Method for the Diagnosis of Cystic Fibrosis
20190128901 ยท 2019-05-02
Assignee
Inventors
Cpc classification
G01N33/92
PHYSICS
International classification
Abstract
The present invention is related to a method for diagnosing cystic fibrosis in a subject comprising a step a), wherein the step a) comprises detecting a biomarker in a sample from the subject, wherein the sample is a sample selected from the group comprising blood, a blood product, urine, saliva, cerebrospinal liquid, stool, tissue and lymph liquid.
Claims
1. A method for diagnosing cystic fibrosis in a subject comprising detecting a biomarker in a sample from the subject, wherein the sample is a sample selected from the group consisting of blood, a blood product, urine, saliva, cerebrospinal liquid, stool, tissue and lymph liquid.
2. The method of claim 1, wherein the method further comprises determining a level of the biomarker present in the sample.
3. The method of claim 1, wherein the level of the biomarker is indicative whether or not the subject is suffering from cystic fibrosis or whether or not the subject is at risk of suffering from cystic fibrosis.
4. A method for diagnosing cystic fibrosis in a subject, wherein the method comprises the following steps: i) adding an internal standard to a sample from the subject, wherein the sample from the subject is selected from the group consisting of blood, a blood product, urine, saliva, cerebrospinal liquid, stool, tissue and lymph liquid; ii) optionally mixing the sample with the internal standard; iii) subjecting the sample to a protein precipitation and/or a biomarker extraction step, whereby protein from the sample is precipitated and/or the biomarker is extracted and a first supernatant of the sample is provided; iv) optionally subjecting the first supernatant of the sample or at least a part thereof to a first separation step which provides a second supernatant; v) subjecting the first supernatant and/or the second supernatant, or at least a part thereof, to a second separation step, wherein the second separation step comprises injecting at least a part of the first supernatant and/or at least a part of the second supernatant into an HPLC-MS/MS system and using an HPLC column with a gradient from acidic water to acetonitrile/acetone; wherein the HPLC column is an HPLC column selected from the group consisting of a C8 HPLC column and a C18 HPLC column, and wherein the second separation step provides a separated sample; vi) subjecting the separated sample to MS/MS, wherein MS/MS comprises collision induced dissociation, in source-fragmentation, electron capture and/or transfer dissociation; and wherein the method comprises a step a), wherein the step a) comprises detecting a biomarker in a or the sample from the subject, and optionally a step b), wherein the step b) comprises determining a level of the biomarker present in the sample.
5. The method of claim 4, wherein the internal standard is selected from the group consisting of N-lauroyl sphingosine, lyso-Gb2, a C17 ceramide, a C19 ceramide, a C21 ceramide, a C23 ceramide, a C25 ceramide and a C27 ceramide.
6. A method of determining the effectiveness of a compound for the treatment of cystic fibrosis, wherein the method comprises the following steps: a) determining a level of a biomarker in a sample from a subject having cystic fibrosis; b) administering to said subject said compound; c) determining the level of the biomarker in a sample from the subject after the compound has been administered to the subject; and d) determining whether the level of the biomarker determined in step c) is lower than the level of the biomarker determined in step a); wherein if a level of the biomarker determined in step c) is lower than the level of the biomarker determined in step a) this indicates the effectiveness of said compound.
7. A method for determining the effectiveness of at least one treatment applied to a subject being positively tested for suffering from or being at risk of suffering from cystic fibrosis comprising a step a), wherein the step a) comprises determining at several points in time a level of a biomarker in a sample from the subject.
8. A method for determining the course of cystic fibrosis in a subject comprising the step of determining at several points in time a level of a biomarker present in a sample from the subject.
9. The method of claim 1, wherein the biomarker is one selected from the group comprising of C26 ceramide, a medium ceramide and a long ceramide, wherein a medium ceramide is a ceramide of formula (2) ##STR00024## wherein n is any integer from: 8, 10, 12, 14 and 16 and a long ceramide is a ceramide of formula (2) ##STR00025## wherein n is any integer from 18, 20, 22, 24, 26, and 28.
10. The method of claim 9, wherein the biomarker is C26 ceramide.
11. The method of claim 9, wherein the biomarker is cis-C26 ceramide.
12. The method of claim 9, wherein the biomarker is total C26 ceramide, preferably total C26 ceramide is cis-C26 ceramide plus trans-C26 ceramide.
13. The method of claim 1, wherein the biomarker is selected from the group comprising isoform 1 compound, isoform 2 compound and the total of isoform 1 compound and isoform 2 compound.
