María Teruel

Identification of novel genetic markers associated with clinical phenotypes of systemic sclerosis through a genome-wide association strategy

The aim of this study was to determine, through a genome-wide association study (GWAS), the genetic components contributing to different clinical sub-phenotypes of systemic sclerosis (SSc). We considered limited (lcSSc) and diffuse (dcSSc) cutaneous involvement, and the relationships with presence of the SSc-specific auto-antibodies, anti-centromere (ACA), and anti-topoisomerase I (ATA). Four GWAS cohorts, comprising 2,296 SSc patients and 5,171 healthy controls, were meta-analyzed looking for associations in the selected subgroups. Eighteen polymorphisms were further tested in nine independent cohorts comprising an additional 3,175 SSc patients and 4,971 controls. Conditional analysis for associated SNPs in the HLA region was performed to explore their independent association in antibody subgroups. Overall analysis showed that non-HLA polymorphism rs11642873 in IRF8 gene to be associated at GWAS level with lcSSc (P = 2.32×10−12, OR = 0.75). Also, rs12540874 in GRB10 gene (P = 1.27 × 10−6, OR = 1.15) and rs11047102 in SOX5 gene (P = 1.39×10−7, OR = 1.36) showed a suggestive association with lcSSc and ACA subgroups respectively. In the HLA region, we observed highly associated allelic combinations in the HLA-DQB1 locus with ACA (P = 1.79×10−61, OR = 2.48), in the HLA-DPA1/B1 loci with ATA (P = 4.57×10−76, OR = 8.84), and in NOTCH4 with ACA P = 8.84×10−21, OR = 0.55) and ATA (P = 1.14×10−8, OR = 0.54). We have identified three new non-HLA genes (IRF8, GRB10, and SOX5) associated with SSc clinical and auto-antibody subgroups. Within the HLA region, HLA-DQB1, HLA-DPA1/B1, and NOTCH4 associations with SSc are likely confined to specific auto-antibodies. These data emphasize the differential genetic components of subphenotypes of SSc.

Immunochip Analysis Identifies Multiple Susceptibility Loci for Systemic Sclerosis

In this study, 1,833 systemic sclerosis (SSc) cases and 3,466 controls were genotyped with the Immunochip array. Classical alleles, amino acid residues, and SNPs across the human leukocyte antigen (HLA) region were imputed and tested. These analyses resulted in a model composed of six polymorphic amino acid positions and seven SNPs that explained the observed significant associations in the region. In addition, a replication step comprising 4,017 SSc cases and 5,935 controls was carried out for several selected non-HLA variants, reaching a total of 5,850 cases and 9,401 controls of European ancestry. Following this strategy, we identified and validated three SSc risk loci, including DNASE1L3 at 3p14, the SCHIP1-IL12A locus at 3q25, and ATG5 at 6q21, as well as a suggested association of the TREH-DDX6 locus at 11q23. The associations of several previously reported SSc risk loci were validated and further refined, and the observed peak of association in PXK was related to DNASE1L3. Our study has increased the number of known genetic associations with SSc, provided further insight into the pleiotropic effects of shared autoimmune risk factors, and highlighted the power of dense mapping for detecting previously overlooked susceptibility loci.

Chromosome mapping of H3 and H4 histone gene clusters in 35 species of acridid grasshoppers

We analyse chromosome location of H3 and H4 histone gene clusters by fluorescence in-situ hybridization (FISH) in 35 species of Acrididae grasshoppers belonging to seven subfamilies. As in other organisms, H3 and H4 co-localized in the same chromosome region in the 11 species where double FISH was performed with the H3 and H4 DNA probes. Chromosome location of H3-H4 histone gene clusters showed high regularity in the species analysed, with all of them carrying a single H3-H4 cluster in an autosome which, in most cases, was located interstitially in the proximal chromosome third. In 17 out of the 21 species with 2n♂ = 23 acrocentric chromosomes, the H3-H4-carrying autosome was about eighth in order of decreasing size. Two of the four exceptions changed H3-H4 localization to proximal (Pezotettix giornae) or distal (Tropidopola graeca) in the eighth-sized autosome, but the remainder (the two Eyprepocnemis species) showed the H3-H4 cluster distally located in the second-sized autosome. All 14 species with 2n♂ = 17 chromosomes (including three long metacentric autosome pairs, five acrocentric autosome pairs and an acrocentric X chromosome) carried an interstitial H3-H4 cluster in the short arm of the smallest of the three long metacentric pairs. These results suggest that chromosome location of H3-H4 histone gene clusters seem to be highly conservative in Acrididae grasshoppers. The change in H3-H4 location from the acrocentric medium-sized autosome in the 2n♂ = 23 karyotype to the long metacentric autosome in the 2n♂ = 17 karyotype is most parsimoniously explained by common ancestry, i.e. by the involvement of the H3-H4-carrying acrocentric in the centric fusion that gave rise to the smallest of the three long metacentric autosomes of 2n♂ = 17 species.

