Targeted sequencing discovers genetic risk alleles associated with break in immune tolerance and production of autoantibodies in healthy individuals

Over 50 loci are associated with SLE based on existing literature from genotyping studies. These loci impact several pathways in the immune response. Production of antinuclear antibodies (ANA) is a hallmark of lupus, which... [ view full abstract ]

Over 50 loci are associated with SLE based on existing literature from genotyping studies. These loci impact several pathways in the immune response. Production of antinuclear antibodies (ANA) is a hallmark of lupus, which precede the onset of clinical symptoms by many years. The genetic risk factors that underlie the initial break in immune tolerance and development of serological autoimmunity are largely unknown. Using SNP genotyping and targeted sequencing methods, we undertook a genome-wide association study approach to understand the genetics of ANA in healthy individuals. Serum and DNA were collected from 2903 healthy individuals with no personal history of autoimmunity. Antinuclear antibodies were detected using an ELISA to human nuclear extract.  Sera from subset (n=724) individuals (healthy ANA-, ANA+, and SLE) were assayed by protein microarray quantifying IgM and IgG responses to 89 previously known human autoantigens.  A nested cohort of 1,969 subjects consisting of all the ANA+ Caucasian individuals and age/gender matched ANA- controls and 1700 SLEs, were genotyped using the ImmunoChipv.1 SNP array. Targeted sequencing was performed in 78 ANA+ and 114 ANA- healthy individuals and 700 SLEs to capture the complete risk haplotypes at SLE risk loci. In apparently healthy population, 16% had moderate and 10% had high levels of IgG antinuclear antibodies.  High titer ANA was almost exclusively seen in female subjects (OR (CI)=1.6 (1.1-2.1), p=0.003). Autoantigen microarray data showed that, ANA+ healthy individuals had a high prevalence of antibodies to non-nuclear and cytoplasmic antigens, while subjects with SLE predictably produced antibodies to a variety of nuclear antigens. A quantitative genetic association test with ANA identified, C11orf30 and HLA, as two major genomic loci associated with high ANA phenotype in healthy population. ANA risk [(p=3.83E-04, OR=2.6)] haplotype at c11orf30 locus was associated with downregulation of its expression in human macrophages and EBV cell lines. Also, healthy carriers of risk haplotype had high titers of anti-Gliadin (p=0.03) and anti-cedar red (p=0.0003) pollen specific IgGs, indicative of susceptibility to food allergies. In case of HLA, ANA risk haplotype was the same that also predispose to lupus (DR3 allele) possibly via upregulation of HLA-class II genes expression on surface of antigen presenting immune cells i.e. human DCs. Healthy individual who carried ANA risk haplotype had higher titers of ANA. Targeted sequencing revealed potentially causal alleles (IRF4, CTCF) sitting on ANA associated regulatory haplotype. This study discovers genetic risk alleles for the development of ANA that includes many of the previously documented SLE risk haplotypes.