Anderson-Fabry disease management: role of the cardiologist.

Anderson-Fabry disease (AFD) is a lysosomal storage disorder characterized by glycolipid accumulation in cardiac cells, associated with a peculiar form of hypertrophic cardiomyopathy (HCM). Up to 1% of patients with a diagnosis of HCM indeed have AFD. With the availability of targeted therapies for sarcomeric HCM and its genocopies, a timely differential diagnosis is essential. Specifically, the therapeutic landscape for AFD is rapidly evolving and offers increasingly effective, disease-modifying treatment options. However, diagnosing AFD may be difficult, particularly in the non-classic phenotype with prominent or isolated cardiac involvement and no systemic red flags. For many AFD patients, the clinical journey from initial clinical manifestations to diagnosis and appropriate treatment remains challenging, due to late recognition or utter neglect. Consequently, late initiation of treatment results in an exacerbation of cardiac involvement, representing the main cause of morbidity and mortality, irrespective of gender. Optimal management of AFD patients requires a dedicated multidisciplinary team, in which the cardiologist plays a decisive role, ranging from the differential diagnosis to the prevention of complications and the evaluation of timing for disease-specific therapies. The present review aims to redefine the role of cardiologists across the main decision nodes in contemporary AFD clinical care and drug discovery.

Restoration of peripheral neuropathy in Fabry mice via intrathecal administration of an adeno-associated virus vector encoding mGLA cDNA.

Anderson-Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a pathological variant of the α-galactosidase A (GLA) gene that results in deficient GLA activity. GLA deficiency leads to the accumulation of globotriaosylceramide (Gb3) and lyso-Gb3 in many tissues. A certain number of FD patients have burning pain or acroparesthesia in the feet and hands since childhood. Enzyme replacement therapy (ERT) is available for FD patients. However, ERT does not dramatically improve these FD-related peripheral neuropathic pain. We generated an adeno-associated virus serotype PHP.eB (AAV-PHP.eB) vector encoding mouse GLA cDNA, which was administered to FD mice intrathecally (it) or intravenously (iv). In the it-administered AAV (it-AAV) FD mice, the GLA enzyme activity in the lumbar dorsal root ganglion (DRG) was significantly greater than that in the untreated (NT) FD mice, and the level of activity was similar to that in wild-type (WT) B6 mice. However, in iv-administered AAV (iv-AAV) FD mice, GLA activity in the DRG did not increase compared to that in NT FD mice. Gb3 storage in the DRG of it-AAV FD mice was reduced compared to that in the DRG of NT FD mice. However, compared with NT FD mice, iv-AAV FD mice did not exhibit a significant reduction in the expression of the Gb3 substrate. Compared with WT mice, FD mice were thermally hyposensitive at 52 °C according to the hot plate test. The it-AAV FD mice showed significant recovery from thermal hyposensitivity. However, the iv-AAV FD mice did not exhibit significant improvement in thermal hyposensitivity. These results suggest that the intrathecal delivery of AAV-PHP.eB-mGLA may be a valuable tool for the treatment of FD-related peripheral neuropathic pain.

Clinical study of left ventricular structure and function in patients with Anderson-Fabry disease before and after enzyme replacement therapy.

AIMS: Cardiac left ventricular hypertrophy (LVH) is the most common manifestation of heart involvement in Anderson-Fabry disease (AFD). Conventional cardiac imaging is not sensitive enough to detect early signs of LVH in AFD. It remains uncertain whether enzyme replacement therapy (ERT) can prevent LVH progression and improve myocardial function. This study aimed to assess the effectiveness of two-dimensional speckle tracking echocardiography (2D-STE) in early detection of cardiac involvement in AFD and monitoring the efficacy of agalsidase alfa and agalsidase beta therapy. METHODS AND RESULTS: Thirteen consecutive AFD patients and 12 healthy controls underwent standard transthoracic 2D, color Doppler, tissue Doppler echocardiography, and 2D strain analysis. Global longitudinal strain (GLS) and global circumferential strain (GCS) were measured. Diastolic strain rate (SR) was extracted. Compared to healthy subjects, AFD patients without LVH showed lower levels of GLS (p < 0.001) and SR (p = 0.01), while there was no difference in GCS (p = 0.82). Following treatment, apical circumferential strain (ACS) showed improvement (p = 0.01). CONCLUSION: In AFD patients without LVH, there was a decrease in global and segmental LS. Higher plasma Lyso-GL-3 concentrations were associated with elevated ACS values after ERT, indicating that ACS in AFD patients without LVH, albeit normal, is involved in early LV dysfunction.

