Method for diagnosing muscular dystrophy

Abstract

A method for the diagnosis, prognosis and therapeutic monitoring of muscular dystrophy, by detecting titin or one or more fragments of titin in a bodily fluid is provided.

Claims

1. A method for the detection of titin or a fragment thereof in a subject, comprising detecting the presence of titin or of one or more fragments of titin in a urine sample from said subject, said detecting comprising: a) contacting said urine sample with antibodies that bind to an epitope of titin and detecting the binding of said antibodies to said epitope; or b) specifically quantifying titin or one or more fragments thereof in said urine sample by mass spectroscopy.

2. The method according to claim 1, said method comprising measuring the level of expression of titin or of one or more fragments thereof in a second urine sample from a subject and comparing the level of titin or of said one or more fragments in said sample to the level of titin or of said one or more fragments of titin in a sample previously taken from the same subject.

3. The method according to claim 1, said method comprising: a) measuring the level of expression of titin or of one or more of its fragments in a urine sample, by means of which a reference level is determined (control level); and b) measuring the level of expression of said titin or of said fragment(s) in a second urine sample taken from the same subject at a time after the administration of a therapeutic treatment (test level); and c) comparing the control level and the test level.

4. The method according to claim 1, wherein the antibody binds to an epitope of the N-terminal titin fragment of approximately 300 amino acids or an epitope of the C-terminal titin fragment of approximately 150 amino acids.

5. A diagnostic kit comprising an antibody for detecting titin and one or more fragments of titin selected from SEQ ID NOs: 12-30 or a fragment between amino acids 12132 and 15880 of SEQ ID NO: 1 or SEQ ID NOs: 31-45 and instructions for implementing the method according to claim 1.

6. The method according to claim 1, wherein said method comprises quantifying titin or one or more fragments thereof in said urine sample by mass spectroscopy.

7. The method according to claim 6, wherein said mass spectroscopy is multiple reaction monitoring mass spectroscopy or MALDI-TOF/TOF mass spectroscopy.

8. The method according to claim 7, wherein said mass spectroscopy is multiple reaction monitoring mass spectroscopy.

9. The method according to claim 7, wherein said mass spectroscopy is MALDI-TOF/TOF mass spectroscopy.

Description

LEGEND OF THE FIGURES

(1) FIG. 1. Identification by two-dimensional electrophoresis of an N-terminal titin fragment in DMD urine.

(2) FIG. 2. Western blot analysis of the presence of the N-terminal titin fragment in urine from DMD and healthy patients.

(3) FIG. 3. Western blot analysis of the presence of the N-terminal titin fragment in urine from other muscular dystrophies.

(4) FIG. 4. Detection of peptides from the various parts of titin in urine from DMD patients, using two-dimensional electrophoresis and LC-MS/MS approaches. Position of the peptides in the titin sequence and the corresponding sequence identifier are as follows: 42-62 (SEQ ID NO:12), 82-98 (SEQ ID NO:13), 99-109 (SEQ ID NO:14), 110-116 (SEQ ID NO:15), 127-136 (SEQ ID NO:16), 932-950 (SEQ ID NO:17), 1004-1012 (SEQ ID NO:18), 11784-11797 (SEQ ID NO:19), 14245-14257 (SEQ ID NO:20), 34253-34271 (SEQ ID NO:21), 34258-34271 (SEQ ID NO:22) and 34272-34295 (SEQ ID NO:23).

(5) FIG. 5. Western blot analysis of the presence of the N-terminal titin fragment in urine from GRMD and healthy dogs.

(6) FIG. 6. Detection of peptides from the various parts of titin in urine from DMD patients, using LC-MS/MS approaches. Position of the peptides in the titin sequence and the corresponding sequence identifier are as follows: 42-62 (SEQ ID NO:12), 82-98 (SEQ ID NO:13), 99-109 (SEQ ID NO:14), 127-136 (SEQ ID NO:16), 185-202 (SEQ ID NO:24), 932-950 (SEQ ID NO: 24), 11958-11982 (SEQ ID NO:25), 16783-16809 (SEQ ID NO:26), 33320-33333 (SEQ ID NO:27), 34097-34111 (SEQ ID NO:28), 34253-34271 (SEQ ID NO:21), 34258-34271 (SEQ ID NO:22), 34272-34294 (SEQ ID NO:29), 34272-34295 (SEQ ID NO:23) and 34304-34322 (SEQ ID NO:30).

(7) FIG. 7. Western blot analysis of the presence of the N-terminal titin fragment in urine from mdx and healthy mice.

