Genetic risk and longitudinal disease activity in systemic lupus erythematosus using targeted maximum likelihood estimation.

Systemic lupus erythematous (SLE) is a chronic autoimmune disease associated with genetic and environmental risk factors. However, the extent to which genetic risk is causally associated with disease activity is unknown. We utilized longitudinal-targeted maximum likelihood estimation to estimate the causal association between a genetic risk score (GRS) comprising 41 established SLE variants and clinically important disease activity as measured by the validated Systemic Lupus Activity Questionnaire (SLAQ) in a multiethnic cohort of 942 individuals with SLE. We did not find evidence of a clinically important SLAQ score difference (>4.0) for individuals with a high GRS compared with those with a low GRS across nine time points after controlling for sex, ancestry, renal status, dialysis, disease duration, treatment, depression, smoking and education, as well as time-dependent confounding of missing visits. Individual single-nucleotide polymorphism (SNP) analyses revealed that 12 of the 41 variants were significantly associated with clinically relevant changes in SLAQ scores across time points eight and nine after controlling for multiple testing. Results based on sophisticated causal modeling of longitudinal data in a large patient cohort suggest that individual SLE risk variants may influence disease activity over time. Our findings also emphasize a role for other biological or environmental factors.

The cumulative effects of known susceptibility variants to predict primary biliary cirrhosis risk.

Multiple genetic variants influence the risk for development of primary biliary cirrhosis (PBC). To explore the cumulative effects of known susceptibility loci on risk, we utilized a weighted genetic risk score (wGRS) to evaluate whether genetic information can predict susceptibility. The wGRS was created using 26 known susceptibility loci and investigated in 1840 UK PBC and 5164 controls. Our data indicate that the wGRS was significantly different between PBC and controls (P=1.61E-142). Moreover, we assessed predictive performance of wGRS on disease status by calculating the area under the receiver operator characteristic curve. The area under curve for the purely genetic model was 0.72 and for gender plus genetic model was 0.82, with confidence limits substantially above random predictions. The risk of PBC using logistic regression was estimated after dividing individuals into quartiles. Individuals in the highest disclosed risk group demonstrated a substantially increased risk for PBC compared with the lowest risk group (odds ratio: 9.3, P=1.91E-084). Finally, we validated our findings in an analysis of an Italian PBC cohort. Our data suggested that the wGRS, utilizing genetic variants, was significantly associated with increased risk for PBC with consistent discriminant ability. Our study is a first step toward risk prediction for PBC.

MHC associations with clinical and autoantibody manifestations in European SLE.

Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease affecting multiple organ systems and characterized by autoantibody formation to nuclear components. Although genetic variation within the major histocompatibility complex (MHC) is associated with SLE, its role in the development of clinical manifestations and autoantibody production is not well defined. We conducted a meta-analysis of four independent European SLE case collections for associations between SLE sub-phenotypes and MHC single-nucleotide polymorphism genotypes, human leukocyte antigen (HLA) alleles and variant HLA amino acids. Of the 11 American College of Rheumatology criteria and 7 autoantibody sub-phenotypes examined, anti-Ro/SSA and anti-La/SSB antibody subsets exhibited the highest number and most statistically significant associations. HLA-DRB1*03:01 was significantly associated with both sub-phenotypes. We found evidence of associations independent of MHC class II variants in the anti-Ro subset alone. Conditional analyses showed that anti-Ro and anti-La subsets are independently associated with HLA-DRB1*0301, and that the HLA-DRB1*03:01 association with SLE is largely but not completely driven by the association of this allele with these sub-phenotypes. Our results provide strong evidence for a multilevel risk model for HLA-DRB1*03:01 in SLE, where the association with anti-Ro and anti-La antibody-positive SLE is much stronger than SLE without these autoantibodies.

Pathway-based analysis of primary biliary cirrhosis genome-wide association studies.

