COMPOUNDS AND METHODS FOR THE DETECTION OF FABRY DISEASE

Abstract

The present invention provides for compounds and methods for the detection and follow-up of Fabry disease (FD). In particular, the present invention relates to a method for detecting or diagnosing FD in a subject, comprising detecting globotriaosylceramide (Gb3) deposits in biomaterial obtained from said subject. The present invention also provides for a method for treatment monitoring of FD in a subject. Further, the present invention relates to the use of a Gb3-specific natural ligand for the detection of Gb3 deposits in biomaterial. Also provided is a kit for detecting Gb3 deposits in biomaterial obtained from a subject.

Claims

1. A method for detecting or diagnosing Fabry disease (FD) in a subject, comprising detecting globotriaosylceramide (Gb3) deposits in biomaterial obtained from said subject, wherein said biomaterial is selected from the group consisting of (i) blood smear prepared from whole blood, (ii) peripheral blood mononuclear cells (PBMCs), and (iii) epithelial cells.

2. The method according to claim 1, wherein said epithelial cells are buccal epithelial cell or bladder epithelial cells.

3. The method according to claim 2, wherein said bladder epithelial cells are preferably present in a urine sample.

4. The method according to claim 1, wherein an increased amount of Gb3 deposit positive cells in said biomaterial as compared to a control is indicative for FD.

5.-6. (canceled)

7. The method according to claim 1, comprising: a) depositing the biomaterial obtained from said subject to a solid support thereby immobilizing said biomaterial, and b) detecting Gb3 deposit positive cells in said biomaterial.

8.-10. (canceled)

11. The method according to claim 7, further comprising contacting the immobilized biomaterial with a Gb3 binding agent or a reagent metabolizing Gb3 to a Gb3 metabolic product, thereby allowing for detection of Gb3 deposit positive cells

12. The method according to claim 11, wherein the Gb3 binding agent is a Gb3 specific antibody or a Gb3 natural ligand

13.-14. (canceled)

15. The method according to claim 11, wherein the reagent metabolizing Gb3 to a Gb3 metabolic product is alpha-galactosidase.

16. The method according to claim 12, wherein the Gb3 natural ligand is a shiga toxin.

17. (canceled)

18. A method for treatment monitoring of Fabry disease (FD) in a patient, comprising comparing the amount of globotriaosylceramide (Gb3) deposits positive cells detected in biomaterial obtained from said patient to the amount of Gb3 deposits positive cells detected in biomaterial obtained from said patient at an earlier date, wherein said biomaterial is one of (i) blood smear prepared from whole blood (ii) peripheral blood mononuclear cells (PBMCs) and (iii) epithelial cells, wherein the comparison provides an evaluation of effect of FD treatment.

19.-20. (canceled)

21. The method according to claim 18, wherein a decreased amount of Gb3 deposit positive cells as compared to the amount of Gb3 deposit positive cells detected at an earlier date indicates a positive treatment effect.

22. The method according to claim 18, wherein no change or an increased amount of Gb3 deposit positive cells as compared to the amount of Gb3 deposit positive cells detected at an earlier date indicates no treatment effect.

23.-24. (canceled)

25. The method according to claim 18, further comprising detecting Gb3 deposit positive cells in said biomaterial.

26.-32. (canceled)

33. The method according to claim 18, comprising the steps of: a) depositing the biomaterial obtained from said patient to a solid support thereby immobilizing said biomaterial, and b) detecting Gb3 deposit positive cells in said biomaterial

34.-36. (canceled)

37. The method according to claim 33, further comprising contacting the immobilized biomaterial with a Gb3 binding agent or a reagent metabolizing Gb3 to a Gb3 metabolic product, thereby allowing for detection of Gb3 deposit positive cells.

38. The method according to claim 37, wherein the Gb3 binding agent is a Gb3 specific antibody or a Gb3 natural ligand.

39.-40. (canceled)

41. The method according to claim 37, wherein the reagent metabolizing Gb3 to a Gb3 metabolic product is alpha-galactosidase.

42. The method according to claim 38, wherein the natural ligand is a shiga toxin.

43.-53. (canceled)

54. A kit comprising: a) a first solid support for depositing biomaterial, and b) a Gb3-binding agent or a reagent metabolizing Gb3 to a Gb3 metabolic product allowing for detection of Gb3 deposits in said biomaterial.

55. (canceled)

56. The kit according to claim 54, wherein the biomaterial is selected from the group consisting of (i) whole blood, (ii) PBMCs, and (iii) epithelial cells.

57.-69. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0113] FIG. 1: A) Nuclear stain (blue) of peripheral blood mononuclear cells (PBMC) of an untreated patient with genetically approved Fabry disease (FD). B) Immunoreaction with an antibody against globotriaosylceramide (Gb3) reveals Gb3 deposits (green) in several PBMC. C) Merged image of A) and B). D) Nuclear stain (blue) of PBMC of a healthy control. E) Immunoreaction with an antibody against Gb3 reveals one cell with Gb3 deposits (green). F) Merged image of D) and E). Yellow arrows indicate Gb3 positive PBMC. Scale bar: 50 μm.

