Genomic mutation and genetic disease

Keren Carss

PhD Student

Stephen Clayton

Bioinformatician

Sarah Fowler

Staff Scientist

Dan King

PhD Student

Raheleh Rahbari

Postdoctoral Fellow

Art Wuster

Postdoctoral Fellow

Margriet van Kogelenberg

mvk1@sanger.ac.ukPostdoctoral Fellow

Keren Carss

- PhD Student

I obtained a Bachelor of Medical Sciences from the University of Birmingham in 2007, where I specialised in cellular and molecular biology (specifically genetics and reproduction). From 2008-2010 I worked as a research assistant at the Clinical Pharmacology Unit of the University of Cambridge, using sequencing to try to find the genetic cause of Familial Hyperaldosteronism Type II, which causes severe hypertension. I started my PhD at the Wellcome Trust Sanger Institute in October 2010.

Research

I use exome sequencing to identify genes that cause developmental disease, and zebrafish embryos to model those genes. Phenotypes I have studied include microcephalic osteodysplastic primordial dwarfism, congenital muscular dystrophy and prenatal structural abnormalities. I am also interested in scientific publishing, and I am an editor for the Cambridge Student Journal of Genetics, and for BlueSci magazine.

References

• Mutations in B3GALNT2 cause congenital muscular dystrophy and hypoglycosylation of α-dystroglycan.

  Stevens E, Carss KJ, Cirak S, Foley AR, Torelli S, Willer T, Tambunan DE, Yau S, Brodd L, Sewry CA, Feng L, Haliloglu G, Orhan D, Dobyns WB, Enns GM, Manning M, Krause A, Salih MA, Walsh CA, Hurles M, Campbell KP, Manzini MC, UK10K Consortium, Stemple D, Lin YY and Muntoni F

  Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, UK.

  Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a β-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement.

  Funded by: Howard Hughes Medical Institute; Medical Research Council; NICHD NIH HHS: K99HD067379, P30HD19655; NIMH NIH HHS: RC2MH089952; NINDS NIH HHS: 1U54NS053672

  American journal of human genetics 2013;92;3;354-65

  PUBMED: 23453667; PMC: 3591840; DOI: 10.1016/j.ajhg.2013.01.016

• Genomic variation in the vomeronasal receptor gene repertoires of inbred mice.

  Wynn EH, Sánchez-Andrade G, Carss KJ and Logan DW

  Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

  Background: Vomeronasal receptors (VRs), expressed in sensory neurons of the vomeronasal organ, are thought to bind pheromones and mediate innate behaviours. The mouse reference genome has over 360 functional VRs arranged in highly homologous clusters, but the vast majority are of unknown function. Differences in these receptors within and between closely related species of mice are likely to underpin a range of behavioural responses. To investigate these differences, we interrogated the VR gene repertoire from 17 inbred strains of mice using massively parallel sequencing.

  Results: Approximately half of the 6222 VR genes that we investigated could be successfully resolved, and those that were unambiguously mapped resulted in an extremely accurate dataset. Collectively VRs have over twice the coding sequence variation of the genome average; but we identify striking non-random distribution of these variants within and between genes, clusters, clades and functional classes of VRs. We show that functional VR gene repertoires differ considerably between different Mus subspecies and species, suggesting these receptors may play a role in mediating behavioural adaptations. Finally, we provide evidence that widely-used, highly inbred laboratory-derived strains have a greatly reduced, but not entirely redundant capacity for differential pheromone-mediated behaviours.

  Conclusions: Together our results suggest that the unusually variable VR repertoires of mice have a significant role in encoding differences in olfactory-mediated responses and behaviours. Our dataset has expanded over nine fold the known number of mouse VR alleles, and will enable mechanistic analyses into the genetics of innate behavioural differences in mice.

