Graves disease (GD, MIM%275000) is the leading cause of hyperthyroidism and thyroid eye disease with both clinical and research importance. Anti-thyroid drug (ATD)-induced agranulocytosis, (TiA, defined as an absolute granulocyte count < 500 per cubic millimeter while taking ATDs), is the most feared adverse effect of ATDs and can occur in 0.1-0.37% of individuals who take these medications. Our long-term goals are to find the susceptibility genes of GD and its related phenotypes (such as TiA), and to further figure out the detailed mechanisms. We have established solid GD research foundation using valuable human samples, and our recent identification of major TiA susceptibility loci is potentially paradigm-shifting. In this proposal, in addition to the “extension of our human study”, we will go one step further to generate humanized major histocompatibility complex (MHC) mice, which will be a powerful tool not only for the diseases we are interested in (such as GD and TiA) but also for numerous other immune-related diseases. For the upcoming 3-year period, we will work on the following 2 aims. First, identification of the susceptibility genes of TiA. Our team recently conducted a two-stage genome-wide association study (GWAS)(in total 42 TiA cases and 1,208 GD controls) and demonstrated a HLA-B allele (Armitage trend Pcombined = 6.75 × 10-32), a HLA-DRB1 allele (Pcombined = 1.83 × 10-9) and a locus at chromosome 3q13 (Pcombined = 7.75 × 10-8) as major independent susceptibility loci of TiA (manuscript under revision at Nature Genetics). For this proposed study, we plan to double our sample size (additional ~40 TiA cases and ~1,000 GD controls) to drastically increase our statistical power and comprehensively identify major TiA association loci. Then we will use next-generation sequencing (NGS)-based approach to narrow down and identify the genuine susceptibility/protective genes/variants. We will take advantage of the humanized MHC mouse model (covered by this grant) for studying TiA pathophysiology. We will also perform other functional study (covered by other grants) on other susceptibility genes identified through GWAS. Second, establishment of humanized MHC mice for study of immune-related diseases. During the past one and half years, our team has made substantial progress in making the humanized MHC class II mice. The overall study design is to replace the mouse MHC class II genes with an “exchangeable” single copy of human class II HLA allele into the homogeneous C57BL/6 inbred mice background. The cassette-like nature of our design makes it possible to exchange the old allele with any new allele of interest using the convenient “transgenic” approach (instead of the time-consuming “knock-in” approach). We have several clever state-of-the-art tricks to make our mouse models groundbreaking, including using pronuclear injection-based mouse targeted transgenesis (PITT), recombineering, C57BL/6N embryonic stem cell lines, and transcription activator-like effector nuclease (TALENs) technologies, etc. For the new proposal, in addition to accomplishing the class II mouse project, we will extend the design to also make humanized class I MHC mouse models. Many immune-related diseases or drug adverse effects (including GD and TiA) have been shown to have susceptibility/protective alleles in “both” class I and class II HLA genes. Our ultimate goal is to put both human class I and class II HLA risk alleles together on the same mouse chromosome/haplotype, which can be extremely powerful for future research.

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