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Cystic fibrosis (CF) was added to the German newborn bloodspot screening (NBS) panel in 2016. This study assesses parental perceptions of CF-NBS and confirmatory testing.Prospective questionnaire-based survey administered to parents of children with positive CF-NBS over 40 months after initiation of CF-NBS in Southwest Germany. Parental perceptions were compared to results from Bavaria and Switzerland.Questionnaires with 29 standardized questions were sent to 343 families with children born between October 2016 and January 2020. A total of 178 (51.9%) replied. Although required by law, only a minority were informed about CF-NBS by a physician. The information provided about NBS was sufficient for 78% of parents. Regarding the information about positive CF-NBS, 52.9% were satisfied but the majority expressed negative emotions (89.5%). While most of these were resolved after confirmatory diagnostics, 17% of parents of children with false-positive CF-NBS and 66.7% of children confirmed with CF remained anxious. Waiting time for sweat testing was3 days in 56.1%, considerably longer than in more centralized screening systems. Parents who waited for a maximum of 3 days were significantly more satisfied. 70.7% of parents were satisfied with the information given during confirmatory diagnostics and 91.4% were satisfied with participating in CF-NBS.CF-NBS stands in high regard with parents. Smooth organization, timely initiation of confirmatory testing, and professional communication are most important to limit parental anxiety. A more centralized system of confirmatory diagnostics appears advantageous in several regards as it reduces time from positive NBS to final diagnosis and increases parental satisfaction.

Vitamin B12 deficiency, mostly of maternal origin in newborns, is a well-treatable condition but can cause severe neurologic sequelae in infants. Early detection of vitamin B12 deficiency allows the pre-symptomatic treatment of affected children. This evaluation assesses the characteristics of maternal vitamin B12 deficiency detected by newborn screening. In a prospective single-center study, a systematic screening strategy for vitamin B12 deficiency using a combination of two second-tier strategies was applied. In addition to confirmatory diagnostics in children, the systematic work-up of vitamin B12 status was also performed for their mothers. Maternal characteristics were assessed including ethnic origin, diet, and vitamin supplementation during pregnancy. For affected mothers, a work-up by internal medicine was recommended. In total, 121 mother–infant couples were analyzed. 66% of mothers adhered to a balanced diet including meat. The cause of maternal vitamin B12 deficiency was unknown in 56% of cases, followed by dietary causes in 32%, and organic causes in 8%. All mothers following a vegan diet and most mothers with a vegetarian diet took vitamin preparations during pregnancy, whereas only 55.8% of mothers with a balanced diet took folic acid or other vitamins. Maternal vitamin B12, folic acid, and homocysteine levels were significantly correlated with the child’s folic acid levels, and with homocysteine, methylmalonic, and methylcitric acid levels in first and second NBS dried blood spots. Most children had normal blood counts and showed normocytosis. Although 36.7% of mothers showed anemia, only one presented with macrocytosis. Adherence to vitamin supplementation in pregnancy is low despite the recommendation for supplementation of folic acid. Ideally, the evaluation of mothers for vitamin B12 levels and appropriate therapy should be initiated in early pregnancy. In infants detected through newborn screening, the multidisciplinary assessment and therapy of both children and mothers should be performed.

Vitamin B

Newborn screening for cystic fibrosis (CF-NBS) was introduced in Germany in 2016. Currently, systematic follow-up of positive CF-NBS results is not implemented or reimbursed in the NBS program. We investigated results of confirmatory testing over 24 months after implementation of CF-NBS for a large German NBS center before and after introduction of an active tracking system and performed a cost calculation for tracking. Results are compared with the federal state of Bavaria, where a centralized tracking system has been in place for many years. At the NBS center, 244 of 281,907 children had a positive CF-NBS result requiring diagnostic confirmation. Before implementation of a telephone tracking system, only 43% of confirmatory results were returned despite repeated written requests. The consecutive strategy including telephone tracking led to an increase of resolved cases to 84%. However, the centralized tracking system in Bavaria, assigning children with positive CF-NBS directly to a responsible CF-center, resolved 99% of cases. The calculated additional cost for a tracking system in Germany including telephone tracking is 1.20€ per newborn screened.Conclusion: The implementation of a tracking system achieves a distinct improvement in CF-NBS with justifiable costs. The effect can be limited by absence of centralized organization of confirmatory testing. What is Known:• Newborn screening for cystic fibrosis (CF-NBS) has been performed for many years in several countries worldwide• While many studies have focused on different CF-NBS strategies, the organization of confirmatory testing and process quality concerning returned information to the NBS center has so far received less attention. What is New:• The implementation of an active tracking system achieves a distinct improvement of clarified cases after positive CF-NBS with justifiable costs.• The effect of a tracking system can be limited by the absence of a centralized organization of confirmatory testing.

