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.

Coherent photo-thermal noise cancellation in a dual-wavelength optical cavity for narrow-linewidth laser frequency stabilisation.

Optical resonators are used for the realisation of ultra-stable frequency lasers. The use of high reflectivity multi-band coatings allows the frequency locking of several lasers of different wavelengths to a single cavity. While the noise processes for single wavelength cavities are well known, the correlation caused by multi-stack coatings has as yet not been analysed experimentally. In our work, we stabilise the frequency of a 729 nm and a 1069 nm laser to one mirror pair and determine the residual-amplitude modulation (RAM) and photo-thermal noise (PTN). We find correlations in PTN between the two lasers and observe coherent cancellation of PTN for the 1069 nm coating. We show that the fractional frequency instability of the 729 nm laser is limited by RAM at 1 × 10-14. The instability of the 1069 nm laser is at 3 × 10-15 close to the thermal noise limit of 1.5 × 10-15.

Drug solubility in biorelevant media in the context of an inhalation-based biopharmaceutics classification system (iBCS).

An inhalation-based Biopharmaceutics Classification System for pulmonary drugs (iBCS) holds the perspective to allow for scientifically sound prediction of differences in the in vivo performance of orally inhaled drug products (OIDPs). A set of nine drug substances were selected, that are administered via both the oral and pulmonary routes. Their solubility was determined in media representative for the oral (Fasted State Simulated Intestinal Fluid (FaSSIF)) and pulmonary (Alveofact medium and Simulated Lung Fluid (SLF)) routes of administration to confirm the need for a novel approach for inhaled drugs. The complexity of these media was then stepwise reduced with the purpose of understanding the contribution of their components to the solubilizing capacity of the media. A second reason for varying the complexity was to identify a medium that would allow robust but accurate dissolution testing. Hence, Hank's balanced salt solution (HBSS) as a medium used in many in vitro biological tests, non-buffered saline solution, and water were included. For some drug substances (salbutamol sulfate, tobramycin, isoniazid, and tiotropium bromide), no significant differences were observed between the solubility in the media used. For other drugs, however, we observed either just small (rifampicin, budesonide, salmeterol) or unexpectedly large differences (beclomethasone dipropionate). Based on the minimum theoretical solubility required for their common pulmonary dose in 10 ml of lung lining fluid, drug solubility was classified as either high or low. Two high solubility and two low solubility compounds were then selected for refined solubility testing in pulmonary relevant media by varying their content of phospholipids, surfactant proteins and other proteins. The solubility of drug substances in simulated lung lining fluids was found to be dependent on the physicochemical properties of the drug substance and the composition of the media. While a pulmonary dissolution medium that would fit all drugs could not be established, our approach may provide guidance for finding the most suitable dissolution medium for a given drug substance and better designing in vitro tests for predicting the in vivo performance of inhalable drug products.

De novo variants in RNF213 are associated with a clinical spectrum ranging from Leigh syndrome to early-onset stroke.

PURPOSE: RNF213, encoding a giant E3 ubiquitin ligase, has been recognized for its role as a key susceptibility gene for moyamoya disease. Case reports have also implicated specific variants in RNF213 with an early-onset form of moyamoya disease with full penetrance. We aimed to expand the phenotypic spectrum of monogenic RNF213-related disease and to evaluate genotype-phenotype correlations. METHODS: Patients were identified through reanalysis of exome sequencing data of an unselected cohort of unsolved pediatric cases and through GeneMatcher or ClinVar. Functional characterization was done by proteomics analysis and oxidative phosphorylation enzyme activities using patient-derived fibroblasts. RESULTS: We identified 14 individuals from 13 unrelated families with (de novo) missense variants in RNF213 clustering within or around the Really Interesting New Gene (RING) domain. Individuals presented either with early-onset stroke (n = 11) or with Leigh syndrome (n = 3). No genotype-phenotype correlation could be established. Proteomics using patient-derived fibroblasts revealed no significant differences between clinical subgroups. 3D modeling revealed a clustering of missense variants in the tertiary structure of RNF213 potentially affecting zinc-binding suggesting a gain-of-function or dominant negative effect. CONCLUSION: De novo missense variants in RNF213 clustering in the E3 RING or other regions affecting zinc-binding lead to an early-onset syndrome characterized by stroke or Leigh syndrome.

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.

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.

Two years of pegvaliase in Germany: Experiences and best practice recommendations.