14. The method of claim 1, wherein the biomarker is detected by means of mass spectrometric analysis.
15. The method of claim 1, wherein the subject is a human being.
16. The method of claim 1, wherein if the level of the biomarker in the sample from the subject is higher than a cut-off value this is indicative that the subject is suffering from cystic fibrosis or is at risk of suffering from cystic fibrosis and/or wherein if the level of the biomarker in the sample from the subject is lower than a cut-off value this is indicative that the subject is not suffering from or is not at risk of suffering from cystic fibrosis, preferably the cut-off value is 69 nmol/L blood, more preferably 69.1 nmol/L blood in case the biomarker is total C26 ceramide and the cut-off value is 27.7 nmol/L blood in case the biomarker is cis-C26 ceramide.
17. The method of claim 6, wherein the sample is selected from the group consisting of blood, a blood product, urine, saliva, cerebrospinal liquid, stool, tissue and lymph liquid.
18. The method of claim 16, wherein the blood product is dried blood spots (DBS).
19. Use of mass spectrometric analysis for the detection of a biomarker, wherein the biomarker is selected from the group consisting of C26 ceramide, a medium ceramide and a long ceramide, wherein a medium ceramide is a ceramide of formula (2) ##STR00026## wherein n is any integer from: 8, 10, 12, 14 and 16 and a long ceramide is a ceramide of formula (2) ##STR00027## wherein n is any integer from 18, 20, 22, 24, 26 and 28, or wherein the biomarker is selected from the group consisting of isoform 1 compound, isoform 2 compound or the total of isoform 1 compound and isoform 2 compound.
20. Use of a biomarker for the diagnosis of cystic fibrosis, in a method of claim 1, wherein the biomarker is selected from the group consisting of C26 ceramide, a medium ceramide and a long ceramide, wherein a medium ceramide is a ceramide of formula (2) ##STR00028## wherein n is any integer from: 8, 10, 12, 14 and 16 and a long ceramide is a ceramide of formula (2) ##STR00029## wherein n is any integer from 18, 20, 22,_24, 26 and 28, or wherein the biomarker is selected from the group consisting of isoform 1 compound, isoform 2 compound or the total of isoform 1 compound and isoform 2 compound.
21. A kit for determining the presence of a biomarker in a sample from a subject, wherein the kit comprises: a) an interaction partner of the biomarker; b) optionally a solid support comprising at least one capture reagent attached thereto, wherein the capture reagent binds the biomarker; and c) instructions for using the solid support to detect the biomarker, wherein the biomarker is C26 ceramide.
22. (canceled)
23. Use of a compound selected from the group consisting of N-lauroyl sphingosine, lyso-Gb2, a C17 ceramide, a C19 ceramide, a C21 ceramide, a C23 ceramide and a C25 ceramide as an internal standard in a) use in a method for diagnosing cystic fibrosis; b) use in a method for determining the course of cystic fibrosis in a subject; and/or c) use in a method for determining the effectiveness of at least one treatment applied to a subject, wherein preferably the method of a), b) and/or c) is a method of claim 1.
Description
[0432] The present invention is now further illustrated by the following figures and examples from which further features, embodiments and advantages may be taken.
[0433] More specifically,
[0434]
[0435]
[0436]
[0437]
EXAMPLES
[0438] In the Examples described in the following a dried blood spot (abbr. DBS) on a filter card was used as a sample from a subject. Nevertheless, a person skilled in the art will acknowledge that depending on the used type of sample from a subject, e.g. comprising saliva, liquor, plasma, serum, full blood, blood on a dry blood filter card or another blood product, the method of the present invention has to be adjusted to the type of sample and furthermore a cut-off value has to be determined for each type of sample according to the method described in the following examples.
Example 1
Method for the Detection of C26 Ceramide in DBS
[0439] Equipment
[0440] For detecting C26 ceramide and/or a substance with molecular weight of 678, detected as MRM transition in positive mode 678.7 m/z to 264.3 m/z, in a sample of plasma from a subject the following equipment was used:
[0441] Apparatus/Piece of Equipment Type/Producer
[0442] UPLC Aquity Series (pumps, autosampler, column manager), Waters, UK
[0443] Mass selective detector TripleQuad 5500, AB SCIEX, Germany
[0444] Multi-tube vortexer DVX-2500 Henry Troemner LLC, USA
[0445] Vortex mixer Vortex Genie 2; Scientific Industries, USA
[0446] Centrifuge Megafuge 1.0; Heraeus, Germany
[0447] Multipette(s), pipette(s) Eppendorf, Germany
[0448] Water bath SW21-C, Julabo, Germany
[0449] Reagents
[0450] For detecting C26 ceramide in a sample from a subject the following reagents were used. To that extent that values depend on temperature (e.g. the pH value) such values were determined at a temperature of 25? C.
