Treatment switch in Fabry disease- a matter of dose?

BACKGROUND: Patients with Fabry disease (FD) on reduced dose of agalsidase-beta or after switch to agalsidase-alfa show a decline in chronic kidney disease epidemiology collaboration-based estimated glomerular filtration rate (eGFR) and a worsened plasma lyso-Gb3 decrease. Hence, the most effective dose is still a matter of debate. METHODS: In this prospective observational study, we assessed end-organ damage and clinical symptoms in 78 patients who had received agalsidase-beta (1.0 mg/kg) for >1 year, which were assigned to continue this treatment (agalsidase-beta, regular-dose group, n=17); received a reduced dose of agalsidase-beta and subsequent switch to agalsidase-alfa (0.2 mg/kg) or a direct switch to 0.2 mg/kg agalsidase-alfa (switch group, n=22); or were re-switched to agalsidase-beta after receiving agalsidase-alfa for 12 months (re-switch group, n=39) with a follow-up of 88±25 months. RESULTS: No differences for clinical events were observed for all groups. Patients within the re-switch group started with the worst eGFR values at baseline (p=0.0217). Overall, eGFR values remained stable in the regular-dose group (p=0.1052) and decreased significantly in the re-switch and switch groups (p<0.0001 and p=0.0052, respectively). However, in all groups males presented with an annual loss of eGFR by -2.9, -2.5 and -3.9 mL/min/1.73 m² (regular-dose, re-switch, switch groups, all p<0.05). In females, eGFR decreased significantly only in the re-switch group by -2.9 mL/min/1.73 m² per year (p<0.01). Lyso-Gb3 decreased in the re-switch group after a change back to agalsidase-beta (p<0.05). CONCLUSIONS: Our data suggest that a re-switch to high dosage of agalsidase results in a better biochemical response, but not in a significant renal amelioration especially in classical males.

Effect of high-intensity interval training in patients with type 1 diabetes on physical fitness and retinal microvascular perfusion determined by optical coherence tomography angiography.

OBJECTIVE: Physical activity may protect from ocular complications of diabetic retinopathy (DR). We investigated exercise training effects on the retinal microvasculature using optical coherence tomography angiography (OCTA) in type 1 diabetes (T1D). METHODS: Twenty T1D patients without clinical signs of DR performed four weeks of high-intensity interval training (HIIT). Cycle ergometry was used for determination of physical fitness. OCTA of the macula and optic nerve head was applied to analyze effects on the foveal avascular zone area, vessel density, vessel diameter index and fractal dimension of the superficial plexus, deep plexus and radial peripapillary capillaries. RESULTS: Large effects for improvement of physical fitness in terms of power output at the individual lactate threshold (+10.7 ± 11.3%, p < .001, ES = 0.95) and maximal power output (+8.2 ± 6.4%, p < .001, ES = 1.4) were detected. Participants presented a reduced increase in heart rate (HR) and lactate (LA) at given exercise intensities at follow-up (p ≤ .0176). Baseline OCTA revealed that HbA1c levels were associated with vessel density in the radial peripapillary capillary and the parafoveal superficial region (p ≤ .014). None of the analyzed microvascular parameters changed in response to the intervention. CONCLUSION: Despite favorable effects of HIIT on physical fitness of T1D patients, disease-specific training protocols may be needed to overcome potentially impaired retinal microvascular adaptations.

Soluble adenylyl cyclase (sAC) regulates calcium signaling in the vascular endothelium.

