal tyrosinemia type I (HT1) based on a determination of succinylacetone is performed in countries worldwide. Recently, biallelic pathogenic variants in GSTZ1 underlying maleylacetoacetate isomerase (MAAI) deficiency have been described as a differential diagnosis in individuals with slightly elevated succinylacetone detected by NBS. We report the experience with NBS for HT1 over 53 months in a large German NBS center and the identification and characterization of additional cases with MAAI deficiency, including one individual with a natural history over 32 years. A total of 516,803 children underwent NBS for HT1 at the NBS center in Heidelberg between August 2016 and December 2020. Of 42 children with elevated succinylacetone, HT1 was confirmed in two cases (1 in 258.401). MAAI deficiency was suspected in two cases and genetically confirmed in one who showed traces of succinylacetone in urine. A previously unreported pathogenic GSTZ1 variant was found in the index in a biallelic state. Segregation analysis revealed monoallelic carriership in the index case's mother and homozygosity in his father. The 32-year-old father had no medical concerns up to that point and the laboratory work-up was unremarkable. MAAI has to be considered a rare differential diagnosis in NBS for HT1 in cases with slight elevations of succinylacetone to allow for correct counselling and treatment decisions. Our observation of natural history over 32 years adds evidence for a benign clinical course of MAAI deficiency without specific treatment.

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.

Influence of Season, Storage Temperature and Time of Sample Collection in Pancreatitis-Associated Protein-Based Algorithms for Newborn Screening for Cystic Fibrosis.

Newborn screening (NBS) for cystic fibrosis (CF) based on pancreatitis-associated protein (PAP) has been performed for several years. While some influencing factors are known, there is currently a lack of information on the influence of seasonal temperature on PAP determination or on the course of PAP blood concentration in infants during the first year of life. Using data from two PAP studies at the Heidelberg NBS centre and storage experiments, we compared PAP determinations in summer and winter and determined the direct influence of temperature. In addition, PAP concentrations measured in CF-NBS, between days 21-35 and 36-365, were compared. Over a 7-year period, we found no significant differences between PAP concentrations determined in summer or winter. We also found no differences in PAP determination after 8 days of storage at 4 °C, room temperature or 37 °C. When stored for up to 3 months, PAP samples remained stable at 4 °C, but not at room temperature (p = 0.007). After birth, PAP in neonatal blood showed a significant increasing trend up to the 96th hour of life (p < 0.0001). During the first year of life, blood PAP concentrations continued to increase in both CF- (36-72 h vs. 36-365 d p < 0.0001) and non-CF infants (36-72 h vs. 36-365 d p < 0.0001). Seasonal effects in central Europe appear to have a limited impact on PAP determination. The impact of the increase in blood PAP during the critical period for CF-NBS and beyond on the applicability and performance of PAP-based CF-NBS algorithms needs to be re-discussed.

Identification of potential interferents of methylmalonic acid: A previously unrecognized pitfall in clinical diagnostics and newborn screening.

OBJECTIVES: Determination of methylmalonic acid (MMA) from dried blood spots (DBS) is commonly performed in clinical diagnostics and newborn screening for propionic acidemia (PA) and methylmalonic acidemia. Isobaric compounds of MMA having the same mass can affect diagnostic reliability and quantitative results, which represents a previously unrecognized pitfall in clinical assays for MMA. We set out to identify interfering substances of MMA in DBS, serum and urine samples from confirmed patients with PA and methylmalonic acidemia. METHODS: Techniques included quadrupole time-of-flight high-resolution mass spectrometry (QTOF HR-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography (LC) and tandem mass spectrometry (MS/MS). RESULTS: The five isobaric metabolites detected in DBS, serum and urine from PA and methylmalonic acidemia patients were confirmed as 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 2-hydroxyisovalerate, 3-hydroxyvalerate and succinate using a series of experiments. An additional unknown substance with low abundance remained unidentified. CONCLUSIONS: The presented results facilitate the diagnostic and quantitative reliability of the MMA determination in clinical assays. Isobaric species should be investigated in assays for MMA to eliminate possible interference in a wide range of conditions including PA, methylmalonic acidemia, a vitamin B12 deficiency, ketosis and lactic acidosis.

