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Unit of Rare Disorders, Department of Internal Medicine, Lozano Blesa; University Hospital. Zaragoza. SpainIISAragon, Hospital, Universitario Lozano Blesa, Zaragoza, Spain
Unit of Rare Disorders, Department of Internal Medicine, Lozano Blesa; University Hospital. Zaragoza. SpainIISAragon, Hospital, Universitario Lozano Blesa, Zaragoza, Spain
Unit of Rare Disorders, Department of Internal Medicine, Lozano Blesa; University Hospital. Zaragoza. SpainIISAragon, Hospital, Universitario Lozano Blesa, Zaragoza, Spain
Unit of Rare Disorders, Department of Internal Medicine, Lozano Blesa; University Hospital. Zaragoza. SpainDepartment of Biochemistry, Lozano Blesa” University Hospital, Zaragoza. Spain
Unit of Rare Disorders, Department of Internal Medicine, Lozano Blesa; University Hospital. Zaragoza. SpainIISAragon, Hospital, Universitario Lozano Blesa, Zaragoza, Spain
LysoGb3 is not elevated in patients whith multiple sclerosis.
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The cut-off point 3.5 ng/mL, for LysoGb3 in DBS, is valid to rule out Fabry disease.
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If demyelinating lesions are present LysoGb3 is enough to discard Fabry disease.
Abstract
Background
The presence of white mater lesions in the central nervous system forces the differential diagnosis between multiple sclerosis (MS) and Anderson–Fabry disease (FD). Due to the type of inheritance, linked to the X chromosome, the diagnosis of FD is especially difficult in women. Tissue´s deposits of globotriaosylceramide (Gb3) are characteristics for FD and the deacylated form of Gb3 (Globotriaosylsphingosine or LysoGb3) is specific for this entity. Our objective is to investigate if concentrations of plasma Lyso-Gb3 are useful for ruling out the FD in a Spanish cohort of patients with a previous diagnosis of MS.
Methods
we evaluated the α-galactosidase A enzymatic activity in 154 patients with a previous diagnosis of MS (93 women and 61 men): 103 Relapsing Remitting MS patients, 19 progressive MS patients and 32 with the clinically isolated syndrome. 116 (75% of the patients) were on MS disease modifying therapy. Enzymatic assay was completed in all cases and done on dried blood spot (DBS) samples. Subsequently the GLA gene was sequenced only in males and females who presented an enzymatic assay significantly lower than standardized controls (<50% for men and <75% for women). For subjects with GLA variants, plasma Lyso-Gb3 levels were performed by Tandem mass spectrometry from DBS, assuming a cut-off point for normality <3.5 ng/mL.
Results
Genetic study was carried out in 30 women and 7 men; 8 of them had non-previous described GLA variants. After a thorough clinical examination no organic disease was found in any of the classical target organs. The study of Lyso-Gb3 concentrations in DBS was lower than 3.5 ng/mL, allowing us to discharge FD in all subjects and to consider these GLA variants like non pathologic.
Conclusions
Lyso-Gb3 concentration in DBS is a useful tool to rule out Fabry disease in patients with MS. A concentration of LysoGb3 < 3.5 ng/mL rules out FD.
Fabry disease (FD) (OMIM 153,000) is a rare X-linked recessive lysosomal storage disorder resulting from the deficiency of α-galactosidase A (α-Gal A) activity (
). The complete or partial lack of enzyme activity causes the accumulation of glycosphingolipids (mostly globotriaosylceramide) in different tissues, including vascular endothelium, renal glomeruli and tubules, dorsal root ganglia, cardiac cells, cornea and skin. The central nervous system (CNS) involvement is clinically manifested by transient ischemic attacks and strokes at an early age, especially in the vertebrobasilar district, probably due to endothelial damage, cardiac involvement and hypertension secondary to kidney disease. Moreover, progressive white matter lesions (WMLs) occurring since early age have been described on brain magnetic resonance imaging (MRI), and these lesions may be confused with inflammatory lesions (
A high prevalence of cerebrovascular events with radiological evidence of CNS involvement in female patients with FD suggests that severity is the same -or more- in female patients compared to men. Neurological manifestations of FD may cause diagnostic difficulties, because can mimics other neurological disorders and this issue should be studied (
). But the disease with which the differential diagnosis should mainly be made is multiple sclerosis (MS), since it is one of the most frequent neurological disorders in young people. A possible error is caused by the similarity in symptoms and the absence of a specific test that allows to confirm the diagnosis of MS. That is why the literature has described cases of patients who were initially diagnosed with MS and subsequently reclassified as Fabry. Because there is increasing evidence that long-term enzyme therapy can halt disease progression, and has been shown that treatment is associated with improved quality of life in FD patients (
Diagnosis of FD is based on different and complementary aspects: 1) clinical suspicion, 2) demonstration of the decreased or absent of α-galactosidase A enzyme activity (Gold standard for males), 3) study of the GLA variants, and 4) evidence of tissue´s deposits of globotriaosylceramide (Gb3) (
Uncertain diagnosis of Fabry disease: consensus recommendation on diagnosis in adults with left ventricular hypertrophy and genetic variants of unknown significance.
