Centro Científico Tecnólogico - Rosario

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease with marked gender and ethnic disparities. We report a large transancestral association study of SLE using Immunochip genotype data from 27,574 individuals of European (EA), African (AA) and Hispanic Amerindian (HA) ancestry. We identify 58 distinct non-HLA regions in EA, 9 in AA and 16 in HA (∼50% of these regions have multiple independent associations); these include 24 novel SLE regions ( P <5 × 10 −8 ), refined association signals in established regions, extended associations to additional ancestries, and a disentangled complex HLA multigenic effect. The risk allele count (genetic load) exhibits an accelerating pattern of SLE risk, leading us to posit a cumulative hit hypothesis for autoimmune disease. Comparing results across the three ancestries identifies both ancestry-dependent and ancestry-independent contributions to SLE risk. Our results are consistent with the unique and complex histories of the populations sampled, and collectively help clarify the genetic architecture and ethnic disparities in SLE.

South America has a complex demographic history shaped by multiple migration and admixture events in pre- and post-colonial times. Settled over 14,000 years ago by Native Americans, South America has experienced migrations of European and African individuals, similar to other regions in the Americas. However, the timing and magnitude of these events resulted in markedly different patterns of admixture throughout Latin America. We use genome-wide SNP data for 437 admixed individuals from 5 countries (Colombia, Ecuador, Peru, Chile, and Argentina) to explore the population structure and demographic history of South American Latinos. We combined these data with population reference panels from Africa, Asia, Europe and the Americas to perform global ancestry analysis and infer the subcontinental origin of the European and Native American ancestry components of the admixed individuals. By applying ancestry-specific PCA analyses we find that most of the European ancestry in South American Latinos is from the Iberian Peninsula; however, many individuals trace their ancestry back to Italy, especially within Argentina. We find a strong gradient in the Native American ancestry component of South American Latinos associated with country of origin and the geography of local indigenous populations. For example, Native American genomic segments in Peruvians show greater affinities with Andean indigenous peoples like Quechua and Aymara, whereas Native American haplotypes from Colombians tend to cluster with Amazonian and coastal tribes from northern South America. Using ancestry tract length analysis we modeled post-colonial South American migration history as the youngest in Latin America during European colonization (9-14 generations ago), with an additional strong pulse of European migration occurring between 3 and 9 generations ago. These genetic footprints can impact our understanding of population-level differences in biomedical traits and, thus, inform future medical genetic studies in the region.

OBJECTIVE: 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. METHODS: Using ultra-performance liquid chromatography analysis of released glycans, we analyzed the composition of the IgG glycome in 261 SLE patients and 247 matched controls of Latin American Mestizo origin (the discovery cohort) and in 2 independent replication cohorts of different ethnicity (108 SLE patients and 193 controls from Trinidad, and 106 SLE patients and 105 controls from China). RESULTS: Multiple statistically significant differences in IgG glycome composition were observed between patients and controls. The most significant changes included decreased galactosylation and sialylation of IgG (which regulate proinflammatory and antiinflammatory actions of IgG) as well as decreased core fucose and increased bisecting N-acetylglucosamine (which affect antibody-dependent cell-mediated cytotoxicity). CONCLUSION: The IgG glycome in SLE patients is significantly altered in a way that decreases immunosuppressive action of circulating immunoglobulins. The magnitude of observed changes is associated with the intensity of the disease, indicating that aberrant IgG glycome composition or changes in IgG glycosylation may be an important molecular mechanism in SLE.

INTRODUCTION: Systemic lupus erythematosus (SLE) is a disease distributed worldwide, which occurs in both genders, and across racial/ethnic and age groups; however, higher rates are observed in adults, in women and in non-Caucasians. Genetic, environmental, sociodemographic and methodological issues are responsible not only for these differences but for the variable course and outcome of the disease. Non-Caucasians have a more severe disease with a higher risk for early mortality and damage accrual. Males also have a more severe disease; however, a negative impact of male gender on lupus outcomes has not been firmly established. Childhood-onset is associated with a more severe disease; moreover, it is also associated with higher damage and diminished survival; finally, late-onset lupus is mild but it is associated with higher damage accrual and a diminished survival. Areas covered: In this review, we discuss the incidence and prevalence of SLE, the impact of age, gender and race/ethnicity in SLE and in the survival of those affected. Expert commentary: Age, gender and race/ethnicity impact disease expression in SLE patients; despite improvements in survival, mortality in SLE remains almost three times higher than in the general population.

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Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, P(meta) = 6.6×10(-8), OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, P(meta) = 2.9×10(-7), OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ~146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (P(meta) = 3.2×10(-7), OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (P(meta) = 3.5×10(-4), OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE.

