Male Fabry Disease Patients Research Articles

Long-term enzyme replacement therapy in Fabry patients protects against oxidative and inflammatory process.

Fabry disease (FD) is an X-linked recessive lysosomal storage disorder, characterized by a deficiency of α-galactosidase, which causes the progressive accumulation of glycosphingolipids, especially globotriaosylsphingosine (Gb3), in lysosomes across multiple organs. Substrate deposition, associated with tissue damage in FD, also contributes to the emergence of a pro-inflammatory state presented by some patients. We investigated pro- and anti-inflammatory cytokines, and the expression of inflammation-associated genes in treated FD patients, as well as oxidative parameters. We found a decrease in the production of cytokines IL-1β, IL-6, IL-10, and TNF-α in male FD patients and a normalization of redox status in male and female FD patients, once the levels of protein, lipid oxidation, and nitrite and nitrate content were like healthy individuals. Our results suggest that long-term ERT in men with FD contributes to the reduction of a pro-inflammatory scenario and a decrease of oxidative damage in patients, reflecting greater control throughout the disease and in the multisystemic changes characteristic of this disorder. These findings lead us to believe that long-term ERT can improve the redox status and protect these individuals against oxidative and nitrative stress, as well as the inflammatory process.

Generation of human induced pluripotent stem cell line MHHi029-A from a male Fabry disease patient carrying c.959A > T mutation

Fabry disease (FD) is a rare and inherited monogenetic disease caused by mutations in the X-chromosomal alpha-galactosidase A gene GLA concomitant with accumulation of its substrate globotriaosylceramide (Gb3) and multi-organ symptoms. We derived an induced pluripotent stem cell line, MHHi029-A, from a male FD patient carrying a c.959A > T missense mutation in the GLA gene. The hiPSCs show a normal karyotype, expression of pluripotency markers and trilineage differentiation capacity. Importantly, they present the patient-specific mutation in the GLA gene and are therefore a valuable resource for investigating the FD mechanism and identifying novel therapies.

Complement activation and cellular inflammation in Fabry disease patients despite enzyme replacement therapy.

Defective α-galactosidase A (AGAL/GLA) due to missense or nonsense mutations in the GLA gene results in accumulation of the glycosphingolipids globotriaosylceramide (Gb3) and its deacylated derivate globotriaosylsphingosine (lyso-Gb3) in cells and body fluids. The aberrant glycosphingolipid metabolism leads to a progressive lysosomal storage disorder, i. e. Fabry disease (FD), characterized by chronic inflammation leading to multiorgan damage. Enzyme replacement therapy (ERT) with agalsidase-alfa or -beta is one of the main treatment options facilitating cellular Gb3 clearance. Proteome studies have shown changes in complement proteins during ERT. However, the direct activation of the complement system during FD has not been explored. Here, we demonstrate strong activation of the complement system in 17 classical male FD patients with either missense or nonsense mutations before and after ERT as evidenced by high C3a and C5a serum levels. In contrast to the strong reduction of lyso-Gb3 under ERT, C3a and C5a markedly increased in FD patients with nonsense mutations, most of whom developed anti-drug antibodies (ADA), whereas FD patients with missense mutations, which were ADA-negative, showed heterogenous C3a and C5a serum levels under treatment. In addition to the complement activation, we found increased IL-6, IL-10 and TGF-ß1 serum levels in FD patients. This increase was most prominent in patients with missense mutations under ERT, most of whom developed mild nephropathy with decreased estimated glomerular filtration rate. Together, our findings demonstrate strong complement activation in FD independent of ERT therapy, especially in males with nonsense mutations and the development of ADAs. In addition, our data suggest kidney cell-associated production of cytokines, which have a strong potential to drive renal damage. Thus, chronic inflammation as a driver of organ damage in FD seems to proceed despite ERT and may prove useful as a target to cope with progressive organ damage.

