Sarcoidosis is associated with a truncating splice site mutation in BTNL2

Abstract

Sarcoidosis is a polygenic immune disorder with predominant manifestation in the lung. Genome-wide linkage analysis previously indicated that the extended major histocompatibility locus on chromosome 6p was linked to susceptibility to sarcoidosis. Here, we carried out a systematic three-stage SNP scan of 16.4 Mb on chromosome 6p21 in as many as 947 independent cases of familial and sporadic sarcoidosis and found that a 15-kb segment of the gene butyrophilin-like 2 (BTNL2) was associated with the disease. The primary disease-associated variant (rs2076530; P(TDT) = 3 x 10(-6), P(case-control) = 1.1 x 10(-8); replication P(TDT) = 0.0018, P(case-control) = 1.8 x 10(-6)) represents a risk factor that is independent of variation in HLA-DRB1. BTNL2 is a member of the immunoglobulin superfamily and has been implicated as a costimulatory molecule involved in T-cell activation on the basis of its homology to B7-1. The G --> A transition constituting rs2076530 leads to the use of a cryptic splice site located 4 bp upstream of the affected wild-type donor site. Transcripts of the risk-associated allele have a premature stop in the spliced mRNA. The resulting protein lacks the C-terminal IgC domain and transmembrane helix, thereby disrupting the membrane localization of the protein, as shown in experiments using green fluorescent protein and V5 fusion proteins.

Toll-like receptor (TLR) 4 polymorphisms are associated with a chronic course of sarcoidosis

“…It has been demonstrated recently that sarcoidosis is associated with a truncating splice site mutation in the butyrophilin-like (BTNL) 2 gene, which leads to T cell activation after stimulation with proinflammatory cytokines such as IL-1β and LPS [56].…”

A short isoform of NOD2/CARD15, NOD2-S, is an endogenous inhibitor of NOD2/receptor-interacting protein kinase 2-induced signaling pathways

“…Determination of the NOD2-S:NOD2 Ratio by Clone Sequencing. To determine the exact ratio of transcripts with an exon 3 skipping, we used a clone sequencing method that has been described to unveil genotype-splicing effects (33,34). PCR products from 30 rounds of PCR (exponential phase) were ligated into a TA-cloning vector (Invitrogen), and the indicated numbers of individual clones were sequenced by using Dye terminator chemistry (Applied Biosystems) on a 3730xL DNA Analyzer (Applied Biosystems).…”

“…Immunoprecipitation and Western blots were performed according to standard protocols (see ref. 33 and Supporting Materials and Methods).…”

Regulation of major histocompatibility complex class II gene expression, genetic variation and disease

“…156 A growing number of reports highlight the role of DNA sequence variation in modulating alternative splicing, which can contribute to disease susceptibility. 157 The MHC class II region has already provided one of the clearest examples of a SNP modulating alternative splicing resulting in common disease through the work of Valentonyte et al (2005) 158 on genetic determinants of sarcoidosis, a chronic granulomatous condition. A genomewide linkage and association analysis resolved variants at butyrophilin-like 2 (a member of the immunoglobulin family) in disease susceptibility and more specifically a G to A nucleotide substitution (rs2076530) within exon 5, which created an alternative splice site, and led to a frameshift and premature stop.…”