GLA Variants Research Articles

Fabry Disease with Genetic Variants of Unknown Significance and Concomitant Immunoglobulin A Nephropathy

Introduction: The diagnosis of Fabry disease (FD) with genetic variants of unknown significance (VUSs) is relatively difficult. We explored patients with novel VUS variants and concomitant immunoglobulin A nephropathy (IgAN) to improve the understanding of VUS. Methods: The study retrospectively investigated patients with genetically confirmed FD. Probands with VUS were selected from the database of FD patients who underwent genetic analysis. Demographic, clinicopathological, and laboratory data from probands and family members were collected and analyzed. Results: Fourteen probands and their family members were included in the study. The probands were divided into group 1 (patients with VUS, n = 5) and group 2 (patients with pathologic/likely pathologic variants, n = 9). The group 1 included 2 missense mutations and 1 deletion mutation, while the group 2 included 6 missense mutations and 2 deletion mutations. There were no significant differences in gender, age, serum creatinine, eGFR, and proteinuria between the two groups. IgA deposition with myeloid bodies was found in all VUS patients. The cardiac involvement in group 2 was more severe than that in group 1. Seven families performed the pedigree analysis, and after the comprehensive evaluation, two GLA variants (c.479C>A, p.Ala160Asp; c.1032-1058 del, p.Ser345_Met353del) were upgraded from VUS to the likely pathogenic. Conclusion: The clinical manifestations of FD are heterogeneous. FD often coexists with nephrotic disorders, such as IgAN and MCD. Comprehensive evaluation, especially tissue-specific biopsy, is necessary for patients with GLA-VUSs. Two GLA variants (c.479C>A, p.Ala160Asp; c.1032-1058 del, p.Ser345_Met353del) were upgraded from VUS to the likely pathogenic after the comprehensive evaluation.

Renal and multisystem effectiveness of 3.9 years of migalastat in a global real-world cohort: Results from the followME Fabry Pathfinders registry.

Fabry disease is a progressive, X-linked lysosomal disorder caused by reduced or absent α-galactosidase A activity due to GLA variants. The effects of migalastat were examined in a cohort of 125 Fabry patients with migalastat-amenable GLA variants in the followME Pathfinders registry (EUPAS20599), an ongoing, prospective, patient-focused registry evaluating outcomes for current Fabry disease treatments. We report annualised estimated glomerular filtration rate (eGFR) and Fabry-associated clinical events (FACEs) in a cohort of patients who had received ≥3 years of migalastat treatment in a real-world setting. As of August 2022, 125 patients (60% male) had a mean migalastat exposure of 3.9 years. At enrolment, median age was 58 years (males, 57; females, 60) with a mean eGFR of 83.7 mL/min/1.73 m2 (n = 122; males, 83.7; females, 83.8) and a median left ventricular mass index of 115.1 g/m2 (n = 61; males, 131.2; females, 98.0). Mean (95% confidence interval) eGFR annualised rate of change in the overall cohort (n = 116) was -0.9 (-10.8, 9.9) mL/min/1.73 m2/year with a similar rate of change observed across patients with varying levels of kidney function at enrolment. Despite population age and baseline morbidity, 80% of patients did not experience a FACE during the mean 3.9 years of migalastat exposure. The incidence of renal, cardiac, and cerebrovascular events was 2.0, 83.2, and 4.1 events per 1000 patient-years, respectively. These data support a role of migalastat in preserving renal function and multisystem effectiveness during ≥3 years of migalastat treatment in this real-world Fabry population.

LysoGb3 quantification facilitates phenotypic categorization of Fabry disease patients: Insights gained by a novel MS/MS method