  Funded by: Wellcome Trust: 098051

  BMC genomics 2012;13;415

  PUBMED: 22908939; PMC: 3460788; DOI: 10.1186/1471-2164-13-415

• Further study of chromosome 7p22 to identify the molecular basis of familial hyperaldosteronism type II.

  Carss KJ, Stowasser M, Gordon RD and O'Shaughnessy KM

  Clinical Pharmacology Unit, Department of Medicine, University of Cambridge, Cambridge, UK.

  Familial hyperaldosteronism type II (FH-II) is an inherited form of hyperaldosteronism associated with hypertension in most patients. The mutations that cause FH-II are unknown, but linkage analysis has mapped them to chromosome 7p22. As FH-II is clinically indistinguishable from sporadic primary aldosteronism, a common and treatable condition, unravelling the cause of FH-II has important implications for these sporadic cases. To investigate whether FH-II is caused by large deletions or insertions, we examined the virtual karyotype of four pairs of affected and unaffected individuals using high-density bead chips. We also sequenced the coding regions of five 7p22 candidate genes that were prioritized because of their putative role in cell growth. We found no evidence of single-nucleotide polymorphism (SNP) copy number variation between pairs, and from the widest gap on the chip, chromosome 7p22 deletions or insertions exceeding ∼50 kb in these pedigrees can be excluded. We found 15 SNPs (two of which were novel), but none of them were non-synonymous and segregated with the disease in the FH-II pedigrees. We have been able to exclude large genomic deletions or insertions at 7p22 and refine the candidate gene list for this locus, but the mutations causing FH-II remain elusive.

  Funded by: British Heart Foundation

  Journal of human hypertension 2011;25;9;560-4

  PUBMED: 20927129; DOI: 10.1038/jhh.2010.93

Stephen Clayton

- Bioinformatician

I studied Biochemistry at the University of York and went on to do a MRes in Bioinformatics.

Research

I am developing pipelines and tools for the DDD project.

Sarah Fowler

- Staff Scientist

- 2004-2007 Research Associate, Genome Dynamics and Evolution, Wellcome Trust Sanger Institute.

- 2001-2004 Research Associate, Sequencing Research and Development, Wellcome Trust Sanger Institute.

- 1997-2001 Ph.D Biomolecular Sciences, UMIST, Manchester.

- 1996-1997 MSc Biomolecular Archaeology, UMIST.

- 1995-1996 Technical Assistant, C.elegans genome sequencing project. Wellcome Trust Sanger Institute.

- 1991-1994 B.A Honours in Archaeology and Prehistory, University of Sheffield.

Research

I currently manage and analyse my own research projects in population genetics, sequence analysis, and human variation, with particular reference to mutation dynamics. I also handle sample logistics for the team and support internal and external collaborative projects. This may involve laboratory work, sequence analysis, or scripting and use of statistical and population genetic analysis programs. In addition, I manage the human genetics laboratory, health and safety, and support the laboratory needs of the team.

References

• Genome-wide SNP and CNV analysis identifies common and low-frequency variants associated with severe early-onset obesity.

  Wheeler E, Huang N, Bochukova EG, Keogh JM, Lindsay S, Garg S, Henning E, Blackburn H, Loos RJ, Wareham NJ, O'Rahilly S, Hurles ME, Barroso I and Farooqi IS

  Wellcome Trust Sanger Institute, Cambridge, UK.

  Common and rare variants associated with body mass index (BMI) and obesity account for <5% of the variance in BMI. We performed SNP and copy number variation (CNV) association analyses in 1,509 children with obesity at the extreme tail (>3 s.d. from the mean) of the BMI distribution and 5,380 controls. Evaluation of 29 SNPs (P < 1 × 10(-5)) in an additional 971 severely obese children and 1,990 controls identified 4 new loci associated with severe obesity (LEPR, PRKCH, PACS1 and RMST). A previously reported 43-kb deletion at the NEGR1 locus was significantly associated with severe obesity (P = 6.6 × 10(-7)). However, this signal was entirely driven by a flanking 8-kb deletion; absence of this deletion increased risk for obesity (P = 6.1 × 10(-11)). We found a significant burden of rare, single CNVs in severely obese cases (P < 0.0001). Integrative gene network pathway analysis of rare deletions indicated enrichment of genes affecting G protein-coupled receptors (GPCRs) involved in the neuronal regulation of energy homeostasis.

