Novel GLA Variants: 10 Experts’ Tips for the Clinical Assessment of Fabry Disease

1. Introduction

Fabry disease (FD) (OMIM #301500) is a rare, X-linked lysosomal storage disorder (LSD) caused by mutations in the GLA gene located on chromosome Xq22 which lead to enzyme α-galactosidase A (α-GalA) deficiency and systemic accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), resulting in multisystem involvement, with clinical manifestations including skin and eye lesions, neuropathic pain, gastrointestinal issues, and life-threatening renal, cerebrovascular, and cardiovascular complications [1]. Prevalence of FD ranges from 1 in 40,000 to 1 in 170,000 and its highly variable phenotypic expression is influenced by factors such as biological sex and the specific GLA mutation, which can lead to either classic or late-onset subtypes [2,3,4]. To date, more than 1000 mutations in the GLA gene have been identified with genotype-phenotype correlations which are not always clearly established [5]. FD male patients often exhibit a more severe clinical course, while heterozygous females carrying the same GLA variant usually show a wider spectrum of symptoms due to X-chromosome skewed inactivation (i.e., lyonization), thereby affecting residual α-GalA activity [6]. Current treatment options include enzyme replacement therapy (ERT) using agalsidase-α, agalsidase-β, and pegunigalsidase alfa, and oral chaperone therapy with migalastat, to mitigate the progression of organ damage [7]. Timely therapy initiation can significantly influence disease trajectories, thus prompt and accurate diagnosis is paramount [8,9]. However, treatment response varies across patients, highlighting the need for a deeper understanding of individual determinants of disease expression, starting from the underlying genetic variant [10]. A significant issue in FD is the interpretation of Variants of Unknown Significance (VUS) which are GLA gene nucleotide variants whose association with the disease remains unclear and sometimes never described before [11]. According to the classification by the American College of Medical Genetics and Genomics (ACMG) revised together with the Association for Molecular Pathology (AMP) and the College of American Pathologists (CAP) genetic variants in Mendelian disorders are classified as pathogenic (P), likely pathogenic (LP), VUS, likely benign (LB), and benign (B) [12]. When a GLA variant is identified, a comprehensive evaluation is recommended in order to decipher its nature not only for guiding treatment or avoiding any useless therapy, but also for risk assessment as to other family members [13]. The term "novel” GLA variants refers to previously unreported alterations in the GLA gene, whereas the use of "de novo” indicate mutations that have already been described and since they arise spontaneously without any apparent parental inheritance their detection involves a careful familial lineage assessment [14]. The topic of GLA variants in FD is rather controversial with a number of them (e.g., p.D313Y, p.E66Q, and probably p.R118C) which were initially described as pathogenic, and were later classified as polymorphisms, therefore having a frequency >1% in the general population [15,16]. Nowadays, technologies such as DNA Next Generation Sequencing (NGS) easily identify gene variants which must be supported by clinical, biochemical, and histopathological evidence in order to determine their clinical impact [17,18]. GLA variants can occur in a single or few families, moreover epigenetic factors can increase variability and complicate the assessment of any genotype-phenotype correlations and in vitro studies such as GLA expression assays are needed [19,20]. However, this type of testing is available only in a limited number of specialized research laboratories which have to deal with an increasing work-load due to more frequent systematic screening protocols of high-risk populations and newborns, leading to more frequent detection of GLA variants and an increasing need for standardized guidelines for their interpretation [21,22,23,24,25]. A multidisciplinary Board of Experts discussed and shared their experiences with respect to the most recent literature findings in order to draw up a list of tips for the assessment of patients presenting with novel GLA variants. The aim of this publication is to provide some practical indications as to which diagnostic path might be the best to accurately investigate these cohorts and, when deemed necessary, to start treatment as soon as possible in order to improve disease outcomes. The statements hereby presented are meant to be the foundation for a more structured diagnostic algorithm to effectively and promptly manage GLA variants in clinical practice.

