Method for the Diagnosis of Gaucher's Disease

Abstract

The present invention is related to an in vitro method for diagnosing Gaucher's disease in a subject comprising a step of a) detecting a biomarker in a sample from the subject, wherein the biomarker is free lyso-Gb1.

Claims

1-16. (canceled)

17. A method for generating quantitative data for a subject comprising the step of determining at several points in time a level of a biomarker present in a sample from the subject, wherein the biomarker is free lyso-Gb1, and wherein the level of the biomarker is indicative of the severity of the disease in the subject.

18. The method of claim 17, wherein the sample is selected from the group consisting of a blood sample, a serum sample, a plasma sample, a whole blood sample and a sample from whole blood collected on a dry blood filter card.

19. The method of claim 17, wherein the subject has been previously treated or diagnosed for Gaucher's disease.

20. The method of claim 17, wherein the level of the biomarker present in the sample from the subject is determined on a regular basis.

21. The method of claim 17, wherein the level of the biomarker present in the sample from the subject is determined every 3 months or every 6 months.

22. The method of claim 17, wherein free lyso-Gb1 is lyso-Gb1 as present in the subject and not the result of manipulating the sample from the subject.

23. The method of claim 17, wherein the blood sample is a full blood sample or a dry blood filter sample.

24. The method of claim 17, wherein the biomarker is detected by means of mass spectrometry analysis, immunoassay, biochip array, functional nucleic acids and/or a fluorescent derivative of free lyso-Gb1.

25. The method of claim 24, wherein the biomarker is detected by means of mass spectrometry.

26. The method of claim 17, wherein at each of the several points in time a separate sample is taken from the subject and the level of the biomarker is determined in the separate sample.

27. The method of claim 17, wherein lyso-Gb1 is of formula (I) ##STR00002##

Description

[0232] The present invention is now further illustrated by the following figures and examples from which further features, embodiments and advantages may be taken.

[0233] More specifically,

[0234] FIG. 1A is a boxplot indicating levels of free lyso-Gb1 in ng/ml plasma;

[0235] FIG. 1B is a boxplot indicating levels of free lyso-Gb1 in ng/ml plasma grouped by gender of the subjects;

[0236] FIG. 2A is a graph showing receiver operating characteristics (ROC) curves of free lyso-Gb1 and chitotriosidase;

[0237] FIG. 2B is a graph showing receiver operating characteristics (ROC) curves of free lyso-Gb1 and CCL18;

[0238] FIG. 3A is a diagram showing free lyso-Gb1 in ng/ml plasma as a function over time for a total of 20 German Gaucher's disease patients;

[0239] FIG. 3B is a diagram showing free lyso-Gb1 in ng/ml plasma as a function over time for a total of 24 non-treated Gaucher's disease patients (10 German, 14 Israeli patients);

[0240] FIG. 3C is a diagram showing free lyso-Gb1 in ng/ml plasma as a function over time for a total of 9 Israeli Gaucher's disease patients before and after start of therapy;

[0241] FIG. 3D is a diagram showing regression based values of free lyso-Gb1 in ng/ml plasma as a function over time for Israeli and German Gaucher's disease patients before and after start of therapy;

[0242] FIG. 4 is a table showing the median level of free lyso-Gb1 for two frequent mutations;

[0243] FIG. 5A is an HPLC-mass spectrometric chromatogram displaying peak intensity of free lyso-Gb1 and IS of a healthy subject;

[0244] FIG. 5B is an HPLC-mass spectrometric chromatogram displaying peak intensity of free lyso-Gb1 and IS of a Gaucher's disease patient;

[0245] FIG. 5C is an HPLC-mass spectrometric chromatogram displaying peak intensity of free lyso-Gb1 and IS of a Gaucher's disease patient;

BRIEF DESCRIPTION OF THE FIGURES

[0246] FIG. 1A is a boxplot indicating levels of free lyso-Gb1; the y-axis demonstrates the logarithmised levels of free lyso-Gb1 in ng/ml determined in plasma of Patients by the method according to the present invention, wherein the x-axis depicts groups of patients, which have been grouped as described in Example 2. The boxplot represents the 25.sup.th and 75.sup.th percentile of each group of patients by the bottom and top of the box, respectively; the band near the middle of the box represents the 50.sup.th percentile (i.e. the median) of each group; The whiskers represent one standard deviation above and below the mean of the data; Any data not included between the whiskers is shown as an outlier with a small circle or star. The horizontal line represents the cut-off level of 5 ng/ml.

[0247] FIG. 1B is a boxplot indicating the levels of free lyso-Gb1 as depicted in FIG. 1A additionally grouped by gender of the subjects; the y-axis represents the logarithmised levels of free lyso-Gb1 in ng/ml determined in plasma of patients by the method according to the present invention, wherein the x-axis represents groups of patients, which have been grouped as described in Example 2 and additionally by the gender of the patients. The boxplot represents the 25.sup.th and 75.sup.th percentile of each group of patients by the bottom and top of the box, respectively; the band near the middle of the box represents the 50.sup.th percentile (e.g. the median) of each group; The whiskers represent one standard deviation above and below the mean of the data; Any data not included between the whiskers is shown as an outlier with a small circle or star. The horizontal line represents the cut-off level of 5 ng/ml.

