Alpha-mannosidosis (AM, OMIM# 248500), an ultra-rare lysosomal storage disorder (LSD), is caused by insufficient activity of alpha-mannosidase, an enzyme involved in degradation of N-glycan oligosaccharides. Hence, specific oligosaccharides accumulate in tissues, resulting in a progressive multi-organ disease. Therapeutic options exist but treatment outcome depends on early diagnosis. Thus, multiple methods have been developed for analysis of these oligosaccharides in urine that are qualitative or semi-quantitative, limiting their use for treatment monitoring. A rapid quantitative method without derivatization was developed for AM biomarkers GlcNAc(Man)2, GlcNAc(Man)3, and GlcNAc(Man)4 in spot urine samples using ultra-performance liquid chromatography coupled to tandem-mass spectrometry. Urine samples of 208 controls, 20 AM patients, and 26 patients with other LSDs were analyzed. Method validation proved high recoveries (88%-108%) and precisions (standard deviation < 8%), low limits of quantification (0.12 μg GlcNAc(Man)2/mL), and high sample stability. We observed a clear separation between controls and AM patients (0.0-9.0 vs 39.4-99.3 μmol GlcNAc(Man)2/mmol creatinine, p < 0.0001). Furthermore, GlcNAc(Man)2 concentration showed significant differences between untreated patients and those treated with enzyme replacement therapy (ERT; n = 13, p = 0.0006) or hematopoietic stem cell transplantation (HSCT; n = 3, p = 0.0143), and between both treatments (p = 0.0143). Thus, the developed method is well-suited for selective screening for AM and offers a simple way to monitor treatment efficacy.

Together with its (3-subunit OSTM1, ClC-7 performs 2Cl-/H+ - /H + exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis and lysosomal storage. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology (hypo- pigmentation, organomegaly, and delayed myelination and development, " HOD syndrome"), " ), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P2 2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl-uptake - uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl-/H+-exchange, - /H +-exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fi broblasts probably due to residual PI(3,5)P2 2 sensitivity. The gain of function caused by the shifted voltage- dependence of either mutant likely is the main pathogenic factor. Loss of PI(3,5)P2 2 inhibition will further increase current amplitudes, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity.

Renal proximal tubulopathy in Fanconi-Bickel syndrome is caused by impaired basolateral glucose transport via GLUT2 and consequently, intracellular accumulation of glucose and glycogen. SGLT2 inhibitors act on apical glucose reabsorption of renal proximal tubular cells. The purpose of this study was to retrospectively describe the first experiences with repurposing the SGLT2 inhibitor empagliflozin to treat the generalized tubulopathy in Fanconi-Bickel syndrome. A case series was conducted of seven persons from five families (five males, two females; three children, who were 14y5m, 2y9m, and 1y6m old) with genetically confirmed Fanconi-Bickel syndrome, off-label treated with empagliflozin. Median (range) age at start of empagliflozin was 27 years (1y6m - 61y) and duration of follow-up under empagliflozin treatment was 169 days (57-344). Under empagliflozin (up to 25 mg/d), biochemical parameters of tubular cell integrity (urinary N-acetyl-glucosaminidase) and/or tubular functions (including urinary α1-microglobulin) improved in all persons with Fanconi-Bickel syndrome, albeit to varying degrees. Clinically, supplementations (i.e., phosphate, alkali, carnitine, and alfacalcidol) could be completely discontinued in the three children, whereas results in the four adult patients were more variable and not as significant. Empagliflozin was well-tolerated and no symptomatic hypoglycemia was observed. In conclusion, SGLT2 inhibitors such as empagliflozin shift the metabolic block in Fanconi-Bickel syndrome, that is, they intervene specifically in the underlying pathophysiology and can thus attenuate renal proximal tubulopathy, especially when started in early childhood.