  Nature genetics 2013;45;5;513-7

  PUBMED: 23563609; DOI: 10.1038/ng.2607

• Variation in genome-wide mutation rates within and between human families.

  Conrad DF, Keebler JE, DePristo MA, Lindsay SJ, Zhang Y, Casals F, Idaghdour Y, Hartl CL, Torroja C, Garimella KV, Zilversmit M, Cartwright R, Rouleau GA, Daly M, Stone EA, Hurles ME, Awadalla P and 1000 Genomes Project

  Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

  J.B.S. Haldane proposed in 1947 that the male germline may be more mutagenic than the female germline. Diverse studies have supported Haldane's contention of a higher average mutation rate in the male germline in a variety of mammals, including humans. Here we present, to our knowledge, the first direct comparative analysis of male and female germline mutation rates from the complete genome sequences of two parent-offspring trios. Through extensive validation, we identified 49 and 35 germline de novo mutations (DNMs) in two trio offspring, as well as 1,586 non-germline DNMs arising either somatically or in the cell lines from which the DNA was derived. Most strikingly, in one family, we observed that 92% of germline DNMs were from the paternal germline, whereas, in contrast, in the other family, 64% of DNMs were from the maternal germline. These observations suggest considerable variation in mutation rates within and between families.

  Funded by: NIGMS NIH HHS: R01 GM070806; Wellcome Trust: 077014, 077014/Z/05/Z

  Nature genetics 2011;43;7;712-4

  PUBMED: 21666693; PMC: 3322360; DOI: 10.1038/ng.862

• Mutation spectrum revealed by breakpoint sequencing of human germline CNVs.

  Conrad DF, Bird C, Blackburne B, Lindsay S, Mamanova L, Lee C, Turner DJ and Hurles ME

  Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

  Precisely characterizing the breakpoints of copy number variants (CNVs) is crucial for assessing their functional impact. However, fewer than 10% of known germline CNVs have been mapped to the single-nucleotide level. We characterized the sequence breakpoints from a dataset of all CNVs detected in three unrelated individuals in previous array-based CNV discovery experiments. We used targeted hybridization-based DNA capture and 454 sequencing to sequence 324 CNV breakpoints, including 315 deletions. We observed two major breakpoint signatures: 70% of the deletion breakpoints have 1-30 bp of microhomology, whereas 33% of deletion breakpoints contain 1-367 bp of inserted sequence. The co-occurrence of microhomology and inserted sequence is low (10%), suggesting that there are at least two different mutational mechanisms. Approximately 5% of the breakpoints represent more complex rearrangements, including local microinversions, suggesting a replication-based strand switching mechanism. Despite a rich literature on DNA repair processes, reconstruction of the molecular events generating each of these mutations is not yet possible.

  Funded by: Wellcome Trust: 077014, 077014/Z/05/Z

  Nature genetics 2010;42;5;385-91

  PUBMED: 20364136; PMC: 3428939; DOI: 10.1038/ng.564

• Ultra-high resolution array painting facilitates breakpoint sequencing.

  Gribble SM, Kalaitzopoulos D, Burford DC, Prigmore E, Selzer RR, Ng BL, Matthews NS, Porter KM, Curley R, Lindsay SJ, Baptista J, Richmond TA and Carter NP

  Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

  Objective: To describe a considerably advanced method of array painting, which allows the rapid, ultra-high resolution mapping of translocation breakpoints such that rearrangement junction fragments can be amplified directly and sequenced.

  Method: Ultra-high resolution array painting involves the hybridisation of probes generated by the amplification of small numbers of flow-sorted derivative chromosomes to oligonucleotide arrays designed to tile breakpoint regions at extremely high resolution.

  How ultra-high resolution array painting of four balanced translocation cases rapidly and efficiently maps breakpoints to a point where junction fragments can be amplified easily and sequenced is demonstrated. With this new development, breakpoints can be mapped using just two array experiments: the first using whole-genome array painting to tiling resolution large insert clone arrays, the second using ultra-high-resolution oligonucleotide arrays targeted to the breakpoint regions. In this way, breakpoints can be mapped and then sequenced in a few weeks.

  Funded by: Wellcome Trust: 077008

  Journal of medical genetics 2007;44;1;51-8

  PUBMED: 16971479; PMC: 2597908; DOI: 10.1136/jmg.2006.044909

• A chromosomal rearrangement hotspot can be identified from population genetic variation and is coincident with a hotspot for allelic recombination.

  Lindsay SJ, Khajavi M, Lupski JR and Hurles ME

  Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom.

  Insights into the origins of structural variation and the mutational mechanisms underlying genomic disorders would be greatly improved by a genomewide map of hotspots of nonallelic homologous recombination (NAHR). Moreover, our understanding of sequence variation within the duplicated sequences that are substrates for NAHR lags far behind that of sequence variation within the single-copy portion of the genome. Perhaps the best-characterized NAHR hotspot lies within the 24-kb-long Charcot-Marie-Tooth disease type 1A (CMT1A)-repeats (REPs) that sponsor deletions and duplications that cause peripheral neuropathies. We investigated structural and sequence diversity within the CMT1A-REPs, both within and between species. We discovered a high frequency of retroelement insertions, accelerated sequence evolution after duplication, extensive paralogous gene conversion, and a greater than twofold enrichment of SNPs in humans relative to the genome average. We identified an allelic recombination hotspot underlying the known NAHR hotspot, which suggests that the two processes are intimately related. Finally, we used our data to develop a novel method for inferring the location of an NAHR hotspot from sequence variation within segmental duplications and applied it to identify a putative NAHR hotspot within the LCR22 repeats that sponsor velocardiofacial syndrome deletions. We propose that a large-scale project to map sequence variation within segmental duplications would reveal a wealth of novel chromosomal-rearrangement hotspots.

  Funded by: Wellcome Trust

  American journal of human genetics 2006;79;5;890-902

  PUBMED: 17033965; PMC: 1698570; DOI: 10.1086/508709

• Shotgun haplotyping: a novel method for surveying allelic sequence variation.

  Lindsay SJ, Bonfield JK and Hurles ME

  Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.

  Haplotypic sequences contain significantly more information than genotypes of genetic markers and are critical for studying disease association and genome evolution. Current methods for obtaining haplotypic sequences require the physical separation of alleles before sequencing, are time consuming and are not scaleable for large surveys of genetic variation. We have developed a novel method for acquiring haplotypic sequences from long PCR products using simple, high-throughput techniques. This method applies modified shotgun sequencing protocols to sequence both alleles concurrently, with read-pair information allowing the two alleles to be separated during sequence assembly. Although the haplotypic sequences can be assembled manually from the resultant data using pre-existing sequence assembly software, we have devised a novel heuristic algorithm to automate assembly and remove human error. We validated the approach on two long PCR products amplified from the human genome and confirmed the accuracy of our sequences against full-length clones of the same alleles. This method presents a simple high-throughput means to obtain full haplotypic sequences potentially up to 20 kb in length and is suitable for surveying genetic variation even in poorly-characterized genomes as it requires no prior information on sequence variation.

  Funded by: Wellcome Trust

  Nucleic acids research 2005;33;18;e152

  PUBMED: 16221968; PMC: 1253838; DOI: 10.1093/nar/gni152

• Finishing the euchromatic sequence of the human genome.

