A novel single-nucleotide polymorphism at the 5'-flanking region of SAA1 associated with risk of type AA amyloidosis secondary to rheumatoid arthritis.

OBJECTIVE: To address whether the gamma haplotype at exon 3 of the SAA1 gene is directly associated with type AA amyloidosis or is merely in linkage with an unknown polymorphism that is primarily associated with disease risk, we examined the SAA1 gene for new polymorphisms. METHODS: We analyzed DNA samples from 44 rheumatoid arthritis (RA) patients with AA amyloidosis (amyloid group), 55 RA patients without AA amyloidosis (RA group), and 58 non-RA healthy subjects (non-RA group). We also examined DNA samples from 50 Caucasians to compare linkage disequilibrium relationships involving SAA1 region polymorphisms between Japanese and Caucasoid populations. RESULTS: We observed 3 novel single-nucleotide polymorphisms (SNPs) in the 5'-flanking region of SAA1: -61C/G, -13T/C, and -2G/A. Comparison of allele frequencies and ratios of individuals with particular alleles between the study groups revealed statistically significant differences between the amyloid and RA groups and between the amyloid and non-RA groups. Statistical analysis revealed that the -13T/C SNP was strongly associated with AA amyloidosis. In addition, we found tight linkage between the -13T allele and the alpha haplotype, rather than the beta haplotype, at exon 3 in the Caucasoid population, while -13T was closely linked to the gamma and beta haplotypes, rather than the alpha haplotype, in the Japanese population. Since the linkage disequilibrium relationship was reversed between the Japanese and Caucasoid populations, different exon 3 haplotypes of SAA1 are found to be associated with the risk of AA amyloidosis in different ethnic groups. CONCLUSION: Our data suggest that the SAA1 -13T allele, rather than SAA1 exon 3 haplotypes, is primarily associated with AA amyloidosis risk.     

SAA1 gene polymorphisms and the risk of AA amyloidosis in Japanese patients with rheumatoid arthritis

To investigate the precise modality of association between SAA1 gene polymorphisms and the development of AA amyloidosis in patients with rheumatoid arthritis (RA), Japanese patients with RA (n = 153), among whom 29 were histologically diagnosed as having amyloidosis, were genotyped for three single nucleotide polymorphisms (SNPs), C-13T, C2995T, and C3010T, in the SAA gene. Pairwise linkage disequilibrium coefficients between each pair of SNPs were calculated and estimated haplotype frequencies were compared between patients with and without amyloidosis. Possible associations between these SNPs and amyloidosis were analyzed by a case–control study and by the Kaplan–Meier method, in which the endpoint was defined as the time of diagnosis of AA amyloidosis. The -13T and 2995C alleles, which were in a tight linkage disequilibrium, were more frequent in the patients with amyloidosis, and the groups with the -13TT and 2995CC genotype had worse survival curves than patients without these genotypes, whereas C3010T was not associated with amyloidosis. Moreover, the haplotype containing −13C and 2995T was found to be protective. Both C-13T and C2995T were associated with the development of amyloidosis. Examining both polymorphisms may be more useful than examining only one of them for estimating the risk of the development of amyloidosis.     

SAA1 gene polymorphisms and the risk of AA amyloidosis in Japanese patients with rheumatoid arthritis

Abstract

To investigate the precise modality of association between SAA1 gene polymorphisms and the development of AA amyloidosis in patients with rheumatoid arthritis (RA), Japanese patients with RA (n = 153), among whom 29 were histologically diagnosed as having amyloidosis, were genotyped for three single nucleotide polymorphisms (SNPs), C-13T, C2995T, and C3010T, in the SAA gene. Pairwise linkage disequilibrium coefficients between each pair of SNPs were calculated and estimated haplotype frequencies were compared between patients with and without amyloidosis. Possible associations between these SNPs and amyloidosis were analyzed by a case–control study and by the Kaplan–Meier method, in which the endpoint was defined as the time of diagnosis of AA amyloidosis. The -13T and 2995C alleles, which were in a tight linkage disequilibrium, were more frequent in the patients with amyloidosis, and the groups with the -13TT and 2995CC genotype had worse survival curves than patients without these genotypes, whereas C3010T was not associated with amyloidosis. Moreover, the haplotype containing ?13C and 2995T was found to be protective. Both C-13T and C2995T were associated with the development of amyloidosis. Examining both polymorphisms may be more useful than examining only one of them for estimating the risk of the development of amyloidosis.     