Phenotypic Expression and Outcomes in Patients with the p.Arg301Gln GLA Variant in Anderson-Fabry Disease.

The p.Arg301Gln variant in the α -galactosidase A gene (GLA) has been poorly described in the literature. The few reports show controversial information, with both classical and nonclassical Anderson-Fabry Disease (AFD) presentation patterns. The aim of this study was to analyze the penetrance, clinical phenotype, and biochemical profile of an international cohort of patients carrying the p.Arg301Gln genetic variant in the GLA gene. This was an observational, international, and retrospective cohort case series study of patients carrying the p.Arg301Gln variant in the GLA gene associated with AFD disease. Forty-nine p.Arg301Gln GLA carriers, 41% male, were analyzed. The penetrance was 63% in the entire cohort and 1.5 times higher in men. The mean age of symptoms onset was 41 years; compared to women, men presented symptoms earlier and with a shorter delay to diagnosis. The typical clinical triad-cornea verticillate, neuropathic pain, and angiokeratomas-affected only 20% of the cohort, with no differences between genders. During follow-up, almost 20% of the patients presented some type of nonfatal cardiovascular and renal event (stroke, need for dialysis, heart failure, and arrhythmias requiring intracardiac devices), predominantly affecting men. Residual levels were the most common finding of α-GAL A enzyme activity, only a few women had a normal level; a small proportion of men had undetectable levels. The incidence of combined outcomes including all causes of death was 33%, and the cumulative incidence of all-cause mortality was 9% at the follow-up. Patients carrying the p.Arg301Gln GLA variant have a high penetrance, with predominantly cardiorenal involvement and clinical onset of the disease in middle age. Only a small proportion showed the classic clinical presentation of AFD. As in other X-linked diseases, males were more affected by severe cardiovascular and renal events. This genotype-phenotype correlation could be useful from a practical clinical point of view and for future decision making.

Inflammation, Oxidative Stress, and Endothelial Dysfunction in the Pathogenesis of Vascular Damage: Unraveling Novel Cardiovascular Risk Factors in Fabry Disease.

Anderson-Fabry disease (AFD), a genetic disorder caused by mutations in the α-galactosidase-A (GLA) gene, disrupts lysosomal function, leading to vascular complications. The accumulation of globotriaosylceramide (Gb3) in arterial walls triggers upregulation of adhesion molecules, decreases endothelial nitric oxide synthesis, and induces reactive oxygen species production. This cascade results in fibrotic thickening, endothelial dysfunction, hypercontractility, vasospasm, and a pro-thrombotic phenotype. AFD patients display increased intima-media thickness (IMT) and reduced flow-mediated dilation (FMD), indicating heightened cardiovascular risk. Nailfold capillaroscopy (NFC) shows promise in diagnosing and monitoring microcirculatory disorders in AFD, though it remains underexplored. Morphological evidence of AFD as a storage disorder can be demonstrated through electron microscopy and immunodetection of Gb3. Secondary pathophysiological disturbances at cellular, tissue, and organ levels contribute to the clinical manifestations, with prominent lysosomal inclusions observed in vascular, cardiac, renal, and neuronal cells. Chronic accumulation of Gb3 represents a state of ongoing toxicity, leading to increased cell turnover, particularly in vascular endothelial cells. AFD-related vascular pathology includes increased renin-angiotensin system activation, endothelial dysfunction, and smooth muscle cell proliferation, resulting in IMT increase. Furthermore, microvascular alterations, such as atypical capillaries observed through NFC, suggest early microvascular involvement. This review aims to unravel the complex interplay between inflammation, oxidative stress, and endothelial dysfunction in AFD, highlighting the potential connections between metabolic disturbances, oxidative stress, inflammation, and fibrosis in vascular and cardiac complications. By exploring novel cardiovascular risk factors and potential diagnostic tools, we can advance our understanding of these mechanisms, which extend beyond sphingolipid accumulation to include other significant contributors to disease pathogenesis. This comprehensive approach can pave the way for innovative therapeutic strategies and improved patient outcomes.