BackgroundFabry disease (FD) is an X-linked lysosomal storage disease resulting from pathogenic variants in the GLA gene coding α-galactosidase A (AGAL) and cleaving terminal alpha-linked galactose. Globotriaosylceramide (Gb3) is the predominantly accumulated sphingolipid. Gb3, deacylated-Gb3 (lysoGb3), and methylated-Gb3 (metGb3) have been suggested as FD biomarkers. Materials and MethodsWe developed a novel LC-MS/MS method for assessing lysoGb3 levels in plasma and Gb3 and metGb3 in urine and tested 62 FD patients, 34 patients with GLA variants of unknown significance (VUS) and 59 healthy controls. AGAL activity in white blood cells (WBCs) and plasma was evaluated in parallel. ResultsIn males, lysoGb3 concentrations in plasma separated classic and late-onset FD patients from each other and from individuals carrying GLA VUS and healthy controls. Calculating AGAL activity/plasmatic lysoGb3 ratio allowed to correctly categorize all females with classic and majority of patients with late-onset FD phenotypes. Correlation of AGAL activity in WBCS with lipid biomarkers identified threshold activity values under which the biomarkers’ concentrations increase. ConclusionWe developed a novel simplified LC-MS/MS method for quantitation of plasma lysoGb3. AGAL activity/plasma lysoGb3 ratio was identified as the best predictor for FD. AGAL activity correlated with plasma lysoGb3 and corresponded to individual FD phenotypes.

Genetic variants of unknown significance in alpha-galactosidase A: Cellular delineation from Fabry disease.

Fabry disease (FD) is an X-linked multiorgan disorder caused by variants in the alpha-galactosidase A gene (GLA). Depending on the variant, disease phenotypes range from benign to life-threatening. More than 1000 GLA variants are known, but a link between genotype and phenotype in FD has not yet been established for all. p.A143T, p.D313Y, and p.S126G are frequent examples of variants of unknown significance (VUS). We have investigated the potential pathogenicity of these VUS combining clinical data with data obtained in human cellular in vitro systems. We have analyzed four different male subject-derived cell types for alpha-galactosidase A enzyme (GLA) activity and intracellular Gb3 load. Additionally, Gb3 load in skin tissue as well as clinical data were studied for correlates of disease manifestations. A reduction of GLA activity was observed in cells carrying p.A143T compared with controls (p < 0.05). In cells carrying the p.D313Y variant, a reduced GLA activity was found only in endothelial cells (p < 0.01) compared with controls. No pathological changes were observed in cells carrying the p.S126G variant. None of the VUS investigated caused intracellular Gb3 accumulation in any cell type. Our data of aberrant GLA activity in cells of p.A143T hemizygotes and overall normal cellular phenotypes in cells of p.D313Y and p.S126G hemizygotes contribute a basic science perspective to the clinically highly relevant discussion on VUS in GLA.

Multinational experience with next-generation sequencing: opportunity to identify transthyretin cardiac amyloidosis and Fabry disease.

Sarcomeric hypertrophic cardiomyopathy (HCM) must be differentiated from phenotypically similar conditions because clinical management and prognosis may greatly differ. Patients with unexplained left ventricular hypertrophy require an early, confirmed genetic diagnosis through diagnostic or predictive genetic testing. We tested the feasibility and practicality of the application of a 17-gene next-generation sequencing (NGS) panel to detect the most common genetic causes of HCM and HCM phenocopies, including treatable phenocopies, and report detection rates. Identification of transthyretin cardiac amyloidosis (ATTR-CA) and Fabry disease (FD) is essential because of the availability of disease-specific therapy. Early initiation of these treatments may lead to better clinical outcomes. In this international, multicenter, cross-sectional pilot study, peripheral dried blood spot samples from patients of cardiology clinics with an unexplained increased left ventricular wall thickness (LVWT) of ≥13 mm in one or more left ventricular myocardial segments (measured by imaging methods) were analyzed at a central laboratory. NGS included the detection of known splice regions and flanking regions of 17 genes using the Illumina NextSeq 500 and NovaSeq 6000 sequencing systems. Samples for NGS screening were collected between May 2019 and October 2020 at cardiology clinics in Colombia, Brazil, Mexico, Turkey, Israel, and Saudi Arabia. Out of 535 samples, 128 (23.9%) samples tested positive for pathogenic/likely pathogenic genetic variants associated with HCM or HCM phenocopies with double pathogenic/likely pathogenic variants detected in four samples. Among the 132 (24.7%) detected variants, 115 (21.5%) variants were associated with HCM and 17 (3.2%) variants with HCM phenocopies. Variants in MYH7 (n=60, 11.2%) and MYBPC3 (n=41, 7.7%) were the most common HCM variants. The HCM phenocopy variants included variants in the TTR (n=7, 1.3%) and GLA (n=2, 0.4%) genes. The mean (standard deviation) ages of patients with HCM or HCM phenocopy variants, including TTR and GLA variants, were 42.8 (17.9), 54.6 (17.0), and 69.0 (1.4) years, respectively. The overall diagnostic yield of 24.7% indicates that the screening strategy effectively identified the most common forms of HCM and HCM phenocopies among geographically dispersed patients. The results underscore the importance of including ATTR-CA (TTR variants) and FD (GLA variants), which are treatable disorders, in the differential diagnosis of patients with increased LVWT of unknown etiology.