Efficacy of etanercept in patients with AA amyloidosis secondary to rheumatoid arthritis

OBJECTIVE: The efficacy of biological therapies in rheumatoid arthritis (RA) is well known, but their hypothetical benefit in amyloid A (AA) amyloidosis secondary to RA still remains to be considered. We evaluated the efficacy and safety of etanercept in serum amyloid A (SAA) 1.3 allele Japanese patients with AA amyloidosis secondary to RA. METHODS: Seven RA patients with histologically confirmed AA amyloidosis and renal involvement who were treated with etanercept were enrolled. They all had the SAA1.3 allele, which has been shown to be a risk factor not only for the association of AA amyloidosis but also for a poor prognosis in Japanese RA patients. Efficacy was assessed as a sustained decrease in RA inflammation and an amelioration of renal function. RESULTS: RA inflammation and AA amyloidosis were improved and stabilized after 43.4 +/- 16.5 weeks. At week 20 the number of tender (p = 0.017) and swollen (p = 0.017) joints, and levels of serum C-reactive protein (p = 0.018) and albumin (p = 0.045) had improved. The values for SAA, serum creatinine, calculated creatinine clearance, and proteinuria also ameliorated. No severe adverse events were observed. One patient eventually had to go on hemodialysis but her tolerance of etanercept remained stable. CONCLUSION: Etanercept can be used safely and effectively in AA amyloidosis secondary to RA with renal involvement, and is of clinical benefit in the short-term, even in patients on hemodialysis. It appears that SAA1.3 allele may be used as a clinical parameter for the introduction of etanercept in Japanese RA with AA amyloidosis.     

HO‐1 promoter polymorphism associated with rheumatoid arthritis

Objective

To investigate the role of the HO‐1 gene as a novel functional candidate gene for rheumatoid arthritis (RA).

Methods

We performed a case–control study including 736 RA patients and 846 healthy controls of Spanish Caucasian origin. Two putative functional HO‐1 promoter polymorphisms, a (GT)_n microsatellite and a −413 A/T single‐nucleotide polymorphism (SNP), were selected as genetic markers and genotyped using polymerase chain reaction–based methods. In addition, the intracellular expression of heme oxygenase 1 (HO‐1) was determined in healthy individuals with different (GT)_n genotypes.

Results

The distribution of HO‐1 (GT)_n short (S) alleles (≤25 GT repeats) and long (L) alleles (>25 GT repeats) revealed a significant protective effect of S (GT)_n alleles (P = 0.019) (odds ratio [OR] 0.8, 95% confidence interval [95% CI] 0.7–0.9) and the SS (GT)_n genotype (P = 0.002) (OR 0.6, 95% CI 0.4–0.9). In contrast, the −413 HO‐1 promoter SNP did not yield any statistically significant deviation between RA patients and controls, considering either allele or genotype frequencies. The haplotype analysis showed a strong protective effect of the S/A haplotype (P = 7 × 10⁻⁷, corrected P [P_corr] = 3 × 10⁻⁶) (OR 0.4, 95% CI 0.3–0.6), whereas the L/A haplotype showed the opposite tendency (P = 0.008, P_corr = 0.03) (OR 1.2, 95% CI 1.0–1.4). In addition, we demonstrated that monocytes from individuals carrying the SS (GT)_n genotype showed a significantly higher percentage of HO‐1 expression than did cells from LL homozygous individuals (P = 0.0003).

Conclusion

In this study, we identified the HO‐1 (GT)_n microsatellite as a new genetic marker involved in RA genetics in our population.

     

Novel genetic markers in the 5′‐flanking region of ANKH are associated with ankylosing spondylitis

Objective

To use a candidate gene approach for the identification of genetic markers that are significantly linked to and associated with ankylosing spondylitis (AS).

Methods

We searched for novel polymorphisms in the ANKH gene (human homolog of the murine progressive ankylosis gene) and genotyped 2 polymorphic sites, one in the 5′‐noncoding region and the other in the promoter region of ANKH, using DNA from affected (n = 273) and unaffected (n = 112) individuals from 124 AS families. We used these ANKH and other nearby polymorphisms to perform linkage and family‐based association analyses.

