RU2780331C2 - Method for diagnostics of gaucher's disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, namely to diagnostics of Gaucher’s disease. A method for identification of an individual suitable for the therapy suitable for the treatment of Gaucher’s disease in him/her includes testing of a sample from the individual for the presence of a biomarker – free lyso-Gb1, where the individual is suitable for the therapy, if the results of sample testing are positive relatively to the biomarker, where the testing results are positive relatively to the biomarker, if a level of the biomarker in the individual’s sample exceeds the threshold level. A method for the treatment of Gaucher’s disease in an individual includes testing of a sample from the individual for the presence of a biomarker.

EFFECT: above-described methods allow for increase in the general therapeutic efficiency in the treatment of Gaucher’s disease.

17 cl, 12 dwg, 6 tbl, 4 ex

Description

Field of invention

The present invention relates to a method for diagnosing Gaucher disease in an individual, a method for determining the course of Gaucher disease in an individual, a method for determining the effectiveness of at least one treatment method used in an individual who is tested positive for the presence or risk of developing Gaucher disease, a method for determining the effectiveness of a compound for treatment Gaucher disease, the use of mass spectrometry to detect a biomarker, the use of a biomarker for Gaucher disease, a kit for determining the presence of a biomarker in a sample from an individual, and a software product where the biomarker is free lyso-Gb1.

The state of the art to which the invention relates

Lysosomal storage diseases, also referred to herein as lysosomal storage disorders or LSD, are a group of rare hereditary metabolic disorders that result from defects in lysosome function. LSD occurs when the functions of a specific organelle in the body's cells, the lysosome, are disrupted. Some of the better known lysosomal storage diseases are Gaucher's disease and Fabry's disease.

LSDs are caused by dysfunction of the lysosomes, usually due to a deficiency of a single enzyme required for the metabolism of lipids, glycoproteins, or so-called mucopolysaccharides. Individually, LSDs occur at a frequency of approximately 1:10,000 to 1:250,000, but as a group, their occurrence is approximately 1:5,000. Most of these disorders are inherited in an autosomal recessive manner; however, some are X-linked, such as Fabry disease and Hunter syndrome (MPS II).

Like other genetic diseases, individuals usually inherit lysosomal storage diseases from their parents. Although each disorder is the result of different gene mutations that cause deficiency of enzyme activity, they all share a common biochemical characteristic—virtually all lysosomal disorders are the result of an abnormal accumulation of substances within the lysosome.

Lysosomal storage diseases mostly affect children, and they often die at an early and unpredictable age, many of them within months or years of birth. Many other children die from this disease after suffering for several years from various symptoms of their particular disorder.

The symptoms of lysosomal storage disease vary, depending on the specific disorder and other variables such as age of onset, and can be mild to severe. These may include developmental delays, movement disorders, seizures, dementia, deafness and/or blindness. Some people with lysosomal storage disease have an enlarged liver (hepatomegaly) and an enlarged spleen (splenomegaly), lung and heart problems, and abnormal bone development.

There are no etiological treatments for lysosomal storage diseases, and treatment is mostly symptomatic, although bone marrow transplantation and enzyme replacement therapy (ERT) have been used in some indications with high success. In addition, for a number of these diseases, cord blood transplantation is performed in specialized centers. In addition, for some of these diseases, substrate reduction therapy (SRT) is currently being evaluated, a method used to reduce the accumulation of accumulated material. Moreover, for some of these disorders, chaperone therapy is used, a method used to stabilize defective enzymes produced in patients. Gene therapy represents another option for treating these diseases.

To date, a definitive diagnosis of Gaucher disease can only be made using a biochemical test that directly measures the defect in the beta-glucosidase enzyme, in conjunction with genetic confirmation. Since various genetic mutations can be the cause of a particular lysosomal storage disease, sequencing of the entire beta-glucosidase gene is used to confirm the diagnosis in Gaucher disease.

Although there have been attempts to use diagnostic methods based on associated biochemical abnormalities, such as high levels of alkaline phosphatase, angiotensin converting enzyme (ACE) and immunoglobulins, or, in the case of Gaucher disease, through cellular analysis demonstrating "corrugated paper" cytoplasm and glycolipid-loaded macrophages, there is an unmet need for a simple biochemical test demonstrating highly specific and highly sensitive early detection of said lysosomal storage disease, monitoring disease progression, and early monitoring of the effectiveness of the treatments used.

Thus, the identification of biomarkers for early detection and diagnosis of Gaucher disease has a high potential to improve clinical outcome in patients. This is particularly important for patients with vague or no symptoms, or to identify patients who are not responding to therapy.

The biomarker must be technically feasible in the hands of many people, easy to measure; suitable, with a constant relative value between experiments/patients and controls, or treated and untreated; reliable, accurate and clinically valid, and classified as highly predictive or predictive.

Several lysosomal enzymes used as indirect biomarkers have been found to be elevated in Gaucher disease, including tartrate-resistant acid phosphatase, hexosaminidase, and human chitinase, chitotriosidase. Thus, attempts are being made to monitor the reduction of accumulation cells in tissues by measuring Gaucher cell substitution markers such as chitotriosidase and CCL18 (C.E. Hollak et al. Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease, J. Clin. Invest. 93 (1994) 1288-1292; R. G. Boot et al. Marked elevation of the chemokine CCL18/PARC in Gaucher disease: a novel surrogate marker for assessing therapeutic intervention, Blood 103 (2004) 33-39). However, in addition to other disadvantages when using chitotriosidase as a biomarker for Gaucher's disease, said enzyme accumulates independently of a direct association with the pathology of Gaucher's disease. Moreover, up to 35% of the listed ethnic groups show a defect in the gene encoding chitotriosidase, resulting in artificially reduced or unmeasurable chitotriosidase activity.

The use of major storage molecules as a biomarker was evaluated for glucosylceramide (Gb1) in the plasma of patients with Gaucher disease and compared with the level of Gb1 in healthy individuals (Groener et al. Biochim Biophys Acta. 2008 Jan-Feb; 1781(1-2):72- 8. Epub 2007 Dec 5.; Plasma glucosylceramide and ceramide in type 1 Gaucher disease patients: correlations with disease severity and response to therapeutic intervention.; Groener JE et al.). However, although Gb1 measured in this trial was increased in the plasma of these patients, this increase in Gb1 was not pronounced and thus the specificity and sensitivity of this method were low, demonstrating that Gb1 is not useful as a biomarker for Gaucher disease. .

As early as 1989, Rosengren et al. (Lysosulfatide (galactosylsphingosine-3-O-sulphate) from metachromatic leukodystrophy and normal human brain, Rosengren B, Fredman P, Mansson JE, Svennerholm L.; J Neurochem. 1989 Apr; 52(4): 1035-41) showed that with lipidosis, not only the catabolism of the main sphingolipid is disturbed, but also its lysocompound. However, this test led to the conclusion that lyso compounds do not play a key role in the pathogenic mechanisms in sphingolipidoses. Thus, these lyso compounds may not be suitable biomarkers for the diagnosis of sphingolipidoses such as Gaucher's disease.

It is important to note that, to date, no highly specific and highly sensitive biomarker and method for diagnosing Gaucher disease has been available, other than the methods described above, which exhibit an unsatisfactory margin of detection, sensitivity and/or specificity, and thus which, as shown, are unsuitable for clinical use.

Thus, there is a need for a fast, simple and, more importantly, reliable method for diagnosing Gaucher's disease.

In view of the above, the problem underlying the present invention is to provide a method for diagnosing Gaucher's disease.

The next problem that serves as the basis for the present invention is to provide a method for determining the course and prognosis of Gaucher's disease.

A further problem underlying the present invention is to provide a method for fairly rapidly determining the efficacy of at least one treatment used in an individual who is tested positive for the presence or risk of developing Gaucher disease.

The next problem that serves as the basis for the present invention is to provide a method for determining the effectiveness of a compound in the treatment of Gaucher's disease.

Another problem underlying the present invention is the provision of a biomarker that allows a specific and sensitive diagnosis of Gaucher's disease. The next problem that serves as the basis for the present invention is a kit that contains a compound that interacts with a biomarker that is specific and sensitive for Gaucher's disease.

These and other problems are addressed by the subject matter of the appended independent claims. Preferred embodiments can be ascertained from the dependent claims.

The problem underlying the present invention is solved in the first aspect, which is also the first embodiment of the first aspect, by a method for diagnosing Gaucher disease in an individual, comprising the step

a) detecting a biomarker in a sample from an individual, where the biomarker is free lyso-Gb1.

In a second embodiment of the first aspect, which is also an embodiment of the first embodiment of the first aspect, the method further includes the step of

b) determining the level of the biomarker present in the sample.

In a third embodiment of the first aspect, which is also an embodiment of the first and second embodiments of the first aspect, the level of the biomarker indicates whether the individual suffers from or is at risk of developing Gaucher disease.

In the fourth embodiment of the first aspect, which is also an embodiment of the first, second and third embodiments of the first aspect, the sample from the individual is a sample from an individual who has previously been treated or previously diagnosed with Gaucher disease.

In the fifth embodiment of the first aspect, which is also an embodiment of the first, second and third embodiments of the first aspect, the sample from the individual is a sample from an individual who has not previously been diagnosed with Gaucher disease.

In the sixth embodiment of the first aspect, which is also an embodiment of the first, second, third, fourth, and fifth embodiments of the first aspect, the method further includes the step of

c) applying, maintaining, reducing, increasing or not using therapy based on the diagnosis of whether the individual is suffering from or at risk of developing Gaucher disease.

In the seventh embodiment of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, and sixth embodiments of the first aspect, the method further includes the step of

d) detection of a biomarker in a sample from an individual after application, maintenance, reduction, increase or no application of therapy in step c).

In an eighth embodiment of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, and seventh embodiments of the first aspect, the method further includes the step of

e) determining the level of the biomarker in the sample from the individual after application, maintenance, reduction, increase or no application of therapy in step c).

In the ninth embodiment of the first aspect, which is also an embodiment of the eighth embodiment of the first aspect, the method further includes the step of

f) determining if the biomarker level determined in step b) is lower than the biomarker level determined in step e).

In the tenth embodiment of the first aspect, which is also an embodiment of the ninth embodiment of the first aspect, the method further includes the step of

g) applying, maintaining, reducing, increasing or not using therapy, based on step f).

In an eleventh embodiment of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth embodiments of the first aspect, the method further comprises detecting at least one additional biomarker in a sample from the individual .

In the twelfth embodiment of the first aspect, which is also an embodiment of the eleventh embodiment of the first aspect, the method further comprises determining the level of at least one additional biomarker in a sample from the individual.

In the thirteenth embodiment of the first aspect, which is also an embodiment of the eleventh and twelfth embodiments of the first aspect, at least one additional biomarker is selected from the group consisting of chitotriosidase and CCL18.

In a fourteenth embodiment of the first aspect, which is also an embodiment of the thirteenth embodiment of the first aspect, at least one additional biomarker is chitotriosidase.

In the fifteenth embodiment of the first aspect, which is also an embodiment of the thirteenth embodiment of the first aspect, at least one additional biomarker is CCL18.

In the sixteenth embodiment of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, and fifteenth embodiments of the first aspect, the method further includes detecting chitotriosidases and CCL18.

In the seventeenth embodiment of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth and sixteenth embodiments of the first aspect, the biomarker and /or at least one additional biomarker is detected using immunoassay, mass spectrometric analysis, biochip, functional nucleic acids and/or fluorescent derivative of free lyso-Gb1.

In the eighteenth embodiment of the first aspect, which is also an embodiment of the seventeenth embodiment of the first aspect, the biomarker is detected by mass spectrometric analysis.

In the nineteenth embodiment of the first aspect, which is also an embodiment of the eighteenth embodiment of the first aspect, the mass spectrometric analysis is selected from the group consisting of SELDI, MALDI, MALDI-Q TOF, MS/MS, TOF-TOF and ESI-O-TOF .

In the twentieth embodiment of the first aspect, which is also an embodiment of the nineteenth embodiment of the first aspect, MS/MS is used in the mass spectrometric analysis.

In the twenty-first embodiment of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth and twentieth embodiments of the first aspect, the method further comprises protein precipitation and/or HPLC.

In the twenty-second embodiment of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth , twentieth and twenty-first embodiments of the first aspect, the method further includes protein precipitation, HPLC, and MS/MS.

In the twenty-third embodiment of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth , twentieth, twenty-first and twenty-second embodiments of the first aspect, the individual is a human.

In the twenty-fourth embodiment of the first aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth of the twentieth, twenty-first, twenty-second and twenty-third embodiments of the first aspect, the step of detecting a biomarker in a sample includes performing a protein precipitation step in the sample, protein precipitation from the sample, where protein precipitation from the sample provides the sample supernatant, HPLC and MS/MS for the sample supernatant; and determining the amount of the biomarker and/or at least one additional biomarker that is present in the sample supernatant.

The problem underlying the present invention is solved in the second aspect, which is also the first embodiment of the second aspect, with a method for diagnosing Gaucher disease in an individual, comprising

i) adding an internal standard to the individual's sample, wherein the individual's sample is selected from the group consisting of plasma, serum, and blood;

ii) optionally mixing the sample containing the internal standard;

iii) carrying out a protein precipitation step in the sample, whereby the protein is precipitated from the sample and the sample supernatant is provided;

iv) optionally performing a first separation step of the sample supernatant which provides the supernatant, preferably the first separation step being a centrifugation step;

v) subjecting the supernatant of step c) or step d), or a portion thereof, to a second separation step, wherein the second separation step comprises injecting a portion of the supernatant into the HPLC-MS/MS system and using an HPLC column with a gradient from acidified water to acetonitrile/acetone ; where the HPLC column is preferably an HPLC column selected from the group consisting of a C8 and C18 HPLC column, and where the second separation step provides a separated sample;

vi) performing MS/MS on the separated sample, where the MS/MS includes electrospray ionization and multiple reaction monitoring;

where the method is preferably the method according to any of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty first, twenty-second, twenty-third and twenty-fourth embodiments of the first aspect;

and, in addition, including the stage

a) detecting a biomarker in a sample from an individual, where the biomarker is free lyso-Gb1;

and optionally a stage

b) determining the level of the biomarker present in the sample.

In the second embodiment of the second aspect, which is also an embodiment of the first embodiment of the second aspect, the internal standard comprises D5-flucatisone propionate and/or lyso-Gb2.

In the third embodiment of the second aspect, which is also an embodiment of the first and second embodiments of the second aspect and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth embodiments of the first aspect, step b) and/or step e) further comprises comparing the biomarker level in a sample from an individual with a borderline level.

In the fourth embodiment of the second aspect, which is also an embodiment of the first, second and third embodiments of the second aspect and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth embodiments of the first aspect, preferably the third embodiment of the second aspect, the level of the biomarker in the sample from the individual that exceeds the threshold level indicates that that the individual suffers from or is at risk of developing Gaucher disease.

In a fifth embodiment of the second aspect, which is also an embodiment of the fourth embodiment of the second aspect, a biomarker level in a sample from an individual that is lower than a threshold level indicates that the individual is not suffering from or at risk of developing Gaucher disease.

In the sixth embodiment of the second aspect, which is also an embodiment of the first, second, third, fourth and fifth embodiments of the second aspect and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, and twenty-fourth embodiments of the first aspect, the threshold level is chosen such that the sensitivity for diagnosing Gaucher disease in an individual is preferably from about 98 .5% to 100%, more preferably 100%, and that the specificity for diagnosing Gaucher disease in an individual is preferably 99.4% to 100%, more preferably 100%.