Newborn genetic screening for Fabry disease: Insights from a retrospective analysis in Nanjing, China

Fabry disease (FD), an X-linked disorder resulting from dysfunction of α-galactosidase A, can result in significant complications. Early intervention yields better outcomes, but misdiagnosis or delayed diagnosis is common, impacting prognosis. Thus, early detection is crucial in the clinical diagnosis and treatment of FD. While newborn screening for FD has been implemented in certain regions, challenges persist in enzyme activity detection techniques, particularly for female and late-onset patients. Further exploration of improved screening strategies is warranted. This study retrospectively analyzed genetic screening results for pathogenic GLA variants in 17,171 newborns. The results indicated an estimated incidence of FD in the Nanjing region of China of approximately 1 in 1321. The most prevalent pathogenic variant among potential FD patients was c.640-801G > A (46.15 %). Furthermore, the residual enzyme activity of the pathogenic variant c.911G > C was marginally higher than that of other variants, and suggesting that genetic screening may be more effective in identifying potential female and late-onset patients compared to enzyme activity testing. This research offers initial insights into the effectiveness of GLA genetic screening and serves as a reference for early diagnosis, treatment, and genetic counseling in FD.

Baseline Characteristics of Fabry Disease "Amenable" Migalastat Patients in Argentinian Cohort.

Fabry disease (FD) is a multisystem lysosomal storage disorder induced by genetic variants in the alpha-galactosidase A (αGalA) gene. Some FD patients have GLA variants with a reduction in overall αGalA enzymatic activity due to mutated proteins with reduced stability, caused by protein misfolding and premature degradation, but the αGalA catalytic activity remains conserved ("amenable" genetic variants). To correct this misfolding and to prevent premature degradation, migalastat, a small iminosugar molecule was developed. We report the clinical characteristics of FD "amenable" cohort patients from Argentina, prior to starting treatment with migalastat. Seventeen Fabry adult patients were recruited from 13 Argentinian Centers; 8 males (47.1%) and 9 females (52.9%) were included. All genotypes included were missense-type "amenables" mutations. Some classic FD typical early manifestations were more frequent in patients with "classic" versus "late-onset" FD phenotype (pain, p=0.002; cornea verticillata, p=0.019). There was a statistically significant difference in estimated glomerular filtration rate in the "classic" versus "late-onset" phenotype (p=0.026) but no difference between genders (p=0.695). Left ventricular mass was similar between genders (p=0.145) and phenotypes (p=0.303). Cardiovascular risk factors were present among "late-onset" females (obesity 50% and smoke 25%). In patients who started "de novo" migalastat, the main indications were (i) heart disease, (ii) kidney damage, and (iii) pain, while in "switched from prior enzyme replacement therapy" patients, the most frequent indication was "patient decision;" this coincides with publications by other authors.

Genetic Diagnosis of Adult Hemodialysis Patients With Unknown Etiology

Kidney disease of unknown etiology accounts for 1 in 10 adult end-stage renal disease (ESRD) cases worldwide. The aim of this study is to clarify the genetic background of patients with chronic kidney disease (CKD) of unknown etiology who initiated renal replacement therapy (RRT) in adulthood. This is a multicenter cross-sectional cohort study. Of the 1164 patients who attended 4 dialysis clinics in Japan, we first selected patients who started RRT between the ages of 20 and 49 years. After excluding patients with apparent causes of CKD (e.g., diabetic nephropathy, polycystic kidney disease (PKD) with family history, patients who underwent renal biopsy), 90 patients with CKD of unknown cause were included. The 298 genes associated with CKD were analyzed using capture-based targeted next-generation sequencing. Of the 90 patients, 10 (11.1%) had pathogenic variants in CKD-causing genes and 17 (18.9%) had variant of unknown significance (VUS). Three patients had PKD1 pathogenic variants, and 1 patient had PKD1 and COL4A4 pathogenic variants. In addition, 2 patients were diagnosed with atypical hemolytic uremic syndrome (aHUS) due to C3 or CFHR5. One patient each was diagnosed with Alport syndrome due to COL4A4 and COL4A3 variants, nephronophthisis due to NPHP1 variants, Fabry disease due to GLA variants, and autosomal-dominant tubulointerstitial kidney disease due to UMOD variants. Genetic diagnoses were not concordant with clinical diagnoses, except for patients with PKD1 variant. This largest study on genetic analysis in hemodialysis-dependent adults revealed the presence of undiagnosed inherited kidney diseases.