  International Human Genome Sequencing Consortium

  A list of authors and their affiliations appears in the Supplementary Information.

  The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers approximately 99% of the euchromatic genome and is accurate to an error rate of approximately 1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human genome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead.

  Nature 2004;431;7011;931-45

  PUBMED: 15496913; DOI: 10.1038/nature03001

• Genome sequence of the nematode C. elegans: a platform for investigating biology.

  C. elegans Sequencing Consortium

  The Washington University Genome Sequencing Center, Box 8501, 4444 Forest Park Parkway, St. Louis, MO 63108, USA. worm@watson.wustl.edu

  The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms. There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly conserved genes provides evidence for a regional organization of the chromosomes.

  Science (New York, N.Y.) 1998;282;5396;2012-8

  PUBMED: 9851916

Dan King

- PhD Student

I grew up on Long Island, New York, just minutes from Cold Spring Harbor Laboratory. I completed my bachelors degree in Molecular and Cellular Biology at the University of Michigan, in Ann Arbor, Michigan and my medical degree at Wayne State University in Detroit, Michigan. During medical school, I joined a project to develop better treatment strategies for patients with glioblastoma (brain cancer). This experience lead me to the HHMI Research Scholars Program at the National Institutes of Health, where I worked for two years with Leslie Biesecker on exome-based copy number variation analysis before coming to the Sanger.

Research

Working with my Ph.D. supervisor, Matt Hurles, my focus has mainly centered on the Deciphering Developmental Disorders project, a large study of rare disease patient trios. Specifically, I developed a software tool, UPDio, which can identify probands of trios with uniparental disomy events. In addition, I'm interested in large-scale mosaic events, such as large UPD and CNVs that affect only a subset of tissues in an individual and have been actively working to detect these events from SNP and exome data. Lastly, I'm is interested in using RNAseq data to assist in highlighting causative genetic variation.

References

• Inhibition of HSP27 alone or in combination with pAKT inhibition as therapeutic approaches to target SPARC-induced glioma cell survival.

  Schultz CR, Golembieski WA, King DA, Brown SL, Brodie C and Rempel SA

  The Barbara Jane Levy Laboratory of Molecular Neuro-Oncology, Henry Ford Hospital, Detroit, MI 48202, USA.

  Background: The current treatment regimen for glioma patients is surgery, followed by radiation therapy plus temozolomide (TMZ), followed by 6 months of adjuvant TMZ. Despite this aggressive treatment regimen, the overall survival of all surgically treated GBM patients remains dismal, and additional or different therapies are required. Depending on the cancer type, SPARC has been proposed both as a therapeutic target and as a therapeutic agent. In glioma, SPARC promotes invasion via upregulation of the p38 MAPK/MAPKAPK2/HSP27 signaling pathway, and promotes tumor cell survival by upregulating pAKT. As HSP27 and AKT interact to regulate the activity of each other, we determined whether inhibition of HSP27 was better than targeting SPARC as a therapeutic approach to inhibit both SPARC-induced glioma cell invasion and survival.

  Results: Our studies found the following. 1) SPARC increases the expression of tumor cell pro-survival and pro-death protein signaling in balance, and, as a net result, tumor cell survival remains unchanged. 2) Suppressing SPARC increases tumor cell survival, indicating it is not a good therapeutic target. 3) Suppressing HSP27 decreases tumor cell survival in all gliomas, but is more effective in SPARC-expressing tumor cells due to the removal of HSP27 inhibition of SPARC-induced pro-apoptotic signaling. 4) Suppressing total AKT1/2 paradoxically enhanced tumor cell survival, indicating that AKT1 or 2 are poor therapeutic targets. 5) However, inhibiting pAKT suppresses tumor cell survival. 6) Inhibiting both HSP27 and pAKT synergistically decreases tumor cell survival. 7) There appears to be a complex feedback system between SPARC, HSP27, and AKT. 8) This interaction is likely influenced by PTEN status. With respect to chemosensitization, we found the following. 1) SPARC enhances pro-apoptotic signaling in cells exposed to TMZ. 2) Despite this enhanced signaling, SPARC protects cells against TMZ. 3) This protection can be reduced by inhibiting pAKT. 4) Combined inhibition of HSP27 and pAKT is more effective than TMZ treatment alone.