OBJECTIVE: More than 80% of autoimmune disease predominantly affects females, but the mechanism for this female bias is poorly understood. We suspected that an X chromosome dose effect accounts for this, and we undertook this study to test our hypothesis that trisomy X (47,XXX; occurring in ∼1 in 1,000 live female births) would be increased in patients with female-predominant diseases (systemic lupus erythematosus [SLE], primary Sjögren's syndrome [SS], primary biliary cirrhosis, and rheumatoid arthritis [RA]) compared to patients with diseases without female predominance (sarcoidosis) and compared to controls. METHODS: All subjects in this study were female. We identified subjects with 47,XXX using aggregate data from single-nucleotide polymorphism arrays, and, when possible, we confirmed the presence of 47,XXX using fluorescence in situ hybridization or quantitative polymerase chain reaction. RESULTS: We found 47,XXX in 7 of 2,826 SLE patients and in 3 of 1,033 SS patients, but in only 2 of 7,074 controls (odds ratio in the SLE and primary SS groups 8.78 [95% confidence interval 1.67-86.79], P = 0.003 and odds ratio 10.29 [95% confidence interval 1.18-123.47], P = 0.02, respectively). One in 404 women with SLE and 1 in 344 women with SS had 47,XXX. There was an excess of 47,XXX among SLE and SS patients. CONCLUSION: The estimated prevalence of SLE and SS in women with 47,XXX was ∼2.5 and ∼2.9 times higher, respectively, than that in women with 46,XX and ∼25 and ∼41 times higher, respectively, than that in men with 46,XY. No statistically significant increase of 47,XXX was observed in other female-biased diseases (primary biliary cirrhosis or RA), supporting the idea of multiple pathways to sex bias in autoimmunity.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a strong genetic component. We undertook the present work to perform the first genome-wide association study on individuals from the Americas who are enriched for Native American heritage. METHODS: We analyzed 3,710 individuals from the US and 4 countries of Latin America who were diagnosed as having SLE, and healthy controls. Samples were genotyped with HumanOmni1 BeadChip. Data on out-of-study controls genotyped with HumanOmni2.5 were also included. Statistical analyses were performed using SNPtest and SNPGWA. Data were adjusted for genomic control and false discovery rate. Imputation was performed using Impute2 and, for classic HLA alleles, HiBag. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. RESULTS: The IRF5-TNPO3 region showed the strongest association and largest OR for SLE (rs10488631: genomic control-adjusted P [Pgcadj ] = 2.61 × 10(-29), OR 2.12 [95% CI 1.88-2.39]), followed by HLA class II on the DQA2-DQB1 loci (rs9275572: Pgcadj = 1.11 × 10(-16), OR 1.62 [95% CI 1.46-1.80] and rs9271366: Pgcadj = 6.46 × 10(-12), OR 2.06 [95% CI 1.71-2.50]). Other known SLE loci found to be associated in this population were ITGAM, STAT4, TNIP1, NCF2, and IRAK1. We identified a novel locus on 10q24.33 (rs4917385: Pgcadj = 1.39 × 10(-8)) with an expression quantitative trait locus (eQTL) effect (Peqtl = 8.0 × 10(-37) at USMG5/miR1307), and several new suggestive loci. SLE risk loci previously identified in Europeans and Asians were corroborated. Local ancestry estimation showed that the HLA allele risk contribution is of European ancestral origin. Imputation of HLA alleles suggested that autochthonous Native American haplotypes provide protection against development of SLE. CONCLUSION: Our results demonstrate that studying admixed populations provides new insights in the delineation of the genetic architecture that underlies autoimmune and complex diseases.

Immune-mediated nephritis contributes to disease in systemic lupus erythematosus, Goodpasture syndrome (caused by antibodies specific for glomerular basement membrane [anti-GBM antibodies]), and spontaneous lupus nephritis. Inbred mouse strains differ in susceptibility to anti-GBM antibody-induced and spontaneous lupus nephritis. This study sought to clarify the genetic and molecular factors that maybe responsible for enhanced immune-mediated renal disease in these models. When the kidneys of 3 mouse strains sensitive to anti-GBM antibody-induced nephritis were compared with those of 2 control strains using microarray analysis, one-fifth of the underexpressed genes belonged to the kallikrein gene family,which encodes serine esterases. Mouse strains that upregulated renal and urinary kallikreins exhibited less evidence of disease. Antagonizing the kallikrein pathway augmented disease, while agonists dampened the severity of anti-GBM antibody-induced nephritis. In addition, nephritis-sensitive mouse strains had kallikrein haplotypes that were distinct from those of control strains, including several regulatory polymorphisms,some of which were associated with functional consequences. Indeed, increased susceptibility to anti-GBM antibody-induced nephritis and spontaneous lupus nephritis was achieved by breeding mice with a genetic interval harboring the kallikrein genes onto a disease-resistant background. Finally, both human SLE and spontaneous lupus nephritis were found to be associated with kallikrein genes, particularly KLK1 and the KLK3 promoter, when DNA SNPs from independent cohorts of SLE patients and controls were compared. Collectively, these studies suggest that kallikreins are protective disease-associated genes in anti-GBM antibody-induced nephritis and lupus.