Prevalence of Fabry disease and GLA variants in young patients with acute stroke: The challenge to widen the screening. The Fabry-Stroke Italian Registry

BackgroundFabry disease (FD) is a treatable X-linked lysosomal storage disorder caused by GLA gene variants leading to alpha-galactosidase A deficiency. FD is a rare cause of stroke, and it is still controversial whether in stroke patients FD should be searched from the beginning or at the end of the diagnostic workup (in cryptogenic strokes). MethodsFabry-Stroke Italian Registry is a prospective, multicentric screening involving 33 stroke units. FD was sought by measuring α-galactosidase A activity (males) and by genetic tests (males with reduced enzyme activity and females) in patients aged 18–60 years hospitalized for TIA, ischemic stroke, or intracerebral hemorrhage. We diagnosed FD in patients with 1) already known pathogenic GLA variants; 2) novel GLA variants if additional clinical, laboratory, or family-derived criteria were present. ResultsOut of 1906 patients, we found a GLA variant in 15 (0.79%; 95%CI 0.44–1.29) with a certain FD diagnosis in 3 (0.16%; 95%CI 0.03–0.46) patients, none of whom had hemorrhage. We identified 1 novel pathogenic GLA variant. Ischemic stroke etiologies in carriers of GLA variants were: cardioaortic embolism (33%), small artery occlusion (27%), other causes (20%), and undetermined (20%). Mild severity, recurrence, previous TIA, acroparesthesias, hearing loss, and small artery occlusion were predictors of GLA variant. ConclusionIn this large multicenter cohort the frequency of FD and GLA variants was consistent with previous reports. Limiting the screening for GLA variants to patients with cryptogenic stroke may miss up to 80% of diagnoses. Some easily recognizable clinical features could help select patients for FD screening.

Investigating Fabry disease - some lessons learned

Despite recent advances, there is still much to be learned about the pathogenesis of Fabry disease. The categorization of GLA gene missense mutations has been complicated by the fact that some missense variants may fall into more than one category. For instance, the A143T variant may cause late-onset Fabry disease in some subjects and not result in Fabry disease in others (pseudo-deficient). Efforts to mitigate the pathobiology of α-galactosidase A deficiency should differentiate between damaging (maladaptive) consequences and compensatory (adaptive) changes. Current therapy leaves a significant unmet need, especially concerning cardiovascular complications and cardiological clinical outcomes. Non-Fabry-specific therapy is necessary and quite beneficial and must be utilized. Its contribution should be considered when trying to assess the net effect of Fabry-specific therapy. Enzyme replacement therapy (ERT) can be administered to patients independently of their GLA genotype, as it slows the decline of kidney function in most patients if initiated sufficiently early in the disease course. Migalastat has better tissue penetration than ERT, but its usefulness is restricted to patients with amenable missense GLA variants. However, it is important to realize that in a substantial proportion of common amenable mutations, migalastat increases α-galactosidase A activity level beyond the disease threshold and thus eliminates the metabolic disturbance that is at the center of Fabry disease. Substrate reduction therapy and gene therapy approaches are being developed, but these therapeutic modalities have their own limitations and difficulties.

Impact of GLA Variant Classification on the Estimated Prevalence of Fabry Disease: A Systematic Review and Meta-Analysis of Screening Studies.