[0114] FIG. 2: Bar graphs illustrate the mean percentage of globotriaosylceramide (Gb3) positive peripheral blood mononuclear cells (PBMC) of men (M) and women (F) with Fabry disease (FD). The mean percentage of Gb3 positive PBMC is higher in men and women with FD compared to healthy controls (Co). ***p<0.001, **p<0.01.

[0115] FIG. 3: Bar graphs illustrate the mean percentage of globotriaosylceramide (Gb3) positive peripheral blood mononuclear cells (PBMC) of men (M) and women (F) with Fabry disease (FD) carrying either classical (CL) or non-classical (NCL) mutations. The mean percentage of Gb3 positive PBMC is higher in men and women with classical FD associated mutations compared to healthy controls (Co). ***p<0.001, **p<0.01.

[0116] FIG. 4: Bar graphs illustrate the mean percentage of globotriaosylceramide (Gb3) positive peripheral blood mononuclear cells (PBMC) of men (M) and women (F) with Fabry disease (FD) carrying classical (CL) mutations and with or without enzyme replacement therapy (ERT). In men, the mean percentage of Gb3 positive PBMC decreased with ERT: the number was highest in untreated men (p<0.001), less in those with ERT>8 days before (p<0.01), and lowest in men with ERT up to eight days before blood withdrawal compared to healthy controls (Co). In women, the mean percentage of Gb3 positive PBMC was also highest without ERT compared to healthy female controls (Co). ***p<0.001, **p<0.01, **p<0.05.

[0117] FIG. 5: A) A negative correlation was found between duration of enzyme replacement therapy (ERT) and the mean percentage of globotriaosylceramide (Gb3) positive peripheral blood mononuclear cells (PBMC) in men with Fabry disease (FD) carrying classical mutations (Spearman correlation coefficient −0.457, p<0.05). B) Of n=15 male (M) and female (F) patients, a second blood sample was obtained at a follow-up visit (visit 1, visit 2). These patients carried classical (CL) and non-classical (NCL) mutations and were either untreated (no ERT) or received ERT. Mean percentage of Gb3 positive PBMC was low at both visits in all patients receiving ERT and dropped in those who started ERT before visit 2. In contrast mean percentage of Gb3 positive PBMC remained high in a male (#6) and female (#15) patients carrying CL mutations without ERT. *ERT was started after visit 1. **ERT was started after visit 1 and one year before visit 2.

[0118] FIG. 6: Correlation of alpha-galactosidase A activity in leucocytes (nmol/min/mg protein) with mean percentage of globotriaosylceramide (Gb3) positive peripheral blood mononuclear cells (PBMC) in men (A) and women (B) with Fabry disease (FD). A negative correlation was found for both genders (men: Spearman correlation coefficient: −0.451, p<0.05; women: Spearman correlation coefficient: −0.423, p<0.05).

[0119] FIG. 7: Bar graphs illustrate alpha-galactosidase A (α-GAL) activity and mean percentage of globotriaosylceramide (Gb3) positive peripheral blood mononuclear cells (PBMC) of men (M) and women (F) with Fabry disease (FD) carrying missense (MS) and nonsense (NS) mutations. In men (A) and women (B) with NS mutations, α-GAL activity was lower compared to those with MS mutations. This was reciprocal to the mean percentage of Gb3 positive PBMC, which was higher in men (A) and women (B) with NS mutations compared to those with MS mutations, ***p<0.01, *p<0.05.

[0120] FIG. 6: Bar graphs illustrate plasma lyso-Gb3 levels in male (M) and female (F) patients with Fabry disease (FD) carrying missense (MS) and nonsense (NS) mutations. In men (A) and women (B), lyso-Gb3 was higher in patients with NS mutations compared to those carrying MS mutations. When investigating plasma lyso-Gb3 levels and patients' mean percentage of globotriaosylceramide (Gb3) positive peripheral blood mononuclear cells (PBMC), a positive correlation was found in men (C; Spearman correlation coefficient: 0.705, p<0.001) and women (Spearman correlation coefficient: 0.499, p<0.01) for high lyso-Gb3 levels with high numbers of Gb3 positive PBMC. ***p<0.001, n.s.: not significant.

[0121] FIG. 9: Bar graphs illustrate the plasma globotriaosysphingosine (tyso-Gb3) levels of men (M) and women (F) with Fabry disease (FD) carrying classical (CL) mutations and with or without enzyme replacement therapy (ERT). ERT did not influence lyso-Gb3 levels.