Screening for health-related quality of life and its determinants in Fabry disease: A cross-sectional multicenter study

BackgroundFabry disease (FD) is a rare X-linked lysosomal storage disorder caused by α-galactosidase A (α-Gal A) deficiency. The progressive accumulation of globotriaosylceramide results in life-threatening complications, including renal, cardiac, and cerebrovascular diseases. In order to improve health care of FD-patients, knowledge of its predictors is important. The aim of our study was to evaluate health-related quality of life (HrQol) in FD and to identify its independent determinants by exploring a wide range of demographic, social and clinical parameters. ResultsIn this cross-sectional multicenter study, 135 adult patients with FD were recruited at three specialized European centers in Germany and Switzerland. Demographics, social status and clinical parameters as well as data on HrQol (EQ5D, EQ VAS) and depression were collected by means of self-reporting questionnaires and confirmed by medical records. HrQol and its predictors were evaluated by univariate and multivariate regression analyses.The study population consisted of 78 female and 57 male FD patients (median age 48 yrs) of whom 80.7% (N = 109) were on enzyme replacement therapy (ERT) and 10.4% (N = 14) were on chaperone treatment. Univariate analysis revealed various factors reducing HrQol such as age > 40 years, classic phenotype, organ involvement (kidney and heart disease, stroke/transient ischemic attack (TIA), gastrointestinal disturbances), depression, and burning limb pain. However, only the following factors were identified as independent predictors of decreased HrQol: classic phenotype, kidney and heart disease, stroke/TIA, depression, and burning limb pain. ERT and chaperone therapy were independent determinants of increased HrQol. ConclusionsModifiable factors, such as burning limb pain and depression, identified as independent predictors of HrQol-deterioration should be addressed in programs aiming to improve HrQol in FD. A multidisciplinary approach is essential in FD-patients since diverse organ involvement prominently compromises HrQol in affected patients. Our findings showed that the classic phenotype is a strong predictor of worsening HrQol.

Investigation of bone mineral density and the changes by enzyme replacement therapy in patients with Fabry disease.

Fabry disease (FD) is an inherited disorder that causes organ dysfunction. However, only a few studies have reported on bone mineral density (BMD) in FD patients, and the relationship between BMD and clinical factors such as globotriaosylsphingosine (lyso-Gb3) remains unclear. Therefore, the current study sought to investigate BMD in FD patients, the relationship between BMD and lyso-Gb3, and the effects of enzyme replacement therapy (ERT) on changes in BMD and lyso-Gb3. This single-center, observational study included 15 patients who visited our facility for FD between January 2008 and June 2021. We assessed BMD and clinical characteristics in study patients, including plasma lyso-Gb3 levels, and examined the relationship between BMD and plasma lyso-Gb3 levels, and changes in BMD after starting ERT. Male patients' BMD had reduced, whereas female patients' BMD was preserved. Male patients had significantly higher plasma lyso-Gb3 levels than female patients. Moreover, plasma lyso-Gb3 levels were found to be significantly related to the lumbar spine and femoral BMD. These were strongly linked with plasma lyso-Gb3 levels in male patients, whereas no strong link was observed in female patients. Furthermore, BMD significantly increased only in male patients although plasma lyso-Gb3 levels significantly decreased by ERT in all patients. BMD decreased possibly due to Gb3 accumulation, and ERT could increase BMD in male FD patients.

Generation and characterization of iPS cell line (CTGUi001-A) from skin fibroblasts of a patient with Fabry disease

We have generated an iPSCs line (CTGUi001-A) from dermal fibroblasts of a 16-year-old male Fabry disease patient with a novel GLA gene mutation (c.156C > A) using Sendai virus encoding the four Yamanaka factors OCT4, SOX2, KLF4, and c-MYC. The CTGUi001-A iPSC line displayed typical embryonic stem cell-like morphology, carried the GLA gene mutation, expressed several pluripotent stem cell makers, retained normal karyotype (46, XY) and was capable of forming teratomas containing three germ layers.

