A common missense variant in BRCA2 predisposes to early onset breast cancer

INTRODUCTION: Mutations in the BRCA2 gene are one of the two major causes of hereditary breast cancer. Protein-truncating mutations of BRCA2 are usually deleterious and increase the risk of breast cancer up to 80% over a lifetime. A few missense mutations in BRCA2 are believed to have a similarly high penetrance, apart from more common neutral polymorphisms. It is often difficult to classify a particular sequence variant as a mutation or a polymorphism. For a deleterious variant, one would expect a greater allele frequency in breast cancer cases than in ethnic-matched controls. In contrast, neutral polymorphic variants should be equally frequent in the two groups. METHODS: We genotyped 3,241 cases of breast cancer diagnosed at under 51 years of age, unselected for family history, from 18 hospitals throughout Poland and 2,791 ethnic-matched controls for a single BRCA2 C5972T variant. RESULTS: The variant was present in approximately 6% of the Polish population. In the study, 13 women (11 cases and two controls (OR = 4.7; p = 0.02)) were homozygous for the variant allele. The overall odds ratio for breast cancer in women with a single copy of the BRCA2 C5972T variant was 1.1 (p = 0.7); however, the effect was significant for patients diagnosed at or before age 40 (OR = 1.4; p = 0.04). We reviewed the association between the BRCA2 variant in different histologic subgroups and found the effect most pronounced in women who had ductal carcinoma in situ (DCIS) with micro-invasion (OR = 2.8; p < 0.0001). CONCLUSION: The BRCA2 C5972T allele is a common variant in Poland that increases the risk of DCIS with micro-invasion. The homozygous state is rare but increases the risk of breast cancer five-fold

Cancer somatic mutations cluster in a subset of regulatory sites predicted from the ENCODE data

Background: Transcriptional regulation of gene expression is essential for cellular differentiation and function, and defects in the process are associated with cancer. The ENCODE project has mapped potential regulatory sites across the complete genome in many cell types, and these regions have been shown to harbour many of the somatic mutations that occur in cancer cells, suggesting that their effects may drive cancer initiation and development. The ENCODE data suggests a very large number of regulatory sites, and methods are needed to identify those that are most relevant and to connect them to the genes that they control. Methods: Predictive models of gene expression were developed by integrating the ENCODE data for regulation, including transcription factor binding and DNase1 hypersensitivity, with RNA-seq data for gene expression. A penalized regression method was used to identify the most predictive potential regulatory sites for each transcript. Known cancer somatic mutations from the COSMIC database were mapped to potential regulatory sites, and we examined differences in the mapping frequencies associated with sites chosen in regulatory models and other (rejected) sites. The effects of potential confounders, for example replication timing, were considered. Results: Cancer somatic mutations preferentially occupy those regulatory regions chosen in our models as most predictive of gene expression. Conclusion: Our methods have identified a significantly reduced set of regulatory sites that are enriched in cancer somatic mutations and are more predictive of gene expression. This has significance for the mechanistic interpretation of cancer mutations, and the understanding of genetic regulation

Early Evolution of Conserved Regulatory Sequences Associated with Development in Vertebrates

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA

Immunohistochemical assessment of Pax8 expression during pancreatic islet development and in human neuroendocrine tumors

The paired box transcription factor Pax8 is critical for development of the eye, thyroid gland as well as the urinary and reproductive organs. In adult, Pax8 overexpression is associated with kidney, ovarian and thyroid tumors and has emerged as a specific marker for these cancers. Recently, Pax8 expression was also reported in human pancreatic islets and in neuroendocrine tumors, identifying Pax8 as a novel member of the Pax family expressed in the pancreas. Herein, we sought to provide a comprehensive analysis of Pax8 expression during pancreogenesis and in adult islets. Immunohistochemical analysis using the most employed Pax8 polyclonal antibody revealed strong nuclear staining in the developing mouse pancreas and in mature human and mouse islets. Astonishingly, Pax8 mRNA in mouse islets was undetectable while human islets exhibited low levels. These discrepancies raised the possibility of antibody cross-reactivity. This premise was confirmed by demonstrating that the polyclonal Pax8 antibody also recognized the islet-enriched Pax6 protein both by Western blotting and immunohistochemistry. Thus, in islets polyclonal Pax8 staining corresponds mainly to Pax6. In order to circumvent this caveat, a novel Pax8 monoclonal antibody was used to re-evaluate whether Pax8 was indeed expressed in islets. Surprisingly, Pax8 was not detected in neither the developing pancreas or in mature islets. Reappraisal of pancreatic neuroendocrine tumors using this Pax8 monoclonal antibody exhibited no immunostaining as compared to the Pax8 polyclonal antibody. In conclusion, Pax8 is not expressed in the pancreas and cast doubts on the value of Pax8 as a pancreatic neuroendocrine tumor marker

