BACKGROUND Congenital disorders of glycosylation are genetic syndromes that result in impaired glycoprotein production. We evaluated patients who had a novel recessive disorder of glycosylation, with a range of clinical manifestations that included hepatopathy, bifid uvula, malignant hyperthermia, hypogonadotropic hypogonadism, growth retardation, hypoglycemia, myopathy, dilated cardiomyopathy, and cardiac arrest. METHODS Homozygosity mapping followed by whole-exome sequencing was used to identify a mutation in the gene for phosphoglucomutase 1 (PGM1) in two siblings. Sequencing identified additional mutations in 15 other families. Phosphoglucomutase 1 enzyme activity was assayed on cell extracts. Analyses of glycosylation efficiency and quantitative studies of sugar metabolites were performed. Galactose supplementation in fibroblast cultures and dietary supplementation in the patients were studied to determine the effect on glycosylation. RESULTS Phosphoglucomutase 1 enzyme activity was markedly diminished in all patients. Mass spectrometry of transferrin showed a loss of complete N-glycans and the presence of truncated glycans lacking galactose. Fibroblasts supplemented with galactose showed restoration of protein glycosylation and no evidence of glycogen accumulation. Dietary supplementation with galactose in six patients resulted in changes suggestive of clinical improvement. A new screening test showed good discrimination between patients and controls. CONCLUSIONS Phosphoglucomutase 1 deficiency, previously identified as a glycogenosis, is also a congenital disorder of glycosylation. Supplementation with galactose leads to biochemical improvement in indexes of glycosylation in cells and patients, and supplementation with complex carbohydrates stabilizes blood glucose. A new screening test has been developed but has not yet been validated. (Funded by the Netherlands Organization for Scientific Research and others.)

CRISPR-Cas-based genome editing holds great promise for targeting genetic disorders, including inborn errors of hepatocyte metabolism. Precise correction of disease-causing mutations in adult tissues in vivo, however, is challenging. It requires repair of Cas9-induced double-stranded DNA (dsDNA) breaks by homology-directed mechanisms, which are highly inefficient in nondividing cells. Here we corrected the disease phenotype of adult phenylalanine hydroxylase (Pah) mice, a model for the human autosomal recessive liver disease phenylketonuria (PKU), using recently developed CRISPR-Cas-associated base editors. These systems enable conversion of CG to TA base pairs and vice versa, independent of dsDNA break formation and homology-directed repair (HDR). We engineered and validated an inteinsplit base editor, which allows splitting of the fusion protein into two parts, thereby circumventing the limited cargo capacity of adeno-associated virus (AAV) vectors. Intravenous injection of AAV-base editor systems resulted in Pah gene correction rates that restored physiological blood phenylalanine (L-Phe) levels below 120 µmol/l [5]. We observed mRNA correction rates up to 63%, restoration of phenylalanine hydroxylase (PAH) enzyme activity, and reversion of the light fur phenotype in Pah mice. Our findings suggest that targeting genetic diseases in vivo using AAV-mediated delivery of base-editing agents is feasible, demonstrating potential for therapeutic application.

