The overall objective of this study is to investigate Fabry-related cardiomyopathy and the use of native T1-mapping, coronary microvascular function, cardiac inflammation, and cardiac injury in an effort to improve the ability to detect disease. The study aims to achieve this by:

1. Investigating the association between cardiac inflammation, fibrosis, and injury against the distribution and degree of microvascular disease in patients with Fabry disease with and without left ventricular hypertrophy (LVH) using cardiac magnetic resonance (CMR) imaging and 82Rubidium Positron emission tomography and computer tomography (82Rb-PET/CT).
2. Using an extensive, in-depth biomarker blood panel to investigate the pathological pathways associated with Fabry disease and Fabry-related cardiomyopathy.

Fabry disease is a rare X-linked lysosomal disorder affecting 1:58,000 in the Danish population (males 1:85,000; females 1:44,000) [1]. A mutation in the gene encoding the enzyme alpha-gal A, an essential enzyme in normal lysosomal function, causes progressive cellular accumulation of the glycosphingolipids, especially globotriaosylceramide (Gb3). This leads to a severe disruption of cellular function. Men with a classic phenotype present with no or very low alpha-gal A-activity and develop early multi-organ involvement, especially renal and cardiac disease, resulting in a severely impaired prognosis [2]. However, both men and women can be affected in the presence of a disease-bearing mutation [2,3]. Females and men with a non-classic phenotype can also present with early organ involvement. However, their presentation is often more heterogeneous. While the classic male phenotype evidently need early initiation of therapy, the need of treatment is less clear in females and in males with a non-classic phenotype [3]. Furthermore, the incidence of new genetic variants of uncertain clinical significance, possibly indicating a Fabry diagnosis, has increased due to the general implementation of genetic screening programs [4-6]. At present, approximately 100 patients in Denmark are diagnosed with a disease-bearing mutation and followed at the Danish National Fabry Centre at Copenhagen University Hospital - Rigshospitalet. The continuing clinical challenge of who will need and when to initiate treatment necessitates close monitoring of patients at risk and, thus, a continued search for precise, reliable methods able to detect early cardiac involvement. Early initiation of therapy prior to the full manifestation of Fabry disease has shown to impede progression while evidence suggests a late initiation of treatment has reduced effects [7-10], further stressing the importance of early detection of Fabry cardiomyopathy and thus, early initiation of treatment.

Cardiomyopathy in Fabry disease In Fabry disease, the complication of greatest prognostic impact is cardiac manifestations, herein including arrhythmias, heart failure and cardiac death [2,3,11]. Although, the progressive deposition of Gb3 accounts for a maximum of 5% of total cardiac volume [12-14], a disproportionate cardiomyocyte hypertrophy, coronary wall thickening and endothelial dysfunction have been noted as general findings [12-14]. Indeed, left ventricular hypertrophy (LVH) has long been a hallmark of Fabry cardiomyopathy [15], however, the disproportionate relationship between a relatively small accumulation of Gb3 and the clinical cardiac manifestation of pronounced LVH has led to the proposal of the accumulation of Gb3 per se causes an early disruption of cellular function by pathways involving oxidative stress and inflammation [14-18]. The stress induced by Gb3 is believed to exacerbate left ventricular mass increase, cellular apoptosis, and cause the irreversible substitution of functioning tissue with reparative fibrosis. A key site and mechanism of stress and perhaps an early indicator of disease may, therefore, be found investigating changes across the vascular wall. Not only does Gb3 accumulation cause structural changes [12-14,19], Gb3 have been shown to induce the production of reactive oxygen species (ROS) through important inflammatory pathways such as transforming growth factor (TGF) β-dependent signaling, a key step in the Fabry-related vasculopathy preceding fibrosis [18]. The early structural changes in the endothelium might, therefore, tie directly to early and detrimental dysfunction [18,19].