Final results of the southwest German pilot study on cystic fibrosis newborn screening - Evaluation of an IRT/PAP protocol with IRT-dependent safety net.

BACKGROUND: Previous studies suggest that PAP-based CF protocols are suitable for newborn screening (NBS) for cystic fibrosis (CF) when newborns designated as CFSPID should not be detected. However, there are still discussions about the performance of IRT/PAP algorithms. We present the final results of a pilot study evaluating a IRT/PAP protocol with an IRT-dependent safety net (SN) conducted from 2008 to 2016 in southwestern Germany on nearly 500,000 newborns. METHODS: To achieve reliable data, all newborns were screened using both the PAP-based and a DNA-based CFNBS algorithm. PAP quantification and genetic analysis of the four most common CFTR mutations in Germany were performed in all newborns with IRT≥99.0 percentile. NBS was rated positive if either PAP was ≥1.6 µg/l and/or at least one CFTR mutation was detected. In addition, an IRT-dependent SN resulted in positive rating for both protocols if IRT was ≥99.9 percentile. To evaluate the IRT/PAP protocol, its performance was compared to that of the IRT/DNA protocol. RESULTS: The IRT/PAP protocol with IRT-based SN used in the study achieved a sensitivity of 94%, if false-negative detected neonates with meconium ileus and those designated as CFSPID were excluded from analysis. CF/CFSPID ratio was 92. However, PPV of the IRT/PAP+SN protocol was with 10.3% very low. CONCLUSIONS: PAP-based CFNBS protocols can be used, if less detection of CFSPID is desired. The IRT/PAP protocol with IRT-dependent SN evaluated here achieved adequate sensitivity but should probably be used in combination with a third-tier test to also achieve an acceptable PPV.

High throughput newborn screening for aromatic ʟ-amino-acid decarboxylase deficiency by analysis of concentrations of 3-O-methyldopa from dried blood spots.

Aromatic l-amino-acid decarboxylase (AADC) deficiency is an inherited disorder of biogenic amine metabolism with a broad neurological phenotype. The clinical symptoms overlap with other diseases resulting in an often delayed diagnosis. Innovative disease-changing treatment options, particularly gene therapy, have emphasised the need for an early diagnosis. We describe the first method for 3-O-methyldopa (3-OMD) analysis in dried blood spots (DBS) suitable for high throughput newborn screening (NBS). We established a novel tandem mass spectrometry method to quantify 3-OMD in DBS and successfully tested it in 38 888 unaffected newborns, 14 heterozygous DDC variant carriers, seven known AADC deficient patients, and 1079 healthy control subjects. 3-OMD concentrations in 38 888 healthy newborns revealed a mean of 1.16 µmol/L (SD = 0.31, range 0.31-4.6 µmol/L). 1079 non-AADC control subjects (0-18 years) showed a mean 3-OMD concentration of 0.78 µmol/L (SD = 1.75, range 0.24-2.36 µmol/L) with a negative correlation with age. Inter- and intra-assay variability was low, and 3-OMD was stable over 32 days under different storage conditions. We identified seven confirmed AADC deficient patients (mean 3-OMD 9.88 µmol/L [SD = 13.42, range 1.82-36.93 µmol/L]). The highest concentration of 3-OMD was found in a NBS filter card of a confirmed AADC deficient patient with a mean 3-OMD of 35.95 µmol/L. 14 DDC variant carriers showed normal 3-OMD concentrations. We demonstrate a novel high-throughput method to measure 3-OMD in DBS, which allows integration in existing NBS programs enabling early diagnosis of AADC deficiency.

A product of immunoreactive trypsinogen and pancreatitis-associated protein as second-tier strategy in cystic fibrosis newborn screening.