In the seventh embodiment of the second aspect, which is also an embodiment of the first, second, third, fourth, fifth and sixth embodiments of the second aspect and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, of the twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth embodiments of the first aspect, step b) and/or step e) further comprises comparing the biomarker level of said an individual with a biomarker level detected in a control sample.

In the eighth embodiment of the second aspect, which is also an embodiment of the seventh embodiment of the second aspect, the control is a sample from an individual who tested positive for the absence of Gaucher disease.

In the ninth embodiment of the second aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh and eighth embodiment of the second aspect and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, and twenty-fourth embodiments of the first aspect, the level of the biomarker in the sample from the individual that exceeds the level of the biomarker in control sample indicates that the individual is suffering from and/or at risk of developing Gaucher disease.

In the tenth embodiment of the second aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth and ninth embodiments of the second aspect and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third, and twenty-fourth embodiments of the first aspect, Gaucher's disease is selected from the group consisting of non-neuronopathic type I , chronic neuronopathic type II and acute neuronopathic type III.

In the eleventh embodiment of the second aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth embodiments of the second aspect and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth embodiments of the first aspect, preferably the tenth embodiment of the second aspect, sample from an individual is selected from the group consisting of blood, blood product, urine, saliva, cerebrospinal fluid, stool, tissue sample and lymph.

In the twelfth embodiment of the second aspect, which is also an embodiment of the eleventh embodiment of the second aspect, the sample from the individual is selected from the group consisting of blood and a blood product.

In the thirteenth embodiment of the second aspect, which is also an embodiment of the eleventh and twelfth embodiments of the second aspect, the blood product is selected from the group consisting of serum and plasma.

In the fourteenth embodiment of the second aspect, which is also the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth embodiments of the second aspect of the first, second, third, fourth, fifth , sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth embodiments of the first aspect, preferably the thirteenth embodiment implementation of the second aspect, the method has a detection limit of 0.2 ng/ml.

In the fourteenth embodiment of the second aspect, which is also the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth embodiments of the second aspect and the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth embodiments of the first aspect, preferably any of the eleventh, twelfth, thirteenth, fourteenth and fifteenth embodiments of the second aspect, the threshold level is 5.0 ng/ml.

In the sixteenth embodiment of the second aspect, which is also an embodiment of the eleventh and twelfth embodiments of the second aspect, the blood is whole blood.

In the seventeenth embodiment of the second aspect, which is also an embodiment of the seventeenth embodiment of the second aspect, whole blood is collected on a dried blood filter card.

In the eighteenth embodiment of the second aspect, which is also an embodiment of the seventeenth and eighteenth embodiments of the second aspect, the method has a detection limit of 0.2 ng/mL.

In the nineteenth embodiment of the second aspect, which is also an embodiment of the seventeenth, eighteenth and nineteenth embodiments of the second aspect, the threshold level is 20.0 ng/mL.

The problem underlying the present invention is solved in the third aspect, which is also the first embodiment of the third aspect, with a method for determining the course of Gaucher disease in an individual, comprising the step

a) determining at multiple time points the level of the biomarker present in the sample from the individual, where the biomarker is free lyso-Gb1.

In the second embodiment of the third aspect, which is also an embodiment of the first embodiment of the third aspect, the individual has been previously treated for Gaucher's disease or has previously been diagnosed with Gaucher's disease.

In the third embodiment of the third aspect, which is also an embodiment of the first embodiment of the third aspect, the individual has not been previously treated for Gaucher's disease or the individual has not previously been diagnosed with Gaucher's disease.

In the fourth embodiment of the third aspect, which is also an embodiment of the first, second and third embodiments of the third aspect, the method further includes the step of

b) applying, maintaining, reducing, increasing or not using therapy based on the diagnosis of whether the individual suffers from or is at risk of developing Gaucher disease.

In the fifth embodiment of the third aspect, which is also an embodiment of the first, second, third and fourth embodiments of the third aspect, the method further includes the step of

c) detection of a biomarker in a sample from an individual after application, maintenance, reduction, increase or no application of therapy in step b).

In the sixth embodiment of the third aspect, which is also an embodiment of the first, second, third, fourth, and fifth embodiments of the third aspect, the method further includes the step of

d) determining the level of the biomarker in the sample from the individual after application, maintenance, reduction, increase or no application of therapy in step b).

In the seventh embodiment of the third aspect, which is also an embodiment of the first, second, third, fourth, fifth, and sixth embodiments of the third aspect, the method further includes the steps of

e) determining if the biomarker level determined in step a) is lower than the biomarker level determined in step d);

In the eighth embodiment of the third aspect, which is also an embodiment of the seventh embodiment of the third aspect, the method further includes the step of

f) applying, maintaining, reducing, increasing or not using therapy, based on step e).

In a ninth embodiment of the third aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, and eighth embodiments of the third aspect, the method further comprises detecting at least one additional biomarker in a sample from the individual.

In the tenth embodiment of the third aspect, which is also an embodiment of the ninth embodiment of the third aspect, the method further comprises determining the level of at least one additional biomarker in a sample from the individual.

In the eleventh embodiment of the third aspect, which is also an embodiment of the ninth and tenth embodiments of the third aspect, at least one additional biomarker is selected from the group consisting of chitotriosidase and CCL18.

In the twelfth embodiment of the third aspect, which is also an embodiment of the eleventh embodiment of the third aspect, at least one additional biomarker is chitotriosidase.

In the thirteenth embodiment of the third aspect, which is also an embodiment of the eleventh embodiment of the third aspect, at least one additional biomarker is CCL18.

In the fourteenth embodiment of the third aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth embodiments of the third aspect, the method further includes detecting chitotriosidase and CCL18.

In the fifteenth embodiment of the third aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth embodiments of the third aspect, the biomarker and/or at least at least one additional biomarker is detected by immunoassay, mass spectrometric analysis, biochip, functional nucleic acids and/or a fluorescent derivative of free lyso-Gb1.

In the sixteenth embodiment of the third aspect, which is also an embodiment of the fifteenth embodiment of the third aspect, the biomarker is detected by mass spectrometric analysis.

In the seventeenth embodiment of the third aspect, which is also the sixteenth embodiment of the third aspect, the mass spectrometry analysis is selected from the group consisting of SELDI, MALDI, MALDI-Q TOF, MS/MS, TOF-TOF, and ESI-O-TOF .

In the eighteenth embodiment of the third aspect, which is also an embodiment of the seventeenth embodiment of the third aspect, MS/MS is used in the mass spectrometric analysis.

In the nineteenth embodiment of the third aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, and eighteenth embodiments of the third aspect, the method further comprises precipitating the protein and/or HPLC.

In the twentieth embodiment of the third aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, and nineteenth embodiments carrying out the third aspect, the method further includes protein precipitation, HPLC and MS/MS.

In the twenty-first embodiment of the third aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth , twentieth and twenty-first embodiments of the third aspect, the individual is a human.

In the twenty-second embodiment of the third aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth of the twentieth, twenty-first and twenty-second embodiments of the third aspect, the step of detecting a biomarker in a sample from an individual comprises precipitating a protein from a sample from an individual, wherein precipitating the protein from the sample provides the sample supernatant, performing HPLC and MS/MS on the sample supernatant, and quantifying the biomarker and/or at least one additional biomarker that is present in a sample from the individual.

In the twenty-third embodiment of the third aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth of the twentieth, twenty-first, twenty-second and twenty-third embodiments of the third aspect, Gaucher's disease is selected from the group consisting of non-neuronopathic type I, chronic neuronopathic type II, and acute neuronopathic type III.

The problem underlying the present invention is solved in the fourth aspect, which is also the first embodiment of the fourth aspect, with a method for determining the effectiveness of at least one method of treatment used in an individual with positive test results for the presence or risk of developing Gaucher disease, including stage

a) determining at several points in time the level of the biomarker present in the sample from the individual,

where the biomarker is free lyso-Gb1.

In the second embodiment of the fourth aspect, which is also an embodiment of the first embodiment of the fourth aspect, the individual has been previously treated for Gaucher's disease or has been diagnosed with Gaucher's disease.

In a third embodiment of the fourth aspect, which is also an embodiment of the first embodiment of the fourth aspect, the individual has not been previously treated for Gaucher's disease or has not previously been diagnosed with Gaucher's disease.

In the fourth embodiment of the fourth aspect, which is also an embodiment of the first, second and third embodiments of the fourth aspect, the method further includes the step of

b) applying, maintaining, reducing, increasing or not using at least one method of treatment used in the individual, based on the decrease in the level of the biomarker.

In the fifth embodiment of the fourth aspect, which is also an embodiment of the first, second, third and fourth embodiments of the fourth aspect, the method further includes the step of

c) detecting a biomarker in a sample from an individual, where the sample is taken before treatment, after application, maintenance, reduction, increase or no application of at least one treatment in step b).

In the sixth embodiment of the fourth aspect, which is also an embodiment of the first, second, third, fourth and fifth embodiments of the fourth aspect, the method of treatment is selected from the group consisting of enzyme replacement therapy, substrate reduction therapy, chaperone therapy, gene therapy, stem cell transplantation. or skipping DNA/RNA.

In the seventh embodiment of the fourth aspect, which is also an embodiment of the first, second, third, fourth, fifth, and sixth embodiments of the fourth aspect, the method further includes the steps of

d) determining if the biomarker level determined in step a) is lower than the biomarker level determined in step c).

In the eighth embodiment of the fourth aspect, which is also an embodiment of the seventh embodiment of the fourth aspect, the method further includes the steps of

e) applying, maintaining, reducing, enhancing or not using at least one treatment used in the individual, based on stage d).

In a ninth embodiment of the fourth aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, and eighth embodiments of the fourth aspect, the method further comprises detecting at least one additional biomarker in a sample from the individual.

In a tenth embodiment of the fourth aspect, which is also an embodiment of the ninth embodiment of the fourth aspect, the method further comprises determining the level of at least one additional biomarker in a sample from the individual.

In the eleventh embodiment of the fourth aspect, which is also an embodiment of the ninth and tenth embodiments of the fourth aspect, at least one additional biomarker is selected from the group consisting of chitotriosidase and CCL18.

In the twelfth embodiment of the fourth aspect, which is also an embodiment of the eleventh embodiment of the fourth aspect, at least one additional biomarker is chitotriosidase.

In the thirteenth embodiment of the fourth aspect, which is also an embodiment of the eleventh embodiment of the fourth aspect, at least one additional biomarker is CCL18.

In the fourteenth embodiment of the fourth aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth embodiments of the fourth aspect, the method further includes detecting chitotriosidase and CCL18.

In the fifteenth embodiment of the fourth aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth and fourteenth embodiments of the fourth aspect, any biomarker is detected by immunoassay , mass spectrometric analysis, biochip, functional nucleic acids and/or fluorescent derivative of free lyso-Gb1.

In the sixteenth embodiment of the fourth aspect, which is also an embodiment of the fifteenth embodiment of the fourth aspect, the biomarker is detected by mass spectrometric analysis.

In the seventeenth embodiment of the fourth aspect, which is also the sixteenth embodiment of the fourth aspect, the mass spectrometry analysis is selected from the group consisting of SELDI, MALDI, MALDI-Q TOF, MS/MS, TOF-TOF and ESI-O-TOF .

In the eighteenth embodiment of the fourth aspect, which is also an embodiment of the seventeenth embodiment of the fourth aspect, MS/MS is used in the mass spectrometric analysis.

In the nineteenth embodiment of the fourth aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, and eighteenth embodiments of the fourth aspect, the method further comprises precipitating the protein and/or HPLC.

In the twentieth embodiment of the fourth aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, and nineteenth embodiments carrying out the fourth aspect, the method further includes protein precipitation, HPLC and MS/MS.

In the twenty-first embodiment of the fourth aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth and twentieth embodiments of the fourth aspect, the individual is a human.

In the twenty-second embodiment of the fourth aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth of the twentieth and twenty-first embodiments of the fourth aspect, the step of detecting a biomarker in a sample from the individual comprises precipitating the protein from the sample from the individual, wherein precipitating the protein from the sample provides a supernatant of the sample; conducting HPLC and MS/MS for the volume of the supernatant and determining the amount of the biomarker and/or at least one additional biomarker that is present in the sample from the individual.

In the twenty-third embodiment of the fourth aspect, which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth , twentieth, twenty-first and twenty-second embodiments of the fourth aspect, Gaucher's disease is selected from the group including non-neuronopathic type I, chronic neuronopathic type II and acute neuronopathic type III.

The problem underlying the present invention is solved in the fifth aspect, which is also the first embodiment of the fifth aspect, with a method for determining the efficacy of a compound in the treatment of Gaucher's disease, comprising the steps of:

a) determining the level of a biomarker in an individual having Gaucher disease;

b) administering said compound to said individual;

c) determining again the level of the biomarker in the specified individual;

d) determining if the level of the biomarker determined in step a) is lower than the level of the biomarker determined in step c),

where the level of the biomarker determined in step c) which is lower than the level of the biomarker determined in step a) indicates the effectiveness of said compound, and where the biomarker is free lyso-Gb1.

In the second embodiment of the fifth aspect, which is also an embodiment of the first embodiment of the fifth aspect, the method further comprises determining the level of a control biomarker.

In a third embodiment of the fifth aspect, which is also an embodiment of the first and second embodiments of the fifth aspect, Gaucher's disease is selected from the group consisting of non-neuronopathic type I, chronic neuronopathic type II, and acute neuronopathic type III.

The problem underlying the present invention is solved in the sixth aspect, which is also the first embodiment of the sixth aspect, using mass spectrometry to detect a biomarker, where the biomarker is free lyso-Gb1.

In the second embodiment of the sixth aspect, which is also an embodiment of the first embodiment of the sixth aspect, the detection includes the use of HPLC.

In the third embodiment of the sixth aspect, which is also an embodiment of the first and second embodiments of the sixth aspect, detection includes MS/MS.

The problem underlying the present invention is solved in the seventh aspect, which is also the first embodiment of the seventh aspect, using a biomarker for Gaucher disease, preferably in the method according to any of the first, second, third, fourth, fifth, sixth, seventh , eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth embodiments of the first aspect, first, second, third, fourth , fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth and nineteenth embodiments of the second aspect, first, second, third, fourth, fifth, sixth, seventh, eighth ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second and twenty-third embodiments of the third aspect, first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth , eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second and twenty-third embodiments of the fourth aspect and the first, second and third embodiments of the fifth aspect, where the biomarker is free lyso- Gb1.

In a second embodiment of the seventh aspect, which is also an embodiment of the first embodiment of the seventh aspect, Gaucher's disease is selected from the group consisting of non-neuronopathic type I, chronic neuronopathic type II, and acute neuronopathic type III.

The problem underlying the present invention is solved in the eighth aspect, which is also the first embodiment of the eighth aspect, with a kit for detecting the presence of a biomarker in a sample from an individual, where the kit includes

a) a biomarker interaction partner;

b) optionally a solid support containing at least one capture reagent attached thereto, where the capture reagent binds the biomarker; and

c) instructions for use of the solid support for biomarker detection, where the biomarker is free lyso-Gb1.

In the second embodiment of the eighth aspect, which is also an embodiment of the first embodiment of the eighth aspect, the set is for

a) diagnosis of Gaucher disease;

b) determining the course of Gaucher's disease in an individual; and/or

c) determining the effectiveness of at least one method of treatment used in an individual,

where the method used in a), b) and/or c), is preferably a method according to any of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth , fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second, twenty-third and twenty-fourth embodiments of the first aspect, first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh , twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth and nineteenth embodiments of the second aspect, first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, two eleven second and twenty third embodiments of the third aspect, first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, twenty-first, twenty-second and twenty-third embodiments of the fourth aspect; and first, second and third embodiments of the fifth aspect.