[0248] FIG. 2A is a graph showing receiver operating characteristics (ROC) curves of free lyso-Gb1 and chitotriosidase; the x-axis represents “1-specificity” and the y-axis represents the sensitivity. Free lyso-Gb1 demonstrates a 100% sensitivity and 100% specificity, wherein chitotriosidase has at the best a sensitivity of 0.9591 or 95.91%, respectively.

[0249] FIG. 2B is a graph showing receiver operating characteristics (ROC) curves of free lyso-Gb1 and CCL18; the x-axis represents “1-specificity” and the y-axis represents the sensitivity. Free lyso-Gb1 demonstrates a sensitivity of 100% and a specificity of 100%, wherein CCL18 has at the best a sensitivity of 0.8658 and 86.58%, respectively.

[0250] FIG. 3A The y-axis represents levels of free lyso-Gb1 as a function over time determined by the method according to the present invention in ng/ml of plasma of 20 German Gaucher's disease patients which were subjected to therapy, more precisely ERT, during the course of the study. Each curve and each patient number, respectively, represents levels determined in plasma collected from the same patient at different time points as indicated on the x-axis. The x-axis represents the time points of plasma collection, wherein time point zero indicates the first measure under therapy for each patient. For the analysis of the change of the level of free lyso-Gb1 over time in Gaucher's disease patients as described in Example 3 non aggregated data was used for those patients for which more than one blood sample has been analysed.

[0251] FIG. 3B is a diagram showing free lyso-Gb1 as a function over time determined by the method according to the present invention in ng/ml of plasma of a total of 24 non-treated Gaucher's disease patients (10 German, 14 Israeli patients); Non-treated as used herein, preferably means that no treatment, e.g. enzyme replacement therapy, has been applied with regard to Gaucher's disease. For the analysis of the change of the level of free lyso-Gb1 over time in Gaucher's disease patients as described in Example 3 non aggregated data was used for those patients for which more than one blood sample has been analysed.

[0252] FIG. 3C is a diagram showing free lyso-Gb1 as a function over time with the free lyso-Gb1 being determined by the method according to the present invention in ng/ml of plasma of a total of 9 Israeli Gaucher's disease patients during time before and after the start of the therapy. The x-axis indicates the time in month, wherein “0” indicates the first point in time after start of therapy. The curve labeled with “overall” depicts the regression based values of free lyso-Gb1.

[0253] FIG. 3D is a diagram showing the regression based values of free lyso-Gb1 as a function over time determined by the method according to the present invention in ng/ml of plasma of a Israeli and German Gaucher's disease patients during time before and after the start of the therapy. The x-axis indicates the time in months, wherein “0” indicates the first point in time after start of therapy. The curve labeled with “overall” depicts the regression based values of free lyso-Gb1.

[0254] FIG. 4 is a table showing the median level of free lyso-Gb1 in patients positively tested for one of two frequent mutations of the glucocerebrosidase gene, namely N370S and L444P in the homozygous as well as in the compound heterozygous situation, wherein compound heterozygosity is the condition of having two heterogeneous recessive alleles at a particular locus that can cause genetic disease in a heterozygous state. A person skilled in the art will acknowledge that patients having a mutation L444P of the glucocerebrosidase gene also face a more malignant prognosis, which is particularly true in the homozygous situation. Accordingly, it is an embodiment of the present invention that the method according to the present invention comprises determining the severity of Gaucher's disease. Said determining the severity of Gaucher's disease comprises determining a level of the biomarker, preferably free lyso-Gb1, present in the sample from the subject and/or comparing said level of said biomarker determined in samples from subjects having different mutations of the glucocerebrosidase gene and/or having no mutation of the glucocerebrosidase gene. The present inventors have found that in a sample from a patient being positively tested for having a homozygous L444P mutation of the cerebrosidase gene the level of free lyso-Gb1 determined by the method according to the present invention is about 194 ng/ml and is elevated compared to the level of free lyso-Gb1 determined in a sample from a patient being positively tested for having a homozygous N370S mutation of the cerebrosidase gene, wherein the level of free lyso-Gb1 determined by the method according to the present invention is about 159 ng/ml). The inventors have also found that in a sample from a patient being positively tested for having a compound heterozygous L444P mutation the level of free lyso-Gb1 determined by the method according to the present invention is 89 ng/ml and is significantly lower compared to the level of free lyso-Gb1 determined by the method according to the present invention in a sample from a patient being positively tested for having a homozygous L444P mutation wherein the level of free lyso-Gb1 determined by the method according to the present invention is about 45.4 ng/ml. Without wishing to be bound by theory the present inventors believe that the level of free lyso-Gb1 in a sample from a subject determined by a method of the present invention is indicative for the severity of Gaucher's disease. It is thus a further embodiment of the present invention that the method of the present invention is for determining the effectiveness of at least one treatment applied to a subject being positively tested for suffering from and/or being at risk for developing Gaucher's disease. The numbers depicted in brackets indicate the ranges of concentration measured in the respective patient group. IQR means interquartile range. All patients that were subjected to a therapy for Gaucher's disease were subjected to ERT.