[0122] FIG. 10: Receiver operating characteristic (ROC) curves are shown for the number of globotriaosylceramide (Gb3) positive peripheral blood mononuclear cells (PBMC) in men (Sensitivity: 91%, specificity 69%, AUC: 0-797, SE: 0.75, Asymptotic sig.: p=0.001. Asymptotic 95% conf, interval: 0.650-0.943) (A) and women (Sensitivity: 91%, specificity 67%, AUC: 0-790, SE: 0.83. Asymptotic sig.: p=0.003. Asymptotic 95% conf. Interval: 0.628-0.952) (B) with Fabry disease (FD) and carrying classical mutations (patients without treatment and patients having received enzyme replacement therapy >8 days before) compared to healthy controls. The relative frequency of “true positive” values on the y-axis (i.e. sensitivity) is plotted against the relative frequency of “true negative” values on the x-axis (i.e. 1−sensitivity),

[0123] FIG. 11: Blood smear prepared using 10 μl venous whole blood of a patient with Fabry disease and a healthy control (B) immunoreacted with antibodies against Gb3. Yellow arrows indicate some of the many cellular Gb3 deposits. No Gb3 deposits are visible in the sample of the healthy control. Scale bar: 50 μm.

[0124] FIG. 12: Blood smear prepared using a drop of finger stick capillary blood of a patient with Fabry disease and a healthy control (B) immunoreacted with antibodies against Gb3. Yellow arrow indicates a cellular Gb3 deposition. No Gb3 deposits are visible in the sample of the healthy control. Scale bar 50 μm. Investigation of blood smears of a male Fabry patient carrying a classical Fabry mutation using the commercial antibody (A) and Shiga toxin (B). While hardly any deposits of Gb3 are seen using the antibody, Shiga toxin reveals dense accumulation of Gb3 in blood cells.

[0125] FIG. 13: The scheme summarizes potential ways how to transfer our idea of detecting Gb3 in blood cells to a test kit for self-administration, named FABRYSTIX.

[0126] FIG. 14: Buccal smear prepared using a buccal swab of a patient with Fabry disease immunoreacted with antibodies against Gb3. Several epithelial cells are visible with a blue nucleus and one shows a green signal potentially indicating Gb3 deposition. Scale bar: 50 μm.

[0127] FIG. 15: The Figure shows the genetic distribution in the study population. Abbreviations: CL: classical mutation (i.e. the mutation is known to be associated with classical symptoms and signs of FD); NCL: non-classical mutation (i.e. the mutation is associated with late onset or predominant involvement of one organ).

EXAMPLES

[0128] The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims.

[0129] Gb3 Detection in Peripheral Blood Mononuclear Cells (PBMCs)

[0130] The inventors investigated 67 consecutive adult FD patient's age 217 years who reported at the Wurzburg Fabry Center for Interdisciplinary Therapy (FAZIT) between 2014 and 2017. The group consisted of 37 men (median age 47 years, range 17-67 years) and 30 women (median age 52 years, range 19.78 years). Additionally, 52 healthy volunteers were recruited as controls. The control group consisted of 26 men (median age 52 years, range 24-77 years) and 26 women (median age 50, range 27-76 years). For the extraction of PBMCs venous blood was obtained in 8×9 ml EDTA containing monovettes. From these blood samples PBMCs were isolated following the protocol described in detail below (Example 1). The PBMCs obtained were then immunoreacted following the protocol also described in Example 1. The results of the immunoreaction were analyzed using a fluorescence microscope (Axiophot 2 microscope. Zeiss, Jena, Germany) that was equipped with a CCD camera (Visitron Systems, Tuchheim) and SPOT Advanced Software (Windows Version 4.5. Diagnostic instruments, Inc, Sterling Heights, USA).

[0131] The protocol for isolation of PBMC comprises the following steps: venous blood is collected in 8×9 ml EDTA-containing monovettes, after mixing, the content of monovettes is transferred into clean Falcon tubes, more precisely, the content of 2 monovettes is used to fill a 50 ml Falcon tube, to reach a maximum volume of 17.5 mL Subsequently, the same volume of 1×PBS buffer (i.e. maximum 17.5 ml) is added to each prepared Falcon and mixed gently. 15 ml of Lymphoprep (RT) are then added into 4 new 50 mi Falcon tubes, and the content of one Falcon tube each (containing the blood-PBS buffer mixture) is included very slowly to the Lymphoprep, in a way that the two solutions do not mix up. These Falcon tubes are then centrifuged for 20 min at 20° C. and 1800 U without breaks. During the centrifugation step, a white ring will form in the each Falcon tube. Two of these rings are to be carefully and entirely collected with a plastic pipette and put in a new 50 ml Falcon tube. Each falcon tube containing said rings is then filled up to 50 ml with 1×PBS and centrifuged for 12 min at 4° C. and 1400 U with breaks. After centrifugation the supernatant is removed up to approximately 5 mi, the cell pellets are resuspended with a sterile pipette and pooled in a new 50 ml Falcon tube, which is again filled up to 50 ml with 1×PBS, and mixed well. The new Falcon tubes are centrifuged for 2 min at 4° C. and 1400 U with breaks, the supernatant is removed with a pipette up to 200 μl. 9.8 ml 1×PBS is added and used to resolve the pellet with a sterile pipette. After pellet resolution, 10 μl trypan blue is added in one well of a plastic well-plate and mixed with 10 μl of the resolved cells. 10 μl of this mixture is added to a Neubauer Improved chamber and 5 squares are diagonally counted (for example: 10.sup.8 cells counted≙10.8×10.sup.7 cells/ml). The rest of the resolved pellets is again centrifuged 10 min at 4° C. and 1400 U with breaks, the supernatant is removed with a pipette. The cells are stored in 1 ml storage medium per 1×10.sup.7 cells at −80° C. OR resolve cells with a dilution of 1×10.sup.6 cells/ml in 1×PBS when directly going on with the staining protocol below.