Background Aromatic L-amino acid decarboxylase (AADC) deficiency is a primary neurotransmitter defect of the biosynthesis of catecholamines and serotonin. The phenotype consists of varying degrees of neurological impairment, including motor and non-motor symptoms. Treatment outcomes correlate with the time point of diagnosis and treatment initiation; therefore, reliable diagnostic markers are necessary. Increased vanillactic acid (VLA) concentrations in the analysis of organic acids in urine have been reported in AADC deficiency. However, this elevation is often subtle and easily missed. In this study, we evaluate the semi-quantitative determination of VLA and vanillylmandelic acid (VMA) concentrations and establish the ratio of a VLA/VMA as a novel diagnostic marker for AADC deficiency. Methods Urine samples obtained from 10,095 non-AADC deficient controls and 14 confirmed AADC deficient patients were used for organic acid analysis by liquid-liquid extraction of the acidified samples and gas chromatographic-mass spectrometric separation after trimethylsilylation. The semi-quantitative determination of VLA and VMA concentrations and the calculation of a VLA/VMA ratio were evaluated as a diagnostic marker for AADC deficiency. Results The mean VLA and VMA concentrations in 10,095 non-AADCD samples was 0.3 mmol/mol creatinine (SD = 1.18, range 0–57.79) and 5.59 mmol/mol creatinine (SD = 3.87, range 0.04–60.62), respectively. The mean concentration of VLA in 14 patient-derived samples was 10.24 mmol/mol creatinine, (SD = 11.58, range = 0.37–33.06) and 0.45 mmol/mol creatinine for VMA (SD = 0.29, range 0.11–1.27). The mean VLA/VMA ratio in non-AADC controls was 0.07 (SD = 0.37, range 0.0–23.24), whereas AADC deficient patients revealed a mean VLA/VMA ratio of 23.16 (SD = 22.83, range 0.97–74.1). The VLA/VMA ratio thus allows a reliable identification of patients with AADC deficiency, especially in the young age cohort as it decreases with age. To take this into account, age-adjusted thresholds have been developed. Conclusion Determination of individual concentrations of VLA and VMA in urine does not allow a reliable diagnosis of AADC deficiency. In this study, we could demonstrate that a semi-quantitative analysis of organic acids in urine allows the formation of metabolite ratios and that the VLA/VMA ratio is a reliable, easily accessible, new parameter for the diagnosis of AADC deficiency.

Newborn screening (NBS) in Germany currently includes 15 target disorders. Recent diagnostic improvements suggest an extension of the screening panel. Since August 2016, a prospective study evaluating 26 additional target disorders (25 metabolic disorders and vitamin B12-deficiency) in addition to the German screening panel is performed at the Newborn Screening Center Heidelberg. First-tier results from tandem-MS screening are complemented by second-tier strategies for 15 of the additional target disorders. NBS results of seven patients diagnosed symptomatically with one of the additional target disorders by selective screening since August 2016 are retrospectively evaluated. Over a 13-month period, 68,418 children participated in the study. Second-tier analyses were performed in 5.4% of samples. Only 59 (0.1%) of study participants had abnormal screening results for one of the additional target disorders. Target disorders from the study panel were confirmed in 12 children: 1 3-hydroxy-3-methylglutaryl coenzyme A (CoA)-lyase deficiency, 1 citrullinemia type I, 1 multiple acyl-CoA dehydrogenase-deficiency, 1 methylenetetrahydrofolate reductase-deficiency, and 8 children with maternal vitamin B12-deficiency. In addition, six of seven patients diagnosed symptomatically outside the study with one of the target disorders would have been identified by the study strategy in their NBS sample. Within 13 months, the study “Newborn Screening 2020” identified additional 12 children with treatable conditions while only marginally increasing the recall rate by 0.1%. Maternal vitamin B12-deficiency was the most frequent finding. Even more children could benefit from screening for the additional target disorders by extending the NBS panel for Germany and/or other countries.

To evaluate a systematic newborn screening (NBS) strategy for vitamin BIn a prospective single-center NBS study, a systematic screening strategy for vitamin BIn a cohort of 176 702 children screened over 27 months, 33 children were detected by NBS in whom (maternal) vitamin BVitamin B

Hydroxyprolinemia is an inborn error of amino acid degradation that is considered a non-disease. Known for more than 50 years, its genetic cause and prevalence have remained unclear. In MS/MS newborn screening, the mass spectrum of hydroxyproline cannot be differentiated from isoleucine and leucine causing false positive newborn screening test results for maple syrup urine disease (MSUD). We studied two siblings with hydroxyprolinemia via exome sequencing and confirmed the candidate gene in five further individuals with hydroxyprolinemia, who were all characterized biochemically and clinically. The prevalence was calculated based on the number of individuals with hydroxyprolinemia detected via MS/MS newborn screening at our centre from 2003 to 2014. In six cases, we identified homozygous or compound heterozygous mutations in PRODH2 as the underlying genetic cause of hydroxyprolinemia. One individual was heterozygous for a deletion in PRODH2 and had an intermittent biochemical phenotype with partial normalization of hydroxyproline concentrations. In one further individual with persistent hydroxyprolinemia no mutation in PRODH2 was found, raising the possibility of another defect of hydroxyproline degradation yet to be identified as the underlying cause of hydroxyprolinemia. Plasma hydroxyproline concentrations were clearly elevated in all individuals with biallelic mutations in PRODH2. All studied individuals remained asymptomatic, giving further evidence that hydroxyprolinemia is a benign condition. The estimated prevalence of hydroxyprolinemia in Germany is about one in 47,300 newborns. Our results establish mutations in PRODH2 as a cause of human hydroxyprolinemia via impaired dehydrogenation of hydroxyproline to delta1-pyroline-3-hydroxy-5-carboxylic acid, and we suggest PRODH2 be renamed HYPDH. Hydroxyprolinemia is an autosomal-recessively inherited benign condition. It is a frequent cause of false positive screening results for MSUD, the prevalence being about 2.5 times higher than that of MSUD.

