The Benefits of Early versus Late Therapeutic Intervention in Fabry Disease

Background Fabry disease (FD) is an X-linked lysosomal storage disorder caused by pathogenic variants of the GLA gene. Heterozygous female patients may show much more variability in clinical manifestations, ranging from asymptomatic to full-blown disease. Because of this heterogeneous clinical picture in women, the diagnosis of FD has typically been delayed for more than a decade, and the optimal time to initiate treatment remains controversial. Case Presentation. Here, we present two unrelated female patients diagnosed with FD harbouring the same pathogenic GLA variant. We discuss the implications of initiating specific therapy at different stages of the disease, with and without organ involvement (early versus late therapeutic intervention). Conclusions These clinical cases suggest that initiating specific treatment at an earlier age in women with FD may prevent organ involvement and associated clinical events.


Introduction
Fabry disease (FD) is a lysosomal storage disorder with Xlinked transmission caused by pathogenic variants in the GLA gene encoding α-galactosidase A (α-Gal A). Defciency or decreased activity of this enzyme results in accumulation of its main unmetabolized substrate (globotriaosylceramide ) and its derivatives (e.g., lyso-Gb3) in various tissues and organs [1][2][3].
Te disease can be divided into a severe, classical phenotype clinically characterised by skin lesions (angiokeratoma), recurrent burning pain (acroparaesthesias), cornea verticillata, hypohidrosis, cardiac and renal injury, and cerebral ischaemia, and a generally milder nonclassical phenotype, also referred to as lateonset FD, characterised by a more variable disease course [4,5]. Heterozygous females have variable clinical manifestations, depending on the inactivation pattern of the X chromosome, ranging from asymptomatic to full-blown disease [6]. α-Gal A levels in females may be within the normal range, so FD diagnosis relies on the sequencing of the GLA gene [7].
Migalastat was approved in 2016. It is an oral pharmacological chaperone, which stabilises α-Gal A when it has an amenable mutation (an estimated 35-50% of patients with FD overall), increasing enzyme trafcking to lysosomes and thus intracellular α-Gal A activity [8][9][10].
Tere are more than 1,000 diferent disease-causing variants known in the GLA gene to be associated with FD (Human Gene Mutation Database, https://www.hgmd. org/). Te occurrence of disease complications is highly age-dependent. Timely diagnosis and early therapeutic intervention are favourable for disease prognosis and can prevent the occurrence of or even partially reverse organ lesions caused by FD [3]. However, much of the published literature describe outcomes after late treatment initiation once substantial organ damage has already occurred.
Here, we report the case of two women with the p. Ile270Tr pathogenic variant who have been treated with migalastat for 11 and 5 years, respectively, and the diferences in functional class deterioration depending on early therapeutic intervention.