We previously reported that the G allele of rs3853839 at 3'untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10(-10), odds ratio (OR) (95%CI) = 1.27 (1.17-1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10(-11), OR = 1.24 [1.18-1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3'UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R(2) = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3'UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta = 2.0×10(-19), OR = 1.25 [1.20-1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

BACKGROUND AND PURPOSE: In a previous study, 0.3 and 0.45 mg/kg of intravenous recombinant tissue plasminogen activator (rt-PA) were safe when combined with eptifibatide 75 mcg/kg bolus and a 2-hour infusion (0.75 mcg/kg per minute). The Combined Approach to Lysis Utilizing Eptifibatide and rt-PA in Acute Ischemic Stroke-Enhanced Regimen (CLEAR-ER) trial sought to determine the safety of a higher-dose regimen and to establish evidence for a phase III trial. METHODS: CLEAR-ER was a multicenter, double-blind, randomized safety study. Ischemic stroke patients were randomized to 0.6 mg/kg rt-PA plus eptifibatide (135 mcg/kg bolus and a 2-hour infusion at 0.75 mcg/kg per minute) versus standard rt-PA (0.9 mg/kg). The primary safety end point was the incidence of symptomatic intracranial hemorrhage within 36 hours. The primary efficacy outcome measure was the modified Rankin Scale (mRS) score ≤1 or return to baseline mRS at 90 days. Analysis of the safety and efficacy outcomes was done with multiple logistic regression. RESULTS: Of 126 subjects, 101 received combination therapy, and 25 received standard rt-PA. Two (2%) patients in the combination group and 3 (12%) in the standard group had symptomatic intracranial hemorrhage (odds ratio, 0.15; 95% confidence interval, 0.01-1.40; P=0.053). At 90 days, 49.5% of the combination group had mRS ≤1 or return to baseline mRS versus 36.0% in the standard group (odds ratio, 1.74; 95% confidence interval, 0.70-4.31; P=0.23). After adjusting for age, baseline National Institutes of Health Stroke Scale, time to intravenous rt-PA, and baseline mRS, the odds ratio was 1.38 (95% confidence interval, 0.51-3.76; P=0.52). CONCLUSIONS: The combined regimen of intravenous rt-PA and eptifibatide studied in this trial was safe and provides evidence that a phase III trial is warranted to determine efficacy of the regimen. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT00894803.

BACKGROUND: Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme β-glucocerebrosidase-1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S-GBA1 mutation on cellular homeostasis and vulnerability in a patient-specific cellular model of PD. METHODS: We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy-lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry. RESULTS: The N370S mutation produced a significant reduction in β-glucocerebrosidase-1 protein and enzyme activity and β-glucocerebrosidase-1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death. CONCLUSIONS: Our results support a connection between the loss of β-glucocerebrosidase-1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1-PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1-PD shares common features with lipid-storage diseases. © 2017 International Parkinson and Movement Disorder Society.

OBJECTIVE: To determine whether specific isoforms of IRF5 are transcribed in patients with systemic lupus erythematosus (SLE) who have risk genotypes in the exon 1B donor splice site at single-nucleotide polymorphism (SNP) no. rs2004640. METHODS: Peripheral blood mononuclear cells were obtained from SLE patients and healthy controls from Argentina, Spain, and Germany and from trio families from Spain and Denmark. A reporter assay was used to investigate the role of SNP no. rs2004640. IRF5 expression in relation to the genotypes of functional SNPs was analyzed using quantitative polymerase chain reaction. Sequencing and genotyping of the IRF5 gene was performed. RESULTS: Sequencing of complementary DNA from individuals with different genotypes showed 4 basic isoforms transcribed from all 5'-untranslated regions (5'-UTRs), suggesting no preferential isoform transcription based on rs2004640 genotypes. Analysis of translation efficiency showed that exon 1A was the most efficient in initiating protein synthesis. We identified a novel polymorphic insertion/deletion that defines the pattern of expression of isoforms of IRF5. The insertion consists of 4 repeats in exon 6 affecting the protein interaction domain. The insertion segregates in the risk haplotype with the high expression allele of a poly(A) site SNP no. rs10954213 and the exon 1B donor splice allele of the 5'-UTR SNP no. rs2004640. The poly(A) polymorphism correlated with levels of IRF5 in cells stimulated with interferon-alpha. The SNP most strongly associated with SLE was SNP no. rs2070197 (P=5.2x10(-11)), which is a proxy of the risk haplotype, but does not appear to be functional. CONCLUSION: None of the functional variants investigated in this study is strongly associated with SLE, with the exception of the exon 1B donor splice site, and its functional importance appears to be small. Our results suggest that there may be other functional polymorphisms, yet to be identified, in IRF5. We did not observe evidence of epistatic interaction between the functional SNPs.