[0255] FIG. 5A is an HPLC-mass spectrometry chromatogram displaying peak intensity in cps of free lyso-Gb1 and IS of a sample from a healthy subject as a function over the retention time in minutes. The retention time of a substance as used herein, preferably is depicted on the x-axis and is the elapsed time between the time of injection of a solute, e.g. a biomarker according to the present invention and/or an internal standard, and the time of elution of the peak maximum of said solute. A person skilled in the art will acknowledge that the retention time of a substance according to the herein described methods is a unique characteristic of said solute and can be used for identification purposes. Internal Standard working solution comprising Lyso-Gb2 as an internal standard was added to the sample as described in Example 1. It is important to understand that by said addition of IS to the sample, i.e. spiking of the sample, to be subjected to the method according to the present invention, the concentration of IS in the sample is known and by determining the area under the peak, i.e. the peak area, of the internal standard in said HPLC-mass spectrometric chromatogram the relation between a peak area and a concentration of a substance, e.g. of IS and/or a biomarker thus can be calculated. More precisely, a person skilled in the art will acknowledge that a peak area of a substance depicted in an HPLC-mass spectrometric chromatogram, such as the HPLC-mass spectrometric chromatogram depicted in FIG. 5A, FIG. 5B or FIG. 5C, represents a measure for an amount of said substance subjected to an HPLC-mass spectrometric analysis. Moreover, a person skilled in the art will be able to calculate the amount of the substance in a sample from a subject subjected to an HPLC-mass spectrometric analysis, e.g. the amount of free lyso-Gb1 in a sample subjected to the method of the present invention, using a ratio of the peak area of free lyso-Gb1, the amount of which is to be determined by said method and the peak area of IS, e.g. free lyso-Gb2; as well as calibration curves generated with said method and said free lyso-Gb1 and/or IS. Accordingly, this allows subsequently for determining a level of free lyso-Gb1.

[0256] FIG. 5B is an HPLC-mass spectrometry chromatogram displaying peak intensity of free lyso-Gb1 and IS of a sample from a Gaucher's disease patient, wherein a level of 17.1 ng/ml free lyso-Gb1 was determined according to the method of the present invention as essentially described in Example 1. Comparing said level of the biomarker in the sample from the subject to a cut-off level of 5 ng/ml, which has been selected such that a sensitivity for diagnosing Gaucher's disease in a subject according to the methods of the present invention is 100% and that a specificity for diagnosing Gaucher's disease in a subject according to the methods of the present invention is 100%, an elevated level of the biomarker in the sample from the subject compared to the cut-off level is indicative for the subject for suffering from Gaucher's disease.

[0257] FIG. 5C is an HPLC-mass spectrometry chromatogram displaying peak intensity of free lyso-Gb1 and IS of a sample from a Gaucher's disease patient, wherein a level of 319 ng/ml free lyso-Gb1 was determined according to the method of the present invention as essentially described in Example 1. Comparing said level of the biomarker in the sample from the subject to a cut-off level of 5 ng/ml, which has been selected such that a sensitivity for diagnosing Gaucher's disease in a subject according to the methods of the present invention is 100% and that a specificity for diagnosing Gaucher's disease in a subject according to the methods of the present invention is 100%, an elevated level of the biomarker in the sample from the subject compared to the cut-off level is indicative that the subject is suffering from Gaucher's disease.

EXAMPLES

[0258] In the Examples described in the following human plasma 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 level has to be determined for each type of sample according to the method described in the following examples. The present inventors have found that using a sample of human serum in the method as described below instead of a sample of human plasma will lead to identical results according to a detection of and a thus determined level of free lyso-Gb1, if the sample of human serum and the sample of human plasma derive from the same subject, and were taken at the same time point; and wherein the samples were measured in parallel; and, more particularly, will lead to the same cut-off level. Without whishing it be bound and in way of illustrative examples, by use of saliva from a human patient a method may be adjusted in dependence of a pH value of the sample; or a cut-off level may be determined being 20 ng/ml if using full blood or blood collected on a dry blood filter card as a sample from a subject.

Example 1: Method for the Detection of Free Lyso-Gb1 in Human Serum

Equipment

[0259] For detecting free lysoGb-1 in a sample of plasma from a subject the following equipment was used.

TABLE-US-00001 Apparatus/Piece of Equipment Type/Producer HPLC pump Series 200, Perkin Elmer, USA Sample injector Series 200, Perkin Elmer, USA Column oven Series 200, Perkin Elmer, USA Mass selective detector API 4000 Q TRAP, AB SCIEX, USA/Canada Multi-tube vortexer Henry Troemner LLC, USA DVX-2500 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

[0260] For detecting free lysoGb-1 in a sample of plasma from a subject the following reagents were used.

[0261] To that extent that values depend on temperature (e.g. the pH value) such values were determined at a temperature of 25° C.

TABLE-US-00002 Reagent Purity Acetonitrile (ACN) HPLC-grade or Gradient grade 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 The abbreviation “p.a.” as used herein means “pro analysis”.

[0262] The term “purum” as used herein, preferably means a commercial grade of a chemical compound having a purity of the above specified value.

[0263] ASTM-I as used herein refers to a water grade standard purity achieved by purification methods comprising Reverse Osmosis and Ultraviolet (UV) Oxidation.

Preparation of Calibration Standards

[0264] A Lyso-Gb1 stock solution was prepared dissolving 1.70 mg Lyso-Gb1 (as delivered by Matreya) in 5 mL of MeOH.