Genome-wide association studies (GWAS) have successfully identified several loci associated with primary biliary cirrhosis (PBC) risk. Pathway analysis complements conventional GWAS analysis. We applied the recently developed linear combination test for pathways to datasets drawn from independent PBC GWAS in Italian and Canadian subjects. Of the Kyoto Encyclopedia of Genes and Genomes and BioCarta pathways tested, 25 pathways in the Italian dataset (449 cases, 940 controls) and 26 pathways in the Canadian dataset (530 cases, 398 controls) were associated with PBC susceptibility (P<0.05). After correcting for multiple comparisons, only the eight most significant pathways in the Italian dataset had FDR <0.25 with tumor necrosis factor/stress-related signaling emerging as the top pathway (P=7.38 × 10⁻4, FDR=0.18). Two pathways, phosphatidylinositol signaling and hedgehog signaling, were replicated in both datasets (P<0.05), and subjected to two additional complementary pathway tests. Both pathway signals remained significant in the Italian dataset on modified gene set enrichment analysis (P<0.05). In both GWAS, variants nominally associated with PBC were significantly overrepresented in the phosphatidylinositol pathway (Fisher exact P<0.05). These results point to established and novel pathway-level associations with inherited predisposition to PBC that, on further independent replication and functional validation, may provide fresh insights into PBC etiology.

Glycine receptor beta-subunit gene mutation in spastic mouse associated with LINE-1 element insertion.

Congenital myoclonus is a widespread neurologic disorder characterized by hyperexcitability, muscular spasticity and myoclonus associated with marked reduction in neural glycine binding sites. The recessive mouse mutation spastic (spa) is a prototype of inherited myoclonus. Here we show that defects in the gene encoding the beta-subunit of the glycine receptor (Glrb) underlie spa: Glrb maps to the same region of mouse chromosome 3 as spa, and Glrb mRNA is markedly reduced throughout brains of spa mice, most likely as a result of an insertional mutation of a 7.1 kilobase LINE-1 element within intron 6 of Glrb. These results provide evidence that Glrb is necessary for postsynaptic expression of glycine receptor complexes, and suggest Glrb as a candidate gene for inherited myoclonus in other species.

Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia.

A mitochondrial protein called uncoupling protein (UCP1) plays an important role in generating heat and burning calories by creating a pathway that allows dissipation of the proton electrochemical gradient across the inner mitochondrial membrane in brown adipose tissue, without coupling to any other energy-consuming process. This pathway has been implicated in the regulation of body temperature, body composition and glucose metabolism. However, UCP1-containing brown adipose tissue is unlikely to be involved in weight regulation in adult large-size animals and humans living in a thermoneutral environment (one where an animal does not have to increase oxygen consumption or energy expenditure to lose or gain heat to maintain body temperature), as there is little brown adipose tissue present. We now report the discovery of a gene that codes for a novel uncoupling protein, designated UCP2, which has 59% amino-acid identity to UCP1, and describe properties consistent with a role in diabetes and obesity. In comparison with UCP1, UCP2 has a greater effect on mitochondrial membrane potential when expressed in yeast. Compared to UCP1, the gene is widely expressed in adult human tissues, including tissues rich in macrophages, and it is upregulated in white fat in response to fat feeding. Finally, UCP2 maps to regions of human chromosome 11 and mouse chromosome 7 that have been linked to hyperinsulinaemia and obesity. Our findings suggest that UCP2 has a unique role in energy balance, body weight regulation and thermoregulation and their responses to inflammatory stimuli.

Classical HLA-DRB1 and DPB1 alleles account for HLA associations with primary biliary cirrhosis.