Fabry-related cardiomyopathy and imaging As one of the most distinguishing factors of Fabry cardiomyopathy, the ability to accurately detect LVH is paramount. Recognizing the improved spatial resolution of CMR imaging, a shift from echocardiography to CMR has recently caused CMR to be recommended as part of routine clinical practice in supplement to echocardiography to improve detection of changes in left ventricular mass [15,20,21]. However, the addition of CMR-based approach has revealed several image-derived parameters of interest, which may provide insight into key aspects of the underlying mechanisms of Fabry disease, such as Gb3 accumulation, changes in fibrotic burden, and inflammation in the early stages of disease [15]. In general, Fabry cardiomyopathy often presents with low native T1 values irrespective of the presence of LVH, which have been suggested as an indirect measure of Gb3 burden [15,16,22]. In comparison, reparative fibrosis increases T1-values [15,16,22]. Furthermore, increased T2-values could be an indirect measure of inflammation [15-17], and interestingly, T2-values have been shown to decrease in concert with decreases in left ventricular mass following enzyme replacement therapy (ERT) [16,21]. Despite its promise, the overall use of T1 and T2 mapping has, however, not yet been implemented in clinical practice.

In comparison, PET/CT-based imaging has shown promise by detecting early Fabry-related changes such as coronary microvascular disease (CMD), which by itself provides important prognostic information [23]. However, use is limited due to radiation. The detection of CMD can elucidate on the progression of vascular endothelial dysfunction and may even be a key step in detecting early disease. Not only is the degree of CMD associated with the degree of LVH [24-26], of note, CMD seem to precede changes in left ventricular mass, as signs of CMD have been found irrespective of sex or the presence of LVH [24-26], suggesting its use is instrumental in detecting the early steps of Fabry-related cardiomyopathy.

Heterogeneity and regional disease progression? In Fabry, the cardiac involvement is believed to progress diffusely throughout the myocardium, with symmetric LVH as a key finding. However, of note, previous reports show great regional heterogeneity in the measured T1- and T2-values as well as regional differences using strain analysis to detect functional decline [15-17,22]. Furthermore, low T1-values, believed to be a pathognomonic feature of Fabry-associated cardiomyopathy, has been proposed to increase and pseudo-normalize with disease progression and the development of fibrosis, making the ability to account for change over time especially important [16]. CMR and PET/CT separately provide global measures of fibrosis, inflammation, and microvascular function, therefore, the combination of modalities may explain the regional differences specific to the individual patien. A combined approach may therefore provide key insights into the pathology of Fabry-associated cardiomyopathy - especially important in distinguishing early and late-stage disease.

Patients with Fabry disease (1)

Inclusion Criteria:

• Male and female individuals with a genetically-verified diagnosis of Fabry disease
• ≥ 18 years of age
• Able to give informed consent

Exclusion Criteria:

• Any contraindication against a pharmacologically induced rest-stress PET/CT protocol according to local safety procedures such as acute coronary syndrome, severe bronchospasm, severe chronic obstructive pulmonary disease, cardiac arrhythmia.
• Any contraindication for MRI according to standard checklist used in clinical routine, including claustrophobia or metallic foreign bodies, metallic implants, internal electrical devices, or permanent makeup/tattoos that cannot be declared MR compatible.
• Pregnancy

Age and sex-matched healthy controls (2)

Inclusion Criteria:

• ≥ 18 years of age
• Able to give informed consent

Exclusion Criteria:

• A genetically-verified diagnosis of Fabry disease.
• Family member to a patient with a genetically-verified diagnosis of Fabry disease
• Cancer expected to influence life expectancy.
• Known heart failure, previous apoplexy or previously established kidney disease.
• Initiation or change of antihypertensive therapy within 3 months of enrollment.
• Known LVH as evaluated on echocardiography
• Any contraindication for a pharmacologically induced stress PET/CT protocol according to local safety procedures such as acute coronary syndrome, severe bronchospasm, severe chronic obstructive pulmonary disease, cardiac arrhythmia.
• Any contraindication for MRI according to standard checklist used in clinical routine, including claustrophobia or metallic foreign bodies, metallic implants, internal electrical devices, or permanent makeup/tattoos that cannot be declared MR compatible.
• Pregnancy

1. Patients (adult) with genetically-verified diagnosis of Fabry disease.
2. Age- and sex-matched healthy controls.

Due to national legistlative restrictions, unrestricted access to individual participant data is not possible. However, data exchange will be possible upon reasonable request under the assurance of data-management in accordance with Danish law.