[0265] Subsequently the solution V1-A-534 was prepared as a mixture of 12 μL of Lyso-Gb1 stock solution and 5 mL DMSO/MeOH (1:1; v/v) as displayed in the following:

TABLE-US-00003 Label of Volume of volume of resulting exp. conc. solution solvent solution [μg/mL] [μL] solution [mL] solvent V1-A-534 0.79968 12 Lyso-Gb1-stock 5 DMSO/MeOH (1:1; v/v)

[0266] Subsequently the Calibration Standards were prepared by spiking solution V1-A-534 or higher concentrated Calibration Standards into the solvent MeOH/water (1:1; v/v).

[0267] A detailed spiking scheme will be displayed in the following.

TABLE-US-00004 Label of Volume of volume of resulting concentration solution solvent Volume solution [ng/mL] [μL] solution [mL] solvent [ml] Std9A-534 102.12 366 V1-A-534 2.5 MeOH/water 2.866 (1:1; v/v) Std8A-534 40.970 162 V1-A-534 3 MeOH/water 3.162 (1:1; v/v) Std7A-534 15.321 353 Std9A-534 2 MeOH/water 2.353 (1:1; v/v) Std6A-534 6.1464 353 Std8A-534 2 MeOH/water 2.353 (1:1; v/v) Std5A-534 2.5906 135 Std8A-534 2 MeOH/water 2.135 (1:1; v/v) Std4A-534 1.0577 53 Std8A-534 2 MeOH/water 2.053 (1:1; v/v) Std3A-534 0.41004 55 Std7A-534 2 MeOH/water 2.055 (1:1; v/v) Std2A-534 0.15868 53 Std6A-534 2 MeOH/water 2.053 (1:1; v/v) Std1A-534 0.050049 39.4 Std5A-534 2 MeOH/water 2.0394 (1:1; v/v)

[0268] For calibration, calibration standards having seven concentration levels between 0.400 and 100 ng/mL were used, namely Calibration Standards Std3A-534, Std4A-534, Std5A-534, Std6A-534, Std7A-534, Std8A-534 and Std9A-534.

Preparation of Control Samples

[0269] Control samples were prepared by spiking solution V1-A-534 or a higher concentrated control sample into a blank matrix.

[0270] A detailed spiking scheme will be displayed in the following.

TABLE-US-00005 Label of Volume of volume of resulting concentration solution blank matrix Volume solution [ng/mL] [μL] solution [mL] [ml] QC-A1- 1.0013 173.6 QC-C1- 8.5 8.6736 534 534 QC-B1- 5.0008 944 QC-C1- 8.5 9.444 534 534 QC-C1- 50.029 634 V1-A- 9.5 10.134 534 534

Blank Matrix

[0271] As a blank matrix, human plasma of a healthy subject was used. A person skilled in the art will acknowledge that said plasma from a healthy subject will contain a native level of free lyso-Gb1. Said native level of free lyso-Gb1 is about 1.4 ng/ml according to the methods of the present invention. It is thus obvious that control samples prepared by spiking of the blank matrix, the blank matrix comprising said native level of free lyso-Gb1, also comprise said native level of free lyso-Gb1 in addition to the level of free lyso-Gb1 obtained by spiking with a concentrated solution or higher concentrated control sample. Accordingly, the level of free lyso-Gb1 in the control samples is as follows:

TABLE-US-00006 QC-A1-534 1 ng/mL + native concentration in blank matrix QC-B1-534 5 ng/mL + native concentration in blank matrix QC-C1-534 50 ng/mL + native concentration in blank matrix 

[0272] A person skilled in the art will acknowledge that human plasma of a healthy subject used as blank matrix can be purchased at any commercial source known to the one skilled in the art. It is important to note that if accidentally plasma of a non-healthy subject, i.e. of a subject having Gaucher's disease, is used as the blank matrix, this will result in unusually high levels of free lyso-Gb1 in the control samples determined by the method according to the present invention and thus will be immediately recognized, as the tolerance of the method is determined as being within a range of 15% above or below the estimated levels of the controls subjected to the method according to the present invention.

Study Samples

Preparation of Internal Standard

[0273] The Internal Standard (IS1) stock solution was prepared dissolving 1.00 mg of Lyso-Gb2 (as delivered by Matreya) in 2 mL of DMSO/MeOH (1/1; vol/vol).

[0274] Subsequently the Internal Standard Working Solution was prepared as a mixture of 410 μL of IS1 stock solution and 500 mL of ethanol. The ethanol may be purchased from any commercial source, wherein the ethanol is absolute ethanol having a grade suitable for the methods described herein. A person skilled in the art will recognize that proteins contained in 50 μl of a sample have to precipitate if 100 μL of said Internal Standard working solution are added to the sample.

Storing of Samples and Solutions

[0275] Control samples or study samples either were immediately stored below −20° C. at once or aliquots were transferred into new glass vials before storing under the same conditions.

[0276] Concentrated solutions (stock solutions, V1-A-534 etc.) as well as Internal Standard stock solutions were frozen below −20° C. pending next spiking.

