The past decade has been particularly glorious for the field of genetics. First, there was the elucidation of the human genome reference sequence in 2001.(1) Genome sequencing was followed by the HapMap project, which aims to detail common genetic variations across different human populations,(2) and more recently by the 1000 Genome Project, which seeks to elucidate new rare genetic variants in the human genome through extensive resequencing of the genome.(3) Collectively, these efforts are rapidly cataloging the genetic blueprint for our species including the common and rare polymorphisms that distinguish one individual from another.

As a result of these efforts, we now have a better understanding of the genetic basis of various human diseases including systemic lupus erythematosus (SLE). Several aspects of SLE genetics have recently been reviewed in greater detail elsewhere,(4)(5)(6) and the focus of this Cyberounds^®; is to highlight key lessons learned from the evolving research.

Epidemiology

SLE is a relatively rare systemic autoimmune disease with varying incidence rates in different ethnic groups. While African-Americans and Chinese have high incidence rates, exceeding 50 per 100,000, SLE is less frequent among Caucasians (<20 per 100,000). African-American, Hispanic and Chinese patients with SLE also have, compared to Caucasians, more severe renal disease.

In contrast to what is we currently know about other autoimmune diseases, the heritability of SLE is relatively high, estimated to be >66%. For example, SLE shows familial aggregation. The sibling risk ratio (λ_s) for SLE is as high as 29, indicating that the frequency of SLE among siblings of SLE patients is 29 times higher than the frequency of SLE among unrelated individuals drawn from the general population. Importantly, while the concordance rate of SLE among dizygotic twins is only 2-5%, the concordance rate of lupus among monozygotic twins is 20-50%, strongly supporting the hypothesis that SLE is a genetic disease, though non-genetic factors, notably environmental elements, must also be contributing.

Common Genetic Polymorphisms Underlying SLE

Given that there is a strong genetic component underlying SLE, much research effort has been directed to illuminating the specific genetic details. It became evident from murine and human studies that SLE is a polygenic disease with multi-factorial origin.(4)(5)(6)(7) More than two dozen genes have been reproducibly and robustly associated with SLE susceptibility, and these can be broadly divided into two categories:

• (i) common genetic polymorphisms, mostly uncovered through genome-wide association studies (GWAS), and
• (ii) rare genetic variants.

Most of the genes that have at present been associated with SLE represent common genetic polymorphisms in the human population, and were uncovered largely through GWAS, particularly over the past three years,(4)(5)(6) as summarized in Table 1. GWAS is an unbiased genome-wide linkage analysis exercise in which large numbers of multi-allelic microsatellite DNA markers positioned at 10-15 kb genomic intervals are screened in order to identify chromosomal regions that are associated with SLE susceptibility. So far, GWAS has helped identify >2000 common genetic variants in a wide spectrum of human diseases.(8) Several of these genes are discussed below.

HLA

There appear to be two independent contributions to SLE that arise from the HLA locus, one from MHC-Class II (notably HLA-DR2 and HLA-DR3), and another from HLA-Class III. These HLA Class II associations with SLE have been documented in multiple ethnic groups.(4)(6)(7) In addition, the pathogenic role of these molecules in disease is supported by humanized mice expressing HLA-DR3 transgenes, as reviewed elsewhere.(4) HLA Class II molecules are expressed on antigen presenting cells (e.g., dendritic cells and B-cells) and serve to present selected antigens to T-lymphocytes.

Although these MHC molecules represent some of the strongest common genetic polymorphisms for SLE (and most other autoimmune diseases), their precise mechanisms of action remain uncertain. It is theorized that aberrant presentation of specific autoantigens by polymorphic disease associated-MHC molecules, or aberrant immune tolerance of thymocytes, may be some of the ways through which MHC might be contributing to disease. The specific genetic contributions from the HLA-Class III locus to SLE pathogenesis are still being worked out. Candidates within this locus that are being currently investigated include the complement genes (C2 and C4), as well as MSH5 and SKIV2L.

