Do genetic mutations and genotypes contribute to onychomycosis?

BACKGROUND: The variability in susceptibility to onychomycosis for individuals exposed to the same environmental risk factors raises the possibility that there may be individuals with a genetic predisposition to dermatophyte infection. OBJECTIVE: To determine whether there are genetic mutations or genotypes which contribute to onychomycosis. METHODS: The PubMed database was searched for examples of immune deficiencies resulting in dermatophyte infections. RESULTS: There are mutations in the innate immune receptors Dectin-1 and its adaptor protein CARD9 which result in familial mucocutaneous infections. There are also specific human leukocyte antigen genotypes that are more common in individuals and families with a high prevalence of onychomycosis. In addition, some patients have been reported with insufficient levels of CD4+CD25+ regulatory T cells. These deficits impair a full innate and adaptive immune response and may result in chronic or recurrent infections. CONCLUSIONS: There are documented mutations and genotypes that contribute to familial and individual susceptibility to onychomycosis.

Preventing and curing citrulline-induced autoimmune arthritis in a humanized mouse model using a Th2-polarizing iNKT cell agonist.

Invariant natural killer T (iNKT) cells are innate lymphocytes with unique reactivity to glycolipid antigens bound to non-polymorphic CD1d molecules. They are capable of rapidly releasing pro- and/or anti-inflammatory cytokines and constitute attractive targets for immunotherapy of a wide range of diseases including autoimmune disorders. In this study, we have explored the beneficial effects of OCH, a Th2-polarizing glycolipid agonist of iNKT cells, in a humanized mouse model of rheumatoid arthritis (RA) in which citrullinated human proteins are targeted by autoaggressive immune responses in mice expressing an RA susceptibility human leukocyte antigen (HLA) DR4 molecule. We found for the first time that treatment with OCH both prevents and cures citrulline-induced autoimmune arthritis as evidenced by resolved ankle swelling and reversed histopathological changes associated with arthritis. Also importantly, OCH treatment blocked the arthritogenic capacity of citrullinated antigen-experienced splenocytes without compromising their global responsiveness or altering the proportion of splenic naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T cells. Interestingly, administering the Th1-promoting iNKT cell glycolipid ligand α-C-galactosylceramide into HLA-DR4 transgenic mice increased the incidence of arthritis in these animals and exacerbated their clinical symptoms, strongly suggesting a role for Th1 responses in the pathogenesis of citrulline-induced arthritis. Therefore, our findings indicate a role for Th1-mediated immunopathology in citrulline-induced arthritis and provide the first evidence that iNKT cell manipulation by Th2-skewing glycolipids may be of therapeutic value in this clinically relevant model, a finding that is potentially translatable to human RA.

Systematic review of nondermatophyte mold onychomycosis: diagnosis, clinical types, epidemiology, and treatment.

Nondermatophyte mold (NDM) onychomycosis is difficult to diagnose given that NDMs are common contaminants of the nails and of the mycology laboratory. Diagnostic criteria and definition of cure are inconsistent between studies, which may affect the quality of published data. We identified 6 major criteria used in the literature: identification of the NDM in the nail by microscopy (using potassium hydroxide preparation), isolation in culture, repeated isolation in culture, inoculum counting, failure to isolate a dermatophyte in culture, and histology. Most studies used 3 or more of these (range = 1-5). We recommend using at least 3 of the criteria to rule out contamination; these should include potassium hydroxide preparation for direct microscopy and isolation of the organism in culture. We review geographic distribution and clinical presentations associated with different NDMs. The treatment with the greatest quantity of data and highest reported cure rates is terbinafine, for the treatment of Scopulariopsis brevicaulis and Aspergillus species infections. Topicals such as ciclopirox nail lacquer may also be effective (data originating from Scopulariopsis brevicaulis and Acremonium species infections), especially when combined with chemical or surgical avulsion of the nail. We recommend that future studies use (and clearly indicate) at least 3 of the main criteria for diagnosis, and report the clinical type of onychomycosis and the isolated organism. When evaluating different treatments, we suggest that authors clearly define their efficacy outcomes.

Immunization with Porphyromonas gingivalis enolase induces autoimmunity to mammalian α-enolase and arthritis in DR4-IE-transgenic mice.