Prevalence of lymphedema among Anderson-Fabry disease patients: A report from the Fabry registry.

BACKGROUND: Anderson-Fabry disease (AFD) is a rare X-linked lysosomal storage disease due to a genetic variation in the α-galactosidase A (GLA) gene. As a result, the activity of the α-galactosidase A (AGAL-A) enzyme is reduced or absent, which causes sphingolipid deposition within different body parts. AFD typically manifests with cardiovascular, renal, cerebrovascular, and dermatologic involvement. Lymphedema is caused by sphingolipid deposition within lymphatics. Lymphedema can cause intolerable pain and limit daily activities. Very limited data exist on lymphedema in AFD patients. METHODS: Using data from the Fabry Registry (NCT00196742) with 7671 patients included (44% males and 56% females), we analyzed the prevalence of lymphedema among AFD patients who were ever assessed for lymphedema and studied the age of first reported lymphedema. Additionally, we assessed whether patients received AFD-specific treatment at some point during their clinical course. The data was stratified by gender and phenotype. RESULTS: Our study showed that lymphedema occurred in 16.5% of the Fabry Registry patients who were ever assessed for lymphedema (n = 5487). Male patients when compared to female patient have higher prevalence (21.7% vs 12.7%) and experienced lymphedema at a younger age (median age at first reported lymphedema of 43.7 vs 51.7 years). When compared to other phenotypes, classic phenotype has the highest prevalence of lymphedema with the earliest reported lymphedema. Among those who reported lymphedema, 84.5% received AFD-specific treatment during their clinical course. CONCLUSIONS: Lymphedema is a common manifestation of AFD in both genders, with a tendency to present later in female patients. Recognition of lymphedema can offer an important opportunity for intervention and potential impact on associated morbidity. Additional future studies are needed to characterize the clinical implications of lymphedema in AFD patients and identify additional treatment options for this growing population.

Left atrial strain correlates with severity of cardiac involvement in Anderson-Fabry disease.

OBJECTIVES: Cardiac involvement in Anderson-Fabry disease (AFD) results in myocardial lipid depositions. An early diagnosis can maximize therapeutic benefit. Thus, this study aims to investigate the potential of cardiac MRI (CMR) based parameters of left atrial (LA) function and strain to detect early stages of AFD. METHODS: Patients (n = 58, age 40 (29-51) years, 31 female) with genetically proven AFD had undergone CMR including left ventricular (LV) volumetry, mass index (LVMi), T1, and late gadolinium enhancement, complemented by LA and LV strain measurements and atrial emptying fractions. Patients were stratified into three disease phases and compared to age and sex-matched healthy controls (HC, n = 58, age 41 [26-56] years, 31 female). RESULTS: A total of 19 early-, 20 intermediate-, and 19 advanced-phase patients were included. LV and LA reservoir strain was significantly impaired in all AFD phases, including early disease (both p < 0.001). In contrast, LA volumetry, T1, and LVMi showed no significant differences between the early phase and HC (p > 0.05). In the intermediate phase, LVMi and T1 demonstrated significant differences. In advanced phase, all parameters except active emptying fractions differed significantly from HC. ROC curve analyses of early disease phases revealed superior diagnostic confidence for the LA reservoir strain (AUC 0.88, sensitivity 89%, specificity 75%) over the LV strain (AUC 0.82). CONCLUSIONS: LA reservoir strain showed impairment in early AFD and significantly correlated with disease severity. The novel approach performed better in identifying early disease than the established approach using LVMi and T1. Further studies are needed to evaluate whether these results justify earlier initiation of therapy and help minimize cardiac complications. KEY POINTS: • Parameters of left atrial function and deformation showed impairments in the early stages of Anderson-Fabry disease and correlated significantly with the severity of Anderson-Fabry disease. • Left atrial reservoir strain performed superior to ventricular strain in detecting early myocardial involvement in Anderson-Fabry disease and improved diagnostic accuracies of approaches already using ventricular strain. • Further studies are needed to evaluate whether earlier initiation of enzyme replacement therapy based on these results can help minimize cardiac complications from Anderson-Fabry disease.

Cardiac magnetic resonance in Fabry disease.