Results

We identified 2 novel polymorphic sites: one in the 5′‐noncoding region of ANKH involving 1–2 copies of an 8‐bp repeat (denoted as ANKH‐OR), and the other in the promoter region involving different copy numbers of a triplet repeat (denoted as ANKH‐TR). ANKH‐OR and ANKH‐TR were in complete linkage disequilibrium. Five markers (D5S1953, ANKH‐TR, ANKH‐OR, D5S1954, and D5S1963) were used for both the linkage and association analyses. Multipoint linkage analysis of 124 AS families showed a modest level of significance (nonparametric linkage score 2.15; P = 0.015) at the ANKH region. The contribution of ANKH to AS susceptibility (λ_s) was 1.9. A family‐based association study on the same AS families revealed that both ANKH‐OR allele 1 and ANKH‐TR allele 7 were significantly associated with disease, assuming an additive model (for ANKH‐OR allele 1, P = 0.03; for ANKH‐TR allele 7, P = 0.04).

Conclusion

Our results indicate that ANKH‐OR and ANKH‐TR are novel genetic markers that are significantly associated with AS.

     

Significance of SAA1.3 allele genotype in Japanese patients with amyloidosis secondary to rheumatoid arthritis

OBJECTIVE: To clarify the clinical significance of the SAA1.3 allele in the development and outcome of AA amyloidosis in Japanese patients with rheumatoid arthritis (RA). METHODS: One hundred and twenty RA patients (60 alive and 60 dead) fulfilling the 1987 ACR criteria and 62 RA patients with biopsy-confirmed amyloid A (AA) amyloidosis (36 alive and 26 dead) were enrolled. The SAA1 genotypes were determined by PCR-based restriction fragment length polymorphism. To predict the clinical outcome of AA amyloidosis, we investigated characteristics and survival, focusing on the SAA1.3 allele retrospectively. RESULTS: The SAA1.3 allele genotype was not only a risk factor for the association of AA amyloidosis but also a poor prognostic factor for the development of AA amyloidosis (P=0.015). Both the association of AA amyloidosis arising early in the RA disease course and symptomatic variety and severity were found in amyloidotic patients with the SAA1.3 allele. The presenting factors adversely influenced were age (P=0.001), lowered serum albumin (P=0.001) and creatinine concentration (P=2.14 x 10(-5)). Renal involvement was associated with poor survival in patients with AA amyloidosis (P=0.011) and the presence of cardiac involvement was likely to be a risk factor for survival (P=0.062). The rate of the causes of death in respect to the category of infection, gastrointestinal diseases, and renal failure was higher in patients with AA amyloidosis than in those without amyloidosis, gastrointestinal diseases and renal failure. Cyclophosphamide was found to be superior to methotrexate in the management of RA patients with AA amyloidosis. CONCLUSION: Our data support the fact that homozygosity for the SAA1.3 allele is a univariate predictor of survival in addition to a risk factor for the association of AA amyloidosis adversely influencing the outcome in Japanese RA patients. Renal involvement is a pivotal clinical manifestation in the development of AA amyloidosis, as is likely to be cardiac involvement in AA amyloidosis secondary to RA.     

A case of AA amyloidosis associated with rheumatoid arthritis effectively treated with Infliximab

We report the case of a 55-year-old Japanese woman with reactive AA amyloidosis associated with rheumatoid arthritis, in which inflammatory disease was completely suppressed with infliximab. Nephrotic syndrome was observed and renal biopsy specimens revealed amyloidosis deposits. Treatment with infliximab normalized the serum amyloid A (SAA) protein level, and subsequently nephritic syndrome disappeared and her creatinine clearance improved. Serial gastrointestinal biopsy specimens showed marked lasting regression of amyloid deposits. Thus treatment with infliximab represents an important therapeutic strategy for AA amyloidosis associated with RA.     

A study of rheumatoid arthritis patients associated with biopsy-proven secondary amyloidosis

Reactive systemic amyloidosis associated with rheumatoid arthritis (RA) was studied clinically in 28 patients (2 men and 26 women). The diagnosis of amyloidosis was established by histological examination of biopsy materials. Upper gastrointestinal tract biopsy was performed in 14 patients, and renal and rectal biopsy in 8 and 4 respectively. The mean age and duration of RA at diagnosis of amyloidosis were 58.6 (range 35-72) years and 15.5 (range 4-44) years respectively. Almost all patients had intractable and progressive courses of RA. Serological activities determined by C-reactive protein (CRP) and erythrocyte sedimentation rates were moderate to high in over 80% of the cases. Renal abnormalities were noticed in 19 cases, and gastrointestinal disorders in 10. Eight patients died from 1 to 54 (mean 15.3) months after the diagnosis of amyloidosis; 5 died of renal failure and 2 of gastrointestinal involvements. Renal impairments progressed frequently and serum creatinine elevated over 1.5 mg/dl in another 8 cases. Five patients progressing to renal failure were treated with hemodialysis. Three died within several weeks after the induction of hemodialysis, although 2 were treated for more than 2 years. Intractable hypotension and pulmonary congestion were frequently observed in these cases. A close relationship was found between serum amyloid A protein (SAA) and CRP concentration, so that the measurement of SAA seemed to be valuable in assessing disease activity. Concerning the treatment of amyloidosis, cyclophosphamide and corticosteroids seemed to be effective in several cases, although it had been unsatisfactory in most cases.     