Susceptibility to primary biliary cirrhosis (PBC) is strongly associated with human leukocyte antigen (HLA)-region polymorphisms. To determine if associations can be explained by classical HLA determinants, we studied Italian, 676 cases and 1440 controls, genotyped with dense single-nucleotide polymorphisms (SNPs) for which classical HLA alleles and amino acids were imputed. Although previous genome-wide association studies and our results show stronger SNP associations near DQB1, we demonstrate that the HLA signals can be attributed to classical DRB1 and DPB1 genes. Strong support for the predominant role of DRB1 is provided by our conditional analyses. We also demonstrate an independent association of DPB1. Specific HLA-DRB1 genes (*08, *11 and *14) account for most of the DRB1 association signal. Consistent with previous studies, DRB1*08 (P=1.59 × 10(-11)) was the strongest predisposing allele, whereas DRB1*11 (P=1.42 × 10(-10)) was protective. Additionally, DRB1*14 and the DPB1 association (DPB1*03:01; P=9.18 × 10(-7)) were predisposing risk alleles. No signal was observed in the HLA class 1 or class 3 regions. These findings better define the association of PBC with HLA and specifically support the role of classical HLA-DRB1 and DPB1 genes and alleles in susceptibility to PBC.

Relationship between diabetes risk and admixture in postmenopausal African-American and Hispanic-American women.

AIMS/HYPOTHESIS: Type 2 diabetes is more prevalent in African-Americans (AFAs) and Hispanic-Americans (HAs) than in European-Americans. We assessed whether continental admixture was correlated with diabetes risk in these high-risk groups. METHODS: We estimated the proportion of sub-Saharan African (AFR), Amerindian (AMI) and European admixture using 92 ancestry-informative marker genotypes in 16,476 AFA and HA women from the Women's Health Initiative. Cox regression models were used to examine the association between admixture and diabetes risk, with and without accounting for socioeconomic status (SES) and adiposity measurements. RESULTS: AFR admixture was significantly associated with diabetes risk in AFA women when adjusting for entry age, neighbourhood SES and BMI or waist/hip ratio (WHR) (all p < 0.0001). In HA women, AMI admixture had significant associations with diabetes risk that remained significant after adjustment for SES and BMI (all p < 0.0005). In both AFAs and HAs, SES showed significant negative associations while BMI or WHR had significant positive associations with diabetes risk, with and without adjustment for genetic admixture. CONCLUSIONS/INTERPRETATION: In AFAs, admixture, SES and BMI/WHR each independently contribute to diabetes risk after accounting for each of the other factors; in HAs, admixture, SES and BMI each independently contribute to diabetes risk after accounting for each of the other factors, whereas admixture is not significantly associated with diabetes risk after accounting for SES and WHR. The findings emphasise the importance of considering both genetic and environmental causes in the aetiology of type 2 diabetes.

Relationship between adiposity and admixture in African-American and Hispanic-American women.

OBJECTIVE: The objective of this study was to investigate whether differences in admixture in African-American (AFA) and Hispanic-American (HA) adult women are associated with adiposity and adipose distribution. DESIGN: The proportion of European, sub-Saharan African and Amerindian admixture was estimated for AFA and HA women in the Women's Heath Initiative using 92 ancestry informative markers. Analyses assessed the relationship between admixture and adiposity indices. SUBJECTS: The subjects included 11 712 AFA and 5088 HA self-identified post-menopausal women. RESULTS: There was a significant positive association between body mass index (BMI) and African admixture when BMI was considered as a continuous variable, and age, education, physical activity, parity, family income and smoking were included covariates (P<10(-4)). A dichotomous model (upper and lower BMI quartiles) showed that African admixture was associated with a high odds ratio (OR=3.27 (for 100% admixture compared with 0% admixture), 95% confidence interval 2.08-5.15). For HA, there was no association between BMI and admixture. In contrast, when waist-to-hip ratio (WHR) was used as a measure of adipose distribution, there was no significant association between WHR and admixture in AFA but there was a strong association in HA (P<10(-4); OR Amerindian admixture=5.93, confidence interval=3.52-9.97). CONCLUSION: These studies show that: (1) African admixture is associated with BMI in AFA women; (2) Amerindian admixture is associated with WHR but not BMI in HA women; and (3) it may be important to consider different measurements of adiposity and adipose distribution in different ethnic population groups.

Evidence for malaria selection of a CR1 haplotype in Sardinia.

