The present invention relates to methods of screening for inflammatory bowel disease. More specifically, this invention relates to microsatellite alleles in the tumor necrosis factor locus of human chromosome 6 which have been found to be associated with Crohn""s disease.
A. Inflammatory Bowel Disease
Inflammatory Bowel Disease (xe2x80x9cIBDxe2x80x9d) is the collective term used to describe two chronic, idiopathic inflammatory diseases of the gastrointestinal tract: ulcerative colitis (xe2x80x9cUCxe2x80x9d) and Crohn""s disease (xe2x80x9cCDxe2x80x9d). UC and CD are considered together because of their overlapping clinical, etiologic, and pathogenetic features. From a therapeutic and prognostic standpoint, however, it is important to distinguish them from one another as well as from non-chronic inflammatory diseases of the bowel.
IBD occurs world-wide and is reported to afflict as many as two million people. Onset has been documented at all ages; however, IBD predominately begins in young adulthood. The three most common presenting symptoms of IBD are diarrhea, abdominal pain, and fever. The diarrhea may range from mild to severe and is often accompanied by urgency and frequency. In UC, the diarrhea is usually bloody and may contain mucus and purulent matter as well. Anemia and weight loss are additional common signs of IBD. Reports of an increasing occurrence of psychological problems, including anxiety and depression, are perhaps not surprising secondary effects of what is often a debilitating disease that occurs in people in the prime of life.
B. The Cause(s) of IBD are Unknown
Although the cause(s) of UC and CD is not known, there is general agreement that genetics is important in a person""s susceptibility to IBD and that the immune system is responsible for mediating the tissue damage in these diseases. Generally speaking, a failure to down regulate the normal self-limited inflammatory response of the bowel is characteristic of IBD. While a wide range of immunologic abnormalities have been reported in these disorders, none has yet been sufficiently reliable to be of diagnostic value. For example, the production of TNF-xcex1 by macrophages and T cells of IBD patients is a point of controversy. Although it has been suggested that patients with IBD, particularly CD, exhibit elevated TNF-xcex1 protein production and gene expression in the cells of the mucosa, others have not been able to document such a phenomena. Therefore, the diagnostic value of assaying for TNF-xcex1 protein production or gene expression is uncertain. Moreover, a suggestion that this immunologic abnormality has a genetic determinant which would be of value in the diagnosis of CD is necessarily speculative in nature.
C. Methods of Diagnosing IBD
A battery of laboratory, radiological, and endoscopic evaluations are combined to derive a diagnosis of IBD and to assess the extent and severity of the disease. Nevertheless, differentiating UC from CD, as well as other types of inflammatory conditions of the bowel, such as irritable bowel syndrome, infectious diarrhea, rectal bleeding, radiation colitis, and the like, is difficult, because the mucosa of the small and large intestines reacts in a similar way to a large number of different insults. Once infectious-types of bowel disorders have been ruled out, the final diagnosis of IBD is often made on the basis of the progression of the disease. In many patients, though, the diagnosis of IBD must still be regarded as indeterminate because of the overlapping features of UC and CD, particularly with CD of the colon.
The leading early symptoms of UC and CD are chronic recurrent diarrhea, bloody diarrhea, recurrent abdominal pain, nausea, weight loss general evidence of inflammation without any obvious explanation (fever, raised ESR, leucocytosis, thrombocytosis and dysproteinenemia or anemia). Among these symptoms, diarrhea and anemia are more characteristic of UC while pain, weight loss and marked evidence of inflammation are more common in CD. While the history and physical examination of a patient can help, the final confirmation of the diagnosis has traditionally been made endoscopically, histologically and, in relation to the small intestine, radiologically as well.
An endoscopic examination of the bowel can reveal important changes in mucosal appearances which can aid the physician in diagnosing IBD.
Unlike CD, UC is a disease of the mucosa and is confined to the large intestine. UC usually begins in the rectum, although it may involve the entire colon at the time of presentation. When UC spreads, it spreads proximally and continuously, without skipping areas. Hence, it is important to take multiple biopsy specimens from different sites of involved and apparent uninvolved mucosa. In some patients, UC remains localized to the rectum or to the left side of the colon.
The mucosa in acutely active UC appears to be hyperemic, granular and friable, while CD shows lymphoid follicles, aphthoid lesions and flat ulcers. Despite its name, inflammation rather than ulceration is the cardinal feature of UC. Ulcerations may or may not be present in UC. Occasionally, inflammation and ulceration vary in severity in different parts of the colon, including the rectum, giving the false impression of skip areas and rectal sparing, the latter of which are features of CD and are of diagnostic importance for that disorder.
The mucosa of CD exhibits patchy involvement with edema, hyperemia, and ulcerations. The ulceration is a prominent feature of CD. Both superficial and deep undermining or cleft-like ulcers occur. They may be linear or serpiginous. Occasionally, the combination of edema with ulcerations creates a cobblestone appearance that is seen radiologically and endoscopically. Inflammatory polyps, as in UC, may occur.
The cardinal histological features in UC include vascular congestion, edema, goblet cell mucin depletion, crypt abscess formation, and inflammatory cell infiltration of the lamina propria. Crypt abscesses are collections of neutrophils that invade the crypt epithelium and accumulate within the lumen of the crypts. Ulcerations, if they occur, are superficial and only become penetrating to the propria muscularis when the disease is fulminant and acute toxic dilatation of the colon occurs.
