We observed a frequency of 1.12% of FD in our cohort. There are no epidemiological studies of FD in Brazil, however, the frequency expected for live male births in the general population is 0.0025% (p= 0.0088) (13). In the pediatric population, a Portuguese group studied a cohort of 292 patients with JIA and its association with FD, however, they did not find a classic pathogenic mutation (10).
The ethnicity of the affected child [admixture of Caucasian and Native South American (Indian)] is not included in the populations of higher incidence for FD (2,3).
In this cohort three exonic GLA variants were found. The first variant, c.1244T>C, was in our index case described as pathogenic and referenced by Serebrinsky et al in 2006 as disease-causing according to ACMG variant classification recommendations (17).
The second variant (c.38C>T p.Ala13Val ) has not been described before, and it is located in a non-conserved nucleotide and weakly conserved amino acid position, with small physicochemical differences between the amino acids alanine and valine (Alamut v.2.4). Software analyses by Polyphen-2, SIFT, MutationTaster and Align-GVGD predict this variant as probably benign according to ACMG recommendations of interpretation of sequence variations (17).
The third variant in GLA exon 1 c.48T>G (rs201449986) was considered likely benign (17). This abnormal sequence is not expected to have clinical significance because it does not alter an amino acid residue and is not located within the splice consensus sequence, according to Sequence Project (http://evs.washington.ed/EVS/;). This allele frequency is 1/6728.
Patients with classic FD have no residual or around 30-35% of ⍺-GalA enzyme activity (14). For diagnosis, an increased levels of Gb3 in lysosomes is required (15). The inheritance is X-linked and recessive, which means that female heterozygous genotype presents with incomplete penetrance, due to X inactivation. The mildest disease allows women to have residual enzyme activity; for that reason, the genetic analysis is the gold standard for diagnosis (16). The enzymatic activity can be measured in peripheral blood cells or dry blood spots. In our study, all analysis was done with dry blood spots. The enzymatic activity is very variable among FD patients, and different organs (18). These variations are a challenge to establish thresholds of FD pathogenicity (18).
In this study, we identified 4 (4.5%) patients with decreased levels of α-GalA activity. Three children had a family history and symptoms suggestive of FD. One patient was asymptomatic.
A defective ⍺-GalA leads to accumulation of undegraded substrates [globotriaosylceramide and globotriaosylsphingosine (Gb3 and Lyso Gb3)] inside lysosomes, acting as damage-associated molecular patterns (DAMPs) or stimulating DAMP production. This production activates an inflammatory pathway, inducing apoptosis and a toll-like receptor- 4 (TLR4) mediated innate immune system pro-inflammatory cytokines secretion (IL-1β and TNF-α) (19). These different cellular mechanisms contribute to the different phenotypic expression of FD (20). These cytokines secretion are a characteristic trace of autoinflammatory disorders (21). The recognition of Gb3 or lyso-Gb3 as antigens also influences the invariant natural killer T cell (iNKTs) to induce the release of others inflammatory cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukins (IL): IL-4, IL-5, IL-9, IL10, IL13, and IL-17. This inflammatory cascade produces a continuous stimulus responsible for the induction and maintenance of the autoimmune response. The activation of the above mentioned inflammatory pathway explains the presence of autoimmune and autoinflammatory features in FD. Our index patient had, beyond the classic FD symptoms, a positive ANA and RNP antibodies, and chronic oligoarthritis (bilateral ankles).
The high frequency of cardiac involvement in our cohort (Table 2) is probably related to JIA, which can involve all of the cardiac structures, including pericardium, myocardium, endocardium; coronary vessels; valves and conduction system (22). However, FD also causes cardiac abnormalities including conduction abnormalities, valvular dysfunction, arrhythmias in childhood, progressing to ventricle concentric hypertrophy in non-treated patients (23). Our index patient had mitral valve prolapse with reflux and left ventricle concentric hypertrophy, cardiac manifestations frequently observed in FD.
The main musculoskeletal symptoms described in early FD is acroparesthesia. However, chronic inflammatory joint and bone diseases (polyarticular, oligo and monoarticular, gout, osteoporosis), degenerative joint conditions, neurologic arthropathy (Charcot's foot) (7), Heberden-like nodules (24) and also myositis have been described (8). Nowadays, the coexistence of FD and autoimmune disease has gained increased visibility in the medical literature, and patients with FD and systemic lupus erythematosus (25,26) rheumatoid arthritis (27), autoimmune hypothyroidism (28), Ig A nephropathy (29) and granulomatosis with polyangiitis (30) have also been described. Patients with FD and rheumatic manifestations have a significant delay in FD diagnosis that can last up to 16 years or more (31). The most common associated mutations observed in FD patients presenting with rheumatic manifestations were R118C and A143T (31).
Other interesting finding in our study was the presence of GLA intronic mutations in patients with JIA. Six intronic variants were identified, c.370-81_-77del, c.548-125C>G and c.548-162A> T, c.640-16A> G, c.1000-22C> T, c.-10C > T and c.-12G> A located in introns 2, 3, 4, 6 and in the 5'UTR region of GLA exon 1, respectively. Three single nucleotide polymorphisms (SNP) [c.370-81_-77del (rs5903184), c.-12G>A (rs3027585) and c.-10C> T (rs2071225)] are relatively common to different ethnic groups, with a frequency of the minor allele about 10% in the British population (32), and 12% in Latin populations (OMIM). These SNP are common to the Portuguese population, the greatest ancestor of the Brazilian citizens (33). Those variants were found in 12 (22.22%) c.370-81_-77del, 8 (14.81%) c.-10C> T and 4 (7.40%) c.-12G> A in the 54 tested patients.