The vascular endothelium acts as a selective barrier between the bloodstream and extravascular tissues. Intracellular [Ca2+]i signaling is essential for vasoactive agonist-induced stimulation of endothelial cells (ECs), typically including Ca2+ release from the endoplasmic reticulum (ER). Although it is known that interactions of Ca2+ and cAMP as ubiquitous messengers are involved in this process, the individual contribution of cAMP-generating adenylyl cyclases (ACs), including the only soluble AC (sAC; ADCY10), remains less clear. Using life-cell microscopy and plate reader-based [Ca2+]i measurements, we found that human immortalized ECs, primary aortic and cardiac microvascular ECs, and primary vascular smooth muscle cells treated with sAC-specific inhibitor KH7 or anti-sAC-small interfering RNA did not show endogenous or exogenous ATP-induced [Ca2+]i elevation. Of note, a transmembrane AC (tmAC) inhibitor did not prevent ATP-induced [Ca2+]i elevation in ECs. Moreover, l-phenylephrine-dependent constriction of ex vivo mouse aortic ring segments was also reduced by KH7. Analysis of the inositol-1,4,5-trisphosphate (IP3) pathway revealed reduced IP3 receptor phosphorylation after KH7 application, which also prevented [Ca2+]i elevation induced by IP3 receptor agonist adenophostin A. Our results suggest that sAC rather than tmAC controls the agonist-induced ER-dependent Ca2+ response in ECs and may represent a treatment target in arterial hypertension and heart failure.-Mewes, M., Lenders, M., Stappers, F., Scharnetzki, D., Nedele, J., Fels, J., Wedlich-Söldner, R., Brand, S.-M., Schmitz, B., Brand, E. Soluble adenylyl cyclase (sAC) regulates calcium signaling in the vascular endothelium.

Neutralising anti-drug antibodies in Fabry disease can inhibit endothelial enzyme uptake and activity.

Fabry disease (FD) is a lysosomal storage disease, treatable by enzyme replacement therapy (ERT) that substitutes deficient α-galactosidase A (AGAL). The formation of neutralising anti-drug antibodies (ADA) inhibiting AGAL activity during infusion is associated with disease progression in affected male patients. In this study we analysed if ADAs also inhibit endothelial enzyme uptake as well as intracellular enzyme activity. Therefore, fluorescence-labelled AGAL in combination with ADA-positive sera from FD patients (n = 8) was used to analyse enzyme uptake in endothelial and FD-specific cells. Furthermore, immune adsorption and a comprehensive ADA epitope mapping were performed. Pre-incubation of AGAL with ADAs significantly inhibited intracellular enzyme activity, which was rescued by immune adsorption (both P < .01). ADAs from some patients also inhibited enzyme uptake. ADA epitope mapping identified an epitope at position 121 to 140 aa potentially responsible for uptake inhibition for these patients. Further analyses revealed the presence of stable AGAL/ADA-immune complexes at pH 4.5 and decreased intracellular enzyme activity in endothelial cells (P < .001). Finally, the pre-incubation of AGAL with ADAs resulted in a reduced depletion of intracellular globotriaosylceramide in patient-derived AGAL-deficient cells, demonstrating a direct negative impact of ADAs on intracellular clearance. Neutralising ADAs may not only inhibit infused AGAL activity, but according to their epitopes can also inhibit endothelial AGAL uptake. Indeed, internalised AGAL/ADA-complexes may not dissociate, underlining the importance of novel therapeutic approaches for ADA reduction and prevention to increase therapy efficiency in affected patients.

Mutation-specific Fabry disease patient-derived cell model to evaluate the amenability to chaperone therapy.