In a third embodiment of the eighth aspect, which is also an embodiment of the first and second embodiments of the eighth aspect, Gaucher's disease is selected from the group consisting of non-neuronopathic type I, chronic neuronopathic type II, and acute neuronopathic type III.

The problem underlying the present invention is solved in the ninth aspect, which is also the first embodiment of the ninth aspect, with a software product including

a) a code that gives access to data attributed to the sample, and the data include the detection of at least one biomarker in the sample, and the biomarker is selected from the group selected from the group including free lyso-Gb1, chitotriosidase and CCL18; and

b) code that executes a classification algorithm that classifies the sample's status for Gaucher disease as a detection function.

In a second embodiment of the ninth aspect, which is also an embodiment of the first embodiment of the ninth aspect, Gaucher's disease is selected from the group consisting of non-neuronopathic type I, chronic neuronopathic type II, and acute neuronopathic type III.

The present inventors unexpectedly discovered that free lyso-Gb1 is a biomarker that provides a method for diagnosing Gaucher disease in an individual, more specifically, diagnosing Gaucher disease in an individual with high specificity and sensitivity using said free lyso-Gb1 as a biomarker.

The present inventors surprisingly found that free lyso-Gb1, which can be detected by the methods of the present invention, circulates in the blood of an individual at a concentration of approximately 1/1000 of total Gb1. Moreover, the present inventors surprisingly found that, unlike total Gb1, free lyso-Gb1 that is present in the blood of an individual is useful in a method for diagnosing Gaucher disease in an individual, comprising the step of detecting a biomarker in a sample from the individual, wherein the biomarker is free lyso-Gb1. The present inventors have also surprisingly discovered that the level of free lyso-Gb1 measured in a sample from an individual by the methods of the present invention provides a diagnosis of Gaucher's disease with high sensitivity and high specificity.

Thus, the present invention differs from the idea of the prior art in that the method of the present invention comprises determining the level of a lyso compound using said lyso compound as a biomarker for the diagnosis of sphingolipidoses. More specifically, the present inventors have surprisingly discovered that determining the level of free lyso-Gb1 in a sample from an individual allows the diagnosis of Gaucher's disease with high sensitivity and high specificity.

It is also to the credit of the authors of the present invention that they realized that part of the total Gb1 that accumulates in Gaucher disease is present as a molecule in its lysoform, i. free lyso-Gb1, and circulates in the blood of the individual in the specified lysoform, in addition to Gb1.

The term "lysosomal storage disorder", also referred to as "lysosomal storage disease" or "LSD" as used herein, preferably refers to genetic diseases and metabolic disorders that result from defects in lysosomal function. Lysosomal storage disorders are caused by lysosomal dysfunction, usually as a result of a deficiency of one enzyme required for the metabolism of lipids, glycoproteins, or so-called mucopolysaccharides. Like other genetic diseases, individuals inherit lysosomal storage diseases from their parents. Although each disorder is the result of distinct gene mutations that cause deficiency of enzyme activity, they all share a common biochemical characteristic - all lysosomal disorders result from abnormal accumulation of substances within the lysosome.

The term "Gaucher's disease" as used herein preferably refers to lysosomal storage disease (LSD), more specifically sphingolipidoses, which are characterized by the deposition of glucocerebroside in cells of the macrophage-monocyte system. Gaucher disease is the most common of the lysosomal storage diseases (James, William D.; Berger, Timothy G.; et al. (2006). Andrews' Diseases of the Skin: clinical Dermatology. Saunders Elsevier. ISBN 0-7216-2921-0 ). It is caused by an inherited deficiency of the enzyme glucocerebrosidase. This deficiency results from a recessive mutation(s) in the gene encoding glucocerebrosidase, a specific lysosomal hydrolase (also known as beta-glucosidase, EC 3.2.1.45, PDB 1OGS), located on chromosome 1 (1q21) and affects both males and females. . Various mutations in beta-glucosidase determine the residual activity of the enzyme, and to a large extent, the phenotype.

Glucocerebrosidase is also referred to herein as β-glucocerebrosidase, beta-glucosidase, acid beta-glucosidase, glucosylceramidase or D-glucosyl-N-acylsphingosingglucohydrolase.

The enzyme is a 55.6 kDa 497 amino acid protein with glucosylceramidase activity, i.e. this enzyme catalyzes the breakdown of a fatty substance called glucocerebroside by splitting, i.e. hydrolysis, the beta-glucosidic bond of glucocerebroside, which is an intermediate in glycolipid metabolism. Glucocerebroside, also referred to herein as glucosylceramide or Gb1, is a component of the cell membrane of erythrocytes and leukocytes. When the enzyme is defective, the substance accumulates, in particular in the cells of the mononuclear cell lineage. This is due to the fact that the macrophages that eliminate these cells are unable to eliminate the by-product that accumulates in fibrils and turn into so-called Gaucher cells, which resemble crumpled paper under light microscopy. Fatty material can accumulate in the spleen, liver, kidneys, lungs, brain, and bone marrow.

Gaucher disease has three general clinical subtypes.

Non-neuropathic type I, also referred to herein as type I, is the most common form of the disease, occurring in approximately 1 in 50,000 live births. It is most common in individuals of Ashkenazi Jewish descent. Symptoms may appear early in life or in adulthood and include an enlarged liver and greatly enlarged spleen (co-hepatosplenomegaly); the spleen can rupture and cause additional complications. Skeletal muscle weakness and bone disease may be prominent. Enlargement of the spleen and displacement of the bone marrow cause anemia, thrombocytopenia, and leukopenia. The brain is not pathologically affected, but lung damage and, rarely, kidney damage may occur. Individuals with disease in this group usually bruise easily (due to low platelet levels) and experience fatigue due to low red blood cell counts. Depending on the onset and severity of the disease, type I patients may live well into adulthood. Many individuals with the disease have a mild form of the disease or may not show any symptoms.

- Chronic neuronopathic type II, also referred to herein as type II, can begin at any time in childhood or even adulthood, and occurs in approximately 1 in 100,000 live births. It is characterized by slowly progressive but milder neurological symptoms compared to the acute variant or type III. The main symptoms include an enlarged spleen and/or liver, seizures, poor coordination, skeletal muscle disorders, eye movement disorders, blood disorders including anemia and respiratory problems. Patients often survive into their early teens and adulthood.

Acute neuronopathic type III, also referred to herein as type III, typically begins within 6 months of birth and has an incidence of approximately 1 in 100,000 live births. Symptoms include an enlarged liver and spleen, extensive and progressive brain damage, eye movement disorders, spasticity, seizures, stiff limbs, and poor sucking and swallowing ability. Affected children usually die before the age of 2 years.

These subtypes have been criticized for not taking into account the full range of observed symptoms. There are also complex heterozygous variants, which greatly increase the complexity of predicting the course of the disease.

In type II and III Gaucher disease, glucocerebroside accumulates in the brain due to the circulation of complex lipids during brain development and the formation of the myelin sheath of the nerves.

Symptoms may include an enlarged spleen and liver, abnormal liver function, skeletal and bone damage that can be painful, severe neurological complications, swelling of the lymph nodes and (sometimes) nearby joints, bloating, brownish skin tone, anemia, low blood platelets. blood and the deposition of yellow fat in the white of the eye (sclera). The most severely affected individuals may also be more susceptible to infection.

Therapy: Enzyme replacement therapy, also referred to herein as ERT, is the preferred therapy of choice. However, successful bone marrow transplantation can cure the non-neurological manifestations of the disease because it provides a population of monocytes with active beta-glucosidase. It is important to mention that this procedure carries significant risks and is rarely performed on patients with Gaucher disease. Surgery to remove the spleen (splenectomy) may be required very rarely if the patient has massive anemia and when the enlarged organ interferes with the patient's comfort. Some anemic patients may benefit from a blood transfusion. Other patients may require joint replacement surgery to improve mobility and quality of life. Other treatment options include antibiotics for infections, antiepileptics for seizures, bisphosphonates for bone damage, and liver transplants.

ERT is based on continuous intravenous administration of recombinant glucocerebrosidase (imiglucerase, Genzyme; velaglucerase, Shire; taliglucerase, Protalix) (G.A. Grabowski et al., Enzyme therapy in type I Gaucher's disease: comparative efficacy of mannose-terminated glucocerebrosidase from natural and recombinant sources, Ann Intern Med 122 (1995) 33-39). For type I patients and most type III patients, ERT with intravenous recombinant glucocerebrosidase (eg, such as imiglucerase) can significantly reduce the size of the liver and spleen, improve skeletal abnormalities, and reverse other manifestations.

More recently, substrate reduction therapy, also referred to herein as SRT, has been developed as an alternative treatment for Gaucher disease (F.M. Piatt et al. N-butyl-desoxynojirirnycin is a novel inhibitor of glycosphingolipid biosynthesis, J. Biol. Chem. 269 (1994) 8362 -8365.). Partial inhibition of glucosphingolipid synthesis by N-butyleneoxynoyrimycin (miglustat, Actelion) has been used in an attempt to balance the reduced catabolic capacity in patients with Gaucher disease. SRT may be effective for type II arrest because it can pass through the blood barrier into the brain. There is currently no effective treatment for the severe brain damage that can occur in patients with type II and III Gaucher disease.

Both ERT and SRT generally result in significant clinical improvements, such as reduction in hepatosplenomegaly, correction of hematologic abnormalities, and stabilization or improvement of skeletal lesions.

Glucocerebroside, also referred to herein as glucosylceramide or Gb1, means any cerebroside in which the monosaccharide end group is glucose.

The person skilled in the art will understand that the term "lyso-Gb1", as used within the scope of the invention, preferably in relation to various methods, preferably means that the molecule is present in its free amino form. More specifically, lyso-Gb1, as used herein, preferably differs from Gb1 in that no fatty acid moiety is linked to the primary amino group of the sphingosine moiety. Moreover, lyso-Gb1 is also referred to herein as glucosylsphingosine or lyso-glucocerebroside and has the formula:

Those skilled in the art will appreciate that the term "free lyso-Gb1" as used herein preferably refers to lyso-Gb1 that is present as such in a sample from an individual, such as blood, and preferably does not result from manipulating a sample of said individual. Such sample manipulation may be the manipulation described by Groener et al. (Groener et al. Plasma glucosylceramide and ceramide in type 1 Gaucher disease patients: Correlations with disease severity and response to therapeutic intervention. Biochimica et Biophysica Acta 1781(2908)72-78, 2007). According to this document, free lyso-Gb1, which is present as such in the blood of the individual from which the sample is taken, more specifically, is not lyso-Gb1, which is formed by chemical, biochemical or physical processing of the sample contained in the blood and the sample , respectively, preferably outside the body of the patient. It will also be understood by those skilled in the art that free lyso-Gb1, as used herein, is preferably present in addition to Gb1, and is a compound produced as a result of the metabolic activity of the individual. Thus, Gb1, which is a molecule that accumulates in association with Gaucher's disease, present in a sample from an individual, has, compared to a molecule in the free lyso form, i.e. free lyso-Gb1, present in the blood of the individual, at least one part of the fatty acid associated with the primary amino group of the part in the form of sphingosine in lyso-Gb1.

The term "sample", as preferably used herein, means a limited amount of an individual's material, where said individual material is part of or taken from an individual and/or an individual's body, and where said material is selected from the group consisting of body fluids such as blood, blood, urine, saliva, cerebrospinal fluid and lymph, as well as stool or any type of tissue and/or cellular material that is part of the individual and/or the body of the individual. The person skilled in the art will understand that it is assumed that the presence and/or level of the biomarker of the invention in the specified sample are similar and reflect the presence and/or level of the biomarker in more of the material of the individual. More precisely, and as an illustrative non-limiting example, the level of a biomarker of the invention, determined in a sample of several ml of an individual's blood, also reflects the level of said biomarker in the blood of the individual's body. Moreover, in one embodiment of the method of the invention for diagnosing Gaucher disease in an individual, the sample from the individual comprises said material of the individual in a form such as processed, fixed and/or stored such that said sample is suitable for use in the method of the invention, wherein such processing, fixation and/or storage preferably do not lead to the formation of lyso-Gb1. The material of the individual in the sample can thus be diluted, for example with a solvent suitable for the process of the invention such as methanol and/or water, can be dried, for example on a filter card, can be dissolved after drying, for example with a solvent suitable for method according to the invention, such as methanol and/or water, or you can add a substance, where the specified substance prevents blood clotting, such as EDTA or heparin. In addition, a person skilled in the art will understand that the method of the invention includes separating said individual material into separate components of said individual material, and/or extracting individual components of said individual material from said individual material, for example, blood is separated into plasma and serum and cellular blood components or protein precipitate from the sample. It will naturally be understood that after such processing, fixation and/or preservation, the sample is subjected to the methods of the invention to detect and/or determine the level of the biomarker contained in the specified sample, and such processing, fixation and/or preservation preferably does not lead to the formation of lyso- Gb1.

In one embodiment of the method of the present invention, where whole blood is collected on a dried blood filter card, preferably approximately 3 μl of whole blood is collected in an area of said dried blood filter card having a diameter of 3 mm. One skilled in the art will appreciate that the total volume collected in this manner may vary, depending on the hematocrit of the individual patient.

Plasma glucosylceramide and ceramide in type 1 Gaucher's disease patients: Correlations with disease severity and response to therapeutic intervention Biochimica et Biophysica Acta 1781(2908)72-78, 2007). Thus, Gb1 levels were found to be different, although ceramide levels did not differ significantly in the plasma of treated and untreated type I Gaucher patients.

In a study described by Groener et al. (Groener et al., supra) the Gb1/ceramide ratio was used to distinguish patients with Gaucher disease from healthy patients. Gb1 and ceramide were measured using high performance liquid chromatography (HPLC), essentially as described in Groener et al. (J.E.M. Groener et al., HPLC for simultaneous quantification of total ceramide, glucosylceramide, and ceramide trihexoside concentrations in plasma, Clin. Chern. 53 (2007) 742-747). In relation to this, it is important to understand that the Gb1 present in plasma is mainly composed of a sugar part and a ceramide part. The ceramide part contains sphingosine and the fatty acid part. According to the prior art method, lipids are extracted and ceramide and glucosylceramide are deacylated by alkaline hydrolysis, thus obtaining a lysoform, i.e. lyso-Gb1 (T. Taketomi et al., Rapid method of preparation of lysoglycosphingolipides and their confirmation by delayed extraction matrix-assisted laser desporption ionization time-of-flight mass spectrometry, J. Biochem. (Tokyo) 120 (1996) 573- 579). The lyso-Gb1 thus obtained is then labeled with a fluorescent dye by derivatization with O-phthaldialdehyde (OPA) at the primary amino group. The derivatized sphingoid bases are then separated by reverse phase HPLC and detected with a fluorescence detector. Thus, this prior art method is able to detect total Gb1 composed of free lyso-Gb1 and Gb1, and is not able to distinguish the level of free lyso-Gb1 from the level of Gb1 in a sample from an individual. The level of said total Gb1 after cleavage of the various fatty acid moieties from the NH2 group of Gb1 is typically in the range of 5 to 30 μg per ml of plasma or serum. It is clear from this that in the method of Groener et al. (Groener et al., supra) total Gb1, which can be prepared and obtained, respectively, from a sample, preferably a blood sample, from an individual, is used as a biomarker instead of free lyso-Gb1 contained in the blood and thus also in image, without carrying out the cleavage of the portion/parts in the form of a fatty acid, preferably a cleavage carried out by a specialist processing the sample. Thus, the present invention relates to the detection of free lyso-Gb1, and not total Gb1, as described in the prior art. An embodiment of the methods of the present invention, including detecting and/or determining the level of free lyso-Gb1 in a sample from an individual, is that free lyso-Gb1 and/or free lyso-Gb1 level is determined separately and/or independently of Gb1 or Gb1 level , which may be present in the blood of an individual. In the following embodiment, Gb1 and/or the level of Gb1 is detected/determined in addition to the detection and/or determination of the level of free lyso-Gb1.

