@article{BreyerGruenerKleinetal.2024, author = {Breyer, Maximilian and Gr{\"u}ner, Julia and Klein, Alexandra and Finke, Laura and Klug, Katharina and Sauer, Markus and {\"U}{\c{c}}eyler, Nurcan}, title = {\(In\) \(vitro\) characterization of cells derived from a patient with the GLA variant c.376A>G (p.S126G) highlights a non-pathogenic role in Fabry disease}, series = {Molecular Genetics and Metabolism Reports}, volume = {38}, journal = {Molecular Genetics and Metabolism Reports}, issn = {22144269}, doi = {10.1016/j.ymgmr.2023.101029}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350295}, year = {2024}, abstract = {Highlights • The GLA variant S126G is not associated with Fabry symptoms in the presented case • S126G has no effect on α-GAL A activity or Gb3 levels in this patient • S126G sensory neurons show no electrophysiological abnormalities Abstract Fabry disease (FD) is a life-limiting disorder characterized by intracellular globotriaosylceramide (Gb3) accumulations. The underlying α-galactosidase A (α-GAL A) deficiency is caused by variants in the gene GLA. Variants of unknown significance (VUS) are frequently found in GLA and challenge clinical management. Here, we investigated a 49-year old man with cryptogenic lacunar cerebral stroke and the chance finding of the VUS S126G, who was sent to our center for diagnosis and initiation of a costly and life-long FD-specific treatment. We combined clinical examination with in vitro investigations of dermal fibroblasts (HDF), induced pluripotent stem cells (iPSC), and iPSC-derived sensory neurons. We analyzed α-GAL A activity in iPSC, Gb3 accumulation in all three cell types, and action potential firing in sensory neurons. Neurological examination and small nerve fiber assessment was normal except for reduced distal skin innervation. S126G iPSC showed normal α-GAL A activity compared to controls and no Gb3 deposits were found in all three cell types. Baseline electrophysiological characteristics of S126G neurons showed no difference compared to healthy controls as investigated by patch-clamp recordings. We pioneer multi-level cellular characterization of the VUS S126G using three cell types derived from a patient and provide further evidence for the benign nature of S126G in GLA, which is of great importance in the management of such cases in clinical practice.}, language = {en} } @phdthesis{Spitzel2023, author = {Spitzel, Marlene}, title = {The impact of inflammation, hypoxia, and vasculopathy on pain development in the α-galactosidase A mouse model of Morbus Fabry}, doi = {10.25972/OPUS-34579}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-345794}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {Fabry disease (FD), an X-linked lysosomal storage disorder, is caused by variants in the gene α-galactosidase A (GLA). As a consequence, the encoded homonymous enzyme GLA is not produced in sufficient amount or does not function properly. Subsequently, globotriaosylceradmide (Gb3), the target substrate of GLA, starts accumulating in several cell types, especially neurons and endothelial cells. FD patients suffer from multiorgan symptoms including cardiomyopathy, nephropathy, stroke, and acral burning pain. It is suggested that the impact of pathological Gb3 accumulation, inflammatory and hypoxic processes, and vasculopathy are contributing to the specific FD pain phenotype. Thus, we investigated the role of inflammation, hypoxia, and vasculopathy on molecular level in dorsal root ganglia (DRG) of the GLA knockout (KO) mouse model. Further, we investigated pain-like characteristics of GLA KO mice at baseline (BS), after capsaicin administration, and after repeated enzyme replacement therapy (ERT) administration for a period of 1.5 years. Acquired data showed disturbances in immune response markers represented by downregulated inflammation-associated genes and lower numbers of CD206+ macrophages in DRG of GLA KO mice. Hypoxic mechanisms were active in DRG of GLA KO mice reflected by increased gene expression of hypoxia- and DNA damage-associated targets, higher numbers of hypoxia-inducible factor 1α-positive (HIF1α+) and carbonic anhydrase 9-positive (CA9+) neurons in DRG of GLA KO mice, and DRG neuronal HIF1α cytosolic-nuclear translocation in GLA KO mice. Vascularization in DRG of GLA KO mice was reduced including lower numbers of blood vessel branches and reduced total blood vessel length. Pain-like behavior of the GLA KO mouse model revealed no mechanical hypersensitivity at BS but age-dependent heat hyposensitivity, which developed also age-matched wild type (WT) mice. Capsaicin administration under isoflurane anesthesia did not elicit the development of nocifensive behavior in GLA KO mice after mechanical or heat stimulation. Repeated ERT administration did not show a clear effect in GLA KO mice in terms of restored heat hyposensitivity to BS paw withdrawal latencies. In summary, we demonstrated the impact of disturbed immune response markers, active hypoxic mechanisms, and reduced vascularization on molecular FD pathophysiology.