Publications by authors named "Lorna Gregory"

12 Publications

  • Page 1 of 1

Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline.

Proc Natl Acad Sci U S A 2013 Dec 20;110(50):20152-7. Epub 2013 Nov 20.

Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom.

The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause Costello syndrome (CS), a congenital disorder associated with predisposition to malignancy. Based on the epidemiology of CS and the occurrence of HRAS mutations in spermatocytic seminoma, we proposed that activating HRAS mutations become enriched in sperm through a process akin to tumorigenesis, termed selfish spermatogonial selection. To test this hypothesis, we quantified the levels, in blood and sperm samples, of HRAS mutations at the p.G12 codon and compared the results to changes at the p.A11 codon, at which activating mutations do not occur. The data strongly support the role of selection in determining HRAS mutation levels in sperm, and hence the occurrence of CS, but we also found differences from the mutation pattern in tumorigenesis. First, the relative prevalence of mutations in sperm correlates weakly with their in vitro activating properties and occurrence in cancers. Second, specific tandem base substitutions (predominantly GC>TT/AA) occur in sperm but not in cancers; genomewide analysis showed that this same mutation is also overrepresented in constitutional pathogenic and polymorphic variants, suggesting a heightened vulnerability to these mutations in the germline. We developed a statistical model to show how both intrinsic mutation rate and selfish selection contribute to the mutational burden borne by the paternal germline.
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http://dx.doi.org/10.1073/pnas.1311381110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3864328PMC
December 2013

Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model.

Brain 2013 Oct 11;136(Pt 10):3106-18. Epub 2013 Sep 11.

1 Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford OX3 9DU, UK.

Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the 'diagnostic odyssey' for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1-3, 6, 7 and Friedrich's ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3-35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US$620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.
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http://dx.doi.org/10.1093/brain/awt236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784284PMC
October 2013

Whole-exome sequencing studies of nonfunctioning pituitary adenomas.

J Clin Endocrinol Metab 2013 Apr 28;98(4):E796-800. Epub 2013 Feb 28.

Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7LJ, United Kingdom.

Context: The tumorigenic role of genetic abnormalities in sporadic pituitary nonfunctioning adenomas (NFAs), which usually originate from gonadotroph cells, is unknown.

Objective: The objective of the study was to identify somatic genetic abnormalities in sporadic pituitary NFAs.

Design: Whole-exome sequencing was performed using DNA from 7 pituitary NFAs and leukocyte samples obtained from the same patients. Somatic variants were confirmed by dideoxynucleotide sequencing, and candidate driver genes were assessed in an additional 24 pituitary NFAs.

Results: Whole-exome sequencing achieved a high degree of coverage such that approximately 97% of targeted bases were represented by more than 10 base reads; 24 somatic variants were identified and confirmed in the discovery set of 7 pituitary NFAs (mean 3.5 variants/tumor; range 1-7). Approximately 80% of variants occurred as missense single nucleotide variants and the remainder were synonymous changes or small frameshift deletions. Each of the 24 mutations occurred in independent genes with no recurrent mutations. Mutations were not observed in genes previously associated with pituitary tumorigenesis, although somatic variants in putative driver genes including platelet-derived growth factor D (PDGFD), N-myc down-regulated gene family member 4 (NDRG4), and Zipper sterile-α-motif kinase (ZAK) were identified; however, DNA sequence analysis of these in the validation set of 24 pituitary NFAs did not reveal any mutations indicating that these genes are unlikely to contribute significantly in the etiology of sporadic pituitary NFAs.

Conclusions: Pituitary NFAs harbor few somatic mutations consistent with their low proliferation rates and benign nature, but mechanisms other than somatic mutation are likely involved in the etiology of sporadic pituitary NFAs.
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http://dx.doi.org/10.1210/jc.2012-4028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447855PMC
April 2013

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer.

Nature 2013 Jan 16;493(7432):406-10. Epub 2012 Dec 16.

Division of Genetics & Epidemiology, The Institute of Cancer Research, Sutton SM2 5NG, UK.

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.
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http://dx.doi.org/10.1038/nature11725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3759028PMC
January 2013

Recessive mutations in SPTBN2 implicate β-III spectrin in both cognitive and motor development.

