F. David Carmona

Identification of CSK as a systemic sclerosis genetic risk factor through Genome Wide Association Study follow-up

Systemic sclerosis (SSc) is complex autoimmune disease affecting the connective tissue; influenced by genetic and environmental components. Recently, we performed the first successful genome-wide association study (GWAS) of SSc. Here, we perform a large replication study to better dissect the genetic component of SSc. We selected 768 polymorphisms from the previous GWAS and genotyped them in seven replication cohorts from Europe. Overall significance was calculated for replicated significant SNPs by meta-analysis of the replication cohorts and replication-GWAS cohorts (3237 cases and 6097 controls). Six SNPs in regions not previously associated with SSc were selected for validation in another five independent cohorts, up to a total of 5270 SSc patients and 8326 controls. We found evidence for replication and overall genome-wide significance for one novel SSc genetic risk locus: CSK [P-value = 5.04 × 10(-12), odds ratio (OR) = 1.20]. Additionally, we found suggestive association in the loci PSD3 (P-value = 3.18 × 10(-7), OR = 1.36) and NFKB1 (P-value = 1.03 × 10(-6), OR = 1.14). Additionally, we strengthened the evidence for previously confirmed associations. This study significantly increases the number of known putative genetic risk factors for SSc, including the genes CSK, PSD3 and NFKB1, and further confirms six previously described ones.

The systemic lupus erythematosus IRF5 risk haplotype is associated with systemic sclerosis

Systemic sclerosis (SSc) is a fibrotic autoimmune disease in which the genetic component plays an important role. One of the strongest SSc association signals outside the human leukocyte antigen (HLA) region corresponds to interferon (IFN) regulatory factor 5 (IRF5), a major regulator of the type I IFN pathway. In this study we aimed to evaluate whether three different haplotypic blocks within this locus, which have been shown to alter the protein function influencing systemic lupus erythematosus (SLE) susceptibility, are involved in SSc susceptibility and clinical phenotypes. For that purpose, we genotyped one representative single-nucleotide polymorphism (SNP) of each block (rs10488631, rs2004640, and rs4728142) in a total of 3,361 SSc patients and 4,012 unaffected controls of Caucasian origin from Spain, Germany, The Netherlands, Italy and United Kingdom. A meta-analysis of the allele frequencies was performed to analyse the overall effect of these IRF5 genetic variants on SSc. Allelic combination and dependency tests were also carried out. The three SNPs showed strong associations with the global disease (rs4728142: P  = 1.34×10−8, OR  = 1.22, CI 95%  = 1.14–1.30; rs2004640: P  = 4.60×10−7, OR  = 0.84, CI 95%  = 0.78–0.90; rs10488631: P  = 7.53×10−20, OR  = 1.63, CI 95%  = 1.47–1.81). However, the association of rs2004640 with SSc was not independent of rs4728142 (conditioned P  = 0.598). The haplotype containing the risk alleles (rs4728142*A-rs2004640*T-rs10488631*C: P  = 9.04×10−22, OR  = 1.75, CI 95%  = 1.56–1.97) better explained the observed association (likelihood P-value  = 1.48×10−4), suggesting an additive effect of the three haplotypic blocks. No statistical significance was observed in the comparisons amongst SSc patients with and without the main clinical characteristics. Our data clearly indicate that the SLE risk haplotype also influences SSc predisposition, and that this association is not sub-phenotype-specific.

Genetic component of giant cell arteritis

Important steps forwards have been taken during recent years towards the understanding of the genetic basis of autoimmunity. The increasing number of study cohorts is allowing better characterization of the genetic component of most autoimmune diseases. However, the molecular mechanisms leading to some less common diseases remain poorly understood. GCA, an antigen-driven systemic vasculitis affecting medium and large blood vessels of elderly people, represents one of these cases. However, although underpowered to detect low to moderate effect sizes and without replication steps, many genetic studies on this disease have been published in the past decade. These reports clearly point to genes located in the MHC region, in particular HLA-DRB1*04 alleles, and other key members of the immune and inflammatory response (including cytokines, adhesion molecules and regulators of innate immunity), as crucial players in the development and progression of GCA. Considering that no literature review has been published so far about the genetic component of this vasculitis, we aimed to summarize here the current knowledge on the genetics underlying GCA predisposition and severity.

