Our investigation in early-diagnosed patients revealed that the modified DS3, with additional items for early diagnosis, namely, uMB numbers, genotype of classical form,, and history of pain attack, was positively correlated with lifetime lyso-Gb3 exposure at diagnosis.
Subjects could be diagnosed in the early phase of the disease, in the absence of organ involvement, by using not only general screening tools such as GAL activity and genetic testing, but also additional tools such as uMBs and family history and data from at-risk relatives.
The MSSI was originally developed for use in males with classical FD, whereas the DS3 was developed for easy use in the general Fabry patient population[18]; the DS3 has a patient-reported domain, as well as clinical domains scored by laboratory data. In females with either form and males with the late-onset form, the DS3 may be more sensitive than the MSSI[19]. The FASTEX was developed to assess the clinical stability or disease progression between two consecutive evaluations. It has been reported to be more sensitive for assessing stability or disease progression than the MSSI and the DS3 [30]. However, these scores were low value and insensitive in our patients without cardiac or renal impairment because the conventional scores consist mainly of organ manifestation items. A useful method of evaluating disease severity in children, adult females, and asymptomatic patients is needed[31] [32].
In FD, early diagnosis—ideally during childhood—is important[21] [32]. Nevertheless, the variable nature of the symptoms can lead to a delay between symptom onset and diagnosis[33]. Particularly, FD in childhood can be mistaken for other diseases such as rheumatological problems because of the nonspecific signs of lethargy and myalgia[34]. In our experience, such children have been misdiagnosed with orthostatic dysregulation or psychological problems because of general fatigue, school refusal, or nonspecific gastrointestinal symptoms. Additionally, adolescents or young adults may not complain of transient pain or hypohidrosis because they may have had those symptoms from early childhood and not consider them a clinically meaningful symptom. To facilitate diagnosis in children, a simple examination or screening test is needed. Potentially useful approaches are interviewing about family history, screening by enzyme assay using a dried blood spot[35],[36], or checking for uMBs. uMBs, which are observed in the urine by light microscopy as laminated bodies with a whorled shape, are a specific finding in FD. uMBs originate from mainly podocytes that have accumulated GL3 and are detected before proteinuria develops [37-39]. In previous report, high renal pathological score was observed in female patients with only uMBs without albuminuria [40]. This suggests that uMBs are potential early biomarkers of renal injury. Furthermore, a recent study showed that degree of uMBs excretion correlated with podocyte vacuolation and ERT reduces uMBs excretion [39, 41]. Podocyturia has also been reported as an early marker of renal injury, but detection requires staining[42]. Although, detectability of uMBs depend on the skill and experience of the technician, it can be convenient indicator because of only using light microscopy. uMBs are not suitable on their own for assessing severity, but they could be useful for assessing severity in children and asymptomatic patients if their scores were added to a conventional scoring system. Other early markers of cardiac involvement include troponin T and cardiac MRI (including T1 mapping) [43, 44]. These were excluded from our study because they were not measured at baseline and their impact on severity and prognosis is unclear.
Lyso-Gb3 has recently been reported to be a pathogenic derivative of GL3 that increases extracellular matrix synthesis in podocytes[45], we speculate that the process may be related to the progression of organ damage. Histological examination to elucidate the pathogenesis of lysosomal storage diseases, including FD, has revealed the direct involvement of GL3 accumulation and defective autophagy[46], as well as of microinflammation caused by cytokine production induced by lyso-Gb3[45, 47]. In FD males, plasma GL3 is high, whereas in symptomatic FD females, plasma GL3 is generally within the normal range[17, 48, 49]. In contrast, plasma lyso-Gb3 levels are high not only in males but also in females[17]. These findings suggest that lyso-Gb3 is more useful than GL3 for diagnosis. We expected that the longer the duration of exposure to lyso-Gb3, the more severe the disease would be. In males, there are reports supporting this hypothesis[17], but few studies of lifetime lyso-Gb3 exposure have focused on females or children. Another report has shown that, in untreated women, disease severity scores increase with age[7], so the above hypothesis might be true for females as well.
To examine this association further, we assessed plasma lyso-Gb3 levels and calculated lifetime lyso-Gb3 exposure values at diagnosis for each patient according to previous reports [15, 17, 50]. Previous study showed that lifetime lyso-Gb3 exposure was correlated with disease manifestations: mildly or moderately male had exposure levels was 1000−5000 U, severely male patients had exposure levels >5000 U [17]. In our patients, lifetime lyso-Gb3 exposures was less than 5000 U and severity score was mild, irrespective of gender or disease subtype. Lyso-Gb3 value might not be simply compared to previous reports because the value vary from laboratory, our results were consistent with previous report[17]. The fact that our patients were relatively young and had received an early diagnosis may explain the low values. They had low exposure to lyso-Gb3 and possibly for this reason had not yet shown signs of progressive or symptomatic renal failure, symptomatic cardiac failure (hypertrophic myopathy), or cerebral infarction. One reason for these early diagnoses could have been that these patients were examined by physicians who were aware of FD and its clinical manifestations.
Our modified scores have given a point for genotype of classical form and early marker such as uMBs, history of past pain attack. Recent study showed that they should be managed according to phenotype because of the clinical course of classical type and late-onset type is different[51]. In this study, we added points to the genotype of classical form, because early-diagnosed patients might not have enough symptoms to determine the phenotype. The modified DS3 was significantly correlated with lifetime lyso-Gb3 exposure in all patients taken together and was useful for assessment in early-diagnosed FD patients. However, in females, no significant correlations were found, even with the modified DS3, despite a trend toward correlation in the case of males. This may have been due to the insufficiency of the sample size for stratification by gender, as well as the heterogeneity of symptoms in females. Generally, FD is milder in female patients than in male patients[7],[8]. At the end of the 1990s, we reported that a female patient with FD showed symptoms that were limited to the kidney because she had lyonization of the X chromosome that included a mutated GLA. At that time, it had not been confirmed that FD could be manifested in heterozygous females and that these individuals were not simply carriers of the pathogenic gene. Subsequent studies have shown that females have a wide spectrum of symptoms ranging from asymptomatic to severe, just as in males[9],[10]. Previous studies have suggested that the skewing of X-chromosome inactivation is part of the reason why females have a wide spectrum of symptoms[52]. However, it has not been determined conclusively whether the degree and distribution of skewing of the organs in which X-chromosome inactivation is present are related to disease severity[31, 52, 53].
Finally, we estimated the longitudinal changes in plasma lyso-Gb3 levels and disease severity scores. Males showed more rapid decreases in plasma lyso-Gb3 concentration after the treatment initiation than did females. The cut-off value of lyso-Gb3 was 0.9 nmol/L (95th percentile of healthy individuals) in a previous study[54]. Note that none of the patients had a plasma lyso-Gb3 concentration lower than the cut-off value at any time during follow-up. However, the reduction in lyso-Gb3 concentration in response to ERT also decreases lyso-Gb3 exposure post-ERT initiation. Indeed, notably, the conventional disease severity scores of our subjects were low and stable, and although uMBs were persistently observed, their presence did not lead to proteinuria. This suggests that decreased exposure to lysoGb3 after ERT initiation may slow the progression of signs and symptoms and improve prognosis. We believe that this possibility should be evaluated in a future study by renal biopsy.
Our study was limited because of the insufficient sample size, follow-up periods and the number of pedigrees. Moreover, there was a lack of weighting of additional items in the modified disease severity scores. Studies that include a large sample size of early-diagnosed FD patients and long-term follow-up period are needed to validate our findings and to determine the effect of lifetime lyso-Gb3 exposure on long-term prognosis.