2. The Issue of Gene Variant Classification

With the advent of NGS, genetic laboratories are performing more complex genetic testing spanning genotyping, single genes, gene panels, exomes, genomes, transcriptomes and epigenetic assays for genetic disorders, with new challenges in sequence interpretation [26,27]. When classifying a variant, laboratories find valuable information in population and disease databases, as well as in published literature, genetic reports often include clinical assertions provided by physicians, while the ClinVar database stores variants with clinical observations and tracking of review status to enable more transparency [28]. Re-analysis of variants is a continuous process, previous gene classifications are constantly modified, and the availability of large population variant frequency data has led a few of them to be re-classified [29]. Optimal results are realized when referring healthcare providers and laboratories work collaboratively, and accurate and complete clinical information sharing is essential for the interpretation of genome-level DNA sequence findings [30,31]. For tests that cover a broad range of phenotypes (i.e., large panels, exome and genome sequencing), laboratories may find candidate causative variants, and further follow-up with physicians for additional evidence to support the classification [32]. Testing other family members is a key-step to establish if a variant is de novo, or it co-segregates with a disease in the family, or it is in trans with a P variant in the same recessive disease-causing gene [33]. Collecting parental samples alongside with that of the proband, i.e., trio testing, is recommended in the setting of exome and genome sequencing, particularly for suspected recessive or de novo causes, and in the absence of one or both parents, the inclusion of affected and unaffected siblings can be valuable [34,35]. A number of genetic variants result in a highly variable phenotypic expression and penetrance, further stressing the importance of providing comprehensive clinical data for their interpretation [36]. A pivotal issue is how to integrate genetic evidence into clinical decision-making, and in newborns or adults enzyme assays and laboratory tests, histological and physical findings, along with imaging studies may allow variants to be reclassified [37]. Therefore, monitoring of the patient is recommended while clinical and instrumental evaluations are ongoing in order to use this information in clinical decision-making [38]. Another challenging issue related to variants’ definition and classification is that of allele frequency (AF), whose value < 1% usually supports pathogenicity, while an AF >1% normally indicates benignity. However, the most frequent pathogenic GLA variants [i.e., p.(Asn215Ser) in Caucasians and IVS4 + 919G>A in Asians] occur at a frequency <1%, while a few GLA variants proven as benign [i.e., p.(Glu66Gln), p.(Arg118Cys), p.(Ser126Gly), p.(Ala143Thr), p.(Asp313Tyr)] do not meet the >1% frequency threshold [9]. A variant should be considered as LB if the AF is > 0.0125% (i.e., FD prevalence: 1/8000) and higher than the frequency of the most common pathogenic allele [39]. However, since FD is considered underdiagnosed, excluding a variant as non-pathogenic solely based on high AF may be misleading. Once a missense variant has been recognized as being pathogenic, the next step is to determine its association with a specific phenotype. The number of gene variants responsible for classic FD is far superior than the number of those associated with late-onset phenotypes which highly segregate within populations and consequently have a higher prevalence, due to their overall lower morbidity and minimal effects on genetic fitness [40,41].

3. Diagnostic Setting

To assess any genotype-phenotype correlation as to novel GLA variants, it is crucial to clarify the diagnostic context: GLA variants may represent the incidental finding of a gene/exome panel, or a wider genome sequencing analysis to assess another Mendelian condition in an adult subject [42,43]; a different scenario is that of an adult or a newborn within a neonatal, prenatal, or familial screening since they belong to a FD-positive or a GLA variant-positive family group [44]; finally, probands may have undergone genetic testing since clinical signs/symptoms strongly suggestive of FD, such as angiokeratomas, cornea verticillata and others are present [45]. Depending on the context in which novel GLA variants emerge and in consideration of any already available data, the aim is to complete the diagnostic framework. If not already available, laboratory tests are initiated or repeated to verify the metabolic profile, and multi-organ or organ-specific clinical, histological, and instrumental evaluations are carried out in order to clarify the nature of the GLA variant. Moreover, the identification of novel GLA variants during newborn screening in asymptomatic subjects might be challenging, since the mother may be the carrier of a skewed inactivation, and if no other male family member carries the same variant the clinical assessment of these GLA variants relies on functional studies.

Experts’ Tips

• Verify whether the genetic test was performed following a clinical sign/symptom suggestive of FD, or it was part of an extended or selective screening (i.e., prenatal, neonatal, familial), or it was due to an incidental finding during the genetic assessment of another Mendelian condition;
• Check for signs/symptoms of FD in relatives up to the third degree to assess family pedigree. Family members who test positive in the genetic assay should follow the diagnostic procedure of the proband.

4. Metabolic Profile

Preliminary laboratory testing can confirm or rule out suspicion of FD when a novel GLA variant is identified, and the assessment of metabolic profile is based on measurements of α-Gal A activity and lysoGb3 levels [46].