OBJECTIVE: To study the effect of the innate cytokine macrophage migration inhibitory factor (MIF) on the susceptibility and severity of systemic lupus erythematosus (SLE) in a multinational population of 1,369 Caucasian and African American patients. METHODS: Two functional polymorphisms in the MIF gene, a -794 CATT(5-8) microsatellite repeat (rs5844572) and a -173 G/C single-nucleotide polymorphism (rs755622), were assessed for association with SLE in 3,195 patients and healthy controls. We also measured MIF plasma levels in relation to genotypes and clinical phenotypes, and assessed Toll-like receptor 7 (TLR-7)-stimulated MIF production in vitro. RESULTS: Both Caucasians and African Americans with the high-expression MIF haplotype -794 CATT(7)/-173*C had a lower incidence of SLE (in Caucasians, odds ratio [OR] 0.63, 95% confidence interval [95% CI] 0.53-0.89, P = 0.001; in African Americans, OR 0.46, 95% CI 0.23-0.95, P = 0.012). In contrast, among patients with established SLE, reduced frequencies of low-expression MIF genotypes (-794 CATT(5)) were observed in those with nephritis, those with serositis, and those with central nervous system (CNS) involvement when compared to patients without end-organ involvement (P = 0.023, P = 0.005, and P = 0.04, respectively). Plasma MIF levels and TLR-7-stimulated MIF production in vitro reflected the underlying MIF genotype of the studied groups. CONCLUSION: These findings suggest that MIF, which has both proinflammatory properties and macrophage and B cell survival functions, exerts a dual influence on the immunopathogenesis of SLE. High-expression MIF genotypes are associated with a reduced susceptibility to SLE and may contribute to an enhanced clearance of infectious pathogens. Once SLE develops, however, low-expression MIF genotypes may protect from ensuing inflammatory end-organ damage.

Significance In agricultural crops, plants are cultivated in close proximity, exposed to mutual shading. Photosensory receptors perceive this light environment and initiate growth responses that adjust the position of plant organs within the canopy to maximize light capture. During the early hours under shade, these responses are mediated by elevated levels of the hormone auxin. Here we show that under prolonged shade, the PHYTOCHROME INTERACTING FACTOR 4 light-signaling transcription factor, selected auxin receptors, their upstream regulatory miRNA, and downstream transcriptional regulators change their abundance to enhance growth responses, while auxin levels return to those observed before shade. Thus, in the presence of persistent shade, plants elicit a system-level rearrangement of auxin signaling, which sustains growth without increased auxin.

Argentine population genetic structure was examined using a set of 78 ancestry informative markers (AIMs) to assess the contributions of European, Amerindian, and African ancestry in 94 individuals members of this population. Using the Bayesian clustering algorithm STRUCTURE, the mean European contribution was 78%, the Amerindian contribution was 19.4%, and the African contribution was 2.5%. Similar results were found using weighted least mean square method: European, 80.2%; Amerindian, 18.1%; and African, 1.7%. Consistent with previous studies the current results showed very few individuals (four of 94) with greater than 10% African admixture. Notably, when individual admixture was examined, the Amerindian and European admixture showed a very large variance and individual Amerindian contribution ranged from 1.5 to 84.5% in the 94 individual Argentine subjects. These results indicate that admixture must be considered when clinical epidemiology or case control genetic analyses are studied in this population. Moreover, the current study provides a set of informative SNPs that can be used to ascertain or control for this potentially hidden stratification. In addition, the large variance in admixture proportions in individual Argentine subjects shown by this study suggests that this population is appropriate for future admixture mapping studies.

