Method for the diagnosis of Farber's disease

Abstract

The present invention is related to a method for diagnosing Farber's disease in a subject, wherein the method comprises detecting C26 ceramide in a sample from the subject.

Claims

1. A method for diagnosing Farber's disease in a subject, wherein the method comprises the following steps: i) adding an internal standard to a sample from the subject, wherein the sample from the subject is selected from the group comprising plasma, serum and blood; ii) optionally mixing the sample containing the internal standard; iii) subjecting the sample to a protein precipitation and/or a biomarker extraction step, whereby protein from the sample is precipitated and/or the biomarker is extracted and a first supernatant of the sample is provided; iv) optionally subjecting the first supernatant of the sample or at least a part thereof to a first separation step which provides a second supernatant, whereby the first separation step is a step of centrifugation; v) subjecting the first supernatant and/or the second supernatant, or at least a part thereof, to a second separation step, wherein the second separation step comprises injecting at least a part of the first supernatant and/or at least a part of the second supernatant into an HPLC-MS/MS system and using an HPLC column with a gradient from acidic water to acetonitrile/acetone; wherein the HPLC column is preferably an HPLC column selected from the group consisting of a C8 HPLC column and a C18 HPLC column, and wherein the second separation step provides a separated sample; and vi) subjecting the separated sample to mass spectrometry analysis, wherein mass spectrometry comprises electrospray ionization multiple reacting monitoring (ESI-MRM MS), wherein the mass spectrometry analysis comprises a step a), wherein the step a) comprises detecting a biomarker in the separated sample from the subject, and optionally a step b), wherein the step b) comprises determining a level of the biomarker present in the separated sample, wherein the level of the biomarker is indicative of whether or not the subject is suffering from Farber's disease or whether or not the subject is at risk of suffering from Farber's disease, and wherein the biomarker is C26 ceramide.

2. The method of claim 1, wherein the internal standard is selected from the group consisting of N-lauroyl sphingosine, lyso-Gb2, a C17 ceramide, a C19 ceramide, a C21 ceramide, a C23 ceramide and a C25 ceramide, deuterated C26 ceramide.

3. A method for the treatment of a subject suffering from or being at risk of developing Farber's disease, wherein the method comprises a) performing an assay to detect a biomarker in a sample from the subject; b) determining a level of the biomarker present in the sample; c) comparing the level of the biomarker present in the sample to a cut-off value; and d) treating the subject with a therapy selected from the group consisting of administration of a corticosteroid to the subject, enzyme replacement therapy and bone marrow transplantation if the level of the biomarker present in the sample is higher than the cut-off value, wherein the biomarker is C26 ceramide.

4. The method according to claim 1, wherein the subject is a human.

Description

(1) The present invention is now further illustrated by the following figures and examples from which further features, embodiments and advantages may be taken.

(2) More specifically,

(3) FIG. 1 is a histogram indicating levels of C26 ceramide in DBS of healthy subjects;

(4) FIG. 2 is a diagram indicating signal of C26 ceramide expressed as peak area in DBS of 4 healthy subjects (the four columns on the left) vs. 4 Farber patients (the four columns on the right);

(5) FIG. 3 is a diagram (whisker plot) indicating signal of C26 ceramide in DBS of 42 healthy subjects (fresh samples), 5 healthy subjects (1 year old samples) and 4 Farber patients;

(6) FIG. 4 is a diagram (whisker plot) indicating the level of total C26 ceramide in DBS of 6 Farber carriers, 6 Farber patients and 79 healthy subjects serving as controls, expressed in ng/ml;

(7) FIG. 5 is a diagram (whisker plot) indicating the level of cis C26 ceramide in DBS of 6 Farber carriers, 6 Farber patients and 79 healthy subjects serving as controls, expressed in ng/ml;

(8) FIG. 6 is a diagram (whisker plot) indicating the level of trans C26 ceramide in DBS of 6 Farber carriers, 6 Farber patients and 79 healthy subjects serving as controls, expressed in ng/ml;

(9) FIG. 7 is a panel of diagrams showing the level of cis-C26 ceramide (isoform 1) as nmol/L blood sample, trans-C26 ceramide (isoform 2) as nmol/L sample, the ratio of both isoforms and the level of total C26 ceramide as nmol/L blood sample for newborns, for juveniles and adults being subjects suffering from Farber's disease (FP), subjects being Farber carriers (FC) or normal controls (NC);

(10) FIG. 8 is a total ion chromatogram (TIC) profile of C26-ceramide isoforms in samples from genetically confirmed Farber patients (Farber patient) vs normal controls (Healthy control), whereby the isoforms were analyzed from were dried blood spots (A) or from in clear plasma (B); and

(11) FIG. 9 is a fragmentation pattern of the two isoforms of Ceramide C26 separated by LC/MS using real tandom mass spectrum (MS/MS).

