Fellow, Pediatric Rheumatology Stanford University School of Medicine Palo Alto, California, United States
Background: Childhood-onset systemic lupus erythematosus (cSLE) is an autoimmune disease with severe course and high burden of inherited risk. Past genetic studies of cSLE have been limited by the difficulty of resolving and determining consequences of variation within disease-relevant loci (e.g., the duplicated C4A and C4B genes) using short-read genomic approaches. New single-molecule sequencing platforms offer a solution to this problem by producing long, kilobase-scale readouts of both DNA sequence and nucleobase modifications such as CpG methylation (5mC). To further exploit this multimodality, we recently developed a method for concurrent single-molecule chromatin accessibility profiling via exogenous adenine methylation (Abdulhay, et al., eLife 2020; PMID 33263279). Objective: To survey haplotype-resolved single nucleotide (SNV), insertion/deletion (indel), structural (SV), and copy number (CNV) variation, 5mC, and chromatin accessibility in cSLE. Design/Methods: Immune cell genomes and epigenomes from a retrospective cSLE cohort will be analyzed in this study. The primary outcome is detection of candidate genetic causes of cSLE. Secondarily, co-assayed 5mC and chromatin accessibility will be analyzed to nominate mechanisms of variant pathogenicity. DNA from EcoGII adenine methyltransferase-treated peripheral blood mononuclear cell (PBMC) nuclei from probands (N=3) and matched unaffected controls (N=2) will be used for 30X coverage Pacific Biosciences sequencing. All probands met 2019 ACR/EULAR lupus classification criteria before age 18 years and had hemoglobin >7.5 g/dL. Phased assembly and SNV, indel, SV, CNV, and 5mC detection will be performed with established methods (hifiasm, DeepVariant, pbsv, HiPhase & jasmine). Chromatin accessibility will be analyzed as described (Abdulhay, et al., 2020). This study has been approved by the Stanford Univ. IRB (#68288). Pilot sequencing of one proband revealed pathogenic complement gene variation. PBMCs have already been cryopreserved for the remaining subjects; data generation and analysis will be completed by Mar. 2024.
