S. Migliaresi

STAT4 associates with systemic lupus erythematosus through two independent effects that correlate with gene expression and act additively with IRF5 to increase risk

Objectives: To confirm and define the genetic association of STAT4 and systemic lupus erythematosus (SLE), investigate the possibility of correlations with differential splicing and/or expression levels, and genetic interaction with IRF5. Methods: 30 tag SNPs were genotyped in an independent set of Spanish cases and controls. SNPs surviving correction for multiple tests were genotyped in five new sets of cases and controls for replication. STAT4 cDNA was analysed by 5′-RACE PCR and sequencing. Expression levels were measured by quantitative PCR. Results: In the fine mapping, four SNPs were significant after correction for multiple testing, with rs3821236 and rs3024866 as the strongest signals, followed by the previously associated rs7574865, and by rs1467199. Association was replicated in all cohorts. After conditional regression analyses, two major independent signals, represented by SNPs rs3821236 and rs7574865, remained significant across the sets. These SNPs belong to separate haplotype blocks. High levels of STAT4 expression correlated with SNPs rs3821236, rs3024866 (both in the same haplotype block) and rs7574865 but not with other SNPs. Transcription of alternative tissue-specific exons 1, indicating the presence of tissue-specific promoters of potential importance in the expression of STAT4, was also detected. No interaction with associated SNPs of IRF5 was observed using regression analysis. Conclusions: These data confirm STAT4 as a susceptibility gene for SLE and suggest the presence of at least two functional variants affecting levels of STAT4. The results also indicate that the genes STAT4 and IRF5 act additively to increase the risk for SLE.

Replication of recently identified systemic lupus erythematosus genetic associations: a case–control study

IntroductionWe aimed to replicate association of newly identified systemic lupus erythematosus (SLE) loci.MethodsWe selected the most associated SNP in 10 SLE loci. These 10 SNPs were analysed in 1,579 patients with SLE and 1,726 controls of European origin by single-base extension. Comparison of allele frequencies between cases and controls was done with the Mantel–Haenszel approach to account for heterogeneity between sample collections.ResultsA previously controversial association with a SNP in the TYK2 gene was replicated (odds ratio (OR) = 0.79, P = 2.5 × 10-5), as well as association with the X chromosome MECP2 gene (OR = 1.26, P = 0.00085 in women), which had only been reported in a single study, and association with four other loci, 1q25.1 (OR = 0.81, P = 0.0001), PXK (OR = 1.19, P = 0.0038), BANK1 (OR = 0.83, P = 0.006) and KIAA1542 (OR = 0.84, P = 0.001), which have been identified in a genome-wide association study, but not found in any other study. All these replications showed the same disease-associated allele as originally reported. No association was found with the LY9 SNP, which had been reported in a single study.ConclusionsOur results confirm nine SLE loci. For six of them, TYK2, MECP2, 1q25.1, PXK, BANK1 and KIAA1542, this replication is important. The other three loci, ITGAM, STAT4 and C8orf13-BLK, were already clearly confirmed. Our results also suggest that MECP2 association has no influence in the sex bias of SLE, contrary to what has been proposed. In addition, none of the other associations seems important in this respect.

Cognitive impairment in systemic lupus erythematosus: a follow-up study

Abstract We evaluated outcome and the clinical value of cognitive impairment in systemic lupus erythematosus (SLE). Fifty-one consecutive SLE subjects with or without overt nervous system involvement received two comprehensive neuropsychiatric and neuropsychological assessments, including the Mental Deterioration Battery, the Mini Mental State Examination (MMSE), and tests from the Wechsler Adult Intelligence Scale. The two neuropsychological assessments were made when subjects were in stable neurological condition. Twenty-seven patients were found to have neuropsychiatric symptoms (NP-SLE) at the first assessment, and three others developed them during the follow-up. Fifteen patients (10 NP-SLE) had cognitive impairment at the first assessment. At retest the cognitive deficit persisted in all patients but one (non-NP-SLE) and had developed in four others. In the cognitively impaired subjects scores on MMSE approached the cutoff for an overt dementing condition. No progressively decreasing scores were found on any of the tests. No relationships were shown between neuropsychological diagnosis and neuropsychiatric disorder, neuroradiological findings, disease activity, or steroid and nonsteroid immunosuppressive therapy. Cognitive impairment thus seems to be a stable symptom of CNS involvement in SLE. It corresponds to the subjective complaint of intellectual difficulties and marginal performance on the MMSE. Intellectual deterioration may occur in patients without other symptoms of NP-SLE. Standardized neuropsychological testing methods should be used routinely to assess SLE patients.