PLoS Genet 2012 6;8(12):e1003074. Epub 2012 Dec 6.

Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

β-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Heterozygous mutations in SPTBN2, the gene encoding β-III spectrin, cause Spinocerebellar Ataxia Type 5 (SCA5), an adult-onset, slowly progressive, autosomal-dominant pure cerebellar ataxia. SCA5 is sometimes known as "Lincoln ataxia," because the largest known family is descended from relatives of the United States President Abraham Lincoln. Using targeted capture and next-generation sequencing, we identified a homozygous stop codon in SPTBN2 in a consanguineous family in which childhood developmental ataxia co-segregates with cognitive impairment. The cognitive impairment could result from mutations in a second gene, but further analysis using whole-genome sequencing combined with SNP array analysis did not reveal any evidence of other mutations. We also examined a mouse knockout of β-III spectrin in which ataxia and progressive degeneration of cerebellar Purkinje cells has been previously reported and found morphological abnormalities in neurons from prefrontal cortex and deficits in object recognition tasks, consistent with the human cognitive phenotype. These data provide the first evidence that β-III spectrin plays an important role in cortical brain development and cognition, in addition to its function in the cerebellum; and we conclude that cognitive impairment is an integral part of this novel recessive ataxic syndrome, Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1 (SPARCA1). In addition, the identification of SPARCA1 and normal heterozygous carriers of the stop codon in SPTBN2 provides insights into the mechanism of molecular dominance in SCA5 and demonstrates that the cell-specific repertoire of spectrin subunits underlies a novel group of disorders, the neuronal spectrinopathies, which includes SCA5, SPARCA1, and a form of West syndrome.
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http://dx.doi.org/10.1371/journal.pgen.1003074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3516553PMC
May 2013

Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3.

Nat Genet 2013 Jan 9;45(1):93-7. Epub 2012 Dec 9.

Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK.

Adaptor protein-2 (AP2), a central component of clathrin-coated vesicles (CCVs), is pivotal in clathrin-mediated endocytosis, which internalizes plasma membrane constituents such as G protein-coupled receptors (GPCRs). AP2, a heterotetramer of α, β, μ and σ subunits, links clathrin to vesicle membranes and binds to tyrosine- and dileucine-based motifs of membrane-associated cargo proteins. Here we show that missense mutations of AP2 σ subunit (AP2S1) affecting Arg15, which forms key contacts with dileucine-based motifs of CCV cargo proteins, result in familial hypocalciuric hypercalcemia type 3 (FHH3), an extracellular calcium homeostasis disorder affecting the parathyroids, kidneys and bone. We found AP2S1 mutations in >20% of cases of FHH without mutations in calcium-sensing GPCR (CASR), which cause FHH1. AP2S1 mutations decreased the sensitivity of CaSR-expressing cells to extracellular calcium and reduced CaSR endocytosis, probably through loss of interaction with a C-terminal CaSR dileucine-based motif, whose disruption also decreased intracellular signaling. Thus, our results identify a new role for AP2 in extracellular calcium homeostasis.
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http://dx.doi.org/10.1038/ng.2492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605788PMC
January 2013

Whole-exome sequencing studies of nonhereditary (sporadic) parathyroid adenomas.

J Clin Endocrinol Metab 2012 Oct 1;97(10):E1995-2005. Epub 2012 Aug 1.

Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, and Department of Surgery, John Radcliffe Hospital, Oxford University Hospitals Trust, Oxford, UK.

Context: Genetic abnormalities, such as those of multiple endocrine neoplasia type 1 (MEN1) and Cyclin D1 (CCND1) genes, occur in <50% of nonhereditary (sporadic) parathyroid adenomas.

Objective: To identify genetic abnormalities in nonhereditary parathyroid adenomas by whole-exome sequence analysis.

Design: Whole-exome sequence analysis was performed on parathyroid adenomas and leukocyte DNA samples from 16 postmenopausal women without a family history of parathyroid tumors or MEN1 and in whom primary hyperparathyroidism due to single-gland disease was cured by surgery. Somatic variants confirmed in this discovery set were assessed in 24 other parathyroid adenomas.

