B-RAF Mutant Alleles Associated with Langerhans Cell Histiocytosis, a Granulomatous Pediatric Disease

TY - JOUR

T1 - B-RAF Mutant Alleles Associated with Langerhans Cell Histiocytosis, a Granulomatous Pediatric Disease

AU - Satoh, Takeshi

AU - Smith, Alexander

AU - Sarde, Aurelien

AU - Lu, Hui-chun

AU - Mian, Sophie

AU - Trouillet, Celine

AU - Mufti, Ghulam

AU - Emile, Jean-Francois

AU - Fraternali, Franca

AU - Donadieu, Jean

AU - Geissmann, Frederic

PY - 2012/4/10

Y1 - 2012/4/10

N2 - 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 B-V600E-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 B-V600E-RAF. In 2 cases novel polymorphisms were identified. A somatic B-600DLAT-RAF insertion mimicked the structural and functional consequences of the B-V600E-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The B-600DLAT-RAF and B-V600E-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 B-T599A-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, B-T599A-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 B-V600E-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that B-V600E-RAF and B-600DLAT-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 B-T599A-RAF allele.

AB - 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 B-V600E-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 B-V600E-RAF. In 2 cases novel polymorphisms were identified. A somatic B-600DLAT-RAF insertion mimicked the structural and functional consequences of the B-V600E-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The B-600DLAT-RAF and B-V600E-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 B-T599A-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, B-T599A-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 B-V600E-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that B-V600E-RAF and B-600DLAT-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 B-T599A-RAF allele.

KW - Alleles

KW - Amino Acid Sequence

KW - Child

KW - Child, Preschool

KW - Extracellular Signal-Regulated MAP Kinases/genetics

KW - Female

KW - Genetic Predisposition to Disease

KW - Granuloma/genetics

KW - HEK293 Cells

KW - Histiocytosis, Langerhans-Cell/enzymology

KW - Humans

KW - Infant

KW - Lymphocytes/metabolism

KW - Male

KW - Molecular Sequence Data

KW - Monocytes/metabolism

KW - Mutation

KW - Phosphorylation/genetics

KW - Polymorphism, Genetic

KW - Proto-Oncogene Proteins B-raf/genetics

KW - Proto-Oncogene Proteins c-raf/genetics

KW - Sequence Homology, Amino Acid

KW - Transcriptional Activation

KW - Young Adult

U2 - 10.1371/journal.pone.0033891

DO - 10.1371/journal.pone.0033891

M3 - Article

C2 - 22506009

SN - 1932-6203

VL - 7

SP - -

JO - PL o S One

JF - PL o S One

IS - 4

M1 - e33891

ER -