Method of diagnosing predisposition for ulcerative colitis in Jewish population by detection of interleukin-1 receptor antagonist polymorphism

A novel, differential association between allele 2 of the variable number of tandem repeats polymorphism at intron 2 of the human IL-1 receptor antagonist gene and ulcerative colitis in humans of Jewish ancestry has been discovered. In accordance with the present invention, there is provided methods of screening for ulcerative colitis in human subjects of Jewish ancestry comprising detecting the presence or absence of nucleic acid of the subject encoding allele 2 of the variable number of tandem repeats polymorphism at intron 2 of the human IL-1 receptor antagonist gene, wherein the presence of nucleic acid encoding allele 2 is indicative of ulcerative colitis. Kits useful for screening for ulcerative colitis in human subjects of Jewish ancestry are also provided.

BACKGROUND OF THE INVENTION 
A. Inflammatory Bowel Disease 
Inflammatory bowel disease (IBD) is the collective term used to describe 
two chronic, idiopathic gastrointestinal disorders: ulcerative colitis 
("UC") and Crohn's disease ("CD"). Although the diseases have distinct 
pathophysiological characteristics, they are frequently considered 
together due to several clinical and therapeutic similarities. Several 
other types of inflammatory conditions of the bowel having known 
infectious, toxic or ischemic etiology, such as irritable bowel syndrome, 
infectious diarrhea, rectal bleeding, radiation colitis, and the like, may 
mimic IBD acutely, because the mucosa of the small and large intestines 
reacts in a similar way to a large number of different insults. However, 
if the disease progression is monitored over time, they can be 
distinguished from IBD by their failure to cause a chronic relapsing and 
remitting syndrome. 
IBD occurs world-wide and is reported to afflict as many as two million 
people. The course and prognosis of IBD is widely variable. 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. Ten to fifteen percent 
of all patients with IBD will require surgery over a 10-year period. The 
risk for the development of cancer is increased in patients with IBD as 
well, particularly in those with UC. The longer the duration of disease, 
the higher the risk of developing carcinoma. Patients with UC regularly 
undergo cancer surveillance by endoscopy after ten years of disease. 
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 etiology of IBD is unknown, a number of studies have suggested 
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, 
but it remains unclear what initiates the pathogenic processes and how it 
may differ, if at all, in UC and CD. 
It has also been suggested that a primary abnormality of the immune system 
and its regulation might serve as primary initiating factors, or that the 
disease process might be initiated by an infectious agent and the injury 
is then perpetuated through immune-mediated or other processes. Although 
the mucosal injury observed during episodes of acute disease can resemble 
the effects of any of a number of recognized infectious agent, no 
transmissible infectious agent has been consistently identified with IBD. 
Autoimmunity has also been suggested in the pathogenesis of IBD. Evidence 
to suggest this hypothesis is based on the existence of circulating 
antibodies that react with unknown alimentary tract antigens of both human 
and animal origin. For example, human fetal and adult colonic, biliary, 
skin and vascular epithelial cells, epithelial cell associated components 
from murine small intestine, rat and human colonic epithelial 
glycoproteins, intestinal bacterial polysaccharide, and antigens from 
germ-free rat feces have been described to react with sera from patients 
with IBD. Other studies demonstrated an increased local IgG response in 
the colonic mucosa of patients with IBD and other colonic inflammations. 
The mechanism of this IgG response, the specific local antigens involved, 
and the role of these antibodies are unknown. 
C. Need for Objective Diagnostic Tools 
Inflammatory bowel disease poses a clinical and scientific challenge to 
physicians and researchers. To date most of the diagnostic tools for IBD 
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, because the mucosa of the small and large intestines reacts in a 
similar way to a large number of different insults. 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 or indeterminate diagnosis even when the IBD 
is suspected. Unfortunately, the patient must often suffer as the disease 
progresses before a definitive diagnosis can be made. In many patients, 
though, the diagnosis of IBD must still be regarded as indeterminate. 
The differentiation between the types of IBD, ulcerative colitis and 
Crohn's disease, 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 for the symptoms of 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. 
Thus, IBD and quite often its treatment affects the lifestyle and 
functional capabilities of those afflicted. Treatment courses often result 
in adverse physiologic manifestations which must be balanced against the 
therapeutic benefit. Any intervention which can improve patients' 
toleration of their disease and therapeutic program is welcome. 