}, subject = {Fabry-Krankheit}, language = {en} } @phdthesis{Hochheimer2022, author = {Hochheimer, Vanessa Christine}, title = {Of cells and enzymes: How dermal fibroblasts can impact pain in Fabry Disease and Why looking at the 3D-structure of α-Galactosidase A may be worthwhile for clinical management of Fabry patients}, doi = {10.25972/OPUS-29660}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-296607}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Fabry Disease (FD) is a genetic lysosomal storage disorder based on mutations in the gene encoding α-Galactosidase A (α-GalA) leading to accumulation of globotriaosylceramide (Gb3). Missense mutations induce an amino acid exchange (AAE) in the α-GalA. Pain is a predominant symptom in FD and the pathophysiology is unclear. Skin punch biopsies were obtained from 40 adult FD patients and ten healthy controls and dermal fibroblast cultures were generated for cell culture experiments to investigate Gb3 load, gene and protein expression patterns and ion channel activity. The 3D-structure of α-GalA was downloaded into Pymol Graphics System and the AAE was depicted and located in order to investigate the correlation between the AAE location type in the α-GalA and the clinical FD phenotype. FD dermal fibroblasts showed high Gb3 load depending on treatment interval and expressed Kca1.1 channels. Activity was reduced in FD cells at baseline, but increased over-proportionately upon Gb3-cleavage by enzyme replacement therapy. Gene and protein expression of Kca1.1 was increased in FD cells. FD dermal fibroblasts showed higher gene expression of Notch1 and several cytokines. Further, it was shown that three different AAE location types can be differentiated: mutations in the active site ('active site'), those buried in the core of α-GalA ('buried') and those at another location, mostly on the protein surface ('other'). FD patients carrying active site or buried mutations showed a severe clinical phenotype with multi-organ manifestation and early disease onset. Patients with other mutations were less severely affected with oligo-organ manifestation sparing the nervous system and later disease onset. These results show that dermal fibroblasts may be involved in FD-associated pain and that stratification of FD patients carrying missense mutations by AAE location type may be an advantageous parameter that can help in the management of FD patients.}, subject = {Fabry-Krankheit}, language = {en} } @article{WagenhaeuserRickertSommeretal.2022, author = {Wagenh{\"a}user, Laura and Rickert, Vanessa and Sommer, Claudia and Wanner, Christoph and Nordbeck, Peter and Rost, Simone and {\"U}{\c{c}}eyler, Nurcan}, title = {X-chromosomal inactivation patterns in women with Fabry disease}, series = {Molecular Genetics \& Genomic Medicine}, volume = {10}, journal = {Molecular Genetics \& Genomic Medicine}, number = {9}, doi = {10.1002/mgg3.2029}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-312795}, year = {2022}, abstract = {Background Although Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA), women may develop severe symptoms. We investigated X-chromosomal inactivation patterns (XCI) as a potential determinant of symptom severity in FD women. Patients and Methods We included 95 women with mutations in GLA (n = 18 with variants of unknown pathogenicity) and 50 related men, and collected mouth epithelial cells, venous blood, and skin fibroblasts for XCI analysis using the methylation status of the androgen receptor gene. The mutated X-chromosome was identified by comparison of samples from relatives. Patients underwent genotype categorization and deep clinical phenotyping of symptom severity. Results 43/95 (45\%) women carried mutations categorized as classic. The XCI pattern was skewed (i.e., ≥75:25\% distribution) in 6/87 (7\%) mouth epithelial cell samples, 31/88 (35\%) blood samples, and 9/27 (33\%) skin fibroblast samples. Clinical phenotype, α-galactosidase A (GAL) activity, and lyso-Gb3 levels did not show intergroup differences when stratified for X-chromosomal skewing and activity status of the mutated X-chromosome. Conclusions X-inactivation patterns alone do not reliably reflect the clinical phenotype of women with FD when investigated in biomaterial not directly affected by FD. However, while XCI patterns may vary between tissues, blood frequently shows skewing of XCI patterns.