The deacylated form of Gb3 (Globotriaosylsphingosine or LysoGb3), is increased approximately 100-fold in the plasma of symptomatic Fabry hemizygotes and also in symptomatic Fabry females (
). There are reports involving women in whom the serum LysoGb3 levels were useful diagnosis markers to identify heterozygotes whereas their α-Gal A activities were in normal range (
). Recently, a study found and excellent correlation between LysoGb3 levels in dried blood spots (DBS) and sera from patients with classic and Later-Onset FD (
), and a cut-off of 3.5 ng/mL comes from a meticulous ROC analysis choosing 100% of specificity. One of the most important aspects in relation to this specific molecule of FD is that its presence in plasma correlates with the appearance of WMLs in both men and women, although the correlation in male sex is greater (
). The main advantage of DBS technique is that the samples can be transferred from different locations at room temperature without risk of degradation.
Because FD and MS share common clinical and radiological features, the aim of this study is to investigate the prevalence of FD in a MS population with a previously confirmed diagnosis of MS according to the McDonald diagnostic criteria (
). Due to the cost and complexity of the biochemical and genetic determinations, we ask ourselves if LysoGb3 in DBS is a useful tool that allows to distinguish between both entities.
2. Material and methods
2.1 Patients and clinical work-up
The study was conducted in accordance with the principles of the Helsinki Declaration. Informed consent for collecting clinical data and blood samples for biobanking was obtained from all patients. 154 MS adult patients (93 women and 61 men) were recruited at the “Lozano Blesa” University Hospital in Zaragoza, Spain, between January 2011 and December 2016. All patients had a confirmed MS and had a comprehensive workup, including medical history.
2.2 α-Galactosidase a activity determination
The enzymatic activity of α-galactosidase A was performed on dried blood spots (DBS) samples using the fluorimetric method described by Chamoles et al. (
). Briefly, 3 mm circles were obtained from DBS filters and incubated at 37 °C during 8 h with 4-methylumbelliferil-α-d-galactopyranoside substrate (4 mM) in phosphate-citrate 0.1 M buffer at pH 4.2 and in presence of N-acetyl-galactosamine to inhibit α-galactiosidase B isoform. The amount of 4-methylumbelliferone released during the reaction was measured on a Twinkle LB 970 fluorimeter, Berthold Technologies (Excitation = =365 nm, Emission = =450 nm). The specific activity of the enzyme was calculated in reference to a standard curve (fluorescence versus concentration) and expressed as nmoles of hydrolyzed substrate per hour and milliliter of blood.
2.3 GLA genetic study
Direct sequencing of GLA was performed in male patients who presented an enzymatic activity lower than the 50% of the value obtained for the negative control, and in females presenting α-galactosidase A activity lower than the 75% of the value obtained for negative control. Genetic sequencing was accomplished on a DNA ABI 310 Sequence Analyzer, Applied Biosystems. Briefly, DBS were used directly in the PCR reaction to amplify the 7 exons of the gene flanked by small regions of intronic sequences (20 to 80 nucleotides). PCR products were purified with Exostar (GE, Healthcare), used in the sequencing reaction, following the manufacturer instructions (Bigdye, Applied Biosystems), and finally separated by capillary electrophoresis. Results obtained were analyzed using ChromasPro 1.42 software.
2.4 LysoGb3 determination
Because the challenge was to know if the found variants in GLA gene were related to FD, the LysoGb3 concentrations in DBS were studied in an independent laboratory (Archimed Life Science®; Vienna. Austria) using a highly-sensitive electrospray ionization liquid chromatography tandem mass spectrometry (modified method described by Nowak et al.) (
). The LysoGb3 determination was carried out only in those subjects with a positive genetic test.
2.5 Statistical aspects
For statistical analysis we used SPSS-PC 22.0. Initially, descriptive study has been performed with the mean, median, and standard deviation frequencies. Subsequently, associations between variables were found using the Pearson Chi-square test with Fisher's exact test. The quantitative variables were analyzed by Student's t-test or analysis of variance. The nonparametric tests U of Mann-Whitney and Kruskal-Walis were applied in the case of non-adjustement to the normal distribution. The analysis of the correlations between the quantitative variables was performed using the Pearson correlation coefficient and the Spearman coefficient as a function of its adjustment to the normal distribution.
