MARKERS FOR ATRIAL FIBRILLATION (AF)

Abstract

The present invention relates to a method for identifying whether or not it may be appropriate to administer to a subject a therapy for alleviating any potential consequences which may arise due to the subject having an atrial fibrillation (AF), the method comprising detecting, in a sample of fluid from the subject, a level of ccl21 and/or ddit4 expression and determining whether or not it may be appropriate to administer to the subject a therapy for alleviating any consequences which may arise due to the subject having AF, based upon the ccl21 and/or ddit4 expression level detected. Also provided are an anticoagulant or other AF therapy and a method of administering an anticoagulant drug or another AF therapy.

Claims

1.-7. (canceled)

8. A method of treating a subject for atrial fibrillation (AF), the method comprising the steps of: identifying a subject who has an atrial fibrillation, wherein identifying the subject comprises detecting a reduced level of ddit4 expression in a sample obtained from the subject, in comparison to a reference level; and administering to the subject an anticoagulant; a rate control therapy; an antiarrhythmic drug; or applying catheter ablation to treat the AF.

9. The method of claim 8, wherein the AF is paroxysmal, persistent or permanent AF.

10. The method of claim 9 wherein the AF is paroxysmal AF.

11. The method of claim 8, wherein the sample is urine, saliva, blood, plasma, serum, sputum, semen, mucus, tears, a vaginal swab, a rectal swab, a cervical smear, a tissue biopsy, or a urethral swab.

12. The method of claim 11 wherein the sample is urine, saliva, blood or sputum.

13. The method of claim 8, wherein detecting a reduced level of ddit4 expression comprises subjecting the sample to an immunoassay, or enzyme-linked immunosorbent assay (ELISA); flow cytometry; electrochemiluminescent assay; or plasmon or surface enhanced resonance assay.

14. The method of claim 8, wherein the immunoassay is a competitive or non-competitive immunoassay, using a solid-phase-bound antibody.

15. An assay system, comprising: a measurement device comprising an antibody that specifically binds ddit4 or a protein fragment thereof and wherein the system is configured to receive a fluid sample from a subject.

16. The assay system of claim 15 further comprising: a data transformation device that acquires a ddit4 expression level from the measurement device and performs data transformation to calculate whether or not the ddit4 expression level is lower than a control, reference or normal value for ddit4 expression in the fluid sample.

17. The assay system of claim 16 further comprising: an output interface device to output data to a user.

18. The assay system of claim 17 further comprising: a database of treatment information, wherein the system identifies treatment information in the database for the level of ddit4 expression determined and outputs the treatment information to the user interface output device.

19. The assay system of claim 17 wherein the user interface output device provides an output to the user, or to the subject, indicating the subject's ddit4 expression level is lower than a control, threshold or reference value and that the user or the subject should administer a therapy suitable for treating atrial fibrillation.

20.-21. (canceled)

22. A method of detecting reduced or elevated ddit4 expression in a subject, the method comprising: (a) obtaining a fluid sample from the subject; and (b) detecting whether the level of expression of ddit4 or a protein fragment thereof is reduced or elevated by contacting the fluid sample with an antibody that specifically binds ddit4 or a protein fragment thereof and detecting binding between ddit4 or the protein fragment thereof and the antibody.

23. The method of claim 22, wherein the fluid sample is urine, saliva, blood or sputum.

24. The method of claim 22, wherein the antibody is a solid phase-bound antibody.

25. The method of claim 22, wherein detecting binding between ddit4 or the protein fragment thereof and the antibody comprises carrying out a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA); flow cytometry; an electrochemiluminescent assay; or a plasmon or surface enhanced resonance assay.

26. The method of claim 22, wherein the antibody is detectably labeled.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention will now be further described by way of example and with reference to the figures which show:

[0040] FIG. 1: Left-right gene expression ratios of “left atrial genes” in atria of wild-type and pitx2+/−mice. All data based on mRNA expression analysis by rtPCR measured in 18 pairs of wt and pitx2+/−mice. This analysis illustrates that all genes show reduced expression in pitx2+/−atria numerically, while ccl21 and ddit4 emerge as the genes with statistically significant differences; shown are mean and SEM;

[0041] FIG. 2: Reduced PITX2, ccl21, and ddit4 mRNA expression in human left and right atria with and without atrial fibrillation. Ccl21 expression is clearly reduced in atria with atrial fibrillation (striped columns, paroxysmal AF; grey columns, chronic AF) compared to those in sinus rhythm (solid black column), confirming observations of pitx2-dependent ccl21 expression in murine left atria shown in FIG. 1. Shown are mean and SEM;

[0042] FIG. 3: a) shows the results of a Western blot of Pitx2 levels in left and right atrial samples from patients with chronic and paroxysmal AF in comparison to patients with normal sinus rhythm; b) shows the results of a) in graphical form;

[0043] FIG. 4: a) shows the results of a Western blot of ddit4 levels in left and right atrial samples from patients with chronic AF in comparison to patients with normal sinus rhythm; b) shows the results of a) in graphical form and further includes the results from patients with paroxysmal AF; and

[0044] FIG. 5: shows that ccl21 plasma levels are significantly lower in left atrial and venous samples from AF patients compared to patients with other forms of cardiac arrhythmias.