Efficacy of cyclophosphamide combined with prednisolone in patients with AA amyloidosis secondary to rheumatoid arthritis

Secondary amyloid A (AA) amyloidosis is an uncommon yet important complication of rheumatoid arthritis (RA). It is one of the most relentless of the extra-articular features of RA, and suitable treatments have not yet been found. We studied the efficacy of cyclophosphamide (CYC) combined with prednisolone (PSL) in amyloidotic patients who had serum amyloid A (SAA) 1.3 genotype, which is a risk factor for secondary amyloidosis in Japanese RA patients. Fifteen RA patients who were SAA1.3 homo- and heterozygotes with biopsy-confirmed AA amyloidosis were treated with a combination of CYC and PSL. Laboratory variables of C-reactive protein (CRP), rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), serum albumin (Alb), serum creatinine (Cre) and Lansburys index were carried out by statistical analysis of changes between before and during the medication. According to the Mann–Whitney rank test, CRP, RF, ESR, Alb and Cre levels improved significantly with the combination treatment (p<0.05). Also, paired t-tests showed significance in Lansburys index between before and during the medication (p=0.007). CYC combined with PSL ameliorated not only laboratory markers but also clinical rheumatoid activity in patients with amyloidosis secondary to RA, whose genotypes were SAA1.3 homo- and heterozygous. CRP, ESR, RF, Alb and Cre will be surrogate markers of therapeutic efficacy. The combination of CYC and PSL appears to be beneficial for Japanese RA patients who are SAA1.3 homo- and heterozygous carriers, associated with secondary AA amyloidosis.Abbreviations AA Amyloid A - CRP C-reactive protein - Ccr Creatinine clearance - CYC Cyclophosphamide - DMARD Disease-modifying antirheumatic drugs - ESR Erythrocyte sedimentation rate - NSAID Non-steroidal anti-inflammatory drugs - PCR Polymerase chain reaction - PSL Prednisolone - RA Rheumatoid arthritis - RF Rheumatoid factor - SAA Serum amyloid A     

Association of the IL4R single‐nucleotide polymorphism I50V with rapidly erosive rheumatoid arthritis

Objective

To examine whether single‐nucleotide polymorphisms (SNPs) of the interleukin‐4 receptor gene IL4R influence susceptibility to, or radiographic progression in, rheumatoid arthritis (RA).

Methods

The contribution of 2 SNPs (I50V and Q551R) in the coding region of IL4R to RA susceptibility was analyzed by allele‐specific polymerase chain reaction in a case–control study of 471 RA patients and 371 healthy controls. Patients with available radiographs of the hands and feet obtained 2 years after disease onset (n = 302) were stratified retrospectively according to radiologic outcome into an erosive and a nonerosive group to evaluate the association between IL4R SNPs and disease progression.

Results

No differences in the genotype and allele frequencies of the I50V or Q551R SNPs were identified between the RA patients and healthy controls. In contrast, significant differences in the distribution of I50V IL4R SNP genotypes between patients with erosive and nonerosive disease were observed (χ² = 15.68, P = 0.0004). Bone erosions at 2 years after disease onset were present in 68.1% of patients homozygous for the V50 allele compared with 37.0% of patients homozygous for the I50 allele (odds ratio 3.86, P < 0.0001). This association was independent of individual factors previously associated with severe disease, such as rheumatoid factor or the HLA–DR shared epitope. On a cellular level, the V50 allele conferred significantly reduced responsiveness to interleukin‐4, providing a possible mechanism for the association of the I50V IL4R polymorphism with early erosions in RA.

Conclusion

Our data identify the I50V IL4R SNP as a novel genetic marker in RA, showing high predictive value for early joint destruction.