3. Results
3.1 Patient characteristics
154 MS patients (93 women (60%) and 61 men (40%) were analyzed: 103 Relapsing Remitting MS patients (67%), 19 progressive MS patients (12%), and 32 with clinical isolated syndrome (21%); The Expanded Disability Status Scale (EDSS) mean was 2.7 ± 2 (0–8.5). Duration of disease was 9.6 years SD 8.4. The 75% of the patients were on MS disease modifying therapy. The most frequent onset symptom was sensory (37 patients). The mean age was 39 years old (19–74). The main age at the onset of disease was 30 years old (13–59). The main time of the disease evolution was 9 years (0.4–53)(Table 1), and features and symptoms at the onset were: sensory (24%), motor (21%), optic neuritis (21%), cerebral or cerebellar symptoms (different from those mentioned above), and multifocal disease.
Table 1Features of multiple sclerosis patients (mean and standard deviation).
Specific enzymatic activity was M 8.3072 nmol/ml.h; SD 2.70239. The proportion of α-galactosidase A activity was significantly lower among MS-females patients than males (Π2 = 8.7; p = 0.003) (Fig. 1). No relationship was found between any of the variables studied in MS and the results of the enzyme determinations.
Genetic study was carry out in 37 patients, 30 women and 7 men. Variants of GLA were found in 8 out of those 37 cases (all of them women). Five of them were variants of normality in intronic regions, and 3 cases nonpathogenic deletions in intronic areas (Table 2). After a careful clinical examination, accompanied by an exhaustive analysis of family segregation and a meticulous ophthalmological, cardiac, cutaneous and renal evaluation, no symptoms or signs of organic involvement of any of the target organs classically affected in FD were found.
Table 2Overview of AGA variants and interpretation after LysoGb3 results.
To conclude, in the eight women with positive genetic variants, lysoGb3 concentrations were determined in DBS. The results obtained in all the samples were lower than the cut-off point 3.5 ng/mL. This allowed us to rule out FD in all our MS patients and confirming that the variants found in the GLA gene were nonpathological (Table 2).
4. Discussion
To date, the relationship between MS and FD is well known (
). A very important fact is the presence of oligoclonal bands in CSF analysis, which could not be reasonably related to FD, while it strongly suggests MS.
We provide a wide range of patients, one of the largest in the literature, with a diagnosis of MS in which no association with FD pathogenic variants was found. However, in our series we did not find any signs or symptoms of systemic involvement corresponding to FD, studying organ by organ in each of the patients. Therefore, in our opinion, screening for FD should only be carried out in subjects with WMLs in the CNS and who also have renal, cardiac or dermatological involvement with absence of oligoclonal bands in the cerebrospinal fluid study, or without involvement of the cervical spinal cord.
One of the most important aspects of our work is that for the first time in the literature we have been able to demonstrate that LysoGb3 in DBS is capable of discriminating patients with WMLs in the central nervous system associated with MS, ruling out FD. LysoGb3 is specific for FD, and is only elevated in subjects affected by this entity. This is especially important in women, since concentrations lower than 3.5 ng/mL were sufficiently discriminative. The DBS technique provides great advantages over the use of plasma determinations, since the samples are easy to handle and transport, being a simple, reproducible and safe method of study. From a practical point of view is it noteworthy that the 3.5 ng/mL cut-off point is capable of identifying 100% of the truly negative subjects for FD. Our work demonstrated the broad applicability of this test for distinguish FD and MS, and in a future it will be especially useful worldwide to “presumptive or possible” MS subjects.
After the study that we have carried out, we think that from this moment and in relation to the screening for FD in patients -especially women- with WMLs lesions in the CNS and doubts about the aetiology of these, a diagnostic algorithm should be applied as shown in Fig. 2.
Fig. 2Diagnostic algorithm for the investigation of FD in “presumptive MS” patients.
The determination of LysoGb3 in DBS (cut-off <3.5 ng/mL) is able to discriminate patients with MS from patients with FD, in those subjects with WMLs lesions in CNS.
Sources of support
The present study has been carried out through grants provided by the companies Shire and Genzyme.
Declaration of Competing Interest
Dr Torralba serves as a consultant for Genzyme Corporation and Shire Company and has received research grants, travel support and honoraria for speaking engagements from both companies. Dr Iñiguez has received research grants, travel support and honoraria for speaking engagements from Bayer, Biogen, Merck, Novartis, Roche, Sanofi-Genzyme and TEVA. The rest of the authors declare the absence of conflict of interests.
References
Aerts J.M.
Groener J.E.
Kulper S.
et al.
Elevated Globotriaosylsphingosine is a hallmark of Fabry disease.
Uncertain diagnosis of Fabry disease: consensus recommendation on diagnosis in adults with left ventricular hypertrophy and genetic variants of unknown significance.
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