Newborn screening (NBS) in Germany currently includes 15 target disorders. Recent diagnostic improvements suggest an extension of the screening panel.Since August 2016, a prospective study evaluating 26 additional target disorders (25 metabolic disorders and vitamin BOver a 13-month period, 68,418 children participated in the study. Second-tier analyses were performed in 5.4% of samples. Only 59 (0.1%) of study participants had abnormal screening results for one of the additional target disorders. Target disorders from the study panel were confirmed in 12 children: 1 3-hydroxy-3-methylglutaryl coenzyme A (CoA)-lyase deficiency, 1 citrullinemia type I, 1 multiple acyl-CoA dehydrogenase-deficiency, 1 methylenetetrahydrofolate reductase-deficiency, and 8 children with maternal vitamin BWithin 13 months, the study "Newborn Screening 2020" identified additional 12 children with treatable conditions while only marginally increasing the recall rate by 0.1%. Maternal vitamin B

Objective To evaluate a systematic newborn screening (NBS) strategy for vitamin B12 deficiency. Study design In a prospective single-center NBS study, a systematic screening strategy for vitamin B12 deficiency was developed and evaluated. Tandem-mass spectrometry screening was complemented by 2 second-tier strategies, measuring methylmalonic/3-OH-propionic/methylcitric acid, and homocysteine from dried blood spots. Results In a cohort of 176 702 children screened over 27 months, 33 children were detected by NBS in whom (maternal) vitamin B12 deficiency was confirmed. Homocysteine was the most sensitive marker for vitamin B12 deficiency, but only combination with a second-tier strategy evaluating methylmalonic acid allowed for detection of all 33 children. Mothers were of various ethnic origins, and 89% adhered to a balanced diet. Treatment in children was performed predominantly by oral vitamin B12 supplementation (84%), and all children remained without clinical symptoms at short-term follow-up. Conclusions Vitamin B12 deficiency is a treatable condition but can cause severe neurologic sequelae in infants if untreated. The proposed screening strategy is feasible and effective to identify moderate and severe cases of vitamin B12 deficiency. With an incidence of 1:5355 newborns, vitamin B12 deficiency is more frequent than inborn errors of metabolism included in NBS panels. Treatment of vitamin B12 deficiency is easy, and additional benefits can be achieved for previously undiagnosed affected mothers. This supports inclusion of vitamin B12 deficiency into NBS but also stresses the need for increased awareness of vitamin B12 deficiency in caregivers of pregnant women.

Organic acids comprise key metabolites of virtually all pathways of intermediary metabolism as well as exogenous compounds (Fig. C.1). Comprehensive quantitative analysis of organic acids in body fluids has therefore the potential of yielding information on the physiological and pathophysiological status of different metabolic pathways as well as their interrelationships. The very complexity and diversity of organic acids has, however, hampered the development of quantitative methods for organic acid analysis, and this methodology has not been and is still not widely available [1–3]. Organic acid analysis is generally performed by capillary gas-liquid chromatography/mass spectrometry of trimethylsilylated or methylated derivatives; identification and quantitation of metabolites by computer-based special user libraries containing both the mass spectra and retention indices. Less commonly used are liquid chromatography-chemical ionization mass spectrometric or automated capillary electrophoretic methods.

Methylmalonic acidurias are biochemically characterized by an accumulation of methylmalonate (MMA) and alternative metabolites. There is growing evidence for basal ganglia degeneration in these patients. The pathomechanisms involved are still unknown, a contribution of toxic organic acids, in particular MMA, has been suggested. Here we report that MMA induces neuronal damage in cultures of embryonic rat striatal cells at a concentration range encountered in affected patients. MMA-induced cell damage was reduced by ionotropic glutamate receptor antagonists, antioxidants, and succinate. These results suggest the involvement of secondary excitotoxic mechanisms in MMA-induced cell damage. MMA has been implicated in inhibition of respiratory chain complex II. However, MMA failed to inhibit complex II activity in submitochondrial particles from bovine heart. To unravel the mechanism underlying neuronal MMA toxicity, we investigated the formation of intracellular metabolites in MMA-loaded striatal neurons. There was a time-dependent intracellular increase in malonate, an inhibitor of complex II, and 2-methylcitrate, a compound with multiple inhibitory effects on the tricarboxylic acid cycle, suggesting their putative implication in MMA neurotoxicity. We propose that neuropathogenesis of methylmalonic aciduria may involve an inhibition of complex II and the tricarboxylic acid cycle by accumulating toxic organic acids, and synergistic secondary excitotoxic mechanisms.