[0132] A protocol recently developed for PBMCs staining comprises the following steps: On the first day. 1×10.sup.5 cells in 25 μl 1×PBS are pipetted as a drop on a glass slide and dried for 2 hours, the cells are then fixed for 10 min at room temperature (RT) with 4% PFA, and washed 3 times for 5 minutes with 1×PBS. Subsequently cells are permeabilized for 5 minutes at room temperature (RT) with 0.3% Triton X-100 in PBS (=0.3% PBST). The solution is allowed to drop off well (i.e not washed), and cells are blocked for 1 h at RT with 10% BSA/PBS. Again the solution is allowed to drop off well (i.e. not washed). The Gb3-antibody is diluted (see below) in 0.01% PBST to 1:250 and pipetted on the cells (final volume: 50-75 μl). Cells are incubated with the antibody at 4° C. over night in humid chamber. On the second day, cells are washed 3 times for 5 minutes with PBS. The secondary antibody (donkey-anti-mouse Alexa Fluor (Nr. Z3)) is diluted 1:150 (a final dilution 1:300) in 0.01% PBST pipetted on cells and incubated for 1 h at RT. The cells are then washed 1×5 min with 1×PBS, Incubated for 10 minutes with DAPI diluted 10.000 in 1×PBS, washed 3 times for 5 min with 1×PBS and covered with Aqua Poly/Mount. A negative control is also prepared by incubating the cells with blocking solution over-night instead of primary antibody and then the regular protocol and incubation with secondary antibody is performed.

[0133] Clinical Characteristics of the Patient Population

Table 1 provides individual data.