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.

Association of systemic lupus erythematosus with decreased immunosuppressive potential of the IgG glycome.

Glycans attached to the Fc portion of IgG are important modulators of IgG effector functions. Interindividual differences in IgG glycome composition are large and they associate strongly with different inflammatory and autoimmune diseases. IKZF1, HLA–DQ2A/B, and BACH2 genetic loci that affect IgG glycome composition show pleiotropy with systemic lupus erythematosus (SLE), indicating a potentially causative role of aberrant IgG glycosylation in SLE. We undertook this large multicenter case–control study to determine whether SLE is associated with altered IgG glycosylation.

The genetic basis of systemic lupus erythematosus: What are the risk factors and what have we learned.

The genome-wide association study is a free-hypothesis approach based on screening of thousands or even millions of genetic variants distributed throughout the whole human genome in relation to a phenotype. The relevant role of the genome-wide association studies in the last decade is undisputed because it has permitted to elucidate multiple risk genetic factors associated with the susceptibility to several human complex diseases. Regarding systemic lupus erythematosus (SLE) this approach has allowed to identify more than 60 risk loci for SLE susceptibility across populations to date, increasing our understanding on the pathogenesis of this disease. We present the latest findings in the genetic of SLE across populations using genome-wide approaches. These studies revealed that most of the genetic risk is shared across borders and ethnicities. Finally, we focus on describing the most important risk loci for SLE attempting to cover the genetic findings in relation to functional polymorphisms, such as missense single nucleotide polymorphisms (SNPs) or regulatory variants involved in the development of the disease. The functional studies try to identify the causality of some GWAS-associated variants, many of which fall in non-coding regions of the genome, suggesting a regulatory role. Many loci show an environmental interaction, another aspect revealed by the studies of epigenetic modifications and those associated with genetic variants. Finally, new-generation sequencing technologies can open other paths in the research on SLE genetics, the role of rare variants and the detailed identification of causal regulatory variation. The clinical relevance of the genetic factors will be shown when we are able to use them or in combination with other molecular measurements to re-classify a heterogeneous disease such as SLE.

Microdissection and chromosome painting of X and B chromosomes in Locusta migratoria.

Acquisition of knowledge of the nature and DNA content of B chromosomes has been triggered by a collection of molecular techniques, one of which, microdissection, has provided interesting results in a number of B chromosome systems. Here we provide the first data on the molecular composition of B chromosomes in Locusta migratoria, after microdissection of the B and X chromosomes, DNA amplification by one (B) or two (X) different methods, and chromosome painting. The results showed that B chromosomes share at least two types of repetitive DNA sequences with the A chromosomes, suggesting that Bs in this species most likely arose intraspecifically. One of these repetitive DNAs is located on the heterochromatic distal half of the B chromosome and in the pericentromeric regions of about half of the A chromosomes, including the X. The other type of repetitive DNA is located interspersedly over the non-centromeric euchromatic regions of all A chromosomes and in an interstitial part of the proximal euchromatic half of the B chromosome. Chromosome painting, however, did not provide results sufficiently reliable to determine, in this species, which A chromosome gave rise to the B; this might be done by detailed analysis of the microdissected DNA sequences

Study of Association of CD40-CD154 Gene Polymorphisms with Disease Susceptibility and Cardiovascular Risk in Spanish Rheumatoid Arthritis Patients