BACKGROUND: In 2019, pegvaliase was approved in Europe for the treatment of phenylketonuria (PKU) in patients aged 16 years and older with blood phenylalanine (Phe) concentrations above 600 µmol/L despite prior management with available treatment options. Since its European approval, German metabolic centres have gained valuable experience, which may be of benefit to other treatment centres managing patients on pegvaliase. METHODS: After a virtual meeting that was attended by nine German physicians, three German dietitians and one American physician, a follow-up discussion was held via an online platform to develop a set of recommendations on the use of pegvaliase in Germany. Eight German physicians contributed to the follow-up discussion and subsequent consensus voting, using a modified Delphi technique. The recommendations were supported by literature and retrospectively collected patient data. RESULTS: Consensus (≥75% agreement) was achieved on 25 recommendations, covering seven topics deemed relevant by the expert panel when considering pegvaliase an option for the treatment of patients with PKU. In addition to the recommendations, a retrospective chart review was conducted in seven of the centres and included 71 patients who initiated treatment with pegvaliase. Twenty-seven patients had been treated for at least 24 months and 23 (85.2%) had achieved blood Phe ≤600 µmol/L with some degree of diet normalisation. Of these patients, 14 had physiological blood Phe on a normalised diet. CONCLUSION: The practical consensus recommendations provide guidance on the different steps along the pegvaliase journey from clinical site requirements to treatment goals and outcomes. The recommendations are intended to support less experienced European metabolic centres with the implementation of pegvaliase, emphasising that a core treatment team consisting of at least a dietitian and metabolic physician is sufficient to initiate pegvaliase and support patients during their treatment journey.

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.

Impact of pregnancy planning and preconceptual dietary training on metabolic control and offspring's outcome in phenylketonuria.

To prevent maternal phenylketonuria (PKU) syndrome low phenylalanine concentrations (target range, 120-360 µmol/L) during pregnancy are recommended for women with PKU. We evaluated the feasibility and effectiveness of current recommendations and identified factors influencing maternal metabolic control and children's outcome. Retrospective study of first successfully completed pregnancies of 85 women with PKU from 12 German centers using historical data and interviews with the women. Children's outcome was evaluated by standardized IQ tests and parental rating of child behavior. Seventy-four percent (63/85) of women started treatment before conception, 64% (54/85) reached the phenylalanine target range before conception. Pregnancy planning resulted in earlier achievement of the phenylalanine target (18 weeks before conception planned vs. 11 weeks of gestation unplanned, p < 0.001) and lower plasma phenylalanine concentrations during pregnancy, particularly in the first trimester (0-7 weeks of gestation: 247 µmol/L planned vs. 467 µmol/L unplanned, p < 0.0001; 8-12 weeks of gestation: 235 µmol/L planned vs. 414 µmol/L unplanned, p < 0.001). Preconceptual dietary training increased the success rate of achieving the phenylalanine target before conception compared to women without training (19 weeks before conception vs. 9 weeks of gestation, p < 0.001). The majority (93%) of children had normal IQ (mean 103, median age 7.3 years); however, IQ decreased with increasing phenylalanine concentration during pregnancy. Good metabolic control during pregnancy is the prerequisite to prevent maternal PKU syndrome in the offspring. This can be achieved by timely provision of detailed information, preconceptual dietary training, and careful planning of pregnancy.

Left Ventricular Strain Analysis During Submaximal Semisupine Bicycle Exercise Stress Echocardiography in Childhood Cancer Survivors.

Background Childhood cancer survivors (CCSs) show relevant cardiac morbidity and mortality throughout life. Early detection is key for optimal support of patients at risk. The aim of this study was to evaluate 2-dimensional speckle-tracking echocardiography strain analysis during semisupine exercise stress in CCSs for detection of subclinical left ventricular dysfunction after cancer treatment. Methods and Results Seventy-seven CCSs ≥1-year postchemotherapy were prospectively examined at rest, low, and submaximal stress level and compared with a cohort of healthy adolescents and young adults (n=50). Global longitudinal strain (GLS), short axis circumferential strain, and corresponding strain rates were analyzed using vendor-independent software. CCSs at median 7.8 years postchemotherapy showed comparable left ventricular GLS, circumferential strain, and strain rate values at all stress stages to healthy controls. Yet, prevalence of abnormal GLS (defined as <2 SD of controls reference) in CCSs was 1.3% at rest, 2.7% at low, and 8.6% at submaximal stress. In CCSs, relative change of circumferential strain from rest to submaximal stress was lower than in healthy controls, median 16.9 (interquartile range [IQR], 3.4; 28.8) % versus 23.3 (IQR, 11.3; 33.3) %, P=0.03, most apparent in the subgroups of CCSs after high-dose anthracycline treatment and cancer diagnosis before the age of 5 years. Conclusions In this prospective 2-dimensional speckle tracking echocardiography strain study, prevalence of abnormal left ventricular GLS increased with stress level reflecting impaired cardiac adaptation to exercise stress in some CCSs. However, relatively early after last chemotherapy, this did not result in significant differences of mean GLS-, circumferential strain-, and strain rate values between CCSs and controls at any stress level.