The production of β-lactamase enzymes is one of the most distributed resistance mechanisms towards β-lactam antibiotics. Metallo-β-lactamases constitute a worrisome group of these kinds of enzymes, since they present a broad spectrum profile, being able to hydrolyze not only penicillins, but also the latest generation of cephalosporins and carbapenems, which constitute at present the last resource antibiotics. The VIM, IMP, and NDM enzymes comprise the main groups of clinically relevant metallo-β-lactamases. Here we present an update of the features of the natural variants that have emerged and of the ones that have been engineered in the laboratory, in an effort to find sequence and structural determinants of substrate preferences. This knowledge is of upmost importance in novel drug design efforts. We also discuss the advances in knowledge achieved by means of in vitro directed evolution experiments, and the potential of this approach to predict natural evolution of metallo-β-lactamases.The production of β-lactamase enzymes is one of the most distributed resistance mechanisms towards β-lactam antibiotics. Metallo-β-lactamases constitute a worrisome group of these kinds of enzymes, since they present a broad spectrum profile, being able to hydrolyze not only penicillins, but also the latest generation of cephalosporins and carbapenems, which constitute at present the last resource antibiotics. The VIM, IMP, and NDM enzymes comprise the main groups of clinically relevant metallo-β-lactamases. Here we present an update of the features of the natural variants that have emerged and of the ones that have been engineered in the laboratory, in an effort to find sequence and structural determinants of substrate preferences. This knowledge is of upmost importance in novel drug design efforts. We also discuss the advances in knowledge achieved by means of in vitro directed evolution experiments, and the potential of this approach to predict natural evolution of metallo-β-lactamases.

Recent reports have associated NCF2, encoding a core component of the multi-protein NADPH oxidase (NADPHO), with systemic lupus erythematosus (SLE) susceptibility in individuals of European ancestry. To identify ethnicity-specific and -robust variants within NCF2, we assessed 145 SNPs in and around the NCF2 gene in 5325 cases and 21 866 controls of European-American (EA), African-American (AA), Hispanic (HS) and Korean (KR) ancestry. Subsequent imputation, conditional, haplotype and bioinformatic analyses identified seven potentially functional SLE-predisposing variants. Association with non-synonymous rs17849502, previously reported in EA, was detected in EA, HS and AA (P(EA) = 1.01 × 10(-54), PHS = 3.68 × 10(-10), P(AA) = 0.03); synonymous rs17849501 was similarly significant. These SNPs were monomorphic in KR. Novel associations were detected with coding variants at rs35937854 in AA (PAA = 1.49 × 10(-9)), and rs13306575 in HS and KR (P(HS) = 7.04 × 10(-7), P(KR) = 3.30 × 10(-3)). In KR, a 3-SNP haplotype was significantly associated (P = 4.20 × 10(-7)), implying that SLE predisposing variants were tagged. Significant SNP-SNP interaction (P = 0.02) was detected between rs13306575 and rs17849502 in HS, and a dramatically increased risk (OR = 6.55) with a risk allele at each locus. Molecular modeling predicts that these non-synonymous mutations could disrupt NADPHO complex assembly. The risk allele of rs17849501, located in a conserved transcriptional regulatory region, increased reporter gene activity, suggesting in vivo enhancer function. Our results not only establish allelic heterogeneity within NCF2 associated with SLE, but also emphasize the utility of multi-ethnic cohorts to identify predisposing variants explaining additional phenotypic variance ('missing heritability') of complex diseases like SLE.

OBJECTIVE: To assess whether genetically determined Amerindian ancestry predicts increased presence of risk alleles of known susceptibility genes for systemic lupus erythematosus (SLE). METHODS: Single-nucleotide polymorphisms (SNPs) within 16 confirmed genetic susceptibility loci for SLE were genotyped in a set of 804 Mestizo lupus patients and 667 Mestizo healthy controls. In addition, 347 admixture informative markers were genotyped. Individual ancestry proportions were determined using STRUCTURE. Association analysis was performed using PLINK, and correlation between ancestry and the presence of risk alleles was analyzed using linear regression. RESULTS: A meta-analysis of the genetic association of the 16 SNPs across populations showed that TNFSF4, STAT4, ITGAM, and IRF5 were associated with lupus in a Hispanic Mestizo cohort enriched for European and Amerindian ancestry. In addition, 2 SNPs within the major histocompatibility complex region, previously shown to be associated in a genome-wide association study in Europeans, were also associated in Mestizos. Using linear regression, we predicted an average increase of 2.34 risk alleles when comparing an SLE patient with 100% Amerindian ancestry versus an SLE patient with 0% Amerindian ancestry (P < 0.0001). SLE patients with 43% more Amerindian ancestry were predicted to carry 1 additional risk allele. CONCLUSION: Our results demonstrate that Amerindian ancestry is associated with an increased number of risk alleles for SLE.