The diagnosis of Fabry disease (FD) has relevant implications related to the management. Thus, a clear assignment of GLA variant pathogenicity is crucial. This systematic review and meta-analysis aimed to investigate the prevalence of FD in high-risk populations and newborns and evaluate the impact of different GLA variant classifications on the estimated prevalence of FD. We searched the EMBASE and PubMed databases on February 21, 2023. Observational studies evaluating the prevalence of FD and reporting the identified GLA variants were included. GLA variants were re-evaluated for their pathogenicity significance using the American College of Medical Genetics and Genomics criteria and the ClinVar database. The pooled prevalence of FD among different settings was calculated. The study was registered on PROSPERO (CRD42023401663) and followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. Of the 3941 studies identified, 110 met the inclusion criteria. The pooled prevalence of FD was significantly different according to the clinical setting and criteria used for the pathogenicity assessment. Using the American College of Medical Genetics and Genomics criteria, the pooled prevalence was 1.2% in patients with left ventricular hypertrophy/hypertrophic cardiomyopathy (26 studies; 10 080 patients screened), 0.3% in end-stage renal disease/chronic kidney disease (38 studies; 62 050 patients screened), 0.7% in stroke (25 studies; 15 295 patients screened), 0.7% in cardiac conduction disturbance requiring pacemaker (3 studies; 1033 patients screened), 1.0% in small-fiber neuropathy (3 studies; 904 patients screened), and 0.01% in newborns (15 studies; 11 108 793 newborns screened). The pooled prevalence was different if the GLA variants were assessed using the ClinVar database, and most patients with a discrepancy in the pathogenicity assignment carried 1 of the following variants: p.A143T, p.D313Y, and p.E66Q. This systematic review and meta-analysis describe the prevalence of FD among newborns and high-risk populations, highlighting the need for a periodic reassessment of the GLA variants in the context of recent clinical, biochemical, and histological data. URL: https://crd.york.ac.uk/PROSPERO/; Unique identifier: CRD42023401663.

Prevalence of Fabry disease in patients with chronic kidney disease: A systematic review and meta-analysis

Fabry disease (FD) is an X-linked lysosomal storage disease caused by pathogenic variants in the GLA gene. It has a wide range of clinical manifestations, typically related to the specific underlying GLA variant. One of the main features of FD is kidney involvement; therefore, several studies have addressed the prevalence of FD in all types of patients with chronic kidney disease. We performed a systematic review and meta-analysis of screening studies in chronic kidney disease patients, including those on dialysis, had undergone a kidney transplantation, and those who did not receive kidney replacement therapy, and assessed the prevalence of pathogenic variants in these cohorts. Fifty-five studies were included, involving a total of 84,062 individuals. Of these, 251 cases were positive for FD; a third of the reported GLA variants were of a benign phenotype (37.8%), followed by classical phenotype (31.7%), late onset (15.5%), and of uncertain significance (14.7%). The overall prevalence among dialysis patients was 0.10% (CI95%, 0.06–0.15), 0.28% (CI95%, 0.06–0.15) among patients with kidney transplantation, and 0.17% (CI95%, 0.11–0.39) among those without kidney replacement therapy. Although the overall prevalence of FD is low in patients with kidney involvement, screening, especially in patients who have not yet undergone kidney replacement therapy, is important, in order to provide timely and effective treatment interventions, including disease modifying therapies. The prevalence of kidney involvement in females with Fabry Disease is lower but this should not lead to inadequate follow up. Further research is also needed on the impact of genetic variants of uncertain significance to elucidate their role in Fabry disease.

B-204 Determination of Lyso-GB3 in Dried Blood Spots: A Useful Biomarker for Fabry Disease

Abstract Background Fabry disease, an X-linked inborn error of metabolism, results from pathogenic mutations in the α-galactosidase A gene (GLA). These mutations reduce or abolish the α-galactosidase A activity, which result in accumulation of glycosphingolipids in the lysosomes, including globotriaosyl-sphingosinein (lyso-GB3), throughout the body, in particular the kidney, heart, and brain. Lyso-GB3 may be elevated in symptomatic patients and supports a diagnosis of Fabry disease. It may also be helpful as a tool for monitoring disease progression, as well as determining treatment response in known patients. Moreover, measurement of Lyso-GB3, may provide additional diagnostic information in the evaluation of uncertain cases, such as in asymptomatic heterozygous female patients, individuals with novel GLA variants of unclear clinical significance, as well as asymptomatic patients identified by family screening. This study aimed to determine Lyso-GB3 in dried blood spot (DBS) samples with a simple, rapid and sensitive LC-MS/MS method, and identify its reference interval in the Brazilian population. Methods All experiments were performed on the Waters TQ-S Micro LC-MS/MS system. The gradient chromatographic separation was carried out using a Waters BEH C18 column in 3.5 min. One DBS punch of 3.1 mm diameter was used for analysis in a microtiter plate and then, a solvent extraction containing stable isotope analogue of Lyso-GB3 was added. After incubation time, the supernatant was directly injected into LC-MS/MS system. The method was validated for linearity, carryover, intra and inter-day precision and the limit of quantification was determined. A reference interval was obtained by analyzing 94 healthy volunteers (49 women and 45 men). The difference of Lyso-GB3 concentration between men and women was investigated (Wilcoxon paired test). The reference interval was determined by the superior limit of the confidence interval (90% CI) of a normal distribution of the results. Results The method was linear between 0.2–100.0 ng.mL−1. The recovery was between 81.0 and 139.7%, which was acceptable considering CLSI guidelines for DBS validation (CLSI NBS04-A). The intra and inter-day precision were below 12.1%, and the limit of quantification was 0.2 ng.mL−1. The reference interval determined was below 0.8 ng/mL. Conclusion In conclusion, the LC-MS/MS method for Lyso-GB3 determination in DBS samples was successfully validated. The sample preparation is simple and the chromatographic method is rapid for routine laboratory. The cutoff determined is comparable with what is available in the literature and adequate for analysis in clinical routine.