BACKGROUND: Patients with Fabry disease (FD) and amenable mutations can be treated with the chaperone migalastat to restore endogenous α-galactosidase A (AGAL) activity. However, certain amenable mutations do not respond biochemically in vivo as expected. Here, we aimed to establish a patient-specific and mutation-specific cell model to evaluate the amenability to chaperone therapy in FD. METHODS: Since current tests to determine amenability are limited to heterologous mutation expression in HEK293T cells with endogenous AGAL activity, we generated CRISPR/Cas9-mediated AGAL-deficient HEK293T cells as a basis for mutant overexpression. Furthermore, primary urinary cells from patients were isolated and immortalised as a patient-specific cell model system to evaluate the amenability to chaperone therapy. RESULTS: Under treatment (>13 months), carriers of p.N215S (n=6) showed a significant reduction of plasma lyso-Gb3 (p<0.05). Lyso-Gb3 levels in carriers of p.L294S increased (p<0.05) and two patients developed severe albuminuria. Both missense mutations were amenable in wild-type HEK293T cells (p<0.05), but presented different responses in CRISPR/Cas9-mediated AGAL knockouts and immortalised urinary cells. Chaperone incubation resulted in increased AGAL activity (p<0.0001) and intracellular globotriaosylceramide (Gb3) reduction (p<0.05) in immortalised p.N215S cells but not in p.L294S and IVS2+1 G>A cells. CONCLUSION: We conclude that repeated AGAL activity measurements in patients' white blood cells are mandatory to assess the in vivo amenability to migalastat. Plasma lyso-Gb3 might be an appropriate tool to measure the biochemical response to migalastat. Patients with low AGAL activities and increasing lyso-Gb3 levels despite in vitro amenability might not benefit sufficiently from chaperone treatment.

Rare non-coding Desmoglein-2 variant contributes to Arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to variants in the coding sequence of desmosomal genes. The potential contribution of non-coding desmoglein-2 (DSG2) variants for development of ARVC is undescribed. We sequenced 1450 base pairs upstream of ATG in the DSG2 gene in 65 unrelated patients diagnosed with ARVC (10 borderline cases). Identified variants was evaluated by cosegregation and allele population frequency analysis, in silico tools, immunohistological investigations of myocardial biopsies, gene reporter assays, electrophoretic mobility shift assays (EMSA), and chromatin immunoprecipitation. The genetic analysis identified one novel, rare heterozygous DSG2 upstream variant (-317G > A) in a genetically unexplained ARVC patient. The variant segregated with signs of disease, was absent in publicly available databases, and affected a predicted binding site for activating protein-1 (AP-1). Immunohistochemical analysis of a myocardial biopsy from the -317G > A patient showed a marked reduction in DSG2 protein levels compared to healthy controls. Luciferase reporter gene assays showed promoter activity of the identified DSG2 upstream region and a general reduction in transcriptional activity in the presence of the minor DSG2_A allele (p < .01). Moreover, the DSG2_A allele reduced DSG2 activation by TGF-beta1 and a protein kinase C pathway activator (PMA; all p < .001 vs. DSG2_G). EMSAs showed altered transcription factor binding in presence of the DSG2_A allele. Chromatin immunoprecipitation assays in wild type epithelial cells identified AP-1 components c-FOS and c-JUN at the -317 locus. In conclusion, the non-coding DSG2 promoter variant -317G > A reduces DSG2 transcription in vitro and reduced myocardial DSG2 protein levels were observed in vivo. Our data support a contribution of non-coding DSG2 variants to the pathogenesis of ARVC.

Effects of high-intensity interval training on microvascular glycocalyx and associated microRNAs.

High-intensity interval training (HIIT) has been proposed to exert vasculoprotective effects. This study aimed to evaluate whether HIIT affects the microvasculature, including the endothelial glycocalyx barrier, and to identify associated microRNAs (miRNAs). Fifty healthy participants (23.1 ± 3.0 yr) performed a 4-wk 4 × 30-s all-out running HIIT. Sidestream dark-field imaging was performed at baseline and follow-up to detect changes of the sublingual microvasculature including the endothelial glycocalyx. Exercise parameters were determined by continuous running field test and documentation of high-intensity runs. miRNAs potentially associated with glycocalyx thickness were selected by structured literature search and blood samples for miRNA, and lactate measurements were drawn at baseline and follow-up HIIT. At baseline, a correlation between maximal exercise performance capacity and glycocalyx thickness (determined by perfused boundary region) was detected (P = 0.045, r = 0.303). Increased exercise performance at follow-up also correlated with glycocalyx thickness (P = 0.031, r = 0.416), and increased high-intensity sprinting speed was associated with an increased number of perfused vessels (P = 0.0129, r = 0.449). Literature search identified miR-143, -96-5p, and -24, which were upregulated by HIIT already at baseline and showed an association with peak blood lactate levels after sprints (all P < 0.05). Moreover, increased baseline miR-143 levels predicted increased glycocalyx thickness at follow-up (AUCmiR-143 = 0.92, 95% confidence interval, 0.81-1.0, P = 0.0008). Elevated resting miR-126 levels after the intervention were associated with cell-free versican mRNA levels. We conclude that HIIT induces changes in the endothelial glycocalyx of the microvasculature. Associated miRNAs such as miR-143 may represent a tool for monitoring early vasculoprotective adaptations to physical activity. NEW & NOTEWORTHY High-intensity interval training is known to improve health-related fitness in general and in lifestyle-induced chronic diseases. To visualize microvasculature structure and to detect exercise-induced changes, sublingual sidestream dark-field imaging microscopy was used, and circulating miRNAs were measured. This study shows that exercise-induced changes correlate with associated circulating miRNA, which might be useful for monitoring vasculoprotective effects. Furthermore, sidestream dark-field imaging may represent a sensitive tool for the early detection of exercise-induced systemic vascular changes.