It is important that each primary amine circulating in plasma that is sufficiently lipophilic to be extracted simultaneously with Gb1 using an organic solvent according to said prior art method is appropriately labeled and thus can interfere with the detection of cleaved lyso-Gb1.

Although total Gb1 measured as lyso-Gb1 in said prior art study was increased in the plasma of said patients, said increase in total Gb1 was not pronounced, and thus the specificity and sensitivity of the method were low, demonstrating that Gb1 is not suitable in as a biomarker for Gaucher disease.

With this in mind, it is important to note that, to the best of the inventors' knowledge, the data described in the Examples section of this specification as applied to the present invention reflects the first systematic specificity and sensitivity analysis for direct comparison of prior art Gaucher biomarkers, i.e. . chitotriosidase and CCL18, and free lyso-Gb1.

Providing sensitivity and/or specificity of >99.0%, free lyso-Gb1 as determined by the methods of the present invention is a biomarker suitable for clinical use in relation to Gaucher's disease. Thus, the biomarker of the present invention and its use clearly outperform the biomarkers known in the art, more specifically one of chitotriosidase and CCL18. It will be well understood that also the method used by Groener et al. (Groener et al., supra) is biased compared to the methods of the present invention in that the specificity and sensitivity of said prior art method are lower and the diagnosis of Gaucher disease based on such prior art method using total Gb1 rather than free lyso-Gb1 are unsuitable for reliable clinical use, i.e. the method does not have sufficient sensitivity and specificity to diagnose Gaucher's disease through reliable statistically reliable prediction.

Chitotriosidase: It was previously discovered that Gaucher cells secrete chitotriosidase and that chitotriosidase in the plasma of patients with symptoms of Gaucher disease is increased on average several hundred times (Hollak et al. Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease. J Clin Invest 1994;93:1288-1292). Thus, plasma chitotriosidase is used as a surrogate marker for manifestations of Gaucher disease and is used for diagnosis, early detection of disease occurrence, and monitoring of therapeutic efficacy (Hollak et al. Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease. J Clin Invest 1994;93: 1288-1292; Mistry et al. A practical approach to diagnosis and management of Gaucher's disease. Baillieres Clin Haematol. 1997; 10: 817-838; Cox et al. Novel oral treatment of Gaucher's disease with N-butyldesoxynojirimycin (OGT 918) to decrease substrate biosynthesis Lancet 2000 355: 1481-1485 Hollak et al Clinically relevant therapeutic endpoints in type I Gaucher disease J Inherit Metab Dis 2001 24: 97-105

However, plasma levels of chitotriosidase do not reflect one specific symptom, but rather they reflect the overall load on the body of Gaucher cells (Aerts et al. Plasma and metabolic abnormalities in Gaucher's disease. Baillieres Clin Haematol. 1997;10: 691-709); moreover, they do not reflect the burden of disease due to bone pathology and brain damage. The level of chitotriosidase is not directly related to the pathophysiology of Gaucher disease. In addition, after treatment, the level of chitotriosidase changes extremely slowly, which makes chitotriosidase unsuitable for rapidly assessing the effectiveness of the treatment to which the patient is or has been subjected, as well as the recurrence of the disease, regardless of the cause of the disease.

Moreover, the use of plasma chitotriosidase as a marker for Gaucher cells is hindered by the fact that patients, including those with Gaucher disease, may be deficient in chitotriosidase activity due to a 24 base pair (bp) duplication in the chitotriosidase gene. Obviously, these individuals cannot be monitored by measuring plasma chitotriosidase activity. chitotriosidase gene Nature of inherited enzyme deficiency J Biol Chem 1998;273:25680-25685). Duplication frequency 24 bp in the chitotriosidase gene depends on ethnicity and can increase almost up to 35% (Prof. Guiliani, Brasil, unpublished data).

CCL18: Glucosylceramide-loaded macrophages or Gaucher cells are the main source of CCL18. Plasma levels of CCL18 in patients with Gaucher disease are significantly elevated (Boot, R.G. et al. 2004. Marked elevation of the chemokine CCL18/PARC in Gaucher disease: a novel surrogate marker for assessing therapeutic intervention. Blood 103:33-39. ). Thus, there are attempts to use the level of CCL18 in plasma as a replacement marker to monitor the success of the applied therapy. However, it has also been discovered that elevated levels of CCL18 are associated with various diseases such as various types of cancer and inflammation of the joints, lungs and skin. For example, ascitic fluids of patients with ovarian carcinoma contain significantly elevated levels of CCL18 compared to patients without ovarian carcinoma (Budd-Chiari syndrome) (Schutyser, E. et al. 2002. Identification of biologically active chemokine isoforms from ascitic fluid and elevated levels of CCL18/pulmonary and activation-regulated chemokine in ovarian carcinoma J Biol Chem 277:24584-24593). CCL18 plays a role in tumor suppression because it attracts and activates certain immune cells. Moreover, children with acute lymphocytic leukemia have been found to have elevated levels of CCL18, while children with acute myeloid leukemia do not show elevated serum levels of CCL18 (Struyf, S et al. 2003. ARC/CCL18 is a plasma CC chemokine with increased levels in childhood acute lymphoblastic leukemia Am J Pathol 163: 2065-2075.). Moreover, plasma levels of CCL18 do not reflect one specific clinical symptom, but instead they reflect the body's overall burden of Gaucher cells. As can be seen from the above, CCL18 exhibits extremely low specificity for diagnosing Gaucher's disease and thus is mainly used as a "helper" substitution marker for chitotriosidase deficient patients.

With regard to the use of chitotriosidase and CCL18, it should be noted that chitotriosidase does not give positive test results in 10-30% of all patients, i.e. the test results of the patients are negative despite the presence of Gaucher disease and thus also refuse to use the therapy. Moreover, in these cases, the marker can no longer be used as a monitoring marker for surveillance purposes, such as ERT. If the patient is suspected to have a chitotriosidase defect, CCL18 is used as a biomarker for diagnosing Gaucher disease, and the method using CCL18 as a biomarker exhibits relatively low specificity and sensitivity, i. gives a false positive or false negative diagnosis in approximately 25% of all patients.

The term "Gaucher disease status" as used herein preferably refers to the disease status of an individual. Examples of types of Gaucher disease statuses include, but are not limited to, an individual's risk of having or developing Gaucher's disease, the individual's stage of disease, and the effectiveness of treatment for the disease. Other statuses and degrees of each status are known in the art. In one embodiment of the present invention, Gaucher's disease status includes Gaucher's disease status: severe, mild, or healthy.

The term "diagnosis" as used herein preferably means determining the presence or absence of a disease or disorder in an individual and/or determining whether the individual is at risk of developing a disease, disorder, or symptoms associated with the disease or disorder, as well as predicting the status of the disease. .

The term "detection" in the context of the present invention means methods that include detecting the presence or absence of a substance in a sample and/or determining the type of said substance. Detection can be performed by methods known in the art and methods further described herein, including, but not limited to, direct measurement of the glucosidase enzyme, such as sequencing the gene encoding glucosidase. To identify one or more of the biomarkers described in the present description, you can use any suitable method. These methods include, but are not limited to, mass spectrometry (eg HPLC-MS/MS), fluorescence (eg sandwich immunoassay), HPLC-fluorescence or HPLC-UV, preferably after conversion to a free lyso-Gb1 derivative.

A biomarker as used within the scope of the invention is preferably any biological compound such as a protein and a fragment thereof, a peptide, a polypeptide, a proteoglycan, a glycoprotein, a lipoprotein, a carbohydrate, a lipid, a nucleic acid, an organic or inorganic chemical compound, a natural polymer, and a small molecular weight compound. , which is differentially present in a sample from an individual with one phenotypic status (eg, having a disease) compared to another phenotypic status (eg, not having a disease) and which can be isolated from a sample from an individual or measured in a sample from an individual. Moreover, the biomarker may be the whole unchanged molecule or it may be a part of it, which is preferably detected by mass spectrometric analysis, an antibody, another protein that specifically binds the biomarker, functional nucleic acids that specifically bind the biomarker and/or a fluorescent label. Moreover, a biomarker is considered informative if a measurable aspect of the biomarker is associated with a given patient status, such as a particular Gaucher disease status. Such a measurable aspect may include, for example, the presence, absence or level of a biomarker in a sample from an individual and/or its presence as part of a biomarker profile. A measurable aspect can also be a ratio of two or more measurable aspects of biomarkers, which biomarkers, for example, may be of a known type or may not be of a known type. The biomarker profile includes at least two such measurable aspects, where the measurable aspects may correspond to the same or different classes of biomarkers, such as nucleic acid and carbohydrate, for example. The biomarker profile may also include at least three, four, five, 10, 20, 30 or more measurable aspects. In one embodiment, the biomarker profile includes hundreds or even thousands of measurable aspects. In another embodiment, the biomarker profile includes at least one measurable aspect of at least one biomarker and at least one measurable aspect of at least one internal standard.

In one embodiment of the method in accordance with the present invention, an internal standard is added to a sample of an individual. It will be appreciated that by said addition of an internal standard, also referred to herein as IS, to a sample, i. addition of the sample to be carried out by the method in accordance with the present invention, the concentration of IS in the sample is known and, for example, by determining the area under the peak, i.e. peak area of an internal standard, e.g. in an HPLC-MS chromatogram, the ratio between the peak area and the concentration of a substance, e.g. free lyso-Gb1 and peak area IS. The person skilled in the art will further understand that various molecules can be used as IS. However, an IS having a similar chemical structure to a molecule such as a biomarker such as free lyso-Gb1 is preferred. Accordingly, the authors of the present invention in one embodiment chose lyso-Gb2, which differs from lyso-Gb1 in that it contains an additional part of the sugar and, in addition, is not present as such in nature. In a preferred embodiment, the IS molecule can be distinguished from free lyso-Gb1 in the method of the present invention. In a further preferred embodiment, the IS is chosen to be a molecule that ideally does not occur or rarely occurs in nature. In one embodiment of the present invention, where an internal standard is added to an individual's sample, it is preferred that IS is added such that it dissolves in a solvent, such as ethanol, prior to said addition to the sample. In a further preferred embodiment, this solvent is chosen such that said solvent is capable of causing protein precipitation, preferably capable of providing a protein precipitation step as a participant in the process of the present invention.

In some embodiments of the present invention, the protein precipitation and/or the protein precipitation step is part of the method of the present invention. It will be understood that precipitation, as used herein, preferably means the formation of a solid in solution, ie, for example, the formation of a protein precipitate in a sample, eg serum, from an individual. When precipitation, such as protein precipitation, occurs in a sample, the resulting solid is referred to as a precipitate, or, when compacted by a centrifuge, a precipitate. The liquid remaining above the solid is in any case called the supernatant. The present invention contemplates various methods for precipitating and/or separating said supernatant and said precipitate or precipitate, including, but not limited to, settling or sedimentation and centrifugation. The person skilled in the art will be aware of other methods for precipitating the protein and/or separating the supernatant and the protein precipitate, however, said skilled person will appreciate that if a method is used, preferably a method according to the invention, where the precipitated protein disables a device such as column or HPLC column used in connection with the present invention, the precipitated protein is preferably isolated from the solvent and/or sample.

In some embodiments of the present invention, the level of a biomarker of the present invention, for example, free lyso-Gb1, determined by the method of the present invention, in a sample is compared with the level of the same or another biomarker of the present invention, determined by the method of the present invention in another sample, for example, from the same patient, from another patient, from a control, and/or at the same or different time points, and/or with a threshold level and/or a control level and/or an IS level. As used herein, "comparison" or "compared to" as used herein preferably means a mathematical comparison of two or more levels of the biomarker(s). Thus, it will be naturally obvious whether one of said values is higher, lower or identical if at least two of such values are compared with each other.

In some embodiments, implementation of the present invention, the level of the biomarker is also determined in the control. As used herein, the control is preferably a sample from an individual, where the Gaucher disease status of said individual is known. In one embodiment, the control is a healthy patient sample. In a further embodiment, an amount of said biomarker is added to said sample from a healthy patient prior to determining the level of said biomarker in said sample from a healthy patient containing said added biomarker using the method of the present invention. In a further embodiment, the control is a sample from at least one individual having Gaucher disease status, such known Gaucher disease status including severe, mild, or healthy Gaucher disease status, such as a control patient. In a further preferred embodiment, the control is a sample from an individual not being treated for Gaucher's disease. In a further preferred embodiment, the control is a sample from a single individual or a collection of samples from different individuals and/or samples taken from the individual(s) at different time points.

The term "level" or "biomarker level" as used herein preferably means the concentration of a substance, preferably a biomarker of the invention and more preferably free lyso-Gb1, in a sample or in an individual. A person skilled in the art will understand that in certain embodiments, the implementation of the specified sample is not necessarily subjected to the method according to the invention as a raw sample, where the method includes determining the level of the specified biomarker, i. said sample can be subjected to, for example, a protein precipitation, separation, eg centrifugation and/or HPLC step, and then subjected to a biomarker level determination step, eg using mass spectrometric analysis. In addition, it should be noted that when the term "level" of a biomarker is used in relation to the level of the biomarker of the invention, which is determined in accordance with the present invention, it means the "level" of the biomarker of the present invention, which is determined by the methods of the present invention and which is contained in the sample, subjected to the method(s) according to the invention.

The level of the biomarker differs between different disease states if the average or median level of the biomarker in the different groups is calculated to be statistically significant. Common tests of statistical significance include, among others, the t-test, ANOVA, Wilcoxon test, Mann-Whitney test, odds ratio, and Kruskal-Wallis test. Biomarkers, alone or in combination, provide indicators of the relative risk that an individual falls into one phenotypic status or another. Thus, the biomarkers of the present invention are useful in one embodiment of the present invention as markers for a disease, therapeutic efficacy of a drug, or method of treatment.

The term "determining the level" of a biomarker, as used herein, preferably means methods that include quantifying at least one substance in a sample from an individual and/or quantifying said substance contained in a body part of an individual, such as saliva, blood, lymph, serum, plasma or cerebrospinal fluid, and/or quantitative determination of the specified substance in the individual, and the substance is selected from the group containing the biomarker.

Those skilled in the art will appreciate that detecting and/or determining the level of free lyso-Gb1 in a sample from an individual thus preferably includes that the Gb1 present in the individual's blood is not chemically converted, transformed or derivatized such that so that free lyso-Gb1 cannot be detected and/or its level cannot be determined separately from and/or in addition to Gb1. One skilled in the art will appreciate that Gb1 present in a sample from an individual who is subjected to a deacetylation step, e.g. by hydrolysis in methanolic sodium hydroxide, will result in the fatty acid portion being cleaved from Gb1, and thus undesirably result in a chemically converted, transformed or derivatized form of Gb1 that cannot be differentiated from free lyso-Gb1. Thus, the inventors of the present invention have the advantage of recognizing that free lyso-Gb1, in addition to Gb1, is useful in a method for diagnosing Gaucher's disease.

