Through the application of genome-wide association methods and subsequent replication studies, it is anticipated that validated markers will emerge that can be used as novel diagnostic and prognostic tools for risk stratification in counseling mothers with anti-Ro/SSA antibodies. In this first genome-wide association study of 116 Caucasian children with cardiac neonatal lupus, the most strongly associated variants (MICB region at class I, NFKBIL1–LTA–TNF–LTB–AIF1 at class III, and C6orf10 at class II) were found in the MHC region, a locus with extensive linkage disequilibrium. Outside the HLA locus, an association was identified at locus 21q22, 22 kb upstream of ERG. With the exception of the HLA region, no locus previously implicated in autoimmune diseases was found to have genome-wide significance in children with cardiac neonatal lupus.
In recent genome-wide association studies of SLE patients, the most significant association was found in the HLA region at 6p21.3. In this 7.05-Mb region, 93 SNPs had a P
value of less than 10−6
, a result which represents the long-range linkage disequilibrium related to the extended HLA–A1;B8;DR3 haplotype (28
). The association of this extended haplotype with the generation of antibodies to Ro/SSA and La/SSB in the context of Sjögren’s syndrome and SLE has been consistently demonstrated (29
). The class III TNFα polymorphism at position −308 (TNFα, rs1800629, TNFα –308A polymorphism-TNF2 allele, proinflammatory), which is also part of the extended haplotype, is associated with a number of autoimmune diseases, including Sjögren’s syndrome (31
), SLE (32
), subacute cutaneous lupus erythematosus (33
), rheumatoid arthritis (34
), and ulcerative colitis (35
). In earlier limited studies of families enrolled in the RRNL (36
), the TNF2 allele was significantly overrepresented in the mothers as well as the affected and unaffected siblings compared with healthy controls. The present study corroborated these findings in children with cardiac neonatal lupus. Specifically, 2 SNPs, which are in proximity to rs1800629, reached genome wide-significance (rs3099844 [Pdom
= 4.5 × 10−10
] and rs2857595 [Padd
= 1.96 × 10−9
]). Notably, there is strong linkage disequilibrium between rs3099844 and rs1800629 (r2
= 0.64) and between rs2857595 and rs1800629 (r2
= 0.86). What is yet to be established is whether the HLA candidates reflect the inheritance of maternal associations expected in women with anti-Ro/SSA and anti-La/SSB antibodies. Association analysis in these families using the transmission disequilibrium test (TDT) will help to elucidate this relationship.
The genetic factors may represent a dual hit by differentially influencing disease in this maternal/fetal dyad. On the maternal side the genes promote the necessary autoantibodies, and on the fetal side the genes promote tissue inflammation in a permissive in utero environment, e.g., hypoxia. The functional biology is supported by an in vitro model of cardiac neonatal lupus in which TNFα was secreted by macrophages cocultured with anti-Ro/SSA–bound human fetal cardiocytes (37
The identification of associations with HLA class I genes in the present study is consistent with the results of a prior study of 40 children with cardiac neonatal lupus that revealed an enrichment of HLA-Cw7 (38
). HLA-Cw7 is inclusive of the PSORS1 region (which is associated with psoriasis [39
]). In the present study, associations in this region were found for rs3130544 and rs7750641. The functional effects of these candidate genes within PSORS1 may relate to stimulation of receptors for class I MHC and dysfunction of natural killer (NK) or T cell self tolerance. A combination of class I MHC and ligand (on NK or T cells) may augment susceptibility to autoimmune injury during pregnancy.
Cardiac neonatal lupus was also associated with non-HLA regions, including loci at 21q22, 12q21, 1q31, and 10p15. A cluster of associated SNPs at 21q22 is in proximity to ERG-ETS2/WDR4. ERG is a transcription factor that serves as a “brake” to both apoptosis and inflammation, components previously described in the cascade to injury and replacement of the atrioventricular node by fibrosis (36
). Yi and coworkers demonstrated that ERG protects fibroblasts against apoptosis induced by serum deprivation (42
). Recently, Yuan and coworkers demonstrated that ERG plays a role in repressing the expression of interleukin-8 (IL-8) (43
), a mediator of inflammatory cell accumulation produced by numerous cell types, including macrophages and fibroblasts. In addition, it has been demonstrated that ERG plays a role in augmenting the expression of transforming growth factor β (TGFβ) receptor type II (44
). Thus, a polymorphism associated with low expression or diminished function of the encoded protein may represent the absence of a needed protective factor for fetuses exposed to maternal anti-Ro/SSA antibodies.