Complement receptor 1 (CR1) levels have been associated with malarial susceptibility and/or severity of the disease in different population groups, and CR1 is a receptor for Plasmodium falciparum. In this study, multiple CR1 single-nucleotide polymorphisms (SNPs) showed strong evidence of population differentiation between Sardinian and other European ethnic groups. Cross population algorithms comparing haplotype structure and differences in haplotype and allele frequency distribution provided additional support for natural selection of CR1 in Sardinia. The predominant Sardinian CR1 haplotype included SNPs that are associated with decreased CR1 levels in Europeans and other population groups. Previous studies have shown that the SNPs within the dominant Sardinian haplotype have a significantly higher frequency in a malaria endemic compared with non-endemic regions in India. Together with the historical evidence of the prevalence of malaria in Sardinia, these data support the role of malaria leading to positive selection of this CR1 haplotype in Sardinia.

CIITA is not associated with risk of developing rheumatoid arthritis.

The major histocompatibility complex (MHC) class II transactivator gene (CIITA) encodes an important transcription factor regulating genes required for human leukocyte antigen (HLA) class II MHC-restricted antigen presentation. MHC genes, particularly HLA class II, are strongly associated with risk of developing rheumatoid arthritis (RA). Given the strong biological relationship between CIITA and HLA class II genes, a comprehensive investigation of CIITA variation in RA was conducted. This study tested 31 CIITA single-nucleotide polymorphisms in 2542 RA cases and 3690 controls (N=6232). All individuals were of European ancestry, as determined by ancestry informative genetic markers. No evidence for association between CIITA variation and RA was observed after a correction for multiple testing was applied. This is the largest study to fully characterize common genetic variation in CIITA, including an assessment of haplotypes. Results exclude even a modest role for common CIITA polymorphisms in susceptibility to RA.

The rs4774 CIITA missense variant is associated with risk of systemic lupus erythematosus.

The major histocompatibility complex (MHC) class II transactivator gene (CIITA) encodes an important transcription factor required for human leukocyte antigens (HLA) class II MHC-restricted antigen presentation. MHC genes, including the HLA class II DRB1*03:01 allele, are strongly associated with systemic lupus erythematosus (SLE). Recently the rs4774 CIITA missense variant (+1632G/C) was reported to be associated with susceptibility to multiple sclerosis. In the current study, we investigated CIITA, DRB1*03:01 and risk of SLE using a multi-stage analysis. In stage 1, 9 CIITA variants were tested in 658 cases and 1363 controls (N=2021). In stage 2, rs4774 was tested in 684 cases and 2938 controls (N=3622). We also performed a meta-analysis of the pooled 1342 cases and 4301 controls (N=5643). In stage 1, rs4774(*)C was associated with SLE (odds ratio (OR)=1.24, 95% confidence interval (95% CI)=1.07-1.44, P=4.2 × 10(-3)). Similar results were observed in stage 2 (OR=1.16, 95% CI=1.02-1.33, P=8.5 × 10(-3)) and the meta-analysis of the combined data set (OR=1.20, 95% CI=1.09-1.33, P(meta)=2.5 × 10(-4)). In all three analyses, the strongest evidence for association between rs4774(*)C and SLE was present in individuals who carried at least one copy of DRB1*03:01 (P(meta)=1.9 × 10(-3)). Results support a role for CIITA in SLE, which appears to be stronger in the presence of DRB1*03:01.

Replicated association of 17q12-21 with susceptibility of primary biliary cirrhosis in a Japanese cohort.

To examine the genetics of susceptibility to primary biliary cirrhosis (PBC), genome-wide association studies GWAS have been performed in patients of European ancestry and have shown the significant associations of IL12-related pathways, SPIB, IRF5-TNPO3, and 17q12-21. We tested whether these findings could be extended to a Japanese cohort, 303 Japanese PBC and 298 controls. We failed to detect significant associations at IL12A (rs574808, rs1075498) and IL12RB2 (rs3790567). There was no genetic variance at IRF5-TNPO3 (rs10488631) in Japanese. A single nucleotide polymorphism (SNP) at SPIB (rs3745516) reached nominal significance, but the corrected P value did not reach significance. For the 17q12-21 region, two SNPs had nominally significant associations [GSDMB (rs2305480, P = 0.022) and ZPBP2 (rs11557467, P = 0.021)] and we noted a significant P value at a SNP in IKZF3 (rs939327, P = 0.0024, P(c) = 0.017) after correction for multiple comparisons. Thus, these results indicate a haplotype on 17q12-21 with a similar association in Japanese and European PBC.