• Normal α-Gal A activity in leukocytes is considered the gold standard to rule out the biochemical diagnosis of FD in males. Enzyme assay on dried blood spot (DBS) is generally used for screening and both plasma and leukocyte enzyme activity should be assayed as some pathogenic variants affect intracellular trafficking or packaging/secretion of the enzyme, therefore the reduction in enzyme activity in plasma is more evident than in leukocytes [47]. Enzyme levels in classical males are generally <1%, values may range between 1% and 20% in late-onset patients, whereas α-Gal A activity can be normal in females due to random X-chromosomal inactivation [48].
• Unlike Gb3, lysoGb3 is a soluble molecule which is more readily excreted from cells [49]. The production of lyso-lipids to reduce cellular accumulation is common strategy in LSDs [49], and in FD patients the excess of Gb3 is converted into lysoGb3, which is transported to the liver and secreted with the bile [50]. Plasma lyso-Gb3 has been increasingly described as a sensitive biomarker of FD [51,52,53,54]. A few studies have pointed out that lyso-Gb3 levels correlate with cardiac disease severity [55,56], and it was reported that individuals with a novel GLA variant and organ involvement consistent with FD had lyso-Gb3 levels ≥2.7ng/mL, whereas in subjects with a novel GLA variant and no organ involvement lyso-Gb3 levels were <2.7 ng/mL [57]. Moreover, lower levels of lyso-Gb3 at treatment initiation are correlated with better long-term outcomes [58,59], and cumulative exposure to lyso-Gb3 during prolonged untreated period appears to predispose to worse response to therapy [48,61].

Experts’ Tip

• Carry out α-Gal A assay on DBS and cells (i.e., leukocytes, fibroblasts), and lysoGb3 measurement on DBS for preliminary metabolic assessment of GLA variants.

5. Renal Profile

In classic patients, Fabry nephropathy is related to a progressive decrease in renal function with deposition of Gb3 in all cell types (i.e. vascular endothelial cells, vascular smooth muscle cells, mesangial cells, interstitial cells, podocytes, and distal tubular epithelial cells) during fetal development [62]. Increased microalbuminuria and proteinuria may already occur before 10 years of age, decline in glomerular filtration rate (GFR) along with hyperfiltration is seen from the adolescence, progressive chronic kidney disease (CKD) with overt albuminuria and proteinuria is described in the second decade of life, while chronic renal insufficiency and end-stage renal disease (ESRD) develop between the 3rd and 5th decades leading to dialysis especially among classic male patients, while kidney dysfunction is less prevalent and milder among heterozygous females [63,64,65].