A vascular Osteonecrosis in Patients with SLE: Relation to Corticosteroid Therapy and Anticardiolipin Antibodies

Sixty-nine unselected SLE patients were studied to evaluate the prevalence of avascular osteonecrosis (AVN) and its relationship with steroid therapy and with anticardiolipin antibodies (aCL). All the patients were under treatment with corticosteroids. AVN occurred in seven of the 69 patients (10. 14%) and was not related to corticosteroid intake. Seventeen of the 69 patients were also treated with methylprednisolone pulse therapy (MPPT) and cumulated the highest corticosteroid doses but none of them suffered from AVN. Excluding the 17 MPPT-treated SLE patients, corticosteroid intake was significantly higher in the AVN-SLE patients. Abnormal IgG and/or IgM aCL serum levels were found in two of the seven AVN-SLE patients and in 24 of the 62 non-AVN SLE, without a statistically significant difference. None of the seven AVN-SLE patients showed features of antiphospholipid syndrome. We conclude that in SLE patients a continuous high-dose steroid treatment may be considered a risk factor for AVN. On the contrary, MPPT regimen may reduce this risk. Anticardiolipin antibodies might represent an added factor which could play a role in some patients but not in all.

Opposed independent effects and epistasis in the complex association of IRF5 to SLE

Genetic variation in the interferon regulatory factor 5 (IRF5) gene affects systemic lupus erythematosus (SLE) susceptibility. However, association is complex and incompletely defined. We obtained fourteen European sample collections with a total of 1383 SLE patients and 1614 controls to better define the role of the different IRF5 variants. Eleven polymorphisms were studied, including nine tag single nucleotide polymorphisms (SNPs) and two extra functional polymorphisms. Two tag SNPs showed independent and opposed associations: susceptibility (rs10488631, P<10−17) and protection (rs729302, P<10−6). Haplotype analyses showed that the susceptibility haplotype, identified by the minor allele of rs10488631, can be due to epistasis between three IRF5 functional polymorphisms. These polymorphisms determine increased mRNA expression, a splice variant with a different exon 1 and a longer proline-rich region in exon 6. This result is striking as none of the three polymorphisms had an independent effect on their own. Protection was independent of these polymorphisms and seemed to reside in the 5′ side of the gene. In conclusion, our results help to understand the role of the IRF5 locus in SLE susceptibility by clearly separating protection from susceptibility as caused by independent polymorphisms. In addition, we have found evidence for epistasis between known functional polymorphisms for the susceptibility effect.

Bias in association studies of systemic lupus erythematosus susceptibility due to geographical variation in the frequency of a programmed cell death 1 polymorphism across Europe

We obtained eight collections of DNA samples from ethnically matched systemic lupus erythematosus (SLE) patients and controls from five European countries totaling 783 patients and 1210 controls. A highly significant cline in the frequency of the PD1.3 A allele was found among controls but not among SLE patients. The frequency of the PD1.3 A allele increased from the Northeast to the Southwest of Europe. The cline was clearly apparent (P=1.2 × 10−6) when data from controls of other five SLE susceptibility studies were included in the analysis. This variation has severely biased SLE association studies owing to the lack of parallel changes in SLE patients. As a consequence, the PD1.3 A allele was more common in SLE patients than in controls in the Northeast and Center of Europe, similar to controls in Southeast Europe, and less frequent than in the controls in the Southwest of the Continent. This dissociation in allele frequencies between SLE patients and controls in different subpopulations indicated that programmed cell death 1 variation and disease susceptibility are not independent but the type of relationship is currently unclear. As allele frequency clines are common in other polymorphisms their impact in genetic epidemiology studies should be carefully considered.