Dose-Dependent Effect of Enzyme Replacement Therapy on Neutralizing Antidrug Antibody Titers and Clinical Outcome in Patients with Fabry Disease.

BACKGROUND: Use of enzyme replacement therapy (ERT) to treat Fabry disease, caused by deficient lysosomal α-galactosidase A activity, can lead to formation of neutralizing antidrug antibodies (ADAs). These antibodies are associated with increased accumulation of plasma globotriaosylceramide (Gb3) and disease progression. Because agalsidase ERT can saturate ADA-binding sites during infusions (achieving agalsidase/antibody equilibrium), we investigated in this open cohort study whether saturated patients (who have excess agalsidase after infusions) experience better clinical outcomes compared with not saturated patients (who have excess ADAs after infusions). METHODS: We isolated ADAs from sera of 26 men with Fabry disease receiving ERT (for a median of 94 months) and determined the amount of agalsidase necessary for antibody saturation. Clinical and biochemical outcomes included measurements of eGFR, interventricular septum thickness, and lyso-Gb3. RESULTS: ADA titers decreased significantly in all patients during infusion. Agalsidase-α and agalsidase-ß had similar ADA-binding capacity and comparable ADA saturation frequency. Fourteen patients with saturated ADAs presented with mild (but significant) loss of eGFR, stable septum thickness, and significantly decreased lyso-Gb3 levels. The 12 not saturated patients had a more pronounced and significant loss of eGFR, increased septum thickness, and a smaller, nonsignificant reduction in lyso-Gb3, over time. In three patients, dose escalation resulted in partially elevated ADA titers, but importantly, also in reduced lyso-Gb3 levels. CONCLUSIONS: A not saturated ADA status during infusion is associated with progressive loss of eGFR and ongoing cardiac hypertrophy. Dose escalation can result in saturation of ADAs and decreasing lyso-Gb3 levels, but may lead to increased ADA titers.

Fabry disease under enzyme replacement therapy-new insights in efficacy of different dosages.