Human GLI3 Intragenic Conserved Non-Coding Sequences Are Tissue-Specific Enhancers

The zinc-finger transcription factor GLI3 is a key regulator of development, acting as a primary transducer of Sonic hedgehog (SHH) signaling in a combinatorial context dependent fashion controlling multiple patterning steps in different tissues/organs. A tight temporal and spatial control of gene expression is indispensable, however, cis-acting sequence elements regulating GLI3 expression have not yet been reported. We show that 11 ancient genomic DNA signatures, conserved from the pufferfish Takifugu (Fugu) rubripes to man, are distributed throughout the introns of human GLI3. They map within larger conserved non-coding elements (CNEs) that are found in the tetrapod lineage. Full length CNEs transiently transfected into human cell cultures acted as cell type specific enhancers of gene transcription. The regulatory potential of these elements is conserved and was exploited to direct tissue specific expression of a reporter gene in zebrafish embryos. Assays of deletion constructs revealed that the human-Fugu conserved sequences within the GLI3 intronic CNEs were essential but not sufficient for full-scale transcriptional activation. The enhancer activity of the CNEs is determined by a combinatorial effect of a core sequence conserved between human and teleosts (Fugu) and flanking tetrapod-specific sequences, suggesting that successive clustering of sequences with regulatory potential around an ancient, highly conserved nucleus might be a possible mechanism for the evolution of cis-acting regulatory elements

Functional Analysis of Conserved Non-Coding Regions Around the Short Stature hox Gene (shox) in Whole Zebrafish Embryos

Background: Mutations in the SHOX gene are responsible for Leri-Weill Dyschondrosteosis, a disorder characterised by mesomelic limb shortening. Recent investigations into regulatory elements surrounding SHOX have shown that deletions of conserved non-coding elements (CNEs) downstream of the SHOX gene produce a phenotype indistinguishable from Leri-Weill Dyschondrosteosis. As this gene is not found in rodents, we used zebrafish as a model to characterise the expression pattern of the shox gene across the whole embryo and characterise the enhancer domains of different CNEs associated with this gene. Methodology/Principal Findings: Expression of the shox gene in zebrafish was identified using in situ hybridization, with embryos showing expression in the blood, putative heart, hatching gland, brain pharyngeal arch, olfactory epithelium, and fin bud apical ectodermal ridge. By identifying sequences showing 65% identity over at least 40 nucleotides between Fugu, human, dog and opossum we uncovered 35 CNEs around the shox gene. These CNEs were compared with CNEs previously discovered by Sabherwal et al. ,resulting in the identification of smaller more deeply conserved sub-sequence. Sabherwal et al.’s CNEs were assayed for regulatory function in whole zebrafish embryos resulting in the identification of additional tissues under the regulatory control of these CNEs. Conclusion/Significance: Our results using whole zebrafish embryos have provided a more comprehensive picture of the expression pattern of the shox gene, and a better understanding of its regulation via deeply conserved noncoding elements. In particular, we identify additional tissues under the regulatory control of previously identified SHOX CNEs. We also demonstrate the importance of these CNEs in evolution by identifying duplicated shox CNEs and more deeply conserved sub-sequences within already identified CNEs

Occupational exposure of workers to pesticides: Toxicogenetics and susceptibility gene polymorphisms

Farm workers are often exposed to pesticides, which are products belonging to a specific chemical group that affects the health of agricultural workers and is mostly recognized as genotoxic and carcinogenic. The exposure of workers from Piauí, Brazil, to these hazardous chemicals was assessed and cytogenetic alterations were evaluated using the buccal micronucleus assay, hematological and lipid parameters, butyrylcholinesterase (BChE) activity and genetic polymorphisms of enzymes involved in the metabolism of pesticides, such as PON1, as well as of the DNA repair system (OGG1, XRCC1 and XRCC4). Two groups of farm workers exposed to different types of pesticides were evaluated and compared to matched non-exposed control groups. A significant increase was observed in the frequencies of micronuclei, kariorrhexis, karyolysis and binucleated cells in the exposed groups (n = 100) compared to controls (n = 100). No differences were detected regarding the hematological parameters, lipid profile and BChE activity. No significant difference was observed either regarding DNA damage or nuclear fragmentation when specific metabolizing and DNA repair genotypes were investigated in the exposed groups