[0451] Acetonitrile (ACN) HPLC-grade or Gradient
[0452] Acetone 99.5%
[0453] Dimethylsulfoxide (DMSO) HPLC grade
[0454] Ethanol (EtOH) p.a., 96%
[0455] Formic acid (FA) p.a., 98-100%
[0456] Methanol (MeOH) Gradient (LiChrosolv)
[0457] Trifluoroacetic acid (TFA) purum>98%
[0458] Water ASTM-I
[0459] The abbreviation p.a. as used herein means pro analysis.
[0460] The term purum as used herein, preferably means a commercial grade of a chemical compound having a purity of the above specified value.
[0461] ASTM-I as used herein refers to a water grade standard purity achieved by purification methods comprising Reverse Osmosis and Ultraviolet (UV) Oxidation.
[0462] Preparation of Calibration Standards
[0463] For calibration, as calibration standards five concentration levels between 2.00 and 200 ng/mL C26 ceramide (as provided by Carbosynth, UK) were used.
[0464] Preparation of Internal Standard
[0465] The Internal Standard (IS 1) stock solution was prepared dissolving 25 ?g of C25 ceramide (as provided by Avanti Polar Lipids, US) in DMSO/EtOH (1/1; vol/vol) to a concentration of 50 ng/mL.
[0466] Storing of Samples and Solutions
[0467] Control samples or study samples either were immediately stored below ?20? C. Standard solutions as well as Internal Standard working solutions were stored between 2? C. and 8? C. until use.
[0468] Sample Preparation for Analysis
[0469] 3 punches ? of 3.2 ?m were cut from the filtercard with dried blood spots and subjected to extraction in presence of internal standard solution in DMSO: water: ethanol vol. 1:1:2 for 60 minutes at 37? C. and sonicated 20 minutes at 40? C. The solution containing internal standard and blood extract was transferred to a PTFE (polytetrafluoroethylene) filter plate (Acroprep?, Pall, Germany) and then to a 96 well plate by centrifugation at 3.500 rpm.
[0470] Methods
[0471] A person skilled in the art will acknowledge that methods for detecting C26 ceramide in a sample from a subject using mass spectrometric analysis may also employ other transitions and fragments which allow for specific detection of and/or quantification of C26 ceramide and its isoforms in said sample from a subject.
[0472] LC/MRM-MS analyses of the C26 ceramide for DBS extracts were performed using a Waters Acquity UPLC (Waters, UK) coupled with an ABSciex 5500 TripleQuad mass spectrometer (ABSciex, Germany). Chromatographic run was performed on a C8 column (ACE columns, Germany). The 10 ?L extract were injected on the column and the compounds were eluted using a linear gradient from 40% A (50 mM formic acid in water) to 100% B (50 mM formic acid in acetonitrile/acetone. The following MRM transitions were monitored: 664.3.fwdarw.264.3 for the internal standard (with DP of 34V, CE of 46 V and CXP of 13 V) and 678.7.fwdarw.264.3 for C26 ceramide isoforms (with DP of 30V, CE of 38.4 V and CXP of 11 V).
[0473] MRM-MS analyses were performed in positive ion mode using the following parameters: CUR gas 10 psi, IS voltage 5 kV, CAD 8 psi, cone temperature 200? C., GS1 45 psi, GS2 60 psi, EP 10 V. For all batches analyzed a standard curve was measured using 5 dilutions of C26 ceramide in ethanol.
[0474] Data analysis was performed using Analyst 1.6.2 and Microsoft Office 2010.
[0475] Software
[0476] Data acquisition, data processing, statistics and calculations were performed using Analyst software 1.6.2 or higher (AB SCIEX, USA/Canada).
Example 2
Determining C26 Ceramide in DBS from Healthy Subjects and Cystic Fibrosis Patients and Cystic Fibrosis Carriers
[0477] Using the methods outlined in Example 1, the content of cis-C26 ceramide (referred to as Isoform 1 in Table 1) and trans-C26 ceramide (referred to as Isoform 2 in Table 1) was determined in DBS from a total of 192 healthy subjects. Similarly, the content of cis-C26 ceramide (referred to as Isoform 1 in Table 1) and trans-C26 ceramide (referred to as Isoform 2 in Table 1) was determined in DBS from a total of 15 previously genetically diagnosed cystic fibrosis patients and in DBS from a total of 40 cystic fibrosis carriers (a priori genetically proven). From such data, the ratio (cis-C26 ceramide/trans-C26 ceramide), in referred to as Ratio in Table 1, and the total content of C26 ceramide (content of cis-C26 ceramide+content of trans-C26 ceramide), referred to as Total in Table 1, were determined. The cut-off value was set at the mean value of the cohort of healthy individuals plus 2? standard deviation.
[0478] The results thereof are shown in Tables 1a, 1b and 1 c.
TABLE-US-00001 TABLE 1a Summary of C26 ceramide levels in healthy controls, cystic fibrosis patients and cystic fibrosis carriers Ceramide C26:0 (nmol/L) Samples N Isoform1 Isoform2 Ratio Total Cut-off 27.7 69.1 Cystic Fibrosis patients 15 67.3 ? 26.0 39.3 ? 29.4 1.9 ? 1.0 106.5 ? 40.74 Cystic Fibrosis carriers 40 49.3 ? 52.8 44.9 ? 20.96 1.5 ? 1.1 93.9 ? 55.28 All Normal Controls 192 18.3 ? 4.7 31.6 ? 8.6 0.6 ? 0.2 49.9 ? 9.6
TABLE-US-00002 TABLE 1b C26 ceramide levels in healthy controls C26Cer C26Cer Total Healthy control Isoform 1 Isoform 2 C26Cer No description nmol/L nmol/L ratio nmol/L 1 Newborn 1 13.1 51.5 0.3 64.6 2 Newborn 2 20.0 45.0 0.4 65.0 3 Newborn 3 15.3 46.4 0.3 61.7 4 Newborn 4 13.6 40.4 0.3 54.0 5 Newborn 5 14.2 22.0 0.6 36.2 6 Newborn 6 19.1 18.4 1.0 37.5 7 Newborn 7 18.8 45 0.4 63.8 8 Newborn 8 18.9 32.6 0.6 51.5 9 Newborn 9 16.9 20.1 0.8 37.0 10 Newborn 10 19.6 32.4 0.6 52.0 11 Newborn 11 27.0 19.7 1.4 46.7 12 Newborn 12 15.9 33.3 0.5 49.2 13 Newborn 13 16.9 25.1 0.7 42.0 14 Newborn 14 25.3 40.4 0.6 65.7 15 Newborn 15 19.3 32.7 0.6 52.0 16 Newborn 16 19.9 23.0 0.9 42.9 17 Newborn 17 19.0 42.8 0.4 61.8 18 Newborn 18 13.2 30.8 0.4 44.0 19 Newborn 19 20.9 45.0 0.4 65.9 20 Newborn 20 9.5 24.1 0.4 33.6 21 Newborn 21 6.9 60.7 0.1 67.6 22 Newborn 22 17.0 34.5 0.5 51.5 23 Newborn 23 12.6 40.0 0.3 52.6 24 Newborn 24 21.1 25.9 0.8 47.0 25 Newborn 25 27.0 17.4 1.6 44.4 26 Newborn 26 9.3 40.0 0.2 49.3 27 Newborn 27 26.0 27.2 1.0 53.2 28 Newborn 28 27.0 42.0 0.6 69.7 29 Newborn 29 25.8 33.5 0.8 59.3 30 Newborn 30 17.0 32.5 0.5 49.5 31 Newborn 31 13.0 42.0 0.3 55 32 Juvenile_0.5-4y_01 22.8 28.0 0.8 50.8 33 Juvenile_0.5-4y_02 