Impact of Structural GLA Protein Changes on Peripheral GLA Activity and Substrate Accumulation in Fabry Disease Patients.

Fabry disease is an X-linked lysosomal storage disorder caused by pathogenic variants in the GLA gene, leading to decreased/absent α-galactosidase activity. In clinical practice, enzyme activity and substrate/byproduct accumulation play a role in diagnosis and disease-monitoring biomarkers. However, interpreting biomarker levels is not straightforward and can change according to the underlying GLA protein abnormality. Our goals were to understand how disrupting specific protein regions changes biomarker behaviour and to establish specific patterns for individual variants. We analysed data from the Biochemical Genetics Laboratory regarding GLA variants, GLA enzyme activity (in dried blood spots, plasma or white blood cells), plasma LysoGb3 accumulation, and urinary Gb3 excretion. We assessed correlations, trends, and potential predictor models of biomarker behaviour. We assessed 169 hemizygous male and 255 heterozygous female patients. For both groups, substrate accumulation correlates inversely with GLA activity. Variants affecting residues buried within the protein core or the active site were associated with more severe biomarker changes, while those affecting residues that establish disulfide bonds or are glycosylated were similar to other variants. For each non-truncating variant, we also established specific profiles of biomarker behaviour. Finally, we also designed predictor models of biomarker behaviour based on structural variant information. This study provides the groundwork for the impact of GLA protein variation on GLA activity and substrate accumulation. This knowledge is of extreme relevance for diagnostic labs and clinicians, as some genetic variants are challenging to interpret regarding pathogenicity. Assessing whether biomarker changes are in the expected range for a specific variant may help diagnostic evaluation. This study also contributes to recognising non-disease-causing variants, considering their overall biochemical impact, and providing a comparative reference for biomarker discovery studies. In the future, the correlation of these findings with disease severity may be of great relevance for diagnosis and monitoring progression.

Phenotypes of undiagnosed adults with actionable OTC and GLA variants

Inherited metabolic disorders (IMDs) are variably expressive, complicating identification of affected individuals. A genotype-first approach can identify individuals at risk for morbidity and mortality from undiagnosed IMDs and can lead to protocols that improve clinical detection, counseling, and management. Using data from 57,340 participants in two hospital biobanks, we assessed the frequency and phenotypes of individuals with pathogenic/likely pathogenic variants (PLPVs) in two IMD genes: GLA, associated with Fabry disease, and OTC, associated with ornithine transcarbamylase deficiency. Approximately 1 in 19,100 participants harbored an undiagnosed PLPV in GLA or OTC. We identified three individuals (2 male, 1 female) with PLPVs in GLA, all of whom were undiagnosed, and three individuals (3 female) with PLPVs in OTC, two of whom were undiagnosed. All three individuals with PLPVs in GLA (100%) had symptoms suggestive of mild Fabry disease, and one individual (14.2%) had an ischemic stroke at age 33, likely indicating the presence of classic disease. No individuals with PLPVs in OTC had documented hyperammonemia despite exposure to catabolic states, but all (100%) had chronic symptoms suggestive of attenuated disease, including mood disorders and migraines. Our findings suggest that GLA and OTC variants identified via a genotype-first approach are of high penetrance and that population screening of these genes can be used to facilitate stepwise phenotyping and appropriate care.