A candidate gene study of CLEC16A does not provide evidence of association with risk for anti-CCP-positive rheumatoid arthritis.

CLEC16A, a putative immunoreceptor, was recently established as a susceptibility locus for type I diabetes and multiple sclerosis. Subsequently, associations between CLEC16A and rheumatoid arthritis (RA), Addison's disease and Crohn's disease have been reported. A large comprehensive and independent investigation of CLEC16A variation in RA was pursued. This study tested 251 CLEC16A single-nucleotide polymorphisms in 2542 RA cases (85% anti-cyclic citrullinated peptide (anti-CCP) positive) and 2210 controls (N=4752). All individuals were of European ancestry, as determined by ancestry informative genetic markers. No evidence for significant association between CLEC16A variation and RA was observed. This is the first study to fully characterize common genetic variation in CLEC16A including assessment of haplotypes and gender-specific effects. The previously reported association between RA and rs6498169 was not replicated. Results show that CLEC16A does not have a prominent function in susceptibility to anti-CCP-positive RA.

Supervised machine learning and logistic regression identifies novel epistatic risk factors with PTPN22 for rheumatoid arthritis.

Investigating genetic interactions (epistasis) has proven difficult despite the recent advances of both laboratory methods and statistical developments. With no 'best' statistical approach available, combining several analytical methods may be optimal for detecting epistatic interactions. Using a multi-stage analysis that incorporated supervised machine learning and methods of association testing, we investigated epistatic interactions with a well-established genetic factor (PTPN22 1858T) in a complex autoimmune disease (rheumatoid arthritis (RA)). Our analysis consisted of four principal stages: Stage I (data reduction)-identifying candidate chromosomal regions in 292 affected sibling pairs, by predicting PTPN22 concordance using multipoint identity-by-descent probabilities and a supervised machine learning algorithm (Random Forests); Stage II (extension analysis)-testing detailed genetic data within candidate chromosomal regions for epistasis with PTPN22 1858T in 677 cases and 750 controls using logistic regression; Stage III (replication analysis)-confirmation of epistatic interactions in 947 cases and 1756 controls; Stage IV (combined analysis)-a pooled analysis including all 1624 RA cases and 2506 control subjects for final estimates of effect size. A total of seven replicating epistatic interactions were identified. SNP variants within CDH13, MYO3A, CEP72 and near WFDC1 showed significant evidence for interaction with PTPN22, affecting susceptibility to RA.

European population substructure correlates with systemic lupus erythematosus endophenotypes in North Americans of European descent.

Previous work has demonstrated that Northern and Southern European ancestries are associated with specific systemic lupus erythematosus (SLE) manifestations. In this study, 1855 SLE cases of European descent were genotyped for 4965 single-nucleotide polymorphisms and principal components analysis of genotype information was used to define population substructure. The first principal component (PC1) distinguished Northern from Southern European ancestry, PC2 differentiated Eastern from Western European ancestry and PC3 delineated Ashkenazi Jewish ancestry. Compared with Northern European ancestry, Southern European ancestry was associated with autoantibody production (odds ratio (OR)=1.40, 95% confidence interval (CI) 1.07-1.83) and renal involvement (OR 1.41, 95% CI 1.06-1.87), and was protective for discoid rash (OR=0.51, 95% CI 0.32-0.82) and photosensitivity (OR=0.74, 95% CI 0.56-0.97). Both serositis (OR=1.46, 95% CI 1.12-1.89) and autoantibody production (OR=1.38, 95% CI 1.06-1.80) were associated with Western compared to Eastern European ancestry. Ashkenazi Jewish ancestry was protective against neurologic manifestations of SLE (OR=0.62, 95% CI 0.40-0.94). Homogeneous clusters of cases defined by multiple PCs demonstrated stronger phenotypic associations. Genetic ancestry may contribute to the development of SLE endophenotypes and should be accounted for in genetic studies of disease characteristics.