MATERIALS AND METHODS

[0045] For mouse, LA and RA from 36 mice were used (18 Pitx2c+/−, 18 littermate wildtype, age 14-20 weeks). For humans, LA and RA tissue from patients in sinus rhythm (n=7) or diagnosed with chronic (n=14) or paroxysmal AF (n=12) was used, all undergoing open heart surgery (CABG or valve surgery, median age 74). RNA was prepared using Qiagen RNeasy fibrous mini-kit and Quiagen QiaShredder Columns; cDNA was generated and RT-PCR reactions were performed using gene specific primers and SYBR Green (Life Technologies) on an ABI 7500 Fast machine.

[0046] Protein lysates were prepared using RIPA protein buffer. Samples were separated by SDS-PAGE (12% resolving/4% stacking) and blotted to membrane. Protein levels were determined by western blotting and normalized to calnexin protein expression.

[0047] Human plasma samples (both left atrial and venous blood) from patients who had undergone catheter ablation (19 with AF, 19 with other forms of cardiac arrhythmias) were used to determine CCL21 levels by ELISA (R&D Systems).

RESULTS

[0048] The earlier work described by Kahr et al, revealed a number of genes which showed preferred expression in left atrial tissue. Comparison of murine gene array data measured in mouse atria from wild type nad pitx2+/−mice suggested that their expression could be regulated by pitx2. However, mRNA levels were not studied in pitx2+/−mouse atria, none of the findings has been validated in human atria, and moreover as the paper highlights, even differences in mRNA concentrations do not always translate into differences in protein concentration. Hence the findings in the Kahr et al paper must be viewed with caution.

[0049] The inventors carried out rtPCR in murine atrial tissue of the 10 genes previously identified in Kahr et al as being differentially expressed between left and right atria and in comparison to pitx2 expression. The results of mouse atrial analyses are shown in FIG. 1. Interesting of the 10 genes studied, only two of the genes, ddit4 and ccl21, were reduced at the mRNA level with a statistically significant difference in human left atrial tissue in AF patients compared to sinus rhythm.

[0050] Furthermore, both ccl21 and ddit4 expression was reduced in human atrial tissue collected from patients with atrial fibrillation compared to those in sinus rhythm. Patients with paroxysmal AF (i.e. with periods of AF alternating with sinus rhythm) showed less reduced ddit4 and ccl21 levels compared to patients in chronic AF (FIG. 2).

[0051] Further work showed that Pitx2c transcripts were more highly expressed in the LA compared to the RA of all patient groups (SR p=0.01; Parox p=0.00; Chronic p=0.00). Whilst there was a general trend towards down-regulation of Pitx2c transcripts in all AF patient samples compared to SR controls, none were significant (see FIG. 3).

[0052] Moreover, Ccl21 was more highly expressed in the LA compared to the RA across all three patient groups (SR p=0.00; Parox p=0.03; Chronic p=0.00). Ddit4l was more highly expressed in the LA compared to the RA in patients with Paroxysmal AF (p=0.01) (see FIG. 4). Furthermore, atrial Ccl21 and Ddit4l protein levels do not differ between patient groups, nor between left and right atria. However, Ccl21 was lower in RA of patients diagnosed with paroxysmal AF compared to those with chronic AF (p=0.05).

[0053] As ccl21 is a secreted chemokine, we did not expect to find a difference in ccl protein levels in atrial tissue, and we could not identify such differences in atrial tissue. Thus, we measured Ccl21 plasma levels in blood. Ccl21 levels were significantly lower in left atrial samples (p=0.004) as well as venous samples (p=0.002) of AF patients (n=19) compared to patients diagnosed with other forms of cardiac arrhythmias (n=19) (see FIG. 5). Moreover, we found that in plasma venous blood ccl21 levels are markedly reduced (61.4 vs. 1.5 pg/ml) in patients with AF compared to controls (FIG. 5).

[0054] In conclusion the present works shows that L-R transcript expression gradients for Ccl21 and Ddit4l are reduced in the LA of Pitx2 heterozygous mice. Pitx2, Ccl21 and Ddit4l are also differentially expressed between the L and RA of the majority of patient samples. Pitx2, Ccl21 and Ddit4l protein levels do not differ between LA and RA tissues samples of patient groups or between patient groups. However Ccl21 plasma levels are significantly lower in patients with AF compared to those with other forms of cardiac arrhythmias. The present data suggest that protein levels of Pitx2, Ccl21 and Dit4l are not reflective of transcript levels. Moreover Ccl21 and ddit4 fluid such as plasma/blood levels could be a marker for patients with atrial fibrillation.