OBJECTIVE: Maple syrup urine disease (MSUD), a life-threatening metabolic disorder, is included in newborn screening (NBS) programs worldwide. The study aims to evaluate the impact of NBS on the long-term outcome of MSUD patients. METHODS: We performed a prospective, national, multicenter, observational study. RESULTS: In the studied NBS cohort (N = 33; 22 classic MSUD [cMSUD], 11 variant MSUD [vMSUD]; median age at last visit 10.4 years), 32 (97%) patients survived, 58% of them had normal cognitive functions (median IQ 87). Initial peak leucine increased linearly with age in cMSUD (median: 1712 mu mol/L), but not in vMSUD. Global IQ correlated inversely with the initial peak leucine concentration (P = .04; beta = -0.0081) and the frequency of decompensations (P = .02; beta = -9.133). A cluster analysis identified 2 subgroups differing in their long-term metabolic control (median leucine concentration: 162 vs 278 mu mol/L; P < .001). In cMSUD, lower leucine concentrations were associated with a higher IQ (95.5 vs 80; P = .008). Liver transplantation (median age 5.8 years) was not associated with better cognitive outcome. NBS is highly sensitive for cMSUD, but vMSUD might be missed (N = 2 missed by NBS). CONCLUSIONS: NBS and the early start of treatment improve survival and long-term outcome in individuals with cMSUD. Disease severity is an important modifier of outcome; however, the time to NBS report and the quality of long-term metabolic control had an independent impact on cognitive outcome, highlighting the importance of an early diagnosis and the quality of treatment.

Newborn screening (NBS) for metachromatic leukodystrophy (MLD) is based on first-tier measurement of sulfatides in dried blood spots (DBS) followed by second-tier measurement of arylsulfatase A in the same DBS. This approach is very precise with 0 -1 false positives per similar to 30,000 newborns tested. Recent data reported here shows that the sulfatide molecular species with an alpha-hydroxyl, 16-carbon, mono-unsaturated fatty acyl group (16:1-OHsulfatide) is superior to the original biomarker 16:0-sulfatide in reducing the number of first-tier false positives. This result is consistent across 4 MLD NBS centers. By measuring 16:1-OH-sulfatide alone or together with 16:0sulfatide, the estimated false positive rate is 0.048% and is reduced essentially to zero with second-tier arylsulfatase A activity assay. The false negative rate is predicted to be extremely low based on the demonstration that 40 out of 40 newborn DBS from clinically-confirmed MLD patients are detected with these methods. The work shows that NBS for MLD is extremely precise and ready for deployment. Furthermore, it can be multiplexed with several other inborn errors of metabolism already tested in NBS centers worldwide.

Background: Congenital heart disease (CHD) is the most common birth defect worldwide. Depending on complexity of the CHD, around 50% of these children require cardiovascular surgery, which may involve removal of the thymus for a better access to the heart. Given the pivotal role of the thymus in shaping a functional adaptive immune response, thymectomy may have detrimental health consequences.

Background: Canavan disease is a devastating neurometabolic disorder caused by accumulation of N acetylaspartate in brain and body fluids due to genetic defects in the aspartoacylase gene ( ASPA ). New gene therapies are on the horizon but will require early presymptomatic diagnosis to be fully effective. Methods: We therefore developed a fast and highly sensitive liquid chromatography mass spectrometry (LC-MS/ MS)-based method for quantification of N -acetylaspartate in dried blood spots and established reference ranges for neonates and older controls. With this test, we investigated 45 samples of 25 Canavan patients including 8 with a neonatal sample. Results: Measuring N -acetylaspartate concentration in dried blood with this novel test, all Canavan patients (with variable severity) were well separated from the control group (median; range: 5.7; 1.6 -13.6 mu mol/L [ n = 45] vs 0.44; 0.24 -0.99 mu mol/L [ n = 59] ( p < 0.05)). There was also no overlap when comparing neonatal samples of Canavan patients (7.3; 5.1 -9.9 mu mol/L [ n = 8]) and neonatal controls (0.93; 0.4 -1.8 mu mol/L [ n = 784]) (p < 0.05). Conclusions: We have developed a new LC-MS/MS-based screening test for early postnatal diagnosis of Canavan disease that should be further evaluated in a population-based study once a promising treatment becomes available. The method meets the general requirements of newborn screening and should be appropriate for multiplexing with other screening approaches that combine chromatographic and mass spectrometry techniques.

We report on liver transplantation in two patients with GSD Ib on treatment with empagliflozin. The use of this SGLT2 inhibitor resulted in a marked decrease of 1,5-anhydroglucitol which has an important role in the development of neutropenia in this condition. As intended, this caused a significant rise of neutrophil numbers. Liver transplantation alone did not produce the desired effect and our observation argues for continuing SGLT2 inhibitor treatment after transplantation.