New insight on the Xq28 association with systemic sclerosis

Objective To evaluate whether the systemic sclerosis (SSc)-associated IRAK1 non-synonymous single-nucleotide polymorphism rs1059702 is responsible for the Xq28 association with SSc or whether there are other independent signals in the nearby methyl-CpG-binding protein 2 gene (MECP2). Methods We analysed a total of 3065 women with SSc and 2630 unaffected controls from five independent Caucasian cohorts. Four tag single-nucleotide polymorphisms of MECP2 (rs3027935, rs17435, rs5987201 and rs5945175) and the IRAK1 variant rs1059702 were genotyped using TaqMan predesigned assays. A meta-analysis including all cohorts was performed to test the overall effect of these Xq28 polymorphisms on SSc. Results IRAK1 rs1059702 and MECP2 rs17435 were associated specifically with diffuse cutaneous SSc (PFDR=4.12×10−3, OR=1.27, 95% CI 1.09 to 1.47, and PFDR=5.26×10−4, OR=1.30, 95% CI 1.14 to 1.48, respectively), but conditional logistic regression analysis showed that the association of IRAK1 rs1059702 with this subtype was explained by that of MECP2 rs17435. On the other hand, IRAK1 rs1059702 was consistently associated with presence of pulmonary fibrosis (PF), because statistical significance was observed when comparing SSc patients PF+ versus controls (PFDR=0.039, OR=1.30, 95% CI 1.07 to 1.58) and SSc patients PF+ versus SSc patients PF− (p=0.025, OR=1.26, 95% CI 1.03 to 1.55). Conclusions Our data clearly suggest the existence of two independent signals within the Xq28 region, one located in IRAK1 related to PF and another in MECP2 related to diffuse cutaneous SSc, indicating that both genes may have an impact on the clinical outcome of the disease.

Novel identification of the IRF7 region as an anticentromere autoantibody propensity locus in systemic sclerosis

Objective Systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) are related chronic autoimmune diseases of complex aetiology in which the interferon (IFN) pathway plays a key role. Recent studies have reported an association between IRF7 and SLE which confers a risk to autoantibody production. A study was undertaken to investigate whether the IRF7 genomic region is also involved in susceptibility to SSc and the main clinical features. Methods Two case-control sets of Caucasian origin from the USA and Spain, comprising a total of 2316 cases of SSc and 2347 healthy controls, were included in the study. Five single nucleotide polymorphisms (SNPs) in the PHRF1-IRF7-CDHR5 locus were genotyped using TaqMan allelic discrimination technology. A meta-analysis was performed to test the overall effect of these genetic variants on SSc. Results Four out of five analysed SNPs were significantly associated with the presence of anticentromere autoantibodies (ACA) in the patients with SSc in the combined analysis (rs1131665: pFDR=6.14 × 10−4, OR=0.78; rs4963128: pFDR=6.14 × 10−4, OR=0.79; rs702966: pFDR=3.83 × 10−3, OR=0.82; and rs2246614: pFDR=3.83 × 10−3, OR=0.83). Significant p values were also obtained when the disease was tested globally; however, the statistical significance was lost when the ACA-positive patients were excluded from the study, suggesting that these associations rely on ACA positivity. Conditional logistic regression and allelic combination analyses suggested that the functional IRF7 SNP rs1131665 is the most likely causal variant. Conclusions The results show that variation in the IRF7 genomic region is associated with the presence of ACA in patients with SSc, supporting other evidence that this locus represents a common risk factor for autoantibody production in autoimmune diseases.

Evidence of association of the NLRP1 gene with giant cell arteritis

Recent studies have focused attention on the involvement of NLRP1 to confer susceptibility for extended autoimmune/inflammatory disorders, being considered a common risk factor in autoimmunity.1–3 NLRP1 provides a scaffold for the assembly of the inflammasome that activates caspases 1 and 5, required for processing and activation of the proinflammatory cytokines interleukin 1β (IL-1β), IL-18 and IL-33 and promoting inflammation.4 In this study, we examined for the first time whether NLRP1 is associated with giant cell arteritis (GCA), a chronic systemic vasculitis affecting large and medium-sized arteries derived from the aorta, in particular the cranial branches of the carotid artery. GCA is the most common vasculitis in the elderly in Western countries with a female predominance.5 To investigate the possible genetic association of NLRP1 with this disease, we genotyped a single-nucleotide polymorphism (rs8182352), which has been reported to confer risk to the development of autoimmune processes in previous studies,1 ,2 in a total of 3583 individuals, comprising a discovery set from Spain (574 patients diagnosed with biopsy-proven GCA and 2366 healthy controls) and a replication set of subjects from Italy (111 biopsy-proven GCA patients and 532 controls) using a predesigned TaqMan allele discrimination assay. All individuals were of …

Association of a non-synonymous functional variant of the ITGAM gene with systemic sclerosis