TABLE-US-00001 TABLE 1 Days Mean % α-GAL Duration since of Gb3 activity Lyso- CL, MS, of ERT last pos. (nmol/min/ Gb3 # Gender Age NCL NS Genotype (years) ERT PBMC mg protein) (ng/ml) 1 M 21 CL NS W349X none none 0.0113 0.01 27.0 2 M 44 CL NS c.679 C > T//R227X none none 0.1810 0.02 25.2 3 M 46 CL NS c. 1196G > A//W399X none none 0.4497 0.02 29.2 4 M 37 CL NS c.1029_1030 del TC fsX30 8 11 0.6880 0.02 5.2 5 M 20 CL NS c. 363 delT//A121fs*8 7 12 0.0263 0.05 4.8 6 M 22 CL NS c. 363 delT//A121fs*8 7 13 0.0017 0.05 7.2 7 M 57 CL NS c. 611 G > A//W204X 4 13 0.4337 0.03 9.0 8 M 32 CL NS c.1069 C > T//p. Q357X 6 3 0.0103 n.d. 6.9 (p.Gln357X) 9 M 47 CL NS c. 1196G > A//W399X 15 4 0.0037 0.03 12.7 10 M 40 CL NS W349X 11 4 0.0197 0.02 75.4 11 M 17 CL NS c.993_994 ins A (fsX338) 7 8 0.0060 0.02 170.0 12 M 47 CL MS c.708G > C//p.W236C none none 0.5540 0.06 27.7 13 M 65 CL MS c.720G > C//p.K240N none none 0.0153 0.03 11.4 14 M 51 CL MS c.406G > C (D136H) none none 0.3600 0.02 22.1 15 M 53 CL MS c.404 C > T//A135V 8 14 0.0010 0.20 24.6 16 M 48 CL MS c.408 T > A//D136E 11 15 0.0053 0.02 27.9 17 M 52 CL MS c.102 G > A//E341K 14 18 0.0083 0.02 14.7 18 M 44 CL MS c.845C > T//T282I 7 1 0.0020 0.02 7.3 19 M 18 CL MS Q321H (c.963G > C) + 3 2 0.0417 0.03 22.6 D322N(c.964G > A) 20 M 33 CL MS c.508 G > A//D170N 11 6 0.0007 0.02 152.0 21 M 35 CL MS c.486G > T//W162C 2 6 0.0077 n.d. n.d. 22 M 50 CL MS c.103 G > A//G35R 10 8 0.0080 n.d. 14.4 23 M 37 CL consensus IVS2 + 1 (G > A) 0.1 13 0.1040 0.03 109.0 splice mutation 24 M 48 CL consensus IVS3 + 1 G > A 16 21 0.0050 n.d. 14.6 splice mutation 25 M 38 CL consensus IVS6 − 10G > A, c.1000 − 4 6 0.0963 0.04 112.0 splice 10G > A mutation 26 M 24 NC MS c.416 A > G//N139S none none 0.0100 0.06 44.3 27 M 44 NC MS c.427 G > A//A143T none none 0.0007 0.11 34.4 28 M 21 NC MS c.416 A > G//N139S 4 12 0 0.06 102.0 29 M 50 NC MS c.1208 del AAG 12 12 0 0.03 46.8 30 M 60 NC MS c.386 T > C//L129P 9 15 0.0007 0.02 23.1 31 M 34 NC MS c.386 T > C//L129P 7 ERT 0.0177 0.03 70.0 32 M 63 NC MS 644 A > G (N215S) 2 6 0.0007 0.06 27.2 33 M 67 NC MS c.644 A > G (N215S) 2 7 0 n.d. 4.8 34 M 62 NC MS c.644 A > G (N215S) 5 9 0.0017 0.05 7.2 35 M 62 NC MS c.644 A > G (N215S) none none 0.0043 0.05 9.0 36 M 51 NC MS c.644 A > G (N215S) none none 0 0.06 6.9 37 M 66 NC MS c.644 A > G (N215S) none none 0.0040 0.04 12.7 38 F 52 CL NS c. 1196G > A//W399X none none 0.0053 0.15 9.4 39 F 22 CL NS c.404 C > T//A135V none none 0.0077 0.18 5.7 40 F 28 CL NS c.1069 C > T//p. Q357X none none 0.0500 0.15 9.5 (p.Gln357X) 41 F 35 CL NS c.718-719delAA (fs248X) none none 0.0100 0.17 19.6 42 F 19 CL NS c.993_994 ins A (fs X 338) none none 0.0663 0.15 7.4 43 F 41 CL NS c.993_994 ins A (fs X 338) 1 7 0.0203 0.20 7.0 44 F 44 CL NS c.363delT//A121fs*8 4 12 0.0017 0.25 10.8 45 F 26 CL MS c.404 C > T//A135V none none 0.0387 0.16 17.0 46 F 41 CL MS c.874G > C (p. none none 0.0130 n.d. 9.7 Ala292Pro)/A292P 47 F 46 CL MS C.860G > C// none none 0.0473 0.23 17.4 p.Trp287Ser 48 F 63 CL MS c.484 T > G//W162G none none 0.0103 0.32 7.8 49 F 53 CL MS c.404 C > T//A135V none none 0.0163 0.36 17.1 50 F 53 CL MS c.1025 G > T//R342L 12 22 0 0.34 4.9 51 F 54 CL MS Q321H (c.963G > C) + 0.1 1 0.0007 0.17 13.3 D322N(c.964G > A) 52 F 52 CL MS c.404 C > T//A135V 3 7 0.0003 0.10 13.0 53 F 78 CL MS c.103 G > A//G35R 9 8 0.0133 0.16 12.1 54 F 56 CL MS c.1025 G > T//R342L 12 8 0.0003 0.43 5.3 55 F 63 NC NS c.1221 del A none none 0.0380 0.22 17.8 56 F 57 NC NS c.756 or 757 del A, none none 0.0087 0.38 25.3 fs268X 57 F 49 NC MS c.386 T > C//L129P 4 ERT 0.0083 0.22 9.5 58 F 52 NC MS c.973 G > A//G325S + none none 0.0023 0.20 8.3 polymorphisms 59 F 75 NC MS c.427 G > A//A143T none none 0.0013 0.49 1.1 60 F 59 NC MS c.644 A > G (N215S) none none 0.0003 n.d. 1.7 61 F 75 NC MS c.644 A > G (N215S) none none 0.0073 0.55 1.5 62 F 23 NC MS c.937G > T//D313Y none none 0.0020 0.80 0.8 63 F 19 NC MS c.937G > T//D313Y none none 0 n.d. 0.5 64 F 45 NC MS c.937G > T, p. none none 0.0100 0.23 0.6 Asp313Tyr (D313Y) 65 F 57 NC MS c.937G > T (D313Y) none none 0 0.57 0.6 66 F 54 NC MS c.1196G > C (bet) 4 5 0 0.30 0.7 p.W399S + IVS2 − 81_77 homo, −10C > T homoz, IVS4 − 16 A > G homoz, IVS6 − 22 C > T homoz α-GAL: α -galactosidase A, CL: classical mutation, ERT: enzyme replacement therapy, F: female, Gb3: globotriaosylceramide, M: male, MS: missense; NS: non-sense, NCL: non-classical mutation, n.d.: no data. *Patients (mother and son) refused sharing the date of the last ERT.
Table 2 gives a baseline data of the patient population:

TABLE-US-00002 TABLE 2 M F N 37 30 Median age 47 (17-67) 52 (19-78) (range) (years) Genotype CL 25 17 Type of mutation NS 11  8 MS 23 21 other.sup.a  3  1 Number of 26/37 (70%)    9/30 (30%)   patients On ERT Median time .sup. 7 (0.1-16) .sup. 4 (0.1-12) since ERT (range) (years) CL, classical mutation; ERT, enzyme replacement therapy; F, female; M, male; MS, missense mutation; NCL, nonclassical mutation; NS, nonsense mutation. .sup.aIntronic consensus splice nutations.
Among men with FD, n=25 had a mutation likely leading to a classic phenotype (i.e. the mutation is known to be associated with typical symptoms and signs of FD) and n=12 had a mutation likely leading to a non-classic phenotype (i.e. the mutation is associated with late onset or predominant involvement of one organ) (van der Tol et al., 2014 JIMD Rep. 17, 83-90). Among women with FD, n=17 had a classical and n=12 a non-classical genotype. Cases were allocated after individual cross-check of the genotype using https://lih16.u.hpc.mssm.edu/pipeline/js/dbFabry/ Mutation.html#. Supplementary FIG. 1 summarizes the genotypic distribution of the study cohort. 19/25 men and 7/17 women with classical mutation and 7/12 men and 2/12 women with non-classical mutation were on ERT (15 men on agalsidase-beta, 10 on agalsidase-alpha, 1 switched from agalsidase-beta to agalsidase-alpha and back; 3 women on agalsidase-beta, 6 on agalsidase-alpha). The median time on ERT was 7 years (0.1-16) in men and 4 years (0.1-12) in women.

[0134] Gb3 Positive PBMC can be Visualized and are More Frequent in Men and Women with FD than in Healthy Controls

Gb3 deposits were distinctly visible in the cytosol of PBMC of patients with FD and to a much lesser extent in healthy controls (FIG. 1). The mean percentage of Gb3 positive PBMC was higher in men (p<0.001) and women (p<0.01) with FD compared to male and female controls (FIG. 2).

[0135] Men and Women Carrying Classical FD Mutations have a Higher Number of Gb3 Positive PBMC, while Patients with Non-Classical Mutations do not Differ from Controls

The mean percentage of Gb3 positive PBMC was sixteen-fold higher in men carrying a classical mutation (0.08) compared to healthy men (0.005; p<0.001), while men carrying a non-classical mutation were not different from male controls (FIG. 3). Similarly, women carrying a classical mutation had four-fold higher load of Gb3 positive PBMC (0.02) than healthy women (0.005; p<0.01), while those with non-classical mutations were not different from controls (FIG. 3).

[0136] Number of Gb3 Positive PBMC Temporarily Decreases Under ERT

The Inventors next investigated, if the number of Gb3 positive PBMC changes with ERT. In men carrying classical mutations, the number of Gb3 positive PBMC consecutively decreased with ERT: the mean percentage of Gb3 positive PBMC was highest in untreated men (p<0.001), lower in those with treatment >8 days before (p<0.01), and close to normal in men with treatment up to eight days before blood withdrawal compared to healthy men (p<0.05; FIG. 4). The mean percentage of Gb3 positive PBMC was higher in untreated men carrying classical mutations compared to those under ERT and carrying classical mutations (p<0.05). In female FD patients with classical mutations, the mean percentage of Gb3 positive PBMC was also highest in untreated women (p<0.001 compared to healthy female controls: FIG. 4). No intergroup difference was found when comparing women with treatment >8 days (n=1) and ≤8 days (n=5) before ERT compared to controls, which may be due to the low number of subjects per subgroup, however, the number of Gb3 positive PMBC was lower in treated women compared to untreated ones (p<0.05). Among men and women carrying non-classical mutations, only n=7 respectively n=2 patients were on ERT, and the number of Gb3 positive PBMC was not different from healthy controls each (data not shown).

[0137] Load of Gb3 Positive PBMC Decreased Under Long-Term ERT

The load of Gb3 positive PBMC decreased with total duration of treatment in men with FD and carrying classical mutations (Spearman correlation coefficient −0.457, p<0.05. FIG. 5A). No correlation was found between the mean percentage of Gb3 positive PBMC and age when investigating untreated male and female FD patients with classical and non-classical mutations (data not shown).

[0138] Of n=15 patients (8 men, B with classical mutation, 5 on ERT at both visits; 7 women, 5 with classical mutation, 6 on ERT at both visits) the inventors obtained a second blood sample after a median time of 25 months in men (11-35) and 30 months in women (24-37). In all patients receiving ERT, the mean percentage of Gb3 positive PBMC was low at both visits. One male patient started ERT Just after visit 1 and 19 months before visit 2 (#3 in FIG. 5B), a second male patient started ERT a year after visit 1 and 12 months before visit 2 (#5 in FIG. 58): both showed a drop of Gb3 load at visit 2. In contrast, the mean percentage of Gb3 positive PBMC remained high in a male patient who had stopped ERT six years before visit 1 (#6 in FIG. 5B) and a female patient who had not yet initiated treatment (#15 in FIG. 58).