• When a GLA variant is identified complete laboratory investigations of kidney function should be carried out, i.e., serum creatinine, estimated GFR (eGFR) along with cystatin C, urinary protein, albumin and/or microalbumin as well as urinary sodium excretion [66]. Assessment of proteinuria and GFR can be used for staging CKD, while urinary protein excretion is strongly associated with renal disease progression in FD, regardless of the gender. In particular, a high value of eGFR (> 135 ml/min/1.73 m2) should be accurately assessed in a suspect of hyperfiltration in a young individual, potentially irreversible histological changes can be observed in renal biopsies of children before the onset of albuminuria [67], and histological evidence of Gb3 accumulation as well as cell and vascular injury in renal tissue can be present prior to microalbuminuria and clinically significant renal events [68]. Therefore, although albuminuria and proteinuria are the most commonly used renal biomarkers, they show low sensitivity in identifying incipient nephropathy, and proteinuria may not be evident in patients with advanced kidney disease and does not necessarily correlate with GFR decline [69].
• Gb3 is elevated in the urine of FD patients especially when no residual enzyme activity is present, while lower or normal levels are reported in female heterozygotes and in cohorts with residual enzyme activity [70]. Therefore urine Gb3 is not an ideal biomarker for FD diagnosis especially when a GLA variant is detected. On the other hand, albuminuria A2 and proteinuria (> 150 mg/day) which are usually the first signs of renal involvement in FD have been described as biomarkers for diagnosis, evaluation of kidney damage and follow-up after therapy initiation along with serum creatinine, cystatin C, beta 2-microglobulin (β2M), and neutrophil gelatinase-associated lipocalin (NGAL), while podocyturia and urinary excretion of CD80 may indicate early, subclinical renal involvement [64,71,73,74,75,76,77].
• Urine microscopy can highlight some basic findings for diagnosing and assessing FD progression, i.e., Maltese cross particles, urinary mulberry cells, and podocyturia, which can be useful in the overall assessment of a GLA variant, even though they are not pathognomonic of the condition [78,79,80]. Maltese cross particles are vacuolated epithelial cells filled with glycosphingolipids visualized through polarized light microscopy [81]. Urinary mulberry cells are distal tubular epithelial cells in which Gb3 has accumulated leading to a lamellar appearance, and they can be detected before kidney damage is identified [82]. Since the loss of urinary podocytes correlates with glomerular injury, it has been suggested as a potential diagnostic tool and to guide treatment strategies in FD nephropathy [83]. In FD patients with normal levels of albuminuria or proteinuria, the amount of urinary podocytes can be significantly higher when compared to healthy individuals, suggesting that this biomarker indicating a clinically silent kidney damage may precede proteinuria [84]. Moreover, podocyturia is described as inversely related to GFR in male patients and it is correlated with clinical severity in FD nephropathy, displaying a potentially prognostic value [64,85].
• Routinely ultrasound (US) renal examination may detect parapelvic cysts (PC) whose presence should prompt to consider FD diagnosis especially in subjects with an unclear family history of renal disease, or when other stigmata of the disease are evident [86]. Even though the presence of PC is not currently regarded as a pathognomonic sign of FD and its pathogenesis is unclear, its prevalence in FD patients ranges from about 30% to 40%, compared with cohorts affected by other renal conditions or with the general population in whom it is reported between 1% and 6% [86,87,88,89].
• Renal biopsy has become a valuable tool in complex genetic cases especially when results of biochemical and molecular analyses are inconclusive [90]. Kidney biopsy provides distinctive features to differentiate renal diseases, especially when clinical context, genetic data, and family history are unclear [91]. The International Study Group of Fabry Nephropathy (ISGFN) highlighted the role of kidney biopsy as part of the baseline assessment of FD nephropathy as renal tissue involvement precedes clinical signs [82]. Early and prominent accumulation of Gb3 leads to cytoplasmic vacuolization and the presence of characteristic concentric lamellated inclusions (i.e., zebra bodies) predominantly observed in podocytes at electron microscopy [92]. Even though zebra bodies are hallmark features of FD, their identification necessitates a thorough evaluation to rule out other potential causes, such as rheumatoid arthritis, lupus nephritis, and hydroxychloroquine-chloroquine, and carbamazepine induced phospholipidosis [92,93]. Proximal and especially distal tubular cells exhibit vacuolization due to Gb3 deposits which are also found in glomerular endothelial and mesangial cells, contributing to glomerular dysfunction [94]. Segmental and global glomerulosclerosis are common findings even in early-stage CKD and correlate with disease progression and renal function decline, as well as interstitial fibrosis and tubular atrophy [95]. Finally, Gb3 accumulation in vascular smooth muscle and endothelial cells leads to arteriolar hyalinosis along with myo-intimal cell proliferation and endothelial thickening, contributing to ischemic injury and hypertension especially in male patients leading to worse renal outcomes [96,97].

Experts’ Tips

• Perform repeated measurements of: a) eGFR (check also for hyperfiltration) with creatinine and cystatin C; b) albuminuria/proteinuria (ACR/PCR), and 24 hrs proteinuria if sample spot samples are positive; c) urinary sediment; d) renal ultrasound to check for the presence of PC.
• Perform kidney biopsy in all subjects with renal symptoms suggestive of FD, and carry out electron microscopy histological examination, along with immunohistochemical investigation for Gb3 deposits. In asymptomatic subjects, evaluate whether to perform a kidney biopsy only in specific cases and based on available evidence of other FD-related organ involvement.

6. Cardiological Profile

Lysosomal sphingolipid storage in FD occurs in all cardiac cell lines, i.e., myocytes, fibroblasts, conduction tissue, and endothelial, smooth muscular, and endocardial cells [98]. Cardiac involvement represents a leading cause of impaired quality of life (QoL) and mortality in both classic and later-onset FD, and the prevalence of FD in adult cohorts with unexplained left ventricular (LV) hypertrophy (LVH) ranges from 0.5 to 1% [99]. Cardiac damage remains subclinical for many years before FD symptom onset. While early extracardiac manifestations may suggest diagnosis in classic patients, in late-onset cardiac phenotype FD diagnosis may be more challenging, and FD cardiomyopathy needs to be differentiated from other more frequent forms of LVH [100].

