Diagnostic value of H3F3A mutations in giant cell tumour of bone compared to osteoclast-rich mimics

Driver mutations in the two histone 3.3 (H3.3) genes, H3F3A and H3F3B, were recently identified by whole genome sequencing in 95% of chondroblastoma (CB) and by targeted gene sequencing in 92% of giant cell tumour of bone (GCT). Given the high prevalence of these driver mutations, it may be possible to utilise these alterations as diagnostic adjuncts in clinical practice. Here, we explored the spectrum of H3.3 mutations in a wide range and large number of bone tumours (n 5 412) to determine if these alterations could be used to distinguish GCT from other osteoclast-rich tumours such as aneurysmal bone cyst, nonossifying fibroma, giant cell granuloma, and osteoclast-rich malignant bone tumours and others. In addition, we explored the driver landscape of GCT through whole genome, exome and targeted sequencing (14 gene panel). We found that H3.3 mutations, namely mutations of glycine 34 in H3F3A, occur in 96% of GCT. We did not find additional driver mutations in GCT, including mutations in IDH1, IDH2, USP6, TP53. The genomes of GCT exhibited few somatic mutations, akin to the picture seen in CB. Overall our observations suggest that the presence of H3F3A p.Gly34 mutations does not entirely exclude malignancy in osteoclast-rich tumours. However, H3F3A p.Gly34 mutations appear to be an almost essential feature of GCT that will aid pathological evaluation of bone tumours, especially when confronted with small needle core biopsies. In the absence of H3F3A p.Gly34 mutations, a diagnosis of GCT should be made with caution.

Klebsiella pneumoniae subsp. pneumoniae–bacteriophage combination from the caecal effluent of a healthy woman

A recent study characterizing bacteriophage populations within human caecal effluent demonstrated the presence of numerous Podoviridae, Siphoviridae and Myoviridae within this material (Hoyles et al., 2014, Res Microbiol 165, 803–812). Further to this work, anaerobic bacteria were isolated on fastidious anaerobe agar from the caecal effluent of a healthy 31-year-old woman. Ten colonies were selected at random, streaked to purity and screened against the remaining caecal effluent (filter-sterilized, 0.45 μm pore size) in an attempt to isolate lytic bacteriophages. Bacteriophages within the effluent [2×105 ± 2.65×103 (n=3) pfu/ml] were active against five of the isolates, all identified by 16S rRNA gene sequence analysis as Klebsiella pneumoniae. One of the five isolates, L4-FAA5, was characterized further and found to be K. pneumoniae subsp. pneumoniae capsule type K2 rmpA+, and was used to propagate a bacteriophage (which we named KLPN1) to purity. Bacteriophage KLPN1 was a member of the Siphoviridae with a rosette-like tail tip and exhibited depolymerase activity, demonstrated by the formation of plaque-surrounding haloes that increased in size over the course of incubation. When screened against a panel of 21 clinical strains representing unknown K. pneumoniae subsp. pneumoniae capsule types and types K1, K2, K5, K20, K54 and K57, KLPN1 infected only K2 strains, but did not exhibit depolymerase activity against these. Whole-genome sequence analysis of KLPN1 showed the bacteriophage to have a genome of 49,037 bp (50.53 GC mol%) comprising 73 predicted ORFs, of which 22 encoded genes associated with structure, host recognition, packaging, DNA replication and cell lysis. The host recognition-associated gene was a potential depolymerase. This is the first report of the isolation of a bacterium–bacteriophage combination from the human caecum, and only the third member of the Siphoviridae known to infect K. pneumoniae subsp. pneumoniae.

Identification of independent association signals and putative functional variants for breast cancer risk through fine-scale mapping of the 12p11 locus

BACKGROUND: Multiple recent genome-wide association studies (GWAS) have identified a single nucleotide polymorphism (SNP), rs10771399, at 12p11 that is associated with breast cancer risk. METHOD: We performed a fine-scale mapping study of a 700 kb region including 441 genotyped and more than 1300 imputed genetic variants in 48,155 cases and 43,612 controls of European descent, 6269 cases and 6624 controls of East Asian descent and 1116 cases and 932 controls of African descent in the Breast Cancer Association Consortium (BCAC; http://bcac.ccge.medschl.cam.ac.uk/ ), and in 15,252 BRCA1 mutation carriers in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Stepwise regression analyses were performed to identify independent association signals. Data from the Encyclopedia of DNA Elements project (ENCODE) and the Cancer Genome Atlas (TCGA) were used for functional annotation. RESULTS: Analysis of data from European descendants found evidence for four independent association signals at 12p11, represented by rs7297051 (odds ratio (OR) = 1.09, 95 % confidence interval (CI) = 1.06-1.12; P = 3 × 10(-9)), rs805510 (OR = 1.08, 95 % CI = 1.04-1.12, P = 2 × 10(-5)), and rs1871152 (OR = 1.04, 95 % CI = 1.02-1.06; P = 2 × 10(-4)) identified in the general populations, and rs113824616 (P = 7 × 10(-5)) identified in the meta-analysis of BCAC ER-negative cases and BRCA1 mutation carriers. SNPs rs7297051, rs805510 and rs113824616 were also associated with breast cancer risk at P < 0.05 in East Asians, but none of the associations were statistically significant in African descendants. Multiple candidate functional variants are located in putative enhancer sequences. Chromatin interaction data suggested that PTHLH was the likely target gene of these enhancers. Of the six variants with the strongest evidence of potential functionality, rs11049453 was statistically significantly associated with the expression of PTHLH and its nearby gene CCDC91 at P < 0.05. CONCLUSION: This study identified four independent association signals at 12p11 and revealed potentially functional variants, providing additional insights into the underlying biological mechanism(s) for the association observed between variants at 12p11 and breast cancer risk