The availability of diagnostic methods that would readily distinguish UC 
from CD as well as other inflammatory disorders of the bowel would 
represent a major clinical advance which would aid in therapeutic 
management of IBD and the design of more specific treatment modalities. In 
addition specific detection of the disease in prospective parents can be 
useful in genetic counseling. Accordingly, there has existed a need for 
convenient and reliable methods of screening for IBD for diagnostic, 
prognostic and therapeutic purposes. 
BRIEF DESCRIPTION OF THE INVENTION 
A powerful association has been discovered in Jews between ulcerative 
colitis ("UC") and the presence of allele 2 of the variable number of 
tandem repeats ("VNTR") polymorphism at intron 2 of the human 
interleukin-1 receptor antagonist ("IL-1ra") gene. This association 
provides the basis for convenient and reliable methods of screening Jewish 
patients for UC, and distinguishing UC from Crohn's disease ("CD") and 
other inflammatory diseases of the bowel. 
The present invention provides novel methods of screening for UC in Jews 
which comprise detecting the presence or absence of nucleic acid encoding 
allele 2 of the VNTR polymorphism at intron 2 of the IL-1ra gene, wherein 
the presence of nucleic acid encoding allele 2 indicates UC. These novel 
methods do not depend upon the presentation of clinical symptoms or the 
activity of the disease and provide a more sensitive method of screening 
for UC within this sub-population than is provided by prior art methods. 
Thus, the present invention has both prognostic and diagnostic value. 
Nucleic acid encoding allele 2 of VNTR polymorphism of the IL-1ra gene can 
be detected in accordance with the present invention by amplifying the 
genomic DNA of a Jewish subject which encodes at least the portion intron 
2 of the IL- 1ra gene encoding the VNTR allele and identifying the allele 
present. Allele 2 can be identified by assaying the nucleic acid sequence 
at intron 2 of the IL-1ra gene of a subject for defining characteristics 
of allele 2 and comparing the results to a positive and or negative 
control. Defining characteristics of nucleic acid encoding allele 2 
include, for example, size, sequence, homology to a probe, and the like. 
Thus there are an array of different formats in which the methods of the 
present invention can be performed. Primers suitable for use in amplifying 
nucleic acid encoding intron 2 of the IL-1ra gene are provided herein. 
Also provided by the present invention are kits for screening for UC in 
Jews by detecting the presence or absence of allele 2 of the VNTR 
polymorphism at intron 2 of the IL-1ra gene. Such kits include reagents, 
primers, sequencing markers, positive and negative controls and the like, 
which are useful in the practice of the present invention. 
DETAILED DESCRIPTION OF THE INVENTION 
Although the clinical features of ulcerative colitis ("UC") and Crohn's 
disease ("CD") have been well characterized, diagnosis of these disease is 
often a protracted and expensive undertaking that requires invasive and 
unpleasant procedures. The etiology and pathogenesis of these chronic, 
relapsing inflammatory bowel diseases ("IBD") remain unknown. Several 
factors have been implicated as possible initiating events in IBD, 
including environmental agents, bacterial or viral organisms, bacterial 
cell wall components or ingested toxins, and dietary products. These 
factors, capable of initiating an inflammatory response in genetically 
predisposed individuals, may initiate a sequence of chronic immunological 
processes that are not appropriately down-regulated. These events 
perpetuate a prolonged cellular and humoral immune response that leads to 
subsequent tissue injury. 
The search for a specific genetic marker which would provide a 
non-invasive, convenient and reliable method of diagnosing IBD has been 
frustrated by the apparent heterogeneity of these diseases. Certainly, 
there can be no group more troubled by this than people of Jewish 
ancestry, particularly Caucasians of Jewish ancestry, who as a group, show 
the highest reported risk of developing IBD than any other ethnic group. 
While UC and CD are both found worldwide, they occur in the highest 
frequency in North America and Northern Europe where the incidence ranges 
from 2 to 15 and the prevalence 35-150 per 1000,000 for UC, and 2-7 and 
30-100 per 100,000 for CD, respectively. The incidence of IBD is highest 
among Caucasians, lower in blacks, and lowest in Asians. For Caucasians, 
the most consistent observation has been that Ashkenazi Jewish populations 
has been shown to be at a higher risk of developing IBD than other ethnic 
groups. 