[0139] The Mean Percentage of Gb3 Positive PBMC Correlates with α-GAL Activity and with Lyso-Gb3

The inventors next investigated, if α-GAL activity is reflected by the number of Gb3 positive PBMC. The median α-GAL activity measured in leucocytes was 0.03 nmol/min/mg protein in men (0.01-0.2) and 0.23 nmol/min/mg protein in women (0.1-0.8). We found a negative correlation in men (Spearman correlation coefficient: −0.451, p<0.05) and women with FD (Spearman correlation coefficient −0.423, p<0.05) with lower α-GAL activity being associated with a higher mean percentage of Gb3 positive PBMC (FIG. 6).

[0140] When stratifying the entire patient cohort for nonsense and missense mutation carriers, the inventors found a higher mean percentage of Gb3 positive PBMC in men (p<0.05) and women (p<0.01) with nonsense mutations (FIG. 7), which was reciprocal to α-GAL activity: men (p<0.05) and women (p<0.05) carrying nonsense mutations had lower α-GAL activity compared to men and women with missense mutations (FIG. 7). Accordingly, men (p<0.001) and women (n.s.) with nonsense mutations had higher plasma levels of lyso-Gb3 than patients carrying missense mutations (FIG. 8A, B), the inventors found a strong positive correlation in men (Spearman correlation coefficient: 0.705, p<0.001) and women with FD (Spearman correlation coefficient: 0.499, p<0.01) of lyso-Gb3 levels and mean percentage of Gb3 positive PBMC (FIG. 8C, D).

[0141] The cohort contained eleven families with two (n=8), three (n=2), and five (n=1) family members. Mean percentages of Gb3 positive PBMC individually varied between family members (Table 3).

TABLE-US-00003 Table 3 Days Mean % α-GAL Duration since of Gb3 activity Lyso- CL, MS, of ERT last pos. (nmol/min/ Gb3 # Gender Age NCL NS Genotype (years) ERT PBMC mg protein) (ng/ml) Family 1 19 M 18 CL MS c.963G > C, Q321H + c.964G > A, 3 2 0.0417 0.03 22.6 D322N 51 F 54 CL MS c.963G > C, Q321H + c.964G > A,   0.1 1 0.0007 0.17 13.3 D322N Family 2 8 M 32 CL NS c.1069 C > T, p. Q357X, 6 3 0.0103 n.d. 6.9 P.Gln357X 40 F 28 CL MS c.1069 C > T, p. Q357X, none none 0.0500 0.15 9.5 P.Gln357X Family 3 50 F 53 CL MS c.1025 G > T, R342L 12  22  0 0.34 4.9 54 F 56 CL MS c.1025 G > T, R342L 12  8 0.0003 0.43 5.3 Family 4 22 M 50 CL MS c.103 G > A, G35R 10  8 0.0080 n.d. 14.4 53 F 78 CL MS c.103 G > A, G35R 9 8 0.0003 0.16 12.1 Family 5 31 M 34 NCL MS c.386 T > C, L129P 7 ERT* 0.0177 0.33 70.0 55 F 49 NCL MS c.386 T > C, L129P 4 ERT* 0.0083 0.22 9.5 Family 6 9 M 47 CL NS c. 1196G > A, W399X 15  4 0.0037 0.03 12.7 38 F 52 CL NS c. 1196G > A, W399X none none 0.0053 0.15 9.4 Family 7 1 M 21 CL NS Exon 7, W349X none none 0.0113 0.01 27.0 10 M 40 CL NS Exon 7, W349X 11  4 0.0197 0.02 75.4 Family 8 26 M 24 NCL MS c.416 A > G, N139S none none 0.0100 0.06 44.3 28 M 21 NCL MS c.416 A > G, N139S 4 12  0 0.06 102.0 Family 9 11 M 17 CL NS c.993_9934 ins A, fsX 338 7 8 0.0060 0.02 170.0 42 F 19 CL NS c.993_9934 ins A, fsX 338 none none 0.0663 0.15 7.4 43 F 41 CL NS c.993_9934 ins A, fsX 338 1 7 0.0203 0.20 7.0 Family 10 5 M 20 CL NS c. 363delT, A121fs*8 7 12  0.0263 0.05 4.8 6 M 22 CL NS c. 363delT, A121fs*8 7 13  0.0017 0.05 7.2 44 F 44 CL NS c. 363delT, A121fs*8 4 12  0.0017 0.25 10.8 Family 11 15 M 53 CL MS c.404 C > T, A135V 8 14  0.0010 0.20 24.6 45 F 26 CL MS c.404 C > T, A135V none none 0.0387 0.16 17.0 52 F 52 CL MS c.404 C > T, A135V 3 7 0.0003 0.10 13.0 49 F 53 CL MS c.404 C > T, A135V none none 0.0163 0.36 17.1 39 F 22 CL NS c.404 C > T, A135V none none 0.0077 0.18 5.7 α-GAL: α -galactosidase A, CL: classical mutation, ERT: enzyme replacement therapy, F: female, Gb3: globotriaosylceramide, M: male, MS: missense; NS: non-sense, NCL: non-classical mutation, n.d.: no data. *Patients (mother and son) refused sharing the date of their last ERT.