• As to cardiovascular biomarkers, the amino terminal fragment of the brain natriuretic propeptide (NT-proBNP) has been studied as a diagnostic and prognostic predictor of heart disease in FD cohorts [101], while sphingosine-1-phosphate (S1P) was identified as a biomarker involved in cardiovascular remodeling in FD with a strong correlation with LVM index. High-sensitivity cardiac troponin (i.e., hs-cTnI and hs-cTnT) serves as a sensitive blood biomarker for detecting and staging Fabry cardiomyopathy, correlating strongly with imaging markers of myocardial involvement such as late gadolinium enhancement and left ventricular hypertrophy. Elevated troponin levels in FD patients reflect ongoing myocardial damage and fibrosis rather than acute coronary events, and a defined low-threshold hs-cTn (<8.5 ng/L) can reliably exclude significant cardiomyopathy. Serial measurements may therefore aid in early diagnosis, risk stratification, and monitoring of disease [102].
• Electrocardiogram (ECG) can show early indicators of FD [103]:
  • PR interval is frequently altered in FD, and PR shortening can be detected in the initial phase of the disease, even with no LVH, while atrioventricular (AV) blocks are more frequent in the advanced phase of the cardiac disease [104].
  • FD also affects the cardiac conduction system at all levels, from the sinoatrial node to the distal ramification of Purkinje fibers leading to cardiac rhythm alterations. The mechanisms of bradyarrhythmia in FD are not completely understood, but post-mortem histological studies described fibrosis and apoptosis of cardiac conduction tissue [105]. On the other hand, factors such as progressive Gb3 and lyso-Gb3 accumulation, atrial remodeling, and diastolic dysfunction may be linked to atrial fibrillation (AFib), which is considered an intermediate-to-late FD complication [106].
  • Similar to PR interval, QRS modifications in FD are correlated with the early or advanced phase of the cardiac disease. During the initial phase of the disease, acceleration of intra-ventricular conduction and QRS narrowing could be observed, whereas in later disease stages a progressive degeneration of myocardial conduction system takes place, leading to QRS prolongation [107]. Other studies demonstrated right bundle branch block (RBBB) more commonly than left bundle branch block (LBBB) in FD patients and the reasons for the peculiar susceptibility of the right bundle branch are unclear [108].
  • FD-related ECG abnormalities also involve LV repolarization, and studies reported ST segment and T wave alterations as frequent findings in these patients [109].
• Bidimensional (2D) echocardiography and cardiac magnetic resonance (CMR) could provide further evidence to confirm or rule out FD diagnosis [110,111,112,113]. Severe LVH in female patients usually develops later, replacement fibrosis can be identified by CMR before hypertrophy appears, and according to the Fabry Outcome Survey (FOS) about 46% of female FD patients develop cardiac symptoms [114,115]. In late-onset FD, cardiomyopathy is often the only manifestation and LVH (i.e., LV maximum wall thickness ≥ 12 mm in males) is typically found at echocardiography [111]. Although diffuse concentric LVH is the most prevalent finding, regional LVH of the septum may cause dynamic LV outflow tract obstruction (LVOTO) with systolic anterior motion of the mitral leaflet [115]. Other presentations include mid-ventricular obstruction, papillary muscle hypertrophy, and distal LVH, while hypertrophy of the right ventricle (RV) and aortic root dilation with preservation of ejection fraction are also ultrasound FD-related findings [116,117,118]. The prevalence of aortic dilatation in FD increases with age due to degenerative changes of the aortic media caused by glycolipid accumulation in the endothelium [119]. The prevalence of RV hypertrophy (RVH), i.e., wall thickness > 5 mm, ranges between 40% and 70% in FD affecting males and females equally, and correlates to disease severity [120].
• CMR including late gadolinium enhancement (LGE) and T1 mapping is advised for further characterization of myocardial tissue in high-risk patients with GLA variants. Moreover, CMR allows assessment of FD structural changes at the inferolateral wall and anteroseptum, early detection of increased myocardial trabeculation, left atrial dilatation, RVH, along with impairment of atrial deformation and compliance likely caused by Gb3 accumulation [121]. LGE imaging enables the detection of myocardial replacement fibrosis showing a mid-wall distribution in the basal-infero-lateral area of the LV, which should raise FD suspicion in patients with unexplained LVH [122]. The prevalence of LGE ranges between 40% and 65% and it correlates with the severity of LVH in male FD patients, and may precede the development of LVH especially in women. LVH has been described in other conditions, i.e., hypertension and aortic stenosis, hypertrophic cardiomyopathy, and cardiac amyloidosis, therefore substantial value to FD differential diagnosis is added by mapping in which individual image pixels are colour-coded for longitudinal (T1) or transverse (T2) magnetism [123]. FD typically lowers myocardial native T1 as a result of intracellular glycosphingolipid storage, with a concentric T1 attenuation pattern in the early phases of the disease before LVH is detected [124,125]. Indeed, reduced T1 is described in approximately 40% of LVH-negative patients and correlates with early electrocardiographic changes and disease progression, becoming prominent in the advanced phases of the condition. However, the signal may become weaker with inflammation and fibrosis and even reverse in the presence of extensive LGE, thus T1 mapping should be interpreted with caution [126]. T2 is typically elevated in areas of LGE and it correlates with troponin rise in FD supporting the role of inflammation in FD cardiomyopathy. In line with positron emission tomography (PET) and endomyocardial biopsy (EMB) findings, Gb3 accumulation leads to myocardial inflammation detected by histological examination in up to 56% of patients [127,128].
• EMB findings in FD show that myocyte histological changes precede imaging findings: cardiomyocyte vacuolization is observed before LVH, Gb3 storage occurs before T1 lowering, and fibrosis is described before LGE [111,129]. Therefore, EMB in high-risk subjects with cardiological symptoms, i.e., angina, dyspnoea, and palpitations needs to be considered when a GLA variant is detected [130,131].