772 SCREENING FOR ANDERSON-FABRY DISEASE IN RELATIVELY YOUNG PATIENTS WITH UNEXPLAINED CONDUCTION DISORDERS REQUIRING PACEMAKER IMPLANTATION

Abstract Background Anderson-Fabry Disease (AFD, OMIM 301500) is a rare X-linked lysosomal storage disorder caused by GLA gene mutation resulting in a deficit or absent activity of the α-galactosidase A enzyme (α-Gal A). This deficiency involves the impossibility of cleavage of glycophospholipids, resulting in an intralisosomal accumulation of them in different tissues. Due to an incidence of 1 in 80000, AFD is considered the second most common glycosphingolipid storage disorder after Gaucher disease. Cardiac manifestations include left ventricular hypertrophy (LVH) and arrhythmias. The rate of pacemaker implantation (PMI) in AFD has been described to be 25 times higher than in the general population, and the requirement of PMI has been reported to be as high as 8% in some AFD series. In this context, the presence of conduction abnormalities in young patients may suggest an undiagnosed AFD.1 Therefore, early diagnosis is important in AFD because appropriate therapies seem to be more effective when initiated promptly. Purpose Our aim is to detect AFD among relatively patients with unexplained conduction disturbances requiring PMI, not submitted to newborn screening. Methods Among 650 patients afferent to our ambulatory for routinary pacemaker follow-up, we considered a selected population with diagnosis of sinus node dysfunction or atrioventricular block (confirmed by atrial pacing rate ≥ 60% or ventricular pacing percentage ≥ 80%) and an age, at the time of PMI, ranging between ≥40 and ≤70 years old. The exclusion criteria were: patients with previous myocardial infarction; patient whit known cardiac disease (such as hypertrophic cardiomyopathy); patients who underwent cardiac surgery and patients with extracardiac disease with cardiac involvement such as autoimmune disorders. For this cohort of 26 adult patients (13 males; 13 females; mean age 63 ± 7 years) a prospective screening study for AFD was performed. After clinical evaluation, transthoracic echocardiography (analyzing signs of left ventricular hypertrophy) and pacemaker check, a dried blood spot sampled in filter paper was analyzed. This filter paper assay was performed in male patients in order to evaluate the α-Galactosidase A enzyme activity through the detection of Fabry disease biomarkers; only in the case of abnormal values, genetic investigation was performed. In female patients, the analysis was exclusively genetic. Results The analyses revealed 58% (15/26) of patients affected by mild LVH (IVS diameter ranging from 11 to 15 mm). No patient had severe LVH (IVS diameter ≥15 mm) or moderate-severe renal dysfunction (more than stage 3B, GFR below than 30-44 mL/min). In the restrict cohort considered, we found one 69 yo female patient with heterozygosis GLA pathogenic mutation, NM_000169.2:c.638A>C p.(Lys213Thr). She had normal value of liso-Gb3 1,1 ng/ml (n.v. ≤ 1,8 ng/ml). She had mild LVH (IVS diameter 12 mm) and no renal dysfunction. Familiar screening was programmed. Conclusion In a highly selected sample of relatively young patients with conduction disturbances requiring pacemaker implantation, a female patient with genetic mutation causing AFD has been identified. Therefore, it seems that screening efforts should be increased in this patient population. 1 Hemelsoet D, De Keyser J, Van Heuverswyn F et al. Screening for Fabry Disease in Male Patients With Arrhythmia Requiring a Pacemaker or an Implantable Cardioverter-Defibrillator. Circulation. 2021 Feb 23;143(8):872-874. doi: 10.1161/CIRCULATIONAHA.120.051400.

The Impact of Kidney Biopsy for Fabry Nephropathy Evaluation on Patients' Management and Long-Term Outcomes: Experience of a Single Center.