(8) FIG. 8. Western blot analysis of the presence of the C-terminal titin fragment in urine from DMD patients, GRMD dogs and mdx mice and their respective healthy controls.

(9) FIG. 9. Western blot analysis of the presence of the N-terminal or C-terminal titin fragment in urine and sera from mdx and healthy mice having undergone physical exercise.

(10) FIG. 10. Identification, using the MALDI-TOF/TOF approach, of an intermediate titin fragment in sera from mdx mice having undergone physical exercise. Position of the peptides in the titin sequence and the corresponding sequence identifier are as follows: 12994-13002 (SEQ ID NO:31), 13352-13362 (SEQ ID NO:32), 13542-13558 (SEQ ID NO:33), 14512-14524 (SEQ ID NO:34), 14867-14883 (SEQ ID NO:35), 14975-14993 (SEQ ID NO:36), 15034-15053 (SEQ ID NO:37), 15203-15214 (SEQ ID NO:38), 15234-15248 (SEQ ID NO:39), 15636-15645 (SEQ ID NO:40), 15746-15759 (SEQ ID NO:41), 15968-15987 (SEQ ID NO:42), 16034-16048 (SEQ ID NO:43), 16133-16148 (SEQ ID NO:44) and 16432-16442 (SEQ ID NO:45).

EXAMPLES

(11) Materials and Methods

(12) Protein Analysis by Two-Dimensional Electrophoresis

(13) Proteins obtained from the urine of three DMD patients and three healthy subjects were compared by two-dimensional electrophoresis. Isoelectric focusing was carried out on an immobilized pH gradient gel (IPG Strip pH 3-10, Bio-Rad, referred to as the strip). Passive rehydration of the gel was carried out in the presence of the sample solubilized in denaturing buffer (7 M urea, 2 M thiourea, 20 mM DTT, 2% CHAPS, 1% ASB-14, 1% TRITON). Migration was carried out according to the following program: 50 V for 5 h, linear increase to 4000 V over the course of 6 h, then 8000 V for a total of 30,000 Vh. The strip was then equilibrated with 3 ml of buffer (0.375 M Tris-HCl, pH 8.8, 6 M urea, 20% glycerol, 2% SDS, 20 mM DTT) for 10 minutes. The equilibration step was repeated twice. For the second dimension, the strips were transferred to a linear gradient acrylamide gel (Criterion TGX 4-20% Precast Gel, Bio-Rad). Electrophoresis was carried out at 50 V for 30 minutes, then 140 V for 1 h 30 in 1TGS buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). The proteins were then stained for 1 hour in a Coomassie blue solution (InstantBlue, Expedeon) and destained by means of successive water baths.

(14) Identification by MALDI-TOF/TOF Mass Spectrometry of the Proteins Separated by Two-Dimensional Electrophoresis

(15) Digestion of the Proteins Separated by Two-Dimensional Electrophoresis

(16) The spots of interest were extracted manually and placed in 1.5 ml Eppendorf tubes. They were washed successively with 500 l of ethanol for 10 minutes, 500 l of water for 5 minutes and 500 l of ethanol for 10 minutes. After removal of the ethanol, digestion was carried out overnight at 37 C. in the presence of 30 ng of porcine trypsin (Promega) per spot in 10 l of NH.sub.4CO.sub.3 buffer, pH 7.9. The peptides were desalted/concentrated on ZipTip C18 tips (Millipore) then washed with 1% formic acid. They were then eluted in 1 l of solution containing 3 mg/ml -cyano-4-hydroxycinnamic acid (CHCA) matrix, 80% acetonitrile and 1% formic acid in order to be analyzed by MALDI-TOF/TOF mass spectrometry.

(17) MALDI-TOF/TOF Mass Spectrometry Analysis

(18) The system used is a MALDI-TOF/TOF ABI 4800+ system (Applied Biosystems, Foster City, Calif.) coupled with a 200 Hz YAG laser (355 nm). The spectra were acquired and the data processed using the 4000 Series Explorer software (version 3.5.1, Applied Biosystems). All the MS and MS/MS spectra were submitted to an in-house Mascot server (Matrix Science, Boston, Mass.) using the Swiss-Prot database. The mass tolerance of the fragment ions was fixed at 100 ppm for the precursor ions and 0.3 daltons for the fragment ions.