Background: Fabry patients on reduced dose of agalsidase-beta or after switch to agalsidase-alfa show a decline in estimated glomerular filtration rate (eGFR) and an increase of the Mainz Severity Score Index. Methods: In this prospective observational study, we assessed end-organ damage and clinical symptoms in 112 patients who had received agalsidase-beta (1.0 mg/kg) for >1 year, who were (i) non-randomly assigned to continue this treatment regime (regular-dose group, n = 37); (ii) received a reduced dose of agalsidase-beta and subsequent switch to agalsidase-alfa (0.2 mg/kg) or a direct switch to 0.2 mg/kg agalsidase-alfa (switch group, n = 38); or (iii) were re-switched to agalsidase-beta after receiving agalsidase-alfa for at least 12 months (re-switch group, n = 37) with a median follow-up of 53 (38-57) months. Results: eGFR of patients in the regular-dose group remained stable. Patients in the switch group showed an annual eGFR loss of - 4.6 ± 9.1 mL/min/1.73 m2 (P < 0.05). Patients in the re-switch group also had an eGFR loss of - 2.2 ± 4.4 mL/min/1.73 m2 after re-switch to agalsidase-beta, but to a lower degree compared with the switch group (P < 0.05). Patients in the re-switch group suffered less frequently from diarrhoea (relative risk 0.42; 95% confidence interval 0.19-0.93; P = 0.02). Lyso-Gb3 remained stable in the switch (P = 0.97) and the regular-dose (P = 0.48) groups, but decreased in the re-switch group after change of the therapy regimen (P < 0.05). Conclusions: After switch to agalsidase-alfa, Fabry patients experienced a continuous decline in eGFR, while this decline was attenuated in patients who were re-switched to agalsidase-beta. Decreasing lyso-Gb3 levels may indicate a better treatment response in the latter group.

Salt-induced Na+/K+-ATPase-α/ß expression involves soluble adenylyl cyclase in endothelial cells.

High dietary salt intake may lead to vascular stiffness, which predicts cardiovascular diseases such as heart failure, and myocardial and cerebral infarctions as well as renal impairment. The vascular endothelium is a primary target for deleterious salt effects leading to dysfunction and endothelial stiffness. We hypothesize that the Ca2+- and bicarbonate-activated soluble adenylyl cyclase (sAC) contributes to Na+/K+-ATPase expression regulation in vascular endothelial cells and is an important regulator of endothelial stiffness. In vitro stimulation of vascular endothelial cells with high sodium (150 mM Na+)-induced Na+/K+-ATPase-α and Na+/K+-ATPase-ß protein expression determined by western blot. Promoter analyses revealed increased cAMP response element (CRE)-mediated Na+/K+-ATPase-α transcriptional activity under high sodium concentrations. Inhibition of sAC by the specific inhibitor KH7 or siRNA reduced the sodium effects. Flame photometry revealed increased intracellular sodium concentrations in response to high sodium stimulations, which were paralleled by elevated ATP levels. Using atomic force microscopy, a nano-technique that measures cellular stiffness and deformability, we detected significant endothelial stiffening under increased sodium concentrations, which was prevented by inhibition of sAC using KH7 and Na+/K+-ATPase using ouabain. Furthermore, analysis of primary aortic endothelial cells in an in vitro aging model revealed an impaired Na+/K+-ATPase-α sodium response and elevated intracellular sodium levels with cellular aging. We conclude that sAC mediates sodium-induced Na+/K+-ATPase expression in vascular endothelium and is an important regulator of endothelial stiffness. The reactivity of Na+/K+-ATPase-α expression regulation in response to high sodium seems to be impaired in aging endothelial cells and might be a component of endothelial dysfunction.

Renal function predicts long-term outcome on enzyme replacement therapy in patients with Fabry disease.

BACKGROUND: Renal and cardiac involvement is responsible for substantial morbidity and mortality in Fabry disease (FD). We analysed the incidence of FD-related renal, cardiac and neurologic end points in patients with FD on long-term enzyme replacement therapy (ERT). METHODS: A retrospective analysis of prospectively collected data from two German FD centres was performed. The impact of renal and cardiac function at ERT-naïve baseline on end point development despite ERT was analysed. RESULTS: Fifty-four patients (28 females) receiving ERT (mean 81 ± 21 months) were investigated. Forty per cent of patients were diagnosed with clinical end points before ERT initiation and 50% of patients on ERT developed new clinical end points. In patients initially diagnosed with an end point before ERT initiation, the risk for an additional end point on ERT was increased {hazard ratio [HR] 3.83 [95% confidence interval (CI) 1.61-9.08]; P = 0.0023}. A decreased glomerular filtration rate (eGFR) ≤75 mL/min/1.73 m2 in ERT-naïve patients at baseline was associated with an increased risk for cardiovascular end points [HR 3.59 (95% CI 1.15-11.18); P = 0.0273] as well as for combined renal, cardiac and neurologic end points on ERT [HR 4.77 (95% CI 1.93-11.81); P = 0.0007]. In patients with normal kidney function, left ventricular hypertrophy at baseline predicted a decreased end point-free survival [HR 6.90 (95% CI 2.04-23.27); P = 0.0018]. The risk to develop an end point was independent of sex. CONCLUSIONS: In addition to age, even moderately impaired renal function determines FD progression on ERT. In patients with FD, renal and cardiac protection is warranted to prevent patients from deleterious manifestations of the disease.

