CA2282062A1 - Diagnostic methods in autoimmune disease - Google Patents
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- CA2282062A1 CA2282062A1 CA 2282062 CA2282062A CA2282062A1 CA 2282062 A1 CA2282062 A1 CA 2282062A1 CA 2282062 CA2282062 CA 2282062 CA 2282062 A CA2282062 A CA 2282062A CA 2282062 A1 CA2282062 A1 CA 2282062A1
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Abstract
In one aspect, the invention provides a method of diagnosis. The diagnostic method may include steps of identifying a patient at risk of an arthritis, the patient having an interferon gamma gene. The patient may be tested to characterize a polymorphism in a first intron of the interferon gamma gene. The polymorphism may comprise a variable length dinucleotide repeat region within the first intron, and the dinucleotide repeat region may be located at least partly between nucleotides 1349 and 1373 in the interferon, gamma gene. The method may be carried out so as to be capable of identifying alleles such as the 126 by allele and the 122 by allele, as further described herein. The polymorphisms may be distinguished based on a difference in the number of CA repeats in a portion of the first intron of the interferon gamma gene.
Description
DIAGNOSTIC METHODS IN AUTOIMMUNE DISEASE
FIELD OF THE INVENTION
The invention is in the field of pharamacogenomics, particularly the utilization of genetic alleles as diagnostic markers.
BACKGROUND OF THE INVENTION
Rheumatoid arthritis is a chronic polyarticular inflammatory disease with a variable course and outcome (Reference No. 1 in the draft paper).
Clinical expression ranges from a mild, non-deforming arthropathy with little long-term disability to severe, incapacitating, erosive articular destruction which may be refractory to conventional disease modifying agents (Reference No. 2 on the draft paper). Prediction of disease progression is inprecise, and is often based on a combination of demographic, clinical and laboratory factors.
Diagnostic indications of arthritic disease may include low socioeconomic status and educational levels, severe initial disease activity, systemic manifestations and extra-articular features, the early appearance of joint erosions, an elevated erythrocyte sedimentation rate, C-reactive protein and the presence of rheumatoid factor. There is epidemiological evidence that there is a genetic relationship between loci in the major histocompatibility complex (MHC) class II region and disease susceptibility and severity (references 4-6 in the draft paper).
Interferon gamma is a homodimeric 34 Kd peptide. IFN gamma may be secreted by T-lymphocytes under certain conditions of activation and by NK cells (Boehm, U., et al. 1997). IFN gamma binds to cell surface receptors on cellular targets including mononuclear phagocytes, endothelial cells and NK cells, and is thought to play an important role in the coordinated regulation and expression of the immune response through the stimulation or repression of genes (Farrar, M.A. et al., 1993; Revel, M. et al., 1986). IFN gamma appears to be produced by T-cells infiltrating the inflamed synovium and may be secreted into the joint space, but the role of this peptide in the progression of the articular injury in arthritis remains controversial (Feldemann, M. et al.
1996).
IFN gamma is encoded by a gene which in humans is mapped to 12q24 on chromosome 12. The known sequence of the gene consists of 4 exons with 3 intervening regions. A variable length dinucleotide repeat polymorphism has been described in humans and lower primates within the first intron of this gene, between positions 1349 and 1373. The number of alleles reported at this microsatellite region appears to vary according to the detection methodology employed to characterize it (Ruiz-Linares A, 1993; Awata, T. et al. 1994;
Pravica, V. et al., 1998).
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method of diagnosis. The diagnostic method may include steps of identifying a patient at risk of an arthritis, the patient having an interferon gamma gene. The patient may be tested to characterize a polymorphism in a first intron of the interferon gamma gene. The polymorphism may comprise a variable length dinucleotide repeat region within the first intron, and the dinucleotide repeat region may be located at least partly between nucleotides 1349 and 1373 in the interferon, gamma gene. The method may be carried out so as to be capable of identifying alleles such as the 126 by allele and the 122 by allele, as further described herein. The polymorphisms may be distinguished based on a difference in the number of CA repeats in a portion of the first intron of the interferon gamma gene.
The methods of the invention may be utilized in patients identified as at risk of rheumatoid arthritis, including Caucasian patients. Patients without symptoms may be diagnosed as at risk of arthritis, and in some cases this may be reflective of epidemiological criteria such as sex, age, socioeconomic factors or family history. The diagnosis of the patient as being at risk of an arthritis may also comprise identifying symptoms such as the following: joint erosions, elevated erythrocyte sedimentation rate, C-reactive protein, polyarticular disease, joint deformities, radiological evidence of subchondral erosions, extra-articular arthritis or the presence of rheumatoid factor.