Systemic sclerosis (SSc) is a chronic fibrotic autoimmune disease of complex aetiology which shares genetic similarities with systemic lupus erythematosus (SLE).1 2 One of the novel risk loci that have been recently associated with SLE is the integrin α M ( ITGAM ) gene, which encodes the α subunit of the αMβ2-integrin.3 4 The most likely causal polymorphism that best explains this association is a non-synonymous single-nucleotide polymorphism (SNP) at the exon 3, rs1143679, which changes the 77th amino acid residue arginine to histidine (R77H). This functional SNP represents one of the highest associated signals with SLE and is predicted to alter the structure and function of the integrin.4 5 To determine whether ITGAM rs1143679 is also associated with SSc susceptibility and clinical manifestations, we genotyped a total of 3735 SSc patients and 3930 matched healthy individuals from seven independent European cohorts of Caucasian origin (Spain, Germany, The Netherlands, Italy, Norway, Sweden and UK) using a predesigned TaqMan® assay (ID: C\__\_|2847895\_1_) in an ABI 7900HT (both from Applied Biosystems, Foster City, California, USA). Case …

Role of the CCR5/Δ32CCR5 polymorphism in biopsy-proven giant cell arteritis

To further explore the potential role of chemokines in giant cell arteritis (GCA), we have studied whether the CCR5/Δ32CCR5 polymorphism is implicated in the susceptibility to the disease and its specific features. A total of 352 Spanish patients with biopsy-proven GCA and 479 matched controls were assessed. DNA was obtained from peripheral blood. Samples were genotyped by PCR with specific primers spanning the 32-bp deletion region. No statistically significant difference in the Δ32CCR5 allele frequency between GCA patients (6.1%) and controls (6.8%) was observed (p = 0.58). This was also the case when the CCR5 /Δ32CCR5 genotype distribution was assessed (p = 0.49). The Δ32CCR5 allele frequency did not differ between patients with or without specific manifestations of the disease, such as polymyalgia rheumatica, visual ischemic manifestations, or irreversible occlusive disease. Hence, our results do not support a potential influence of Δ32CCR5 in the susceptibility to or clinical spectrum of GCA.

SOX9 is not required for the cellular events of testicular organogenesis in XX mole ovotestes

Mammalian sex determination is the genetic process that commits the undifferentiated bipotential gonads to develop as either testes or ovaries. The differentiation of SOX9-expressing Sertoli cells is assumed to be necessary to initiate testis development. Insectivorous moles of the genus Talpa represent a unique case of generalized true hermaphroditism, as XX female moles constitutively develop two ovotestes instead of normal ovaries. In this work, we have investigated the expression patterns of a number of genes known to play key roles in gonad organogenesis, throughout the entire process of ovotestis development in female moles. Molecular and morphological evidence are provided that these ovotestes contain primary medullary testis-like cords, Leydig cells, peritubular myoid cells, and a testis-specific vasculature, but no Sertoli cells. Our results show for the first time that SOX9 is not required for the formation of the primary testis cords, but it is necessary for the maintenance and subsequent development of these cords. In addition, the expression pattern of WNT4 in male and female moles indicates that this gene inhibits Leydig cell differentiation and, contrary to the proposed scenario in the mouse, it is not required for the colonization and survival of primordial germ cells. According to our data, mole ovotestes result from a process of PDGFRalpha-mediated mesonephric cell migration, which occurs simultaneously in both sexes. The fact that FST remains inactive during the critical stages of female gonad development, explains the lack of migration inhibition, and may be a consequence of improper WNT4 signalling in the mole.

Retinal development and function in a ‘blind’ mole

Animals adapted to dark ecotopes may experience selective pressure for retinal reduction. No previous studies have explicitly addressed the molecular basis of retinal development in any fossorial mammal. We studied retinal development and function in the Iberian mole Talpa occidentalis, which was presumed to be blind because of its permanently closed eyes. Prenatal retina development was relatively normal, with specification of all cell types and evidence of dorsoventral regionalization. Severe developmental defects occurred after birth, subsequent to lens abnormalities. ‘Blind’ Iberian moles had rods, cones and rod nuclear ultrastructure typical of diurnal mammals. DiI staining revealed only contralateral projections through the optic chiasm. Y-maze experiments demonstrated that moles retain a photoavoidance response. Over-representation of melanopsin-positive retinal ganglion cells that mediate photoperiodicity was observed. Hence, molecular pathways of eye development in Iberian moles retain the adaptive function of rod/cone primary vision and photoperiodicity, with no evidence that moles are likely to completely lose their eyes on an evolutionary time scale.