(19) Identification of Total Proteins by LC-MS/MS Mass Spectrometry

(20) Digestion of the Proteins in Solution

(21) All the urine or blood (serum) proteins underwent enzymatic digestion followed by LC-MS/MS analysis. Protein digestion was carried out with two enzymes successively: endoproteinase Lys-C and trypsin. For this purpose, 50 g of proteins were solubilized in a 50 l reaction volume containing 50 mM Tris-HCl, pH 8.3, 6 M urea and 2 M thiourea. The proteins were reduced with 1 l of 500 mM DTT (final concentration: 10 mM) at room temperature for 30 minutes. Then the proteins were alkylated with 6 l of 550 mM iodoacetamide (final concentration: 55 mM) for 20 minutes. The proteins were digested with 5 l of endoproteinase Lys-C (1 g) for 3 hours at room temperature in darkness. The mixture was diluted 4 times with 3 volumes of MQ water (195 l for a final volume of 260 l; final concentration of urea/thiourea: 1.5 M/0.5 M) and the proteins were digested by 10 l of trypsin (1 g) for 16 hours at room temperature in darkness. Digestion was ended by adding 8.4 l of 100% formic acid (final concentration: 3%).

(22) LC-MS/MS Mass Spectrometry Analysis

(23) The peptides were analyzed by liquid chromatography coupled to a tandem mass spectrometer (LC-MS/MS). The system used was an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, San Jose, Calif.) coupled to an Easy nano-LC Proxeon chromatography system (Thermo Fisher Scientific, San Jose, Calif.). The peptides in solution were desalted/concentrated in ZipTip C18 tips (Millipore), then washed with 1% formic acid. They were then eluted by a 50% acetonitrile and 0.1% formic acid solution, dried, then taken up again in 0.1% formic acid. Chromatographic separation of the peptides was carried out with the following parameters: C18 Proxeon Easy Column (10 cm, 75 m i.d, 120 ), with a flow rate of 300 nl/min progressing from 95% solvent A (water0.1% formic acid) to 25% solvent B (100% acetonitrile, 0.1% formic acid) in 20 minutes, then to 45% B in 40 min and finally to 80% B in 10 min. The peptides were analyzed in the Orbitrap in full ion scan mode with a resolution of 30,000 and a mass range from 300 to 2000 m/z. The fragments were obtained by collision-induced dissociation (CID) with a collision activation energy of 40% and were analyzed in the LTQ. The MS/MS data were acquired in a mode in which the 20 most intense precursor ions were isolated, with a dynamic exclusion of 15 seconds. The data were processed with the Proteome Discoverer 1.3 software (Thermo Fisher Scientific, San Jose, Calif.) coupled to an in-house Mascot search server (Matrix Science, Boston, Mass.; version 2.3.2). The mass tolerance of the fragment ions was fixed at 10 ppm for the precursor ions and 0.6 daltons for the fragments. Oxidation (M) and carbamidomethylation (C) were considered as possible variable modifications. The maximum number of cleavage errors was limited to two for trypsin digestion. The MS-MS data were analyzed with reference to the Swiss-Prot database.

(24) Western Blot Protein Analysis

(25) The samples of interest were loaded on a 1D SDS-PAGE 4-12% Bis-tris gel (Invitrogen), and migration was carried out for 30 minutes at 50 volts, then 1 hour 40 at 140 volts in 1MOPS buffer (Invitrogen). Then the proteins were transferred onto a PVDF membrane (Millipore) for 2 hours at 40 volts. The membrane was saturated in Odyssey buffer (LI-COR Biosciences) overnight at 4 C. and incubated with the primary antibody for 1 hour at room temperature with agitation, followed by three 10-minute washes with PBS-0.1% TWEEN. Visualizing was carried out by incubating the membrane with a secondary antibody, labeled with a fluorophore emitting in the infrared range at 800 nm, for 1 hour at room temperature with agitation, away from light, followed by three 10-minute PBS-0.1% TWEEN washes, then 2 PBS washes. The fluorescent signal was revealed by scanning the membrane with the Odyssey imager (LI-COR Biosciences).

(26) Primary antibody (1): Mouse anti-titin monoclonal IgG at a concentration of 1 g/ml (#H00007273-M07 clone 2F12, from Abnova, directed against the N-terminal part (amino acids 1 to 111))

(27) Primary antibody (2): Rabbit anti-titin monoclonal IgG diluted to 1/300th directed against the C-terminal part (sequence: NEFGSDSATVNINIRSMC SEQ ID NO: 46); amino acids 35197 to 35213 in the mouse corresponding to amino acids 34334 to 34349 in humans) (Charton et al., 2010)

(28) Secondary antibody (1): Goat anti-mouse polyclonal antibody at a concentration of 0.1 g/ml (IRDye 800CW Goat anti-mouse, LI-COR Biosciences)

(29) Secondary antibody (2): Goat anti-rabbit polyclonal antibody at a concentration of 0.1 g/ml (IRDye 800CW Goat anti-rabbit, LI-COR Biosciences)

(30) Physical Exercise in Mice

(31) Two groups of 6 male mice (healthy and mdx) were placed on a treadmill inclined downwards (15) so as to perform a session of running for 30 minutes (8 m/min for 5 minutes, then 12 m/min for 25 minutes). The urine and sera of the mice were collected 7 days before exercise and also 3 h, 24 h and 48 h after exercise.

