Background BRAF (v raf murine sarcoma viral oncogene homologue B1) is an attractive new autoantigen with the ability to identify rheumatoid arthritis (RA) patients who do not have the classical ACPAs (anti citrullinated protein antibodies) in their sera. BRAF is a kinase involved in the mitogen-activated protein kinase (MAPK) signalling pathway. BRAF is also mutated at high frequency in cancers. Because mutations can alter protein function and conformation, they may trigger autoantibody production. Here, we tested whether RA patients with anti BRAF had BRAF mutations in peripheral blood cells (PBMCs).
Materials and Methods Genomic DNA from PBMCs from 61 RA patients and 30 controls were screened for germline mutations through the 7 exons of BRAF gene (exons 6, 11, 12, 13, 14, 15 and 16) containing the 26 known mutations of BRAF by direct sequencing of polymerase chain reaction products. An enriched real time polymerase chain reaction that amplifies variant but blocks wild type sequence was used to detect the predominant BRAF mutation at nucleotide 1799 which converts valine 600 to glutamic acid and accounts for 90% of BRAF mutations in human cancers. After melting curve analysis, variant was then identified by sequencing.
Results No BRAF mutation was detected by classical PCR and sequencing (10% of sensitivity). However, by using enriched PCR (0.1% of sensitivity), we found that 51% (23/45) of RA patients have V600 mutation versus 18% (9/49) of controls. In most patients the valine residue at position 600 was substituted by an alanine. In RA patients, BRAF was mutated in a fraction (1%) of PBMCs. The presence of V600A mutation was not correlated with the presence of autoantibodies to BRAF. Of interest, 68% (19/28) of RA patients positive for ACPAs have V600A mutation versus 24% (4/17) of RA patients negative for ACPAs.
Conclusions Most RA patients positive for ACPAs have V600A mutation in BRAF gene. The presence of V600A mutation may explain BRAF overexpression and BRAF tolerance defect in RA patients.
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