Das NF1-Mikrodeletions-Syndrom: Die frühzeitige genetische Diagnose erleichtert den Umgang mit einer klinisch definierten Erkrankung.

Neurofibromatose Typ-1 (NF1) ist ein Genodermatose, die häufig in der Dermatologie behandelt wird. Bei vielen Patienten mit NF1 wird die Diagnose aufgrund klinischer Merkmale erstellt wie Café-au-Lait-Flecken, Freckling und plexiformen Neurofibromen, die schon während der frühen Kindheit auftreten können. Später im Leben sind oft kutane Neurofibrome weitere wichtige diagnostische Merkmale. Die NF1 ist durch ausgeprägte klinische Variabilität und eine breite Heterogenität der NF1-Genmutationen charakterisiert, was Genotyp/Phänotyp-Korrelationen erschwert. Wichtige Ausnahmen sind NF1-Mikrodeletionen, die bei 5-11 % aller NF1-Patienten auftreten. Patienten mit NF1-Mikrodeletionen zeigen häufig spezifische Merkmale wie Gesichtsdysmorphien und sind von großer Statur. Zudem sind früh auftretende kutane und subkutane Neurofibrome, schwere Entwicklungsverzögerungen in multiplen Bereichen sowie kognitive Einschränkungen pathognomonisch für das NF1-Mikrodeletions-Syndrom. Darüber hinaus sind NF1-Mikrodeletionen mit einem Risiko für maligne periphere Nervenscheidentumoren assoziiert, das etwa zweifach höher ist als bei intragenischen NF1-Mutationen. Die schweren klinischen Manifestationen bei Patienten mit NF1-Mikrodeletionen machen eine frühe multidisziplinäre klinische Betreuung und häufige Tumor-Überwachung der Patienten notwendig. Wenn bei einem Patienten Red-Flag-Symptome für das NF1-Mikrodeletions-Syndrom auftreten, ist eine frühzeitige genetische Untersuchung notwendig, um eine NF1-Mikrodeletion zu bestätigen oder auszuschließen.

The NF1 microdeletion syndrome: early genetic diagnosis facilitates the management of a clinically defined disease.

Neurofibromatosis type-1 (NF1) is a genodermatosis frequently encountered in general dermatology. In many patients, the diagnosis of NF1 is made clinically based on the presence of café-au-lait macules and skinfold freckling, as well as plexiform neurofibromas detectable during early childhood. Later in life, cutaneous neurofibromas often represent important diagnostic features. NF1 is characterized by extreme clinical variability and a broad heterogeneity of NF1 gene mutations which impede genotype/phenotype correlations. Notable exceptions are NF1 microdeletions observed in 5-11 % of all NF1 patients. Patients with NF1 microdeletions frequently exhibit facial dysmorphic features and a tall stature as rather specific clinical signs. Furthermore, cutaneous and subcutaneous neurofibromas present at an early age, severe global developmental delay and cognitive disability are pathognomonic for the "NF1 microdeletion syndrome". Importantly, NF1 microdeletions are associated with an approximately twofold higher risk for malignant peripheral nerve sheath tumors than intragenic NF1 gene mutations. The severe clinical manifestations of patients with NF1 microdeletions require early multidisciplinary clinical care and frequent tumor surveillance. Therefore, when red flag features for the "NF1 microdeletion syndrome" are present in a patient, genetic testing is necessary to confirm or exclude an NF1 microdeletion.

The biochemical subtype is a predictor for cognitive function in glutaric aciduria type 1: a national prospective follow-up study.

The aim of the study was a systematic evaluation of cognitive development in individuals with glutaric aciduria type 1 (GA1), a rare neurometabolic disorder, identified by newborn screening in Germany. This national, prospective, observational, multi-centre study includes 107 individuals with confirmed GA1 identified by newborn screening between 1999 and 2020 in Germany. Clinical status, development, and IQ were assessed using standardized tests. Impact of interventional and non-interventional parameters on cognitive outcome was evaluated. The majority of tested individuals (n = 72) showed stable IQ values with age (n = 56 with IQ test; median test age 11 years) but a significantly lower performance (median [IQR] IQ 87 [78-98]) than in general population, particularly in individuals with a biochemical high excreter phenotype (84 [75-96]) compared to the low excreter group (98 [92-105]; p = 0.0164). For all patients, IQ results were homogenous on subscale levels. Sex, clinical motor phenotype and quality of metabolic treatment had no impact on cognitive functions. Long-term neurologic outcome in GA1 involves both motor and cognitive functions. The biochemical high excreter phenotype is the major risk factor for cognitive impairment while cognitive functions do not appear to be impacted by current therapy and striatal damage. These findings implicate the necessity of new treatment concepts.