Experts’ Tips

• Perform a complete cardiological evaluation with measurement of cardiac biomarkers, evaluation of heart function by ECG, Holter, echocardiogram, and CMR with T1 and T2 mapping to check for evidence of FD signs.
• Perform endomyocardial biopsy in high-risk patients with heart symptoms and signs suggestive of FD, when the heart appears to be the only organ involved.

7. Neurological Profile

The neurological involvement of FD refers to both the peripheral (PNS) and central nervous system (CNS) and this latter includes the VIII cranial nerve and the neuro-enteric plexus.

• PNS involvement in FD presents as a ganglionopathy due to Gb3 accumulation in the dorsal root ganglion neurons and Schwann cells, which causes loss of cell functions with secondary degeneration of the afferent bundles of nerve fibers in their distal site. Studies have shown reduction of both myelinated and non-myelinated fibers, and decreased intraepidermal nerve fiber density [132,133]. Small fiber neuropathy (SFN) in FD does not display specific features and involves autonomic fibers, responsible for acroparesthesias, neuropathic pain, abnormal thermal sensation, impaired sweating, and GI dysmotility [134]. The VIII cranial nerve is also involved leading to hearing loss and imbalance [135]. Fabry-related neuropathic pain due to SFN is often the earliest manifestation of FD in children, being described in boys as early as 3 years of age or less, and later on in girls [136]. Fabry crises are characterized by an agonizing burning sensation originating in the acral segments of the limbs, radiating in a distal-to-proximal direction, which may be precipitated by an increase in body temperature due to exercise, fever, warm environments secondary to impaired sweating [137]. In adulthood, pain is usually less severe, even though its prevalence is described as high as 80% and heavily conditioned by comorbidities, e.g., chronic kidney disease [138]. However, FD is only one among multiple causes of SFN and in patients without any systemic involvement a pure SFN phenotype never leads to a straightforward FD diagnosis [139,140].
• CNS involvement in FD includes a more commonly reported cerebrovascular phenotype, due to the damage in the endothelial lining and in the smooth muscle cells of the tunica media, and a less frequently described direct neuronal involvement, showing a prodromal neurodegenerative clinical phenotype [141]. Cerebrovascular manifestations in FD may include both ischemic and hemorrhagic stroke, as well as chronic small vessel disease (SVD) without any acute stroke, and the large artery involvement usually presents as dilatative arteriopathy which is a rather common finding also in other monogenic diseases and in the general population due to ageing and other risk factors [142,143,144]. Neuroradiological findings of FD on magnetic resonance imaging are non-specific and include white matter hyperintensities (WMHs) with variable severity degree, small vessel infarcts (SBI), lacunae, enlarged perivascular spaces (EPVs), and cerebral microbleeds [145].

Experts’ Tips

• Carry out a thorough case history to assess the presence of other systemic signs and symptoms of FD and a complete neurological examination to select the subjects to be referred to further specific instrumental investigations.

8. Systemic Signs and Symptoms

Systemic signs and symptoms sometimes represent the earliest manifestations of FD and can be the reason for genetic assessment, or they can help to decipher genetically unclear results in subjects who presents cardiac, renal or neurological findings raising FD suspicion.