[0142] Gb3 Deposits in PBMC Reflect Treatment Effects, but Lyso-Gb3 does not

The inventors then investigated, if lyso-Gb3 reflects the effect of ERT. In contrast to our results obtained for mean percentage of Gb3 positive PBMC (Figure. 4), plasma lyso-Gb3 levels were not influenced by ERT (shown for untreated and treated men and women carrying classical mutations; (FIG. 9).

[0143] Gb3 Deposits in PBMC are of Diagnostic Value in Men and Women with FD

The sensitivity/specificity of the mean percentage of Gb3 positive PBMC for the detection of FD was 91%/69% in men and 91%/67% in women carrying classical FD mutations (untreated patients and patients having received ERT ≥8 d before) and when setting the cut-off value at 0 Gb3 positive PBMC (FIG. 10).

Gb3 Visualization in Blood Smears

[0144] To facilitate the procedure of Gb3 visualization in blood samples, the inventors investigated if blood smears immunoreacted with antibodies against Gb3 would also allow the detection of Gb3 positive blood cells. The qualitative assessment of blood samples obtained from two FD patients and two healthy controls as 10 μl whole venous blood (FIG. 11) or few drops of finger stick capillary blood (FIG. 12) revealed that Gb3 deposits were also unequivocally visible in blood smear preparations. These results are further expected to improve when using Gb3 specific staining with e.g. its natural ligands such as shiga toxin instead of the commercial antibody.

[0145] A newly established protocol for staining Gb3 in blood smears comprises the following steps: On the first day, few drops of blood are placed on a glass slide, these drops are then thinly streaked/smeared out over the glass slide (over approx. 1.7 cm×3 cm area). The streak is then allowed to dry at room temperature for 30 minutes and subsequently is fixed for 10 minutes using 4% PFA at room temperature. The cells of the blood smear are then permeabilized with 0.3% triton X-100 in PBS (=0.3% PBST) at room temperature. The fixing solution is dropped off well (i.e. not washed) and the smear is blocked with 1 h at room temperature with 10% BSA/PBS. The blocking solution is allowed to drop off well (i.e not washed) and the smear is incubated with anti Gb-3 antibody (1:250 dilution) in 0.01% PBST and pipetted on the cells to a final volume of 50-75 μl. The smear is incubated with the antibody over night in humid chamber. On the second day, after the incubation cells are washed 3 times for 5 minutes with PBS. The secondary antibody (donkey-anti-mouse Alexa Fluor (Nr. Z3)) is diluted 1:150 (a final dilution 1:300) in 0.01% PBST, pipetted on the smear and incubated for 1 h at RT. The cells are then washed 1×5 min with 1×PBS, Incubated for 10 minutes with DAPI diluted 10.000 in 1×PBS, washed 3 times for 5 min with 1×PBS and covered with Aqua Poly/Mount. A negative control is also prepared by incubating the smear with blocking solution over-night instead of primary antibody and then the regular protocol and incubation with secondary antibody is performed.

[0146] Gb3 Visualization is Also Possible in Buccal Swabs

Another example shows the use of buccal epithelial cells for Gb3 detection. FIG. 14 shows the first result of Gb3 visualization in buccal epithelial cells, used to create a buccal swab then immunoreacted with antibodies against Gb3. Also in this case the inventors prove that Gb3 deposits are unequivocally visible in buccal swab preparations. Analogously to the blood smear preparations, these results are further expected to improve when using Gb3 specific natural ligands e.g. shiga toxin.

[0147] Materials Used in the Described Protocols:

The anti Gb3-antibodies used were the following commercial antibodies: Anti-Gb3 monoclonal antibody (M. Kotani et al. 1994 Biochem. Biophys. 310, 89); Anti-Gb3 monoclonal antibody, Cat #: A2506: company: TC (Tokyo Chemical Industry Co.) http://www.tcichemicals.com/eshop/deidelcommodity/A2506/

[0148] The buffers and mediums used were composed as follows: 1% PBS (0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4); 4% PFA (Distilled water, HCl, 1 N NaOH, Paraformaldehyde, 1×PBS): Storage medium (50 ml heat inactivated fetal bovine serum, 40 ml RPIM without additives, 10 ml DMSO).

[0149] The composition of 10×PBS stock solution is depicted in Table 4 and the pH has been titrated to 6.7.

TABLE-US-00004 TABLE 4 5 liters 10 liters 2 liters NaCl 400 g  800 g  160 g KCl 10 g  20 g   4 g Na.sub.2HPO.sub.4 71 g 142 g 28.4 g Na.sub.2HPO.sub.4 × 1H.sub.2o 69 g 138 g 27.6 g

[0150] For the preparation of 1 liter of 4% PFA; heat up to approximately 60° C. 800 ml 1×PBS in a glass bottle on a heat plate under the hood, add 40 g paraformaldehyde and mix with a magnetic stirring rod. Add 1 N NaOH dropwise until the paraformaldehyde is completely dissolved. After cooing, titrate pH to 6.9 with HCl and add 1×PBS to a final volume of 1 liter.