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

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 ^V600EB-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 ^V600EB-RAF. In 2 cases novel polymorphisms were identified. A somatic ^600DLATB-RAF insertion mimicked the structural and functional consequences of the ^V600EB-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The ^600DLATB-RAF and ^V600EB-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 ^T599AB-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, ^T599AB-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 ^V600EB-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that ^V600EB-RAF and ^600DLATB-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 ^T599AB-RAF allele.     

Inferring loss-of-heterozygosity from unpaired tumors using high-density oligonucleotide SNP arrays

Loss of heterozygosity (LOH) of chromosomal regions bearing tumor suppressors is a key event in the evolution of epithelial and mesenchymal tumors. Identification of these regions usually relies on genotyping tumor and counterpart normal DNA and noting regions where heterozygous alleles in the normal DNA become homozygous in the tumor. However, paired normal samples for tumors and cell lines are often not available. With the advent of oligonucleotide arrays that simultaneously assay thousands of single-nucleotide polymorphism (SNP) markers, genotyping can now be done at high enough resolution to allow identification of LOH events by the absence of heterozygous loci, without comparison to normal controls. Here we describe a hidden Markov model-based method to identify LOH from unpaired tumor samples, taking into account SNP intermarker distances, SNP-specific heterozygosity rates, and the haplotype structure of the human genome. When we applied the method to data genotyped on 100 K arrays, we correctly identified 99% of SNP markers as either retention or loss. We also correctly identified 81% of the regions of LOH, including 98% of regions greater than 3 megabases. By integrating copy number analysis into the method, we were able to distinguish LOH from allelic imbalance. Application of this method to data from a set of prostate samples without paired normals identified known regions of prevalent LOH. We have developed a method for analyzing high-density oligonucleotide SNP array data to accurately identify of regions of LOH and retention in tumors without the need for paired normal samples.     

The AHL- and BDSF-Dependent Quorum Sensing Systems Control Specific and Overlapping Sets of Genes in Burkholderia cenocepacia H111

Quorum sensing in Burkholderia cenocepacia H111 involves two signalling systems that depend on different signal molecules, namely N-acyl homoserine lactones (AHLs) and the diffusible signal factor cis-2-dodecenoic acid (BDSF). Previous studies have shown that AHLs and BDSF control similar phenotypic traits, including biofilm formation, proteolytic activity and pathogenicity. In this study we mapped the BDSF stimulon by RNA-Seq and shotgun proteomics analysis. We demonstrate that a set of the identified BDSF-regulated genes or proteins are also controlled by AHLs, suggesting that the two regulons partially overlap. The detailed analysis of two mutually regulated operons, one encoding three lectins and the other one encoding the large surface protein BapA and its type I secretion machinery, revealed that both AHLs and BDSF are required for full expression, suggesting that the two signalling systems operate in parallel. In accordance with this, we show that both AHLs and BDSF are required for biofilm formation and protease production.     

Inter-population variation in the behaviour of adult and juvenile Red-footed Boobies Sula sula

Early life is a critical phase of the life cycle of animals and is attracting increased attention because little information is available on the behaviour of young individuals during this period. Behaviour during early life is probably influenced by the environmental conditions encountered by young animals, but data on intraspecific variation between breeding sites during this crucial period of life are limited. Here we study variability in the foraging behaviour of juveniles and adults in three colonies of a pantropical seabird, the Red-footed Booby Sula sula. Both adults and juveniles were measured and fitted with GPS loggers in three remote islands: Genovesa (Galapagos, Eastern Pacific Ocean), Europa (Western Indian Ocean) and Surprise (New Caledonia, Western Pacific Ocean). Foraging behaviour was compared between age-classes, sex and colonies by examining trip characteristics, different behaviours at sea, potential associations between individuals and morphological characteristics. Compared with adults, juveniles conducted shorter trips that were restricted to around the colony, especially on Genovesa (max. range: 203.4 ± 125.1 km and 3.6 ± 3.1 km, respectively). Juveniles appeared more constrained by poor flight skills and experience rather than by their morphology. Adults travelled 45% of the time during at-sea trips, whereas juveniles spent a a lower proportion of time travelling but foraged more often using an ‘area-restricted search’ behaviour, potentially training to catch prey. Associations between juveniles were commonly detected in the three colonies and occurred mostly during foraging, suggesting that social learning is an important strategy. Variability of morphometric measurements in both adults and juveniles was high between sites, with larger birds found on Genovesa. These results suggest that adaptations to local environmental conditions are already visible in their early life. Future studies should continue to investigate the behavioural flexibility of juvenile birds to better understand the effect of local environmental conditions during this critical stage of life.     

Blood proteome of acute intracranial hemorrhage in infant victims of abusive head trauma

Abusive head trauma (AHT) is a leading cause of mortality and morbidity in infants. While the reported incidence is close to 40 cases per 100′000 births/year, misdiagnoses are commonly observed in cases with atypical, subacute, or chronic presentation. Currently, standard clinical evaluation of inflicted intracranial hemorrhagic injury (ICH) in infants urgently requires a screening test able to identify infants who need additional investigations. Blood biomarkers characteristic of AHT may assist in detecting these infants, improving prognosis through early medical care. To date, the application of innovative omics technologies in retrospective studies of AHT in infants is rare, due also to the blood serum and cerebrospinal fluid of AHT cases being scarce and not systematically accessible. Here, we explored the circulating blood proteomes of infants with severe AHT and their atraumatic controls. We discovered 165 circulating serum proteins that display differential changes in AHT cases compared with atraumatic controls. The peripheral blood proteomes of pediatric AHT commonly reflect: (i) potentially secreted proteome from injured brain, and (ii) proteome dysregulated in the system's circulation by successive biological events following acute ICH. This study opens up a novel opportunity for research efforts in clinical screening of AHT cases.