Histology in CD shows the characteristic findings of granuloma formation with epithelioid and giant cells. However, these features are found in only 20-40% of biopsies. Transmural inflammation is also typical of CD, and even more typical is its disproportionate distribution (submucosa greater than mucosa). The mucosa shows infiltration by granulocytes with preservation of normal numbers of goblet cells. Lymphocytes and plasma cells are found in the lamina propria, and lymphoid aggregates are present. Lastly, aphthoid lesions are a typical histological feature in the early stages.
The presence of anti-neutrophil cytoplasmic antibodies (xe2x80x9cANCAsxe2x80x9d) can easily be detected in a blood sample, for example, by immunofluorescence assay or a fixed neutrophil ELISA as detailed in Saxon, et al., J. Allergy Clin. Immunol., Vol. 86 No. 2, pp. 202-210 (1990) and incorporated herein by reference. The prevalence of positive ANCA in patients with UC ranges from about 50 to 86%. This UC-associated ANCA has perinuclear immunofluorescence staining pattern which is different from other ANCAs. Moreover, the presence of ANCA is highly specific for UC compared with other forms of colitis. Although a proportion of CD exhibit ANCA, it is at a much lower titre than UC.
Thus the ANCA status of a patient (positive indicating UC and negative indicating CD) is another factor that aids the physician in the diagnosis of IBD. ANCAs also have an increased frequency among the clinically healthy relatives of UC patients compared with environmental and ethnically matched controls. Therefore, ANCA status, in combination with family history of IBD, has also aided physicians in predicting a subject""s susceptibility to IBD.
D. Need for Objective Diagnostic Tools
To date most of the diagnostic tools for UC and CD, with the exception of ANCA status, are quite subjective. Diagnosis depends upon a host of procedures aimed at confirming the suspected diagnosis. The initial symptoms are often confused for non-chronic bowel disorders by physicians unfamiliar with IBD. Consequently, IBD often goes mistreated and undiagnosed until the disease shows its chronicity which results in referral of the patient to a specialist. The imprecise and subjective nature of endoscopic and radiologic examination can result in a misdiagnosis between UC and CD or indeterminate diagnosis even when the IBD is suspected.
Histological examination does provide greater certainty of an accurate diagnosis, but the problems of differentiating between the two diseases based on the histological findings are often underestimated. There is no single histological criterion which is proof of one or the other disease. The epithelial cell granuloma for example, which is often accorded a key role in the diagnosis of CD is only to be found in about 20% of biopsy specimens from such patients. They can also occur in other diseases. Unfortunately, the patient must often suffer as the disease progresses before a definitive diagnosis can be made.
The selective identification of CD as opposed to UC or other inflammatory conditions of the intestines carries important prognostic and therapeutic implications. For example, when colectomy is indicated, the type of IBD involved determines which surgical options are appropriate. Surgery (total colectomy) does represent a cure in UC, though a dramatic one. In CD, surgery is never curative. Continent procedures such as the ileorectal pull-through (mucosal proctectomy) or the Kock pouch may be desirable in UC, but are contraindicated in CD.
The availability of a genetic marker that would readily distinguish CD from UC and/or non-chronic inflammatory diseases of the bowel, independent of or in combination with existing diagnostic tools, would represent a major clinical advance which would aid in therapeutic management of IBD and the design of more specific treatment modalities. Accordingly, there has existed a need for convenient and reliable methods of screening for CD and distinguishing CD from UC for diagnostic, prognostic and therapeutic purposes.
A novel association between Crohn""s disease (xe2x80x9cCDxe2x80x9d) and the presence of specific microsatellite alleles at the tumor necrosis factor (xe2x80x9cTNFxe2x80x9d) locus of human chromosome 6 has. been discovered. This association provides the basis for convenient and reliable methods of screening for CD and distinguishing CD from ulcerative colitis (xe2x80x9cUCxe2x80x9d).
In accordance with the present invention, there is provided methods of screening for CD comprising detecting the presence or absence of nucleic acid of a subject encoding TNF microsatellite alleles associated with CD, wherein the presence of nucleic acid encoding two or more of the alleles is indicative of CD. TNF microsatellite allele associated with CD include the a2, b1, c2, d4 and e1 TNF microsatellite alleles. Preferably, CD is indicated by the detection of nucleic acid encoding TNF microsatellite alleles associated with CD including the a2, b1, and c2 TNF microsatellite alleles. More preferably, nucleic acid is detected which also encodes the d4 or e1 TNF microsatellite alleles, or both.
Nucleic acid encoding TNF microsatellite alleles associated with CD may be detected in accordance with the present invention by amplifying the nucleic acid and identifying the TNF microsatellite alleles. TNF microsatellite allele may be identified by assaying nucleic acid of a subject for defining characteristics of TNF microsatellite alleles associated with CD and comparing the results to a positive and or negative control. Defining characteristics of nucleic acid encoding TNF microsatellite allele associated with CD include, for example, size, sequence, type of sequence repeats, and the like. Primers suitable for use in amplifying nucleic acid encoding TNF microsatellite alleles are provided herein.
Also provided are methods of screening for CD which comprise determining the TNF microsatellite alleles encoded by nucleic acid of a subject at TNF microsatellite loci selected from the group consisting of a, b, c, d and e, and identifying CD by the presence of three or more TNF microsatellite alleles selected from the group consisting of a2, b1, c2, d4 and e1.
Kits for screening for CD and for distinguishing CD from UC are also described which comprise nucleic acid encoding TNF microsatellite alleles associated with CD, for example, the a2, b1, c2, d4 and e1 TNF microsatellite alleles. Kits of the present invention may also include reagents, primers, sequencing markers, positive and negative controls and the like, which are useful in the practice of the present invention.