[0277] Internal Standard working solutions were stored between 2° C. and 8° C. until use. The present inventors have found that free lyso-Gb1 is stable in the above mentioned solutions. More precisely, the level of free lyso-Gb1 of a plasma and/or a serum sample of a Gaucher's disease patient determined by the methods according to the present invention were found to be identical, if the level of free lyso-Gb1 was determined in said samples prior to and after storage at 37° C. for 2 days. Accordingly, the solutions and samples of the present invention can be transported in a number of ways well known to one skilled in the art, wherein the use of a cold chain for transportation of patient material is preferred but not necessarily required. A person skilled in the art will also know methods and their respective conditions for appropriate storage of solutions and samples, wherein, for example, said solutions and samples may be stored for several weeks.

Sample Preparation for Analysis

[0278] All samples used in an analytical batch are prepared for analysis as follows: [0279] Frozen samples were thawed at approximately 20 to 25° C. in a water bath taking from ambient conditions. After thawing the samples were mixed. [0280] 50 μL of the sample were transferred into a sample vial. [0281] 100 μL of Internal Standard working solution (in EtOH) was added to the sample. [0282] The thus obtained mixture was subsequently mixed using a DVX-2500 Multi-tube vortex device at 2500 rpm for about 30 seconds. [0283] The thus obtained mixture was centrifuged for phase separation at 4000 rpm for 2 minutes. [0284] Transfer of a volume of the supernatant adequate to injection purposes (approx. 100 μL) into appropriate (conical) auto-sampler vials.

Methods

Chromatographic and Auto-Sampler Parameters

[0285] The samples prepared for analysis as described above were subsequently subjected to the method described in the following:

TABLE-US-00007 Parameter Scheduled range/description Mobile phase solvent A 50 mM FA in water Mobile phase solvent B 50 mM FA in ACN/acetone (1:1; vol/vol) Chromatographic run 0.0-4.0 min linear gradient: 5% B .fwdarw. 66% B 4.1-5.1 min isocratic: 100% B 5.1-5.9 min isocratic: 5% B Flow 0.9 mL/min Injection volume 5 μL Injector flush 0.1% TFA in 70% MeOH Column + Precolumn ACE 3 C8, 50 × 2.1 mm ID + Security Guard C8 Column temperature 60° C. Retention time approx. 3.4 to 3.6 min: lyso-Gb1 and lyso-Gb 2 (IS)

[0286] The ACE 3 C8 column (ACE C8 column Nr. ACE-112-0502) used herein has been purchased from Advanced Chromatography Technologies, Aberdeen.

[0287] It will be appreciated by a person skilled in the art that parameters where a “±” range is indicated represent parameters which may be adjusted between sequences. A sequence as used herein, preferably is a batch of defined numbers of samples, preferably 250 in maximum analyzed sequentially, wherein parameters comprising flow and temperature remain unchanged. Adjustments and calibrations performed between sequences are known to those skilled in the art and comprise exchange of the column.

[0288] These adjustments within the specified limits are minor changes and are recorded within the raw data of the study at the measuring station.

Detection

[0289] The thus prepared samples were subsequently subjected to the detection method the parameters of which are described in the following:

MS Ionisation mode: Electrospray Ionisation (ESI)
MS polarity: positive
MS detection mode: Multiple reaction monitoring (MRM)
Vaporizer temperature: 500° C.±50° C.
Ionisation voltage: 5.5 kV
Collisionally activated dissociation low

(CAD) gas:

Gas 1: Pressure=45 psi

Gas 2: Pressure=60 psi

[0290] Curtain gas: pressure=40 psi
Lateral position: 5 units
Vertical position: 4 units
Quadrupole resolution unit unit
Transitions 462.4.fwdarw.282.2 m/z lyso-Gb1 [0291] 624.5.fwdarw.282.2 m/z lyso-Gb2 (Internal Standard)
DP (declustering potential) 40 V
CXP (collision cell exit potential) 8 V

[0292] A person skilled in the art will acknowledge that methods for detecting free lyso-Gb1 and/or determining the level of free lyso-Gb1 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 free lyso-Gb1 in said sample from a subject.

Evaluation and Calculation of Results

[0293] To evaluate and to calculate results obtained with the above specified methods the following protocol were applied.

Rounding Procedure

[0294] Concentration data fed into and retrieved from the chromatographic data system (CDS) were rounded to five significant digits. Further calculations in the spreadsheet were performed to full computational accuracy and subsequently rounded to the significant digits/decimal places to be reported. Hence, deviations of intermediate results might occur caused by rounding. Accuracy and coefficients of variation (CV) will be reported with one and two decimal places, respectively.

[0295] Note referring to the rounding procedure: The last digit reported would be up-rounded if the subsequent digit was equal or greater than “5”.

Regression and Statistics

[0296] Based on Calibration Standards the calibration curve fitting were established using the data processing software by means of peak area ratios (peak area of free lyso-substance contained in the sample from the subject/peak area of Internal Standard). Free lyso-substance concentrations were evaluated using an Internal Standard method A quadratic (y=ax.sup.2+bx+c) regression model using the weighting factor 1/conc. will be used to calculate the concentration of each analyte in every batch to be evaluated. The concentrations were calculated by means of the following formula:

[00001]$\mathrm{concentration}=\frac{-b\pm \sqrt{b^2-4a\left(c-\mathrm{peak}\mathrm{area}\mathrm{ratio}\right)}}{2a}$

[0297] Based thereon mean values, precision results (in terms of CVs) and accuracies (formula shown below) will be calculated using the program “Lotus 123”.