Chromosome 17p12-q11 harbors susceptibility loci for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies against intracellular components, the formation of immune complexes, and inflammation in various organs, typically the skin and kidney glomeruli. The etiology of the disease is not well understood but is most likely the result of the interaction between genetic and environmental factors. In order to identify susceptibility loci for SLE, we have performed genome scans with microsatellite markers covering the whole genome in families from Argentina, Italy, and Europe. The results reveal a heterogeneous disease with different susceptibility loci in different family sets. We have found significant linkage to chromosome 17p12-q11 in the Argentine set of families. The maximum LOD score was given by marker D17S1294 in combination with D17S1293, when assuming a dominant inheritance model (Z=3.88). We also analyzed a repeat in the promoter region of the NOS2A gene, a strong candidate gene in the region, but no association was found. The locus on chromosome 17 has previously been identified in genetic studies of multiple sclerosis families. Several other interesting regions were found at 1p35, 1q31, 3q26, 5p15, 11q23 and 19q13, confirming previously identified loci for SLE or other autoimmune diseases.

Viral RNA in nerve tissues of patients with hepatitis C infection and peripheral neuropathy.

To assess the presence of viral ribonucleic acid (RNA) in nerve tissues of 15 patients with hepatitis C virus (HCV) infection and peripheral neuropathy with (11) or without (4) mixed cryoglobulinemia, nested reverse transcription–polymerase chain reaction (RT‐PCR) was performed. Amplification of HCV‐RNA was successful in 7 patients with and 3 without mixed cryoglobulinemia. This study demonstrates that the nested RT‐PCR technique is a sensitive method to detect viral RNA in nerve tissue, and offers further evidence that in patients with HCV infection peripheral neuropathy can occur in the absence of mixed cryoglobulinemia. Muscle Nerve 27:102–104, 2003

Peripheral neuropathy in hepatitis-related mixed cryoglobulinemia: electrophysiologic follow-up study.

A retrospective study was performed on 27 patients with hepatitis C (HCV)–related mixed cryoglobulinemia (purpura, arthralgia, hepatitis, glomerulonephritis, peripheral neuropathy) to assess peripheral nerve involvement during follow‐up of up to 8 years. All patients had the same degree of organ/system involvement initially and were clinically evaluated at least annually. All 27 patients received steroids; 15 also received recombinant interferon‐α2b (rIFN‐α2b). At first examination, neurological signs and electrodiagnostic findings consistent with peripheral neuropathy were found in 20 (74%) and in 24 (88.8%) patients, respectively. Neurological evaluation and electrodiagnostic data at 3 and 8 years revealed worsening of neuropathy, whereas the other manifestations of mixed cryoglobulinemia (MC) were stable. At the last examination, clinical and electrodiagnostic signs of neuropathy were found in 25 patients (92.5%), occurring in 1 of 3 patients with normal initial findings, and worsened in 8. A more severe neuropathy was observed in 3 (25%) of the patients treated with prednisone alone and in 6 (40%) of the patients additionally treated with rIFN‐α2b. Our data confirm that in patients with HCV‐related MC, peripheral nerve involvement is frequent, is progressive, and does not seem to benefit by addition of rIFN‐α2b to steroid treatment. Muscle Nerve, 2005

Further Evidence of Subphenotype Association with Systemic Lupus Erythematosus Susceptibility Loci: A European Cases Only Study

Introduction Systemic Lupus Erythematosus (SLE) shows a spectrum of clinical manifestations that complicate its diagnosis, treatment and research. This variability is likely related with environmental exposures and genetic factors among which known SLE susceptibility loci are prime candidates. The first published analyses seem to indicate that this is the case for some of them, but results are still inconclusive and we aimed to further explore this question. Methods European SLE patients, 1444, recruited at 17 centres from 10 countries were analyzed. Genotypes for 26 SLE associated SNPs were compared between patients with and without each of 11 clinical features: ten of the American College of Rheumatology (ACR) classification criteria (except ANAs) and age of disease onset. These analyses were adjusted for centre of recruitment, top ancestry informative markers, gender and time of follow-up. Overlap of samples with previous studies was excluded for assessing replication. Results There were three new associations: the SNPs in XKR6 and in FAM167A-BLK were associated with lupus nephritis (OR = 0.76 and 1.30, Pcorr = 0.007 and 0.03, respectively) and the SNP of MECP2, which is in chromosome X, with earlier age of disease onset in men. The previously reported association of STAT4 with early age of disease onset was replicated. Some other results were suggestive of the presence of additional associations. Together, the association signals provided support to some previous findings and to the characterization of lupus nephritis, autoantibodies and age of disease onset as the clinical features more associated with SLE loci. Conclusion Some of the SLE loci shape the disease phenotype in addition to increase susceptibility to SLE. This influence is more prominent for some clinical features than for others. However, results are only partially consistent between studies and subphenotype specific GWAS are needed to unravel their genetic component.