Background: Fabry disease (FD) is a rare lysosomal storage disease causing progressive loss of target organ function. All renal cell types are involved from the early stages, even before clinical signs can be detected. FD-specific therapies can stop/mitigate disease progression. Thus, it is important to validate early markers of renal lesions so that they can be adopted as criteria for timely treatment initiation. Materials and methods: We retrospectively analyzed and extensively evaluated 21 FD case patients; this evaluation included a kidney biopsy. We looked for the influence of pathological findings on the management of FD patients. In addition, we investigated the association between general and FD-specific features and long-term patients’ outcomes. We defined a combined endpoint as being at least one of the following: 50% decrease of estimated glomerular filtration rate (eGFR) from baseline, kidney failure (KF), end-stage kidney disease (ESKD), or death and mortality. Results: Our cohort of 21 FD patients (11 males and 10 females) was stratified according to the presence of the combined endpoint: group 1 (n = 15) included patients without the combined endpoint, while group 2 (n = 6) patients reached the combined endpoint outcome. Patients from group 2 presented lower mean baseline eGFR (72.2 ± 38.7 mL/min/1.73 m2 vs. 82.5 ± 26.4 mL/min/1.73 m2) without statistical significance (p = 0.44), but significantly (p = 0.22) higher median baseline proteinuria (2.7 g/24 h vs. 0.4 g/24 h). Specific lysosomal deposits were identified in all patients. Segmental sclerosis was present in all patients with the combined endpoint and in only 33% of patients without the combined endpoint (p = 0.009). Global sclerosis and interstitial fibrosis were present in both groups, with no significant differences. A total of 15 out of the 16 treatment-naïve patients (7 males and 9 females) started FD-specific therapy after kidney biopsy. Treatment was initiated in all male FD patients and in 8 female patients. In 2 females, pathological findings in kidney biopsy offered important reasons to start FD treatment, although specific criteria of the Romanian protocol for prescription of FD-specific therapy were still not fulfilled. Cox univariate analysis showed that every increase in 24 h proteinuria with 1 g is associated with a 65% risk of developing the combined endpoint (HR = 1.65; 95%CI: 1.05–2.58; p = 0.02), and that the presence of segmental sclerosis increased the risk of developing the combined endpoint by 51.3 times (HR = 51.3; 95% CI: 95% CI: 1.67–103.5; p = 0.01). Kaplan–Meier analysis showed that the cumulative risk of developing the combined endpoint was higher in patients in whom segmental sclerosis (100% vs. 0%, log-rank test, p = 0.03) was present. Conclusions: Histological evaluation is an important tool for the detection of early kidney involvement and provides additional support to the early initiation of FD-specific therapy. Presence of segmental sclerosis can predict the long-term outcomes of kidney disease deterioration and mortality and may be used as an early indicator of disease progression. Additionally, in the absence of other criteria according to current guidelines, specific FD renal lesions as revealed by kidney biopsy might become a distinct criterion to initiate FD therapy.

The potential consequences of bidirectional promoter methylation on GLA and HNRNPH2 expression in Fabry disease phenotypes in a family of patients carrying a GLA deletion variant.

Fabry disease (FD) is a rare inherited disease characterized by a wide range of symptoms attributed to GLA mutations resulting in defective α-galactosidase A (α-Gal A) and accumulation of glycosphingolipids. The GLA locus is paired in a divergent manner with the heterogeneous nuclear ribonucleoprotein HNRNPH2 locus mapped in the RPL36A-HNRNPH2 readthrough locus. As a follow-up to our recent finding of the co-regulation of GLA and HNRNPH2 via a bidirectional promoter (BDP) in normal kidney and skin cells, the potential accumulative influence of BDP methylation and GLA mutation on the severity of FD in patients from the same family, two males and two females carrying a GLA deletion mutation, c.1033_1034delTC (p.Ser345Argfs) was addressed in the present study. The molecular analyses of the FD patients compared with the control revealed that the expression of GLA was significantly low (P<0.05), and HNRNPH2 showed a tendency of low expression (P=0.1) when BDP methylation was elevated in FD patients, compared with low BDP methylation and high GLA expression (P<0.05), and a high trend of HNRNPH2 expression in normal individuals. The accumulative effects of the mutation and BDP methylation with the severity of the disease were observed in three patients. One male FD patient, a member of the FD family diagnosed with progressive loss of kidney function, hypertension, and eventually a stroke, and the lowest level of α-Gal A enzyme activity showed the highest BDP DNA methylation level. It is concluded that the DNA methylation of GLA-HNRNPH2 BDP may serve a role in diagnosing and treating FD.