[00002]$\mathrm{accuracy}\left(\%\right)=\frac{\mathrm{calculated}\mathrm{concentration}}{\mathrm{expected}\mathrm{concentration}}\mathrm{•100}$

Appropriate statistical models are described in e.g. [0298] Green, J. R., Statistical Treatment of Experimental Data (Elsevier, New York, 1977), page 210 ff. [0299] Lothar Sachs, Angewandte Statistik—Anwendung statistischer Methoden (Springer, Berlin, Heidelberg, N.Y., Tokyo 1984)

Software

[0300] Data acquisition, data processing, statistics and calculations were performed using Analyst® software 1.4.2 or higher (AB SCIEX, USA/Canada) as well as Lotus 1-2-3 97 or higher (Lotus Corp, USA).

Handbooks

[0301]

TABLE-US-00008 Handbook Arbeiten mit SmartSuite 97 (Lotus Development Corp., 1997) Documentation of Documentation of Analyst ® Software (AB SCIEX, software used USA/Canada): Operator's Manual & Operator's Manual Addendum “New Functionality in Analyst 1.2” and Online Help System Analyst 1.4 (or higher)

Example 2: Genetic Testing and Classification of Study Participants

[0302] After consenting of patients to participation in the study, patients were subjected to a genetic testing for mutations of the glucocerebrosidase gene. Accordingly, 5 to 10 ml of EDTA blood were sequenced according to Seeman et al. (Seeman et al., 1995). Were appropriate other genes beside the glucocerebrosidase gene were sequenced in addition, particularly in controls. Furthermore the chitotriosidase gene was sequenced for detection of the 24 bp duplication as mentioned above. Said genetic testing was controlled using test samples of age and sex matched control patients.

[0303] 253 subjects were tested.

[0304] According to the result of the above described genetic testing, patients participating in the study were classified into the following groups:

1.) Patients having Gaucher's disease: gold standard for the diagnosis was the detection of two pathogenic mutations within the glucocerebrosidase gene, either homozygous or compound heterozygous (group is named in the figures as “Gaucher”);
2.) Patients being heterozygous carriers of one mutation within the glucocerebrosidase gene (typically relatives of affected patients) (group is named in the figures as “heterozygote”)
3.) patients with other lysosomal storage disorders as control (group is named in the figures as “other LSD”); this comprises patients with sphingomyelinase deficiency (Niemann Pick A/B), Krabbe disease and Niemann Pick Cl; all diagnoses have been proven by the detection of two pathogenic mutations
4.) healthy age and gender matched controls (group is named in the figures as “control”) The following table 1a shows the classifying of patients into the above described groups according to the results of the above described genetic testing.

TABLE-US-00009 TABLE 1a Subjects classified by results of genetic analysis cases valid missing total Groups (Dgn) N percentage N percentage N percentage control 140 100.0% 0 0% 140 100.0% Heterozygous 13 100.0% 0 0% 13 100.0% (carrier) Gaucher 59 100.0% 0 0% 59 100.0% other LSD 20 100.0% 0 0% 20 100.0%

[0305] The distribution of the gender of the 232 German patients as well as the distribution of the gender of 21 Israeli patients are depicted in Table 1b.

TABLE-US-00010 TABLE 1b 232 German subjects and 21 Israeli classified by gender Germans Israel N % N % total 232 21 Sex male 146 57.0 11 52.4 female 110 43.0 10 47.6

[0306] The following table 1c shows the distribution of the age of the 232 German patients and the classification of said patients based on the results of the above described genetic testing as well as the gender of said patients.

TABLE-US-00011 TABLE 1c Patient characteristics of 253 subjects Healthy Heterozygous controls carrier Gaucher Other LSD N subjects 140 13 80 20 N samples 155 15 287 28 Age in years 28.5 35.0 30.0 23.5 (median, (4.8-47.3) (30.5-58.5) (8.0-48.0) (4.0-43.5) interquartile (n = 134) (n = 13) (n = 79) (n = 14) range) (number of cases) male female male female male female male female n 79 61 8 5 45 35 12 8 Age (median, 25.5 34.0 33.5 39.0 22.0 32.5 21.0 34 interquartile (5.3-47.0) (3.8-48.8) (26.0-51.8) (33.0-69.5) (7.5-50.0) (12.8-43.3) (3.3-30.3) (8.8-45.8) range)

[0307] The level of free lyso-Gb1 in samples of said 253 subjects was determined according to the method described in Example 1. The level of free lyso-Gb1 in samples from said patients depending on the classification by genetic analysis is shown in FIG. 1A. FIG. 1B shows the level of free lyso-Gb1 in samples from said patients depending on the classification based on the genetic analyses and on the gender of the patients.

[0308] The type of mutation and the distribution of the types of mutations of the glucocerebrosidase gene in patients classified as Gaucher's disease patients according to the results obtained in the genetic testing as described above are depicted in Table 2 below.