OBJECTIVE: To examine the hypothesis that the subset of rheumatoid arthritis (RA) characterized by antibodies to citrullinated α-enolase is mediated by Porphyromonas gingivalis enolase in the context of DR4 alleles. METHODS: Recombinant human α-enolase and P gingivalis enolase, either citrullinated or uncitrullinated, were used to immunize DR4-IE-transgenic mice and control mice (class II major histocompatibility complex-deficient [class II MHC(-/-)] and C57BL/6 wild-type mice). Arthritis was quantified by measurement of ankle swelling in the hind paws and histologic examination. Serum IgG reactivity with α-enolase and citrullinated α-enolase was assayed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Antibodies to peptide 1 of citrullinated α-enolase (CEP-1) and its arginine-bearing control peptide, REP-1, were also assessed by ELISA. RESULTS: Significant hind-ankle swelling (≥0.3 mm) occurred in DR4-IE-transgenic mice immunized with citrullinated human α-enolase (9 of 12 mice), uncitrullinated human α-enolase (9 of 12 mice), citrullinated P gingivalis enolase (6 of 6 mice), and uncitrullinated P gingivalis enolase (6 of 6 mice). Swelling peaked on day 24. None of the control groups developed arthritis. The arthritic joints showed synovial hyperplasia and erosions, but there was a paucity of leukocyte infiltration. Antibodies to human α-enolase, both citrullinated and unmodified, and to CEP-1 and REP-1 were detectable in all immunized mice except the class II MHC(-/-) control mice. CONCLUSION: This is the first animal model that links an immune response to P gingivalis enolase to an important subset of RA, defined by antibodies to citrullinated α-enolase in the context of DR4. The fact that arthritis and anti-CEP-1 antibodies were induced independent of citrullination of the immunizing antigen suggests that the unmodified form of α-enolase may be important in initiating the corresponding subset of human RA.

Interventions for actinic keratoses.

BACKGROUND: Actinic keratoses are a skin disease caused by long-term sun exposure, and their lesions have the potential to develop into squamous cell carcinoma. Treatments for actinic keratoses are sought for cosmetic reasons, for the relief of associated symptoms, or for the prevention of skin cancer development. Detectable lesions are often associated with alteration of the surrounding skin (field) where subclinical lesions might be present. The interventions available for the treatment of actinic keratoses include individual lesion-based (e.g. cryotherapy) or field-directed (e.g. topical) treatments. These might vary in terms of efficacy, safety, and cosmetic outcomes. OBJECTIVES: To assess the effects of topical, oral, mechanical, and chemical interventions for actinic keratosis. SEARCH METHODS: We searched the following databases up to March 2011: the Cochrane Skin Group Specialised Register, CENTRAL in The Cochrane Library, MEDLINE (from 2005), EMBASE (from 2010), and LILACS (from 1982). We also searched trials registers, conference proceedings, and grey literature sources. SELECTION CRITERIA: Randomised controlled trials (RCTs) comparing the treatment of actinic keratoses with either placebo, vehicle, or another active therapy. DATA COLLECTION AND ANALYSIS: At least two authors independently abstracted data, which included adverse events, and assessed the quality of evidence. We performed meta-analysis to calculate a weighted treatment effect across trials, and we expressed the results as risk ratios (RR) and 95% confidence intervals (CI) for dichotomous outcomes (e.g. participant complete clearance rates), and mean difference (MD) and 95% CI for continuous outcomes (e.g. mean reduction in lesion counts). MAIN RESULTS: We included 83 RCTs in this review, with a total of 10,036 participants. The RCTs covered 18 topical treatments, 1 oral treatment, 2 mechanical interventions, and 3 chemical interventions, including photodynamic therapy (PDT). Most of the studies lacked descriptions of some methodological details, such as the generation of the randomisation sequence or allocation concealment, and half of the studies had a high risk of reporting bias. Study comparison was difficult because of the multiple parameters used to report efficacy and safety outcomes, as well as statistical limitations. We found no data on the possible reduction of squamous cell carcinoma.The primary outcome 'participant complete clearance' significantly favoured four field-directed treatments compared to vehicle or placebo: 3% diclofenac in 2.5% hyaluronic acid (RR 2.46, 95% CI 1.66 to 3.66; 3 studies with 420 participants), 0.5% 5-fluorouracil (RR 8.86, 95% CI: 3.67 to 21.44; 3 studies with 522 participants), 5% imiquimod (RR 7.70, 95% CI 4.63 to 12.79; 9 studies with1871 participants), and 0.025% to 0.05% ingenol mebutate (RR 4.50, 95% CI 2.61 to 7.74; 2 studies with 456 participants).It also significantly favoured the treatment of individual lesions with photodynamic therapy (PDT) compared to placebo-PDT with the following photosensitisers: aminolevulinic acid (ALA) (blue light: RR 6.22, 95% CI 2.88 to 13.43; 1 study with 243 participants, aminolevulinic acid (ALA) (red light: RR 5.94, 95% CI 3.35 to 10.54; 3 studies with 422 participants), and methyl aminolevulinate (MAL) (red light: RR 4.46, 95% CI 3.17 to 6.28; 5 studies with 482 participants). ALA-PDT was also significantly favoured compared to cryotherapy (RR 1.31, 95% CI 1.05 to 1.64).The corresponding comparative risks in terms of number of participants completely cleared per 1000 were as follows: 313 with 3% diclofenac compared to 127 with 2.5% hyaluronic acid; 136 with 0.5% 5-fluorouracil compared to 15 with placebo; 371 with 5% imiquimod compared to 48 with placebo; 331 with ingenol mebutate compared to 73 with vehicle; 527 to 656 with ALA/MAL-PDT treatment compared to 89 to 147 for placebo-PDT; and 580 with ALA-PDT compared to 443 with cryotherapy.5% 5-fluorouracil efficacy was not compared to placebo, but it was comparable to 5% imiquimod (RR 1.85, 95% Cl 0.41 to 8.33).A significant number of participants withdrew because of adverse events with 144 participants affected out of 1000 taking 3% diclofenac in 2.5% hyaluronic acid, compared to 40 participants affected out of 1000 taking 2.5% hyaluronic acid alone, and 56 participants affected out of 1000 taking 5% imiquimod compared to 21 participants affected out of 1000 taking placebo.Based on investigator and participant evaluation, imiquimod treatment and photodynamic therapy resulted in better cosmetic outcomes than cryotherapy and 5-fluorouracil. AUTHORS' CONCLUSIONS: For individual lesions, photodynamic therapy appears more effective and has a better cosmetic outcome than cryotherapy. For field-directed treatments, diclofenac, 5-fluorouracil, imiquimod, and ingenol mebutate had similar efficacy, but their associated adverse events and cosmetic outcomes are different. More direct comparisons between these treatments are needed to determine the best therapeutic approach.