}, language = {en} } @article{SpitzelWagnerBreyeretal.2022, author = {Spitzel, Marlene and Wagner, Elise and Breyer, Maximilian and Henniger, Dorothea and Bayin, Mehtap and Hofmann, Lukas and Mauceri, Daniela and Sommer, Claudia and {\"U}{\c{c}}eyler, Nurcan}, title = {Dysregulation of immune response mediators and pain-related ion channels is associated with pain-like behavior in the GLA KO mouse model of Fabry disease}, series = {Cells}, volume = {11}, journal = {Cells}, number = {11}, issn = {2073-4409}, doi = {10.3390/cells11111730}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-275186}, year = {2022}, abstract = {Fabry disease (FD) is a rare life-threatening disorder caused by deficiency of the alpha-galactosidase A (GLA) enzyme with a characteristic pain phenotype. Impaired GLA production or function leads to the accumulation of the cell membrane compound globotriaosylceramide (Gb3) in the neurons of the dorsal root ganglia (DRG) of FD patients. Applying immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT PCR) analysis on DRG tissue of the GLA knockout (KO) mouse model of FD, we address the question of how Gb3 accumulation may contribute to FD pain and focus on the immune system and pain-associated ion channel gene expression. We show a higher Gb3 load in the DRG of young (<6 months) (p < 0.01) and old (≥12 months) (p < 0.001) GLA KO mice compared to old wildtype (WT) littermates, and an overall suppressed immune response in the DRG of old GLA KO mice, represented by a reduced number of CD206\(^+\) macrophages (p < 0.01) and lower gene expression levels of the inflammation-associated targets interleukin(IL)1b (p < 0.05), IL10 (p < 0.001), glial fibrillary acidic protein (GFAP) (p < 0.05), and leucine rich alpha-2-glycoprotein 1 (LRG1) (p < 0.01) in the DRG of old GLA KO mice compared to old WT. Dysregulation of immune-related genes may be linked to lower gene expression levels of the pain-associated ion channels calcium-activated potassium channel 3.1 (KCa3.1) and transient receptor potential ankyrin 1 channel (TRPA1). Ion channel expression might further be disturbed by impaired sphingolipid recruitment mediated via the lipid raft marker flotillin-1 (FLOT1). This impairment is represented by an increased number of FLOT1\(^+\) DRG neurons with a membranous expression pattern in old GLA KO mice compared to young GLA KO, young WT, and old WT mice (p < 0.001 each). Further, we provide evidence for aberrant behavior of GLA KO mice, which might be linked to dysregulated ion channel gene expression levels and disturbed FLOT1 distribution patterns. Behavioral testing revealed mechanical hypersensitivity in young (p < 0.01) and old (p < 0.001) GLA KO mice compared to WT, heat hypersensitivity in young GLA KO mice (p < 0.001) compared to WT, age-dependent heat hyposensitivity in old GLA KO mice (p < 0.001) compared to young GLA KO mice, and cold hyposensitivity in young (p < 0.001) and old (p < 0.001) GLA KO mice compared to WT, which well reflects the clinical phenotype observed in FD patients.}, language = {en} } @article{LauUeceylerCairnsetal.2022, author = {Lau, Kolja and {\"U}{\c{c}}eyler, Nurcan and Cairns, Tereza and Lorenz, Lora and Sommer, Claudia and Schindeh{\"u}tte, Magnus and Amann, Kerstin and Wanner, Christoph and Nordbeck, Peter}, title = {Gene variants of unknown significance in Fabry disease: Clinical characteristics of c.376AG (p.Ser126Gly)}, series = {Molecular Genetics \& Genomic Medicine}, volume = {10}, journal = {Molecular Genetics \& Genomic Medicine}, number = {5}, doi = {10.1002/mgg3.1912}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-312817}, year = {2022}, abstract = {Background Anderson-Fabry disease (FD) is an X-linked lysosomal storage disorder with varying organ involvement and symptoms, depending on the underlying mutation in the alpha-galactosidase A gene (HGNC: GLA). With genetic testing becoming more readily available, it is crucial to precisely evaluate pathogenicity of each genetic variant, in order to determine whether there is or might be not a need for FD-specific therapy in affected patients and relatives at the time point of presentation or in the future. Methods This case series investigates the clinical impact of the specific GLA gene variant c.376A>G (p.Ser126Gly) in five (one heterozygous and one homozygous female, three males) individuals from different families, who visited our center between 2009 and 2021. Comprehensive neurological, nephrological and cardiac examinations were performed in all cases. One patient received a follow-up examination after 12 years. Results Index events leading to suspicion of FD were mainly unspecific neurological symptoms. However, FD-specific biomarkers, imaging examinations (i.e., brain MRI, heart MRI), and tissue-specific diagnostics, including kidney and skin biopsies, did not reveal evidence for FD-specific symptoms or organ involvement but showed normal results in all cases. This includes findings from 12-year follow-up in one patient with renal biopsy. Conclusion These findings suggest that p.Ser126Gly represents a benign GLA gene variant which per se does not cause FD. Precise clinical evaluation in individuals diagnosed with genetic variations of unknown significance should be performed to distinguish common symptoms broadly prevalent in the general population from those secondary to FD.