To characterize the polymorphism, a region of the first intron may be amplified, such as a region comprising a variable length dinucleotide repeat.
The use of an allele of an interferon, gamma gene as described herein may provide prognostic information with respect to the likelihood of particular clinical outcomes for the patient, and as a result may be utilized to modify treatment regimens. In particular, the presence of alleles associated with relatively severe disease, such as the 126 by allele, may be taken as an indication that aggressive therapy should be pursued relatively early in the progression of the disease.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the invention discloses a correlation between two alleles, designated 126 by and 122 bp, and the occurrence of particular disease states in arthritis. There is a positive correlation between the occurrence of the by allele and severe rheumatoid arthritis. There is a negative correlation between the occurrence of the 122 by allele and severe rheumatoid arthritis.
For each allele, there is a corresponding and reverse correlation with relatively mild rheumatoid arthritis. One aspect of the present invention therefore provides pharmacogenomic methods to assist in diagnosis of arthritis disease, including prediction of disease severity and selection of therapy regimens in particular patients.
EXAMPLE
48 adult Caucasian patients with severe rheumatoid arthritis and 39 patients with mild rheumatoid arthritis were selected sequentially from a hospital patient population. 50 patients that did not present with symptoms of arthritic disease were selected as a control comparator group. Patients with severe rheumatoid arthritis were aged 58+ 12 years and were predominantly female and had a mean disease duration of 19+ 12 years. All such patients had clinically severe polyarticular disease with joint deformities and radiological evidence of subchondral erosions. 75% of such patients had extra-articular manifestations of disease other than the sicca syndrome. 87% of patients with S severe rheumatoid arthritis were rheumatoid factor positive. All such patients had not responded favourably to therapy with conventional disease-modifying anti-rheumatic drugs (DMRDS), and had been maintained on cyclosporine treatment for a mean period of 26 months.
Patients with mild disease were aged 61+ 13 years, predominantly female, and had a mean disease duration of 12+ 7 years. All such patients had clinically mild disease, which had been controlled for a mean period of 90 months by antimalarials alone without prior or current use of DMRDS. Only 26% of such patients had joint deformities, and 36% had extra-articular disease manifestations other than sicca syndrome. 34% of such patients were rheumatoid factor positive.
Peripheral blood was obtained from patients and control subjects, and genomic DNA extracted by proteinase K digestion and by salting out.
Molecular typing at the IFN gamma (12q24.12) microsatellite polymorphism was performed by PCR followed by use of a DNA sequencer and gene analysis software. Locus or group-specific amplification was performed using 5' and 3' oligonucleotide amplification primers as follows:
5'6 FAMAG ACA TTC ACA ATT GAT TTT ATT CTT AC 3' 5' CCT TCC TGT AGG GTA TTA TTA TAC G3' The primers were designed with a high annealing temperature to enhance specificity. Both primers were obtained from Perkin-Elmer-ABI-PRISM and the forward primer was fluorescently labelled at the 5' end.
Genomic DNA (100 ng) was amplified using 50 pmoles each of the oligonucleotide primers, 100 uM, each of dNTP, 1.5 mM MgCl2 and 0.8u of TAQ polymerase in a Perkin-Elmer PCR cycler. Cycling conditions included a 5-minute hot start at 95° C. followed by 32 cycles of 95° C
for 45 seconds (denaturation) and 62° C for one minute (annealing and extension) with a final extension of S minutes at 62° C in the last cycle. The amplified product was run on a 1.5% agarose gel for detection of positive amplification and then on a long-range gel on a 377 DNA sequencer ABI-PRISM) data were collected using 377 collection software and size analysis was performed using Genescan 2Ø2 software and Genescan 2Ø2 software and Genescan-500 ROX as a size standard (ABI-PRISM).
A total of six alleles were documented in the patients and controls, ranging in length from 120 by to 130 bp, as shown in Table 1.
TABLE 1: Proportion of subjects expressing individual alleles in the first intron of the IFNy gene. Controls (n=50), patients with severe RA (n=48), patients with mild RA (n=39).