A retrospective investigation to establish new screening approach for the detection of patients at high risk of Fabry disease in male left ventricular hypertrophy patients

BackgroundThe prevalence of Fabry disease (FD) in male patients with left ventricular hypertrophy (LVH) is about 1%. From the perspective of performing more efficient screening with measurement of α-galactosidase (α-Gal) activity, it is important to raise the pretest probability. MethodsWe retrospectively investigated the prevalence of FD in 701 male patients with LVH who already had been screened by measurement of α-Gal activity in eight hospitals. From the viewpoint of enzymatic screening, we validated previously reported clinical features of FD including the electrocardiographic and echocardiographic characteristics with comparing each clinical determinant between patients with FD and non-FD patients. We finally aimed to establish a new screening approach for the detection of patients at high risk of FD. ResultsThere were five FD patients (0.7%) in the 701 male patients with LVH. Those five patients with FD all had the cardiac variant type and age at detection of LVH was ≥35 years in all patients. In LVH patients with LV ejection fraction (EF) ≥ 50%, Pend-Q interval < 40 msec, SV1 + RV5 > 4.0 mV, and diffuse LVH were important determinants of FD. In LVH patients with LVEF < 50%, asymmetric septal hypertrophy and posterior wall motion abnormality seemed to be associated with FD. ConclusionsIn our retrospective study, the prevalence of FD in male patients with LVH was found to be 0.7%. We established the efficient combinations of clinical determinants using age at detection of LVH, Pend-Q interval, high voltage, and LVH pattern in an echocardiogram.

Reduced hip bone mineral density is associated with high levels of calciprotein particles in patients with Fabry disease

SummaryCalciprotein particles (CPP) are nanoscale mineralo-protein aggregates that help stabilize excess mineral in the circulation. We examined the relationship between CPP and bone mineral density in Fabry disease patients. We found an inverse correlation with total hip and femoral neck density, but none with lumbar spine.PurposeCalciprotein particles (CPP) are colloidal mineral-protein complexes made up primarily of the circulating glycoprotein fetuin-A, calcium, and phosphate. They form in extracellular fluid and facilitate the stabilization, transport, and clearance of excess minerals from the circulation. While most are monomers, they also exist in larger primary (CPP-I) and secondary (CPP-II) form, both of which are reported to be raised in pathological states. This study sought to investigate CPP levels in the serum of patients with Fabry disease, an X-linked systemic lysosomal storage disorder that is associated with generalized inflammation and low bone mineral density (BMD).MethodsWe compared serum CPP-I and CPP-II levels in 59 patients with Fabry disease (37 female) with levels in an age-matched healthy adult cohort (n=28) and evaluated their association with BMD and biochemical data obtained from routine clinical review.ResultsCPP-I and CPP-II levels were higher in male Fabry disease patients than female sufferers as well as their corresponding sex- and age-matched controls. CPP-II levels were inversely correlated with BMD at the total hip and femoral neck, but not the lumbar spine. Regression analyses revealed that these associations were independent of common determinants of BMD, but at the femoral neck, a significant association was only found in female patients.ConclusionLow hip BMD was associated with high CPP-II in patients with Fabry disease, but further work is needed to investigate the relevance of sex-related differences and to establish whether CPP measurement may aid assessment of bone disease in this setting.

Early start of enzyme replacement therapy in pediatric male patients with classical Fabry disease is associated with attenuated disease progression.

Enzyme replacement therapy (ERT) slows disease progression of Fabry disease (FD), especially when initiated before the onset of irreversible organ damage. However, with the clinically asymptomatic progression of renal, cardiac and cerebral disease manifestations spanning decades, optimal timing of ERT initiation remains unclear. In this cross-sectional retrospective study, seven male FD patients with a classical disease phenotype (cFD) who started treatment with agalsidase-beta in childhood were evaluated after 10years of treatment (median age at evaluation 24years, range 14-26). Cardiac imaging (echocardiography and MRI), electrophysiological and biochemical data of these patients were compared to those of untreated male cFD patients (n=23, median age 22years, range 13-27). Albuminuria was less common and less severe in treated patients (albumin to creatinine ratio, ACR 0-8.8mg/mmol, median 0.4) compared to untreated patients (ACR 0-248mg/mmol, median 3.7, p=0.02). The treated group had a lower left ventricular mass, measured using echocardiography (median 80g/m2 versus 94g/m2, p=0.02) and MRI (median 53g/m2 versus 68g/m2, p=0.02). Myocardial fibrosis was absent in all included patients. eGFR was normal in all treated patients whereas 7/23 (30%) of untreated patients had abnormal eGFR. Cerebral manifestations did not differ. Start of treatment with ERT before age 16, in male cFD patients is associated with reduced occurrence of renal and cardiac manifestations of FD, as assessed by intermediate endpoints. Confirmation that this approach delays or even prevents renal failure and cardiac events requires another decade of follow-up.