Objective Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased cardiovascular (CV) mortality. Since CD40-CD154 binding has direct consequences on inflammation process initiation, we aimed to replicate previous findings related to disease susceptibility in Spanish RA population. Furthermore, as the major complication in RA disease patients is the development of CV events due to accelerated atherosclerosis, and elevated levels of CD40L/CD154 are present in patients with acute myocardial infarction, we assessed the potential association of CD40 and CD154/CD40L gene variants with CV risk in Spanish RA patients. Methods One thousand five hundred and seventy-five patients fulfilling the 1987 ACR classification criteria for RA and 1600 matched controls were genotyped for the CD40 rs1883832, rs4810485 and rs1535045 and CD154 rs3092952 and rs3092920 gene polymorphisms, using predesigned TaqMan single nucleotide polymorphism genotyping assays. Afterwards, we investigated the influence of CD40-CD154 gene variants in the development of CV events. Also, in a subgroup of 273 patients without history of CV events, we assessed the influence of these polymorphisms in the risk of subclinical atherosclerosis determined by carotid ultrasonography. Results Nominally significant differences in the allele frequencies for the rs1883832 CD40 gene polymorphism between RA patients and controls were found (p = 0.038). Although we did not observe a significant association of CD40-CD154 gene variants with the development of CV events, an ANCOVA model adjusted for sex, age at the time of the ultrasonography assessment, follow-up time, traditional CV risk factors and anti-cyclic citrullinated peptide antibodies disclosed a significant association (p = 0.0047) between CD40 rs1535045 polymorphism and carotid intima media thickness, a surrogate marker of atherosclerosis. Conclusion Data from our pilot study indicate a potential association of rs1883832 CD40 gene polymorphism with susceptibility to RA. Also, the CD40 rs1535045 gene variant may influence development of subclinical atherosclerosis in RA patients.

A Single, Recent Origin of the Accessory B Chromosome of the Grasshopper Eyprepocnemis plorans

B chromosomes are dispensable chromosomes found in >2000 eukaryotic species, usually behaving as genomic parasites. Most B chromosomes seem to be made up of the same kind of DNA sequences present in the A chromosomes. This sequence similarity makes it difficult to obtain specific molecular probes that may permit B-presence diagnosis without cytogenetic analysis. We have developed a sequence-characterized amplified region (SCAR) marker for B chromosomes in the grasshopper Eyprepocnemis plorans, which specifically amplifies a 1510-bp DNA fragment exclusively in B-carrying individuals. Fluorescent in situ hybridization and fiber FISH analyses showed that this marker is a tandemly repeated DNA sequence closely intermingled with 45S rDNA. PCR reactions showed the presence of SCAR-like sequences in the A chromosomes, but in two separate fragments, supporting the intraspecific origin of B chromosomes in this species. SCAR marker DNA sequence showed to be identical in B chromosome variants from several localities from Spain and Morocco, and it was very similar to those found in B chromosome variants from Greece and Armenia. This strongly suggests that this sequence was already present in the ancestral B chromosome of this species. In addition, the scarce sequence variation observed among several B variants from very distant populations suggests either a functional constraint or, more likely, a recent and unique origin for B chromosomes in this species.

Histone H3 lysine 9 acetylation pattern suggests that X and B chromosomes are silenced during entire male meiosis in a grasshopper

The facultative heterochromatic X chromosome in leptotene spermatocytes of the grasshopper Eyprepocnemis plorans showed marked hypoacetylation for lysine 9 in the H3 histone (H3-K9) with no sign of histone H2AX phosphorylation. Since H3-K9 hypoacetylation precedes the meiotic appearance of phosphorylated H2AX (γ-H2AX), which marks the beginning of recombinational DNA double-strand breaks (DSBs), it seems that meiotic sex-chromosome inactivation (MSCI) in this grasshopper occurs prior to the beginning of recombination and hence synapsis (which in this species begins later than recombination). In addition, all constitutively heterochromatic chromosome regions harbouring a 180-bp tandem-repeat DNA and rDNA (B chromosomes and pericentromeric regions of A chromosomes) were H3-K9 hypoacetylated at early leptotene even though they will synapse at subsequent stages. This also suggests that meiotic silencing in this grasshopper might be independent of synapsis. The H3-K9 hypoacetylated state of facultative and constitutive heterochromatin persisted during subsequent meiotic stages and was even apparent in round spermatids. Finally, the fact that B chromosomes are differentially hypoacetylated in testis and embryo interphase cells suggests that they might be silenced early in development and remain this way for most (or all) life-cycle stages.