Sex Chromosome Aneuploidies in Autoimmune Disease

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Abstract Systemic lupus erythematosus (SLE) and Sjögren’s syndrome (SS) are chronic, inflammatory, autoimmune diseases. Some features of these illnesses are similar. These include aspects of clinical manifestations, serology and epidemiology. Most critical for this proposal is that there is a strong female bias found for both diseases such that at least 90% of patients are women. The mechanism for this sex bias was unexplained until the work of the PI, which began more than a decade ago and was funded in part by this VA Merit Review for which a renewal is sought. The PI and his colleagues found that men with 47,XXY (Klinefelter’s syndrome) are at the same risk of SLE or SS as women. In addition, 47,XXX women are at increased risk of either disease compared to women carrying a 46,XX chromosomal complement. This X chromosome dose effect was found for SLE and SS but not rheumatoid arthritis, primary biliary cirrhosis or sarcoidosis. Thus, our data indicate multiple molecular pathways to female-biased disease. In cells with more than one X chromosome, all but one undergoes X inactivation. However, X inactivation in humans is not an all-or-none process such that up to 15% of genes on the X chromosome escape inactivation. That is, these genes are transcribed and translated from both X chromosomes (biallelically) in normal (46,XX) women. We have identified two X chromosomes genes that escape X inactivation in immune cells and are both involved in toll like receptor (TLR) 7 signaling to produce interferon, and have a lupus risk allele identified in genome-wide association studies. Namely, these genes are TLR7 itself and CXorf21. The protein product of CXorf21 physically interacts with the protein product of another lupus risk gene – SLC15a4. In studies performed in the previous cycle of this award, and published in 2019, we find that CXorf21 and SLC15a4 regulate endolysosomal pH and are critical for TLR7 signaling. Knockdown of CXorf21 by CRISPR-Cas9 raises endolysosomal pH (less conducive to TLR7 signaling), and impairs TLR7 signaling with marked reduction of interferon as well as other cytokine production (see Progress Report and Preliminary Data). Of course, interferon production through TLR7 signaling is known to be a critical aspect of lupus pathogenesis in both human and murine lupus. Thus, these data constitute a strong premise for the work proposed herein. Much of the genetics of SLE has not been identified by GWAS, and much of this missing genetics may be related to presently unidentified genetic synergism. In the first aim, we will determine the degree of genetic synergism found in the interaction of the lupus-associated alleles of these genes involved in the TLR7 pathway. This aim will use our cohort of >300 men with SLE, the largest cohort ever assembled by more than 3-fold. In the second aim, we will define the physiological roles of the protein products of these genes in the TLR signaling pathway with particular attention to the associated alleles using cutting edge molecular techniques. The final aim we will approach precision medicine by studying the role of these genes in determining the efficacy of hydroxychloroquine. Since the genes and hydroxychloroquine are involved in TLR7 signaling and lysosomal pH, we hypothesize that the associated alleles will reduce the efficacy. This aim will utilize a unique resource of SLE patients on hydroxychloroquine and followed for flare. Thus, these related but independent aims will advance the science of the X chromosome gene dose effect that explains much of the sex bias of SLE and SS.