(32) Moreover, since the mdx mice could not perform physical exercise lasting longer than 30 mins, a group of 6 healthy male mice underwent the same exercise but for 1 h 30 (8 m/min for 5 minutes, then 12 m/min for 1 h 25 minutes). The urine and sera of the mice were collected 7 days before exercise and also 3 h, 24 h and 48 h after exercise.

(33) Results

(34) The proteins were separated as described in Materials and Methods. FIG. 1 corresponds to a representative gel from the experiment. The top gel shows the results from separating the proteins from a healthy subject, and the bottom gel the proteins from a DMD individual. The framed spot was identified by MALDI-TOF/TOF mass spectrometry as an N-terminal titin fragment. This fragment was only detected in the urine from DMD patients. The results are representative for three different DMD patients and three different healthy subjects.

(35) The proteins were analyzed by Western blot as described in Materials and Methods. FIG. 2 corresponds to a representative result in which panel (A) corresponds to the results from the analysis of the DMD patients and (B) the results from the healthy subjects. The N-terminal titin fragment (marked as Titin on the Western blot) was detected in 11 out of 12 DMD patients, and was undetectable in the 10 healthy subjects analyzed. C negative control: urine from the healthy subject previously analyzed by two-dimensional electrophoresis; C+ positive control: urine from the DMD patient analyzed by two-dimensional electrophoresis.

(36) In total, 23 DMD patients were tested using this method and the N-terminal titin fragment was detected in 21 of them. This fragment was absent in the urine from healthy individuals (13 subjects tested).

(37) The proteins were analyzed by Western blot as described in Materials and Methods. FIG. 3 shows the results from the analysis of urine from patients suffering from the following muscular dystrophies: Duchenne muscular dystrophy (DMD); a distinctive case of DMD (patient walking at the age of 21) (special DMD); -sarcoglycanopathy (-Sarco); and Becker muscular dystrophy (Becker). The N-terminal titin fragment (marked as Titin on the Western blot) was detected in the distinctive case of DMD, in those patients suffering from -sarcoglycanopathy (2 out of 2) and in one patient suffering from Becker muscular dystrophy (1 out of 2). C negative control: urine sample from the healthy subject analyzed by two-dimensional electrophoresis; C+ positive control: urine sample from the DMD patient analyzed by two-dimensional electrophoresis.

(38) The peptides obtained from the digestion of proteins from urine or blood of DMD patients and healthy patients were analyzed by the two-dimensional electrophoresis and LC-MS/MS approaches as described in Materials and Methods. The peptides sequenced by MS/MS are shown in FIG. 4. By means of the two-dimensional electrophoresis approach, the three peptides of the N-terminal part of titin were identified in the three DMD patients. A pool of 13 urine samples from DMD patients was also analyzed by the LC-MS/MS approach (three different analyses). The same peptides from the N-terminal part of titin and also peptides from other parts of titin, including the C-terminal part (after amino acid 34253), were identified in these analyses. No titin peptide was identified in the urine from healthy subjects. These results show that other parts of titin can be used as biomarkers. The analysis of the blood (serum) from 4 DMD patients using the LC-MS/MS approach demonstrated the presence of peptide 82-98 in the serum of just one patient. No titin peptide was identified in the sera of 6 healthy subjects tested using this same approach (data not shown).

(39) The proteins were analyzed by western blot as described in Materials and Methods. The N-terminal titin fragment (marked as Titin on the Western blot) was detected in 2 out of 2 GRMD dogs, and was undetectable in the 2 healthy dogs analyzed (FIG. 5). C negative control: urine sample from the healthy subject analyzed by two-dimensional electrophoresis; C+ positive control: urine sample from the DMD patient analyzed by two-dimensional electrophoresis.

(40) Supplementary Studies

(41) The peptides obtained from the digestion of proteins from urine of DMD patients and healthy patients were analyzed using the LC-MS/MS approach as described in Materials and Methods. Urine from 5 healthy individuals and 5 DMD patients was analyzed individually using the LC-MS/MS approach. The individual analysis enabled the detection of more titin peptides than in the pooled analysis as described previously and also demonstrated that a majority of the peptides identified corresponded to the N-terminal and C-terminal parts of titin. The peptides sequenced by MS/MS are shown in FIG. 6 with the identification score for each DMD patient.