BACKGROUND: In cystic fibrosis newborn screening (CFNBS), immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP) can be used as screening parameters. We evaluated the IRT×PAP product as second-tier parameter in CFNBS in newborns with elevated IRT. METHODS: Data on 410,111 screened newborns including 78 patients with classical cystic fibrosis (CF) from two European centers were retrospectively analyzed by discrimination analysis to identify a screening protocol with optimal cutoffs. We also studied differences in PAP measurement methods and the association of IRT and PAP with age. RESULTS: PAP values differed systematically between fluorometric and photometric assays. The IRT×PAP product showed better discrimination for classical CF than PAP only as second-tier screening parameter (p<0.001). In CF patients, IRT decreased while PAP values remained high over years. In newborns without CF, IRT decreased after birth over weeks while PAP increased within days. CONCLUSIONS: The IRT×PAP product performs well as second-tier cutoff parameter for CFNBS. Screening quality parameters depend on the analytic method and on age at blood collection.

Quantitative acylcarnitine profiling in peripheral blood mononuclear cells using in vitro loading with palmitic and 2-oxoadipic acids: biochemical confirmation of fatty acid oxidation and organic acid disorders.

Organic acid (OAD) and fatty acid oxidation disorders (FAOD) are inborn errors of metabolism often presenting with life-threatening metabolic decompensation followed by (irreversible) organ failure, and even death during catabolic state. Most of these diseases are considered as treatable, and metabolic decompensations can be avoided by early diagnosis and start of therapy. Confirmation of suspected diagnosis currently relies on enzymatic and mutation analyses and in vitro loading of palmitic acid in human skin fibroblast cultures. Furthermore, in some cases potentially life-threatening in vivo loading or fasting tests are still performed. In this study, we established a standardized in vitro loading test in peripheral blood mononuclear cells (PBMC) that allows reliable biochemical confirmation of a suspected diagnosis within 1 week. Patients with confirmed diagnosis of short-, medium-, very-long-chain, and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiencies, methylmalonic, propionic, isovaleric acidurias, and glutaric aciduria type I were included in the study. PBMC, isolated from heparinized venous blood samples of these individuals were incubated for 5 days with palmitic acid or 2-oxoadipic acid (glutaric aciduria type I), respectively, and quantitative acylcarnitine profiling was subsequently performed in supernatants using electrospray ionization tandem mass spectrometry. All patients were clearly identified, including those with mild biochemical phenotypes who, in particular, are at risk to be missed under balanced metabolic conditions. In glutaric aciduria type I, the same results were also obtained using lymphoblasts. In conclusion, our assay allows biochemical confirmation of a number of FAOD and OAD and could easily be implemented into the confirmatory diagnostic work-up.

Inactivation of the glucocorticoid receptor in hepatocytes leads to fasting hypoglycemia and ameliorates hyperglycemia in streptozotocin-induced diabetes mellitus.

Hepatic glucose production by gluconeogenesis is the main source of glucose during fasting and contributes significantly to hyperglycemia in diabetes mellitus. Accordingly, glucose metabolism is tightly controlled by a variety of hormones including insulin, epinephrine, glucagon, and glucocorticoids (GCs) acting on various cell types. GC effects are mediated by the GC receptor (GR), a ligand-dependent transcription factor, which in the liver and kidney controls gluconeogenesis by induction of gluconeogenic enzymes. To specifically study the contribution of GC on liver carbohydrate metabolism, we generated mice with an inactivation of the GR gene exclusively in hepatocytes using the Cre/loxP technology. Half of the mutant mice die within the first 2 d after birth most likely due to hypoglycemia. Adult mice have normal blood sugar under basal conditions but show hypoglycemia after prolonged starvation due to reduced expression of genes involved in gluconeogenesis. We further demonstrate that absence of GR in hepatocytes limits the development of hyperglycemia in streptozotocin-induced diabetes mellitus probably due to impaired induction of gluconeogenesis. These findings show the essential role of GR function in liver glucose metabolism during fasting and in diabetic mice and indicate that liver-specific GC antagonists could be beneficial in control of diabetic hyperglycemia.