In a preferred embodiment of the methods of the present invention, the method is for detecting and/or determining the level of free lyso-Gb1 in a sample from an individual, where the Gb1 present in the sample is not subjected to a step leading to Gb1 deacetylation, preferably not subjected to a step leading to cleavage parts in the form of a fatty acid from Gb1 contained in the sample. In a further preferred embodiment of the method of the present invention, Gb1 present in a sample from an individual is not chemically converted, transformed or derivatized. In a further preferred embodiment of the method of the present invention, free lyso-Gb1 present in the sample from the individual is separated from the Gb1 present in the sample from the individual before the step which results in the elimination of a fatty acid portion from the Gb1 and/or before the step in which Gb1 is chemically converted, transformed or derivatized. In a further preferred embodiment, the step of detecting and/or determining the level of a biomarker in a sample from an individual, where the biomarker is free lyso-Gb1, is carried out after separation using HPLC by applying mass spectrometric analysis.

An individual is considered a healthy individual with respect to Gaucher's disease if the individual does not suffer from symptoms associated with Gaucher's disease. Moreover, in one embodiment of the methods of the invention, an individual is considered healthy if he does not have a mutation of the functional parts of the cerebrosidase gene and/or does not have a mutation in the cerebrosidase gene, resulting in a decrease or deficiency of the glucocerebrosidase enzyme or its activity, which leads to symptoms associated with Gaucher disease. These mutations will be detected if a sample from an individual is subjected to genetic testing for such mutations as described herein. In a further embodiment of the present invention, a sample from a healthy individual is used as a control sample or as an empty matrix in the methods of the present invention. The empty matrix, as mentioned herein, is preferably a sample from a healthy individual. However, it will be understood that such an empty matrix may contain a native level of free lyso-Gb1.

In one embodiment of the present invention, the level of the biomarker indicates that the individual is suffering from or at risk of developing a disease or disorder. The biomarker level determined by the method of the present invention is compared to a control biomarker level, where the result of said comparison allows the diagnosis of the disease.

More specifically, comparing the level of a biomarker in a sample from an individual with a control level of the biomarker includes comparing the level of a biomarker in a sample from an individual with a threshold level, where if the level of the biomarker in the sample from the individual is increased or higher compared to the threshold level, this indicates that that the individual suffers from or is at risk of developing Gaucher disease and/or where, if the level of the biomarker in the sample from the individual is reduced or lower than the threshold level, this indicates that the individual is not suffering from or is not at risk of developing Gaucher disease . It is also within the scope of the present invention that comparison of the level of a biomarker in a sample from an individual with a control level allows the determination of the severity of Gaucher disease, where if the level of a biomarker in a sample from an individual is elevated, increased or higher compared to the control level, then this indicates that the individual suffers from or is at risk of developing Gaucher disease with a more severe status or progression; and where, if the level of the biomarker in the sample from the individual is reduced or lower than the control level, then this indicates that the individual suffers from or is at risk of developing Gaucher disease with a less severe status or progression. In a further embodiment of the present invention, this comparison of the level of a biomarker in a sample from an individual with a control level comprises comparing the level of a biomarker in said individual with the level of a biomarker detected in a control sample, where if the level of a biomarker in a sample from an individual is increased, increased or is higher in compared with a control sample, this indicates that the individual is suffering from and/or at risk of developing Gaucher disease; and/or if the level of the biomarker in the sample from the individual is increased, increased or higher than the control sample, then this indicates that the individual suffers from or is at risk of developing Gaucher disease with a more severe status or progression. Said control is preferably selected from the group consisting of healthy individuals, individuals suffering from Gaucher disease or at risk for symptoms of Gaucher disease, individuals testing positive for a mutation or combination of mutations in the cerebrosidase gene, where the mutation or combination of mutations in the cerebrosidase gene is indicative of the prospect of development in an individual with Gaucher disease with a more severe or less severe status or progression. In a further embodiment of the present invention, this control level is determined in the control sample, where optionally free lyso-Gb1 is added to the control sample in a specific amount before determining the level of free lyso-Gb1 in the control sample.

The merit of the authors of the present invention is that they were able to develop a method for diagnosing Gaucher disease in an individual, where the method includes detecting a biomarker in a sample from an individual, where the biomarker is free lyso-Gb1, and preferably further includes determining the level of a biomarker in a sample from an individual, and more preferably further comprises comparing the level of the biomarker in the sample from the individual to a threshold level that exhibits high sensitivity, ie. sensitivity at least 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99 .9% or 100%, and high specificity of at least 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%. In a further embodiment of the present invention, these methods in accordance with the present invention allow the diagnosis of Gaucher disease in an individual, regardless of the progression status of Gaucher disease in the individual. More specifically, the methods of the present invention allow the diagnosis of Gaucher disease in an individual having an early Gaucher disease status as well as an individual having an advanced or advanced Gaucher disease status.

The effectiveness of a method in correctly diagnosing Gaucher's disease is typically measured as method sensitivity, method specificity, or area under the true/false detection ratio curve (also referred to herein as the "ROC curve"). The ROC curve is a plot of true positive rates versus false positive rates for various possible threshold levels of the diagnostic method. The ROC curve shows the relationship between sensitivity and specificity. Sensitivity is the percentage of true positive results that are predicted by the test to be positive, while specificity is the percentage of true negative results that are predicted to be negative by the test. The ROC curve provides the sensitivity of the test as a function of 1-specificity. The larger the area under the ROC curve, the more powerful the predictive value of the test. Thus, an increase in sensitivity will be accompanied by a decrease in specificity. The closer the curve passes from the left axis and then to the top edge of the ROC space, the more accurate the test is. In contrast, the closer the curve is to the 45-degree diagonal of the ROC plot, the less accurate the test is. Thus, the area under the ROC is a measure of the accuracy of the test. The accuracy of a test depends on how well the test separates the study group into those with the disease in question and those who do not have the disease in question. An area under the curve (also referred to herein as "AUC") of 1 represents an ideal method, while an area of 0.5 corresponds to a less suitable method. Thus, preferred diagnostic methods of the present invention have an AUC greater than 0.50, more preferred methods have an AUC greater than 0.9, and most preferred methods have an AUC greater than 0.998.

Other useful and suitable indicators of the applicability of the method are positive predictive value and negative predictive value. The positive predictor is the percentage of true positives that are positive in the test. The negative predictor is the percentage of true negatives that are negative in the test.

Methods for determining the status of Gaucher disease in an individual using prior art biomarkers, eg, chitotriosidase and/or CCL18, exhibit sensitivity and specificity typically not exceeding 90%.

One of skill in the art will appreciate that although the specificity and sensitivity of the methods of the present invention are as high as described above and were determined as described in the Examples section below, isolated instances where a patient having Gaucher's disease, will be determined as a false negative in the test, or where a patient without Gaucher's disease will be determined as a false positive, using the method of the invention. Considering these cases, when determining the specificity and sensitivity of the method in accordance with the present invention, the specificity and sensitivity will be lower than the values described above. However, one of skill in the art will also appreciate that such high specificity and such high sensitivity as described above have never been described before for a method for diagnosing Gaucher's disease. Thus, it is important to note that while the sensitivity and specificity of the method of the present invention may vary, if patient populations other than those described in the Examples, for example, differing in the number of patients, are subject to the methods of the present invention, the authors of the present invention firmly believe that none of the methods known in this field, using biomarkers will achieve higher specificity and higher sensitivity compared to the methods in accordance with the present invention. This is especially true because the detection limit of the methods of the present invention allows the determination of the level of free lyso-Gb1 in many healthy individuals. Thus, an individual with a disease with false negative test results using the methods of the present invention is defined as a false negative in the test because the level of the biomarker in the sample of the individual with the disease with false negative test results is the same as the level of the biomarker in the sample of a healthy individual. . In particular, it is important to note that said individual with false negative test results is not determined to be negative for the reason that the biomarker level is too low to be detected by the method of the present invention.

The "limit of detection" of a substance, such as free lyso-Gb1, as used herein is preferably the level of the substance as determined by the method for determining the level of the substance, where a level less than or lower than the specified limit of detection cannot be determined by the method. . Thus, it is naturally understood that "cut-off level" and "limit of detection" as used herein are preferably not necessarily identical, although both of them reflect a certain level of a substance, for example, a biomarker of the present invention. It will naturally be understood that, on the contrary, the threshold level will preferably be chosen so that the selectivity and sensitivity of the method are as high as possible. In contrast, the limit of detection reflects the absolute level of the biomarker of the present invention, which reflects the minimum level of the biomarker that can be detected using the method for determining the level of the specified biomarker. Thus, it is naturally understood that the limit of detection depends on the method for determining the level of the substance and on the substance, the level of which is to be determined using the method. It will be well understood by one of skill in the art that a high detection limit, for example higher than the ideal threshold level, will likely result in low sensitivity of the method, since the percentage of true positive results predicted to be positive in the test also depends on whether one can whether the biomarker level should be determined for the indicated true positive results. In other words, if the detection limit is higher than the ideal cut-off level, true positive results having a biomarker level slightly above the cut-off level may not differ from true negative results having a biomarker level lower than the cut-off level, since it cannot a biomarker level should be determined for both true positive results having a biomarker level above the cut-off level and negative results having a biomarker level lower than the cut-off level. Thus, it is well understood that a low detection rate is an advantage. Thus, it is also the credit of the inventors that they have shown that a lower level of detection provides a method for diagnosing Gaucher disease in an individual, including the step of determining the level of the biomarker present in the sample with higher selectivity and sensitivity. An "ideal threshold" as used herein is preferably a threshold as described herein, a method using said ideal threshold having the highest selectivity and sensitivity.

An embodiment of the methods according to the present invention is that they include the step of confirming said method by diagnosing a disease or disorder, preferably Gaucher disease, in an individual using the method of the present invention; the step of diagnosing a disease or disorder, preferably Gaucher disease, in an individual by genetic testing, including gene sequencing, preferably sequencing a gene known to a person skilled in the art that a mutation in it causes the disease or disorder, more preferably cerebrosidase gene sequencing in the case of Gaucher disease ; and comparing the results of said method and said genetic testing. As used herein, a healthy individual is preferably considered healthy with respect to a disease or disorder if said individual does not suffer from symptoms associated with said disease or disorder and if the result of the genetic testing does not reveal mutations in a gene of which one of skill in the art is aware that a mutation in it causes a disease or disorder. Also imply that a healthy individual has positive test results for the absence of Gaucher disease.

The term "determining the status of Gaucher disease" in an individual, as used herein, preferably means classifying a biomarker profile of an individual selected from the group including identifying or detecting the presence or absence of Gaucher disease in an individual, predicting the occurrence or risk of developing Gaucher disease in an individual, determining course of an individual's Gaucher disease, determining and/or predicting the severity of Gaucher disease in an individual, determining whether the individual is suffering from early Gaucher disease status or advanced or advanced Gaucher disease status, or determining whether the level of a biomarker in an individual changes over time .

The term "treatment management in an individual" or "individual management" as used herein preferably refers to the behavior of a clinician or clinician following determination of the status of Gaucher's disease. For example, if the result of the method according to the present invention is inconclusive or there is a reason why confirmation of status is necessary, the physician may order new tests such as testing for glucocerebrosidase function and/or sequencing of the gene encoding glucocerebrosidase. Alternatively, if the status indicates that treatment for Gaucher's disease is warranted, the physician may prescribe the individual for treatment for Gaucher's disease. Likewise, if the status is negative or if the results indicate that treatment is successful, further management may not be necessary. However, one of skill in the art will appreciate that, in addition to gene therapy, any therapy used, such as ERT and/or SRT, must be used throughout the life of a Gaucher patient. Moreover, an embodiment of the present invention is that the management of treatment in an individual includes titrating the dose of a drug used as a method of treating Gaucher's disease, for example, recombinant enzyme units used in an ERT performed on a patient. In some embodiments of the methods of the present invention, where the level of a biomarker present in a sample from an individual is determined at multiple time points or compared to other levels of the biomarker, a threshold level, and/or a control level of said biomarker, the skilled artisan will or will not use therapy, or change the therapy already used, for treatment or no treatment, or to continue the treatment of Gaucher's disease.

It is within the scope of the present invention that the skilled artisan will apply the dosage and/or maintain the dosage or change the dosage, eg, apply the dosage or a higher dosage, i.e. increase the dosage if such a biomarker level comparison demonstrates, for example, that the level of said biomarker is higher than, for example, a threshold level, i.e. the patient is diagnosed with Gaucher disease; or that the level determined earlier in the same patient is lower or the same, ie. the therapy used is insufficient, i.e. does not result in lower levels. On the other hand, one skilled in the art will use or not use a dosage, or maintain or decrease a dosage, eg, not using a dosage, or using a lower dosage, i.e. reduce the dosage if such comparison of the biomarker level indicates, for example, that the level of said biomarker is lower than, for example, a threshold level, i.e. the patient is diagnosed as having no Gaucher disease; or that the level determined earlier in the same patient is higher, ie. the therapy used is sufficient, i.e. leads to lower levels. In one embodiment of the present invention, a relatively high level of free Lyso-Gb1, based on such a comparison, indicates the use of a high dose of the recombinant enzyme used in ERT, and/or a relatively low level of free Lyso-Gb1, based on such a comparison, indicates the use low dosage of recombinant enzyme used in ERT. However, it is also well understood that a qualified practitioner will take into account the patient's history, i.e. a qualified person managing the treatment of a patient suffering from Gaucher disease and being treated so that the biomarker level is lower than the threshold level, for example, decides not to stop treatment, but to reduce the dosage and increase the time between further applications of the methods of the present invention.

The course of Gaucher's disease can be determined by the method of the present invention by determining the level of a biomarker in a sample from an individual at various time points during the course of the disease. It is important to note that a single application of the method for diagnosing Gaucher disease in accordance with the present invention allows the diagnosis of Gaucher disease and in certain embodiments includes the step of determining the course of treatment in an individual, based on diagnosing whether the individual suffers from or is at risk of developing Gaucher disease. If an individual whose sample is thus subjected to the method of the present invention has positive test results for the presence or risk of developing Gaucher disease, the skilled artisan will know what decision to make regarding the management of treatment in the individual, i.e. how the individual will be treated, for example, using a certain dose of enzyme in relation to ERT. It is well understood that, regardless of the judgment of the skilled artisan as to how to administer treatment in an individual, the skilled artisan may decide on at least one additional use of the method in accordance with the present invention at a later point in time. Thus, an embodiment of the present invention is that it is possible to compare biomarker levels determined at different time points, where different time points mean at least two time points. Without wishing to be bound by theory, the inventors of the present invention have discovered that the level of the biomarker of the present invention in samples from one particular patient can correlate with the severity of the disease in that individual at the time the patient is sampled. Thus, it is well understood that an elevated biomarker level determined in a sample at a later time point compared to a biomarker level determined in a sample at an earlier time point indicates a more severe status in an individual at a later time point compared to status of an individual at an earlier point in time. A lower level of a biomarker measured in a sample at a later time point compared to a biomarker level determined in a sample at an earlier time point indicates a less severe status in the individual at the later time point compared to the status of the individual at the earlier time point. . Thus, in one aspect, the present invention relates to a method for determining the course of Gaucher disease in an individual, comprising the step of determining at several points in time the level of a biomarker present in a sample from the individual, where the biomarker is free lyso-Gb1. In a further aspect, the invention relates to a method for determining the effectiveness of at least one treatment used in an individual with positive test results for the presence or risk of developing Gaucher disease, comprising the step of determining at several points in time the level of a biomarker present in a sample from an individual, where the biomarker is free lyso-Gb1. It will be well understood by those skilled in the art that the methods of the present invention thus allow the selection of therapy and/or adjustment of the doses and/or dosage of the selected therapy based on the results of the method of the invention. If, for example, an individual is being treated for Gaucher disease, the method for diagnosing Gaucher disease in an individual in accordance with the present invention can be used every 3 months and the biomarker levels thus determined will be compared to determine the effectiveness of the treatment(s) and/or therapy/therapies. used by the individual. If the individual reaches a status where a stable level of the biomarker is maintained over time, the frequency of using the method for diagnosing Gaucher disease in an individual in accordance with the present invention can be reduced to a frequency of once every 6 months. If the dosage of therapy is changed, for example, the units of the recombinant enzyme used in ERT are reduced or increased, the frequency of using the method for diagnosing Gaucher disease in an individual according to the present invention can be set back to every 3 months. By comparing certain levels of the biomarker in samples from the individual, the skilled artisan will determine whether the level of the biomarker is increasing, decreasing, or maintaining a stable level of the biomarker over time. Thus, a qualified physician may decide to reduce the dosage of therapy, for example, the number of units of the recombinant enzyme used in ERT; increase dosage of therapy; or maintain the dosage of therapy, depending on the comparison of biomarker levels determined by the method in accordance with the present invention. A decrease in the level of free lyso-Gb1 by approximately 60% over a period of 12 months indicates successful therapy for Gaucher disease, where the decrease, as used in the framework of the invention, preferably means that the level of free lyso-Gb1 determined by the method of the present invention at the end of the time period , compared with the level of free lyso-Gb1, defined by the method of the present invention at the beginning of the specified period of time. Thus, the qualified physician may decide to reduce the dosage of the therapy used or to maintain the dosage of the therapy used. If the decrease in the level of free lyso-Gb1 is significantly weaker, a qualified specialist may decide to increase the dosage of therapy. Also merit of the authors of the present invention is the understanding that a decrease in the level of free lyso-Gb1 correlates with the effectiveness of therapy. The more severe the reduction in free lyso-Gb1 over a period of time, eg 12 months, the more successful is therapy, such as eg ERT, SRT or chaperone therapy. Thus, a further embodiment of the present invention is that the method of the present invention is for comparing the efficacy of a therapy or at least two therapies used in an individual.