IRF5

IRF5 is a gene that plays a critical role in the activation of innate immune cells (e.g., myeloid cells); it encodes a transcription factor in the Type I interferon pathway that regulates the expression of IFN-dependent genes and various inflammatory cytokines. Given that the latter cytokines play a central role in SLE pathogenesis, and the fact that Type I interferons are currently being targeted therapeutically in ongoing clinical trials, IRF5 emerges as a very exciting candidate gene for SLE. It has indeed been elegantly documented that the functional disease-associated variant of IRF5 is associated with increased expression of IRF5 and IFN-I, and the modulation of this gene mitigates lupus in a mouse model, as reviewed.(4)

ITGAM

ITGAM (also known as CD11b, CR3 or Mac-1) is a gene that expresses an integrin adhesion molecule that plays an important role in the activation of myeloid cells, including macrophages, dendritic cells and neutrophils. This gene is also associated with lupus nephritis, possibly because of heightened activation of myeloid cells within the kidneys. Though the causal variants in this gene are predicted to affect ligand binding, exactly which ligands and cells are implicated remains a hotbed of active investigation.(9)(10)

As detailed in Table 1, several other common alleles confer SLE susceptibility including genes that affect inflammation (IRAK1, TNFAIP3, TNIP1), cell signaling (BANK1, BLK, PTPN22, STAT4), cell:cell interactions (TNFSF4/OX40L and PDCD1), and immune complex handling (FCGR2 and FCGR3). It is also interesting to note that some of the implicated genes (e.g., STAT4 and PTPN22) are associated with multiple autoimmune diseases including psoriasis, rheumatoid arthritis and Crohn’s disease, underscoring the shared pathogenic pathways for autoimmunity in general.

TNFAIP3 and its interacting protein, TNIP1, both regulate NFkB signaling and inflammatory pathways. TNFAIP3 has been implicated in several different ethnic groups, though its functional impact on inflammation has not yet been systematically studied. BANK1 is a B-cell adaptor protein that regulates the interaction between the src family of tyrosine kinases and the calcium channel IP3R, and the release of intracellular calcium following B-cell signaling. Blk is a member of the src family of tyrosine kinases that regulates activation of B-cells. Ongoing studies are aimed at elucidating how these two molecules impact B-cell tolerance and activation in the context of SLE.

In general, common genetic variants have low "effect sizes" — i.e., the possession of any given disease-associated gene polymorphism escalates disease susceptibility only marginally. As is evident from Table 1, most of the strongest "common genetic variants" that confer lupus susceptibility have odds ratios in the range of 1.2-1.5, while several others have marginal odds ratios below 1.2. Hence, the predictive potential (or diagnostic capacity) of any one of these common genetic variants is very low. Taken in concert, the predictive potential of selected combinations of these susceptibility genes is more promising. Thus, it has been estimated that a composite genetic panel comprised of seven genes (including HLA, IRF5 and ITGAM) may have a reasonable predictive value for SLE, accounting for ~15% of the sibling risk ratio for the disease, with a degree of specificity and sensitivity that parallels the predictive potential of the PSA test for prostate cancer.(8)

Although this estimate awaits independent validation, these statistics are highly encouraging. This is the first such estimate using a composite genetic panel for predicting SLE, and these panels are likely to undergo further refinement in the near future as we gain a better understanding of which common genetic polymorphisms are best associated with SLE, or its complications. As Rhodes and Vyse have pointed out, the combination of high heritability and low prevalence of SLE could augur well for future genetic profiling and prediction of SLE.(5)

Rare Genetic Triggers of SLE

A second class of genes that confer SLE susceptibility encompasses rare genetic variations that are present in the human population at very low frequencies.(11) As summarized in Table 2, these rare genes have significantly larger effect sizes, with odds ratios exceeding 2.