IFNy AllelesControlsSevere Mild RA RA b b (Size) % % % p OR OR OR
P p A2 (130 0 6 7.77 NS 0 - - 6.08 NS
bp) A3 (128 16 15 0.90 NS 15 0.95 NS 0.94 NS
bp) A4 (126 12 73 19.74p<0.000121 1.89 NS 10.43p<0.0001 bp) AS (124 68 77 1.58 NS 67 0.94 NS 1.68 NS
bp) A6 (122 68 6 0.019p<0.000164 0.50 NS 0.037p<0.0001 bp) A7 (120 0 0 - - 3 3.93 NS 0.27 NS
bp) ° severe or mild KA compared with controls severe compared with mild RA
NS: not statistically significant OR: odds ratio The frequency of the polymorphisms in normal subjects ranged from 0% for the 130 by and 120 by alleles, to 68% for the 122 by allele. The genotype frequencies did not deviate from the expected value by Hardy-Weinberg equilibrium. The alleles identified herein appear to correspond closely in length to those initially reported by Ruiz-Linares, which ranged from 122-134 by (Ruiz-Linares, 1994 Hum. Mol. Genet. 2(9):1508). Such alleles may vary, for example, depending upon the ethnic origin of the subjects and the methodology of characterization (see for example Awata, T. et al.
1994 Diabetologia 37:1159). In some populations, 6-8 alleles encompassing at least 11-15 CA repeats may exist at this site, including the intermediate polymorphism of 128 bp.
As shown in this example, patients with severe rheumatoid arthritis differ significantly in the frequency of 2 alleles. The 126 by allele was present in 73% of patients with severe rheumatoid arthritis compared with 21% of patients with mild rheumatoid arthritis (OR: 10.43, p < 0.0001) and 12% of normal subj ects (OR: 19.74, p < 0.0001 ). In contrast, the 122 by allele was detected in only 6% of patients with severe rheumatoid arthritis compared with 64% of patients with mild disease (OR: 0.037, p < 0.0001) and 68% of normal subjects (OR: 0.019, p < 0.0001). There was no significant difference in the frequencies of the other microsatellite polymorphisms between the three groups of individuals.
Table 2 shows a grouping of the subjects into one of four categories, depending upon their expression of the 126 by allele and the 122 by allele.
Almost three quarters (73%) of patients with severe rheumatoid arthritis expressed the 126 by allele without the 122 by allele, compared with 10% of patients with mild rheumatoid arthritis (OR: 23.56, p < 0.0001) and 4% of normal subjects (OR: 60.62, p < 0.0001). In contrast, only 6% of patients with severe rheumatoid arthritis expressed the 122 by allele without the 126 by allele compared with 54% of patients with mild rheumatoid arthritis (OR:
0.057, p < 0.0001) and 70% of normal subjects (OR: 0.029, p < 0.0001). The 126 by and 122 by alleles were not conjointly expressed by any patients with severe disease compared with conjoint expression in 10% of patients with mild disease (OR: 0.081, p = NS) and conjoint expression in 8% of normal subjects (OR: 0.11, p = NS).
FIELD OF THE INVENTION
The invention is in the field of pharamacogenomics, particularly the utilization of genetic alleles as diagnostic markers.
BACKGROUND OF THE INVENTION
Rheumatoid arthritis is a chronic polyarticular inflammatory disease with a variable course and outcome (Reference No. 1 in the draft paper).
Clinical expression ranges from a mild, non-deforming arthropathy with little long-term disability to severe, incapacitating, erosive articular destruction which may be refractory to conventional disease modifying agents (Reference No. 2 on the draft paper). Prediction of disease progression is inprecise, and is often based on a combination of demographic, clinical and laboratory factors.
Diagnostic indications of arthritic disease may include low socioeconomic status and educational levels, severe initial disease activity, systemic manifestations and extra-articular features, the early appearance of joint erosions, an elevated erythrocyte sedimentation rate, C-reactive protein and the presence of rheumatoid factor. There is epidemiological evidence that there is a genetic relationship between loci in the major histocompatibility complex (MHC) class II region and disease susceptibility and severity (references 4-6 in the draft paper).
Interferon gamma is a homodimeric 34 Kd peptide. IFN gamma may be secreted by T-lymphocytes under certain conditions of activation and by NK cells (Boehm, U., et al. 1997). IFN gamma binds to cell surface receptors on cellular targets including mononuclear phagocytes, endothelial cells and NK cells, and is thought to play an important role in the coordinated regulation and expression of the immune response through the stimulation or repression of genes (Farrar, M.A. et al., 1993; Revel, M. et al., 1986). IFN gamma appears to be produced by T-cells infiltrating the inflamed synovium and may be secreted into the joint space, but the role of this peptide in the progression of the articular injury in arthritis remains controversial (Feldemann, M. et al.