TABLE-US-00012 TABLE 2 Distribution of mutations being detected in the German Gaucher population (166 alleles) type of mutation n % N370S 54 32.5% L444P 33 19.9% RecNciI 15 9.0% G202R 4 2.4% D409H 3 1.8% Rec 3 1.8% G355A 2 1.2% IVS2+1A>G 2 1.2% L335V 2 1.2% L444R 2 1.2% R120W 2 1.2% R285H 2 1.2% RecAP2 2 1.2% T226I 2 1.2% T231R 2 1.2% T491I 2 1.2% V398L 2 1.2% A46term 1 0.6% A495P 1 0.6% A88P 1 0.6% C287F 1 0.6% F216Y 1 0.6% G82A 1 0.6% H255Q 1 0.6% I93F 1 0.6% IVS3+1G>A 1 0.6% L324Q 1 0.6% N234S 1 0.6% N409S 1 0.6% P161R 1 0.6% P178S 1 0.6% P29X 1 0.6% P68fs 1 0.6% Q326K 1 0.6% R120Q 1 0.6% R257ter 1 0.6% R359Q 1 0.6% R502C 1 0.6% R502H 1 0.6% RecAF3 1 0.6% RecAF4 1 0.6% RecAH3 1 0.6% RecTL 1 0.6% S13L 1 0.6% S146L 1 0.6% S237F 1 0.6% S364N 1 0.6% V398L 1 0.6% W184R 1 0.6%

Measurement of Chitotriosidase Activity

[0309] Chitotriosidase activity was measured as essentially described in Hollak et al. (Hollak C E, van Weely S, van Oers M H, Aerts J M. Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease. J Clin Invest. 1994 March; 93(3):1288-92) by incubating 10 μl of EDTA plasma or serum with 100 μl of 0.022 mM fluorogenic substrate 4-methylumbelliferyl-fl-D-NN,N′-triacetylchitotriose (4 MU-chitotrioside; Sigma Aldrich, ST. Louis, Mo., USA) as substrate in McIlvain buffer (0.1 M citric acid/0.2 M sodium phosphate, pH 5.2) at 37° C. In Gaucher's disease patients, samples were diluted 50× in demineralized water before incubation. After 30 min the reaction was stopped with 200 μl of 0.5 M glycine/NaOH buffer (pH 10.5) by mixing at room temperature. The substrate hydrolysis by chitotriosidase produces the fluorescent molecule 4-methylumbelliferone, which was quantified with a fluorimeter (Tecan Group Ltd., Månnedorf, Switzerland), excitation at 366 nm and emission at 446 nm, and compared with a standard 4-methylumbelliferone calibration curve. Chitotriosidase activity was expressed as nanomoles of substrate hydrolyzed per hour per milliliter of incubated serum.

Quantification of CCL18

[0310] CCL18 in plasma was quantified with a DuoSet ELISA Development kit purchased from R&D Systems, Minneapolis, Minn., USA in accordance with the manufacturer's instructions. The sensitivity of the method was 5 pg/ml.

Example 3: Diagnosis of Gaucher's Disease Using Free Lyso-Gb1 as a Biomarker

[0311] The protocols described in Example 1 above were used to generate HPLC-mass spectrometric chromatograms of 485 blood samples derived from the 253 subjects. Exemplary HPLC-mass spectrometric chromatograms displaying peak intensity of free lyso-Gb1 and IS of three samples from two Gaucher's disease patients and one healthy control person are depicted in FIG. 5A, FIG. 5B and FIG. 5C.

[0312] Gold standard for the classification of patients into the group “Gaucher”, was based on the sequencing of the entire coding area as well as the the intron-exon-boundaries of the glucocerebrosidase gene according to the genetic testing as described in Example 2 resulting in the detection of either a homozygous mutation or a compound heterozygosity.

[0313] The results of a determination of the levels of Chitotriosidase or CCL18 in samples from patients were available in 58 or 44 Gaucher's disease patients, respectively. Said results were obtained as described in Example 2.

[0314] For comparing the diagnostic value of the different biomarkers and for the calculation of correlations between the biomarkers the data obtained by the method described above was first aggregated by using the earliest measured level of every marker for Gaucher's disease patients before therapy and the highest level for non-Gauchers for a particular patient if more than one blood sample was available.

[0315] Paired sample statistical techniques were used for the comparison of two biomarkers. The method exploits the mathematical equivalence of the AUC to the Mann-Whitney U-statistic (Delong E. R., Delong D. M., Clarke-Pearson D. L. (1988) Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach, Biometrics, 44, 837-45.).

[0316] The accuracy of levels of the different biomarkers (free lyso-Gb1, Chitotriosidase and CCL18) obtained by the method described in Example 1 above was evaluated to discriminate patients with Gaucher's disease from patients without having Gaucher's disease using Receiver Operating Characteristic (ROC) curve analysis (Metz C. E. (1978) Basic principles of ROC analysis, Semin Nucl Med, 8, 283-98; Zweig M. H., Campbell G. (1993) Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine, Clin Chem, 39, 561-77). Measurement of Chitotriosidase activity and CCL18 was performed as described in Example 2 herein.

[0317] The ROC curves were calculated using PASW Statistics 18, Release Version 18.0.2 (© SPSS, Inc., 2009, Chicago, Ill., www.spss.com). The comparisons of ROC curves and the linear mixed models were done using SAS software, Version 9.2 of the SAS System for Windows. (© 2008 SAS Institute Inc., Cary, N.C., USA).