As opposed to the common genetic polymorphisms discussed previously, rare variants have significantly higher predictive value for SLE. Perhaps the best known examples of rare genes for SLE include the complement deficiencies, which rank among the earliest genes associated with SLE. Although these are very rare, deficiencies of complement components C1q, C2 and C4 also have very high predictive value for SLE. Complement proteins likely play an important role in immune complex clearance and B-cell tolerance induction. The latest addition to this group of rare genes is SIAE, which controls the production of sialyl O-acetyl esterase, a molecule that promotes the binding of sialic acid residues to CD22, a negative regulator of B-cell signaling. Thus, SIAE could potentially regulate the threshold for B-cell signaling and immune tolerance (Table 2). It is predicted that we will see a steady expansion of rare variants for various polygenic diseases, including SLE, in the near future, thanks to the tremendous potential of current sequencing technologies that are rapid, highly multiplexed and far more accurate, but very cost-effective.

Despite these recent advances, the field of SLE genetics has a long way to go before the genes uncovered can be factored into routine diagnostic panels. With respect to most of the genes listed in Table 1, the causative molecular changes responsible for disease (and their mode of action) are currently unknown. Furthermore, it is estimated that the currently uncovered common genetic variants collectively account for only 15-20% of the heritability of SLE — thus, a substantial fraction of the genetic basis of SLE awaits elucidation. Although some of the genes associated with SLE susceptibility have been uncovered (as listed in Tables 1 and 2), we know little about the genes responsible for specific lupus-associated end-organ manifestations such as lupus nephritis or CNS lupus. Most of the completed genetic studies have focused on Caucasian SLE patients, and much less information is available concerning the genetic basis of SLE in other ethnic groups. Finally, we have limited understanding of how the different genes work together to cause lupus — in this regard murine studies have been quite informative, as discussed below.

Insights From Murine Studies

Several mouse strains develop lupus and its associated end-organ complications spontaneously including the NZB/NZW F1, MRL/lpr and NZM2410 strains. In particular, "genetic dissection" studies using the latter strain have uncovered at least three distinct checkpoints for lupus development, as discussed elsewhere.(12) Briefly, linkage analysis studies in this strain uncovered three strong non-H2 chromosomal intervals associated with lupus, notably, Sle1^z on chromosome 1, Sle2^z on chromosome 4 and Sle3^z on chromosome 7. The ensuing series of studies were quite unique in that they could only be done in experimental animals. The researchers bred each of these three disease intervals onto an otherwise healthy C57BL/6 (B6) background to generate a novel series of "congenic strains" bearing Sle1^z, Sle2^z or Sle3^z individually. As a result, researchers were able, for the first time, to study ‘monogenic’ models of murine lupus, as opposed to studying ‘polygenic’ strains.

This genetic simplification through ‘congenic dissection’ has been instrumental in demonstrating that each lupus susceptibility locus in this model infringed a different "checkpoint" in disease development. The B6.Sle1^z congenic strain exhibited a breach of immune tolerance to nuclear antigens. The immune breach provoked the production of autoantibodies to chromatin and autoreactive T cells responding to histone epitopes, with increased expression of activation markers on T cells and B cells. It has subsequently been shown that the culprit gene within this interval responsible for these phenotypes, namely Ly108, plays an important role in infringing B-cell tolerance, acting at very early stages in B-cell development. B6.Sle2^z mice exhibited B cell hyperactivity and elevated B1 cell numbers, leading to polyclonal and/or polyreactive hyper-gammaglobulinemia. Thus, both these genetic loci harbored genes that shaped the functioning of the adaptive arm of the immune system, which is comprised of B-cells and T-cells. These murine studies have taught us that polymorphic variants in genes that patrol lymphocyte tolerance checkpoints in the adaptive arm of the immune system could contribute to lupus pathogenesis.

In contrast, studies of B6 mice bearing the third interval, Sle3^z, indicated that this locus was impinging upon a second checkpoint in lupus development — innate immunity. Congenics harboring this disease locus exhibited pro-inflammatory myeloid cells, which had the capacity to elaborate more IL-12, IL-1β and TNF-α and also better co-stimulate T-cells. More recent studies have revealed additional lupus-associated genes/molecules that impact innate immunity including Type I interferons and TLR signaling molecules in various mouse models of lupus, as reviewed.(10)

The congenic dissection studies in mice demonstrated that the consequence of breaching both above checkpoints, in the adaptive and innate arms of the immune system, respectively, was the generation of potentially pathogenic mediators including autoantibodies, immune complexes, T-cells and myeloid cells, but not necessarily end organ disease. It turns out that the eventual development of end organ pathology is contingent upon the activity of molecules that are operative within the end-organs. Indeed, the congenic mouse studies also led to the identification of a third class of molecules that could affect end organ pathology, as detailed below.

