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.


Introduction
Pathogenic genetic variants in the alpha-galactosidase A (αGalA) gene (GLA), located on region q22.1 X chromosome, cause the multisystem disorder called Fabry disease (FD) [1].Reduced or defcient αGalA activity is leading to progressive intracellular globotriaosylceramide (Gb3) storage in numerous cells and organs, including the nervous system, kidneys, and heart [1].
"Classic" FD phenotype is presented in men with (i) severely reduced (<1-3% of mean normal) αGalA activity; (ii) marked Gb3 accumulation in vascular endothelial cells, cardiomyocytes, smooth muscle cells, and podocytes; and (iii) childhood or adolescent symptoms onset, followed by progressive multiorgan failure with shortening of life expectancy due to premature death [1,2].Te frst clinical manifestations, including neuropathic pain in the extremities (chronic paresthesia and episodic severe pain crises), gastrointestinal (GI) discomfort, hypohidrosis, angiokeratomas, and cornea verticillata typically emerge during childhood or early adulthood [1,2].
Men with a "Late-onset" FD phenotype present with (i) varying residual αGalA activity levels reduced (>3% of the mean normal) and (ii) clinical manifestations usually limited to a single organ, with symptoms onset around the 5 th decade of life [1,2].
In heterozygous FD female patients, disease spectrum severity ranges from asymptomatic to severe "classic" phenotype and is in part dependent on the GLA type variant (genotype) and the X chromosome inactivation (lyonization) profle [1][2][3].
In Argentina, therapeutic strategies for FD currently include (i) enzyme replacement therapy (ERT): intravenous administration of agalsidase (recombinant αGalA; α or β) and (ii) chaperone therapy which involves the stabilization of the conformation of αGalA protein in the endoplasmic reticulum to improve its catalytic ability by oral administration of migalastat, an iminosugar αGalA competitive inhibitor.Other therapeutic strategies in development include gene therapy and substrate inhibition therapy.
Te real prevalence and incidence of FD are uncertain.While initially reported incidences ranged from 1/476000 to 1/117000 in the general population, newborn screening initiatives have found an unexpectedly high prevalence, as high as 1/3100 newborns in Italy [1].On the other hand, a high prevalence of FD has been reported in at-risk populations, both in adult men and women on hemodialysis (1/ 476 and 1/667, respectively), hypertrophic cardiomyopathy (1/476 and 1/667, respectively), and cryptogenetic stroke (1/ 769 and 1/714, respectively).In Argentina, there are no databases with accurate epidemiological records.
A FD patients subgroup with "missense" type GLA genetic variants can be "amenables" (as explained below) which cause both (i) misfolding and (ii) reduced stability of αGalA, so it is prematurely degraded in the trans-Golgi network and cannot reach the cytoplasmic lysosome.
Despite this, αGalA catalytic activity remains conserved [4,5].1-Deoxygalactonojirimycin (migalastat), a small iminosugar molecule, was developed to prevent αGalA premature degradation.Migalastat is capable of binding to the αGalA active site, correcting the protein structure misfolding, improving stability, and thus being able to cross the trans-Golgi network without being retained and fnally reach the lysosome to fulfll its physiological catalytic function [4,5].Tis pharmacological mechanism, by which a small molecule can help a protein fold correctly, allowing it to enter physiological processing pathways properly was enunciated as "chemical chaperone" [6,7].Certain GLA genetic variants specifcally cause this αGalA structure alteration that can be corrected with migalastat and are called "amenables" (or migalastat responders) variants.In 2016, migalastat was approved by the FDA, and today, it is available as an alternative to ERT to treat FD patients with "amenable" GLA genetic variants [5,8].
To date, there are no cohorts of "amenable" FD patients reported in Latin America; rather, most studies include these patients from other geographic latitudes.For this reason, in the present work, we report the results of "amenable" FD patients from a Latin American country for the frst time.
In the present study, we report the clinical characteristics of FD "amenable" cohort patients from Argentina prior to starting treatment with migalastat.Tis is the initial report, and the follow-up of this cohort receiving migalastat every 6 months will be reported later.
Inclusion criteria were as follows: FD patients with confrmed diagnosis by the genetic test and "amenable" GLA variants.Te study of the genetic variant was carried out by PCR amplifcation and sequencing of all coding exons and fanking intronic regions from previous DNA extraction from Dried Blood Spot.Reference sequence: NM_000169.2(ENST00000218516).Exclusion criteria were as follows: (i) FD patients with confrmed diagnosis by genetic test and NO "amenable" GLA variant and (ii) FD patients with inclusion criteria who refused to participate in the study.Quantifcation of αGalA enzymatic activity was performed by the fuorometric method.Decreased or normal enzyme activity was considered at values < than or > than 4.0 nmol/h/l, respectively.Plasma globotriaosylsphingosine (Lyso-Gb3) was quantifed by the liquid chromatography method with tandem mass spectrometry.
Te presence/absence of a neuropathic pain crisis and/or typical acroparesthesias was evaluated by questioning (Brief Pain Inventory-Short Form) and quantitative sensory testing (QST) [1,2,11].Te presence/absence of dyshidrosis and typical GI symptoms were evaluated by questioning (Gastrointestinal Symptom Rating Scale) and physical examination [1,2,12].Te presence/absence of angiokeratomas was evaluated by a dermatologist specialist in FD [1,2].Te presence/absence of hearing loss was defned by alterations in the logo-audiometry test [1,2].Te presence/ absence of cornea verticillata was evaluated by ophthalmological examination with a slit lamp, performed by an ophthalmologist with experience in FD [1,2].FD cardiac involvement was defned by (i) cardiac arrhythmia typical of FD (electrophysiological disorders in 12-lead electrocardiogram) and/or (ii) left ventricular hypertrophy (LVH) assessed by tissue Doppler echocardiogram and/or cardiac magnetic resonance imaging (MRI); causes of cardiomyopathy other than FD were ruled out [1,2].FD central nervous system (CNS) involvement was defned by (i) cerebral white matter lesions in brain MRI angiography and/or (ii) clinical stroke by antecedents during the interrogatory and/or physical examination [1,2].Left ventricular mass index (LVMI) was performed by cardiac MRI, and the results expressed in g/m 2 .