[0318] The ROC-curves comparing accuracy of levels of chitotriosidase and free lyso-Gb1 are shown in FIG. 2A and the ROC-curves comparing accuracy of levels of CCL18 and free lyso-Gb1 are shown in FIG. 2B, respectively.

[0319] The results depicted in the ROC-curves shown in FIG. 2A and FIG. 2B also show the specificity and the sensitivity of the method according to the present invention depending on different cut-off levels of free lyso-Gb1. Table 3 below shows accordingly the Sensitivity and the Specificity of the method according to the present invention depending on different cut-off levels of free lyso-Gb1.

TABLE-US-00013 TABLE 3 Sensitivity and Specificity of the method for diagnosing Gaucher's disease depending on the cut-off level of free lyso-Gb1 in the German subjects (n = 232) Cut-off level >2.8 [ng/mL] >4.1 [ng/mL] >5 [ng/mL] Sensitivity 100.0% 100.0% 100.0% Specificity 97.7% 99.4% 100.0%

[0320] Comparing the level of the biomarker in a sample from a subject determined by the method according to the present invention to a cut-off level, preferably a cut-off level allowing for a diagnosis having high specificity and high sensitivity thus allows for diagnosing Gaucher's disease in said subject, wherein an elevated level of the biomarker in the sample from the subject compared to the cut-off level is indicative for the subject for suffering from or for being at risk for developing Gaucher's disease and wherein a lower level of the biomarker in the sample from the subject compared to the cut-off level is indicative for the subject for not suffering from or for not being at risk for developing Gaucher's disease.

[0321] The area under the curve (AUC) and the 95% confidence limits for the different biomarkers are reported in table 4.

TABLE-US-00014 TABLE 4 Sensitivity and specificity for different biomarkers with regard to diagnose Gaucher. Chitotriosidase CCL18 free lyso-Gb1 (n = 228/58 (n = 210/44 (n = 232/59 Gaucher) Gaucher) Gaucher) Cut-off level >145 [nmolMU/h/ml] >166 [ng/ml] >5 [ng/mL] Sensitivity 93.1% 79.5% 100.0% Specificity 90.0% 79.5% 100.0% AUC and 95% CI in 0.96 (0.92-1.00) 0.87 (0.80-0.93) 1.00 (1.00-1.00) ROC Analysis

[0322] Accordingly, in table 3 the sensitivity and the specificity of the depicted biomarkers used in a method for diagnosing Gaucher's disease in a sample from a subject is compared using a cut-off level having the highest AUC in the respective method using the respective biomarker. Depicted is the ideal cut-off level of the respective method. Measurement of Chitotriosidase activity and CCL18 was performed as described in Example 2 herein. Free lyso-Gb1 was determined according to the method of the present invention. The ideal cut-off level is 5 ng/ml.

[0323] A person skilled in the art will acknowledge that the method according to the present invention using free lyso-Gb1 as a biomarker for diagnosing Gaucher's disease is clearly advantageous over methods using CCL18 or Chitotriosidase. This is especially true since at least 6% of the Caucasian population and up to 35% e.g. of the Latin American population, including those with Gaucher's disease, are deficient in chitotriosidase activity.

[0324] Accordingly, levels of free lyso-Gb1 determined in a sample from a subject according to the method of the instant application higher than 5.0 ng/mL allow for diagnosing that the subject is suffering from or is at risk for developing Gaucher's disease with a sensitivity of and with a specificity of 100%.

Example 4: Analysis of Change of Biomarkers Over Time

[0325] The method and patients used in connection with this Example were those as described in Examples 1 to 3.

[0326] For analyzing how the level of biomarkers changed over time in patients having Gaucher's disease non aggregated data was analyzed for those patients for whom more than one blood sample was analyzed. A time point zero was set to the first measure under therapy for every patient.

[0327] The levels of free lyso-Gb1 over time for individual patients are shown in FIG. 3A, FIGS. 3B and 3C.

[0328] To test for the significance of a time dependent reduction of free lyso-Gb1 levels indicative for a successful therapy, free lyso-Gb1 levels after start of a therapy were compared to free lyso-Gb1 levels before start of a therapy using linear mixed models. Untreated patients demonstrate no significant reduction of free lyso-Gb1 over the time.

[0329] Therefore the values of free lyso-Gb1 levels were logarithmised to overcome the skewness in the distribution of the values. To account for the heterogeneity between patients in the starting values as well as in the rate of change random intercept and slope models were used. In all models the observed heterogeneity was statistically significant. Only p-values for the linear time reduction are reported.

[0330] The values for time and those values which incorporated a squared term for time were centered to test for a curvilinear relation between time and marker level for Chitotriosidase and for CCL18. For free lyso-Gb1 the squared term did not improve the model and was not incorporated in the final model.

[0331] As a therapy German patients have been treated with 40 U/kg body weight in the mean, wherein units refers to units of recombinant glucocerebrosidase in ERT. The reduction in free lyso-Gb1 is specifically intense after start of therapy (after 6 months<0.0001). But also the reduction over time is significant (<0.0001). There is a reduction of free lyso-Gb1 after 12 months of treatment in a range of 60% in the mean.

[0332] 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.