14.0 16.9 0.8 30.9 34 Juvenile_0.5-4y_03 10.4 23.5 0.4 33.9 35 Juvenile_0.5-4y_04 22.1 34.2 0.6 56.3 36 Juvenile_0.5-4y_05 21.4 15.5 1.4 36.9 37 Juvenile_0.5-4y_06 24.6 13.5 0.9 38.1 38 Juvenile_0.5-4y_07 13.0 20.4 0.6 33.4 39 Juvenile_0.5-4y_08 24.9 27.8 0.9 52.7 40 Juvenile_0.5-4y_09 10.5 23.9 0.4 34.4 41 Juvenile_0.5-4y_10 18.4 36.0 0.5 54.4 42 Juvenile_0.5-4y_11 17.6 24.6 0.7 42.2 43 Juvenile_0.5-4y_12 17.5 27.3 0.6 44.8 44 Juvenile_0.5-4y_13 18.7 21.6 0.9 40.3 45 Juvenile_0.5-4y_14 10.9 20.7 0.5 31.6 46 Juvenile_0.5-4y_15 27.0 26.6 1.1 53.6 47 Juvenile_0.5-4y_16 17.7 43.6 0.4 61.3 48 Juvenile_0.5-4y_17 20.7 31.3 0.7 52 49 Juvenile_0.5-4y_18 19.2 23.6 0.8 42.8 50 Juvenile_0.5-4y_19 19.6 34.8 0.6 54.4 51 Juvenile_0.5-4y_20 17.4 24.7 0.7 42.1 52 Juvenile_0.5-4y_21 26.1 32.9 0.8 59 53 Juvenile_0.5-4y_22 22.6 29.2 0.8 51.8 54 Juvenile_0.5-4y_23 21.4 28.8 0.7 50.2 55 Juvenile_0.5-4y_24 23.8 19.1 1.2 42.9 56 Juvenile_0.5-4y_25 18.9 16.4 1.1 35.3 57 Juvenile_0.5-4y_26 23.0 31.2 0.7 54.2 58 Juvenile_0.5-4y_27 23.2 33.4 0.7 56.6 59 Juvenile_0.5-4y_28 27.0 40.4 0.8 67.4 60 Juvenile_0.5-4y_29 7.1 23.3 0.3 30.4 61 Juvenile_0.5-4y_30 14.4 46.8 0.3 61.2 62 Juvenile_0.5-4y_31 20.9 28.4 0.7 49.3 63 Juvenile_0.5-4y_32 8.7 29.5 0.3 38.2 64 Juvenile_0.5-4y_33 27.0 16.1 2.0 43.1 65 Juvenile_0.5-4y_34 27.0 31.4 0.9 58.4 66 Juvenile_0.5-4y_35 23.0 17.7 1.6 40.7 67 Juvenile_0.5-4y_36 26.3 38.5 0.7 64.8 68 Juvenile_4-17y_01 19.5 37.2 0.5 56.7 69 Juvenile_4-17y_02 13.3 36.2 0.4 49.5 70 Juvenile_4-17y_03 25.8 33.6 0.8 59.4 71 Juvenile_4-17y_04 22.0 45.0 0.5 67.0 72 Juvenile_4-17y_05 24.8 29.6 0.8 54.4 73 Juvenile_4-17y_06 23.6 36.4 0.6 60.0 74 Juvenile_4-17y_07 16.0 50.8 0.4 66.8 75 Juvenile_4-17y_08 23.8 31.6 0.8 55.4 76 Juvenile_4-17y_09 21.5 40.8 0.5 62.3 77 Juvenile_4-17y_10 18.4 43.0 0.3 61.4 78 Juvenile_4-17y_11 11.8 50.4 0.2 62.2 79 Juvenile_4-17y_12 19.0 31.3 0.6 50.3 80 Juvenile_4-17y_13 20.7 17.0 1.2 37.7 81 Juvenile_4-17y_14 22.9 35.3 0.6 58.2 82 Juvenile_4-17y_15 16.9 38.7 0.4 55.6 83 Juvenile_4-17y_16 16.0 38.5 0.4 54.5 84 Juvenile_4-17y_17 23.0 29.8 0.8 52.8 85 Juvenile_4-17y_18 24.8 38.0 0.7 62.8 86 Juvenile_4-17y_19 19.0 28.4 0.7 47.4 87 Juvenile_4-17y_20 13.4 22.5 0.6 35.9 88 Juvenile_4-17y_21 23.9 19.0 1.3 42.9 89 Juvenile_4-17y_22 20.9 39.3 0.5 60.2 90 Juvenile_4-17y_23 14.7 35.3 0.4 50.0 91 Juvenile_4-17y_24 10.3 30.0 0.3 40.3 92 Juvenile_4-17y_25 17.0 21.6 0.8 38.6 93 Juvenile_4-17y_26 18.4 35.6 0.5 54.0 94 Juvenile_4-17y_27 20.2 40.8 0.5 61.0 95 Juvenile_4-17y_28 26.6 38.9 0.7 65.5 96 Juvenile_4-17y_29 14.9 48.0 0.3 62.9 97 Juvenile_4-17y_30 18.2 31.6 0.6 49.8 98 Juvenile_4-17y_31 21.0 30.5 0.7 51.5 99 Juvenile_4-17y_32 25.9 28.0 0.9 53.9 100 Juvenile_4-17y_33 16.1 37.0 0.4 53.1 101 Juvenile_4-17y_34 24.4 27.9 0.9 52.3 