EXAMPLES

(12) In the Examples described in the following DBS was used as a sample from a subject. Nevertheless, a person skilled in the art will acknowledge that depending on the used type of sample from a subject, e.g. comprising saliva, liquor, plasma, serum, full blood, blood on a dry blood filter card or another blood product, the method of the present invention has to be adjusted to the type of sample and furthermore a cut-off value has to be determined for each type of sample according to the method described in the following examples.

Example 1: Method for the Detection of C26 Ceramide in DBS

(13) Equipment

(14) For detecting C26 ceramide and/or a substance with molecular weight of 678, detected as MRM transition in positive mode 679 m/z to 264 m/z, in a sample of plasma from a subject the following equipment was used: Apparatus/Piece of Equipment Type/Producer UPLC Aquity Series (pumps, autosampler, column manager), Waters, UK Mass selective detector TripleQuad 5500, AB SCIEX, Germany Multi-tube vortexer DVX-2500 Henry Troemner LLC, USA Vortex mixer Vortex Genie 2; Scientific Industries, USA Centrifuge Megafuge 1.0; Heraeus, Germany Multipette(s), pipette(s) Eppendorf, Germany Water bath SW21-C, Julabo, Germany
Reagents

(15) For detecting C26 ceramide in a sample from a subject the following reagents were used. To that extent that values depend on temperature (e.g. the pH value) such values were determined at a temperature of 25° C. Acetonitrile (ACN) HPLC-grade or Gradient Acetone 99.5% Dimethylsulfoxide (DMSO) HPLC grade Ethanol (EtOH) p.a., 96% Formic acid (FA) p.a., 98-100% Methanol (MeOH) Gradient (LiChrosolv) Trifluoroacetic acid (TFA) purum >98% Water ASTM-I

(16) The abbreviation “p.a.” as used herein means “pro analysis”.

(17) The term “purum” as used herein, preferably means a commercial grade of a chemical compound having a purity of the above specified value.

(18) ASTM-I as used herein refers to a water grade standard purity achieved by purification methods comprising Reverse Osmosis and Ultraviolet (UV) Oxidation.

(19) Preparation of Calibration Standards

(20) For calibration, all calibration standard C26 ceramide mentioned above having five concentration levels between 2.00 and 200 ng/mL were used.

(21) Preparation of Internal Standard

(22) The Internal Standard (IS 1) stock solution was prepared dissolving 25 μg of C25 ceramide (as delivered by Avanti) in DMSO/MeOH (1/1; vol/vol) to a concentration of 50 ng/mL.

(23) Storing of Samples and Solutions

(24) Control samples or study samples either were immediately stored below −20° C. Standard solutions as well as Internal Standard working solutions were stored between 2° C. and 8° C. until use.

(25) Sample Preparation for Analysis

(26) 3 punches Ø of 3.2 μm were cut from the filtercard with dried blood spots and subjected to extraction in internal standard solution in DMSO: water: ethanol vol. 1:1:2 for 60 minutes at 37° C. and sonicated 20 minutes at 40° C. The solution containing internal standard and blood extract was transferred to a filter plate and then to a 96 well plate by centrifugation at 3.500 rpm.

(27) Methods

(28) A person skilled in the art will acknowledge that methods for detecting C26 ceramide in a sample from a subject using mass spectrometric analysis may also employ other transitions and fragments which allow for specific detection of and/or quantification of C26 ceramide in said sample from a subject.

(29) Software

(30) Data acquisition, data processing, statistics and calculations were performed using Analyst software 1.6.2 or higher (AB SCIEX, USA/Canada).

Example 2: Determining C26 Ceramide in DBS from Healthy Subjects and Farber's Disease Patients and Farber's Disease Carriers

(31) TABLE-US-00003 TABLE 1 The samples used in this example were as follows: Sample description Number of samples Healthy controls fresh samples 42 Healthy controls 1 year old 5 samples kept at RT Healthy controls random samples 59 Farber's disease patients 5 Farber's disease carriers 7

(32) The following experiments were performed using the methods described at Example 1: a) C26 ceramide level was determined in DBS from 79 random healthy controls. The results of the analyses are described in Table 2 and visualized in FIG. 1 showing a median level of 88.6 ng/mL.