Fabry disease (FD) is a rare X-linked inherited lysosomal storage disorder caused by deficient a-galactosidase A activity that leads to an accumulation of glycolipids, mainly globotriaosylceramide (Gb3) and globotriaosylsphingosine, in affected tissues, including the heart. Cardiovascular involvement usually manifests as left ventricular hypertrophy (LVH), myocardial fibrosis, heart failure, and arrhythmias, which limit the quality of life and represent the most common causes of death. Following the introduction of enzyme replacement therapy, early diagnosis and treatment have become essential in slowing down the disease progression and preventing major cardiac complications. Recent advances in the understanding of FD pathophysiology suggest that in addition to Gb3 accumulation, other mechanisms contribute to the development of cardiac damage. FD cardiomyopathy is characterized by an earlier stage of glycosphingolipid accumulation and a later one of hypertrophy. Morphological and functional aspects are not specific in the echocardiographic evaluation of Anderson-Fabry disease. Cardiac magnetic resonance with tissue characterization capability is an accurate technique for the differential diagnosis of LVH. Progress in imaging techniques has improved the diagnosis and staging of FD-related cardiac disease: a decreased myocardial T1 value is specific of FD. Late gadolinium enhancement is typical of the later stage of cardiac involvement but as in other cardiomyopathy is also valuable to predict the outcome and cardiac response to therapy.

Molecular Pathogenesis of Central and Peripheral Nervous System Complications in Anderson-Fabry Disease.

Fabry disease (FD) is a recessive monogenic disease linked to chromosome X due to more than two hundred mutations in the alfa-galactosidase A (GLA) gene. Modifications of the GLA gene may cause the progressive accumulation of globotriaosylceramide (Gb3) and its deacylated form, globotriasylsphingosine (lyso-Gb3), in lysosomes of several types of cells of the heart, kidneys, skin, eyes, peripheral and central nervous system (not clearly and fully demonstrated), and gut with different and pleiotropic clinical symptoms. Among the main symptoms are acroparesthesias and pain crisis (involving the peripheral nervous system), hypohidrosis, abdominal pain, gut motility abnormalities (involving the autonomic system), and finally, cerebrovascular ischemic events due to macrovascular involvement (TIA and stroke) and lacunar strokes and white matter abnormalities due to a small vessel disease (SVS). Gb3 lysosomal accumulation causes cytoplasmatic disruption and subsequent cell death. Additional consequences of Gb3 deposits are inflammatory processes, abnormalities of leukocyte function, and impaired trafficking of some types of immune cells, including lymphocytes, monocytes, CD8+ cells, B cells, and dendritic cells. The involvement of inflammation in AFD pathogenesis conflicts with the reported poor correlation between CRP levels as an inflammation marker and clinical scores such as the Mainz Severity Score Index (MSSI). Also, some authors have suggested an autoimmune reaction is involved in the disease's pathogenetic mechanism after the α-galactosidase A deficiency. Some studies have reported a high degree of neuronal apoptosis inhibiting protein as a critical anti-apoptotic mediator in children with Fabry disease compared to healthy controls. Notably, this apoptotic upregulation did not change after treatment with enzymatic replacement therapy (ERT), with a further upregulation of the apoptosis-inducing factor after ERT started. Gb3-accumulation has been reported to increase the degree of oxidative stress indexes and the production of reactive oxygen species (ROS). Lipids and proteins have been reported as oxidized and not functioning. Thus, neurological complications are linked to different pathogenetic molecular mechanisms. Progressive accumulation of Gb3 represents a possible pathogenetic event of peripheral nerve involvement. In contrast, central nervous system participation in the clinical setting of cerebrovascular ischemic events seems to be due to the epitheliopathy of Anderson-Fabry disease with lacunar lesions and white matter hyperintensities (WMHs). In this review manuscript, we revised molecular mechanisms of peripheral and central neurological complications of Anderson-Fabry Disease. The management of Fabry disease may be improved by the identification of biomarkers that reflect the clinical course, severity, and progression of the disease. Intensive research on biomarkers has been conducted over the years to detect novel markers that may potentially be used in clinical practice as a screening tool, in the context of the diagnostic process and as an indicator of response to treatment. Recent proteomic or metabolomic studies are in progress, investigating plasma proteome profiles in Fabry patients: these assessments may be useful to characterize the molecular pathology of the disease, improve the diagnostic process, and monitor the response to treatment.

Prevalence and predictors of bradyarrhythmias requiring permanent pacing in patients with Anderson-Fabry disease.

