B-RAF mutant alleles associated with Langerhans cell histiocytosis, a granulomatous pediatric disease

Abstract

Background: Langerhans cell histiocytosis (LCH) features inflammatory granuloma characterised by the presence of CD1a+ dendritic cells or 'LCH cells'. Badalian-Very et al. recently reported the presence of a canonical (V600E)B-RAF mutation in 57% of paraffin-embedded biopsies from LCH granuloma. Here we confirm their findings and report the identification of two novel B-RAF mutations detected in LCH patients.

Methods and results: Mutations of B-RAF were observed in granuloma samples from 11 out of 16 patients using 'next generation' pyrosequencing. In 9 cases the mutation identified was (V600E)B-RAF. In 2 cases novel polymorphisms were identified. A somatic (600DLAT)B-RAF insertion mimicked the structural and functional consequences of the (V600E)B-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The (600DLAT)B-RAF and (V600E)B-RAF mutations were found enriched in DNA and mRNA from the CD1a+ fraction of granuloma. They were absent from the blood and monocytes of 58 LCH patients, with a lower threshold of sequencing sensitivity of 1%-2% relative mutation abundance. A novel germ line (T599A)B-RAF mutant allele was detected in one patient, at a relative mutation abundance close to 50% in the LCH granuloma, blood monocytes and lymphocytes. However, (T599A)B-RAF did not destabilize the inactive conformation of the B-RAF kinase, and did not induce increased ERK phosphorylation or C-RAF transactivation.

Conclusions: Our data confirmed presence of the (V600E)B-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that (V600E)B-RAF and (600DLAT)B-RAF mutations are somatic mutants enriched in LCH CD1a(+) cells and absent from the patient blood. Further studies are needed to assess the functional consequences of the germ-line (T599A)B-RAF allele.

Figure 1. Analysis of B-RAF mutant.

A. Sequence alignment, results from 454 pyrosequencing of granuloma cells from patients 1–10 and 16. B. ‘Sanger’ sequencing of patient 16 blood; A/G transition at nucleotide 1795. C. Pedigree of patient 16. Both the patient and his mother carry a ^T599AB-RAF allele, while his father is ^wtB-RAF. D–E. Comparison between ^wtB-RAF 5P_15056 (D, purple), ^V600EB-RAF structure (E, cyan) and the modeled mutant ^600DLATB-RAF (F, grey). In D, Val600 (yellow) forms a hydrophobic contact with Phe468 (red arrow). In E and F charged residues Asp and Glu (in orange) disrupt the hydrophobic network of interactions, stabilising the active conformation of the P-loop. In F, insertion Asp-Leu-Ala-Thr shifted Val600 and disrupt the hydrophobic cluster. G, H. MEK phosphorylation in 293 T cells. 293 T cells were transiently transfected with with mock or B-RAF mutant expressing vectors (WT, V600E, T599A, 600DLAT, D594A, G596R), and with (H) or without (G) wtC-RAF. Twenty-four hours after transfection, the medium was changed to serum-free DMEM, followed by further 18 hours culture. Total cell lysates were immunoblotted with the indicated antibodies.

Figure 2. Analysis of ^T599AB-RAF.

(A, B) Comparison between models of ^WTB-RAF 5P_15056 (A, violet) and ^T599AB-RAF (B, gold). ^T599AB-RAF substitutes a polar uncharged residue with a hydrophobic residue, causing the loss of short-ranged interactions with residues D576 and D594. C. Analysis of MEK and ERK phosphorylation in THP1 cell lines stably transfected with ^WTB-RAF-FLAG and ^T599AB-RAF-FLAG. Experiment was repeated twice with similar results. (D–F) Analysis of MEK and ERK phosphorylation and IL-8 production in U937 cell lines stably transfected with ^WTB-RAF, ^T599AB-RAF, and ^D594AB-RAF. Experiment was repeated twice with similar results.