Josep Ordi-Ros

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.

DNA methylation and systemic lupus erythematosus.

Abstract:  Several studies have indicated the importance of DNA hypomethylation in the etiology of systemic lupus erythematosus (SLE). Different enzymes linked to the DNA methylation process have been described. The identification of all these enzymes means that cells have the capacity to modify their methylation patterns. Therefore, to obtain a deeper understanding of the role this epigenetic mechanism may have on SLE, the enzymes involved in the DNA methylation mechanism must be thoughtfully analyzed. In fact, studies of enzymes (other than DNMT1) in this autoimmune disease are still lacking. We have recently investigated the simultaneous gene expression of DNMT1, DNMT3A, DNMT3B, MBD2, and MBD4 in SLE patients. Here we review some of the studies that focus on the relationship between DNA methylation and SLE as well as we report our recent findings in this field. We suggest some alternative hypothesis that could help to understand the causes of the global DNA hypomethylation observed in the CD4+ T cells of these patients.

Molecular mechanisms mediated by human endogenous retroviruses (HERVs) in autoimmunity.

Eight per cent of the human genome is derived from the integration of retroviral sequences that were incorporated in our DNA more than 25 million years ago. Although some of these elements show mutations and deletions, some HERVs are transcriptionally active and produce functional proteins. Different mechanisms have been described which link HERVs to some chronic diseases such as several cancers, nervous system diseases and autoimmune rheumatic and connective tissue diseases. They could cause disease because of their capacity for being moved and inserted next to certain genes whose expression would be consequentially altered. Another way in which disease could potentially arise is when HERV‐encoded proteins are expressed. These proteins would be considered as [foreign] and they could trigger B‐cells to produce antibodies against them, which, in turn, might cross‐react with other proteins of our bodies. This mechanism could give rise to autoimmune diseases such as rheumatoid arthritis (RA), lupus erythematosus, Sjögrens syndrome (SJS), mixed connective tissue diseases and inflammatory neurological disease. Furthermore, it should be pointed out that HERV‐proteins may act as superantigens. Interestingly, some environmental agents seem to induce the expression of HERVs. Thus, ultraviolet light and several chemical agents could reactivate such sequences by altering their structure without modifying their nucleotide composition when the methylation pattern is changed. Therefore, the epigenetic changes observed in pathological conditions such as systemic lupus erythematosus (SLE) or cancer could be translated into an effect on the activation of some of the retroelements present in our genome which ultimately could have a direct or indirect role on the initiation and clinical evolution of certain chronic diseases. Copyright

DNase 1 and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown aetiology with a complex genetic basis that includes many susceptibility genes on multiple chromosomes. As many complex human diseases, SLE involves multiple, interacting genetic and environmental determinants, and identifying genes and enzymes for complex traits is challenging and has had limited success so far. DNase1 has been implicated in the pathophysiology of SLE since the 1950s. The importance of DNase1 has grown up since the description that apoptotic cells can be the source of self-antigens in SLE. Many articles have focused in disturbed apoptosis and in the defects of the apoptotic cell debris as the origin of nucleosomes against which the immune response can be induced. The enzyme DNase1 plays a role in the clearance of apoptotic debris, and is therefore of capital interest in this process. In this review we highlight the current findings in the pathophysiology, genetics, and therapeutical role of DNase1 in SLE.

Transcript overexpression of the MBD2 and MBD4 genes in CD4+ T cells from systemic lupus erythematosus patients

Global DNA hypomethylation in CD4+ T cells has been detected in systemic lupus erythematosus (SLE), and it seems to be linked to its pathogenesis. We investigated the relationship between overall DNA methylation and the expression of two methyl CpG‐binding domain (MBD) proteins. DNA deoxymethylcytosine (dmC) content of purified CD4+ T cells from 29 SLE patients and 30 healthy controls was measured by means of an ELISA. Transcript levels of two methyl CpG‐binding proteins (MBD2 and MBD4) were quantified by real‐time RT‐PCR. Association studies were also carried out with several laboratory parameters, as well as with the patients’ clinical manifestations. SLE patients had significantly less CD4+ T cell DNA dmC content than controls (0.802±0.134 vs. 0.901±0.133; P=0.007). MBD2 and MBD4 mRNA levels were considerably higher in the patients’ group: 0.975 ± 0683 versus 0.604 ± 0.614 (P=0.004) and 0.359 ± 0.330 versus 0.092 ± 0.169, respectively (P<0.0005). It is interesting that SLE patients showed a negative correlation between methylation indices and MBD2 (r=–0.609, P<0.0005) and MBD4 (r=–0.395, P=0.034) transcript levels. MBD2 and MBD4 transcript overexpression and inverse correlations with DNA methylation indices indicate that both enzymes may really have a direct and active role on the genome‐wide DNA hypomethylation observed in CD4+ T cells from SLE patients.

Transcript levels of DNA methyltransferases DNMT1, DNMT3A and DNMT3B in CD4+ T cells from patients with systemic lupus erythematosus.