A differential association between UC and the presence of allele 2 of the 
variable number tandem repeat polymorphism at intron 2 of the 
interleukin-1 receptor antagonist gene in human subjects of Jewish 
ancestry has been discovered. This association provides the basis for 
convenient and reliable methods of screening for UC in human subjects of 
Jewish ancestry, providing valuable information in the diagnosis of IBD 
and the determination of susceptibility to UC. 
Interleukin-1 ("IL-1") is a pro-inflammatory cytokine produced 
predominately by activated macrophages. IL-1 induces fever, release of 
acute phase reactants, production of arachidonic acid metabolites, and 
activation of other immune cells. 
The cytokine interleukin-1 receptor antagonist ("IL-1ra ") is a potent 
anti-inflammatory protein that appears to play a role in several chronic 
inflammatory bowel diseases including IBD. The inflammatory response to 
IL-1 is modulated by IL-1ra, a 22 kilodalton protein secreted by the same 
cells that produce IL-1. The IL-1ra is structurally related to IL-1.alpha. 
and IL-1.beta. and competes with these molecules for occupancy of IL-1 
cell surface receptors. Since IL-1ra does not trigger signal transduction, 
it acts as a competitive inhibitor. It may therefore play a crucial role 
in many IL-1 mediated diseases, acting as an important endogenous 
regulator of inflammation. 
The gene encoding IL-1ra is located on human chromosome 2 (2q13-14). The 
published gene sequence of IL-1ra shows four copies of an 86-base pair 
sequence in intron 2 of the IL-1ra gene. See, Lennard, A. et al., Cytokine 
4:83-89 (1992), incorporated herein by reference in its entirety. 
Recently, a variable length polymorphism was reported at intron 2 of the 
IL-1ra gene, comprising five alleles. See, Tarlow, J., et. al., Human 
Genetics 91:403-404 (1993), incorporated herein by reference in its 
entirety. This polymorphism is referred to herein as the variable number 
of tandem repeats ("VNTR") polymorphism of the IL-1ra gene. 
Alleles 1 through 5 (A1-A5) of the VNTR polymorphism are characterized by 
the number of copies of the 86-base pair sequence present at the loci as 
described in Table 1. These alleles can be easily distinguished from one 
another on the basis of their unique lengths, by direct sequencing and the 
like. For example, primers flanking this region of the VNTR polymorphism 
can be used to amplify the polymorphic region by PCR. The PCR products can 
then be analyzed by electrophoresis to determine the allele present. The 
primers defined as SEQ ID NO 1 and SEQ ID NO 2 have been used to amplify 
genomic DNA encoding the VNTR polymorphism of IL-1ra and the five alleles 
characterized as follows in Table 1: 
TABLE 1 
______________________________________ 
Five alleles identified from DNA amplified from 70 unrelated 
individuals Tarlow, J., et. al., Human Genetics 91:403-404 (1993). 
Allele Frequency Size (bp) 
Number of repeats 
______________________________________ 
A1 0.736 410 4 
A2 0.214 240 2 
A3 0.036 500 5 
A4 0.007 325 3 
A5 0.007 595 6 
______________________________________ 
While investigators have observed a moderate association (Odds ratio 
.about.2) between allele 2 of the VNTR polymorphism of IL-1ra and UC 
patients in Caucasian populations from North America and Europe, this 
association was too weak to be of diagnostic or prognostic value (allele 2 
of IL-1ra present in 35% of UC versus 24% of controls). See, Mansfield, J. 
et al., Gastroenterology 106:637-642 (1994). 
For the first time, it has been discovered that allele 2 of the VNTR 
polymorphism at intron 2 of IL-1ra can be used as a diagnostic and 
prognostic genetic marker of UC in humans of Jewish ancestry. Jewish UC 
patients have a significantly increased frequency of allele 2 compared 
with Jewish controls (Odds ratio=5), while non-Jewish UC patients have a 
frequency of allele 2 similar to non-Jewish controls. 