Accurate measurement of free carnitine in dried blood spots by isotope-dilution electrospray tandem mass spectrometry without butylation.

BACKGROUND: To test the feasibility of free carnitine (FC) determination in dried blood spot specimens (DBS) by stable isotope-dilution electrospray-ionisation tandem mass spectrometry (MS/MS). METHODS: The MS/MS method established for newborn screening, measuring acylcarnitines by positive precursor ion scan of m/z 85 in DBS, was adapted by omitting the butylation and heating step during sample preparation. FC measurement in DBS by this non-butylating MS/MS assay was compared with the butylating MS/MS method and the spectrophotometric Cobas method. RESULTS: FC measurement by the non-butylating MS/MS method meets the demands for a bioanalytical microassay with respect to linearity, detection limit (LOD), accuracy, and precision. Formation of FC was 0-1% and 1-4% in liquid samples and in DBS by the non-butylating MS/MS method, while 3-10% and 8-16% by the butylating method, respectively. Acid-catalysed hydrolysis (butanolysis) in liquid samples was higher for short-chain acylcarnitines (acetyl- and propionylcarnitine). Hydrolysis in DBS was more pronounced for long-chain acylcarnitines. FC concentrations in healthy newborns without butylation were 35% lower than those measured by the established newborn screening assay. CONCLUSIONS: The non-butylating MS/MS assay provides a simple and accurate method for FC determination in DBS and represents a trivial but important adaptation of a method already used in many laboratories.

Expanded newborn screening for inborn errors of metabolism by electrospray ionization-tandem mass spectrometry: results, outcome, and implications.

OBJECTIVE: The aims of this study were to determine the impact of expanded newborn screening using tandem mass spectrometry (MS/MS) on the overall detection rate of inborn errors of metabolism in Germany and to assess the outcome for the patients that were diagnosed. METHODS: During the period of study, 250,000 neonates in a German population were investigated for 23 inborn errors of metabolism by electrospray ionization-MS/MS. The overall value of the screening program was estimated by 1) complete ascertainment of all positive tests; 2) definite assignment of all diagnoses including reconfirmation at 12 months; and 3) clinical follow-up of all detected patients in an overall interval of 42 months. The mean observation period was 13.5 months per child. RESULTS: In 106 newborns, confirmed inborn errors of metabolism were found. The disorders were classified as 50 classic forms and 56 variants. A total of 825 tests (0.33%) were false-positives. Seventy of the 106 newborns with confirmed disorders were judged to require treatment. Six children developed symptoms despite treatment. Three children had died. Among 9 children who became symptomatic before report of the results of screening, in 6 the diagnosis had been made in advance of the screening report. In evaluation of the screening program, 61 of the 106 identified children (58% of true-positives, or 1 of 4100 healthy newborns) were judged to have benefited from screening and treatment, because the diagnosis had not been made before screening. None of these infants had died and none developed psychomotor retardation or metabolic crisis during the follow-up period. CONCLUSIONS: The screening by MS/MS for up to 23 additional disorders has approximately doubled the detection rate compared with that achieved by the conventional methods used in Germany. This strategy represents valuable preventive medicine by enabling diagnosis and treatment before the onset of symptoms.

The endogeneous formation of highly chlorinated tetrahydro-beta-carbolines as a possible causative mechanism in idiopathic Parkinson's disease.

The causative interrelationship between long-term, low level exposure to chlorinated volatile organic solvents (VOSs) and neurodegenerative diseases (polyneuropathy, encephalopathy) are still an issue of controversial debate. Endogeneously formed chlorinated tetrahydro-beta-carbolines found by Bringmann 1995 (TaClo hypothesis) may contribute, in particular, to the development of (idiopathic) Parkinson's disease (PD) in the presence of the sufficient amount of trichloroacetaldehyde, an intermediate in metabolism of trichloroethylene (TRI). Long-term storage of specific VOSs over years, evident frrom exhalation pattern during the postexposure period, may serve as a promoting factor to form continuously TaClo non-enzymatically from tryptamine and trichloroacetaldehyde. Thus, the induction of TaClo-mediated neurotoxic processes extends over years. The onset of Parkinson's disease in three chronic TRI-exposed individuals during the postexposure period could be associated with the presence of TaClo in ng-range. Consequently, determination of TaClo and its derivatives in blood of humans exposed to chlorinated VOSs may serve as a marker of risk indicating either causative or supportive processes of neurodegeneration that may lead to manifestation of PD after many years.