1996).
IFN gamma is encoded by a gene which in humans is mapped to 12q24 on chromosome 12. The known sequence of the gene consists of 4 exons with 3 intervening regions. A variable length dinucleotide repeat polymorphism has been described in humans and lower primates within the first intron of this gene, between positions 1349 and 1373. The number of alleles reported at this microsatellite region appears to vary according to the detection methodology employed to characterize it (Ruiz-Linares A, 1993; Awata, T. et al. 1994;
Pravica, V. et al., 1998).
SUMMARY OF THE INVENTION
In one aspect, the invention provides a method of diagnosis. The diagnostic method may include steps of identifying a patient at risk of an arthritis, the patient having an interferon gamma gene. The patient may be tested to characterize a polymorphism in a first intron of the interferon gamma gene. The polymorphism may comprise a variable length dinucleotide repeat region within the first intron, and the dinucleotide repeat region may be located at least partly between nucleotides 1349 and 1373 in the interferon, gamma gene. The method may be carried out so as to be capable of identifying alleles such as the 126 by allele and the 122 by allele, as further described herein. The polymorphisms may be distinguished based on a difference in the number of CA repeats in a portion of the first intron of the interferon gamma gene.
The methods of the invention may be utilized in patients identified as at risk of rheumatoid arthritis, including Caucasian patients. Patients without symptoms may be diagnosed as at risk of arthritis, and in some cases this may be reflective of epidemiological criteria such as sex, age, socioeconomic factors or family history. The diagnosis of the patient as being at risk of an arthritis may also comprise identifying symptoms such as the following: joint erosions, elevated erythrocyte sedimentation rate, C-reactive protein, polyarticular disease, joint deformities, radiological evidence of subchondral erosions, extra-articular arthritis or the presence of rheumatoid factor.
To characterize the polymorphism, a region of the first intron may be amplified, such as a region comprising a variable length dinucleotide repeat.
The use of an allele of an interferon, gamma gene as described herein may provide prognostic information with respect to the likelihood of particular clinical outcomes for the patient, and as a result may be utilized to modify treatment regimens. In particular, the presence of alleles associated with relatively severe disease, such as the 126 by allele, may be taken as an indication that aggressive therapy should be pursued relatively early in the progression of the disease.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the invention discloses a correlation between two alleles, designated 126 by and 122 bp, and the occurrence of particular disease states in arthritis. There is a positive correlation between the occurrence of the by allele and severe rheumatoid arthritis. There is a negative correlation between the occurrence of the 122 by allele and severe rheumatoid arthritis.
For each allele, there is a corresponding and reverse correlation with relatively mild rheumatoid arthritis. One aspect of the present invention therefore provides pharmacogenomic methods to assist in diagnosis of arthritis disease, including prediction of disease severity and selection of therapy regimens in particular patients.
EXAMPLE
48 adult Caucasian patients with severe rheumatoid arthritis and 39 patients with mild rheumatoid arthritis were selected sequentially from a hospital patient population. 50 patients that did not present with symptoms of arthritic disease were selected as a control comparator group. Patients with severe rheumatoid arthritis were aged 58+ 12 years and were predominantly female and had a mean disease duration of 19+ 12 years. All such patients had clinically severe polyarticular disease with joint deformities and radiological evidence of subchondral erosions. 75% of such patients had extra-articular manifestations of disease other than the sicca syndrome. 87% of patients with S severe rheumatoid arthritis were rheumatoid factor positive. All such patients had not responded favourably to therapy with conventional disease-modifying anti-rheumatic drugs (DMRDS), and had been maintained on cyclosporine treatment for a mean period of 26 months.
Patients with mild disease were aged 61+ 13 years, predominantly female, and had a mean disease duration of 12+ 7 years. All such patients had clinically mild disease, which had been controlled for a mean period of 90 months by antimalarials alone without prior or current use of DMRDS. Only 26% of such patients had joint deformities, and 36% had extra-articular disease manifestations other than sicca syndrome. 34% of such patients were rheumatoid factor positive.
Peripheral blood was obtained from patients and control subjects, and genomic DNA extracted by proteinase K digestion and by salting out.