Thus, the person skilled in the art knows that progression monitoring, i. the course of Gaucher's disease, as well as the effectiveness of therapy in an individual, can be carried out by frequently determining the level of free lyso-Gb1 in samples from the individual.

In a further aspect, the invention relates to a method for determining the efficacy of at least one treatment used in an individual with positive test results for the presence or risk of developing Gaucher disease, comprising the step of determining at several points in time the level of a biomarker present in a sample from an individual, where the biomarker is free lyso-Gb1. With regard to what is described above in relation to the management of treatment in an individual, one skilled in the art will appreciate that the effectiveness of a single treatment or a combination of at least two treatments can be compared using the methods of the present invention. Thus, using the method of the present invention, it is possible to study and compare several new drugs, dosage forms, dosages or methods of treating Gaucher's disease.

An embodiment of the present invention is that the method for diagnosing Gaucher's disease according to the present invention is independent of whether or not the individual has been previously treated for Gaucher's disease. Thus, a sample from an individual may be a sample from an individual who has previously been treated for Gaucher's disease, as well as a sample from an individual who has not previously been treated for Gaucher's disease. Thus, a further embodiment of the present invention is that the method of the present invention comprises the step of administering treatment to an individual and/or determining the level of a biomarker in a sample from the individual after controlling the course of the individual. Said treatment of an individual may be based on a diagnosis of whether the individual is suffering from or at risk of developing Gaucher disease; on the detection of a biomarker in a sample from an individual after controlling the course of the individual; or on determining the level of a biomarker in a sample from an individual after controlling the course of the individual. However, one of skill in the art will appreciate that a sample of some non-Gaucher patients or some patients successfully treated for Gaucher disease will show a free lyso-Gb1 level below the detection limit.

Without wishing to be bound by any theory, the present inventors believe that the level of free lyso-Gb1 present in a sample from an individual furthermore correlates with disease severity in an individual suffering from Gaucher's disease. In this regard, the authors of the present invention found by evaluating the results presented in the present description (for example, shown in figure 4 of the present description), that despite the fact that, in principle, the level of free lyso-Gb1 differs in specific individuals, and , more specifically, may differ in particular individuals having the same mutation(s), the higher the level of free lyso-Gb1, the greater the severity of Gaucher disease in terms of a statistical mean for a clinical score. Thus, the level of free lyso-Gb1 correlates with the severity of Gaucher disease in that patients who test positive for individual glucocerebrosidase gene mutations known to cause mild (eg, N370S mutation) or more severe (eg, N370S mutation) L444P) course of Gaucher's disease, the level of free lyso-Gb1 determined in these patients was statistically correlated with the severity usually associated with such a mutation.

Thus, a further embodiment of various aspects of the present invention relates to a method for determining the severity of Gaucher disease in an individual, comprising the step of

a) determining the level of the biomarker present in the sample from the individual, where the biomarker is free lyso-Gb1, and step

b) determining the severity of Gaucher disease, for example, by comparing the level of free lyso-Gb1 in an individual, preferably determined by the method of the present invention, with a clinical indicator.

In relation to this, it is important to note that if the level of free lyso-Gb1 is determined in samples from patients suffering from Gaucher disease with the L444P mutation when sequencing the cerebrosidase gene (homozygous and complex heterozygous) who were subjected to the method of the present invention, the average level of free lyso -Gb1 exceeds the average level of free lyso-Gb1, determined in samples from patients suffering from Gaucher disease with the N370S mutation when sequencing the cerebrosidase gene using the same method (figure 4). It is known that the L444P mutation causes a more severe course of Gaucher's disease - this is especially true when the individual is homozygous for this mutation. Consistently, a higher mean level of free lyso-Gb1 is found in homozygotes compared to the homozygous N370S mutation (194 ng/ml and 159 ng/ml, respectively, see Figure 4). Moreover, patients with the complex heterozygous L444P mutation have significantly lower levels of free lyso-Gb1 than homozygotes (89 ng/ml and 45.4 ng/ml, respectively). The person skilled in the art will know the clinical scores to categorize the severity of Gaucher's disease or symptoms or the severity of its symptoms. Thus, an embodiment of the method of the present invention is that the course of Gaucher disease in a patient is predicted, and more specifically, the severity of Gaucher disease is determined based on the level of a biomarker determined in accordance with the method of the present invention.

An embodiment of the present invention is that chitotriosidase levels determined in patients not having a chitotriosidase gene mutation, in particular not having a 24 bp duplication as described herein, serve as a basis for correlation with the severity of Gaucher disease, since the average level of chitotriosidase, determined in the sample from these patients, as described in the present description, correlates with the severity of Gaucher disease. Thus, for example, a chitotriosidase level below 200 nmol IU/hr/mL correlates with Gaucher disease status in a non-Gaucher patient. In relation to this, it is important to note that a patient being treated for Gaucher disease may also exhibit chitotriosidase levels below 200 nmol IU/hr/mL. Levels greater than 2000 nmol IU/hr/mL correlate with Gaucher disease status "extensive" or "severe" and chitotriosidase levels between 200 and 2000 nmol IU/hr/mL correlate with Gaucher disease status "mild".

In this regard, it is important to note that chitotriosidase levels of 200 to 1000 nmol IU/h/mL can also occur in a sample from an individual suffering from another LSD such as Niemann-Pick type C disease or Krabbe disease, thereby making the use of chitotriosidase unsuitable for diagnosis. Gaucher disease. Thus, the factors described above in relation to the use of chitotriosidase levels to correlate with the severity of Gaucher disease are generally only applicable to patients where the presence or absence of Gaucher disease and/or another LSD known to exhibit elevated chitotriosidase levels has been proven with using mutational analysis.

If the level of free lyso-Gb1 is determined in accordance with the methods of the present invention in said patients who do not have a mutation in the chitotriosidase gene, in particular, do not have a 24 bp duplication, as described herein, the indicated level of free lyso-Gb1 determined in a sample from each of said patients correlate with the level of chitotriosidase in said patients and/or with the severity of Gaucher disease and/or the status of Gaucher disease in said patient. Thus, the severity of Gaucher disease and/or the status of Gaucher disease, including healthy, mild or severe, is determined and more preferably correlated with chitotriosidase levels and/or ranges of chitotriosidase levels, as described above.

One skilled in the art will recognize that a level of a biomarker of the present invention, determined in a sample from an individual, where said biomarker level is correlated with the severity of Gaucher disease, as described above, will be indicative of the use of a specific therapy and/or dose or dosage of said therapy. For example, if the level of a biomarker determined in accordance with the methods of the invention correlates with a "severe" or "advanced" Gaucher disease status, the individual is treated for Gaucher disease, and the method for diagnosing Gaucher disease in the individual in accordance with the present invention can be used every 3 months and the levels of the biomarker thus determined can be compared to determine the effectiveness of the treatment(s) and/or therapy/therapies used in the individual. If an individual reaches a status where the biomarker level correlates with "mild" Gaucher disease, or where a stable biomarker level is maintained over time, the frequency of using the method for diagnosing Gaucher disease in an individual in accordance with the present invention can be reduced to a frequency of once every 6 months.

In another aspect, the present invention relates to a method for determining the effectiveness of a composition for the treatment of Gaucher's disease. Such a method may include the steps of determining the level of free lyso-Gb1 in an individual having Gaucher's disease; administering to said individual said compound in an amount sufficient to determine the effectiveness of said compound; re-determining the level of free lyso-Gb1 in the specified individual; comparison of the level of free lyso-Gb1 determined before and after administration of said composition, where the lower level of free lyso-Gb1 determined after administration of said composition compared to the level of free lyso-Gb1 determined after administration of said composition indicates the effectiveness of said compound for the treatment of Gaucher's disease.

The present invention is further further illustrated by means of the following figures and examples, from which further features, embodiments and advantages can be established.

More specific,

1A is a box plot showing free lyso-Gb1 levels in ng/ml plasma;

1B is a box plot showing free lyso-Gb1 levels in ng/mL plasma grouped by gender of individuals;

2A is a graph showing true/false detection ratio (ROC) curves for free lyso-Gb1 and chitotriosidase;

2B is a graph showing true/false detection (ROC) ratio curves for free lyso-Gb1 and CCL18;

3A is a graph showing free lyso-Gb1 in ng/ml plasma as a function of time for a total of 20 German patients with Gaucher disease;

3B is a graph showing free lyso-Gb1 in ng/ml plasma as a function of time for a total of 24 untreated patients with Gaucher disease (10 German, 14 Israeli patients);

3C is a graph showing free lyso-Gb1 in ng/ml plasma as a function of time for a total of 9 Israeli patients with Gaucher disease before and after initiation of therapy;

3D is a graph showing regression-based values for free lyso-Gb1 in ng/ml plasma as a function of time for Israeli and German Gaucher patients before and after initiation of therapy;

Figure 4 is a table showing the median level of free lyso-Gb1 for two common mutations;

5A is a HPLC-MS chromatogram showing the peak intensity of free lyso-Gb1 and IS in a healthy individual;

5B is an HPLC-MS chromatogram showing the peak intensity of free lyso-Gb1 and IS of a patient with Gaucher disease;

5C is an HPLC mass spectrometry chromatogram showing the peak intensity of free lyso-Gb1 and IS in a patient with Gaucher disease.

Brief description of the drawings

Figure 1A is a box plot showing free lyso-Gb1 levels; the y-axis shows the logarithmic levels of free lyso-Gb1 in ng/ml, determined in the plasma of patients by the method in accordance with the present invention, where the x-axis represents the groups of patients who were divided into groups, as described in example 2. The box plot reflects 25 and 75th percentile of each patient group using the bottom and top of the drawers, respectively; the line near the middle of the box represents the 50th percentile (i.e. median) of each group; whiskers represent one standard deviation above and below the mean of the data; any data not included between the whiskers is represented as isolated points using a small circle or star. The horizontal line represents the threshold level of 5 ng/mL.

FIG. 1B is a box plot showing free lyso-Gb1 levels as shown in FIG. 1A, further grouped by gender of individuals; the y-axis represents the logarithmic levels of free lyso-Gb1 in ng/ml, determined in the plasma of patients by the method in accordance with the present invention, where the x-axis represents the groups of patients who were divided into groups as described in example 2, and, in addition by sex of patients. The box plot depicts the 25th and 75th percentiles of each patient group using the bottom and top of the boxes, respectively; a line near the middle of the box represents the 50th percentile (eg median) of each group; whiskers represent one standard deviation above and below the mean of the data; any data not included between the whiskers is represented as isolated points using a small circle or star. The horizontal line represents the threshold level of 5 ng/mL.

FIG. 2A is a graph showing true/false detection ratio (ROC) curves for free lyso-Gb1 and chitotriosidase; the x-axis shows "1-specificity" and the y-axis shows sensitivity. Free lyso-Gb1 shows 100% sensitivity and 100% specificity, with chitotriosidase at best having a sensitivity of 0.9591 or 95.91%, respectively.

FIG. 2B is a graph showing true/false detection (ROC) ratio curves for free lyso-Gb1 and CCL18; the x-axis shows "1-specificity" and the y-axis shows sensitivity. Free lyso-Gb1 shows 100% sensitivity and 100% specificity, where CCL18 at best has a sensitivity of 0.8658 and 86.58%, respectively.

In FIG. 3A, the y-axis plots free lyso-Gb1 levels as a function of time, determined by the method of the present invention in ng/ml, in the plasma of 20 German Gaucher patients who were treated, more specifically ERT, during the trial. Each curve and each patient number, respectively, reflect the levels determined in plasma taken from the same patient at different time points, as indicated on the x-axis. The x-axis represents the times of plasma collection, where the zero point indicates the first measurement in each patient's therapy. To analyze the change in the level of free lyso-Gb1 over time in patients with Gaucher disease, as described in example 3, non-aggregated data were used for those patients for whom more than one blood sample was analyzed.

3B is a graph showing free lyso-Gb1 as a function of time determined by the method according to the present invention in ng/ml plasma of a total of 24 untreated patients with Gaucher disease (10 German, 14 Israeli patients); untreated, as used herein, preferably means that no treatment, eg enzyme replacement therapy, has been used for Gaucher's disease. To analyze the change in the level of free lyso-Gb1 over time in patients with Gaucher disease, as described in example 3, non-aggregated data were used for those patients for whom more than one blood sample was analyzed.

3C is a graph showing free lyso-Gb1 as a function of time, with free lyso-Gb1 determined by the method of the present invention in ng/mL plasma of a total of 9 Israeli Gaucher patients during the time before and after onset. therapy. The x-axis indicates the time in months, where "0" indicates the first point in time after the start of therapy. The curve labeled "overall" reflects regression-based free lyso-Gb1 values.

3D is a graph showing free lyso-Gb1 regression-based values as a function of time determined by the method of the present invention in ng/ml of plasma from Israeli and German Gaucher patients during the time before and after the start of therapy. . The x-axis indicates the time in months, where "0" indicates the first point in time after the start of therapy. The curve labeled "overall" represents regression-based free lyso-Gb1 values.