INTRODUCTION: Bradyarrhythmias are an established red flag for storage cardiac conditions including Anderson-Fabry disease (AFD). The prevalence of bradyarrhythmias requiring a pacemaker (PM) and their timing in AFD is unresolved. METHODS: We evaluated the prevalence and predictors of PM requirement in a large AFD cohort, investigating the occurrence of bradyarrhythmias as initial versus late manifestation. We retrospectively evaluated 82 consecutive AFD patients referred to our multidisciplinary referral center from 1994 to 2020 with a median follow-up of 6.9 years, identifying those requiring pacing. Univariable analysis was performed to identify cardiac features associated with PM implantation. RESULTS: Five of 82 (6%) AFD patients required PM implantation (5/39, i.e., 13% of those with cardiac involvement), always in the context of advanced cardiomyopathy. In none, bradyarrhythmias were the presenting feature. Indications included sick sinus syndrome in three patients, advanced atrioventricular block in two patients. QRS prolongation during follow-up strongly correlated with the onset of bradyarrhythmias. CONCLUSION: Severe bradyarrhythmias are relatively frequent in patients with AFD cardiomyopathy, but do not represent a mode of presentation, occurring late in the disease course and always in the context of advanced cardiac involvement. Monitoring QRS variations over time may help to identify patients requiring pacing.

Cardiovascular magnetic resonance native T1 mapping in Anderson-Fabry disease: a systematic review and meta-analysis.

BACKGROUND: T1 mapping is an established cardiovascular magnetic resonance (CMR) technique that can characterize myocardial tissue. We aimed to determine the weighted mean native T1 values of Anderson-Fabry disease (AFD) patients and the standardized mean differences (SMD) as compared to healthy control subjects. METHODS: A comprehensive literature search of the PubMed, Scopus and Web of Science databases was conducted according to the PRISMA statement to retrieve original studies reporting myocardial native T1 values in AFD patients and healthy controls. A random effects model was used to calculate SMD, and meta-regression analysis was conducted to explore heterogeneity sources. Subgroup analysis was also performed according to scanner field strength and sequence type. RESULTS: From a total of 151 items, 14 articles were included in the final analysis accounting for a total population of 982 subjects. Overall, the weighted mean native T1 values was 984 ± 47 ms in AFD patients and 1016 ± 26 ms in controls (P < 0.0001) with a pooled SMD of - 2.38. In AFD patients there was an inverse correlation between native T1 values and male gender (P = 0.002) and left ventricular hypertrophy (LVH) (P < 0.001). Subgroup analyses confirmed lower T1 values in AFD patients compared to controls with a pooled SMD of -  2.54, -  2.28, -  2.46 for studies performed on 1.5T with modified Look-Locker inversion recovery (MOLLI), shortened MOLLI and saturation-recovery single-shot acquisition, respectively and of -  2.41 for studies conducted on 3T. CONCLUSIONS: Our findings confirm a reduction of native T1 values in AFD patients compared to healthy controls and point out that the degree of T1 shortening in AFD is influenced by gender and LVH. Although T1 mapping is useful in proving cardiac involvement in AFD patients, there is need to standardize shreshold values according to imaging equipment and protocols.

Proteomics for the study of new biomarkers in Fabry disease: State of the art.

Nephropathy represents a major complication of Fabry Disease and its accurate characterization is of paramount importance in predicting the disease progression and assessing the therapeutic responses. The diagnostic process still relies on performing renal biopsy, nevertheless many efforts have been made to discover early reliable biomarkers allowing us to avoid invasive procedures. In this field, proteomics offers a sensitive and fast method leading to an accurate detection of specific pathological proteins and the discovery of diagnostic and prognostic biomarkers that reflect disease progression and facilitate the evaluation of therapeutic responses. Here, we report a review of selected literature focusing on the investigation of several proteomic techniques highlighting their advantages, limitations and future perspectives in their application in the routine study of Fabry Nephropathy.

Pathogenesis and Molecular Mechanisms of Anderson-Fabry Disease and Possible New Molecular Addressed Therapeutic Strategies.