CTLA-4Ig blocks the development and progression of citrullinated fibrinogen-induced arthritis in DR4-transgenic mice.

OBJECTIVE: To assess the role of T cells in the mouse model of citrullinated human fibrinogen-induced rheumatoid arthritis (RA) using CTLA-4Ig, an agent that blocks T cell costimulation, which is required for T cell activation. METHODS: Humanized HLA-DRß1*0401-transgenic (DR4-Tg) mice were immunized with Cit-human fibrinogen to induce arthritis. Prior to, and at the onset or peak of, arthritis, the DR4-Tg mice were treated with CTLA-4Ig or control human IgG1 or were left untreated. Arthritis development and progression were monitored by measuring ankle swelling with calipers and by assessing histopathologic changes. The immune responses to the citrullinated antigens and the corresponding unmodified antigens, as well as the arthritogenicity of lymphocytes from these mice, were examined. The latter was performed using lymphocyte transfers from CTLA-4Ig-treated or control mice via intraperitoneal injection into naive DR4-Tg mice. Recipient mice also received an intraarticular injection of Cit-human fibrinogen, unmodified human fibrinogen, or vehicle. RESULTS: CTLA-4Ig-treated, but not human IgG1-treated, arthritic mice had significantly reduced ankle swelling and pathologic joint damage. Treatment with CTLA-4Ig, but not human IgG1, suppressed Cit-human fibrinogen-induced T cell activation, including citrulline-specific T cell activation, when given prior to disease onset. Transfer of splenic lymphocytes from untreated or human IgG1-treated arthritic mice caused arthritis in recipients, and this occurred when Cit-human fibrinogen, but not unmodified fibrinogen, was deposited into the joint. Splenocytes from CTLA-4Ig-treated mice were unable to transfer arthritis. CONCLUSION: Activated citrulline-specific T cells play a direct role in the development and progression of arthritis in this model of Cit-human fibrinogen-induced RA.

Aberrant IgG galactosylation precedes disease onset, correlates with disease activity, and is prevalent in autoantibodies in rheumatoid arthritis.