Electrocardiographic Characteristics and Their Correlation with Echocardiographic Alterations in Fabry Disease.

Fabry disease (FD) is an X-linked disorder with α-galactosidase A deficiency. Males (>30 years) and females (>40 years) often present with cardiac manifestations, predominantly left ventricular hypertrophy (LVH). The aim of this study was to evaluate electrocardiographic (ECG) characteristics within FD patients to identify gender related differences, and to additionally explore the association of ECG parameters with structural and functional alterations on transthoracic echocardiography (TTE). Retrospective cross-sectional analysis of 45 FD patients with contemporaneous ECG and TTE was performed and compared to age and gender matched healthy controls. FD patients demonstrated alterations in several ECG parameters particularly in males, including prolonged P-wave duration (91 vs. 81 ms, p = 0.022), prolonged QRS duration (96 vs. 84 ms, p < 0.001), increased R-wave amplitude in lead I (8.1 vs. 5.7 mV, p = 0.047), increased Sokolow–Lyon index (25 vs. 19 mV, p = 0.002) and were more likely to meet LVH criteria (31% vs. 7%, p = 0.006). FD patients with impaired basal longitudinal strain (LS) on TTE were more likely to meet LVH criteria (41% vs. 0%, p = 0.018). Those with more advanced FD (increased LV wall thickness on TTE) were more likely to meet LVH criteria but additionally demonstrated prolonged ventricular depolarization (QRS duration 101 vs. 88 ms, p = 0.044). Therefore, alterations on ECG demonstrating delayed atrial activation, delayed ventricular depolarization and evidence of LVH were more often seen in male FD patients. Impaired basal LS, a TTE marker of early cardiac involvement, correlated with ECG abnormalities. Increased LV wall thickness on TTE, a marker of more advanced FD, was associated with more severe ECG abnormalities.

Defective Function of KCa3.1 Channels in Lysosomal Storage Disorders

Background: The calcium-calmodulin-gated KCa3.1 is considered a key regulator of membrane hyperpolarization, maintenance of cellular electrolyte balance, Ca2+-signalling, secretion of pro-inflammatory cytokines, neurodegeneration, pathological tissue remodelling. Osteonecrosis/-penia, vasculopathies, and neurodegeneration are chronic and progressive inflammatory complications of several inherited lysosomal storage diseases, such as Gaucher Disease (GD1/GD3), Fabry Disease(FD), and Niemann-Pick disease type C1 (NPC1). Here we hypothesized that KCa3.1 functions are impaired in a LSD-related fashion in patients' fibroblasts or the monocyte/macrophages lineage.Methods: A total of25 subjects were analyzed (5 FD, 5 NPC, 5 GD1, 5 GD2, 5 healthy controls). Fibroblasts from patients' skin biopsies were cultured in DMEM supplemented with 10% FCS (Biochrom KG, Berlin, Germany) and cells were used at passages 1-4. For patch-clamp experiments, cells were seeded on coverslips and used within 24 h. Monocytes isolated and purified from peripheral blood from age-matched GD patients (GD1 and GD3) and healthy volunteers (Ctrl). Measurements were repeated in patient's and Ctrl's macrophages that were differentiated from monocyte precursors in vitro by exposure to patient's or Ctrl's erythrocyte lysates for 4 days, respectively.Ca2+-activated K+ currents were measured in the whole-cell configuration using an EPC10-USB amplifier (HEKA, Electronics, Lambrecht-Pfalz, Germany) and a K pipette solution (intracellular) containing 1 μM Ca2+ free (in mM): 140 KCl, 1 MgCl2, 2 EGTA, 1.71 CaCl2 (1 μM [Ca2+]free), and 5 HEPES (adjusted to pH 7.2 with KOH). For data acquisition and analysis we used the patch-master program (HEKA). Ohmic leak currents of up to 1 nS were subtracted where appropriate. We quantified outward currents at a 0 mV holding potential (to avoid contamination with non-selective cation currents and chloride currents). Capacitance values (a measure of cell surface and thus cell size)Results: Patch-clamp demonstrated lower fibroblast KCa3.1 membrane functions in NPC1 and male FD patients. KCa3.1-currents in monocytes were similar in GD-1/3 and Ctrls. In differentiated and activated macrophages, the overall larger KCa3.1 currents in GD-1 macrophages were reduced to 40% of controls while in GD-3 macrophages KCa3.1 currents were barely detectable. Glucosylceramide was found to produce KCa3.1-current desensitization. At the gene level, KCa3.1-mRNA expression was impaired in NPC1, GD1/3, and male FD patients.Conclusions: Defective KCa3.1 functions are a feature and biomarker of cellular dysfunction in the LSD, GD1/3, FD, and NPC1 and supports the concept that biased lipid metabolism harms ion channel functions. Since KCa3.1 is involved in the mechanism of cellular differentiation, neurodegeneration, and inflammation, the impaired KCa3.1 -activity could be of pathomechanistic relevance in LSD cellular malfunction by promoting differentiation into pro-inflammatory cell types. Pharmacological targeting of KCa3.1 could be therapeutic utility to achieve neuroprotection in GD3 and NPC1 patients and bone complications in GD1. DisclosuresNo relevant conflicts of interest to declare.