     

Two single‐nucleotide polymorphisms in the 5′ and 3′ ends of the osteopontin gene contribute to susceptibility to systemic lupus erythematosus

Objective

To test the association of osteopontin (OPN) polymorphisms with systemic lupus erythematosus (SLE).

Methods

The coding 5′ and 3′ flanking regions of the OPN gene were scanned for polymorphisms by denaturing high‐performance liquid chromatography. A case–control association study was performed in 394 Italian SLE patients and 479 matched controls. OPN serum levels were determined by enzyme‐linked immunosorbent assay in 40 patients and 124 controls, and the mean levels were compared between the different OPN genotypes.

Results

Among the 13 detected single‐nucleotide polymorphisms (SNPs), alleles −156G (frequency 0.714 versus 0.651; P = 0.006, corrected P [P_corr] = 0.036) and +1239C (0.377 versus 0.297; P = 0.00094, P_corr = 0.0056) were significantly increased in the SLE patients compared with the controls. The presence of the associated allele in single or double dose conferred an odds ratio (OR) of 2.35 (95% confidence interval [95% CI] 1.38–4.02) for SNP −156 and an OR of 1.57 (95% CI 1.16–2.13) for SNP +1239. These effects were independent of each other, i.e., not a consequence of linkage disequilibrium between the 2 alleles. The risk associated with a double dose of susceptibility alleles at both SNPs was 3.8‐fold higher (95% CI 2.0–7.4) relative to the complete absence of susceptibility alleles. With regard to individual clinical and immunologic features, a significant association was seen between lymphadenopathy and −156 genotypes (overall P = 0.0011, P_corr = 0.046). A significantly increased OPN serum level was detected in healthy individuals carrying +1239C (P = 0.002), which is indicative of an association between the SLE susceptibility allele and OPN levels.

Conclusion

These data suggest the independent effect of a promoter (−156) and a 3′‐untranslated region (+1239) SNP in SLE susceptibility. We can speculate that these sequence variants (or others in perfect linkage disequilibrium) create a predisposition to high production of OPN, and that this in turn may confer susceptibility to SLE.

     

Spontaneous pneumoperitoneum: an unusual complication of systemic reactive AA amyloidosis secondary to rheumatoid arthritis.

We report a 71-year-old man with reactive AA amyloidosis secondary to rheumatoid arthritis who developed spontaneous pneumoperitoneum with intestinal pseudo-obstruction as an initial symptom. Severe deposition of amyloid in the intestinal wall was considered to play an important role in the pathogenesis of this unusual symptom. The patient has been successfully treated with total parenteral alimentation and intermediate-dose prednisolone (30 mg/day). Although pneumoperitoneum usually suggests gastrointestinal perforation requiring emergency surgery, conservative therapy should be seriously considered in amyloidosis-related cases with no associated peritonitis, since multiple vital organs are probably involved by severe amyloid deposition, thus increasing the risks of surgery.     

A genetic association between juvenile rheumatoid arthritis and a novel interleukin-1α polymorphism

Objective. The genetic factors that predispose to the development of juvenile rheumatoid arthritis (JRA) and its complications are not completely understood. The cytokine interleukin-1 (IL-1) has been implicated in the pathogenesis of JRA and other inflammatory diseases. This study was performed to test whether polymorphisms of the IL-1α gene might be associated with JRA. Methods. We sequenced the 5' regulatory region (containing the promoter) of the human IL-1α gene in 18 normal subjects. This revealed a C (IL-1A1) to T (IL-1A2) transition polymorphism at position -889. We studied the frequencies of both alleles in patients with JRA (n = 269) and controls (n = 99). Results. An increased gene carriage of IL-1A2 was found in patients with early-onset, pauciarticular JRA (EOPA-JRA; n = 103) compared with controls (0.66 versus 0.49; P = 0.01, odds ratio [OR] = 2.1). Within this subset of JRA, the association with IL-1A2 was particularly strong in the patients in whom chronic iridocyclitis developed (n = 28) compared with those without chronic iridocyclitis (0.89 versus 0.57; P = 0.002, OR = 6.2). Within the group of EOPA-JRA patients, IL-1A2 was also associated with elevation of the erythrocyte sedimentation rate (P < 0.0025). Conclusion. This is the first report of a cytokine gene association with JRA, and we conclude that IL-1α itself, or a gene for which the IL-1α polymorphism is a marker, may contribute to the pathogenesis of EOPA-JRA and the ocular complications found in this group.