102 Juvenile_4-17y_35 19.2 26.7 0.7 45.9 103 Juvenile_4-17y_36 24.7 33.2 0.7 57.9 104 Juvenile_4-17y_37 21.0 31.5 0.7 52.5 105 Juvenile_4-17y_38 19.1 38.4 0.5 57.5 106 Juvenile_4-17y_39 21.4 47.0 0.4 69.4 107 Juvenile_4-17y_40 14.8 22.3 0.7 37.1 108 Juvenile_4-17y_41 10.2 18.0 0.6 28.2 109 Juvenile_4-17y_42 17.2 17.6 1 34.8 110 Adult_01 19.3 46.0 0.4 65.3 111 Adult_02 13.5 33.2 0.4 46.7 112 Adult_03 11.3 26.4 0.4 37.7 113 Adult_04 15.1 38.6 0.4 53.7 114 Adult_05 12.3 32.2 0.4 44.5 115 Adult_06 21.3 44.0 0.5 65.3 116 Adult_07 14.5 48.4 0.3 62.9 117 Adult_08 14.4 31.0 0.5 45.4 118 Adult_09 10.9 29.5 0.4 40.4 119 Adult_10 22.0 45.0 0.6 67.0 120 Adult_11 15.3 37.3 0.4 52.6 121 Adult_12 14.6 44.4 0.3 59.0 122 Adult_13 22.3 35.9 0.6 58.2 123 Adult_14 18.4 24.3 0.8 42.7 124 Adult_15 14.9 21.1 0.7 36.0 125 Adult_16 15.9 28.0 0.6 43.9 126 Adult_17 15.3 25.3 0.6 40.6 127 Adult_18 25.1 35.1 0.7 60.2 128 Adult_19 14.7 42.0 0.3 56.7 129 Adult_20 17.6 28.7 0.6 46.3 130 Adult_21 15.4 24.3 0.6 39.7 131 Adult_22 24.9 39.6 0.6 64.5 132 Adult_23 18.1 26.7 0.7 44.8 133 Adult_24 17.4 34.7 0.5 52.1 134 Adult_25 20.6 31.8 0.6 52.4 135 Adult_26 15.7 28.4 0.6 44.1 136 Adult_27 16.5 29.7 0.6 46.2 137 Adult_28 13.1 34.2 0.4 47.3 138 Adult_29 18.5 27.2 0.7 45.7 139 Adult_30 22.9 36.5 0.6 59.4 140 Adult_31 14.8 28.7 0.5 43.5 141 Adult_32 11.0 34.6 0.3 45.6 142 Adult_33 18.8 30.4 0.6 49.2 143 Adult_34 17.8 36.8 0.5 54.6 144 Adult_35 15.9 40.4 0.4 56.3 145 Adult_36 16.5 43.2 0.4 59.7 146 Adult_37 25.2 36.3 0.7 61.5 147 Adult_38 15.1 24.1 0.6 39.2 148 Adult_39 27.9 36.5 0.8 64.1 149 Adult_40 17.6 32.1 0.5 49.7 150 Adult_41 18.3 21.9 0.8 40.2 151 Adult_42 16.9 25.9 0.7 42.8 152 Adult_43 15.4 19.7 0.8 35.1 153 Adult_44 19.7 27.0 0.7 46.7 154 Adult_45 16.1 28.4 0.6 44.5 155 Adult_46 14.0 28.7 0.5 42.7 156 Adult_47 19.4 25.6 0.8 45.0 157 Adult_48 16.6 32.1 0.5 48.7 158 Adult_49 22.6 34.3 0.7 56.9 159 Adult_50 19.4 37.0 0.5 56.4 160 Adult_51 15.0 32.5 0.5 47.5 161 Adult_52 13.7 31.8 0.4 45.5 162 Adult_53 19.2 23.4 0.8 42.6 163 Adult_54 15.4 26.5 0.6 41.9 164 Adult_55 16.1 18.8 0.9 34.9 165 Adult_56 26.1 27.7 0.9 53.8 166 Adult_57 25.7 31.4 0.8 57.1 167 Adult_58 14.2 25.8 0.5 40.0 168 Adult_59 17.7 32.0 0.6 49.7 169 Adult_60 19.2 32.6 0.6 51.8 170 Adult_61 17.8 28.8 0.6 46.6 171 Adult_62 15.4 32.7 0.5 48.1 172 Adult_63 19.5 32.6 0.6 52.1 173 Adult_64 13.3 28.3 0.5 41.6 174 Adult_65 13.7 31.0 0.4 44.7 175 Adult_66 21.1 32.9 0.6 54.0 176 Adult_67 18.9 33.1 0.6 52.0 177 Adult_68 14.5 32.7 0.4 47.2 178 Adult_69 14.2 21.8 0.7 36.0 179 Adult_70 22.2 22.9 1.0 45.1 180 Adult_71 17.7 31.4 0.6 49.1 181 Adult_72 17.5 30.0 0.6 47.5 182 Adult_73 16.9 33.6 0.5 50.5 183 Adult_74 27.0 40.0 0.7 67.0 184 Adult_75 11.7 24.6 0.5 36.3 185 Adult_76 12.6 28.9 0.4 41.5 186 Adult_77 20.4 39.3 0.5 59.7 187 Adult_78 19.9 28.3 0.7 48.2 188 Adult_79 12.6 40.0 0.3 52.6 189 Adult_80 15.2 24.2 0.6 39.4 190 Adult_81 11.1 27.6 0.4 38.7 191 Adult_82 16.0 24.9 0.6 40.9 192 Adult_83 15.6 25.2 0.6 40.8 minimum 6.9 3.5 0.1 28.1 maximum 27.9 60.7 2.0 69.7 mean 18.3 31.6 0.6 49.9 Standard Deviation 4.7 9.6 Cut-off 27.7 69.1
