This article is available online at http://www.jlr.org Niemann-Pick type C (NPC; MIM#257220) is a progressive neurodegenerative disease caused by a disorder in the intracellular traffi cking of cholesterol that leads to a lysosomal/endosomal accumulation of unesterifi ed cholesterol and glycolipids in many tissues ( 1-3 ). The detection of free cholesterol accumulation by fi lipin staining in fibroblasts has been for many years the gold standard methodology for the biochemical diagnosis of the disease. This method has good sensitivity and specifi city. However, juvenile or adult onset forms sometimes present interpretation diffi culties ( 4 ). In addition, the method involves an invasive skin biopsy and the culture of fi broblasts that requires several weeks for the cellular growth, which delays the diagnosis. Some studies in cellular and animal models showed a correlation between lipid accumulation and cellular oxidative stress that produces an increase of reactive oxygen species and lipid peroxidation ( 5-8 ). In the NPC murine model, an increase of two oxysterols, cholestane-3 b ,5 a ,6 b -triol (CT) and 7-ketocholesterol (7-KC), has been observed ( 8 ). This evidence was later confi rmed in NPC patients (8)(9)(10)(11)(12)(13), showing a correlation between the oxysterol profi le and the age of onset and severity of the disease ( 8,14 ). Moreover, CT specifi cally has been found to be increased in NPC disease compared with other lysosomal and neurodegenerative diseases ( 8 ). These results Abstract Niemann-Pick type C (NPC) is a progressive neurodegenerative disease characterized by lysosomal/endosomal accumulation of unesterifi ed cholesterol and glycolipids. Recent studies have shown that plasma cholestane-3 ␤ ,5 ␣ ,6 ␤ -triol (CT) and 7-ketocholesterol (7-KC) could be potential biomarkers for the diagnosis of NPC patients. We aimed to know the sensitivity and specifi city of these biomarkers for the diagnosis of NPC compared with other diseases that can potentially lead to oxysterol alterations. We studied 107 controls and 122 pa- 26,26,26,27,27, ; AUC, area under receiver-operating characteristic curve; BHT, butylhydroxytoluene; CI, confi dence interval; CT, cholestane-3 b ,5 a ,6 b -triol; 5 a ,26,26,26,27,27, ; CTX, cerebrotendinous xanthomatosis; % CV, percentage coeffi cient of variation; LAL, lysosomal acid lipase; LOD, limit of detection; LOQ, limit of quantifi cation; LSD, lysosomal storage disease; MRM, multiple reaction monitoring; NPC, Niemann-Pick type C; ROC, receiver-operating characteristic; SLO, Smith-Lemli-Opitz.

Morquio A (Mucopolysaccharidosis IVA; MPS IVA) is an autosomal recessive lysosomal storage disorder caused by partial or total deficiency of the enzyme galactosamine-6-sulfate sulfatase (GALNS; also known as N-acetylgalactosamine-6-sulfate sulfatase) encoded by the GALNS gene. Patients who inherit two mutated GALNS gene alleles produce protein with decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing GAG accumulation within lysosomes and consequently pleiotropic disease. GALNS mutations occur throughout the gene and many mutations are identified only in single patients or families, causing difficulties both in mutation detection and interpretation. In this study, molecular analysis of 163 patients with Morquio A identified 99 unique mutations in the GALNS gene believed to negatively impact GALNS protein function, of which 39 are previously unpublished, together with 26 single-nucleotide polymorphisms. Recommendations for the molecular testing of patients, clear reporting of sequence findings, and interpretation of sequencing data are provided.

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Background Alteration of vitamin B12 metabolism can be genetic or acquired, and can result in anemia, failure to thrive, developmental regression and even irreversible neurologic damage. Therefore, early diagnosis and intervention is critical. Most of the neonatal cases with acquired vitamin B12 deficiency have been detected by clinical symptoms and only few of them trough NBS programs. We aim to assess the usefulness of the second-tier test: methylmalonic acid (MMA), methylcitric acid (MCA) and homocysteine (Hcys) in our newborn screening program and explore the implications on the detection of cobalamin (vitamin B12) related disorders, both genetic and acquired conditions. Methods A screening strategy using the usual primary markers followed by the analysis of MMA, MCA and Hcys as second tier-test in the first dried blood spot (DBS) was developed and evaluated. Results During the period 2015–2018 a total of 258,637 newborns were screened resulting in 130 newborns with acquired vitamin B12 deficiency (incidence 1:1989), 19 with genetic disorders (incidence 1:13,613) and 13 were false positive. No false negatives were notified. Concerning the second-tier test, the percentage of cases with MMA above the cut-off levels, both for genetic and acquired conditions was very similar (58% and 60%, respectively). Interestingly, the percentage of cases with increased levels of Hcys was higher in acquired conditions than in genetic disorders (87% and 47%, respectively). In contrast, MCA was high only in 5% of the acquired conditions versus in 53% of the genetic disorders, and it was always very high in all patients with propionic acidemia. Conclusions When screening for methylmalonic acidemia and homocystinuria, differential diagnosis with acquired vitamin B12 deficiency should be done. The results of our strategy support the inclusion of this acquired condition in the NBS programs, as it is easily detectable and allows the adoption of corrective measures to avoid the consequences of its deficiency.

