Variation in the matrix metalloproteinase-3, -7, -12 and -13 genes is associated with functional status in rheumatoid arthritisS. Ye, N. Patodi, K. Walker-Bone, I. Reading, C. Cooper

& E. DennisonInternational Journal of Immunogenetics 2006: 34, 81–85

Rheumatoid arthritis Rheumatoid arthritis (RA) is a

multifactorial, polygenic disorder with an estimated heritability of 60% (Jirholt et al., 2001; Svendsen et al., 2002).

Susceptibility to and severity of the disease has been associated with variations of human leukocyte antigen genes on chromosome 6

The pathogenesis of RA is characterized by chronic joint inflammation, and in most cases the synovitis will lead to permanent damage of the articular cartilage and bone

Matrix metalloproteinasesMatrix metalloproteinases

(MMPs), a group of proteases expressed in rheumatoid synovium, play an important role in the above pathological processes

The genes for MMP1, MMP3, MMP7, MMP12 and MMP13 contain single nucleotide polymorphisms that appear to also have an influence on their expression

Aim In this study, the authors examined whether in RA

patients, these MMP gene polymorphisms are associated with the degree of physical impairment as assessed by the Steinbrocker index (Steinbrocker et al., 1949) and the Health Assessment Questionnaire (HAQ) score (Kirwan & Reeback, 1986), both of which are indicators of the degree and extent of joint inflammation and tissue damage and have been shown to be associated with other parameters including Ritchie articular index (a measure of joint inflammation), circulating inflammatory markers such as the erythrocyte sedimentation rate and C-reactive protein, and the presence of erosive disease on hand radiographs

Subjects Eighty two British Caucasian patients (27 men and

55 women) who fulfilled the American College of Rheumatology 1987 criteria for RA and who were seropositive for rheumatoid factor were recruited to the study.

Each subject was approached by a trained rheumatologist when they attended outpatients as part of their normal rheumatology follow-up, and written consent was obtained.

Review of case notes was performed to obtain information on duration of disease, presence of erosive disease, current erythrocyte sedimentation rate, prednisolone therapy and second-line medication (with duration).

Steinbrocker score & HAQ score The patient was then invited to complete

the Steinbrocker criteria (which divides patients into four broad functional classes according to their ability to perform usual self-care, vocational and avocational activities) (Steinbrocker et al., 1949) and

HAQ (a short self-report questionnaire of 20 questions in 8 categories that asks about the difficulties patients have with certain activities) (Kirwan & Reeback, 1986) and the Ritchie articular index (Ritchie et al., 1968) was calculated. The study conformed to the Declaration of Helskinki guidelines.

Determination of genotypesThe genotyping for the MMP1-

1607 1G > 2G, MMP3-1612 5A > 6A, MMP7-181G > A, MMP12- 82A > G and MMP13-77 A > G polymorphisms were done using PCR-PFLP

The digests were fractionated by gel electrophoresis, stained with Vistra Green and visualized using a fluorimager.

Statistical analysis

The STATA statistical software package was used for the analyses.

Univariate analysis was performed to examine relationships between functional outcome (Steinbrocker score or HAQ score) and the following variables: age, sex, erythrocyte sedimentation rate, presence of erosive disease, Ritchie score and prednisolone therapy.

Statistical analysis Allele and genotype frequencies were calculated by

the gene counting method. The Hardy–Weinberg equilibrium (HWE) programme

(ftp://linkage.rockefeller.edu/software/utilities) was used to assess whether the genotype distribution of the polymorphisms was in HWE.

Linear regression analyses were performed to explore the associations between the MMP gene polymorphisms and functional outcome (Steinbrocker index and HAQ score) of RA, first without and then with, adjustment for age, sex, erythrocyte sedimentation rate, presence of erosive disease, Ritchie score, prednisolone therapy and years of diagnosis.