[0479] The normal distribution of cis-C26 ceramide and total C26 ceramide for healthy controls is shown in
TABLE-US-00003 TABLE 1c C26 ceramide levels in cystic fibrosis patients and carriers C26Cer C26Cer Total Sample Isoform Isoform ra- C26Cer No description 1 nmol/L 2 nmol/L tio nmol/L Cystic Fibrosis - Affected patients 1 Cystic Fibrosis_01 patient 110.7 61.3 1.8 171.9 2 Cystic Fibrosis_02 patient 53.5 34.2 1.6 87.7 3 Cystic Fibrosis_03 patient 82.8 52.7 1.6 135.5 4 Cystic Fibrosis_04 patient 78.7 45.7 1.8 124.4 5 Cystic Fibrosis_05 patient 40.7 58.3 0.7 99.0 6 Cystic Fibrosis_06 patient 55.0 31.2 1.8 86.2 7 Cystic Fibrosis_07 patient 69.7 19.7 3.6 89.5 8 Cystic Fibrosis_08 patient 49.7 31.6 1.6 81.3 9 Cystic Fibrosis_09 patient 38.8 31.8 1.2 70.7 10 Cystic Fibrosis_10 patient 47.6 23.3 2.1 70.9 11 Cystic Fibrosis_11 patient 78.3 47.3 1.7 125.6 12 Cystic Fibrosis_12 patient 57.5 24.9 2.3 82.4 13 Cystic Fibrosis_13 patient 95.4 27.5 3.5 122.9 14 Cystic Fibrosis_14 patient 64.0 63.0 1.0 127.0 15 Cystic Fibrosis_15 patient 86.3 36.4 2.5 122.7 minimum 38.8 19.7 0.7 70.7 maximum 110.7 63.0 3.6 171.9 mean 67.3 39.3 1.9 106.5 Cystic Fibrosis - Carriers 1 CF Carrier_01 carrier 48.4 60.6 0.8 109.0 2 CF Carrier_02 carrier 97.6 22.2 4.5 119.7 3 CF Carrier_03 carrier 15.8 33.8 0.5 49.5 4 CF Carrier_04 carrier 10.9 55.1 0.2 66.1 5 CF Carrier_05 carrier 13.8 33.7 0.4 47.4 6 CF Carrier_06 carrier 28.7 67.9 0.4 96.6 7 CF Carrier_07 carrier 47.3 48.0 1.0 95.4 8 CF Carrier_08 carrier 69.2 49.1 1.8 107.9 9 CF Carrier_09 carrier 73.5 58.2 1.3 131.8 10 CF Carrier_10 carrier 58.0 32.7 1.8 90.7 11 CF Carrier_11 carrier 12.6 51.1 0.3 63.7 12 CF Carrier_12 carrier 94.5 31.8 3.1 126.4 13 CF Carrier_13 carrier 33.9 31.2 1.1 65.1 14 CF Carrier_14 carrier 92.5 43.1 2.2 135.6 15 CF Carrier_15 carrier 10.8 37.4 0.3 48.2 16 CF Carrier_16 carrier 110.5 38.2 2.9 148.7 17 CF Carrier_17 carrier 10.7 37.4 0.3 48.1 18 CF Carrier_18 carrier 13.8 47.9 0.3 61.7 19 CF Carrier_19 carrier 14.6 52.0 0.3 66.6 20 CF Carrier_20 carrier 103.9 64.0 1.7 167.8 21 CF Carrier_21 carrier 12.9 31.3 0.4 44.1 22 CF Carrier_22 carrier 16.4 55.7 0.3 72.2 23 CF Carrier_23 carrier 28.9 35.5 0.8 64.3 24 CF Carrier_24 carrier 36.6 34.1 1.1 70.7 25 CF Carrier_25 carrier 14.6 27.5 0.5 42.0 26 CF Carrier_26 carrier 60.1 41.3 1.5 101.4 27 CF Carrier_27 carrier 66.3 40.0 1.7 106.2 28 CF Carrier_28 carrier 67.1 28.8 2.3 95.9 29 CF Carrier_29 carrier 50.5 5.1 