Abstract Newborn screening (NBS) for isovaleric acidemia (IVA) is performed by flow injection tandem mass spectrometry quantifying C5 carnitines (C5). Isovalerylcarnitine, however, is isomeric with pivaloylcarnitine which can be present in blood due to maternal use of pivaloylester‐containing antibiotics, available in Germany since late 2016. During a 36‐month period (January 19–December 21), all newborns screened in Hamburg with a C5 above cutoff (NeoGram®: 0.50 μmol/L or Neobase®2: 0.45 μmol/L) were included in the study. As a second‐tier test, a simple ultra performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) method was developed to differentiate the C5 isomers pivaloyl‐, 2‐methylbutyryl‐, isovaleryl‐, and valerylcarnitine. Out of 156 772 newborns tested, one turned out to have genetically proven IVA while 99 were false positive (C5: 0.5–8.2 μmol/L) due to the presence of pivaloylcarnitine. These cases have increased year by year and show local clusters. Retrospective analysis of another 39 cases from 287 206 neonates tested at the NBS center in Heidelberg with C5 elevation (0.9–10.6 μmol/L) but clinical and biochemical exclusion of IVA yielded evidence of pivaloylcarnitine in all cases. Inclusion of a second‐tier test into NBS significantly reduces the high and increasing false‐positive rate of IVA screening. This avoids further diagnostic steps, prevents unnecessary stress and anxiety of parents in a remarkably high number of cases. If Hamburg data of 2021 are extrapolated to all of Germany, one can assume around 800 (1‰) false‐positive cases in comparison to an average of two classic IVA cases per year. Unless licensing of pivaloylester‐containing drugs for use during pregnancy is reconsidered, a second‐tier test for C5 determination is indispensable.

Fanconi-Bickel syndrome (FBS) is a very rare but distinct clinical entity with the combined features of hepatic glycogen storage disease, generalized proximal renal tubular dysfunction with disproportionately severe glucosuria, and impaired galactose tolerance. Here, we report five cases (out of 93 diagnosed in our lab) with pathogenic variants on both GLUT2 (SLC2A2) alleles. They come from 3 families and presented with an exceptionally mild clinical course. This course was correlated to data from old and most recent expression and transport studies in Xenopus oocytes. GLUT2 genotype in patients 1 and 2 was p.[153_4delLI];[P417R] with the first variant exhibiting normal membrane expression and partially retained transport activity (5.8%) for 2-deoxyglucose. In patient 3, the very first GLUT2 variant ever detected (p.V197I) was found, but for the first time it was present in a patient in the homozygous state. This variant had also shown unaffected membrane expression and remarkable residual activity (8%). The genotype in patient 4, p.[153_4delLI];[(E440A)], again included the 2-amino-acid deletion with residual transporter function, and patient 5 is the first found to be homozygous for this variant. Our results provide further evidence for a genotype-phenotype correlation in patients with GLUT2 variants; non-functional variants result in the full picture of FBS while dysfunctional variants may result in milder presentations, even glucosuria only, without other typical signs of FBS.

CLN2 disease is a lysosomal storage disorder that belongs to the neuronal ceroid lipofuscinoses (NCL) and progressively leads to dementia, blindness and early death. It is caused by a deficiency of the lysosomal enzyme tripeptidyl peptidase 1 (TPP1). The disease has recently gained interest as an enzyme replacement therapy has become available. For this therapy to be effective, the diagnosis must be made early. We have investigated the diagnostic reliability of a test for TPP1 deficiency in dried blood specimens (DBS), previously developed by us, to detect CLN2 disease. During a 12-year period we have received 3882 DBS for testing TPP1 activity. To check for quality of samples, we measured activities of two additional lysosomal enzymes as controls, palmitoyl peptidase 1 and ß-galactosidase. Of all samples, 1.7% were excluded from the study because of subnormal activity of more than one enzyme. For all samples with subnormal TPP1 activity and good sample quality, we reviewed clinical information. Consequently, we obtained adequate clinical and molecular genetic data for 51 patients. All of those had doubtless evidence of CLN2 disease (including seven atypical patients) as shown by symptoms of a progressive brain disease and presence of known pathogenic CLN2variants. The sensitivity of the test could not be evaluated directly. However, as our NCL clinic is a major reference center for these disorders and we have never received feedback information that a patient with normal TPP1 activity in our DBS test was later diagnosed with CLN2 disease, this constitutes convincing circumstantial evidence of a high sensitivity. The TPP1 test on DBS as used in this study was shown to be a reliable, convenient and inexpensive tool for a first diagnostic step in a suspected case of CLN2 disease and, can be applied with minor modifications for mass throughput, like newborn screening programs.