Serum-Mediated Inhibition of Enzyme Replacement Therapy in Fabry Disease.

Fabry disease (FD) is a progressive multisystemic disorder, treatable with recombinant enzyme replacement therapy (agalsidase). However, recent studies suggest an endogenous inhibition of agalsidase in patients with FD, as reported for other lysosomal storage diseases. To assess the clinical consequences of serum-mediated agalsidase inhibition in affected patients, we determined the agalsidase inhibition status of 168 patients (68 male) with FD and compared outcomes of inhibition-positive patients with those of inhibition-negative patients. The assessment included clinical events during time on agalsidase, determination of renal and cardiac function, and evaluation of FD-related symptoms. The frequency of serum-mediated agalsidase inhibition was 40% in agalsidase-treated males. Inhibition did not depend on the compound initially used (agalsidase-α or -ß). Agalsidase inhibition was associated with higher lyso-globotriaosylceramide levels and worse disease severity scores in patients. Compared with agalsidase inhibition-negative men, agalsidase inhibition-positive men showed greater left ventricular mass (P=0.02) and substantially lower renal function (difference in eGFR of about -30 ml/min per 1.73 m(2); P=0.04), which was confirmed by a longitudinal 5-year retrospective analysis. Additionally, affected patients presented more often with FD-typical symptoms, such as diarrhea, fatigue, and neuropathic pain, among others. Therefore, patients with poor clinical outcome on agalsidase should be tested for agalsidase inhibition. Future studies are warranted to determine if affected patients with FD benefit from acute reduction of anti-agalsidase antibodies or long-term immune modulation therapies to suppress agalsidase inhibition and to identify mechanisms that minimize antibody generation against agalsidase.

Patients with Fabry Disease after Enzyme Replacement Therapy Dose Reduction and Switch-2-Year Follow-Up.

Because of the shortage of agalsidase-ß supply between 2009 and 2012, patients with Fabry disease either were treated with reduced doses or were switched to agalsidase-α. In this observational study, we assessed end organ damage and clinical symptoms with special focus on renal outcome after 2 years of dose-reduction and/or switch to agalsidase-α. A total of 89 adult patients with Fabry disease who had received agalsidase-ß (1.0 mg/kg body wt) for >1 year were nonrandomly assigned to continue this treatment regimen (regular-dose group, n=24), to receive a reduced dose of 0.3-0.5 mg/kg and a subsequent switch to 0.2 mg/kg agalsidase-α (dose-reduction-switch group, n=28), or to directly switch to 0.2 mg/kg agalsidase-α (switch group, n=37) and were followed-up for 2 years. We assessed clinical events (death, myocardial infarction, severe arrhythmia, stroke, progression to ESRD), changes in cardiac and renal function, Fabry-related symptoms (pain, hypohidrosis, diarrhea), and disease severity scores. Determination of renal function by creatinine and cystatin C-based eGFR revealed decreasing eGFRs in the dose-reduction-switch group and the switch group. The Mainz Severity Score Index increased significantly in these two groups (P=0.02 and P<0.001, respectively), and higher frequencies of gastrointestinal pain occurred during follow-up. In conclusion, after 2 years of observation, all groups showed a stable clinical disease course with respect to serious clinical events. However, patients under agalsidase-ß dose-reduction and switch or a direct switch to agalsidase-α showed a decline of renal function independent of the eGFR formula used.