Results: Over 90% of targeted exons were captured and represented by more than 10 base reads. Analysis identified 212 somatic variants (median eight per tumor; range, 2-110), with the majority being heterozygous nonsynonymous single-nucleotide variants that predicted missense amino acid substitutions. Somatic MEN1 mutations occurred in six of 16 (∼35%) parathyroid adenomas, in association with loss of heterozygosity on chromosome 11. However, no other gene was mutated in more than one tumor. Mutations in several genes that may represent low-frequency driver mutations were identified, including a protection of telomeres 1 (POT1) mutation that resulted in exon skipping and disruption to the single-stranded DNA-binding domain, which may contribute to increased genomic instability and the observed high mutation rate in one tumor.

Conclusions: Parathyroid adenomas typically harbor few somatic variants, consistent with their low proliferation rates. MEN1 mutation represents the major driver in sporadic parathyroid tumorigenesis although multiple low-frequency driver mutations likely account for tumors not harboring somatic MEN1 mutations.
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http://dx.doi.org/10.1210/jc.2012-2303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446457PMC
October 2012

The human pancreatic islet transcriptome: expression of candidate genes for type 1 diabetes and the impact of pro-inflammatory cytokines.

PLoS Genet 2012 8;8(3):e1002552. Epub 2012 Mar 8.

Laboratory of Experimental Medicine, Université Libre de Bruxelles (ULB), Brussels, Belgium.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic beta cells are killed by infiltrating immune cells and by cytokines released by these cells. Signaling events occurring in the pancreatic beta cells are decisive for their survival or death in diabetes. We have used RNA sequencing (RNA-seq) to identify transcripts, including splice variants, expressed in human islets of Langerhans under control conditions or following exposure to the pro-inflammatory cytokines interleukin-1β (IL-1β) and interferon-γ (IFN-γ). Based on this unique dataset, we examined whether putative candidate genes for T1D, previously identified by GWAS, are expressed in human islets. A total of 29,776 transcripts were identified as expressed in human islets. Expression of around 20% of these transcripts was modified by pro-inflammatory cytokines, including apoptosis- and inflammation-related genes. Chemokines were among the transcripts most modified by cytokines, a finding confirmed at the protein level by ELISA. Interestingly, 35% of the genes expressed in human islets undergo alternative splicing as annotated in RefSeq, and cytokines caused substantial changes in spliced transcripts. Nova1, previously considered a brain-specific regulator of mRNA splicing, is expressed in islets and its knockdown modified splicing. 25/41 of the candidate genes for T1D are expressed in islets, and cytokines modified expression of several of these transcripts. The present study doubles the number of known genes expressed in human islets and shows that cytokines modify alternative splicing in human islet cells. Importantly, it indicates that more than half of the known T1D candidate genes are expressed in human islets. This, and the production of a large number of chemokines and cytokines by cytokine-exposed islets, reinforces the concept of a dialog between pancreatic islets and the immune system in T1D. This dialog is modulated by candidate genes for the disease at both the immune system and beta cell level.
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http://dx.doi.org/10.1371/journal.pgen.1002552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3297576PMC
September 2012

Preparation of high-quality next-generation sequencing libraries from picogram quantities of target DNA.

Genome Res 2012 Jan 16;22(1):125-33. Epub 2011 Nov 16.

Systems Biology Laboratory UK, Abingdon, Oxfordshire OX14 4SA, United Kingdom.

New sequencing technologies can address diverse biomedical questions but are limited by a minimum required DNA input of typically 1 μg. We describe how sequencing libraries can be reproducibly created from 20 pg of input DNA using a modified transpososome-mediated fragmentation technique. Resulting libraries incorporate in-line bar-coding, which facilitates sample multiplexes that can be sequenced using Illumina platforms with the manufacturer's sequencing primer. We demonstrate this technique by providing deep coverage sequence of the Escherichia coli K-12 genome that shows equivalent target coverage to a 1-μg input library prepared using standard Illumina methods. Reducing template quantity does, however, increase the proportion of duplicate reads and enriches coverage in low-GC regions. This finding was confirmed with exhaustive resequencing of a mouse library constructed from 20 pg of gDNA input (about seven haploid genomes) resulting in ∼0.4-fold statistical coverage of uniquely mapped fragments. This implies that a near-complete coverage of the mouse genome is obtainable with this approach using 20 genomes as input. Application of this new method now allows genomic studies from low mass samples and routine preparation of sequencing libraries from enrichment procedures.
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http://dx.doi.org/10.1101/gr.124016.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246199PMC
January 2012

Substantial histone reduction modulates genomewide nucleosomal occupancy and global transcriptional output.