• Absence and decreased ability to sweat, i.e., anhidrosis and hypohidrosis, which leads to hyperthermia, poor exercise tolerance, and altered fever manifestation are reported in 53% of classic males and 28% of females with FD. Sweating impairment is due to sympathetic sudomotor fibres dysfunction and it can be measured by means of Quantitative Sudomotor Axon Reflex Tests (QSART) [146].
• Auditory and vestibular abnormalities due to involvement of cranial nerve VIII are frequently reported in classic phenotype of FD ranging from hearing loss, to tinnitus and vertigo, and hearing impairment has been described as an early indicator of an underlying and undetected FD in females and otherwise asymptomatic individuals, even though these are very common complaints also in the overall general population [147].
• Gastrointestinal (GI) dysfunction is common in FD and it is associated with deposition of Gb3 in the autonomic ganglia and mesenteric blood vessels of the gut [148]. GI symptoms include abdominal pain, bloating, diarrhea, constipation, nausea, and vomiting, as well as failure to gain weight and often appear early in classic patients [149]. In order to carry out differentiation with Irritable Bowel Syndrome (IBS), techniques such as standard and video capsule endoscopy, scintigraphy, and intestinal biopsy can be used [150]. Radiographic studies may reveal thickened, edematous colonic folds, mild dilatation of the small bowel, granular-appearing ileum, and the loss of haustral markings throughout the colon [151].
• Ophthalmological signs in FD occur as early as the first decade of life and patients can display cornea verticillata, Fabry cataract, as well as increased retinal vessel tortuosity, conjunctival telangiectasias and aneurysm-like formations, but none of these signs affects visual function [152]. Cornea verticillata (i.e., whorl-like, linear pigmentation in the inferior part of the cornea observed by slit-lamp microscopy) is considered a pathognomonic sign of FD occurring in the majority of classical males, even though therapy with amiodarone or chloroquine can result in similar findings [153]. Fabry cataract is characterized by posterior capsular whitish spoke-like deposits of granular material and it is considered to be another pathognomonic sign of FD, even though mannosidosis might cause similar lens opacities, while increased ocular vessel tortuosity has also been reported in fucosidosis as well as in healthy individuals [154]. Due to the early onset of ocular signs, ophthalmologists have the opportunity to identify FD before the condition is advanced, therefore any patient with cornea verticillata, or Fabry-like cataract especially in combination with retinal vascular tortuosity, conjunctival vascular telangiectasia, or lenticular opacities, should undergo a comprehensive review of symptoms, family history and genetic testing, after a careful exclusion of other causes [155].
• Skin abnormalities have been reported in 78% of males and 50% of females with classic FD [156]. Angiokeratomas are the most common dermatological sign occurring in 66% of males and in 36% of females with classic FD, even though they can also appear in other LSD (i.e., mannosidosis, fucosidosis, sialidosis, b-galactosidase deficiency, and Schindler disease) [157]. Angiokeratomas are described as 1-5 mm, pinkish, dark red, blue-black, non-blanching macules or papules which increase in number and size with age and appear on the umbilicus, hands, knees, elbows, and upper back and chest regions especially in female subjects, spreading to the genitals mainly in males during adolescence [158]. Telangiectasia in sun-exposed areas is the second-most common dermatological sign in FD, and they appear as small superficial angiomas due to cumulative damage of the vascular endothelial cells with vessel dilatation [159]. Although skin biopsy may be a useful additional diagnostic test, findings can be normal in heterozygous females [160]. Nonetheless, FD should be considered in patients when angiokeratoma corporis diffusum is accompanied by other dermatological signs, such as linear telangiectasia and sweating impairment [161].

Experts’ Tips

• Assess the presence of early-onset systemic symptoms, e.g., GI alterations, acroparesthesias, and carry out a differential diagnosis;
• Assess the presence of early-onset pathognomonic or highly suggestive signs of FD, i.e., cornea verticillata, angiokeratomas, hypoacusia, hypohidrosis/anhidrosis, hypertension.

Table 1. Complete list of Experts’ tips for assessment of GLA variants in Fabry disease.