Table 1. Characteristics of subjects

Table 2. Steinbrocker index and HAQ score in different genotype groups

Results The genotype distributions of all polymorphisms were in

agreement with HWE, and the allele frequencies were similar to those previously reported in Caucasian samples

Steinbrocker and HAQ scores were highly correlated (r = 0.78, P < 0.0001) and higher in women than men (P < 0.05).

There was no association of Steinbrocker score or HAQ score with age. Similarly, patients with a longer duration of disease had higher Steinbrocker (r = 0.27, P = 0.01) and HAQ scores (r = 0.29, P = 0.009), respectively.

There was no significant association of Steinbrocker or HAQ score with erythrocyte sedimentation rate, presence of erosive disease or prednisolone therapy.

However, both Steinbrocker and HAQ scores were higher in patients with a higher Ritchie index (P < 0.001).

Results There was an association between the MMP13 - 77A

> G polymorphism and Steinbrocker index, with patients of the A/A genotype having highest mean score, and the A allele appeared to have a recessive effect (P = 0.005).

The association remained significant after adjusting for age, sex, erythrocyte sedimentation rate, presence of erosive disease, Ritchie score, prednisolone therapy and years of diagnosis (P = 0.003).

Results There was also an association between the MMP3-

1612 5A/6A polymorphism and Steinbrocker index, with patients of the 5A/5A genotype having the highest mean score. The 5A allele appeared to have a dose effect (P = 0.021 under an additive genetic model) or a recessive effect (5A/5A compared with 5A/6A and 6A/ 6A, P = 0.020). The statistical significance of the association was reduced after adjusting for age, sex, erythrocyte sedimentation rate, presence of erosive disease, Ritchie score, prednisolone therapy and years of diagnosis (P = 0.065 under the additive genetic model and P = 0.082 under the recessive model).

Results

An association between the MMP7-181A > G polymorphism and Steinbrocker score was observed, with patients of the A/A genotype having lower score than patients of the A/G or G/G genotypes, after adjusting for the covariates mentioned previously (P = 0.037).

Results

An association between the MMP12-82A > G polymorphism and Steinbrocker score was also detected, with patients of the A/A genotype having higher score than patients of the A/G genotype (none of the patients was of the G/G genotype), after adjusting for the covariates mentioned previously (P = 0.043).

Discussion The degree of disability expressed by a

patient with RA is known to be related to a number of factors, such as the degree and extent of tissue inflammation and the amount of structural damage.

MMPs that can degrade various structural proteins and whose expression is increased in synovial cells and chondrocytes in arthritic joints are known to play an important role in recruitment of inflammatory cells and in cartilage and bone degradation

Discussion These high-expression alleles were found in

this study to be associated with more severe physical functional impairment indicated by higher Steinbroker score in RA patients.

The finding of an association between MMP gene promoter polymorphisms and RA phenotypes suggest that genetic polymorphisms could also be a factor that influences MMP expression during the pathogenesis of RA.

Discussion Six polymorphisms versus two phenotypes

(Steinbrocker and HAQ scores) were tested in this study, and a Bonferroni correction for the number of test (n = 12) would require that the P value is < 0.0041 for a test to be considered significant at the α = 0.05 level. If this correction is applied, the MMP13-77A > G with Steinbrocker score (P = 0.002) would be significant, whereas the effect of the MMP3-1612 5A > 6A, MMP7-181A > G and MMP12-82A > G polymorphisms would fail to reach significance.

This correction, however, is likely to be conservative, as the polymorphisms were correlated and so were the two phenotypes.

Summary The results of this hypothesis-generating

study indicate that the MMP3, MMP12 and MMP13 gene polymorphisms are likely to be among genetic factors that underlie inter-individual variability in the functional outcome of RA.

As identifying the underlying genetic factors could enhance our understanding of the pathogenesis of this complex disorder that could eventually lead to improved prevention and treatment of this common disease, further studies of MMP gene polymorphisms to include the MMP3, MMP12 and MMP13 polymorphisms, in large cohorts of RA patients would be warranted.