Screening for Fabry disease in male patients with end-stage renal disease in western France

ContextFabry disease is a rare X-linked genetic disease due to pathogenic variants in the GLA gene. Classic Fabry disease is characterized by glycosphingolipids accumulation in all organs including the kidney, resulting in end-stage renal disease in a subset of male patients. Fabry disease should therefore be considered in the differential diagnosis of patients with unexplained end-stage renal disease. ObjectiveWe performed a prospective screening study in Western France to determine the prevalence of Fabry disease in a large population of dialyzed and transplanted patients. Patients and methodsPatients meeting the inclusion criteria (males, 18-70 years with end-stage renal disease of unknown or vascular origin) were selected from the REIN® registry and the CRISTAL® database. Screening on filter papers was performed after patient consent was obtained during either a dialysis session or a transplantation follow-up visit. ResultsOne thousand five hundred and sixty-one end-stage renal disease male patients were screened and 819 consented (dialysis: n=242; transplant: n=577). One single patient was found with decreased alpha-galactosidase levels <25%. GLA sequencing identified the p.Phe113Leu variant in favor of an unknown superimposed kidney disease responsible for end-stage renal disease since this GLA pathogenic variant is associated with a later-onset cardiac form of Fabry disease with minimal kidney involvement. Family cascade genotyping revealed a previously undiagnosed affected brother. ConclusionThe prevalence of Fabry disease in end-stage renal disease patients was 0.12%, questioning the efficacy of this screening strategy with respect to the low prevalence. However, beside the benefit for the patient and his family, the increased awareness of Fabry disease among participating nephrologists may be of interest for future patients.

A late-onset male Fabry disease patient with somatic mosaicism of a classical GLA mutation: a case report

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA). Male patients of FD develop early sign and symptoms in childhood or adolescence. However, "de novo somatic mosaicism" is rare and might be developed a relatively mild phenotype despite carrying a classic type. A 34-year-old male patient visited with foamy urine. Renal biopsy findings were consistent with FD. Leukocyte α-galactosidase activity was markedly reduced at 5.3 nmol/hr/mg (normal range, 25-126). Sequence analysis of the patient's GLA gene identified mosaicism for the mutation GLA[NM_000169.2] c.820=/G>C. This mutation results in a substitution of the amino acid in position 274 from glycine to arginine. However, no family members showed FD-related symptoms, and the daughter of the patient was also tested for paternity and was identified as a real biological daughter, but DNA sequence analysis for FD showed no mutations. Based on these results, we diagnosed the patients as de novo mutation with somatic mosaicism. Next generation sequencing turned out that 58% of the readings had the mutated allele in buccal cells, 84% in blood, and 85% in urine, when 100% should be expected in a hemizygous affected male confirming the presence of somatic mosaicisms. The patient has been on treatment for enzyme replacement therapy (agalsidase-β, 1.0 mg/kg biweekly) for past 9 years and has maintained normal renal function (serum creatinine 1.0 mg/dL) with mild albuminuria (123 mg/g Cr). Therefore, this case suggests somatic mosaicism is one of important phenotype modifiers.

