Dysregulated immunity in PID patients with low GARP expression on Tregs due to mutations in LRRC32

摘要

Identification of patients with mutant LRRC32 variants. A The filtering strategy for exome sequencing data with indication of variant numbers at each step is demonstrated. freq., individual frequency in our internal cohort and in gnomAD exomes and genomes; hi/mo, predicted high or moderate impact of a mutation on gene function/structure based on the type of variant (e.g., a “stop_gained” variant is predicted as “high”, whereas a missense variant is predicted as “moderate”); AF1 frequency of reads with the alternative allele, DP read depth (number of reads covering a variant), MOI mode of inheritance, AD autosomal dominant, AR autosomal recessive, XLR X-linked recessive. B Schematic presentation of LRRC32 alleles in patient 1 and patient 2. Affected nucleotides and amino acids and their positions are indicated and denoted by green and red for the wt and mutant variants, respectively. C The predicted protein structure of each mutant GARP proteins in complex with TGFβ1 and LAP is depicted. GARP is indicated in green, TGFβ1 is indicated in orange, and LAP is indicated in gray. Mutated amino acids are indicated in red and noted with arrows. The dashed line indicates part of GARP with a nonresolved crystal structure. N-ter N-terminus, C-ter C-terminus, er extracellular, ic intracellular. D Expression of mutated GARP proteins on the surface of transfected HEK293 cells after staining for GARP. Representative staining out of three independent experiments with similar results is shown. E Analysis of allelic LRRC32 expression in patients. Total mRNA was purified from Tregs and analyzed by allele-specific PCRs for c.741G>A and c.1262G>A in triplicate. Mean values are indicated. Results from patients are indicated in red. Individual results from seven healthy controls are indicated in gray. The probes for the wt and mutant alleles were labeled with VIC and FAM, respectively, and duplexed in one reaction. Shaded areas indicate background fluorescence