OBJECTIVE: To examine the association between aberrant IgG galactosylation and disease parameters in rheumatoid arthritis (RA). METHODS: Analysis of N-glycan in serum samples from multiple cohorts was performed. The IgG N-glycan content and the timing of N-glycan aberrancy relative to disease onset were compared in healthy subjects and in patients with RA. Correlations between aberrant galactosylation and disease activity were assessed in the RA cohorts. The impact of disease activity, sex, age, anti-cyclic citrullinated peptide (anti-CCP) antibody titer, disease duration, and C-reactive protein level on aberrant galactosylation was determined using multivariate analysis. The N-glycan content was also compared between epitope affinity-purified autoantibodies and the remaining IgG repertoire in RA patients. RESULTS: Our results confirm the aberrant galactosylation of IgG in RA patients as compared with healthy controls (mean +/- SD 1.36 +/- 0.43 versus 1.01 +/- 0.23; P < 0.0001). We observed a significant correlation between levels of aberrant IgG galactosylation and disease activity (Spearman's rho = 0.37, P < 0.0001). This correlation was higher in women (Spearman's rho = 0.60, P < 0.0001) than in men (Spearman's rho = 0.16, P = 0.10). Further, aberrant IgG galactosylation substantially predated the onset of arthritis and the diagnosis of RA (3.5 years) and resided selectively in the anticitrullinated antigen fraction. CONCLUSION: Our findings identify aberrant IgG galactosylation as a dysregulated component of the humoral immune response in RA that begins prior to disease onset, associates with disease activity in a sex-specific manner, and resides preferentially in autoantibodies.

Severe necrotizing pancreatitis following combined hepatitis A and B vaccination.

Necrotizing pancreatitis is a severe form of pancreatitis and is associated with substantial morbidity and mortality. We report a case of necrotizing pancreatitis that developed following combined hepatitis A and B vaccination. No other causes of pancreatitis could be determined. Although confirming the diagnosis is challenging, 3 main factors suggest a possible link to the vaccine: the chronology of the events, the patient's human leukocyte antigen genotype and the incongruent immune response to the vaccine components. This report serves to alert physicians to the possible development of necrotizing pancreatitis after vaccination.

Clostridium difficile infection in rural Ontario: a retrospective multisite population-based study.

INTRODUCTION: We conducted a retrospective, population-based study to assess the prevalence of Clostridium difficile infections and the associated risk factors among inpatients and outpatients in our region. METHODS: We used laboratory data over a 2-year period to identify inpatient and outpatient cases of C. difficile infection. Data were collected from 3 local catchment areas for rural hospital laboratories in Sioux Lookout, Mount Forest and the South Huron Hospital Association in Exeter. We gathered demographic data and infection-specific information, including recent antibiotic use and recent or current hospital admission or nursing home stay. RESULTS: During the study period, 34 cases of C. difficile infection occurred in 29 patients, with an estimated crude annual rate of 24.3/100,000 population. Of the cases, 47.1% were diagnosed in outpatients. Most patients (76.5%) had taken antibiotics within the previous 90 days, and antibiotic use and hospital admission accounted for 47.1% of cases. Clindamycin was more commonly associated with C. difficile infections at the northern site and ciprofloxacin at the southern sites. There were 2 deaths from comorbidities. CONCLUSION: The estimated annual incidence of C. difficile infection in our study is similar to urban-based estimates. Almost half of the cases involved outpatients, indicating a need to recognize this illness as a serious outpatient condition. Antibiotic stewardship is an ongoing consideration, as most patients were exposed to antibiotic use before infection.

INTRODUCTION: Nous avons effectué une étude rétrospective basée dans la population pour évaluer la prévalence des infections à Clostridium difficile et les facteurs associés chez les patients hospitalisés et non hospitalisés de notre région.. MÉTHODES: Nous avons utilisé les données de laboratoire sur une période de 2 ans pour recenser les cas d'infections à C. difficile chez les patients hospitalisés et non hospitalisés. Les données ont été recueillies à partir de 3 bassins de population locaux pour les laboratoires hospitaliers ruraux de Sioux Lookout, de Mount Forest et de la South Huron Hospital Association à Exeter. Nous avons colligé les données démographiques et les renseignements spécifiques aux infections, y compris l'utilisation récente de l'antibiothérapie et les hospitalisations ou séjours en foyers de soins infirmiers récents ou en cours. RÉSULTATS: Au cours de la période de l'étude, 34 infections à C. difficile ont été dénombrées chez 29 patients, pour un taux annuel brut estimé de 24,3/100 000 habitants. Parmi ces cas, 47,1 % n'étaient pas hospitalisés au moment du diagnostic. La plupart des patients (76,5 %) avaient pris des antibiotiques au cours des 90 jours précédents et l'antibiothérapie et l'hospitalisation caractérisaient 47,1 % des cas. La clindamycine a le plus souvent été associée aux infections à C. difficile dans le site le plus au Nord et la ciprofloxacine, dans les deux sites plus au Sud. On a déploré 2 décès par suite de comorbidités. CONCLUSION: L'incidence annuelle estimée de l'infection à C. difficile au cours de notre étude a été similaire aux estimations obtenues en milieu urbain. Près de la moitié des cas s'observaient chez des patients non hospitalisés, rappelant la nécessité de considérer cette infection comme un grave problème de santé chez les patients externes. La bonne gestion de l'utilisation des antibiotiques demeure un enjeu constant puisque la plupart des patients avaient été exposés à des antibiotiques avant leur infection.