In psoriasis, risk alleles also reside at non-HLA genes such as chromosomes 1 and 5 (45
), which include IL23R and IL13, respectively. Although they did not reach genome-wide significance, SNPs associated with each of these genes were associated with cardiac neonatal lupus. IL-13 is a prototypic Th2 cytokine that is also strongly profibrotic. Mice deficient in IL-13 are protected against a fluorescein isothiocyanate–induced model of lung fibrosis, and IL-13 can stimulate fibroblast collagen production independently of TGFβ (46
). However, it has recently been demonstrated that signaling through IL-13Rα2, initially thought to be a decoy receptor for IL-13, results in the production of TGFβ (47
). Moreover, in vivo gene silencing of IL-13Rα2 using small interfering RNA attenuates bleomycin-induced lung fibrosis, with decreases in TGFβ1 secretion and collagen formation (48
In this passively acquired autoimmune disease, with the exception of HLA, there were no significant genome-wide associations with identified polymorphisms that have been implicated in other autoimmune diseases, such as SLE, in which there are genetic contributions relating to dysfunction of the acquired immune system. Overall, however, there was an inflation factor of 1.22 for these autoimmune SNPs in cardiac neonatal lupus, compared with an inflation factor of 1.01 for the entire genome. Specifically, the enrichment of a variant of IRF5 is intriguing, and the variation at 7q32 has attracted a substantial amount of attention (21
). However, it is acknowledged that maternal inheritance may account for some of these genes for which the associations are just below the level of significance. There are several possible and non–mutually exclusive interpretations. First, the associations may simply reflect the maternal enrichment of the genome for autoimmunity risk alleles, given the autoantibody state of the mother. Second, there may be an enrichment in autoimmunity-predisposing risk alleles in children with cardiac neonatal lupus independent of the maternal state. Third, there may be no significant enrichment, consistent with the relative immaturity of the human fetal immune system, which relies almost exclusively on adaptive maternal immunity, and the genetic contribution to cardiac injury may be restricted to the innate immune system and tissue reactivity.
These data require careful consideration and recognition of their inferential limits. As discussed above, maternal inheritance may be a limitation. Specifically, at 6p21, it is difficult to distinguish whether inheritance reflects transmission of genes related to maternal autoimmunity or whether these genes represent enrichment of genes that are biologically important. TDT will be informative. The limited power of the study is another limitation. However, the strength of the study is in the precise clinical phenotyping of the cases, with 96% having advanced atrioventricular block, the most characteristic cardiac manifestation associated with maternal anti-Ro/SSA antibodies. Given the rarity of the disease, the study included 4 cases of first-degree block, persisting after birth in 3 as shown by EKG, suggesting sustained injury, and 1 case of isolated cardiomyopathy. Arguably, combining cases expressing full progression of disease with those having less advanced disease may also represent a limitation.
Given the current interest in identifying a biomarker to predict more serious lifelong injury, isolated in utero prolongation of the PR interval has become an important focus of attention (10
). As such, these cases could provide potential candidates to increase the power of the study of a rare disease. However, given the uncertainty as to the pathologic significance of transient first-degree block, we did not include these children in the analysis due to concerns of underestimating the genetic influence. Thus, unlike SLE, in which the manifestations are heterogeneous with regard to organ system, severity, and temporal course, cardiac neonatal lupus represents a more homogeneous phenotype.
This study represents the first large-scale investigation of genes associated with cardiac neonatal lupus. Identification of risk alleles is an incremental step toward the discovery of a fetal genetic component that contributes to the development of lifelong cardiac damage in newborns exposed to maternal anti-Ro/SSA antibodies. These analyses support the potential of this first cohort to provide clues that are immediate and of high impact in the study of the genetics of cardiac neonatal lupus, with candidates identified in several pathways relevant to the pathogenesis of disease, antigen presentation, apoptosis, and inflammation. The absence of a significant association with genes previously identified as being associated with autoimmune diseases emphasizes the passive nature of this unique disease.