Establishing phased assemblies and fine mapping of complex genomic regions encoding NK cell receptors and their implication in skin disease - PROJECT SUMMARY/ABSTRACT Pemphigus is an autoimmune skin blistering disease that causes painful blisters and affects thousands of Americans and individuals worldwide. Although variation within the natural killer cell complex (NKC) and the leukocyte receptor complex (LRC) has been associated with susceptibility to pemphigus foliaceus (PF), their structure, diversity, and functional roles in autoimmunity remain poorly characterized. Extreme polymorphism, extensive sequence homology, and complex structural variation across these regions have posed significant technical challenges, limiting accurate sequencing, variant calling, and genome assembly. The main aim of this project is to integrate novel long-read library preparation and sequencing techniques with custom variant calling and bioinformatic tools to characterize variation within the NKC and LRC that confer differential risk to PF, to describe the complex structure of these regions through the creation of a graph reference, and to interrogate and detail the variation within KLR genes and their association with PF risk. A discovery and a replication cohort will be prepared utilizing stringent size selection protocols and ~5,000 custom designed biotinylated probes to enrich for the NKC and LRC, and sequenced using PacBio HiFi technology, yielding reads with a median length of 10-15 kilobases. These targeted long-read data permit long-range phasing and identification of single nucleotide polymorphisms (SNP), copy number variations (CNV), and haplotypes for the highest fidelity association study of these regions to date, identifying loci within these regions that confer differential susceptibility to PF. To facilitate the discovery of novel variation within the NKC and LRC and to increase the accuracy of variant calling, a panel of individuals from varied ancestries will be sequenced using the method described above and used for the creation of a graph reference. Next, we will interrogate the structure and diversity of the KLR gene family, which has been implicated in a plethora of diseases, becoming the first group to comprehensively describe the alleles, haplotypes, and structural variation of these genes. Leveraging new insights, we will conduct the most specific association study of these genes ever reported, analyzing both alleles and structural variation and their relationship to PF risk. This project will report the most complete description of the regions encoding NK cell receptors and their contribution to PF risk and will enhance understanding of two highly complex and health-relevant regions; providing insight into this disease, and other autoimmune diseases and cancers, which share a multitude of associations in these regions. The fellowship training plan developed to accompany this project has been created to facilitate the acquisition of several key skills that will ensure my successful transition to a biomedical scientist focused on identifying variation within immune genes that are associated with disease, namely: improved critical thinking, mastery of bioinformatic skills in immunogenomics, and molecular biology techniques.