Molecular typing at the IFN gamma (12q24.12) microsatellite polymorphism was performed by PCR followed by use of a DNA sequencer and gene analysis software. Locus or group-specific amplification was performed using 5' and 3' oligonucleotide amplification primers as follows:
5'6 FAMAG ACA TTC ACA ATT GAT TTT ATT CTT AC 3' 5' CCT TCC TGT AGG GTA TTA TTA TAC G3' The primers were designed with a high annealing temperature to enhance specificity. Both primers were obtained from Perkin-Elmer-ABI-PRISM and the forward primer was fluorescently labelled at the 5' end.
Genomic DNA (100 ng) was amplified using 50 pmoles each of the oligonucleotide primers, 100 uM, each of dNTP, 1.5 mM MgCl2 and 0.8u of TAQ polymerase in a Perkin-Elmer PCR cycler. Cycling conditions included a 5-minute hot start at 95° C. followed by 32 cycles of 95° C
for 45 seconds (denaturation) and 62° C for one minute (annealing and extension) with a final extension of S minutes at 62° C in the last cycle. The amplified product was run on a 1.5% agarose gel for detection of positive amplification and then on a long-range gel on a 377 DNA sequencer ABI-PRISM) data were collected using 377 collection software and size analysis was performed using Genescan 2Ø2 software and Genescan 2Ø2 software and Genescan-500 ROX as a size standard (ABI-PRISM).
A total of six alleles were documented in the patients and controls, ranging in length from 120 by to 130 bp, as shown in Table 1.
TABLE 1: Proportion of subjects expressing individual alleles in the first intron of the IFNy gene. Controls (n=50), patients with severe RA (n=48), patients with mild RA (n=39).
IFNy AllelesControlsSevere Mild RA RA b b (Size) % % % p OR OR OR
P p A2 (130 0 6 7.77 NS 0 - - 6.08 NS
bp) A3 (128 16 15 0.90 NS 15 0.95 NS 0.94 NS
bp) A4 (126 12 73 19.74p<0.000121 1.89 NS 10.43p<0.0001 bp) AS (124 68 77 1.58 NS 67 0.94 NS 1.68 NS
bp) A6 (122 68 6 0.019p<0.000164 0.50 NS 0.037p<0.0001 bp) A7 (120 0 0 - - 3 3.93 NS 0.27 NS
bp) ° severe or mild KA compared with controls severe compared with mild RA
NS: not statistically significant OR: odds ratio The frequency of the polymorphisms in normal subjects ranged from 0% for the 130 by and 120 by alleles, to 68% for the 122 by allele. The genotype frequencies did not deviate from the expected value by Hardy-Weinberg equilibrium. The alleles identified herein appear to correspond closely in length to those initially reported by Ruiz-Linares, which ranged from 122-134 by (Ruiz-Linares, 1994 Hum. Mol. Genet. 2(9):1508). Such alleles may vary, for example, depending upon the ethnic origin of the subjects and the methodology of characterization (see for example Awata, T. et al.
1994 Diabetologia 37:1159). In some populations, 6-8 alleles encompassing at least 11-15 CA repeats may exist at this site, including the intermediate polymorphism of 128 bp.
As shown in this example, patients with severe rheumatoid arthritis differ significantly in the frequency of 2 alleles. The 126 by allele was present in 73% of patients with severe rheumatoid arthritis compared with 21% of patients with mild rheumatoid arthritis (OR: 10.43, p < 0.0001) and 12% of normal subj ects (OR: 19.74, p < 0.0001 ). In contrast, the 122 by allele was detected in only 6% of patients with severe rheumatoid arthritis compared with 64% of patients with mild disease (OR: 0.037, p < 0.0001) and 68% of normal subjects (OR: 0.019, p < 0.0001). There was no significant difference in the frequencies of the other microsatellite polymorphisms between the three groups of individuals.
Table 2 shows a grouping of the subjects into one of four categories, depending upon their expression of the 126 by allele and the 122 by allele.
Almost three quarters (73%) of patients with severe rheumatoid arthritis expressed the 126 by allele without the 122 by allele, compared with 10% of patients with mild rheumatoid arthritis (OR: 23.56, p < 0.0001) and 4% of normal subjects (OR: 60.62, p < 0.0001). In contrast, only 6% of patients with severe rheumatoid arthritis expressed the 122 by allele without the 126 by allele compared with 54% of patients with mild rheumatoid arthritis (OR:
0.057, p < 0.0001) and 70% of normal subjects (OR: 0.029, p < 0.0001). The 126 by and 122 by alleles were not conjointly expressed by any patients with severe disease compared with conjoint expression in 10% of patients with mild disease (OR: 0.081, p = NS) and conjoint expression in 8% of normal subjects (OR: 0.11, p = NS).