Case presentation
2.1. Case 1. We present the case of a 38-year-old female with 10 years of follow-up since migalastat initiation. Her previous medical history included gastrointestinal symptoms, including episodes of diarrhoea at 18 years of age. An intestinal biopsy was performed to relate the gastrointestinal symptoms to FD, but no GL-3 deposits were detected. Terefore, the patient was diagnosed with irritable bowel syndrome. She also noticed acroparaesthesia and whorled opacities in the cornea (cornea verticillata). Sequence analysis of the GLA gene in the patient, in comparison with the wild-type sequence (Figure 1), revealed a single nucleotide point mutation in heterozygosis at nucleotide c.809T >C p.(Ile270Tr) (GLA variant: NM_000169.3). Tis variant results in the change from isoleucine to threonine at position 270 of the exon 6 [11] Tis variant has not been found in the population database gnomAD, and it is predicted to be pathogenic by most computational predictive programs (BayesDel_addAF, DANN, DEOGEN2, FATHMM-MKL, LIST-S2, M-CAP, MVP, MutationAssessor, Muta-tionTaster, SIFT) as well as by the meta-predictor REVEL (score = 0.97). Tis variant has been associated with the classic Fabry phenotype, and its amenability to migalastat therapy has been confrmed in the GLP HEK cell-based assay [12]. Taking into account all this evidence and following the American College of Medical Genetics and Genomics guidelines for the interpretation of sequence variants [13], the variant GLA c.809T >C p. (Ile270Tr) is pathogenic.
Regarding her family history, her father had classical FD and, after undergoing a kidney and heart transplant, fnally died at age 42. Her 43-year-old sister was also diagnosed with the disease and remains asymptomatic. Her 13-year-old son was found to be unafected in a prenatal diagnosis ( Figure 2).
In October 2010, after confrming eligibility criteria, she was included in a clinical trial to receive migalastat for 24 months (FACETS study [8]) ( Figure 3).
Her glomerular fltration rate (GFR) at baseline, measured by plasma clearance of iohexol, was normal, and her GL-3 in urine was >4 times the upper limit of normal, fulflling the criteria for the clinical trial. At enrolment, the number of GL-3 inclusions in interstitial capillary cells (KIC GL-3) obtained from a kidney biopsy sample was 0.236. Likewise, GL-3 inclusions were documented in podocytes endothelial cells (data not shown). α-Gal A activity in white blood cells was decreased by almost 40% [8] and plasma lyso-Gb3 was increased (Figure 4(a)). Te protein excretion rate was within the normal reference values (Figure 4(b)). Te echocardiographic evaluation confrmed preserved dimension and function (Figure 4(c)).
After 6 months, migalastat treatment reduced KIC GL-3 inclusions, urine GL-3, and plasma lyso-Gb3. Importantly, enzyme activity was fully restored (Figure 4(a)). Renal and cardiac parameters were kept at physiological values (Figures 4(b) and 4(c)). Six months later (12 months after migalastat initiation), plasma lyso-Gb3 levels remained stable without further reduction, while urine GL-3 concentration showed a meaningful decrease. In addition, α-Gal A activity remained stable. Renal biopsy revealed a slight increase in KIC GL-3 inclusions (Figure 4 In 2012, after completing the pivotal study, the patient was enrolled in the open-label extension (OLE) study (AT1001-041) to receive migalastat for an additional 24 months ( Figure 3). Forty-eight months (4 years) after migalastat initiation, all scores related to renal and cardiac parameters fell within normal reference values (Figures 4(b) and 4(c)).
In 2014, at age 30. Te patient was included in the second extension of the study (AT1001-042), where she received migalastat for another 36 months (Figure 3). At the end of this period (7 years on migalastat treatment), renal and cardiac values were still within physiological ranges (Figures 4(b) and 4(c)).
Since 2017, our patient has continued treatment with migalastat in its marketed form. Currently, she remains stable, with no signifcant albuminuria and no organ involvement ( Figures 3, 4(b), and 4(c)).

Case 2.
We present the case of a 65-year-old woman diagnosed with FD and severe organ involvement treated with migalastat for the last 3 years. Te patient had a long medical history, including multiple conditions such as cardiac hypertrophy with preserved left ventricular ejection fraction (LVEF) and diastolic dysfunction, exertional dyspnoea, cutaneous lupus disease, primary antiphospholipid syndrome, dyslipidaemia, hypertension, and atrophic gastritis. She is currently being treated with torasemide, amiodarone, enalapril, bisoprolol, acenocoumarol, and pravastatin. In 2016, she sufered a stroke of the right middle cerebral artery along with paroxysmal atrial fbrillation. At that time, typical FD manifestations (severe sensorineural hypoacusis, cornea verticillata, and angiokeratomas) were also detected ( Figure 3). Genetic testing demonstrated the presence of a pathogenic sequence variant in the GLA gene: c.809T >C p.(Ile270Tr), the same as the previous case although not a relative ( Figure 1). Plasma α-Gal A activity was decreased (less than 30%) (Figure 4(a)). Cardiac hypertrophy was then attributed to FD. Fabryspecifc therapy was immediately initiated with enzyme replacement therapy (ERT) (agalsidase beta) (Figure 3). Regarding her family history, although her mother had a hypertrophic cardiomyopathy and her father and brother had ischaemic heart disease and sensorineural hearing loss, genetic testing was negative for FD ( Figure 2).
In 2018, the patient requested to be switched from ERT to migalastat. During follow-up, the patient experienced unexplained syncopes and conduction disturbances (frstdegree auriculoventricular blockade with right ventricle bundle branch block and left anterior hemiblock), which were confrmed by a pathological electrophysiological study showing a prolonged Hiss-Ventricular (HV) interval, and the patient was ftted with a permanent dual chamber pacemaker in 2019 ( Figure 3).