(33) TABLE-US-00004 TABLE 2 Results of the C26 ceramide determination in DBS from healthy controls Descriptive statistics Mean 90.9 Standard Error 2.2 Median 88.6 Standard Deviation (STD) 19.5 Range 81.9 Minimum 53.4 Maximum 135.3 CUT OFF (Mean + 2 * STD) 129.8 Count 79.0 b) C26 ceramide signal was measured in DBS from 4 random healthy controls and 4 Farber's disease patients. The results of the analyses are visualized in FIG. 2. showing that the individual and average C26 levels in Farber' disease patients are higher compared with the levels found in healthy controls. c) C26 ceramide signal was measured in DBS from 42 freshly collected samples from healthy controls, 5 old samples from healthy controls and 4 Farber's disease patients. The results of the analyses are visualized in FIG. 3. showing that C26 levels healthy controls are lower than the levels found in Farber's disease patients, irrespectively if the samples are fresh or have been stored. d) C26 ceramide levels were determined in DBS from 79 healthy controls, 6 Farber's disease carriers and 6 Farber's disease patients. The results of the analyses are visualized in FIG. 4. and listed in Table 3. The results show a good separation between Farber's disease patients/carriers on the one hand and healthy controls on the other hand.

(34) TABLE-US-00005 TABLE 3 Farber's disease Farber's disease Healthy carriers patients controls Number of samples 6 6 79 Minimum 88.3 155.5 53.4 25% Percentile 94.73 183.0 75.2 Median 228.1 247.7 88.6 75% Percentile 375.0 433.6 106.7 Maximum 590.6 787.5 135.3 Mean 255.9 324.4 90.1 Std. Deviation 185.2 234.5 19.5 Std. Error 75.6 95.75 2.1

Example 3: Determining Cis-C26 Ceramide and Trans-C26 Ceramide in DBS from Healthy Subjects and Farber's Disease Patients and Farber's Disease Carriers

(35) The levels of Ceramide C26 isomers were tested in 6 genetically confirmed Farber patients, 6 genetically confirmed Farber carriers and 79 normal controls. In the experiments a standard curve with Ceramide C26 was used and the results were expressed in ng/mL of blood. The results are summarized in the table 4.

(36) TABLE-US-00006 TABLE 4 Farber patients Farber Carriers Healthy controls (N = 6) (N = 6) (n = 79) Total Trans Cis Total Trans Cis Total Trans Cis C26 C26 C26 C26 C26 C26 C26 C26 C26 mean 324 18 307 256 28 228 91 29 62 maxi- 788 20 768 591 45 571 135 44 103 mum mini- 155 11 139 88 20 43 53 18 134 mum Standard deviation 19 6 18 Cut-off 130 41 97

(37) The results are also illustrated in FIGS. 5 and 6.

(38) The experiments show that the cis-isomer rather than the trans-isomer of C26 ceramide is mainly accumulating in the lysosomes of Farber patients. Cis-C26 ceramide and total C26 ceramide can be used as biomarkers for Farber disease to distinguish between healthy persons and Farber patients. Trans-C26 ceramide levels are slightly decreased in patient samples.

Example 4: Standardization and Validation of Cis-C26 Ceramide and Trans-C26 Ceramide Assay

(39) The normal range of C26 ceramides levels in blood was determined on a group of 192 normal controls with ages from 3 months to 65 years. The results show that total C26—ceramide has a value of 49.8±9.6 nmol/L blood, from which 18.5±4.9 nmol/L is isoform 1 blood and 33.8±8.6 nmol/L isoform 2. For the pathological range, 10 clinically confirmed Farber patients with ages from 2 months to 22 years were found to have a total C26 ceramide concentration of 134.8±52.2 nmol/L blood, from which 94.6±55.4 nmol/L blood is isoform 1 (which is cis-C26 ceramide) and 40.1±19.6 nmol/L isoform 2 (which is trans-C26 ceramide).

(40) The cut-off for total C26—ceramide was set at 69.0 nmol/L and at 28.3 nmol/L for isoform 1 (mean value of the normal controls+2*standard deviation).