PLoS Biol 2011 Jun 28;9(6):e1001086. Epub 2011 Jun 28.

San Raffaele University, Milan, Italy.

The basic unit of genome packaging is the nucleosome, and nucleosomes have long been proposed to restrict DNA accessibility both to damage and to transcription. Nucleosome number in cells was considered fixed, but recently aging yeast and mammalian cells were shown to contain fewer nucleosomes. We show here that mammalian cells lacking High Mobility Group Box 1 protein (HMGB1) contain a reduced amount of core, linker, and variant histones, and a correspondingly reduced number of nucleosomes, possibly because HMGB1 facilitates nucleosome assembly. Yeast nhp6 mutants lacking Nhp6a and -b proteins, which are related to HMGB1, also have a reduced amount of histones and fewer nucleosomes. Nucleosome limitation in both mammalian and yeast cells increases the sensitivity of DNA to damage, increases transcription globally, and affects the relative expression of about 10% of genes. In yeast nhp6 cells the loss of more than one nucleosome in four does not affect the location of nucleosomes and their spacing, but nucleosomal occupancy. The decrease in nucleosomal occupancy is non-uniform and can be modelled assuming that different nucleosomal sites compete for available histones. Sites with a high propensity to occupation are almost always packaged into nucleosomes both in wild type and nucleosome-depleted cells; nucleosomes on sites with low propensity to occupation are disproportionately lost in nucleosome-depleted cells. We suggest that variation in nucleosome number, by affecting nucleosomal occupancy both genomewide and gene-specifically, constitutes a novel layer of epigenetic regulation.
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http://dx.doi.org/10.1371/journal.pbio.1001086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3125158PMC
June 2011

Identification and characterization of enhancers controlling the inflammatory gene expression program in macrophages.

Immunity 2010 Mar 4;32(3):317-28. Epub 2010 Mar 4.

Department of Experimental Oncology, European Institute of Oncology (IEO), IFOM-IEO Campus, Via Adamello 16, Milan, Italy.

Enhancers determine tissue-specific gene expression programs. Enhancers are marked by high histone H3 lysine 4 mono-methylation (H3K4me1) and by the acetyl-transferase p300, which has allowed genome-wide enhancer identification. However, the regulatory principles by which subsets of enhancers become active in specific developmental and/or environmental contexts are unknown. We exploited inducible p300 binding to chromatin to identify, and then mechanistically dissect, enhancers controlling endotoxin-stimulated gene expression in macrophages. In these enhancers, binding sites for the lineage-restricted and constitutive Ets protein PU.1 coexisted with those for ubiquitous stress-inducible transcription factors such as NF-kappaB, IRF, and AP-1. PU.1 was required for maintaining H3K4me1 at macrophage-specific enhancers. Reciprocally, ectopic expression of PU.1 reactivated these enhancers in fibroblasts. Thus, the combinatorial assembly of tissue- and signal-specific transcription factors determines the activity of a distinct group of enhancers. We suggest that this may represent a general paradigm in tissue-restricted and stimulus-responsive gene regulation.
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http://dx.doi.org/10.1016/j.immuni.2010.02.008DOI Listing
March 2010

Stem cell regulation by JAK/STAT signaling in Drosophila.

Semin Cell Dev Biol 2008 Aug 18;19(4):407-13. Epub 2008 Jun 18.

Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.

Stem cells have become one of the "buzz" topics in the last decade or so. One of the best systems to study adult stem cells in vivo is in the model organism, Drosophila melanogaster. One hundred years of genetic analysis, a sequenced and highly annotated genome and genomics makes this a difficult organism to avoid. The JAK/STAT pathway has been shown to regulate stem cells during haematopoiesis and gametogenesis in Drosophila. In this review we cover the current literature and contrast each group of stem cells with respect to JAK/STAT signaling.
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http://dx.doi.org/10.1016/j.semcdb.2008.06.003DOI Listing
August 2008