More specifically, when the genotype at the IL-1ra locus was determined for 
unrelated Jewish and non-Jewish Caucasian human subjects with UC (n=106), 
CD (n=158), and controls (n=114) by PCR using the primers and procedures 
described below in greater detail, it was determined that the frequency of 
individuals carrying the allele 2 was significantly increased in the UC 
compared with controls (p=0.04; OR=1.7; 95% CI=1.0-3.1). CD patients had a 
similar frequency as controls (p=0.66). However, when this population was 
divided into Jewish and non-Jewish groups, a significant difference was 
only observed in Jews (p=0.003; OR=5.0; 95%CI=1.5-17.5), but not in 
non-Jews (p=0.85) as reported in detail in Table 2. The OR of 5 is the 
highest reported in any population. 
TABLE 2 
______________________________________ 
UC CD Controls 
Ethnic Groups 
N % of 2 N % of 2 
N % of 2 
______________________________________ 
Total Caucasian 
106* 58.5 158* 47.5 114* 44.7 
Jews 39 76.9 68 48.5 25 40.0 
Non-Jews 66 48.5 88 46.6 81 46.9 
______________________________________ 
*unknown ethnicity for 1 UC, 2 CD, and 8 controls 
The .sub..chi. 2 test was used for the statistical test. Both Odds Ratio 
(OR) and its 95% confidence interval (CI) were calculated to measure the 
strength of associations. 
Thus, in accordance with the present invention, there is provided methods 
of screening for UC in a human subject of Jewish ancestry, comprising 
detecting the presence or absence of nucleic acid encoding allele 2 of 
VNTR polymorphism at intron 2 of the IL-1ra gene in said subject, wherein 
the presence of nucleic acid encoding allele 2 is indicative of UC. 
Nucleic acid of a subject which is suitable for screening in accordance 
with the present invention may be derived from any nucleated cell sample, 
and preferably from peripheral mononuclear blood cells. 
"A human subject of Jewish ancestry" or "a person of Jewish ancestry" 
refers to a person having had at least one biologically-related 
grandparent who is Jewish. Preferably, the human subject is Caucasian of 
Jewish ancestry. Even more preferably, the human subject is an Caucasian 
of Ashkenazi Jewish ancestry. 
Detecting the presence or absence of nucleic acid encoding allele 2 of the 
VNTR polymorphism of the IL-1ra gene may be accomplished by determining 
whether or not genomic DNA a the human subject possesses a defining 
characteristic of nucleic acid encoding allele 2 of the VNTR polymorphism 
of the IL-1ra gene. One of skill in the art will understand that there are 
many means available to make such a determination, e.g., electrophoresis, 
automated sequencing, allele-specific oligonucleotide probing, 
differential restriction endonuclease digestion, ligase-mediated gene 
detection, and the like. 
For example, genomic DNA encoding at least intron 2 of the IL-1ra human 
gene can be isolated from nucleated cells of a human subject of Jewish 
ancestry and assayed for such characteristics as size, specific sequence, 
number of sequence repeats, ability to hybridize with a labeled probe 
under specific hybridization parameters, ability to bind with an antibody 
specific for a particular allele and the like, and then compared to a 
positive control which defines the same characteristic for allele 2 of the 
VNTR polymorphism and/or a negative control which defines the same 
characteristic for an allele of the VNTR polymorphism not known to be 
associated with UC in persons of Jewish ancestry. 
A positive control for allele 2 of the VNTR polymorphism of the IL-1ra gene 
using the primers described herein for PCR amplification of genomic DNA 
and fragment length as the defining characteristic, a fragment of 240 base 
pairs would be a positive control and a negative control would be a 
fragments of 325, 410, 500 and/or 595 base pairs. Of course, the greatest 
likelihood of accurately detecting the presence of allele 2 of the VNTR 
polymorphism of the IL-1ra gene in this example would be to compare the 
results of the assay to the positive and negative control. 
To increase the accuracy of detecting allele 2 of the VNTR polymorphism, 
the control should be subjected to the same test procedures and parameters 
as the nucleic acid of the subject being assayed. Likewise, assays to 
detect the presence or absence of nucleic acid encoding allele 2 of the 
VNTR polymorphism should be calibrated against a standard. For example, in 
a presently preferred embodiment using fragment length as the defining 
characteristic of alleles of the VNTR polymorphism, a positive control (a 
nucleic acid sequence known to encode allele 2 of the VNTR polymorphism) 
is amplified and electrophoresed using the same reagents, primers and 
parameters as that used for the nucleic acid of the subject being tested. 