(41) When the results of C26 ceramide analyses are grouped according to the patients ages into newborns (up to 6 months, Farber phenotype); juveniles (6 months to 17 years, Farber phenotype) and adults (>17 years, SMA phenotype), several trends emerge: (i.) the concentration ranges of C26 ceramide and its isoforms in normal controls are not changing with age; (ii.) isoform 1 is extremely high in the newborn Farber patients (187.5±6.4 nmol/L) and decreases in juvenile (80.8±27.1 nmol/L) and even more in the patients that reach adulthood (though still above the normal range, 43.0±12.7 nmol/L); (iii.) isoform 2 is in normal range in newborn Farber patients (31.0±2.0 nmol/L) and increases to pathological levels in patients that reach adulthood (72.0±21.2 nmol/L); (iv.) both isoform 1 and isoform 2 are low (in normal range) in the newborn Farber carriers and could increase in time to pathological levels although the carriers included in this study do not present clinical symptoms characteristic to FD; moreover the levels in adult carriers seem to decrease compared with the juvenile carriers. The results are also illustrated in FIG. 7 Said FIG. 7 shows C26 ceramide levels in the blood of Farber patients referred to as FP, Farber carriers referred to as FC and normal controls referred to as NC. All the patients, carriers and normal controls were grouped according to the donors ages in newborns (up to 6 months), juveniles (6 months to 17 years) and adults (>17 years).

(42) TABLE-US-00007 TABLE 5 Concentration of C26 ceramide isoforms in the blood of Farber patients, Farber carriers and normal controls. Ceramide C26:0 (nmol/L) (mean ± STD) Samples N Isoform1 Isoform2 ratio Total Farber Patients Newborns (0-6 months) 2 187.5 ± 6.4  31.0 ± 2.0  6.1 ± 0.2 218.5 ± 9.1  Juvenile (0.5-17 years) 6 80.8 ± 27.1 32.5 ± 9.4  2.6 ± 1.0 113.5 ± 34.05 Adults (>17 years) 2 43.0 ± 12.7 72.0 ± 21.2 0.6 115.0 ± 33.9  All Farber patients 10 94.6 ± 55.4 40.1 ± 19.6 2.9 ± 2.0 134.8 ± 52.2  Farber Carriers Newborns (0-6 months) 1 34.0 28.0 1.2 62.0 Juvenile (0.5-17 years) 2 193.0 ± 36.0  53.0 ± 33.9 4.3 ± 2.1 246.5 ± 71.4  Adults (>17 years) 8 59.63 ± 31.89 40.4 ± 13.0 1.6 ± 0.7 99.9 ± 34.1 All Farber carriers 11 81.6 ± 62.8 41.6 ± 16.7 2.0 ± 1.4 123.1 ± 71.92 Normal Controls Newborns (0-6 months) 31 18.3 ± 5.8  35.3 ± 11.8 0.6 ± 0.3 53.6 ± 11.7 Juvenile (0.5-17 years) 78 19.7 ± 5.1   25.9 ± 10.43 0.6 ± 0.2 50.5 ± 10.7 Adults (>17 years) 83 17.4 ± 4.1  31.5 ± 6.5  0.6 ± 0.2 48.6 ± 8.1  All normal controls 192 18.5 ± 4.9  33.8 ± 8.6  0.6 ± 0.2 49.8 ± 9.6  Cut-off (mean.sub.controls + 2 * STD) 28.3 — — 69.0

(43) These results indicate that in classical Farber phenotype isoform 1 is dominant from the first months of life while for SMA phenotype isoform 2 is accumulating dominantly. Nevertheless the concentration of the total C26 ceramide and the concentration of isoform 1 can be used as specific biomarkers to clearly distinguish between the normal controls and Farber patients.

(44) Characteristics of the C26 Ceramide Assay for Farber Disease Diagnosis

(45) Precision of the assay was checked by determining the C26 ceramides levels in one Farber sample and in one normal control sample 6 times in the same batch (intra-assay) in two different batches (inter-assay). The coefficient of variation (CV %) for within-run precision was found to be between 3.1% and 4.2% and between-runs 3.7% and 6.1%. Accuracy of the quantification method was investigated using 7 solutions of trans-C26 ceramide of different concentrations, covering different areas of the calibration line (low, medium, high and above the highest point in the calibration line) 6 times on the same batch in 2 different batches. For accuracy, within-run CV % was found to be between 1.7 to 3.8% and between-runs 0.3-2.7%. Linearity of the standard curve was determined 5 times in the same batch using 7 solutions of trans-C26 ceramide of different concentrations (0 ng/mL to 100 ng/mL).

