title: 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.
  
  
  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.
authors.affiliation: School of Medicine, Centre for Molecular and Cellular Biology of Inflammation (CMCBI), King's College London, London, United Kingdom.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Satoh
authors.forename: Alexander
authors.initials: A
authors.lastname: Smith
authors.forename: Aurelien
authors.initials: A
authors.lastname: Sarde
authors.forename: Hui-chun
authors.initials: HC
authors.lastname: Lu
authors.forename: Syed
authors.initials: S
authors.lastname: Mian
authors.forename: Sophie
authors.initials: S
authors.lastname: Mian
authors.forename: Celine
authors.initials: C
authors.lastname: Trouillet
authors.forename: Ghulam
authors.initials: G
authors.lastname: Mufti
authors.forename: Jean-Francois
authors.initials: JF
authors.lastname: Emile
authors.forename: Franca
authors.initials: F
authors.lastname: Fraternali
authors.forename: Jean
authors.initials: J
authors.lastname: Donadieu
authors.forename: Frederic
authors.initials: F
authors.lastname: Geissmann
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D002648:Child; D002675:Child, Preschool; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D005260:Female; D020022:Genetic Predisposition to Disease; D006099:Granuloma / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006646:Histiocytosis, Langerhans-Cell / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007223:Infant; D008214:Lymphocytes / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation / Q000235:genetics; D011110:Polymorphism, Genetic; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation; D055815:Young Adult
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases