Serum Neurofilament Light Chain Is Not A Useful Biomarker Of Central Nervous System Involvement in Women With Fabry Disease Ⅱ

3. Results and Discussion

Serum NfL concentration did not significantly differ between groups (0,053 ± 0,1 vs. 0,048 ± 0,09 ng/ mL; p = 0.9). Women with FD also had similar blood hemoglobin, serum phosphate, and PTH. Serum calcium concentration was higher in the FD group than in healthy women (2.38 ± 0.08 vs. 2.28 ± 0.11 mmol/ L, respectively; p = 0.03). MMSE, MoCA, and SF-36 scores were also similar in each group.

In women with FD, there was a significant positive correlation between age and serum PTH concentration (R = 0.62, p = 0.03). The same correlation was not seen in the control group. In the FD group, there was also a significant positive correlation between NfL and lyso-Gb3 concentration (R = 0.69, p = 0.01).

In the control group, a significant negative correlation was found between serum NfL and hemoglobin (R = -0.8, p = 0.001) MoCA score (R = -0.6, p = 0.04), and a positive correlation between NfL and serum PTH concentration (R = 0.8, p = 0.0009). These correlations were not present in women with FD.

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In the control group, there was a strong negative correlation between age and MoCA score (R = -0.83, p = 0.0009) and a positive correlation between age and SF-36 score (R = 0.6, p = 0.04). In the FD group, only a significant correlation between age and MoCA score was observed (R = -0.85, p = 0.0004). ROC analysis showed that the best predictor for MCI in both groups was eGFR. The area under the curve (AUC) for women with FD was 0.938 (95% CI: 0.792 - 1.083) and in the control group 0.857 (95% CI: 0.628 - 1.086). Detailed information on ROC analysis results is provided in Figure 2.

Our cross-sectional single-center study was designed to prove the concept that serum NfL levels could reflect the severity of cognitive impairment and indirectly, the level of CNS involvement in women at earlier stages of FD without previous clinical symptoms of nervous system involvement. The hypothesis behind the study was that damage to the neurons in FD patients would result in an increase in the concentration of NfL in cerebrospinal fluid and its penetration into the peripheral circulation, which can be assessed by measuring their serum levels. Loeffler T, et al. in a mouse model showed that NfL concentration can be a valuable biomarker not only in typical neurodegenerative diseases but also in other diseases that have an additional neuronal component, like lysosomal storage diseases, e.g., Gaucher disease (15). Erante D et al. confirmed that NfL is a good biomarker of neurodegeneration in Niemann-Pick disease (16). Ru Y, et al. described NfL as a biomarker of neurodegeneration in neuronal ceroid lipofuscinosis type 2 (CLN2 disease), another lysosomal storage disease. These authors showed in a canine model a significant correlation between serum NfL concentration and disease progression. Differently, in the human part of their study, they were unable to show correlations between NfL concentration and CLN2 severity or age of the patients. However, they showed, that NfL concentration decreased by 50% after enzyme replacement therapy was initiated (17).

The above-cited studies assessed the utility of NfL as a biomarker for the nervous system involvement in some lysosomal storage diseases. To our knowledge, there have been no similar studies in patients with FD. In FD women usually have a milder disease course than men, which is due to a random inactivation of the X chromosome. However, severe involvement of such target organs as the heart or kidneys is quite common (18). Despite residual enzyme activity, women with FD develop characteristic symptoms with age, including central nervous system symptoms. However, the clinical manifestation is more varied and the symptoms appear about 10 years later compared to men. The median interval between the onset of early FD symptoms and correct diagnosis is even more delayed in women, with a delay of 19 years on average (19). The identification of the marker of early nervous system involvement in women with FD could be particularly clinically relevant since women with this disease develop symptoms much later than men but the nervous system is most frequently involved. The symptoms of FD significantly interfere with patients' daily functioning, which contributes to a significantly reduced lifespan. Studies show that the life expectancy of men with Fabry disease is 15 to even 20 years shorter, and that of women is 6 to 10 years shorter compared to the average life expectancy in the population (20). In our study, we were not able to confirm that NfL concentration is a clinically useful biomarker for an assessment of the degree of nervous system involvement in women with FD. It may be due to the young age of the patients included in the study and the fact that most of them had no or moderate typical symptoms of the disease from other organs than the nervous system. In most of our study subjects genetic tests were performed due to diagnosis of FD in their relatives.

The α-Gal activities and the lyso-Gb3 concentration are the serum markers commonly used in the diagnosis and monitoring of FD. Measurement of α-Gal activity in plasma or leukocytes, which is the reference method for laboratory confirmation of the diagnosis in men, is often inconclusive in female patients whose enzymatic activity can range from low to normal values. In our study, 25% of women with FD had normal α-Gal activity and the lyso-Gb3 concentration was above normal in all cases. The basis underlying variability of the phenotype in women is still poorly understood, but the role of X chromosome inactivation appears to be most important (21). In FD, also-Gb3 levels are always elevated in men, but only between 40 and 60% in women. Lyso-Gb3 levels in women increase with age and are within the normal range in childhood. However, when symptomatic FD is suspected in adult women, both measurements of α-Gal A and plasma also-Gb3 activity improve the diagnostic value (18).

In our study, the concentration of lyso-Gb3 did not correlate with the results of the tests validated for diagnosis of cognitive dysfunction and with the results of the SF-36. Despite no difference in NfL concentration between the study and control group, a positive significant correlation between the concentration of lyso-Gb3 and NfL in the populations of women with FD was seen. An accumulation of lyso-Gb3 deposits as a result of α-Gal A deficiency causes damage to nerve cells with subsequent pro-inflammatory activity (22,23). It is possible that due to early diagnosis of the disease and lack of previous neurologic symptoms in our study group, the risk of neurodegeneration was low and thus no difference in serum NfL concentration between the two groups was observed

Studies conducted so far have shown that patients with FD have significant cognitive deficits (24). Most of the studies attributed the changes in cognitive functioning in the course of FD to the accumulation of glycosphingolipids in the cerebral circulation (25). In our study, however, no relationship was found between also-Gb3 concentration and cognitive impairment. Many psychological tests have been developed for screening for cognitive impairment in clinical practice. They differ in their sensitivity and specificity. The most commonly used screening test in the diagnosis of cognitive impairment is MMSE. Körver S, et al. showed that MMSE did not allow screening for MCI in patients with FD and it may lose its predictive value when the cognitive impairment is milder, less prevalent, and occurs predominantly in the executive domain, as appears to be the case in FD (14). Our results are consistent with the finding that MMSE cannot accurately distinguish patients with subtle cognitive impairment from patients without clinically detectable cognitive impairment. Therefore MoCA should be preferred to MMSE in populations at risk for MCI or with early-stage dementia. In a study conducted by Körver S, et al. The MoCA questionnaire classified 21% of patients with FD as having MCI, compared with 11% in the reference group (14). These data are consistent with our results. In our study, more patients were identified as having MCI with the MoCA test compared to MMSE.

In our study, the mean score in the MMSE and MoCA test was similar in FD women and control subjects. Löhle M, et al. also did not find any significant difference in the tests accessing cognitive functions between the patients with FD and healthy subjects. Their study group was larger and included both women and men in different stages of FD and with both classic and late type (11). In contrast, our study included only women without any signs of central nervous system involvement. Our study results indicate that neither depression nor disease severity, time since FD symptoms, and enzyme activity predicted a cognitive dysfunction. The analysis found no association between cognitive impairment and test scores.

Kidney disease is one of the major complications of FD and is associated with a continuous accumulation of glycosphingolipids throughout the nephron. This leads to progressive loss of GFR and eventually to renal failure. The kidney and brain share similar hemodynamic abilities, such as vasoregulation of the microcirculation. Studies have shown that lower eGFR was associated with increasing severity of chronic white matter hyperintensities (CWMH), and patients with more stable eGFR had fewer strokes than those with rapidly progressive kidney disease (26). Our study also suggests the importance of eGFR levels as a predictor of MCI in this patient group. Therefore, further research into the relationship between eGFR levels and MCI in FD patients is warranted (9).

In our study, we did not show an association between elevated serum calcium levels in women with FD and serum PTH. In one previous study, the authors tried to elucidate the pathomechanism of calcium-phosphate disturbances in patients with FD using a mouse model GlatmTg (CAG-A4GALT). The study results showed a relationship between hypercalcemia hypercalciuria and secondary hyperthyroidism (27). This may suggest that patients with FD are at increased risk of accelerated bone resorption and osteomalacia (28).

Our study has limitations because we have assessed the concentration of NfL only in the per circulation. However, previous studies showed that the concentration of NfL in the blood strongly correlates with its concentration in the cerebrospinal fluid (29,30). Another limitation is a small study group, which is a result of the ultra-low incidence of the disease in the population and of the selection of only female patients without any previous signs of central nervous system involvement typical for FD.

4. Conclusions 

The results of our study did not confirm the relation between the degree of nervous system involvement in women with FD and serum NfL levels. Therefore serum NfL cannot be considered as a useful marker of cognitive impairment in this disease. Our study also showed that MoCA is the preferred test to detect mild cognitive impairment in FD. Funding: The study was supported by the Medical University of Lodz grant No. 503/1-151-02/503-01. Conflict of Interest: The authors have no conflicts of interest to disclose. 

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