Population Frequency of Undiagnosed Fabry Disease in the General Population

Fabry disease is an X-linked disorder that results from pathogenic GLA variants and can now be treated. Most studies of its population frequency have examined only males or attendees at kidney failure or cardiac clinics. This study determined the prevalence of undiagnosed Fabry disease from predicted pathogenic GLA variants in the general population. The Genome Aggregation Database (gnomAD) was examined for predicted pathogenic GLA variants based on variant rarity (≤5), and transcript effect in 4 computational tools (CADD >20, PP2 >0.95, SIFT<0.05, Mutation Taster - Disease-causing) and amino acid conservation in vertebrates in a Clustal. Predicted pathogenic variants in GLA occurred in 1 in 3225 of the gnomAD population and 1 in 3478 of its control subset. Predicted pathogenic variants were more common in women than expected (3.1:1), which is consistent with men being excluded from gnomAD because of Fabry complications. Predicted pathogenic variants were not found in members of this cohort with South Asian, Ashkenazim, or Finnish ancestries. Variants identified as pathogenic in the Fabry database were found in 1 in 2651 individuals of the gnomAD database and pathogenic variants from ClinVar in 1 in4420. The population frequency of 1 in 3225 for undiagnosed men and women with Fabry disease still represents an underestimate because our pathogenicity criteria were rigorous, the cohort did not include already-diagnosed individuals, and whole exome sequencing does not detect intronic variants and large deletions. This study confirms that Fabry disease is more common than previously recognized and still underdiagnosed especially in women.

Optimizing human α-galactosidase for treatment of Fabry disease

Fabry disease is caused by a deficiency of α-galactosidase A (GLA) leading to the lysosomal accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids. Fabry patients experience significant damage to the heart, kidney, and blood vessels that can be fatal. Here we apply directed evolution to generate more stable GLA variants as potential next generation treatments for Fabry disease. GLAv05 and GLAv09 were identified after screening more than 12,000 GLA variants through 8 rounds of directed evolution. Both GLAv05 and GLAv09 exhibit increased stability at both lysosomal and blood pH, stability to serum, and elevated enzyme activity in treated Fabry fibroblasts (19-fold) and GLA–/– podocytes (10-fold). GLAv05 and GLAv09 show improved pharmacokinetics in mouse and non-human primates. In a Fabry mouse model, the optimized variants showed prolonged half-lives in serum and relevant tissues, and a decrease of accumulated Gb3 in heart and kidney. To explore the possibility of diminishing the immunogenic potential of rhGLA, amino acid residues in sequences predicted to bind MHC II were targeted in late rounds of GLAv09 directed evolution. An MHC II-associated peptide proteomics assay confirmed a reduction in displayed peptides for GLAv09. Collectively, our findings highlight the promise of using directed evolution to generate enzyme variants for more effective treatment of lysosomal storage diseases.

Clinical outcomes among young patients with Fabry disease who initiated agalsidase beta treatment before 30 years of age: An analysis from the Fabry Registry.