As the previous variants, other SNP and its combination, denominated CIH, have been described as associated with Fabry-similar symptoms.
The two SNP of the α-GalA gene c.1000-22C> T [rs2071228] and c.640-16A> G (rs2071397) were associated with the presence of angiokeratomas and acroparesthesias in patients with hypertrophic cardiomyopathies without FD (34). In vitro and in vivo analyzes have shown that polymorphisms in the 5'UTR region can alter the expression of the α-GalA gene, with possible clinical relevance, particularly in male patients with GLA variants associated with a high reduction in enzyme activity (35). The c.-10T allele, found in 15% of the positive results of this study, was previously associated with a decrease in α-GalA activity in leukocytes (33). It has a possible correlation with neurological injuries such as stroke, transient ischemic attack, white matter injury and fine fiber neuropathy, in patients with peripheral neuropathy (36), as well as in patients with FD (35). In our study, this variant was associated with the difficulty of weight gain and ocular changes. Classic ocular manifestations in FD are observed by the age of 4, while heterozygotes present it later, around the age of 10 (37).
The c.-10T allele, located in the 5' non-coding region, has been associated with a decrease in the expression of α-GalA (34), altering in the promoter gene the nuclear protein binding site (38). Studies are still needed to determine the real role of this variant in GLA. Recent data suggested that reduced enzyme activity, with normal levels, may be a risk factor in Parkinson's disease (39). There are numerous descriptions of the c.-10T allele and Fabry-simile manifestations (34).
The most frequent intronic variant in our cohort was c.1000-22C> T (rs2071228), observed in 24 (44.44%) of 54 patients. This variant, located in intron 6, is phenotypically associated with FD, and idiopathic hypertrophic cardiomyopathy by the bank Vep Ensembl. This allele is also associated with some CIH that seem to translate enzymatic alteration with the accumulation of glycosphingolipids (38). Haplotypes are a combination of inherited alleles at adjacent loci. There are numerous reports of groups of alleles causing Fabry simile changes and FD per se. Gervas-Arruga et al, studied a ICH (c.-10C> T , c.369 + 990C> A, c.370-81_370-77delCAGCC, c.640-16A> G, c.1000-22C> T) in the GLA gene. They evaluated the enzymatic levels in cells (fibroblasts and leukocytes) in the plasma and the enzyme's quantitative expression. The results suggested an altered expression pattern of the studied gene, without sufficient abnormality of enzyme levels in plasma, leukocytes, and skin fibroblasts to cause FD. However, glycosphingolipids accumulation in fibroblasts, renal and glomerular tubular cells has been described (38).
Another study described a similar CIH on a GLA in a patient with the early systemic onset of FD. The patient carried only the haplotype (-10C> T, c.370-77_-81del, c.640-16A> G, c.1000-22C> T), suggesting that those variants located in a promoter and intronic regulatory region, could cause disease even without the presence of exonic abnormalities (40). In our cohort, we had 8 (14.8%) of the 54 tests, that presented this same haplotype (-10C> T, c.370-77_-81del, c.640-16A> G, c.1000-22C> T ), including the patient with FD and the patient with c.38C>T variant who presented cornea verticillata. Haplotypes 1 and 2 were associated with visual changes and corneal abnormalities, and haplotype 3 had a positive association with valve changes (table 1).
There was no association of acroparesthesias/peripheral neuropathies with ICHs, despite their incidence in half of our sample. A limited number of genetic tests in our cohort may have influenced the possible positive associations between the variants found and reported clinical signs.
In our JIA cohort we observed a variety of clinical symptoms related to FD. Almost 50% described acroparesthesias, and a third of the patients had difficulty of weight gain, while 42% had family history of stroke. All these features could be associated with chronic arthritis and its treatment. Our index patient was initially treated with enzyme replacement, considering FD was misdiagnosed as JIA. Despite a significant improvement of anhidrosis, muscular, abdominal pain and GB3 levels, he had persistent chronic arthritis course with synovial thickening, suggesting the co-existence of JIA. Methotrexate significantly improved his symptoms. We showed another patient with a VOUS for FD, unfortunately, young age is an obstacle for identification of FD or FD-like symptoms. These patients need extensive follow-up to determine if these mutations are pathogenic or not, especially in females.
We observed FD as a comorbidity in 1.12% of our JIA cohort but the small number of JIA patients in this cohort was a limitation for this study. We only included 50% of our cohort, mostly due to logistic issue (missing appointments, incomplete clinical evaluation).
Other limitation is the genetic test, which should have been done for all patients. Although we can assume that patients with normal enzyme levels do not present exonic pathogenic changes, the same cannot be concluded for intronic variants and GLA haplotypes. We also had no access to patient’s family medical records. Therefore, we could not explain the high incidence of familiar history of vascular events and confirm other possible confounders such as diabetes, obesity or antiphospholipid syndrome.