Anderson-Fabry disease (AFD) is a rare disease with an incidenceof approximately 1:117,000 male births. Lysosomal accumulation of globotriaosylceramide (Gb3) is the element characterizing Fabry disease due to a hereditary deficiency α-galactosidase A (GLA) enzyme. The accumulation of Gb3 causes lysosomal dysfunction that compromises cell signaling pathways. Deposition of sphingolipids occurs in the autonomic nervous system, dorsal root ganglia, kidney epithelial cells, vascular system cells, and myocardial cells, resulting in organ failure. This manuscript will review the molecular pathogenetic pathways involved in Anderson-Fabry disease and in its organ damage. Some studies reported that inhibition of mitochondrial function and energy metabolism plays a significant role in AFD cardiomyopathy and in kidney disease of AFD patients. Furthermore, mitochondrial dysfunction has been reported as linked to the dysregulation of the autophagy-lysosomal pathway which inhibits the mechanistic target of rapamycin kinase (mTOR) mediated control of mitochondrial metabolism in AFD cells. Cerebrovascular complications due to AFD are caused by cerebral micro vessel stenosis. These are caused by wall thickening resulting from the intramural accumulation of glycolipids, luminal occlusion or thrombosis. Other pathogenetic mechanisms involved in organ damage linked to Gb3 accumulation are endocytosis and lysosomal degradation of endothelial calcium-activated intermediate-conductance potassium ion channel 3.1 (KCa3.1) via a clathrin-dependent process. This process represents a crucial event in endothelial dysfunction. Several studies have identified the deacylated form of Gb3, globotriaosylsphingosine (Lyso-Gb3), as the main catabolite that increases in plasma and urine in patients with AFD. The mean concentrations of Gb3 in all organs and plasma of Galactosidase A knockout mice were significantly higher than those of wild-type mice. The distributions of Gb3 isoforms vary from organ to organ. Various Gb3 isoforms were observed mainly in the kidneys, and kidney-specific Gb3 isoforms were hydroxylated. Furthermore, the action of Gb3 on the KCa3.1 channel suggests a possible contribution of this interaction to the Fabry disease process, as this channel is expressed in various cells, including endothelial cells, fibroblasts, smooth muscle cells in proliferation, microglia, and lymphocytes. These molecular pathways could be considered a potential therapeutic target to correct the enzyme in addition to the traditional enzyme replacement therapies (ERT) or drug chaperone therapy.

Cardiac involvement in Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) include a heterogeneous group of rare, inborn, metabolic diseases characterized by deficiency of lysosomal enzymes or of other proteins involved in lysosomal function, leading to multi organ system substrates accumulation, with consequent multi systemic clinical presentation. Cardiac disease is particularly important in some group of LSDs as glycogen storage diseases (Pompe), mucopolysaccharidoses and in glycosphingolipidoses (Anderson-Fabry disease and less frequently Gaucher disease). Various cardiac manifestations may be observed including hypertrophic and dilated cardiomyopathy, coronary artery disease and valvular disease. The availability of enzyme replacement therapy (ERT) has changed the natural history of some LSDs such as Pompe disease, thanks to the significant effects on cardiological involvement. In other LSDs such as MPSs or Fabry disease, ERT has been shown to stabilize or slow the progression of heart damage. This imposes the need for a timely diagnosis that allows a rapid onset of ERT.

Unexpectedly High Prevalence of Coronary Spastic Angina in Patients With Anderson-Fabry Disease.

BACKGROUND: Although we and others have reported cases of patients with Anderson-Fabry disease (AFD) complicated by coronary spastic angina (CSA), the prevalence of CSA in these patients remains unknown. Methods and Results: We performed the acetylcholine-induced provocation test, according to the Japanese guidelines for the diagnosis and treatment of patients with CSA, in 9 consecutive patients having 5 independent AFD pedigrees. Coronary spasms were provoked in conjunction with symptoms and ECG ischemic changes in 8 of 9 (89%) patients with AFD. CONCLUSIONS: We found an unexpectedly high prevalence of CSA in patients with AFD.

Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A.