Global DNA hypomethylation in CD4+ T cells has been detected in systemic lupus erythematosus (SLE) and it seems to be linked to its pathogenesis. We investigated the relationship between overall DNA methylation and the expression of three DNA (cytosine‐5) methyltransferases involved in the DNA methylation process. The DNA deoxymethylcytosine (dmC) content of purified CD4+ T cells from 29 SLE patients and 30 healthy controls was measured by means of an enzyme‐linked immunosorbent assay (ELISA). The transcript levels of DNA cytosine‐5‐methyltransferase 1 (DNMT1), DNA cytosine‐5‐methyltransferase 3A (DNMT3A) and DNA cytosine‐5‐methyltransferase 3B (DNMT3B) were quantified by real‐time reverse transcription–polymerase chain reaction (RT‐PCR). Association studies were also carried out with several laboratory parameters, as well as with the patients’ clinical manifestations. SLE patients had a significantly lower CD4+ T‐cell DNA dmC content than controls (0·802 ± 0·134 versus 0·901 ± 0·133) (P = 0·007). No differences in transcript levels were observed for DNMT1, DNMT3A and DNMT3B between patients and controls. The simultaneous association of low complement counts with lymphopenia, high titres of anti‐double‐stranded DNA (anti‐dsDNA), or an SLE disease activity index (SLEDAI) of > 5, resulted in the increase of at least one of the three DNA methyltransferases. It is possible that patients were reacting indirectly to an underlying DNA hypomethylation status by increasing the mRNA levels of DNA methyltransferases when the disease was being definitely active.

Implication of Human Endogenous Retroviruses in the Development of Autoimmune Diseases

Retroviruses can exist in an endogenous form, in which viral sequences are integrated into the human germ line and are vertically transmitted in a Mendelian fashion. Human endogenous retroviruses (HERVs), probably representing footprints of ancient germ-cell retroviral infections, occupy about 1% of the human genome. Some HERVs emerged in the genome over 25 million years ago, while others have appeared rather recently, at about the time of hominid and ape lineages divergence. Although some of these elements show mutations and deletions, some HERVs are transcriptionally active and produce functional proteins. Some medical conditions, such as cancer and autoimmune diseases, are linked to the transcription of some of the HERVs genes, to the expression of HERVs proteins (that may act as superantigens, for example), and/or to the development of antibodies against them that might cross-react with our own proteins. Their genetic sequences may also be, totally or partially, integrated into genes that regulate the immune response. These mechanisms could give rise to autoimmune diseases, such as lupus erythematosus, insulin-dependent diabetes mellitus, multiple sclerosis, Sjögrens syndrome, and rheumatoid arthritis, among others. This review is aimed at discussing evidence for a possible role of HERVs in the etiopathogenesis of different autoimmune diseases.

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.

Contribution of the initial features of systemic lupus erythematosus to the clinical evolution and survival of a cohort of Mediterranean patients

Background: Systemic lupus erythematosus has a wide spectrum of immunological and clinical manifestations. Its course is characterised by exacerbations which may result in mortality or morbidity to vital organs/systems. Objective: To determine clear and early prognostic markers to avoid further complications. Methods: 245 adult patients diagnosed between January 1978 and March 2001 were studied. Clinical manifestations and laboratory findings both at onset and during the clinical course were collected. The number, type, and severity of the flares were also noted. Statistical analyses between disease features at onset, subsequent flares, and mortality were performed. Results: 239 patients entered the study. Their mean age at onset was 30 years. The mean time between onset and diagnosis was 36 months and the mean evolution time was 114 months. 205 patients developed 915 flares; 205 (22.4%) of these flares were major flares, and affected 110 patients. Cardiac, neurological, or renal affection at onset were associated with a higher probability of developing cardiac (p=0.022), neurological (p<0.001), and renal (p<0.001) exacerbations, respectively, during the evolution. Lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) were predictors of stroke (aCL, p=0.000; LA, p=0.001). Age at diagnosis (p=0.003) and valvular disease at onset (p=0.008) were independent predictors of low survival. Conclusions: Renal, cardiac, or neurological involvement and the presence of LA or aCL positivity at onset were predictors of renal, cardiac, or neurological flares, respectively. Age and valvular involvement at onset were found to be independent adverse outcome predictors for low survival.

Long-term outcomes—mycophenolate mofetil treatment for lupus nephritis with addition of tacrolimus for resistant cases

BACKGROUND Although mycophenolate mofetil (MMF) is being increasingly used to manage lupus nephritis (LN), long-term experience is limited. Despite treatment, a significant proportion of patients will be refractory to this regime. METHODS We report, in this observational study, our long-term experience treating 70 patients with biopsy-proven LN, with MMF as continuous induction-maintenance therapy, who were followed up prospectively over a 5-year period. As rescue therapy for MMF-resistant cases, tacrolimus (0.075 mg/kg/day) was added. The study primary end point was complete response (CR). Secondary end points included partial response (PR), treatment failure, relapse and side effects. Predictor factors associated to renal outcome were analysed by Cox regression analysis. RESULTS Thirty-six MMF-treated patients (51%) remained in CR, and 23 (33%) failed treatment at last follow-up. Time to treatment failure was associated with persistent hypoalbuminaemia (hazard ratio (HR) = 0.87; 95%CI, 0.81-0.95; P = 0.001), higher proteinuria (HR = 1.29; 95%CI, 1.03-1.62; P = 0.030) and fewer early responses (HR 0.28; 95%CI, 0.10-0.77; P = 0.014). Renal relapse occurred in 24 (34%) patients. Time to flare was associated with persistent anti-dsDNA titres (HR = 1.001; 95%CI, 1.001-1.003; P = 0.005) and younger age at inclusion (HR = 0.36; 95%CI, 0.14-0.90; P = 0.029). Tacrolimus was added to 17 (24%) patients. A significant reduction of proteinuria was already observed at 3 months (P = 0.002). After 2 years follow-up, 12 (70%) of them achieved clinical response (six CR and six PR). Conclusions. MMF is an effective treatment for LN. Combination therapy with tacrolimus is an effective and safe alternative for MMF-resistant patients.