Discussion
Tis report describes two unrelated female patients with FD harbouring the same GLA p. Ile270Tr pathogenic variant and the efect of early treatment, not only on symptoms and surrogates, including LVM and GFR, but also on clinical events. Te literature has extensively described this variant in association with the classic Fabry phenotype [11,[16][17][18][19][20][21]. Specifc features of advanced disease, such as cardiac fbrosis or severe renal dysfunction, are hallmarks of irreversible organ damage. Tus, it is mandatory to start treatment before the occurrence of irreversible manifestations to achieve better outcomes [22][23][24]. Tis is in line with the two cases described here. In the frst case, treatment was initiated at an early stage of the disease, before renal or cardiac involvement, while in the second case, treatment was started at a late stage, with severe and irreversible cardiac involvement. As previously mentioned, the patient in the frst case initiated FD-specifc therapy upon inclusion in the phase III FACETS study at the age of 26 years, before any evident organ damage. After 6 months of migalastat treatment, KIC GL-3 inclusions, urine GL-3, and plasma lyso-Gb3 were reduced, and α-Gal A activity was fully restored. Tese results are consistent with recent fndings from the migalastat phase III ATTRACT study [8,25]. Moreover, the patient remained asymptomatic with no organ involvement for the whole follow-up. Tis is doubly remarkable when talking about a variant associated with a classic phenotype, given that these females are more likely to develop complications than nonclassical ones [5]. Te second patient was diagnosed late in the course of the disease, when cardiac hypertrophy was already severe. Although she started ERT immediately upon diagnosis and was later switched to migalastat, her cardiac hypertrophy did not improve, and she even required a pacemaker. Te patient's many comorbidities may have also played a role in her poorer outcome. However, she has not developed Fabry nephropathy, as although her eGFR is not optimal, she shows no microalbuminuria, a hallmark of the disease. Tis may be an example of how FD therapy does not reverse organ involvement but may prevent damage to other organs, such as the kidney.
Even so, it cannot be ruled out that the diferent disease severity in the two cases stems from a diferent X inactivation pattern. According to previous studies, preferential inactivation of the wild-type GLA allele is associated with an early onset of disease and rapid progression, compared to a milder disease when the mutated allele is preferentially inactivated [26,27]. Conversely, other studies [28,29] reported that X inactivation patterns in symptomatic females did not difer from those of female controls of the same age. Some of these discrepancies may arise from the fact that disease progression is also related to a cross-correction mechanism that degrades over time, explaining why women with FD develop more symptoms with age [28]. Tis highlights the need for a larger series to support our fndings.      In conclusion, these clinical cases at diferent stages of disease progression highlight the importance of early diagnosis and timely treatment initiation in patients with FD and provide further evidence of the long-term efcacy and safety of migalastat in patients with amenable GLA mutations.

Data Availability
Te datasets during and/or analysed during the current study are available from the corresponding author upon request.

Conflicts of Interest
MF has received honoraria for lectures, presentations, speaker bureaus, manuscript writing, or educational events from Reata and Otsuka, and for attending meetings and/or traveling from Otsuka, Sanof Genzyme, and Amicus. EA, AM, VB, JM, MPV, JC, and ER declare that they have no conficts of interest. RT has received medical writing support for the present manuscript from Amicus, consulting fees from Amicus, Takeda, Chiesi, and Sanof Genzyme, honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Amicus, Takeda, Chiesi, and Sanof Genzyme, and is a council member of the European Renal Association (ERA).

Authors' Contributions
MF investigated and visualized the study and reviewed and edited the article. EA conceptualized, validated, investigated, and visualized the study, developed methodology, performed formal analysis and data curation, collected resources, and reviewed and edited the article. AM validated the study, collected resources, and wrote the article. VB wrote the original draft of the manuscript and reviewed and edited the article. JM investigated the study, collected resources, and reviewed and edited the article.. MPV conceptualized, investigated, and visualized the study, developed methodology, and collected resources. JC investigated the study and wrote and reviewed the article. ER visualized the study, collected resources, and reviewed and edited the article. RT conceptualized, validated, investigated, supervised, and visualized the study, developed methodology, performed formal analysis and data curation, collected resources, and reviewed and edited the article.