Table 2: Proportion of subjects expressing the 126 by allele, 122 by allele, both or neither in the first intros of the IFNy gene. Controls (n=50), patients with severe RA (n=48) patients with mild RA (n=39).
ControlsSevere Mild RA RA
Categories% % % ORb ORa ORa Pb pa pa 126 by 4 73 60.62<0.000110 2.74 NS 23.56 <0.0001 allele 122 by 70 6 0.029<0.000154 0.50 NS 0.057 <0.0001 allele Both 8 0 0.11 NS 10 1.31 NS 0.081 NS
Neither 18 21 1.20 NS 26 1.57 NS 0.76 NS
compares wltn controls severe compared with mild NS: not statistically significant OR: odds ratio Table 3: Results of logistic regression for the effects of HLA-DR, IFN-g, and clinical measures on the odds of severe disease. Odds ratios and chi-square statistics are marginal (i.e. have been adjusted for all other factors). Odds ratios here reflect the distribution of patients observed rather than underlying prevalence of mild and severe RA. Controls (n=50), patients with severe RA
(n=48) patients with mild RA (n=39).
Severe vs. Severe vs. Severe vs.
Factor d.f. Control Mild Mild xz xz x2 HLA-DR 3 11.23* 13.04** 10.66*
IFN-y 3 65.88** 43.91*** 28.09***
RF 2 -- -- 9.33 Age 1 -- -- 0.46 Duration1 -- -- 6.57*
Gender 1 -- -- 3.84**
Factor Effect O.R. O.R. O.R.
HLA-DR H vs. 23.95* 14.55 48.27 L
B vs. 7.23 0.43 0.63 L
N vs. 2.10 2.15 2.52 L
IFN-'y H vs. 327.09*** 107.57*** 278.02**
L
B vs. 0.03 0.00 0.00 L
N vs. 19.26*** 5.13* 9.95 L
RF NA vs. 2.10 neg pos vs. 25.07*
neg Age 10 years 0.77 Duration 10 years 4.71 Gender m vs. 11.16 f ~p<u.u~; ~°p<u.m ievei; TTTp<u.um ievei.
HLA-DR: H = QKRRA/QRRAA, L = DERAA, B = Both, N = Neither IFN-g: H = 126 by allele; L = 122 by allele; B = Both; N = Neither These striking results were confirmed in a subsequent and independent group of 12 patients with severe RA who were selected according to the same clinical and laboratory criteria. Seventy-five percent of these patients expressed the 126 by allele and 8% the 122 by allele; when all subjects with severe RA were combined (n=60) the patient frequencies were unchanged from those reported in tables 1 and 2.
Logistic regression was used to examine the influences of IFN-y polymorphism, HLA DR-B 1 genotype (Wayland and Goronzy, 1997, J. Mol.
Med. 75:772) and other prognostic factors. The results are shown in table 3.
Inheritance of the INF-'y 126 by allele is strongly associated with the presence of severe RA even after accounting for HLA-DRB 1 polymorphism, while possession of the IFNy 122 by polymorphism is highly negatively associated with the presence of severe disease. The association of these IFN~y alleles with severe RA is considerably greater than that noted for the most tightly associated MHC class II alleles or other clinical predictors including gender, age at onset, duration of disease, or rheumatoid factor positivity.
_g-In accordance with one aspect of the present invention, the diagnostic test for the presence of IFN gamma alleles may be carried out on asymptomatic individuals to assess the individual's susceptibility to rheumatoid arthritis. In individuals presenting with arthritic symptoms, the S test may be utilized to assess the likelihood of progression to the severe form of the disease. In accordance with these aspects of the invention, the presence of the 126 by allele may be taken as an indication of increased susceptibility to rheumatoid arthritis, including increased susceptibility to progression of the arthritis to the severe form of the disease, as set out in Table 3.
The diagnostic test may in some embodiments be utilized to determine whether the IFN gamma alleles are homozygous or heterozygous for example, in the exemplary embodiment, 10% (6/60) of patients with severe rheumatoid arthritis were homozygous for the 126 by allele, compared with 3% (1/39) of those with mild disease and 0% (0/50) of normal controls. In contrast, none of the 60 patients with severe rheumatoid arthritis were homozygous for the 122 by allele, compared with 8% (3/39) of those with mild disease and 14% (7/50) of normal controls.
