Simona Murko

Background: Alpha-mannosidosis (AM) is a debilitating lysosomal storage disorder (LSD) caused by insufficient activity of alpha-mannosidase, an enzyme responsible for cleavage of alpha-linked mannose residues from N-glycan-type oligosaccharides during their degradation. As a result, specific oligosaccharides accumulate in tissues and are also detectable in body fluids. AM is a progressive multi-organ disease for which hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) are therapeutic options, however, treatment results depend considerably on early diagnosis. Therefore, inclusion of AM into newborn screening panels would significantly contribute to improving therapeutic outcome. Methods: A rapid and fully quantitative method for the specific AM oligosaccharide biomarker Man ₂ GlcNAc in dried blood spots (DBS) using ultra-performance liquid chromatography coupled to tandem-mass spectrometry was developed. DBS from 420 controls, 27 AM patients (including five newborn screening samples), and 35 patients with other LSDs were analyzed. Results: A distinct separation in biomarker concentrations was observed between unaffected individuals and untreated AM patients (0.1 – 0.8 vs 3.2 – 11.3 µmol Man ₂ GlcNAc/L, p 2 GlcNAc concentrations significantly differed between untreated patients and those on ERT ( n = 17, p n = 15, p p vs 0.1 – 0.8 µmol Man ₂ GlcNAc/L, p =0.0001). Conclusion: We present a simple and rapid method for quantitative analysis of the specific AM oligosaccharide biomarker Man ₂ GlcNAc, which is remarkably stable compared to enzymatic assays. Thus, the method is useful for selective screening in suspected cases as well as for monitoring the response to different forms of treatment. Furthermore, Man ₂ GlcNAc has the potential to serve as a useful biomarker for newborn screening offering the prospect of earlier treatment initiation, improved therapeutic outcome and higher quality of life for AM patients.

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.

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.

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.