Items	Experts’ tips
DIAGNOSTIC SETTING	1	Verify whether the diagnostic procedure is started following a clinical sign/symptom suggestive of FD, or if it is part of an extended or selective screening (i.e., prenatal, neonatal, familial), or if it is an incidental finding during genetic assessment for another Mendelian condition
FAMILY HISTORY	2	Check for signs/symptoms of FD in relatives up to the third degree to thoroughly assess family pedigree. Family members who test positive in the genetic assay should follow the diagnostic procedure of the proband
METABOLIC PROFILE	3	Carry out α-Gal A assay on cells (i.e., leukocytes, fibroblasts) and Lyso-Gb3 measurement on DBS
RENAL PROFILE	4	Perform kidney biopsy and histological examination in subjects with progressive renal disease of unknown origin along with immunohistochemical investigation for Lyso-Gb3. In asymptomatic women with a highly suspicious family history for FD, evaluate whether to perform a biopsy based on available literature data for therapy initiation
	5	Perform a) measurement of glomerular filtration rate to check for hyperfiltration and proteinuria, b) analysis of urinary sediment, c) renal ultrasound to check for the presence of parapelvic cysts
CARDIOLOGICAL PROFILE	6	Perform endomyocardial biopsy in patients with heart signs/symptoms suggestive of FD, or in the absence of cardiac hypertrophy but with early systemic findings that point to a genetic disorder.
	7	Perform measurement of serological biomarkers and evaluation of heart function by ECG, Holter, echocardiogram, and CMR to check for imaging signs of FD
NEUROLOGICAL PROFILE	8	Carry out a thorough case history to assess the presence of other systemic signs and symptoms of FD and complete neurological examination to select subjects to be referred for further instrumental investigations
SYSTEMIC SIGNS AND SYMPTOMS	9	Assess the presence of early-onset systemic symptoms (e.g., GI alterations, acroparesthesias) and carry out differential diagnosis
	10	Assess the presence of early-onset pathognomonic or highly suggestive signs of FD, i.e., corneal verticillata, angiokeratomas, hypoacusis, hypohidrosis/anhidrosis, hypertension

Table 1. Complete list of Experts’ tips for assessment of GLA variants in Fabry disease.

Items	Experts’ tips
DIAGNOSTIC SETTING	1	Verify whether the diagnostic procedure is started following a clinical sign/symptom suggestive of FD, or if it is part of an extended or selective screening (i.e., prenatal, neonatal, familial), or if it is an incidental finding during genetic assessment for another Mendelian condition
FAMILY HISTORY	2	Check for signs/symptoms of FD in relatives up to the third degree to thoroughly assess family pedigree. Family members who test positive in the genetic assay should follow the diagnostic procedure of the proband
METABOLIC PROFILE	3	Carry out α-Gal A assay on cells (i.e., leukocytes, fibroblasts) and Lyso-Gb3 measurement on DBS
RENAL PROFILE	4	Perform kidney biopsy and histological examination in subjects with progressive renal disease of unknown origin along with immunohistochemical investigation for Lyso-Gb3. In asymptomatic women with a highly suspicious family history for FD, evaluate whether to perform a biopsy based on available literature data for therapy initiation
	5	Perform a) measurement of glomerular filtration rate to check for hyperfiltration and proteinuria, b) analysis of urinary sediment, c) renal ultrasound to check for the presence of parapelvic cysts
CARDIOLOGICAL PROFILE	6	Perform endomyocardial biopsy in patients with heart signs/symptoms suggestive of FD, or in the absence of cardiac hypertrophy but with early systemic findings that point to a genetic disorder.
	7	Perform measurement of serological biomarkers and evaluation of heart function by ECG, Holter, echocardiogram, and CMR to check for imaging signs of FD
NEUROLOGICAL PROFILE	8	Carry out a thorough case history to assess the presence of other systemic signs and symptoms of FD and complete neurological examination to select subjects to be referred for further instrumental investigations
SYSTEMIC SIGNS AND SYMPTOMS	9	Assess the presence of early-onset systemic symptoms (e.g., GI alterations, acroparesthesias) and carry out differential diagnosis
	10	Assess the presence of early-onset pathognomonic or highly suggestive signs of FD, i.e., corneal verticillata, angiokeratomas, hypoacusis, hypohidrosis/anhidrosis, hypertension

9. Conclusions

The increasing detection of novel GLA variants makes it necessary to determine their nature since their misinterpretation can lead to either missed opportunities for early treatment or unnecessary and potentially harmful interventions. The timely start of ERT in FD subjects with the most severe form of the condition makes it possible to slow down and limit damage to vital organs, considerably improving QoL. Furthermore, the identification of new GLA variants makes it possible to extend the tests to other family members. Since FD is a systemic disease characterized by an extremely variable clinical presentation and long-term evolution, the development of recommendations for speeding up the diagnostic process and timely management of the condition requires a complex collaboration among several specialists. The Authors consider the set of tips hereby presented to be an initial stage that will be further optimized with the development of a system for evaluating GLA variants in the form of a detailed algorithm covering the most common clinical scenarios thus providing better guidance for diagnostic procedures.