}, language = {en} } @article{vanderVeenVlietstravanDussenetal.2020, author = {van der Veen, Sanne J. and Vlietstra, Wytze J. and van Dussen, Laura and van Kuilenburg, Andr{\´e} B.P. and Dijkgraaf, Marcel G. W. and Lenders, Malte and Brand, Eva and Wanner, Christoph and Hughes, Derralynn and Elliott, Perry M. and Hollak, Carla E. M. and Langeveld, Mirjam}, title = {Predicting the development of anti-drug antibodies against recombinant alpha-galactosidase A in male patients with classical Fabry disease}, series = {International Journal of Molecular Sciences}, volume = {21}, journal = {International Journal of Molecular Sciences}, number = {16}, issn = {1422-0067}, doi = {10.3390/ijms21165784}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285687}, year = {2020}, abstract = {Fabry Disease (FD) is a rare, X-linked, lysosomal storage disease that mainly causes renal, cardiac and cerebral complications. Enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A is available, but approximately 50\% of male patients with classical FD develop inhibiting anti-drug antibodies (iADAs) that lead to reduced biochemical responses and an accelerated loss of renal function. Once immunization has occurred, iADAs tend to persist and tolerization is hard to achieve. Here we developed a pre-treatment prediction model for iADA development in FD using existing data from 120 classical male FD patients from three European centers, treated with ERT. We found that nonsense and frameshift mutations in the α-galactosidase A gene (p = 0.05), higher plasma lysoGb3 at baseline (p < 0.001) and agalsidase beta as first treatment (p = 0.006) were significantly associated with iADA development. Prediction performance of a Random Forest model, using multiple variables (AUC-ROC: 0.77) was compared to a logistic regression (LR) model using the three significantly associated variables (AUC-ROC: 0.77). The LR model can be used to determine iADA risk in individual FD patients prior to treatment initiation. This helps to determine in which patients adjusted treatment and/or immunomodulatory regimes may be considered to minimize iADA development risk.}, language = {en} } @article{JovanovicKlassenHeuschmannetal.2020, author = {Jovanovic, Ana and Klassen, Philipp and Heuschmann, Peter and Sommer, Claudia and Roberts, Mark and {\"U}{\c{c}}eyler, Nurcan}, title = {English version of the self-administered Fabry Pain Questionnaire for adult patients}, series = {Orphanet Journal of Rare Diseases}, volume = {15}, journal = {Orphanet Journal of Rare Diseases}, doi = {10.1186/s13023-020-01580-9}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230298}, year = {2020}, abstract = {Background Pain is an early symptom of Fabry disease (FD) and is characterized by a unique phenotype with mainly episodic acral and triggerable burning pain. Recently, we designed and validated the first pain questionnaire for adult FD patients in an interview and a self-administered version in German: the Wurzburg Fabry Pain Questionnaire (FPQ). We now report the validation of the English version of the self-administered FPQ (enFPQ). Methods After two forward-backward translations of the FPQ by native German and native English speakers, the enFPQ was applied at The Mark Holland Metabolic Unit, Manchester, UK for validation. Consecutive patients with genetically ascertained FD and current or previous FD pain underwent a face-to-face interview using the enFPQ. Two weeks later, patients filled in the self-administered enFPQ at home. The agreement between entries collected by supervised administration and self-administration of the enFPQ was assessed via Gwet's AC1-statistics (AC1) for nominal-scaled scores and intraclass correlation coefficient (ICC) for interval-scaled elements. Results Eighty-three FD patients underwent the face-to-face interview and 54 patients sent back a completed self-administered version of the enFPQ 2 weeks later. We found high agreement with a mean AC1-statistics of 0.725 for 55 items, and very high agreement with a mean ICC of 0.811 for 9 items. Conclusions We provide the validated English version of the FPQ for self-administration in adult FD patients. The enFPQ collects detailed information on the individual FD pain phenotype and thus builds a solid basis for better pain classification and treatment in patients with FD.