9.8 55.6 30 CF Carrier_30 carrier 89.9 31.8 2.9 121.7 31 CF Carrier_31 carrier 43.1 56.8 0.8 99.8 32 CF Carrier_32 carrier 56.1 56.0 1.0 112.1 33 CF Carrier_33 carrier 12.5 105.1 0.1 117.5 34 CF Carrier_34 carrier 19.4 72.2 0.3 91.6 35 CF Carrier_35 carrier 94.7 37.6 2.5 132.4 36 CF Carrier_36 carrier 102.5 78.4 1.3 181.0 37 CF Carrier_37 carrier 45.2 41.3 1.1 86.5 38 CF Carrier_38 carrier 131.0 40.1 3.3 171.2 39 CF Carrier_39 carrier 51.8 33.6 1.6 85.4 40 CF Carrier_40 carrier 12.0 47.7 0.3 59.8 minimum 10.7 5.1 0.1 42.0 maximum 131.0 105.1 9.8 181.0 mean 49.3 44.9 1.5 93.9
Example 3
Determining the Cut-Off Value for C26 Ceramide for the Diagnosis of Cystic Fibrosis
[0480] Based on the results presented in Examples 1 and 2, the cut-off values for the diagnosis of cystic fibrosis patients and cystic fibrosis carrier based on cis-C26 ceramide and total C26 ceramide were determined. Using dried blood spots, the cut-off value for the diagnosis of cystic fibrosis based on the level of cis-C26 ceramide is 27.7 nmol/L blood of the subject to be tested and, respectively, diagnosed, and the cut-off value for the diagnosis of cystic fibrosis based on the level of total C26 ceramide is 69.1 nmol/L blood of the subject to be tested and, respectively, diagnosed.
[0481] These results are also shown as a whisker plot in
Example 4
Positive Predictive Value and Negative Predictive Value of C26 Ceramide in the Diagnosis of Cystic Fibrosis
[0482] All of the samples described in Example 1 were used for calculating the positive predictive value (PPV) and the negative predictive value (NPV) versus the healthy controls. The results are shown in Table 2.
TABLE-US-00004 TABLE 2 PPV and NPV for C26 ceramide for the diagnosis of cystic fibrosis Patients with Cystic fibrosis (as confirmed on molecular genetics) Condition Condition positive negative C26Cer Test True False Positive Isoform 1 outcome positive positive predictive (cis-C26 positive (TP) = 15 (FP) = 1 value = ceramide) TP/(TP + FP) = 0.937 Test False True Negative outcome negative negative predictive negative (FN) = 0 (TN) = 192 value = TN/(FN + TN) = 1 Sensitivity = Specificity = TP/(TP + TN/(FP + FN) = 1 TN) = 0.995 Patients with cystic fibrosis (as confirmed on molecular genetics) Condition Condition positive negative Total Test True False Positive C26Cer outcome positive positive predictive positive (TP) = 15 (FP) = 2 value = TP/(TP + FP) = Test False True Negative outcome negative negative predictive negative (FN) = 0 (TN) = 192 value = TN/(FN + TN) = 1 Sensitivity = Specificity = TP/(TP + TN/(FP + FN) = 1 TN) = 0.989
[0483] The features of the present invention disclosed in the specification, the claims, the sequence listing and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.