Aldosterone signaling and soluble adenylyl cyclase-a nexus for the kidney and vascular endothelium.

The steroid hormone aldosterone regulates the reabsorption of water and ions in the kidney and plays a central role in blood pressure regulation and homeostasis. In recent years, the vascular endothelium has been established as an important aldosterone target organ with major implications in renal and cardiovascular health and disease. Different lines of evidence suggest that the calcium- and bicarbonate-activated soluble adenylyl cyclase (sAC) is a novel mediator of aldosterone signaling in both the kidney and vascular endothelium. This review summarizes our current understanding of the molecular mechanisms of sAC gene expression regulation in the kidney and vascular endothelium and outlines the potential clinical implications of sAC in chronic kidney disease and cardiovascular disease. This review is part of a special issue entitled: The role of soluble adenylyl cyclase in health and disease. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

Patients with Fabry disease after enzyme replacement therapy dose reduction versus treatment switch.

Because of the shortage of agalsidase-beta in 2009, many patients with Fabry disease were treated with lower doses or were switched to agalsidase-alfa. This observational study assessed end-organ damage and clinical symptoms during dose reduction or switch to agalsidase-alfa. A total of 105 adult patients with Fabry disease who had received agalsidase-beta (1.0 mg/kg body weight) for ≥1 year were nonrandomly assigned to continue this treatment regimen (regular-dose group, n=38), receive a reduced dose of 0.3-0.5 mg/kg (dose-reduction group, n=29), or switch to 0.2 mg/kg agalsidase-alfa (switch group) and were followed prospectively for 1 year. We assessed clinical events (death, myocardial infarction, severe arrhythmia, stroke, progression to ESRD); changes in cardiac, renal, and neurologic function; and Fabry-related symptoms (neuropathic pain, hypohidrosis, diarrhea, and disease severity scores). Organ function and Fabry-related symptoms remained stable in the regular-dose group. In contrast, estimated GFR decreased by about 3 ml/min per 1.73 m(2) (P=0.01) in the dose-reduction group, and the median albumin-to-creatinine ratio increased from 114 (0-606) mg/g to 216 (0-2062) mg/g (P=0.03) in the switch group. Furthermore, mean Mainz Severity Score Index scores and frequencies of pain attacks, chronic pain, gastrointestinal pain, and diarrhea increased significantly in the dose-reduction and switch groups. In conclusion, patients receiving regular agalsidase-beta dose had a stable disease course, but dose reduction led to worsening of renal function and symptoms. Switching to agalsidase-alfa is safe, but microalbuminuria may progress and Fabry-related symptoms may deteriorate.

Interindividual transcriptional regulation of the human biglycan gene involves three common molecular haplotypes.

OBJECTIVE: The extracellular matrix proteoglycan biglycan (BGN) is involved in cardiovascular disease pathophysiology, as it mediates the subendothelial retention of atherogenic apolipoprotein B-containing lipoproteins, affects adaptive remodeling after myocardial infarction, and exerts proinflammatory effects in macrophages. In a cardiovascular disease-related setting of vascular endothelial cells and human monocytes, we examined the molecular mechanisms of common molecular haplotypes affecting human BGN transcriptional regulation. APPROACH AND RESULTS: After the molecular characterization of the BGN promoter, we determined the prevalence of BGN promoter variants (1199 base pair portion) in 87 individuals of European ancestry, and identified 3 molecular haplotypes by subcloning and sequencing of subjects' single DNA strands: MolHap1 [G(-578)-G(-151)-G(+94)] MolHap2 [G(-578)-A(-151)-T(+94)] and MolHap3 [A(-578)-G(-151)-G(+94)]. By 5' rapid amplification of cDNA-ends, we detected 1 additional upstream transcription start site at position -46 in EA.hy926 endothelial cells. Reporter gene assays located the BGN core promoter to the region spanning positions -39 and +162. Strongest promoter activity was mapped to the region between -1231 and -935. The introduction of MolHap2 and MolHap3 into the active BGN promoter led to a significant loss of transcriptional activity (all probability values <0.05), compared with MolHap1. By use of electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and cotransfection of transcription factors, we identified specificity protein 1, v-ets erythroblastosis virus E26 oncogene homolog (ETS) family members, and an activator protein-1 complex to interact differentially with the BGN promoter in the context of each individual MolHap. CONCLUSIONS: Our results indicate that molecular haplotypes within the BGN promoter may contribute to the molecular basis of interindividually different transcriptional BGN regulation, possibly modulating the predisposition to cardiovascular disease-related phenotypes.