Moyamoya disease associated with pediatric pulmonary hypertension-a case report.

Moyamoya disease (MD) is a rare vaso-occlusive disorder that primarily affects intracranial cerebral arteries. The involvement of extracranial vessels is unusual. However, there are previous reports suggesting MD to be a systemic disorder, causing disease manifestations in vessels of other parts of the body. We report the case of a female patient with MD and multiple episodes of ischemic strokes followed by bypass surgery of cerebral arteries during infancy. Due to corresponding ischemic lesions the girl showed global retardation of psychomotor development and central right sided movement disorder. At the age of 10 years the girl was admitted to our hospital with recurrent syncope. While cranial MRI excluded any newly added ischemic lesions, electrocardiography revealed evidence of right ventricular hypertrophy, and subsequent echocardiography then indicated pulmonary hypertension, which was confirmed by cardiac catheterization. Despite an upfront combination pulmonary vasodilating therapy, the pulmonary vascular disease appeared to be progressive. Genetic analysis showed heterozygous c.12341C>T mutation in the RNF213 gene. This case presentation demonstrates that pulmonary arterial hypertension is a rare comorbidity in patients with MD, especially in patients with genetic predictors such as the RNF213 mutation. Thus, regular echocardiographic screening for early signs of pulmonary arterial hypertension in patients with MD should be part of regular clinical work-up. Early detection and treatment of pulmonary arterial hypertension in MD might help to improve the long-term outcome in the individual patient.

Physiologically relevant dissolution conditions towards improved in vitro - in vivo relationship - A case study with enteric coated pantoprazole tablets.

There are many hurdles in the development of generic formulations. In vitro biopredictive dissolution conditions together with alternative in vitro - in vivo relationship (IVIVR) approaches can be a powerful tool to support the development of such formulations. In this study, we hypothesized that the release profile of enteric coated (EC) formulations of pantoprazole in physiologically relevant bicarbonate buffer (BCB) would detect possible performance differences between test and reference formulations resulting in more accurate IVIVR results and predictability when compared to a pharmacopeial dissolution test. We correlated the in vitro performance of test and reference formulations (both in BCB and pharmacopeial phosphate buffer) with the in vivo data from a failed bioequivalence study. Test and reference formulations of EC pantoprazole tablets passed the USP dissolution criteria. However, they failed statistical similarity in vitro both in compendial and BCB. Bicarbonate buffer was additionally more discriminative while being more physiologically relevant. Having BCB as an additional test to evaluate EC products in vitro might improve the comparison of formulations. This can de-risk the development of generic EC formulations.

Nanomedicine at the crossroads - A quick guide for IVIVC.

For many years, nanomedicine is pushing the boundaries of drug delivery. When applying these novel therapeutics, safety considerations are not only a key concern when entering clinical trials but also an important decision point in product development. Standing at the crossroads, nanomedicine may be able to escape the niche markets and achieve wider acceptance by the pharmaceutical industry. While there is a new generation of drug delivery systems, the extracellular vesicles, standing on the starting line, unresolved issues and new challenges emerge from their translation from bench to bedside. Some key features of injectable nanomedicines contribute to the predictability of the pharmacological and toxicological effects. So far, only a few of the physicochemical attributes of nanomedicines can be justified by a direct mathematical relationship between the in vitro and the in vivo responses. To further develop extracellular vesicles as drug carriers, we have to learn from more than 40 years of clinical experience in liposomal delivery and pass on this knowledge to the next generation. Our quick guide discusses relationships between physicochemical characteristics and the in vivo response, commonly referred to as in vitro-in vivo correlation. Further, we highlight the key role of computational methods, lay open current knowledge gaps, and question the established design strategies. Has the recent progress improved the predictability of targeted delivery or do we need another change in perspective?

Retraction notice to "Case-control study about the acceptance of Pegvaliase in Phenylketonuria" Molecular Genetics and Metabolism Reports 22 (2020) 100557.

[This retracts the article DOI: 10.1016/j.ymgmr.2019.100557.].

Retraction notice to "Sustaining benefits of nutritional therapy in young adults with phenylketonuria - A 2 year prospective study" [Molecular Genetics and Metabolism Reports 22 (2020) 100573].

[This retracts the article DOI: 10.1016/j.ymgmr.2020.100573.].