A sequencing marker equal in size to the control is also subjected to 
electrophoresis using the same reagents and parameters as those used with 
the test and control nucleic acid. Sequencing markers useful in the 
practice of the present invention are available from a variety of 
commercial sources. 
Genomic DNA of a subject encoding the sequence of interest at intron 2 of 
the IL-1ra gene can be amplified to make detection of the VNTR allele 
easier. Amplification of nucleic acid may be achieved using conventional 
methods, see, e.g., Maniatis, et al., Molecular Cloning: A Laboratory 
Manual 187-210 (Cold Spring Harbour Laboratory, 1982) which is 
incorporated herein by reference. Amplification, however, is preferably 
accomplished via the polymerase chain reaction ("PCR") method disclosed by 
U.S. Pat. Nos. 4,698,195 and 4,800,159, the respective contents of which 
are incorporated herein by reference. Application of PCR to detect alleles 
of the VNTR polymorphism of IL-1ra gene requires less DNA and is faster 
than standard Southern blotting and hybridization techniques. 
Thus, oligonucleotide primer pairs can be constructed that allow enzymatic 
amplification of a subject's nucleic acid that encodes the VNTR 
polymorphism at intron 2 of the IL-1ra gene. The amplified nucleic acid 
can then be assayed to detect the presence or absence of allele 2. 
Primer pairs suitable for use in the practice of the present invention are 
linear oligonucleotides ranging in length from about ten to about thirty 
nucleotides in length. One of the primers in the pair should be 
complementary to a nucleotide sequence upstream of the nucleic acid 
encoding the VNTR polymorphism at intron 2 of the IL-1ra gene targeted for 
amplification. The other primer should be complementary to a sequence 
located down stream of this target site. Preferably, the primers suitable 
for use in the present invention are specific for amplification of nucleic 
acid encoding VNTR polymorphism at intron 2 of the IL-1ra gene and do not 
prime amplification of nucleic acid which does not encode VNTR 
polymorphism of the IL-1ra gene. The sequences complementary to the primer 
pairs may be separated by as many nucleotides as the PCR technique and the 
other technique(s) for detecting the presence or absence of VNTR 
polymorphism will allow, provided that an appropriate control is used. 
Primers suitable for use in amplifying genomic DNA encoding the VNTR 
polymorphism at intron 2 of the IL-1ra gene can be constructed using the 
oligonucleotide primer sequences described herein as well as the genomic 
sequence of the IL-1ra gene provided at Lennard, A. et al., Cytokine 
4:83-89 (1992) and incorporated herein by reference . 
A pair of primers suitable for use in the practice of the present invention 
is set forth in SEQ ID NOS 1 and 2. These primers are suitable for use in 
amplifying genomic DNA encoding the alleles of the VNTR polymorphism of 
intron 2 of the IL-1ra gene, and may be used as a pair or each in 
combination with another suitable primer. 
The novel methods for screening for UC and for distinguishing UC from CD 
disclosed herein include the use of traditional diagnostic tests for UC in 
combination with the detection of nucleic acid of a subject encoding 
allele 2 of the VNTR polymorphism of IL-1ra. Thus, for example, a positive 
test for HLA DR2 and/or a positive pANCA status may be used in combination 
with detecting the presence of nucleic acid encoding allele 2 of the VNTR 
polymorphism. 
Kits for use in screening for UC in human subjects of Jewish ancestry are 
also provided by the present invention. Such kits can include all or some 
of the positive controls, negative controls, reagents, primers, sequencing 
markers, probes and antibodies described herein for determining the 
presence or absence of nucleic acid encoding allele 2 of the VNTR 
polymorphism of the IL-1ra gene. Kits of the present invention may 
contain, for example, nucleic acid encoding allele 2 and/or alleles 1,3,4 
and 5 of the VNTR polymorphism of the IL-1ra gene, the nucleic acid 
sequence of any one or more of these alleles, schedules of the number and 
type of nucleotide sequence repeats and characteristics of one or more of 
these alleles, one or more labeled oligonucleotide probes specific for one 
or more alleles of the VNTR alleles, one or more primers for amplification 
of nucleic acid encoding the VNTR polymorphism at intron 2 of the IL-1ra 
gene, reagents commonly used for amplification, polymerase, antibody 
specific for, or which binds particular VNTR alleles and combinations of 
any of the above. 