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Short-chain enoyl-CoA hydratase (ECHS1) is a mitochondrial beta-oxidation enzyme involved in the metabolism of acyl-CoA fatty acid esters, as well as in valine metabolism. ECHS1 deficiency has multiple manifestations, including Leigh syndrome early at birth or in childhood with poor prognosis, to cutis laxa, exercise-induced dystonia and congenital lactic acidosis.Here we describe the case of a newborn with mutations in ECHS1 that caught our attention after the incidental finding of 3-hydroxy-butyryl\3-hydroxy-isobutyryl\malonylcarnitine (C4OH\C3DC) and tiglylcarnitine (C5:1) on blood spot in the newborn screening (NBS) program. Diagnosis was suspected based on the analysis of organic acids on dried urine spot. A moderate increase of 2-methyl-2,3-dihydroxybutyric acid, was detected, which is a known marker of this disease. Exome analysis showed c.404A>G (p.Asn135Ser) mutation in homozygosis in the ECHS1 gene. The child was therefore admitted to the hospital. Initial examination showed little response to auditory stimuli and mild hypertonia of the extremities. Clinical deterioration was evident at 4 months of age, including neurological and cardiac involvement, and the patient died at 5 months of age. This case illustrates how an incidental detection in the NBS Program can lead to the diagnosis ECHS1 deficiency. Although it is a severe disease, with no treatment available, early detection would allow adequate genetic counseling avoiding the odyssey that suffered most of these families.

There are few causes of treatable neurodevelopmental diseases described to date. Branched Chain Ketoacid Dehydrogenase Kinase (BCKDK) deficiency causes branched-chain amino acid (BCAA) depletion and is linked to a neurodevelopmental disorder characterized by autism, intellectual disability, and microcephaly. We report the largest cohort of patients studied, broadening the phenotypic and genotypic spectrum. Moreover, this is the first study to present newborn screening findings and mid-term clinical outcome. In this cross-sectional study, patients with a diagnosis of BCKDK deficiency were recruited via investigators’ practices through a MetabERN initiative. Clinical, biochemical and genetic data were collected. Dried blood spot (DBS) newborn screening (NBS) amino acid profiles were retrieved from collaborating centers and compared to a healthy newborn reference population. Twenty-one patients with BCKDK mutations were included from 13 families. Patients were diagnosed between 8 months and 16 years (mean: 5.8 years, 43% female). At diagnosis, BCAA levels (leucine, valine, and isoleucine) were below reference values in plasma and in cerebrospinal fluid. All patients had global neurodevelopmental delay; 18/21 had gross motor function (GMF) impairment with GMF III or worse in 5/18, 16/16 intellectual disability, 17/17 language impairment, 12/17 autism spectrum disorder, 9/21 epilepsy, 12/15 clumsiness, 3/21 had sensorineural hearing loss and 4/20 feeding difficulties. No microcephaly was observed at birth, but 17/20 developed microcephaly during follow-up. Regression was reported in 6 patients. Movement disorder was observed in 3/21 patients: hyperkinetic movements (1), truncal ataxia (1) and dystonia (2). After treatment with high protein diet (≥ 2 g/kg/day) and BCAA supplementation (100-250 mg/kg/day), plasma BCAA increased significantly (p < 0.001), motor functions and head circumference stabilized/improved in 13/13 and in 11/15 patients, respectively. Amongst cases with follow-up data, none of the 3 patients starting treatment before 2 years of age developed autism at follow-up. The patient with the earliest age of treatment initiation (8 months) showed normal development at 3 years of age. NBS in DBS identified BCAA levels significantly lower than those of the normal population. This work highlights the potential benefits of dietetic treatment, in particular early introduction of BCAA. Therefore, it is of utmost importance to increase awareness about this treatable disease and consider it as a candidate for early detection by NBS programs.

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