Fluorescence-based live-cell imaging (LCI) of lysosomal glycosidases is often hampered by unfavorable pH and redox conditions that reduce fluorescence output. Moreover, most lysosomal glycosidases are low-mass soluble proteins that do not allow for bulky fluorescent protein fusions. We selected α-galactosidase A (GALA) as a model lysosomal glycosidase involved in Anderson-Fabry disease (AFD) for the current LCI approach. Examination of the subcellular localization of AFD-causing mutants can reveal the mechanism underlying cellular trafficking deficits. To minimize genetic GALA modification, we employed a biarsenical labeling protocol with tetracysteine (TC-tag) detection. We tested the efficiency of halogen-substituted biarsenical probes to interact with C-terminally TC-tagged GALA peptide at pH 4.5 in vitro and identified F2FlAsH-EDT2 as a superior detection reagent for GALA. This probe provides improved signal/noise ratio in labeled COS-7 cells transiently expressing TC-tagged GALA. The investigated fluorescence-based LCI technology of TC-tagged lysosomal protein using an improved biarsenical probe can be used to identify novel compounds that promote proper trafficking of mutant GALA to lysosomal compartments and rescue the mutant phenotype.-Bohl, C., Pomorski, A., Seemann, S., Knospe, A.-M., Zheng, C., Krezel, A., Rolfs, A., Lukas, J. Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A.

Impact of immunosuppressive therapy on therapy-neutralizing antibodies in transplanted patients with Fabry disease.

BACKGROUND: Inhibitory antibodies towards enzyme replacement therapy (ERT) are associated with disease progression and poor outcome in affected male patients with lysosomal disorders such as Fabry disease (FD). However, little is known about the impact of immunosuppressive therapy on ERT inhibition in these patients with FD. METHODS: In this retrospective study, we investigated the effect of long-term immunosuppression on ERT inhibition in male patients with FD (n = 26) receiving immunosuppressive therapy due to kidney (n = 24) or heart (n = 2) transplantation. RESULTS: No ERT-naïve transplanted patient (n = 8) developed antibodies within follow-up (80 ±72 months) after ERT initiation. Seven (26.9%) patients were tested ERT inhibition positive prior to transplantation. No de novo ERT inhibition was observed after transplantation (n = 18). In patients treated with high dosages of immunosuppressive medication such as prednisolone, tacrolimus and mycophenolate-mofetil/mycophenolate acid, ERT inhibition decreased after transplantation (n = 12; P = 0.0160). Tapering of immunosuppression (especially prednisolone) seemed to re-increase ERT inhibition (n = 4, median [range]: 16.6 [6.9; 36.9] %; P = 0.0972) over time. One ERT inhibition-positive patient required interventions with steroid therapy and increased doses of tacrolimus, which also lowered ERT inhibition. CONCLUSION: We conclude that the immunosuppressive maintenance therapy after transplantations seems to be sufficient to prevent de novo ERT inhibition in ERT-naïve patients. Intensified high dosages of immunosuppressive drugs are associated with decreased antibody titres and decreased ERT inhibition in affected patients, but did not result in long-term protection. Future studies are needed to establish ERT inhibition-specific immunosuppressive protocols with long-term modulating properties to warrant an improved disease course in ERT inhibition-positive males.

Patterns of CMR measured longitudinal strain and its association with late gadolinium enhancement in patients with cardiac amyloidosis and its mimics.

BACKGROUND: Regional variability of longitudinal strain (LS) has been previously described with echocardiography in patients with cardiac amyloidosis (CA), however, the reason for this variability is not completely evident. We sought to describe regional patterns in LS using feature-tracking software applied to cardiovascular magnetic resonance (CMR) cine images in patients with CA, hypertrophic cardiomyopathy (HCM), and Anderson-Fabry's disease (AFD) and to relate these patterns to the distribution of late gadolinium enhancement (LGE). METHODS: Patients with CA (n = 45) were compared to LV mass indexed matched patients with HCM (n = 19) and AFD (n = 19). Peak systolic LS measurements were obtained using Velocity Vector Imaging (VVI) software on CMR cine images. A relative regional LS ratio (RRSR) was calculated as the ratio of the average of the apical segmental LS divided by the sum of the average basal and mid-ventricular segmental LS. LGE was quantified for the basal, mid, and apical segments using a threshold of 5SD above remote myocardium. A regional LGE ratio was calculated similar to RRSR. RESULTS: Patients with CA had significantly had worse global LS (-15.7 ± 4.6%) than those with HCM (-18.0 ± 4.6%, p = 0.046) and AFD (-21.9 ± 5.1%, p < 0.001). The RRSR was higher in patients with CA (1.00 ± 0.31) than in AFD (0.79 ± 0.24; p = 0.018) but not HCM (0.84 ± 0.32; p = 0.114). In CA, a regional difference in LGE burden was noted, with lower LGE in the apex (31.5 ± 19.1%) compared to the mid (38.2 ± 19.0%) and basal (53.7 ± 22.7%; p < 0.001 for both) segments. The regional LGE ratio was not significantly different between patients with CA (0.33 ± 0.15) and AFD (0.47 ± 0.58; p = 0.14) but lower compared to those with HCM (0.72 ± 0.43; p < 0.0001). LGE percentage showed a significant impact on LS (p < 0.0001), with a 0.9% decrease in absolute LS for every 10% increase in LGE percentage. CONCLUSION: The presence of marked "relative apical sparing" of LS along with a significant reduction in global LS seen in patients with CA on CMR cine analysis may provide an additional tool to differentiate CA from other cause of LVH. The concomitant presence of a base to apex gradient in quantitative LGE burden suggests that the regional strain gradient may be at least partially explained by the burden of amyloid deposition and fibrosis.