Statistical
Analyses.Categorical variables were expressed as numbers (percentages) and continuous variables as the mean ± standard deviation (SD).Parametric or nonparametric tests were used according to nominal or continuous variables.Te contingency tables were analyzed with the Fisher exact or Chi 2 test.Diferences were considered signifcant if p < 0.05.Statistical analyses were performed with IBM SPSS Statistic Editor 1.8 version.
All genotypes included were missense-type "amenables" mutations.Figure 1 shows the distribution of genotype frequencies by gender (a) and FD phenotype (b).
Te frequency of cardiac arrhythmia and CNS involvement were similar in patients classifed by gender and phenotypes (cardiac arrhythmia between genders p � 0.522 and between phenotypes p � 0.375, respectively; CNS involvement between genders p � 0.929 and between phenotypes p � 0.929, respectively).
Figure 5 shows the frequency distribution of the variables "switch from ERT to migalastat" and "migalastat de novo" in patients classifed by gender and phenotype (a) and start criterion treatment (b).

Discussion
Results from a cohort of "classic" and "late-onset" male and female "amenables" FD patients of similar age, diagnosed in adulthood, are presented.
A major methodological limitation of this study is the small number of patients included and its cross-sectional design.Te small sample size may afect the distribution of patients in the "classic" versus "late-onset" and "male" versus "female" groups; this could have conditioned the diferences in clinical manifestations found in our results.On the other Global Health, Epidemiology and Genomics hand, some FD biomarkers can be modifed over time; therefore, the cross-sectional design can condition the results; this is the case of Lyso-Gb3.
Lyso-Gb3 has been previously correlated with FD severity and phenotype [13].Contrary to expectations, in our population, plasma Lyso-Gb3 was similar between both phenotypes (among patients grouped by gender), probably due to an efect of the subjects included distribution, with more patients in the "late-onset" group and within the "classical" population, a greater number of females.
Te signifcant diference in the onset of symptoms is an expected result, since the "classic" phenotype typically presents early symptoms and the "late-onset" not [1,2].Te signifcant eGFR diference between "classic" and "late-onset" is an expected result because the classic phenotype typically afects kidney function earlier [1,2,14].Because they present earlier and more severe renal damage, patients with the classic phenotype typically present greater urinary protein excretion [1,2,14,15].Te albuminuria found, similar between both phenotypes, can be explained by the high prevalence of cardiovascular risk factors present in the "late-onset" female group, who theoretically should have less albuminuria due to Fabry nephropathy but have kidney damage from causes other than Fabry [16].FD cardiac involvement can occur both among adult patients with "classic" phenotypes and in "late-onset" phenotypes with cardiac presentation [1,2].Our population did not  4 Global Health, Epidemiology and Genomics present diferences in the variables "myocardial mass" and "cardiac arrhythmia."Central nervous system involvement was similar in patients classifed by gender and phenotype.CNS involvement typically occurs in "classic" phenotypes and not in "late-onset" patients, probably the higher cardiovascular risk factors frequency present among "late-onset" females may have modifed this result.
In patients who started "de novo" migalastat, the main indications were (i) heart disease, (ii) kidney damage, and  6 Global Health, Epidemiology and Genomics (iii) pain, while in the "switched from prior ERT" group, the most frequent indication was "patient decision."Tis coincides with publications by other authors [17,18].

Conclusions
In Argentina, the indications for starting "de novo" migalastat treatment were heart disease, kidney damage, and pain, while in "switched from prior ERT" patients, the most frequent indication was "patient decision."Among Fabry "amenables" patients from Argentina, NOT all major clinical disease manifestations are more frequent among "classic" versus "late-onset" phenotypes.Global Health, Epidemiology and Genomics

Figure 5 :
Figure 5: Frequency distribution of variables "switch from ERT to migalastat" and "migalastat de novo" in patients classifed by gender and phenotype and start criterion treatment.M: males; F: females.