Defective lysosomal storage in Fabry disease modifies mitochondrial structure, metabolism and turnover in renal epithelial cells

Fabry disease (FD) is an X-linked lysosomal storage disorder. Deficiency of the lysosomal enzyme alpha-galactosidase (GLA) leads to accumulation of potentially toxic globotriaosylceramide (Gb3) on a multisystem level. Cardiac and cerebrovascular abnormalities as well as progressive renal failure are severe, life-threatening long-term complications. The complete pathophysiology of chronic kidney disease (CKD) in FD and the role of tubular involvement for its progression are unclear. We established human renal tubular epithelial cell lines from the urine of male FD patients and male controls. The renal tubular system is rich in mitochondria and involved in transport processes at high-energy costs. Our studies revealed fragmented mitochondria with disrupted cristae structure in FD patient cells. Oxidative stress levels were elevated and oxidative phosphorylation was upregulated in FD pointing at enhanced energetic needs. Mitochondrial homeostasis and energy metabolism revealed major changes as evidenced by differences in mitochondrial number, energy production and fuel consumption. The changes were accompanied by activation of the autophagy machinery in FD. Sirtuin1, an important sensor of (renal) metabolic stress and modifier of different defense pathways, was highly expressed in FD. Our data show that lysosomal FD impairs mitochondrial function and results in severe disturbance of mitochondrial energy metabolism in renal cells. This insight on a tissue-specific level points to new therapeutic targets which might enhance treatment efficacy.

Predicting the Development of Anti-Drug Antibodies against Recombinant alpha-Galactosidase A in Male Patients with Classical Fabry Disease.

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.

P0062GLUCOSYLCERAMIDE SYNTHASE INHIBITION WITH VENGLUSTAT IN CLASSIC FABRY DISEASE PATIENTS LEADS TO PROGRESSIVE REDUCTION OF ENDOTHELIAL CELL GLOBOTRIAOSYLCERAMIDE INCLUSION VOLUME

Abstract Background and Aims Fabry disease (FD) is a rare, X-linked, genetic disorder caused by mutations in the gene for the lysosomal enzyme alpha-galactosidase A (αGal-A). The progressive accumulation of glycosphingolipids, most notably globotriaosylceramide (GL-3), in lysosomes of a variety of organs and cell types leads, over decades, to renal, cardiovascular, and other clinical manifestations. In a phase 2 study, glucosylceramide synthase inhibition with venglustat led to a progressive reduction in plasma GL-3 to low-normal levels. Scoring by light microscopy showed a reduction in lysosomal GL-3 inclusions in skin capillary endothelial cells (EC) after 3 years of therapy, although not after 6 months. To further characterize the effect of venglustat using more precise methods, we applied unbiased stereological methods to quantitate GL-3 inclusions in skin ECs. Methods Skin biopsies were obtained from classic male Fabry disease patients (N = 11) at baseline and during daily treatment with venglustat in a phase 2 study (NCT02228460, NCT02489344). Images from at least 50 superficial skin capillaries were obtained using transmission electron microscopy at 7,500 X magnification. The fraction of the volume (Vv) of cytoplasm occupied by GL-3 inclusions [Vv(Inc/Endo)] in all ECs in these capillaries was estimated using point counting by a masked reader. Two-sided paired t tests were used to evaluate differences between baseline and post-treatment values at each time point. Results Venglustat therapy led to a significant reduction from baseline in Vv(Inc/Endo) of 21.1% after 6 months and 38.7% after 3 years of treatment (Figure). Conclusions Treatment with venglustat led to a reduction in GL-3 inclusion fractional volume in skin capillary ECs which was detectable after 6 months using precise quantitative methods. This was followed by further reduction over the next 2 1/2 years. We posit that, in the absence of αGal-A activity, inhibition of GL-3 production with venglustat allowed other enzymatic or non-enzymatic mechanisms to progressively reduce EC lysosomal GL-3 content, and that long-term venglustat therapy may therefore prevent or reverse progressive tissue injury in Fabry disease.

Lyso-Gb3: A valid follow-up marker of treatment in male Fabry disease patients?

Lyso-Gb3: A valid follow-up marker of treatment in male Fabry disease patients?