Promoter-like sequences regulating transcriptional activity in neurexin and neuroligin genes.

Synapse function requires the cell-adhesion molecules neurexins (Nrxn) and neuroligins (Nlgn). Although these molecules are essential for neurotransmission and prefer distinct isoform combinations for interaction, little is known about their transcriptional regulation. Here, we started to explore this important aspect because expression of Nrxn1-3 and Nlgn1-3 genes is altered in mice lacking the transcriptional regulator methyl-CpG-binding protein2 (MeCP2). Since MeCP2 can bind to methylated CpG-dinucleotides and Nrxn/Nlgn contain CpG-islands, we tested genomic sequences for transcriptional activity in reporter gene assays. We found that their influence on transcription are differentially activating or inhibiting. As we observed an activity difference between heterologous and neuronal cell lines for distinct Nrxn1 and Nlgn2 sequences, we dissected their putative promoter regions. In both genes, we identify regions in exon1 that can induce transcription, in addition to the alternative transcriptional start points in exon2. While the 5'-regions of Nrxn1 and Nlgn2 contain two CpG-rich elements that show distinct methylation frequency and binding to MeCP2, other regions may act independently of this transcriptional regulator. These data provide first insights into regulatory sequences of Nrxn and Nlgn genes that may represent an important aspect of their function at synapses in health and disease.

Characterization of pre-existing anti-PEG and anti-AGAL antibodies towards PRX-102 in patients with Fabry disease.

Polyethylene glycol (PEG)ylated drugs are used for medical treatment, since PEGylation either decreases drug clearance or/and shields the protein from undesirable immunogenicity. PEGylation was implemented in a new enzyme replacement therapy for Fabry disease (FD), pegunigalsidase-alfa (PRX-102). However, exposure to PEG via life-style products and vaccination can result in the formation of anti-PEG antibodies. We demonstrate the de novo formation of functional anti-PEG antibodies in a healthy male after the second mRNA-based vaccination against SARS-CoV-2. Consequently, we analyzed the frequency and inhibitory function of anti-PEG and anti-α-Galactosidase A (AGAL) antibodies in 102 FD patients (46.9% males). We identified 29 out of 87 (33.3%) patients with low anti-PEG titers. Sera from patients without anti-AGAL antibodies [n=70] showed a higher rescued AGAL activity of agalsidase-beta and PRX-102 [both p<0.0001] compared to those with anti-AGAL antibodies [n=15]. Sera from anti-AGAL antibody-negative and -positive patients had less inhibitory effects on PRX-102 (rescued activity: 89 ± 6% versus 85 ± 7% and 49 ± 26% versus 25 ± 32%; both p<0.0001). Enzyme stability assays demonstrated that AUCs in anti-AGAL-negative sera (n=20) were 7.6-fold higher for PRX-102, while AUCs of both enzymes in anti-AGAL-positive sera (n=6) were decreased. However, AUC for PRX-102 was 33% of non-anti-AGAL-positive sera treated PRX-102 and 5-fold higher compared to agalsidase-beta. Anti-PEG antibodies had no significant effects on serum half-life of PRX-102, probably due to low titers. Conceivably, therapy efficacy may be superior under next-generation PRX-102 therapy compared to current enzyme replacement therapies in terms of reduced inhibitory effects of anti-AGAL and minor inhibitory effects of anti-PEG antibodies.