Sanitization of contaminated footwear from onychomycosis patients using ozone gas: a novel adjunct therapy for treating onychomycosis and tinea pedis?

BACKGROUND: Ozone gas possesses antimicrobial properties against bacteria, viruses, and yeasts. Previously, we demonstrated the efficacy of ozone in killing ATCC strains of the dermatophyte fungi Trichophyton rubrum and Trichophyton mentagrophytes. OBJECTIVE: To test the efficacy of ozone gas in sanitizing onychomycosis patient footwear contaminated with fungal material as a means of minimizing the risk of reinfection. METHODS: Swabs of footwear from onychomycosis patients were cultured prior to and after ozone exposure to test the ability of ozone to sanitize these items. RESULTS: We identified contamination of footwear from most onychomycosis patients, a potential source of reinfection in these individuals. Furthermore, ozone gas was effective in sanitizing contaminated footwear. CONCLUSION: Ozone gas is effective in sanitizing footwear and represents a novel adjunct therapy to be used in conjunction with antifungal medications and/or devices to better treat onychomycosis and tinea pedis patients in both the short and the long term.

Association of severe inflammatory polyarthritis in primary Sjögren's syndrome: clinical, serologic, and HLA analysis.

OBJECTIVE: To evaluate the clinical, serologic, and MHC class II antigen characteristics of a group of patients with primary Sjögren's syndrome (SS) and severe arthritis. METHODS: A case-control study comparing 35 patients with primary SS: 17 with inflammatory arthritis, 18 without arthritis. RESULTS: All patients fulfilled criteria for primary SS. There were no demographic or clinical features other than inflammatory arthritis, often erosive, that distinguished patients with arthritis from those without. All patients had anti-Ro/SSA autoantibodies, most had anti-La/SSB autoantibodies, and a high percentage of these patients had anti-citrullinated peptide antibodies absent in those without inflammatory arthritis. HLA typing revealed that most patients with anti-citrulline antibodies expressed MHC class II molecules with the shared epitope (SE). The presence of DRB1*0301 linked to the expression of anti-Ro/SSA autoantibodies did not influence the level or frequency of anti-citrulline antibodies in these patients. CONCLUSION: Severe arthritis with features resembling rheumatoid arthritis including erosive disease can occur in primary SS, particularly among those with anti-citrulline antibodies and the SE.

Toll-like receptor 2 ligands on the staphylococcal cell wall downregulate superantigen-induced T cell activation and prevent toxic shock syndrome.

Staphylococcal superantigens are pyrogenic exotoxins that cause massive T cell activation leading to toxic shock syndrome and death. Despite the strong adaptive immune response induced by these toxins, infections by superantigen-producing staphylococci are very common clinical events. We hypothesized that this may be partly a result of staphylococcal strains having developed strategies that downregulate the T cell response to these toxins. Here we show that the human interleukin-2 response to staphylococcal superantigens is inhibited by the simultaneous presence of bacteria. Such a downregulatory effect is the result of peptidoglycan-embedded molecules binding to Toll-like receptor 2 and inducing interleukin-10 production and apoptosis of antigen-presenting cells. We corroborated these findings in vivo by showing substantial prevention of mortality after simultaneous administration of staphylococcal enterotoxin B with either heat-killed staphylococci or Staphylococcus aureus peptidoglycan in mouse models of superantigen-induced toxic shock syndrome.

Arthritis induced by posttranslationally modified (citrullinated) fibrinogen in DR4-IE transgenic mice.