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to".
ControlsSevere Mild RA RA
Categories% % % ORb ORa ORa Pb pa pa 126 by 4 73 60.62<0.000110 2.74 NS 23.56 <0.0001 allele 122 by 70 6 0.029<0.000154 0.50 NS 0.057 <0.0001 allele Both 8 0 0.11 NS 10 1.31 NS 0.081 NS
Neither 18 21 1.20 NS 26 1.57 NS 0.76 NS
compares wltn controls severe compared with mild NS: not statistically significant OR: odds ratio Table 3: Results of logistic regression for the effects of HLA-DR, IFN-g, and clinical measures on the odds of severe disease. Odds ratios and chi-square statistics are marginal (i.e. have been adjusted for all other factors). Odds ratios here reflect the distribution of patients observed rather than underlying prevalence of mild and severe RA. Controls (n=50), patients with severe RA
(n=48) patients with mild RA (n=39).
Severe vs. Severe vs. Severe vs.
Factor d.f. Control Mild Mild xz xz x2 HLA-DR 3 11.23* 13.04** 10.66*
IFN-y 3 65.88** 43.91*** 28.09***
RF 2 -- -- 9.33 Age 1 -- -- 0.46 Duration1 -- -- 6.57*
Gender 1 -- -- 3.84**
Factor Effect O.R. O.R. O.R.
HLA-DR H vs. 23.95* 14.55 48.27 L
B vs. 7.23 0.43 0.63 L
N vs. 2.10 2.15 2.52 L
IFN-'y H vs. 327.09*** 107.57*** 278.02**
L
B vs. 0.03 0.00 0.00 L
N vs. 19.26*** 5.13* 9.95 L
RF NA vs. 2.10 neg pos vs. 25.07*
neg Age 10 years 0.77 Duration 10 years 4.71 Gender m vs. 11.16 f ~p<u.u~; ~°p<u.m ievei; TTTp<u.um ievei.
HLA-DR: H = QKRRA/QRRAA, L = DERAA, B = Both, N = Neither IFN-g: H = 126 by allele; L = 122 by allele; B = Both; N = Neither These striking results were confirmed in a subsequent and independent group of 12 patients with severe RA who were selected according to the same clinical and laboratory criteria. Seventy-five percent of these patients expressed the 126 by allele and 8% the 122 by allele; when all subjects with severe RA were combined (n=60) the patient frequencies were unchanged from those reported in tables 1 and 2.
Logistic regression was used to examine the influences of IFN-y polymorphism, HLA DR-B 1 genotype (Wayland and Goronzy, 1997, J. Mol.
Med. 75:772) and other prognostic factors. The results are shown in table 3.
Inheritance of the INF-'y 126 by allele is strongly associated with the presence of severe RA even after accounting for HLA-DRB 1 polymorphism, while possession of the IFNy 122 by polymorphism is highly negatively associated with the presence of severe disease. The association of these IFN~y alleles with severe RA is considerably greater than that noted for the most tightly associated MHC class II alleles or other clinical predictors including gender, age at onset, duration of disease, or rheumatoid factor positivity.
_g-In accordance with one aspect of the present invention, the diagnostic test for the presence of IFN gamma alleles may be carried out on asymptomatic individuals to assess the individual's susceptibility to rheumatoid arthritis. In individuals presenting with arthritic symptoms, the S test may be utilized to assess the likelihood of progression to the severe form of the disease. In accordance with these aspects of the invention, the presence of the 126 by allele may be taken as an indication of increased susceptibility to rheumatoid arthritis, including increased susceptibility to progression of the arthritis to the severe form of the disease, as set out in Table 3.
The diagnostic test may in some embodiments be utilized to determine whether the IFN gamma alleles are homozygous or heterozygous for example, in the exemplary embodiment, 10% (6/60) of patients with severe rheumatoid arthritis were homozygous for the 126 by allele, compared with 3% (1/39) of those with mild disease and 0% (0/50) of normal controls. In contrast, none of the 60 patients with severe rheumatoid arthritis were homozygous for the 122 by allele, compared with 8% (3/39) of those with mild disease and 14% (7/50) of normal controls.
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to".