A method for quantitative acylcarnitine profiling in human skin fibroblasts using unlabelled palmitic acid: diagnosis of fatty acid oxidation disorders and differentiation between biochemical phenotypes of MCAD deficiency.

Inherited disorders of fatty acid oxidation are a group of acute life-threatening but treatable disorders, clinically complicated by severe hypoketotic hypoglycemia precipitated by prolonged fasting. Among them, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is by far the most frequent disorder. Here we report a modified method for quantitative acylcarnitine profiling by electrospray ionisation-tandem mass spectrometry (ESI-MS-MS) in human skin fibroblasts using unlabelled palmitic acid as substrate. The reliability of this method was tested in cultured skin fibroblasts from previously diagnosed patients with specific carnitine cycle and fatty acid beta-oxidation defects. Furthermore, acylcarnitine profiling was investigated in fibroblasts and dried blood spots from patients with different variants of MCAD deficiency. ESI-MS-MS-based investigation of cultured skin fibroblasts from patients with disorders of fatty acid oxidation revealed a pathognomonic acylcarnitine profiling. In addition, this method delineated different variants of MCAD deficiency, i.e. mild and classical. The octanoylcarnitine (C8)-to-decanoylcarnitine (C10) and C8-to-acetylcarnitine (C2) ratios were the most specific markers to differentiate mild and classical forms of MCAD deficiency in fibroblasts. Similar results were obtained by quantitative acylcarnitine profiling in dried blood spots. In conclusion, this novel technique is a powerful tool for the investigation of fatty acid oxidation disorders under standardized conditions in fibroblasts.

Chronic treatment with glutaric acid induces partial tolerance to excitotoxicity in neuronal cultures from chick embryo telencephalons.

Glutaryl-CoA dehydrogenase deficiency (GDD) is characterized biochemically by an accumulation of glutaric (GA) and 3-hydroxyglutaric (3-OH-GA) acids and clinically by the development of acute striatal degeneration. 3-OH-GA was recently shown to induce neuronal damage via N-methyl-D-aspartate (NMDA) receptors. The pathogenetic role of GA, however, remains unclear. We demonstrate that GA exerts a dual action in cultured chick embryo neurons. Short-term incubation with millimolar concentrations of GA induces a weak neuronal damage, adding to 3-OH-GA neurotoxicity. In contrast, chronic treatment with subtoxic, micromolar concentrations of GA results in partial tolerance to 3-OH-GA- and NMDA-induced cell damage. A downregulation of NMDA receptors, in particular of the NR2B subunit, is critically involved in this GA-induced effect, resulting in a reduced Ca(2+) increase and generation of reactive oxygen species after acute exposure to NMDA or 3-OH-GA. Furthermore, GA decreases Na(+)/K(+)-ATPase activity, which is prevented by glutathione, suggesting a modulation of NMDA receptor function via resting membrane potential and Na(+)-dependent glutamate transport. In contrast, GA does not inhibit mitochondrial respiratory chain and beta-oxidation of fatty acids, virtually excluding an activation of NMDA receptors secondary to ATP depletion. These results strongly suggest that GA modulates the NMDA receptor-mediated neurotoxicity of 3-OH-GA, providing an explanatory basis for the non-linear relationship between organic acid concentrations and disease progression in GDD patients. Furthermore, GA-induced downregulation of NMDA receptors might be involved in the delayed cerebral maturation of GDD patients, resulting in frontotemporal atrophy and a reduced opercularization, which are common neuroradiological findings in GDD patients.