Rheumatoid arthritis (RA) is a common autoimmune disease that afflicts the synovium of diarthrodial joints. The pathogenic mechanisms inciting this disease are not fully characterized, but may involve the loss of tolerance to posttranslationally modified (citrullinated) antigens. We have demonstrated that this modification leads to a selective increase in antigenic peptide affinity for major histocompatibility complex (MHC) class II molecules that carry the RA-associated shared epitope, such as HLA-DRB1*0401 (DR4). We describe the induction of arthritis in DR4-IE transgenic (tg) mice with citrullinated fibrinogen, a protein commonly found in inflamed synovial tissue and a frequent target of autoantibodies in RA patients. The disease induced in these mice was characterized by synovial hyperplasia followed by ankylosis, but lacked a conspicuous polymorphonuclear cell infiltrate. Immunological analysis of these mice through T cell epitope scanning and antibody microarray analysis identified a unique profile of citrulline-specific reactivity that was not found in DR4-IE tg mice immunized with unmodified fibrinogen or in wild-type C57BL/6 mice immunized with citrullinated fibrinogen, two conditions where arthritis was not observed. These observations directly implicate citrullinated fibrinogen as arthritogenic in the context of RA-associated MHC class II molecules.

Evidence for a novel rheumatoid arthritis susceptibility locus on chromosome 6p.

OBJECTIVE: To investigate whether the large linkage peak on chromosome 6p harbors rheumatoid arthritis (RA) susceptibility loci in addition to the well-characterized HLA-DRB1 gene. METHODS: DNA samples obtained from 377 UK RA affected sibling pair (ASP) families, comprising test (181 ASPs) and replication (196 ASPs) cohorts, were used for linkage analysis. Three hundred eighty-four patients with RA derived from a subset of 192 ASPs were compared with a panel of 288 unrelated healthy controls for association studies. Samples were genotyped for 35 microsatellites and 25 single-nucleotide polymorphisms (SNPs). RESULTS: In the test cohort, the maximum logarithm of odds (LOD) score was obtained over D6S1260 (LOD 7.1). Evidence for linkage to the telomeric portion of the peak was increased in subsets of ASPs in which both individuals had erosive disease or both carried 2 copies of the shared epitope. HLA-A, HLA-DRB1, and 8 additional markers showed evidence of linkage in the presence of association with RA (using the extended transmission disequilibrium test [ETDT]). The positive ETDT result for 2 adjacent markers (D6S1665 and 210901-4) mapping to the telomeric end of the linked region ( approximately 11 Mb from DRB1) was replicated (for D6S1665) in the second cohort of ASPs. Haplotypic overtransmission of pairwise combinations between D6S1665*7 and 210901-4*4 was identified through the TDTPhase program. Multipoint conditional analysis showed this effect to be independent of HLA-DRB1. SNP-based association studies of the region identified a 4-marker haplotype in the DEK gene that was significantly associated with RA (P = 0.009). CONCLUSION: Evidence has been presented for an RA susceptibility locus mapping under the linkage peak on 6p, 11 Mb telomeric of HLA-DRB1. Preliminary association data implicate the gene DEK.

Investigation of susceptibility loci identified in the UK rheumatoid arthritis whole-genome scan in a further series of 217 UK affected sibling pairs.

OBJECTIVE: A previous whole-genome scan (WGS) of 182 UK rheumatoid arthritis (RA) affected sibling pair (ASP) families suggested linkage to HLA and 11 other chromosome regions. Replication of such findings in an independent cohort can help to distinguish true linkages from false-positive linkages. Since RA is a heterogeneous disease, some loci may be linked only in subsets of patients. Thus, the aim of this study was to investigate in an additional set of RA ASP families linkage to regions showing deviation in expected allele-sharing ratios in the UK WGS and to perform subset analysis on the combined cohort. METHODS: Twenty loci were investigated for linkage in 217 Caucasian UK RA ASPs. Stratification analysis was performed on the combined cohort of 377 RA ASP families to account for sex, RA severity, and the shared epitope (SE). RESULTS: None of the regions of linkage identified in the initial WGS achieved statistical significance in the second cohort. In contrast, after stratification analysis, 14 regions showed nominal evidence of linkage (logarithm of odds score >0.8) in one or more subgroups. In particular, the strength of evidence for linkage to chromosome 16p was increased in subsets of ASPs with younger age at disease onset (LOD score 2.38) and for linkage to chromosome 6q in female-female ASPs (LOD score 2.31) and in ASPs in which both siblings had 2 copies of the SE (LOD score 3.03). CONCLUSION: These results support the evidence for heterogeneity of RA. This information will inform the future design of association-based investigations as the search for disease genes in the linked regions begins.