When different congenic strains were challenged with a pathogenic anti-glomerular antibody insult, it became evident that B6 mice bearing a second gene within the Sle3 congenic interval exhibited significantly increased renal disease. Mapping studies using recombinant congenics revealed that a sublocus harboring the kallikrein gene complex on chromosome 7 was responsible for this phenotype. Functional studies indicated that increased renal kallikrein played a protective role in nephritis. Collectively these studies provided evidence for a third checkpoint in the development of lupus nephritis that could possibly modulate the degree of end-organ pathology in the face of potentially pathogenic autoantibodies and autoaggressor lymphocytes.(10)

These ‘congenic dissection’ studies illustrate that the genesis of fatal lupus is the end-result of multiple genes and pathways acting in concert. This multi-step pathogenic process also explains the contrasting phenotypes of the monocongenic versus the polycongenic mouse strains. Thus, although the individual loci, Sle1^z, Sle2^z or Sle3^z, in isolation were not sufficient for the development of fatal lupus but only elicited modest serological and cellular features of autoreactivity, the epistatic interaction of these loci with each other led to highly penetrant lupus nephritis.(10) Though these studies had originated with the NZM2410 mouse model of lupus, parallel findings have also been reported in other mouse models of lupus.(13)(14)

The 3-Checkpoint Model

Although this 3-checkpoint model may be an over-simplification of the molecular events that lead to lupus, it provides us with a working framework for classifying and understanding how different human lupus genes might be operating and interfacing with each other. Based on the known functions of the implicated human SLE genes (that are listed in Table 1 and 2), one can readily identify genes that may potentially infringe each of the three checkpoints identified in mice.

Genes such as Bank1, Blk and the complement molecules are candidates that could be envisioned to breach checkpoint I (patrolling the adaptive arm of the immune system) based on the published properties of these molecules. In fact, there is already functional evidence from studies that profile the entire B-cell (or immunoglobulin) antigen specificity repertoire that demonstrate that early B-cell tolerance checkpoints may also be infringed in human SLE. (15)

A relatively extensive subset of the genes implicated in human SLE could be envisioned to upregulate myeloid cell activity or enhance T-cell:B-cell or T-cell:dendritic cell interactions, leading in essence to a breach of checkpoint II (patrolling the innate arm of the immune system).

Finally, genes such as ITGAM, complement, and the various F_cR molecules could potentially play a role at the third checkpoint in lupus development, to regulate the degree of end-organ inflammation, again based on the published properties of these molecules. Clearly, these provisional assignments to the respective checkpoints may have to be revised once we learn more about the functional attributes of these genes.

Concluding Thoughts

The fact that the concordance rate of SLE among monozygotic twins rarely exceeds 50% points to the important contribution of the environment. Though some of the environmental insults have been documented (e.g., smoking, UV light, EBV infections, some chemicals), the precise environmental triggers for SLE remain largely unexplored. It is likely that a certain threshold of genetic susceptibility (possibly dictated by genetically determined infringements in various key pathogenic pathways leading to lupus) and environmental triggers must be exceeded before the disease manifests itself. Thus, in addition to defining the genetic basis of SLE, future efforts in this field have to delve deeper into potential environmental interactions that can modify lupus susceptibility.

These pressing challenges are likely to reshape our knowledge over the next several years concerning the genetic basis of SLE. As the genetic blueprint for SLE slowly takes form, we will gain a clearer understanding of how this mysterious disease originates, and we will be better positioned to formulate more effective strategies to predict and manage the disease. Tailoring the therapy according to the patient’s genetic makeup may also become a reality.