Figure 4 is a table showing the median level of free lyso-Gb1 in patients with positive test results for one of the two common mutations of the glucocerebrosidase gene, namely N370S and L444P, in the homozygous, as well as in the complex heterozygous case, where the complex heterozygosity is the state of having two heterozygous recessive alleles at a particular locus that can cause a genetic disorder when heterozygous. One of skill in the art will appreciate that patients with the L444P mutation in the glucocerebrosidase gene also have a more malignant prognosis, which is especially true in a homozygous situation. Thus, an embodiment of the present invention is that the method of the present invention comprises determining the severity of Gaucher disease. Said determination of the severity of Gaucher disease includes determining the level of a biomarker, preferably free lyso-Gb1, present in a sample from an individual, and/or comparing said level of said biomarker, determined in a sample from individuals having various mutations in the glucocerebrosidase gene and/or not having a mutation in the glucocerebrosidase gene. The present inventors have discovered that in a sample from a patient testing positive for the homozygous L444P mutation in the cerebrosidase gene, the level of free lyso-Gb1 determined by the method of the present invention is approximately 194 ng/mL and is elevated compared to the level of free lyso-Gb1 detected in a sample from a patient testing positive for the homozygous N370S mutation in the cerebrosidase gene, where the free lyso-Gb1 level, as determined by the method of the present invention, is approximately 159 ng/mL. The inventors also discovered that in a sample from a patient testing positive for the complex heterozygous L444P mutation, the level of free lyso-Gb1, determined by the method in accordance with the present invention, is 89 ng/ml, which is significantly lower than the level of free lyso-Gb1. -Gb1 as determined by the method of the present invention in a sample from a patient tested positive for homozygous L444P mutation, wherein the level of free lyso-Gb1 as determined by the method of the present invention is approximately 45.4 ng/mL. Without wishing to be bound by any theory, the present inventors believe that the level of free lyso-Gb1 in a sample from an individual, determined by the method of the present invention, is indicative of the severity of Gaucher disease. Thus, a further embodiment of the present invention is that the method of the present invention is for determining the effectiveness of at least one treatment used in an individual who is tested positive for the presence and/or risk of developing Gaucher disease. The numbers in parentheses indicate the concentration ranges measured in the respective patient group. IQR stands for interquartile range. All patients who were treated for Gaucher's disease were subjected to ERT.

FIG. 5A is an HPLC-MS chromatogram showing the maximum intensity in cps of free lyso-Gb1 and IS of a sample from a healthy individual as a function of retention time in minutes. Retention time, as used within the scope of the invention, is preferably represented on the x-axis and represents the elapsed time between the time of injection of a solute, such as a biomarker according to the present invention and/or internal standard, and the elution time of the maximum peak of said solute. One of skill in the art will appreciate that the retention time of a substance according to the methods described herein is a unique characteristic of said solute and can be used for identification purposes. An internal standard working solution containing 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. addition of the sample to be subjected to the method according to the present invention, the concentration of IS in the sample is known and thus by determining the area under the peak, i.e. peak area, internal standard on the specified chromatogram HPLC-mass spectrometry can be calculated the ratio between the peak area and the concentration of a substance, such as IS and/or biomarker. More precisely, one skilled in the art will recognize that the peak area of a substance represented in an HPLC mass spectrometry chromatogram, such as the HPLC mass spectrometry chromatogram presented in FIG. 5A, FIG. 5B, or FIG. substance subjected to analysis by HPLC-mass spectrometry. Moreover, a person skilled in the art will be able to calculate the amount of a substance in a sample from an individual subjected to HPLC-MS analysis, for example, the amount of free lyso-Gb1 in a sample subjected to the method of the present invention, using the peak area ratio of free lyso-Gb1 , the amount of which is to be determined by this method, and the peak area IS, for example, free lyso-Gb2; as well as calibration curves obtained using the specified method, and the specified free lyso-Gb1 and/or IS. Thus, it allows the subsequent determination of the level of free lysoGb1.

FIG. 5B is an HPLC-MS chromatogram showing the peak intensity of free lyso-Gb1 and IS of a sample from a patient with Gaucher's disease where a free lyso-Gb1 level of 17.1 ng/mL was determined according to the method of the present of the invention, essentially as described in Example 1. Comparison of the indicated level of the biomarker in a sample from an individual with a threshold level of 5 ng/ml, which was chosen such that the sensitivity for diagnosing Gaucher disease in an individual in accordance with the methods of the present invention was 100% and that the specificity of diagnosing Gaucher disease in an individual according to the methods of the present invention was 100%, an elevated level of the biomarker in the sample from the individual compared to the threshold level indicates that the individual suffers from Gaucher disease.

FIG. 5C is an HPLC-MS chromatogram showing the peak intensity of free lyso-Gb1 and IS of a sample from a patient with Gaucher's disease, where a free lyso-Gb1 level of 319 ng/mL was determined according to the method of the present invention, essentially as described in Example 1. Comparison of the indicated level of biomarker in a sample from an individual with a threshold level of 5 ng/ml, which was chosen such that the sensitivity of the diagnosis of Gaucher disease in an individual in accordance with the methods of the present invention was 100% and that the specificity of diagnosis Gaucher disease in an individual in accordance with the methods of the present invention was 100%, an increased level of the biomarker in the sample from the individual compared to the threshold level indicates that the individual suffers from Gaucher disease.

Examples

In the examples described below, human plasma was used as a sample from an individual. However, one skilled in the art will appreciate that, depending on the type of sample used from the individual, such as saliva, CSF, plasma, serum, whole blood, filter card blood with dried blood, or other blood product, the method of the present the invention needs to be adjusted depending on the type of sample and, moreover, for each type of sample, it is necessary to measure the threshold level in accordance with the method described in the examples below. The present inventors have discovered that using a human serum sample in the method as described below, instead of a human plasma sample, will result in identical detection results and thus a determined level of free lyso-Gb1 if the human serum sample and the human plasma sample come from the same and the same individual and they were taken at the same time; and where the samples were measured in parallel; and, more specifically, will lead to the same threshold level. Without reference, and as illustrative examples, by using saliva from a human patient, the method can be adjusted depending on the pH of the sample; or the threshold level can be defined as being 20 ng/mL using whole blood or blood collected on a filter card with dried blood as a sample from an individual.

Example 1 Method for Detecting Free Lyso-Gb1 in Human Serum

Equipment

The following equipment was used to detect free lyso-Gb-1 in a plasma sample from an individual.

Device/unit of equipment Type/manufacturer HPLC pump Series 200, Perkin Elmer, USA Sample injector Series 200, Perkin Elmer, USA column thermostat Series 200, Perkin Elmer, USA Mass selective detector API 4000 Q TRAP, AB SCIEX, USA/Canada DVX-2500 Multi-Tube Shaker Henry Troemner LLC, USA Shaking mixer Vortex Genie 2; Scientific Industries, USA Centrifuge Megafuge 1.0; Heraeus, Germany Multipipette(s), pipette(s) Eppendorf, Germany Water bath SW21-C, Julabo, Germany

Reagents

The following reagents were used to detect free lyso-Gb-1 in a plasma sample from an individual.

Since the values depend on temperature (for example, the pH value), such values were determined at a temperature of 25ºC.

Reagent Purity Acetonitrile (ACN) HPLC category or gradient category Acetone 99.5% Dimethyl sulfoxide (DMSO) HPLC category 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 "for analysis".

The term "purum" as used herein preferably denotes a commercial category of a chemical compound having a purity greater than the stated value.

ASTM-I, as used herein, refers to a water category standard purity achieved by purification methods including reverse osmosis and ultraviolet (UV) oxidation.

Obtaining Calibration Standards

Lyso-Gb1 stock solution was prepared by dissolving 1.70 mg Lyso-Gb1 (provided by Matreya) in 5 ml MeOH.

A solution of V1-A-534 was then prepared as a mixture of 12 μl of Lyso-Gb1 stock solution and 5 ml of DMSO/MeOH (1:1; v/v) as follows:

Designation of the resulting solution Experimental concentration [µg/ml] Solution volume [µl] Solution Solvent volume [ml] Solvent V1-A-534 0.79968 12 lyso-Gb1 stock solution 5 DMSO/MeOH (1:1; v/v)

Calibration standards were then prepared by adding a solution of Vl-A-534 or more highly concentrated calibration standards to MeOH/water (1:1; v/v) solvent.

The detailed addition scheme is presented below.

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

Calibration standards were used for calibration, having seven concentration levels between 0.400 and 100 ng/ml, namely the calibration standards Std3A-534, Std4A-534, Std5A-534, Std6A-534, Std7A-534, Std8A-534 and Std9A-534.

Obtaining Control Samples

Control samples were prepared by adding a solution of V1-A-534 or a more highly concentrated control sample to an empty matrix.

The detailed addition scheme is presented below.

Designation of the resulting solution Concentration [ng/ml] Solution volume [µl] Solution Empty matrix volume [ml] Volume [ml] QC-A1-534 1.0013 173.6 QC-C1-534 8.5 8.6736 QC-B1-534 5.0008 944 QC-C1-534 9.5 9.444 QC-C1-534 50.029 634 V1-A-534 9.5 10.134

empty matrix

Human plasma from a healthy individual was used as an empty matrix. One of skill in the art will recognize that said plasma from a healthy individual will contain a native level of free lyso-Gb1. The indicated native level of free lyso-Gb1 is approximately 1.4 ng/ml in accordance with the methods of the present invention. Thus, it is clear that the controls obtained by adding an empty matrix, where the empty matrix contains the indicated native level of free lyso-Gb1, also include the indicated native level of free lyso-Gb1 in addition to the level of free lyso-Gb1 obtained by adding a concentrated solution or more highly concentrated control sample. Thus, the level of free lyso-Gb1 in the control samples is as follows:

QC-A1-534 1 ng/ml + native concentration in empty matrix QC-B1-534 5 ng/ml + native concentration in empty matrix QC-C1-534 50 ng/ml + native concentration in empty matrix

One of skill in the art will appreciate that human plasma from a healthy individual used as an empty matrix can be purchased from any commercial source known to one of skill in the art. It is important to note that if by chance the plasma of an unhealthy individual, i.e. an individual with Gaucher disease is used as an empty matrix, this will result in unusually high levels of free lyso-Gb1 in controls determined by the method of the present invention, and thus this will be immediately ascertained, since the error of the method is defined as being in range of 15% above or below the estimated levels of controls subjected to the method in accordance with the present invention.

Samples under study

Obtaining an internal standard

An internal standard stock solution (IS1) was prepared by dissolving 1.00 mg Lyso-Gb2 (supplied by Matreya) in 2 ml DMSO/MeOH (1/1; v/v).

A working solution of the internal standard was then prepared as a mixture of 410 μl of stock solution IS1 and 500 ml of ethanol. Ethanol can be purchased from any commercial source where the ethanol is an absolute ethanol of a category suitable for the methods described herein. One skilled in the art will recognize that proteins contained in 50 µl of sample should precipitate if 100 µl of said internal standard working solution is added to the sample.

Storage of samples and solutions

Control or test samples were either immediately stored below -20ºC or aliquots were transferred to new glass vials until stored under the same conditions.

Concentrated solutions (stock solutions, V1-A-534, etc.) as well as stock solutions of internal standards were frozen below -20°C until the next addition.

Internal standard working solutions were stored at 2ºC to 8ºC until use.

The present inventors have discovered that free lyso-Gb1 is stable under the conditions mentioned above. More specifically, the levels of free lyso-Gb1 in a plasma and/or serum sample of a patient with Gaucher disease determined by the methods of the present invention were found to be identical if the level of free lyso-Gb1 was determined in said samples before and after storage at 37ºC for 2 days. Thus, the solutions and samples of the present invention can be transported in a number of ways well known to one of skill in the art, where the use of a cold chain for transporting patient material is preferred but not necessarily required. The person skilled in the art will also be aware of the methods and their respective conditions for proper storage of solutions and samples, where, for example, these solutions and samples can be stored for several weeks.

Sample preparation for analysis

All samples used in the analysis lot are obtained for analysis as follows:

Frozen samples were thawed at approximately 20 to 25° C. in a water bath under ambient conditions. After thawing, the samples were mixed.

50 μl of the sample was transferred to a sample vial.

100 µl of an internal standard working solution (in EtOH) was added to the sample.

Then, the mixture thus obtained was mixed using a DVX-2500 multi-tube shaker at 2500 rpm. for approximately 30 seconds.

The mixture thus obtained was centrifuged to separate the phases at 4000 rpm. within 2 minutes.

A volume of supernatant sufficient for injection (approximately 100 μl) was transferred into suitable (conical) autosampler vials.

Ways

Chromatography Options and Autosampler Options

The samples obtained for analysis as described above were then subjected to the method described below:

Parameter Intended Range/Description Mobile phase solvent A 50 mM FA in water Mobile phase solvent B 50 mM FA in ACN/acetone (1:1; v/v) Chromatographic run 0.0-4.0 min, linear gradient: 5% B → 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 Flushing the injector 0.1% TFA in 70% MeOH Column + pre-column ACE 3 C8, 50x2.1mm ID + Security Guard C8 Column temperature 60ºC retention time approximately 3.4-3.6 min: lyso-Gb1 and lyso-Gb2 (IS)

The ACE 3 C8 column (ACE C8 column no. ACE-112-0502) used in this experiment was purchased from Advanced Chromatography Technologies, Aberdeen.

Those skilled in the art will appreciate that parameters where "±" range is indicated reflect parameters that can be adjusted between sequences. The sequence, as used within the scope of the invention, is preferably a batch of specific sample numbers, preferably a maximum of 250, analyzed sequentially, where the parameters, including flow and temperature, remain unchanged. Intersequence adjustments and calibrations are known to those skilled in the art and include column replacement.

These changes within the specified limits are minor changes and are registered in the original data of the study at the measurement station.

detection

Then, the samples thus obtained were subjected to a detection method, the parameters of which are described below:

MS ionization mode: Electrospray ionization (ESI) MS polarity: positive MS detection mode: multiple response monitoring (MRM) Evaporator temperature: 500ºC±50ºC Ionization voltage: 5.5 kV Collision-activated dissociation (CAD) gas: short Gas 1: pressure = 45 psi inch Gas 2: pressure = 60 psi inch Gas curtain: pressure = 40 psi inch Side position: 5 units Vertical position: 4 units Quadrupole resolution: unit → unit Transitions 462.4 → 282.2 m/z lyso-Gb1 624.5 → 282.2 m/z lyso-Gb2 (internal standard) DP (Declustering Potential): 40 V CXP (Collision Cell Exit Potential): 8 V

One skilled in the art will appreciate that methods for detecting free lyso-Gb1 and/or determining the level of free lyso-Gb1 in a sample from an individual using mass spectrometric analysis will also use other transitions and fragments that allow specific detection and/or quantitation. determination of free lyso-Gb1 in the specified sample from the individual.

Evaluation and calculation of results

The following protocol was used to evaluate and calculate the results obtained by the methods described above.

Rounding procedure

Concentration data entered and retrieved from the chromatographic data system (CDS) were rounded to five significant figures. Further calculations in the spreadsheet were performed for full accuracy of the calculations, and then rounded to significant figures/decimal places for reporting. Thus, there may be deviations in intermediate results due to rounding. Accuracy and Variance Coefficients (CV) are reported to one or two decimal places, respectively.

With regard to the rounding procedure, it should be noted that the last digit provided is rounded up if the next digit is equal to or greater than "5".

Regression and statistics

From the calibration standards, the calibration curve was fitted using data processing software by peak area ratios (peak area of free lyso contained in the individual sample/peak area of internal standard). Free lyso-substance concentrations were assessed using the internal standard method. A quadratic regression model (y=ax ² +bx+c) using a weighting factor of 1/concentration is used to calculate the concentration of each analyte in each batch to be evaluated. The concentrations were calculated using the following formula:

Based on these averages, exact results (in CV values) and error (formula below) are calculated using the Lotus 123 program.

Suitable statistical models are described, for example, in

Green, J.R., Statistical Treatment of Experimental Data (Elsevier, New York, 1977), page 210 ff.