Clinical manifestations of classic Fabry disease (α-galactosidase A deficiency) usually occur in childhood, while complications involving major organs typically develop in adulthood. Outcomes of Fabry-specific treatment among young patients have not been extensively reported. Our aim was to analyze clinical outcomes among patients aged 5-30 years at initiation of treatment with agalsidase beta using data from the Fabry Registry (NCT00196742, sponsor: Sanofi). Reported GLA variants were predicted to be associated with the classic phenotype or not classified in fabry-database.org. Linear mixed models were conducted to assess changes over ≥2-year follow-up in the estimated glomerular filtration rate (eGFR) stratified by low (LRI) and high (HRI) renal involvement (defined by proteinuria/albuminuria levels), and changes in interventricular septal thickness (IVST) and left ventricular posterior wall thickness (LVPWT) Z-scores stratified by median age at first treatment. Self-reports ('yes'/'no') of abdominal pain, diarrhea, chronic peripheral pain (denoting neuropathic pain), and acute pain crises at baseline were compared with reports after ≥0.5-year and ≥2.5-year follow-up using McNemar's test. Male (n = 117) and female patients (n = 59) with LRI initiated treatment at a median age of 19.9 and 23.6 years, respectively, and were followed for a median of 6.3 and 5.0 years, respectively. The eGFR slopes were -1.18 (Pfrom 0 <0.001) and -0.92 mL/min/1.73 m2/year (Pfrom 0 = 0.040), respectively. Males with HRI (n = 23, median UPCR 1.0 g/g), who started treatment at a median age of 26.7 years, had an eGFR slope of -2.39 mL/min/1.73 m2/year (Pfrom 0 <0.001; Pdifference = 0.055, as compared with the slope of -1.18 mL/min/1.73 m2/year for LRI males) during a median follow-up of 5.6 years. Echocardiographic variables were stable among males, regardless of age, and among young females (median follow-up >5.5 years and ≥4.5 years, respectively). Older females (treatment initiation at median age 27.5 years) had a slope of LVPWT Z-scores of 0.18/year (n = 12, Pfrom 0 = 0.028), whereas IVST Z-scores remained stable (n = 13, 0.10/year, Pfrom 0 = 0.304) during a median follow-up of ≥3.7 years. These slopes did not significantly differ from slopes of younger females. Reports of chronic peripheral pain and acute pain crises by males, and of diarrhea and acute pain crises by females, significantly reduced after a median follow-up of ≥4.0 years. After a median follow-up of ≥5.4 years, reports of all four symptoms significantly decreased among males, whereas among females only reports of abdominal pain significantly decreased. During sustained treatment with agalsidase beta in young Fabry patients with a predicted classic phenotype or with unclassified GLA variants with similar characteristics, the decline in eGFR was modest among male and female patients with LRI. The greater decline in eGFR among older, proteinuric (i.e., HRI) males may suggest a benefit of earlier treatment. Overall, echocardiographic variables remained stable, particularly among males and younger females. Significant reductions in symptom reports occurred primarily among males after longer follow-up and were less noticeable among females. These observed trends are suggestive of an overall improvement after treatment in young patients, but warrant larger longitudinal studies.

The Spanish Fabry women study: a retrospective observational study describing the phenotype of females with GLA variants

BackgroundFabry disease (FD) is an X-linked condition caused by variants in the GLA gene. Since females have two X chromosomes, they were historically thought to be carriers. Although increased knowledge has shown that females often develop the disease, data from Spain and other countries reported that females were undertreated. The aim of this study was to provide a wider and more recent description of the disease characteristics and associated management of females with a GLA variant in a Spanish cohort.ResultsNinety-seven females from 12 hospitals were included in this retrospective study. Mean age was 50.1 ± 17.2 years. Median follow-up time from GLA variant identification was 36.1 months, and most (70.1%) were identified through family screening. Variants associated with classic/non-classic phenotypes were similarly distributed (40.2%/53.6%). Missense variants were the most prevalent (n = 84, 86.6%). In the overall group, 70.4% had major organ involvement (i.e., cardiac, renal, cerebrovascular, peripheral nervous system or gastrointestinal), and 47.3% also had typical Fabry signs (angiokeratoma, cornea verticillata or increased plasma lyso-Gb3). Cardiac involvement was the most prevalent (49.5%) and the main reason for treatment initiation. A total of 33 (34%) patients received disease-specific therapy, 55% of whom were diagnosed by family screening. Females carrying variants associated with a classic phenotype had higher frequencies of clinical manifestations (92.3%) and were predominant in the treated subgroup (69.7%). Despite this, there were 34 untreated females (56.7% of total untreated), with both phenotypes represented, who had major organ involvement, with 27 of cardiac, renal or cerebrovascular nature. Age or comorbidities in this subgroup were comparable to the treated subgroup (P = 0.8 and P = 0.8, respectively).ConclusionsEfforts have been made in recent years to diagnose and treat timely Fabry females in Spain. A high percentage of females with pathogenic variants, regardless of their associated phenotype, will likely develop disease. A proportion of females with severe disease in this cohort received specific treatment. Still a significant number of females, even with same profile as the treated ones, who may be eligible for treatment according to European recommendations, remained untreated. Reasons for this merit further investigation.

Long-term multisystemic efficacy of migalastat on Fabry-associated clinical events, including renal, cardiac and cerebrovascular outcomes

BackgroundFabry disease is a rare, multisystemic disorder caused by GLA gene variants that lead to alpha galactosidase A deficiency, resulting in accumulation of glycosphingolipids and cellular dysfunction. Fabry-associated clinical events...