}, language = {en} } @article{WannerFeldtRasmussenJovanovicetal.2020, author = {Wanner, Christoph and Feldt-Rasmussen, Ulla and Jovanovic, Ana and Linhart, Aleš and Yang, Meng and Ponce, Elvira and Brand, Eva and Germain, Dominique P. and Hughes, Derralynn A. and Jefferies, John L. and Martins, Anna Maria and Nowak, Albina and Vujkovac, Bojan and Weidemann, Frank and West, Michael L. and Ortiz, Alberto}, title = {Cardiomyopathy and kidney function in agalsidase beta-treated female Fabry patients: a pre-treatment vs. post-treatment analysis}, series = {ESC Heart Failure}, volume = {7}, journal = {ESC Heart Failure}, number = {3}, doi = {10.1002/ehf2.12647}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-235963}, pages = {825-834}, year = {2020}, abstract = {Long-term treatment effect studies in large female Fabry patient groups are challenging to design because of phenotype heterogeneity and lack of appropriate comparison groups, and have not been reported. We compared long-term cardiomyopathy and kidney function outcomes after agalsidase beta treatment with preceding treatment-naive outcomes. Methods and results Self-controlled pretreatment and post-treatment comparison (piecewise mixed linear modelling) included Fabry female patients ≥18 years at treatment initiation who received agalsidase beta (0.9-1.1 mg/kg every other week) for ≥2 years, with ≥2 pretreatment and ≥2 post-treatment outcome measurements during 10-year follow-up. Left ventricular posterior wall thickness (LVPWT)/interventricular septal thickness (IVST) and estimated glomerular filtration rate (eGFR, Chronic Kidney Disease Epidemiology Collaboration creatinine equation) analyses included 42 and 86 patients, respectively, aged 50.0 and 46.3 years at treatment initiation, respectively. LVPWT and IVST increased pretreatment (follow-up 3.5 years) but stabilized during 3.6 years of treatment (LVPWT: n = 38, slope difference [95\% confidence interval (CI)] = - 0.41 [ - 0.68, - 0.15] mm/year, P\(_{pre-post difference}\)<0.01; IVST: n = 38, slope difference =-0.32 [-0.67, 0.02] mm/year, P\(_{pre-post difference}\) = 0.07). These findings were not modified by renal involvement or antiproteinuric agent use. Compared with the treatment-naive period (follow-up 3.6 years), eGFR decline remained modest and stabilized within normal ranges during 4.1 years of treatment (slope difference, 95\% CI: -0.13 [-1.15, 0.89] mL/min/1.73m\(^2\)/year, P\(_{pre-post difference}\) = 0.80). Conclusions Cardiac hypertrophy, progressing during pretreatment follow-up, appeared to stabilize during sustained agalsidase beta treatment. eGFR decline remained within normal ranges. This suggests that treatment may prevent further Fabry-related progression of cardiomyopathy in female patients and maintain normal kidney function.}, language = {en} } @article{RickertWagenhaeuserNordbecketal.2020, author = {Rickert, V. and Wagenh{\"a}user, L. and Nordbeck, P. and Wanner, C. and Sommer, C. and Rost, S. and {\"U}{\c{c}}eyler, N.}, title = {Stratification of Fabry mutations in clinical practice: a closer look at α-galactosidase A-3D structure}, series = {Journal of Internal Medicine}, volume = {288}, journal = {Journal of Internal Medicine}, number = {5}, doi = {10.1111/joim.13125}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-218125}, pages = {593 -- 604}, year = {2020}, abstract = {Background Fabry disease (FD) is an X-linked lysosomal storage and multi-system disorder due to mutations in the α-galactosidase A (α-GalA) gene. We investigated the impact of individual amino acid exchanges in the α-GalA 3D-structure on the clinical phenotype of FD patients. Patients and methods We enrolled 80 adult FD patients with α-GalA missense mutations and stratified them into three groups based on the amino acid exchange location in the α-GalA 3D-structure: patients with active site mutations, buried mutations and other mutations. Patient subgroups were deep phenotyped for clinical and laboratory parameters and FD-specific treatment. Results Patients with active site or buried mutations showed a severe phenotype with multi-organ involvement and early disease manifestation. Patients with other mutations had a milder phenotype with less organ impairment and later disease onset. α-GalA activity was lower in patients with active site or buried mutations than in those with other mutations (P < 0.01 in men; P < 0.05 in women) whilst lyso-Gb3 levels were higher (P < 0.01 in men; <0.05 in women). Conclusions The type of amino acid exchange location in the α-GalA 3D-structure determines disease severity and temporal course of symptom onset. Patient stratification using this parameter may become a useful tool in the management of FD patients.}, language = {en} }