Human immune responses to hapten-conjugated cells. I. Primary and secondary proliferative responses in vitro.

An in vitro model was developed to study both primary and secondary proliferative responses of human lymphocytes to hapten-conjugated peripheral blood mononuclear cells. Coculture of human lymphocytes with autologous trinitrophenyl (TNP)-conjugated stimulator cells resulted in primary proliferative responses. Subjects segregated into high and low primary responders with mean stimulation indices of 11 and 2.1, respectively. Restimulation of primed cells from high responder subjects 3 wk after initial sensitization generated secondary proliferative responses. To investigate the antigenic requirements for secondary stimulation, autologous TNP-conjugate primed responders were restimulated with both autologous and allogeneic TNP-conjugated stimulators. In all experiments restimulation with autologous conjugated cells yielded substantially greater proliferative responses than with allogeneic conjugates. Experiments were then performed to ascertain whether HLA determinant homology between primed responder and stimulator cells influenced the level of secondary responsiveness. Homology for HLA-A and B locus serologic determinants was not associated with enhanced responsiveness. In contrast, D region determinant homology, detected by B-cell antigen typing, showed a highly significant positive correlation with the magnitude of secondary responses. The data thus strongly suggest that for secondary proliferative responses to TNP, human T cells recognize hapten in association with HLA-D region determinants.

Clonal expansion of abnormal B cells in old NZB mice.

The spleens of old NZB mice have an abnormal population of B cells with extra chromosomes. These hyperdiploid B cells manifest increased proliferative capacity; they grow in (NZB X DBA/2)F1 spleens after intravenous injections. Molecular analysis of individual old NZB and F1 passaged spleens demonstrate that hyperdiploid cells represent a clonal or oligoclonal expansion of B cells. All spleens with at least 10% hyperdiploid cells demonstrated both heavy and kappa light chain immunoglobulin gene rearrangements by Southern blot hybridization. None of the hyperdiploid spleens from old NZB mice had lambda rearrangements and only one of five showed evidence of clonal rearrangement of the TCR-beta gene. One also had a VK10 clonal rearrangement. Elevated p53 oncogene protein was observed in NZB hyperdiploid spleen cells; however, no p53 or other oncogene rearrangements or amplifications were seen. Hyperdiploid cells were IgM-bright, IgD-dull, Ia+, dull B220, Thy-1-, and Ly-1-dull. Spleens with hyperdiploid B cells had increased percentages of Ly-1 B cells. The data suggest that hyperdiploid cells in old NZB mice represent clonal expansion of B cells and that they may represent an intermediate stage between autoimmunity and malignancy.

Role of Ia-positive cells in induction of secondary human immune responses to haptens in vitro.

We have described techniques for induction of primary and secondary human immune responses in vitro to lymphoid cells modified with trinitrophenyl, dinitrophenyl, and fluorescein isothiocyanate. Optimal secondary proliferative responses required the presentation of hapten on stimulator cells that shared HLA-D region determinants with the responder cell and/or the original stimulator cell. In contrast, hapten-specific cytotoxic responses assessed on modified allogeneic targets with no detected HLA homology with the responder were comparable in magnitude to those detected on modified autologous targets. Furthermore, secondary proliferative, but not cytotoxic, responses required presentation of Ia+ stimulator populations. Modified B cells, surface-immunoglobulin-negative, non T cells (null-cells), and Ia+ activated T cells all induced proliferative responses at least as effectively as equal numbers of hapten-conjugated macrophage/monocytes. Conversely, Ia(-) null cells and macrophages were entirely unable to stimulate. The data thus suggest that for proliferative responses, primed human T cells respond to modified lymphoid cells only when hapten is recognized in the context of Ia molecules.