(46) The curve was found to be linear with R values between 0.9981 and 0.9996. The selectivity of the measurement was insured by using LC-MRM-MS, cross-contamination was checked for each batch by injecting IS solution after the highest concentration of the standard curve. Limit of detection was found to be 0.12 ng/mL and limit of quantification 0.4 ng/mL in blank filter paper. Robustness of the method was checked by measurement of the DBS extract immediately after the sample preparation, at 12 h after the sample preparation and at 24 h after the sample preparation. Total C26 ceramide and isoform 1 were increased above cutoff for all the investigated Farber samples, for this cohort (100% sensitivity).

(47) The specificity of the total C26 ceramide and isoform 1 as biomarkers for Farber, C26 Ceramide content was investigated using the 10 Farber patients, 192 normal controls, 2 JIA (juvenile idiopathic arthritis) patients and 30 genetically confirmed LSD patients (5 GD (Gaucher disease), 5 PD (Pompe disease), 5 NPA/B (Niemann-Pick type A/B, 5 FD and 5 MPS 2 (mucopolysaccharidosis type 2). The results show that the two biomarkers are increased above the cutoff for the Farber patients and below the cut-off for all the normal controls, JIA patients and patients affected by LSDs characterized by an impaired enzyme.

(48) TABLE-US-00008 TABLE 6 Concentration of C26 ceramide isoforms in the blood of Farber patients, JIA patients, patients affected by other LSD and normal controls. Ceramide C26:0 (nmol/L) Samples N Isoform1 Isoform2 Ratio Total Cut-off 28.3 — — 69.0 Farber's Patients 10  94.6 ± 55.4  40.1 ± 19.6 2.9 ± 2.0 134.8 ± 52.2 Juvenile idiopathic arthritis Patients 2 17.5 ± 0.7  37.5 ± 14.8 0.5 ± 0.3  55.0 ± 14.1 Gaucher Patients 5 24.0 ± 1.4 35.6 ± 6.8 0.7 ± 0.2 59.3 ± 6.8 Pompe Patients 5 21.3 ± 3.0 28.8 ± 2.3 0.8 ± 0.1 50.0 ± 5.0 Niemann-Pick Type A/B Patients 5 19.5 ± 7.8 28.5 ± 9.9 0.8 ± 0.3  48.5 ± 13.3 Fabry Patients 5 25.5 ± 3.8 32.5 ± 1.0 0.8 ± 0.2 57.5 ± 3.0 Hunter Patients 5 23.3 ± 5.6 35.8 ± 7.0 0.7 ± 0.1  59.0 ± 10.8 All Normal Controls 192 18.5 ± 4.9 33.8 ± 8.6 0.6 ± 0.2 49.8 ± 9.6

Example 5: Confirmation of the C26 Ceramide Structure as Biomarker for Farber's Disease

(49) Further MS/MS analyses confirmed the presence of the same compound in both peaks corresponding to the C26 Ceramide transition. The two peaks correspond to the cis- and trans-isomers of the C26 ceramide. This is based on the fact that pure trans-C26 ceramide has the same retention time (2.17 min) and fragmentation pattern as the Isoform 2 found in biological samples (see FIG. 8 and FIG. 9).

(50) More specifically, FIG. 8 shows the total ion chromatogram (TIC) profile of C26-ceramide isoforms in samples from genetically confirmed Farber patients vs normal controls, whereby the isoforms were analyzed from were dried blood spots (A) or from in clear plasma (B) (clear plasma was prepared from whole blood with no visible signs of hemloysis. In DBS both isoforms are present in both samples from healthy controls and Farber patients; while the isoform 1 is dominant in the Farber patients samples, the isoform 2 is dominant in healthy controls. Isoform 1 is not present in the samples from healthy controls, while in the samples of Farber patients it can be quantified.

(51) More specifically, FIG. 9. shows the fragmentation patterns of the two isoforms of Ceramide C26 separable by LC/MS which are the cis- and trans-isomers of the C26. This is based on two facts: (i.) the analysis of pure trans-C26 ceramide reveals that it elutes at RT 2.17 min (second peak) and (ii.) the fragmentation pattern is identical for the two peaks.

(52) The features of the present invention disclosed in the specification, the claims, the sequence listing and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.