As amenable, these suggested kit components may be packaged in a manner 
customary for use by those of skill in the art. For example, these 
suggested kit components may be provided in solution or as a liquid 
dispersion or the like. 
A presently preferred embodiment of the inventive kits for use in screening 
for UC comprises DNA encoding allele 2 of the VNTR polymorphism of the 
IL-1ra gene in Tris-EDTA buffer solution preferably kept at 4.degree. C. 
Another embodiment of the inventive kits for use in screening for UC 
further comprises one or more primers specific for amplification of 
nucleic acid encoding the VNTR polymorphism at intron 2 of the IL-1ra 
gene, for example, primers selected from the group comprising SEQ ID NO 1 
and SEQ ID NO 2. 
Yet another embodiment of the inventive kits for use in screening for UC 
further comprises sequencing markers ranging in size from about 100 to 
about 600 base pairs.

EXAMPLES 
Blood samples were obtained from 106 human patients diagnosed as having 
ulcerative colitis ("UC"), 158 human patients diagnosed as having Crohn's 
disease ("CD"), and 114 healthy humans ("controls"). The control group was 
selected to be ethnically and socioeconomically matched to the UC and CD 
patients. An individual was used as control only if he/she did not have 
inflammatory bowel disease, multiple sclerosis, systemic lupus 
erythematosus, or other recognized autoimmune diseases. The distribution 
of age, gender, and ethnicity (Jewish/non-Jewish) were comparable between 
patients and controls. A patient was considered to be Jewish if he/she was 
of Jewish ancestry, i.e., at least one of the patient's biological 
grandparents was Jewish. All patients and controls analyzed were 
Caucasians. 
A. Isolation of Genomic DNA 
Genomic DNA is isolated from blood samples of each patient and each of the 
control group by methods well known in the art, for example, by methods 
described in J. Sambrook, et al., Molecular Cloning: A Laboratory Manual, 
2nd Edition, Cold Spring Harbor Laboratory Press (1989), incorporated 
herein by reference. 
B. Amplification Of DNA Encoding Intron 2 of the IL-1ra Gene By PCR 
The polymerase chain reaction ("PCR") was performed to amplify genomic DNA 
encoding intron 2 of the IL-1ra gene. The primers identified as SEQ ID 
NOs. 1 and 2 were used. The PCR reaction is performed in a total volume of 
2 .mu.l using 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, 0.15 
.mu.M primers, 200 .mu.M each dATP, dCTP, dGTP, dTTP; 100 to 300 ng 
genomic, and two units Taq DNA polymerase. 
PCR conditions are as follows: Denaturing of the nucleic acid sample in the 
first cycle of amplification is at 96.degree. C. for one minute, followed 
by annealing primers at 60.degree. C. for one minute, and polymerization 
at 70.degree. C. for two minutes. Twenty nine subsequent cycles of 
amplification were carried out, denaturing at 94.degree. C. for one 
minute, followed by annealing primers at 60.degree. C. for one minute, and 
polymerization at 70.degree. C. for two minutes. After the final round of 
amplification, the final PCR products are analyzed on a 2% agarose gel. 
C. Electrophoresis of Amplified Genomic DNA Encoding Intron 2 of the IL-1ra 
Gene 
20 .mu.l amplified DNA is electrophoresed on a 2% agarose gel and stained 
with ethidium bromide. Gels are run with a 100 base pair ladder control 
DNA sequencing marker to size fragments. Although extra fragments may 
shadow the specific DNA bands, results are unambiguously interpretable, 
and confirmed by comparison to nucleic acid derived from human patients 
known to encode the specified alleles at Intron 2 of the IL-1ra gene and 
subjected to the same amplification and electrophoresis procedures 
described above. 
Although the invention has been described with reference to presently 
preferred embodiments, it should be understood that various modifications 
can be made without departing from the spirit of the invention. 
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