(42) The proteins were analyzed by Western blot as described in Materials and Methods. The N-terminal titin fragment (marked as Titin on the Western blot) was detected in 2 out of 2 mdx mice, and was undetectable in the 2 healthy mice analyzed (FIG. 7A). Several non-specific bands can be seen and are linked to the secondary antibody (FIG. 7B). C+ positive control: urine sample from the DMD patient analyzed by two-dimensional electrophoresis.

(43) The proteins were analyzed by Western blot as described in Materials and Methods. The C-terminal titin fragment (marked as Titin on the Western blot) was detected in 5 out of 5 DMD patients, and was undetectable in the 4 healthy subjects analyzed (FIG. 8A). The C-terminal titin fragment (marked as Titin on the Western blot) was detected in 2 out of 2 dogs, and was undetectable in the 2 healthy dogs analyzed (FIG. 8B). The C-terminal titin fragment (marked as Titin on the Western blot) was detected in 2 out of 2 mdx mice, and was undetectable in the 2 healthy mice analyzed (FIG. 8C). C+ positive control: urine sample from the DMD patient analyzed by two-dimensional electrophoresis.

(44) The proteins were analyzed by Western blot as described in Materials and Methods. The N-terminal titin fragment (marked as N-ter titin on the Western blot) was detected in all the mdx mice which had undergone physical exercise, with an increase 3 h after exercise (FIG. 9A). Moreover, the C-terminal titin fragment was only detected in the serum of mdx mice 3 h after exercise. This fragment was undetectable in the human control and in the sera of mice which did not undergo physical exercise (FIG. 9B). (NB: detecting the N-terminal titin fragment in the serum of mdx mice is not possible owing to the presence of only one primary antibody on the market (mouse anti-N-terminal titin) and hence the obligation to use an anti-mouse secondary antibody on mouse serum.) Moreover, the N-terminal titin fragment is still undetectable in the urine of healthy mice having undergone physical exercise for 30 minutes, or even for 1 h 30 min (FIG. 9C-D). C human: human negative control: urine sample from a healthy subject; C+ human: human positive control: urine or serum sample from a DMD patient; C+ mouse: mouse positive control: urine sample from mdx mouse.

(45) The serum proteins of mdx mice having undergone physical for exercise 30 min were separated on a 1D SDS-PAGE gel as described in Materials and Methods. The bands of interest (bands framed in FIG. 10A) were extracted manually and identified by MALDI-TOF/TOF mass spectrometry as an intermediate titin fragment (from amino acids 12994 to 16442; corresponding to amino acids 12132 to 15580 in the human sequence). This fragment was only detected in the sera of mdx mice having undergone physical exercise for 30 min and was undetected in healthy mice having undergone physical exercise for 30 min or even for 1 h 30 min (data not shown). The peptides sequenced by MALDI-TOF/TOF are shown in FIG. 10B with the identification score for each mdx mouse. Moreover, the size of this intermediate fragment (3000 amino acids, i.e., 300 kDa) identified by MALDI-TOF/TOF mass spectrometry corresponds to the size observed on the SDS-PAGE gels (300 kDa).

(46) In summary, all these experiments demonstrate that several fragments of titin (for example: N-terminal, C-terminal or intermediate) are detected in both the serum and the urine of DMD patients, GRMD dogs, and mdx mice, and are undetected in the serum or the urine of healthy subjects, healthy dogs and healthy mice.

REFERENCES

(47) Batchelor, C. L. and S. J. Winder (2006). Sparks, signals and shock absorbers: how dystrophin loss causes muscular dystrophy. Trends Cell Biol 16(4): 198-205. Bushby, K., R. Finkel, et al. Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management. Lancet Neurol 9(1): 77-93. Charton, K., Danile N., et al. (2010). Removal of the calpain 3 protease reverses the myopathology in a mouse model for titinopathies. Hum Mol Genet 19(23): 4608-24 Cirak, S., V. Arechavala-Gomeza, et al. (2011). Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet 378(9791): 595-605. Le Rumeur, E., S. J. Winder, et al. Dystrophin: more than just the sum of its parts. Biochim Biophys Acta 1804(9): 1713-22. Lu, Q. L., T. Yokota, et al. The status of exon skipping as a therapeutic approach to duchenne muscular dystrophy. Mol Ther 19(1): 9-15. Muntoni, F., S. Torelli, et al. (2003). Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol 2(12): 731-40. Nicholson, G. A., G. J. Morgan, et al. (1986). The effect of aerobic exercise on serum creatine kinase activities. Muscle Nerve 9(9): 820-4.