Claims (16)
1. A method of diagnosis, comprising:
a) identifying a patient at risk of an arthritis, the patient having an interferon gamma gene;
b) testing the patient to characterize a polymorphism in a first intron of the interferon gamma gene.
a) identifying a patient at risk of an arthritis, the patient having an interferon gamma gene;
b) testing the patient to characterize a polymorphism in a first intron of the interferon gamma gene.
2. The method of claim 1, wherein the polymorphism comprises a variable length dinucleotide repeat region within the first intron.
3. The method of claim 2, wherein the variable length dinucleotide repeat region is at least partly located between nucleotides 1349 and 1373 in the interferon gamma gene.
4. The method of claim 1, 2 or 3 wherein the characterization of the polymorphism is carried out so as to be capable of identifying alleles selected from the group consisting of a 126bp allele and a 122bp allele.
5. The method of any one of claims 1 through 4 wherein the characterization of the polymorphism is carried out so as to be capable of resolving alleles having a different number of CA repeats in a portion of the first intron of the interferon gamma gene.
6. The method of any one of claims 1 through 5, wherein the arthritis is rheumatoid arthritis.
7. The method of any one of claims 1 through 6 wherein the patient is caucasian.
8. The method of any one of claims 1 through 7 wherein the step of identifying the patient at risk of the arthritis comprises diagnosing the patient with rheumatoid arthritis.
9. The method of any one of claims 1 through 8, wherein the step of identifying the patient at risk of the arthritis comprises diagnosing the patient with a symptom selected from the group consisting of joint erosions, elevated erythrocyte sedimentation rate, C-reactive protein, polyarticular disease, joint deformities, radiological evidence of subchondral erosions, extra-articular arthritis, and the presence of rheumatoid factor.
10. The method of any one of claims 1 through 9, wherein the characterization of the polymorphism comprises amplification of a variable length dinucleotide repeat region.
11. The use an allele of an interferon gamma gene to diagnose a patient at risk of an arthritis.
12. The use of the allele according to claim 11, wherein the allele is selected from the group consisting of alleles comprising a variable length dinucleotide repeat region within a first intron of the interferon gamma gene.
13. The use of the allele according to claim 12, wherein the variable length dinucleotide repeat region comprises a variable number of CA repeats.
14. The use of the allele according to claim 11, 12 or 13, wherein the allele is selected from the group consisting of a 126bp allele and a 122bp allele.
15. A method of diagnosing the susceptibility of a patient to arthritis, the patient having an interferon gamma gene, comprising testing the patient to characterize a polymorphism in a first intron of the interferon gamma gene.
16. A method of treating a patient having an interferon gamma gene, comprising testing the patient to characterize a polymorphism in a first intron of the interferon gamma gene; and, treating the patient for arthritis if the polymorphism is indicative of susceptibility to arthritis.
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2282062 CA2282062A1 (en) | 1999-09-08 | 1999-09-08 | Diagnostic methods in autoimmune disease |
| CA 2308049 CA2308049A1 (en) | 1999-09-08 | 2000-04-07 | Diagnostic and therapeutic methods in autoimmune disease |
| CA 2303504 CA2303504A1 (en) | 1999-09-08 | 2000-04-10 | Diagnostic methods in autoimmune disease |
| EP00960244A EP1212463A2 (en) | 1999-09-08 | 2000-09-08 | Diagnostic and therapeutic methods in autoimmune disease |
| CA002384029A CA2384029A1 (en) | 1999-09-08 | 2000-09-08 | Diagnostic and therapeutic methods in autoimmune disease |
| JP2001521775A JP2003508081A (en) | 1999-09-08 | 2000-09-08 | Diagnosis and treatment methods for autoimmune diseases |
| PCT/CA2000/001043 WO2001018240A2 (en) | 1999-09-08 | 2000-09-08 | Diagnostic and therapeutic methods in autoimmune disease |
| AU72628/00A AU7262800A (en) | 1999-09-08 | 2000-09-08 | Diagnostic and therapeutic methods in autoimmune disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2282062 CA2282062A1 (en) | 1999-09-08 | 1999-09-08 | Diagnostic methods in autoimmune disease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2282062A1 true CA2282062A1 (en) | 2001-03-08 |
Family
ID=4164112
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2282062 Abandoned CA2282062A1 (en) | 1999-09-08 | 1999-09-08 | Diagnostic methods in autoimmune disease |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2282062A1 (en) |
-
1999
- 1999-09-08 CA CA 2282062 patent/CA2282062A1/en not_active Abandoned
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