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

The (R)-and (S)-isomers of a-methyl-branched fatty acids were shown to be rapidly interconverted as coenzyme A thioesters, by an a-methylacyl-CoA racemase. The enzyme was purified some 5600-fold from rat liver, to apparent homogeneity. It is a monomer of 45 kDa with an isolectric point of pH 6.1 and is optimally active between pH 6 and pH 7. It acts only on coenzyme A thioesters, not on free fatty acids, and accepts as substrates a wide range of a-methylacyl-CoAs, including pristanoyl-CoA and trihydroxycoprostanoyl-CoA (an intermediate in bile acid synthesis), but neither 3-methyl-branched nor linear-chain acyl-CoAs. The racemase catalyzes a rapid exchange of the H atom in the a-position of the fatty acid against a proton from water, indicating that the mechanism involves abstraction of a proton. Based on this observation, a very sensitive and convenient radiometric assay, with 2-methy1[2-3H]acyl-CoAs as substrates, was developed. The enzyme was inactivated by micromolar concentrations of Hg2' and to a lesser extent by Cu2+ but not by iodoacetamide and only slightly by N-ethylmaleimide and thimerosal.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent inherited defect of fatty acid oxidation, with a significant morbidity and mortality in undiagnosed patients. Adverse outcomes can effectively be prevented by avoiding metabolic stress and following simple dietary measures. Therefore, prospective newborn screening (NBS) is being proposed for this condition. However, technical validation of MCADD population screening and assessment of its overall benefit require broadening of the as-yet-scarce knowledge of the MCADD genetic heterogeneity unraveled by NBS and its phenotypic consequences. Here, we describe the entire spectrum of sequence variations occurring in newborns with MCADD in the population of Bavaria, Germany, in relation to the biochemical phenotype. Among 524,287 newborns, we identified 62 cases of MCADD, indicating a birth incidence of 1 in 8,456. In all of the 57 newborns available for analysis, two alterations within the MCADD gene (ACADM) were identified. The most prevalent alteration c.985A>G (Lys329Glu) occurred in 27 (47%) newborns in the homozygous and in 18 (32%) in the heterozygous state (63% of defective alleles). The mild folding variant c.199T>C (Tyr67His) was identified in nine individuals, six of them being compound heterozygous with c.985A>G (Lys329Glu). Neither of the prevalent alterations were found in the remaining nine newborns. A total of 18 sequence variations were identified; 13 of them were novel: eight missense mutations, one nonsense mutation, two splice variants, and two small deletions. The remaining five were previously reported in MCADD patients. The ACADM heterogeneity uncovered was larger as anticipated from previous c.985A>G (Lys329Glu) carrier screening data. In addition, we show that MCADD appears to occur as frequently in Turkish newborns as in the native German population. Our data validate that biochemical NBS for MCADD is a highly specific procedure for disease detection, with the identification of a significant share of milder biochemical phenotypes, such as c.199T>C (Tyr67His). These show statistically lower acylcarnitine markers, allowing us to distinguish subgroups within the spectrum of ACADM sequence variations that correlate to biochemical MCADD disease expression. Our data might provide technical and medical guidance for decision making in the worldwide efforts to introduce MCADD population screening.

Background: Newborn screening for CF started 01/2011 in Switzerland. We investigated the parents' opinions about the information received, their feelings, and overall approval of the screening. Methods: This is a prospective questionnaire survey of all parents of positively screened children. Parents were phoned by CF-centres and invited for diagnostic investigations. They completed a questionnaire after the visit to the CF-centre. Results: From 2011-2013, 246 families received the questionnaire and 138 (56%) replied. Of these 77 (60%) found the information received at birth satisfactory; 124 (91%) found the information provided in the CF-centre satisfactory. Most parents (n = 98, 78%) felt troubled or anxious when the CF-centre called, 51 (38%) remained anxious after the visit. Most parents (n = 122; 88%) were satisfied with the screening, 4 (3%) were not, and 12 (9%) were unsure. Conclusions: The smooth organisation of the screening process, with personal information by a CF specialist and short delays between this information and the final diagnostic testing, might have contributed to reduce anxiety among parents. Most families were grateful that their child had been screened, and are happy with the process.

A specific racemase for α‐methylacyl‐CoAs, which had previously been studied in rat liver [W. Schmitz, R. Fingerhut, E. Conzelmann (1994) Eur. J. Biochem. 222, 313–323], has now been demonstrated also in human tissues. The human enzyme cross‐reacts with a polyclonal antiserum against the rat liver racemase. The racemase was purified from human liver some 3600‐fold. It is a monomer of 47 kDa with an isolectric point of pH 6.1 and is optimally active between pH 7–8. It acts only on coenzyme A thioesters, not on free fatty acids, and accepts as substrates a wide range of α‐methylacyl‐CoAs, including pristanoyl‐CoA and trihydroxycoprostanoyl‐CoA (an intermediate in bile acid synthesis), but neither 3‐methyl‐branched nor linear‐chain acyl‐CoAs. A clear difference in subcellular localization of the enzyme was found between humans and rats: the rat enzyme co‐distributed exclusively with mitochondrial marker enzymes whereas in human cells, only 10–30% of the activity was found in mitochondria, the bulk activity was located in peroxisomes. Cells from patients with general deficiency of peroxisome assembly (Zellweger syndrome) showed strongly reduced racemase activity, with only the mitochondrial share being present while the peroxisomal form was absent.

Summary Sickle Cell Disease (SCD) is an increasing global health problem and presents significant challenges to European health care systems. Newborn screening (NBS) for SCD enables early initiation of preventive measures and has contributed to a reduction in childhood mortality from SCD. Policies and methodologies for NBS vary in different countries, and this might have consequences for the quality of care and clinical outcomes for SCD across Europe. A two‐day Pan‐European consensus conference was held in Berlin in April 2017 in order to appraise the current status of NBS for SCD and to develop consensus‐based statements on indications and methodology for NBS for SCD in Europe. More than 50 SCD experts from 13 European countries participated in the conference. This paper aims to summarise the discussions and present consensus recommendations which can be used to support the development of NBS programmes in European countries where they do not yet exist, and to review existing programmes.