Segment-by-segment assessment of left ventricular myocardial affection in Anderson-Fabry disease by non-enhanced T1-mapping.

Background Anderson-Fabry disease (AFD) is an X-linked lysosomal enzyme disorder associated with an intracellular accumulation of sphingolipids, which shorten myocardial T1 relaxation times. Myocardial affection, however, varies between different segments. Purpose To evaluate the specific segmental distribution and degree of segmental affection in AFD patients. Material and Methods Twenty-five patients with AFD, 14 patients with hypertrophic cardiomyopathy (HCM), and 21 controls were included. A Modified Look-Locker Inversion Recovery sequence (MOLLI) was used for non-enhanced T1 mapping at 1.5 T in addition to standard cardiac imaging in 10-12 short axis views. T1 values were evaluated with a mixed model ANOVA and regression analysis to determine the best diagnostic cutoff values for T1 for each myocardial segment. Results Regression analysis showed the best diagnostic cutoff compared to controls in cardiac segments 1-4, 8-9, and 14. Mean differences between T1 for AFD versus HCM were greatest in segment 3, 4, and 9 (99 ms, 103 ms, 86 ms, respectively). Overall T1 times were 888 ± 70 ms and 903 ± 14 ms (AFD with and without LVH); 1014 ± 17 ms and 1001 ± 22 ms (HCM and controls, P < 0.05). Conclusion Myocardial segments are affected by a varying degree of T1 shortening in AFD patients. Segment-specific cutoff values allow the most specific detection and quantification of the extent of myocardial affection.

Relationship between left ventricular diastolic function and myocardial sympathetic denervation measured by (123)I-meta-iodobenzylguanidine imaging in Anderson-Fabry disease.

PURPOSE: Whether cardiac sympathetic nervous function abnormalities may be present in patients with Anderson-Fabry disease (AFD) remains unexplored. We investigated the relationship between left ventricular (LV) function and cardiac sympathetic nervous function in patients with AFD. METHODS: Twenty-five patients (12 men, mean age 43 ± 13 years) with genetically proved AFD and preserved LV ejection fraction and ten age and gender-matched control subjects underwent speckle tracking echocardiography and (123)I-meta-iodobenzylguanidine (MIBG) imaging from which early and late heart to mediastinum (H/M) ratios and myocardial washout rate values were calculated. RESULTS: In AFD patients, a significant correlation between late H/M ratio and LV mass index (r = -61, p = 0.001), left atrial volume (r = -0.72, p < 0.001), systolic pulmonary artery pressure (r = -0.75, p < 0.001), and early diastolic untwisting rate (r = -0.66, p < 0.001) was found. Ten AFD patients exhibited a late H/M ratio below two fold standard deviation of control subjects (≤1.75). Patients showing late H/M ratio ≤ 1.75 had significantly higher LV mass index, relative wall thickness, left atrial volume and systolic pulmonary artery pressure, lower systolic longitudinal strain and an early diastolic untwisting rate compared to patients with late H/M ratio > 1.75. At multivariable linear regression analysis, early diastolic untwisting rate was the only independent predictor of late H/M ratio ≤ 1.75 (odds ratio 1.15, 95 % confidence interval 1.07-1.31, p < 0.05). CONCLUSION: The present findings provide the first demonstration of a cardiac sympathetic derangement in AFD patients with preserved LV ejection fraction, which is mostly related to LV diastolic dysfunction.