Dissecting the Transcriptional Regulatory Properties of Human Chromosome 16 Highly Conserved Non-Coding Regions

Non-coding DNA conservation across species has been often used as a predictor for transcriptional enhancer activity. However, only a few systematic analyses of the function of these highly conserved non-coding regions (HCNRs) have been performed. Here we use zebrafish transgenic assays to perform a systematic study of 113 HCNRs from human chromosome 16. By comparing transient and stable transgenesis, we show that the first method is highly inefficient, leading to 40% of false positives and 20% of false negatives. When analyzed in stable transgenic lines, a great majority of HCNRs were active in the central nervous system, although some of them drove expression in other organs such as the eye and the excretory system. Finally, by testing a fraction of the HCNRs lacking enhancer activity for in vivo insulator activity, we find that 20% of them may contain enhancer-blocking function. Altogether our data indicate that HCNRs may contain different types of cis-regulatory activity, including enhancer, insulators as well as other not yet discovered functions

Resolving early mesoderm diversification through single-cell expression profiling.

In mammals, specification of the three major germ layers occurs during gastrulation, when cells ingressing through the primitive streak differentiate into the precursor cells of major organ systems. However, the molecular mechanisms underlying this process remain unclear, as numbers of gastrulating cells are very limited. In the mouse embryo at embryonic day 6.5, cells located at the junction between the extra-embryonic region and the epiblast on the posterior side of the embryo undergo an epithelial-to-mesenchymal transition and ingress through the primitive streak. Subsequently, cells migrate, either surrounding the prospective ectoderm contributing to the embryo proper, or into the extra-embryonic region to form the yolk sac, umbilical cord and placenta. Fate mapping has shown that mature tissues such as blood and heart originate from specific regions of the pre-gastrula epiblast, but the plasticity of cells within the embryo and the function of key cell-type-specific transcription factors remain unclear. Here we analyse 1,205 cells from the epiblast and nascent Flk1(+) mesoderm of gastrulating mouse embryos using single-cell RNA sequencing, representing the first transcriptome-wide in vivo view of early mesoderm formation during mammalian gastrulation. Additionally, using knockout mice, we study the function of Tal1, a key haematopoietic transcription factor, and demonstrate, contrary to previous studies performed using retrospective assays, that Tal1 knockout does not immediately bias precursor cells towards a cardiac fate.We thank M. de Bruijn, A. Martinez-Arias, J. Nichols and C. Mulas for discussion, the Cambridge Institute for Medical Research Flow Cytometry facility for their expertise in single-cell index sorting, and S. Lorenz from the Sanger Single Cell Genomics Core for supervising purification of Tal1−/− sequencing libraries. ChIP-seq reads were processed by R. Hannah. Research in the authors’ laboratories is supported by the Medical Research Council, Cancer Research UK, the Biotechnology and Biological Sciences Research Council, Bloodwise, the Leukemia and Lymphoma Society, and the Sanger-EBI Single Cell Centre, and by core support grants from the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust - MRC Cambridge Stem Cell Institute and by core funding from Cancer Research UK and the European Molecular Biology Laboratory. Y.T. was supported by a fellowship from the Japan Society for the Promotion of Science. W.J. is a Wellcome Trust Clinical Research Fellow. A.S. is supported by the Sanger-EBI Single Cell Centre. This work was funded as part of Wellcome Trust Strategic Award 105031/D/14/Z ‘Tracing early mammalian lineage decisions by single-cell genomics’ awarded to W. Reik, S. Teichmann, J. Nichols, B. Simons, T. Voet, S. Srinivas, L. Vallier, B. Göttgens and J. Marioni.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nature1863

Long-range evolutionary constraints reveal cis-regulatory interactions on the human X chromosome

Enhancers can regulate the transcription of genes over long genomic distances. This is thought to lead to selection against genomic rearrangements within such regions that may disrupt this functional linkage. Here we test this concept experimentally using the human X chromosome. We describe a scoring method to identify evolutionary maintenance of linkage between conserved noncoding elements and neighbouring genes. Chromatin marks associated with enhancer function are strongly correlated with this linkage score. We test >1,000 putative enhancers by transgenesis assays in zebrafish to ascertain the identity of the target gene. The majority of active enhancers drive a transgenic expression in a pattern consistent with the known expression of a linked gene. These results show that evolutionary maintenance of linkage is a reliable predictor of an enhancer's function, and provide new information to discover the genetic basis of diseases caused by the mis-regulation of gene expression