Purpose: CLN2 disease is a genetic disorder caused by dysfunction of the lysosomal enzyme tripeptidyl peptidase 1 (TPP1) that belongs to the neuronal ceroid lipofuscinoses (NCL) and leads to epilepsy, dementia, and death in young persons. CLN2 disease has recently become treatable by enzyme replacement, which can only be effective when the disease is diagnosed early. We have investigated the reliability of a test for TPP1 deficiency in dried blood specimens (DBS) to detect CLN2 disease. Results: During a 12-year period we have received 3882 samples for testing TPP1. Quality of samples was checked by measuring two additional lysosomal enzyme activities. For 50 samples with subnormal TPP1 activity and good sample quality, we obtained adequate clinical and molecular genetic data. All 50 patients had doubtless evidence of CLN2 disease (including seven atypical patients) as shown by clinical findings and the presence of known pathogenic CLN2 variants. Our institution is a major reference center for NCL, and we have never received information that a patient with a normal DBS test was later diagnosed with CLN2 disease. Conclusions: We consider our TPP1 test on DBS to be a reliable, convenient and inexpensive tool for a first diagnostic step in suspected CLN2 disease.

Objectives: Precise quantification of amino acids (AAs) is mandatory for successful diagnosis and monitoring of patients with metabolic diseases. We compared ion-exchange chromatography (IEC) and liquid chromatography with tandem mass spectrometry (LC-MS/MS), the two methods most commonly used in clinical laboratories for the quantification of AAs in physiological samples. Design & methods: 123 apparently healthy children were selected for the study. The plasma samples for LC-MS/MS were prepared accordingly to the aTRAQ Kit for Physiological Fluids on Sciex 3200 Qtrap, for IEC according to the protocol from Pickering laboratories on the AA analyzer Pinnacle PCX. Results were interpreted using the Pearson correlation coefficient and the percent difference Bland-Altman test. Results: The Spearman correlation coefficients of the 14 AAs that we evaluated varied from 0.67 in Tau to 0.89 in Leu and Thr. The mean differences in measurements (IEC compared to LC-MS/MS) of 11 AAs complied with our acceptance criterion of < 15%, the differences of Ser and Tyr were higher (19.5% and -19.0%, respectively), and the measured concentrations of Cit were much lower in LC-MS/MS than IEC (31% difference). Conclusion: The two methods are sufficiently comparable for most AAs and the reference values for individual AAs did not have to be refined, with the exception of citrulline. For the monitoring of patients on therapy (e.g. patients with phenylketonuria), it is still advisable to always use the same analytical method for the quantification of AAs.

Caloric restriction (CR) promotes longevity and exerts anti-aging effects by increasing Sirtuin production and activation. Gastric inhibitory polypeptide (GIP), a gastrointestinal peptide hormone, exerts various effects on pancreatic β-cells and extra-pancreatic tissues. GIP promotes glucose-dependent augmentation of insulin secretion and uptake of nutrients into the adipose tissue.Gipr-/- and Gipr+/+ mice were used for lifespan analysis, behavior experiments and gene expression of adipose tissue and muscles. 3T3-L1 differentiated adipocytes were used for Sirt1 and Nampt expression followed by treatment with GIP and α-lipoic acid.We observed that GIP receptor-knockout (Gipr-/-) mice fed normal diet showed an extended lifespan, increased exploratory and decreased anxiety-based behaviors, which are characteristic behavioral changes under CR. Moreover, Gipr-/- mice showed increased Sirt1 and Nampt expression in the adipose tissue. GIP suppressed α-lipoic acid-induced Sirt1 expression and activity in differentiated adipocytes.Although maintenance of CR is difficult, food intake and muscle endurance of Gipr-/- mice were similar to those of wild-type mice. Inhibition of GIP signaling may be a novel strategy to extend the lifespan of diabetic patients.

The availability of three enzyme replacement therapy (ERT) drugs and two substrate reduction therapy (SRT) drugs to treat Gaucher disease provides an opportunity to tailor therapies to a patient's specific clinical concerns. However, there is a gap in the literature regarding individual drug effectiveness in treating particular symptoms and the potential benefits of combination treatment.This report details treatment of a patient with Gaucher disease type 1 whose main clinical concern was profound thrombocytopenia (around 20 × 109/L, normal range: 150–450 × 109/L) with several episodes of bleeding with minimal trauma and bruises. The patient was treated with ERT at doses up to 60 units/kg weekly, with no improvement in platelet levels for 6 years. Subsequently, the patient transitioned to SRT and platelet levels increased almost two fold within the first month, and have remained stable at safe levels (30–60 × 109/L) for almost 2.5 years at the time of publication.This report demonstrates a possible therapeutic benefit of SRT in individual patients who do not meet therapeutic goals in terms of thrombocytopenia after a considerable period on first-line ERT treatment. Oral administration of SRT also improved this patient's quality of life allowing discontinuation of weekly ERT infusions, which better accommodated her demanding career and busy lifestyle.

Background: Inborn errors of metabolism (IEM) are disorders with a block in the metabolic pathway caused by a genetic defect of a specific enzyme. Although each of these diseases is quite rare, as a group they account for a significant proportion of newborn and childhood morbidity and mortality. Early diagnosis is important to prevent complications or even death of the child. Selective screening is an important diagnostic tool for the diagnosis of IEM.Methods: In Slovenia, symptomatic patients with suspected IEM are referred to the University Children's Hospital Ljubljana. Techniques used for selective screening are gas chromatography-mass spectrometry, ion exchange chromatography- post-column derivatization, liquid chromatography- tandem mass spectrometry and isoelectric focusing. Fluorimetric method is used for enzyme activity measurement.Results: There are 168 patients with amino and organic acidemias, 5 patients with disorders in fatty acids metabolism, 1 patient with a congenital disorder of glycosylation, 42 patients with Fabry disease (of which 37 are adult) and 20 patients with Gaucher disease (of which 18 are adult) in the Slovenian Register for Rare Diseases.Conclusions: In Slovenia, management of patients with IEM is centralized at the University Children's Hospital, with the exception of adult patients with Fabry and Gaucher disease. The team work is well organized with close cooperation between the laboratory and pediatricians specialized in metabolic disorders. According to the known frequencies of IEM from the literature, we would expect more positive results than obtained. To evaluate these results, we are planning to perform a pilot study on expanded newborn screening.

Introduction. Newborn screening in whole Slovenia started in 1979 with screening for phenylketonuria (PKU). Congenital hypothyroidism (CH) was added into the programme in 1981. The aim of this study was to analyse the data of neonatal screening in Slovenia from 1993 to 2012 for PKU, and from 1991 to 2012 for CH.Methods. Blood samples were collected from the heels of newborns between the third and the fifth day after birth. Fluorometric method was used for screening for PKU, CH screening was done by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA).Results. From 1993 to 2012, from 385,831 newborns 57 were identified with PKU. 184 newborns out of 427,396 screened from 1991 to 2012, were confirmed for CH. Incidences of PKU and CH in the periods stated are 1:6769 and 1:2323, respectively.Conclusions. Successful implementation of newborn screening for PKU and CH has helped in preventing serious disabilities of the affected children. Adding screening for new metabolic diseases in the future would be beneficial.

Background Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare inherited mitochondrial fatty acid oxidation disorder associated with variations in the ACADS (Acyl-CoA dehydrogenase, C-2 to C-3 short chain) gene. SCADD has highly variable biochemical, genetic and clinical characteristics. Phenotypes vary from fatal metabolic decompensation to asymptomatic individuals. Subject and methods A Romani boy presented at 3 days after birth with hypoglycaemia, hypotonia and respiratory pauses with brief generalized seizures. Afterwards the failure to thrive and developmental delay were present. Organic acids analysis with gas chromatography-mass spectrometry (GS/MS) in urine and acylcarnitines analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in dried blood spot were measured. Deoxyribonucleic acid (DNA) was isolated from blood and polymerase chain reactions (PCRs) were performed for all exons. Sequence analysis of all exons and flanking intron sequences of ACADS gene was performed. Results Organic acids analysis revealed increased concentration of ethylmalonic acid. Acylcarnitines analysis showed increase of butyrylcarnitine, C4-carnitine. C4-carnitine was 3.5 times above the reference range (<0.68 µmol/L). Confirmation analysis for organic acids and acylcarnitine profile was performed on the second independent sample and showed the same pattern of increased metabolites. Sequence analysis revealed 3-bp deletion at position 310-312 in homozygous state (c.310_312delGAG). Mutation was previously described as pathogenic in heterozygous state, while it is in homozygous state in our patient. Conclusions In our case clinical features of a patient, biochemical parameters and genetic data were consistent and showed definitely SCAD deficiency.