Lothar Sachs, Angewandte Statistik - Anwendung statistischer Capable (Springer, Berlin, Heidelberg, New York, Tokyo 1984)

Software

Data acquisition, data processing, statistical processing, and calculations were performed using Analyst software 1.4.2 or higher (AB SCIEX, USA/Canada) and Lotus 1-2-3 97 or higher (Lotus Corp, USA).

Reference books

Arbeiten mit SmartSuite 97 (Lotus Development Corp., Handbook 1997)

Documentation for the software used

Documentation for Analyst® Software (AB SCIEX, 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

After obtaining consent from patients to participate in the study, patients were subjected to genetic testing for mutations in the glucocerebrosidase gene. Thus, 5-10 ml of EDTA blood was sequenced according to Seeman et al. (Seeman et al., 1995). If necessary, additionally sequenced other genes, in addition to glucocerebrosidase, in particular in controls.

Moreover, the chitotriosidase gene was sequenced to detect a 24 bp duplication as mentioned above. Said genetic testing was controlled using study samples of age and sex matched control patients.

253 individuals were studied.

According to the result of the genetic study described above, the patients participating in the trial were divided into the following groups:

1.) Patients with Gaucher disease: The gold standard for diagnosis was the detection of two pathogenic mutations in the glucocerebrosidase gene, either homozygous or compound heterozygous (in the figures, the group is named "Gaucher");

2.) Patients who are heterozygous carriers of a mutation in the glucocerebrosidase gene (usually relatives of affected patients) (in the figures the group is referred to as "heterozygotes")

3.) Patients with other lysosomal storage disorders as controls (in the figures the group is referred to as "other LSD"); it includes patients with sphingomyelinase deficiency (Niemann Pick A/B), Krabbe disease and Niemann-Pick type C1 disease; all diagnoses were confirmed by identifying two pathogenic mutations.

4.) Healthy age and sex matched controls (group referred to as "control" in the figures). The following table 1a presents the classification of patients into the groups described above, according to the results of the genetic testing described above.

Table 1a
Individuals
classified according to the results of genetic analysis Groups (Dgn) cases reliable missing Total N percent N percent N percent control 140 100.0% 0 0% 140 100.0% Heterozygous (carriers) 13 100.0% 0 0% 13 100.0% Gaucher 59 100.0% 0 0% 59 100.0% other LSDs twenty 100.0% 0 0% twenty 100.0%

The gender distribution of 232 German patients, as well as the gender distribution of 21 Israeli patients, is presented in Table 1b.

Table 1b
232 German individuals and 21 Israeli individuals classified by sex Germanic Israeli N % N % Total 232 21 Floor male 146 57.0 eleven 52.4 feminine 110 43.0 ten 47.6

Table 1c below shows the age distribution of 232 German patients and the classification of these patients based on the results of the genetic testing described above, as well as the sex of these patients.

Table 1c
Patient characteristics for 253 individuals Health control Heterozygous carriers Gaucher Other LSDs N individuals 140 13 80 twenty N samples 155 fifteen 287 28 Age, in years 28.5 35.0 30.0 23.5 (mean - interquartile range) (4.8-47.3) (30.5-58.5) (8.0-48.0) (4.0-43.5) (number of cases) (n=134) (n=13) (n=79) (n=14) men women men women men women men women n 79 61 eight 5 45 35 12 eight Age 25.5 34.0 33.5 39.0 22.0 32.5 21.0 34 (mean - interquartile range) (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)

The level of free lyso-Gb1 in samples of these 253 individuals was determined by the method described in example 1. The level of free lyso-Gb1 in samples from these patients, depending on the classification by genetic analysis, is presented in figa. On figv presents the level of free lyso-Gb1 in samples from these patients, depending on the classification based on genetic analyzes and sex of patients.

The type of mutation and the distribution of types of mutations of the glucocerebrosidase gene in patients classified as patients with Gaucher disease according to the results obtained from genetic testing as described above are shown in Table 2 below.

table 2
Distribution of mutations detected in the Germanic group
with Gaucher disease (166 alleles) type of mutation n % type of mutation n % N370S 54 32.5% IVS3+1G>A one 0.6% L444P 33 19.9% L324Q one 0.6% RecNciI fifteen 9.0% N234S one 0.6% G202R four 2.4% N409S one 0.6% D409H 3 1.8% P161R one 0.6% Rec 3 1.8% P178S one 0.6% G355A 2 1.2% P29X one 0.6% IVS2+1A>G 2 1.2% P68fs one 0.6% L335V 2 1.2% Q326K one 0.6% L444R 2 1.2% R120Q one 0.6% R120W 2 1.2% R257ter one 0.6% R285H 2 1.2% R359Q one 0.6% RecAP2 2 1.2% R502C one 0.6% T226I 2 1.2% R502H one 0.6% T231R 2 1.2% RecAF3 one 0.6% T491I 2 1.2% RecAF4 one 0.6% V398L 2 1.2% RecAH3 one 0.6% A46 term one 0.6% RecTL one 0.6% A495P one 0.6% S13L one 0.6% A88P one 0.6% S146L one 0.6% C287F one 0.6% S237F one 0.6% F216Y one 0.6% S364N one 0.6% G82A one 0.6% V398L one 0.6% H255Q one 0.6% W184R one 0.6% I93F one 0.6%

Measurement of chitotriosidase activity

Chitotriosidase activity was measured essentially as described in Hollak et al. (Hollak CE, van Weely S, van Oers MH, Aerts JM. Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease. J Clin Invest. 1994 Mar;93(3): 1288-92) by incubating 10 µl of plasma or EDTA serum with 100 µl of 0.022 mM fluorogenic substrate 4-methylumbelliferyl-f1-D-N,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 37ºC. In patients with Gaucher disease, samples were diluted 50× in demineralized water prior to incubation. After 30 minutes, the reaction was stopped with 200 μl of 0.5 M glycine/NaOH buffer (pH 10.5) by mixing at room temperature. Hydrolysis of the substrate with chitotriosidase yielded a fluorescent molecule, 4-methylumbelliferone, which was quantified with a fluorimeter (Tecan Group Ltd., Mannedorf, Switzerland), excitation at 366 nm and emission at 446 nm, and compared with a calibration curve for a 4-methylumbelliferone standard. Chitotriosidase activity was expressed as nanomoles of substrate hydrolysed per hour per milliliter of incubated serum.

Quantification of CCL18

Plasma CCL18 was quantified using the DuoSet ELISA Development kit purchased from R&D Systems, Minneapolis, MN, USA, following the manufacturer's instructions. The sensitivity of the method was 5 pg/ml.

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

The protocols described in example 1 above were used to obtain HPLC-mass spectrometry chromatograms for 485 blood samples taken from 253 individuals. Exemplary HPLC mass spectrometric chromatograms showing free lyso-Gb1 peak intensity and IS for three samples from two Gaucher patients and one healthy control are shown in FIGS. 5A, 5B and 5C.

The gold standard for classifying patients into the Gaucher group was based on sequencing of the entire coding region as well as the intron-exon boundaries of the glucocerebrosidase gene according to genetic testing as described in Example 2, which resulted in the detection of either a homozygous mutation or compound heterozygosity.

The results of determining the levels of chitotriosidase or CCL18 in samples from patients were available for 58 or 44 patients with Gaucher disease, respectively. These results were obtained as described in example 2.

To compare the diagnostic value of different biomarkers and to calculate correlations between biomarkers, the data obtained by the method described above were first pooled using the earliest measured level of each marker for patients with Gaucher disease before therapy and the highest level for individuals without Gaucher disease for a particular patient if more than one blood sample was available.

Paired statistical methods were used to compare the two biomarkers. The method uses the mathematical equivalence of AUC with Mann-Whitney U-statistics (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.)

The accuracy of the levels of various biomarkers (free lyso-Gb1, chitotriosidase and CCL18) obtained by the method described in Example 1 was evaluated to distinguish patients with Gaucher's disease from patients without Gaucher's disease using true/false detection ratio (ROC) curve analysis (Metz 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 the activity of chitotriosidase and CCL18 was carried out as described in example 2 of the present description.

ROC curves were calculated using PASW Statistics 18, Release Version 18.0.2 (© SPSS, Inc., 2009, Chicago, IL, www.spss.com). Comparison of ROC curves and linear mixed models was performed using SAS software, Version 9.2 SAS System for Windows. (© 2008 SAS Institute Inc., Cary, NC, USA).

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

The results presented in the ROC-curves presented in figa and figv also demonstrate the specificity and sensitivity of the method in accordance with the present invention, depending on the different threshold levels of free lyso-Gb1. Table 3 below shows, respectively, the sensitivity and specificity of the method in accordance with the present invention, depending on the different threshold levels of free lyso-Gb1.

Table 3
Sensitivity and specificity of the diagnostic method
Gaucher disease, depending on the threshold level
free lyso-Gb1 in Germanic individuals (n=232) Threshold 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%

Comparing the level of a biomarker in a sample from an individual determined by the method according to the present invention to a cut-off level, preferably a cut-off level, allowing a diagnosis having high specificity and high sensitivity, thus allowing the diagnosis of Gaucher disease in said individual, where an elevated level of the biomarker in where a lower level of the biomarker in a sample from an individual compared to the cut-off level indicates that the individual is suffering from or at risk of developing Gaucher disease, and where a lower biomarker level in the individual sample compared to the cut-off level indicates that the individual is not suffering from or at risk of developing Gaucher disease.

Area under the curve (AUC) and 95% confidence intervals for various biomarkers are presented in Table 4.

Table 4
Sensitivity and specificity for various biomarkers in relation to the diagnosis of Gaucher disease Chitotriosidase
(n=228/58 Gaucher) CCL18
(n=210/44 Gaucher) free lyso-Gb1
(n=232/59 Gaucher) Threshold level >145 [nmolIU/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 ROC analysis 0.96
(0.92-1.00) 0.87
(0.80-0.93) 1.00
(1.00-1.00)

Thus, in Table 3, the sensitivity and specificity of the presented biomarkers used in the method for diagnosing Gaucher disease in a sample from an individual are compared using the threshold level having the highest AUC in the corresponding method using the corresponding biomarker. The ideal threshold level of the corresponding method is presented. Measurement of the activity of chitotriosidase and CCL18 was carried out as described in example 2 of the present description. Free lyso-Gb1 was determined in accordance with the method of the present invention. The ideal threshold level is 5 ng/mL.

One skilled in the art will appreciate that the method of the present invention using free lyso-Gb1 as a biomarker for diagnosing Gaucher disease is clearly superior to methods using CCL18 or chitotriosidase. This is especially true since at least 6% of the Europioid population and up to 35% of, for example, Hispanic populations, including individuals with Gole's disease, are deficient in chitotriosidase activity.

Thus, levels of free lyso-Gb1 determined in a sample from an individual in accordance with the method of the present application, greater than 5.0 ng/ml, allow the diagnosis that the individual suffers from or is at risk of developing Gaucher disease with a sensitivity and specificity of 100%.

Example 4: Analysis of biomarker changes over time

The method and patients used in this example were the method and patients as described in examples 1-3.

To analyze how biomarker levels changed over time in patients with Gaucher disease, non-aggregated data were analyzed for those patients for whom more than one blood sample was analyzed. Time zero was set for the first measurement during each patient's therapy.

Free lyso-Gb1 levels over time for individual patients are shown in Figure 3A, Figure 3B and Figure 3C.

To investigate the significance of a time-dependent decrease in free lyso-Gb1 levels indicative of successful therapy, post-therapy free lyso-Gb1 levels were compared to pre-therapy free lyso-Gb1 levels using linear mixed models. Untreated patients did not show a significant decrease in free lyso-Gb1 levels over time.

Thus, the levels of free lyso-Gb1 were converted to logarithmic to overcome deviations in the distribution of values. Random cut and slope models were used to account for interpatient heterogeneity in baseline values as well as in rate of change. In all models, the observed heterogeneity was statistically significant. Only the p-values for the linear decrease in time are described.

Values for time and magnitude, which included a squared value for time, were centered to investigate the non-linear relationship between time and marker levels for chitotriosidase and for CCL18. For free lyso-Gb1, the squared value did not improve the model and was not included in the final model.

As therapy, German patients were administered 40 U/kg body weight on average, where units refer to units of recombinant glucocerebrosidase in ERT. The decrease in free lyso-Gb1 is especially intense after the start of therapy (after 6 months <0.0001). However, the decrease over time is also significant (<0.0001). There was a decrease in free lyso-Gb1 after treatment for 12 months, on average in the range of 60%.

The features of the present invention described in the description, claims, sequence listing and/or drawings may, either alone or in combination, be material for carrying out the invention in its various forms.

Claims (19)

1. A method for identifying an individual suitable for therapy suitable for treating his Gaucher disease, where the method includes testing a sample from an individual for the presence of a biomarker, where the biomarker is free lyso-Gb1, where the sample is selected from the group consisting of a blood sample, a serum sample and a plasma sample, where the individual is eligible for therapy if the test results of the sample are positive for the biomarker, where the test results are positive for the biomarker, if the level of the biomarker in the sample from the individual exceeds the cut-off level, and the cut-off level is 20 ng/mL if the sample is a blood sample and the cutoff is 5.0 ng/mL if the sample is a serum sample or a plasma sample. 2. The method of claim 1, wherein the therapy is selected from the group consisting of enzyme replacement therapy, substrate reduction therapy, chaperone therapy, gene therapy, stem cell transplantation, and DNA/RNA skipping. 3. The method according to claim 1, where the free lyso-Gb1 has the formula (I):

(I). 4. The method of claim 1 wherein the free lyso-Gb1 is a lyso-Gb1 that is present in the individual as such as a result of the individual's metabolic activity and is not generated by chemical, biochemical or physical processing of the sample. 5. The method of claim 1, wherein the blood sample is a whole blood sample or a whole blood sample collected on a dried blood filter card. 6. The method according to any one of claims 1 to 5, wherein the biomarker is detected by immunoassay, mass spectrometric analysis, biochip, functional nucleic acids and/or a fluorescent derivative of free lyso-Gb1. 7. The method according to claim 6, where the biomarker is detected using mass spectrometric analysis. 8. The method of claim 1, wherein the subject is an individual suspected of having Gaucher disease. 9. The method of claim 1, wherein the subject is an individual with Gaucher disease. 10. A method for treating Gaucher disease in an individual, comprising testing a sample from an individual for the presence of a biomarker, where the sample is selected from the group consisting of a blood sample, a serum sample, and a plasma sample, and administering therapy to the individual for the treatment of Gaucher disease, if the results of testing the sample are positive in biomarker, where the biomarker is free lyso-Gb1, where the test results are positive for the biomarker if the level of the biomarker in the sample from the individual exceeds the cut-off level, and the cut-off level is 20 ng/ml if the sample is a blood sample, and the cut-off the level is 5.0 ng/mL if the sample is a serum sample or a plasma sample. 11. The method of claim 10, wherein the therapy is selected from the group consisting of enzyme replacement therapy, substrate reduction therapy, chaperone therapy, gene therapy, stem cell transplantation, and DNA/RNA skipping. 12. The method according to claim 10, where free lyso-Gb1 has the formula (I):

(I). 13. The method of claim 10 wherein free lyso-Gb1 is lyso-Gb1 that is present in the individual as such as a result of the individual's metabolic activity and is not generated by chemical, biochemical or physical processing of the sample. 14. The method according to any one of claims 10-13, wherein the biomarker is detected by immunoassay, mass spectrometric analysis, biochip, functional nucleic acids and/or a fluorescent derivative of free lyso-Gb1. 15. The method of claim 14 wherein the biomarker is detected by mass spectrometric analysis. 16. The method of claim 10, wherein the subject is an individual suspected of having Gaucher disease. 17. The method of claim 10, wherein the subject is an individual with Gaucher disease.

Images