  Conclusions: We conclude that inhibition of HSP27 alone, or in combination with pAKT inhibitor IV, may be an effective therapeutic approach to inhibit SPARC-induced glioma cell invasion and survival in SPARC-positive/PTEN-wildtype and SPARC-positive/PTEN-null tumors, respectively.

  Funded by: NCI NIH HHS: CA138401, CA86997, R01 CA086997, R01 CA138401

  Molecular cancer 2012;11;20

  PUBMED: 22480225; PMC: 3349587; DOI: 10.1186/1476-4598-11-20

Raheleh Rahbari

- Postdoctoral Fellow

- Postdoctoral Research Fellow, January 2013 – Present , Wellcome Trust Sanger Institute Investigating the germline whole genome mutation rate

- Postdoctoral Research Associate, June 2012 – January 2013, University of Leicester Investigating the role of copy number variation in autoimmune disease

- Bioinformatics, October 2011- April 2012, University of Leicester Data identification import and curation for the GWAS cetral database

- PhD research, October 2007 – September 2011, University of Leicester Investigating the endogenous activity of L1 retrotransposons during human embryogenesis, by estimating the rate of de novo L1 retrotransposition in early human development

Research

One of the aim of our team is to characterise the fundamental mutational mechanisms that underpin genetic variation. My project aims to estimate the effect of age and sex on mutation rates by identifying de novo mutations in children that are not found in either parent, ascribing these mutations to the paternal or maternal germline, and investigating whether the numbers of paternal or maternal germline mutations changes with increasing parental age. The outcome will help us to control for potential genetic or environmental influences on mutation rate and maximise our power to detect the influence of age on mutation rate.

References

• Transduction-Specific ATLAS Reveals a Cohort of Highly Active L1 Retrotransposons in Human Populations.

  Macfarlane CM, Collier P, Rahbari R, Beck CR, Wagstaff JF, Igoe S, Moran JV and Badge RM

  Department of Genetics,, University of Leicester, University Road, Leicester, UK.

  Long INterspersed Element-1 (LINE-1 or L1) retrotransposons are the only autonomously active transposable elements in the human genome. The average human genome contains ∼80-100 active L1s, but only a subset of these L1s are highly active or 'hot'. Human L1s are closely related in sequence, making it difficult to decipher progenitor/offspring relationships using traditional phylogenetic methods. However, L1 mRNAs can sometimes bypass their own polyadenylation signal and instead utilize fortuitous polyadenylation signals in 3' flanking genomic DNA. Retrotransposition of the resultant mRNAs then results in lineage specific sequence "tags" (i.e., 3' transductions) that mark the descendants of active L1 progenitors. Here, we developed a method (Transduction-Specific Amplification Typing of L1 Active Subfamilies or TS-ATLAS) that exploits L1 3' transductions to identify active L1 lineages in a genome-wide context. TS-ATLAS enabled the characterization of a putative active progenitor of one L1 lineage that includes the disease causing L1 insertion L1RP , and the identification of new retrotransposition events within two other "hot" L1 lineages. Intriguingly, the analysis of the newly discovered transduction lineage members suggests that L1 polyadenylation, even within a lineage, is highly stochastic. Thus, TS-ATLAS provides a new tool to explore the dynamics of L1 lineage evolution and retrotransposon biology.

  Human mutation 2013

  PUBMED: 23553801; DOI: 10.1002/humu.22327

• IAP display: a simple method to identify mouse strain specific IAP insertions.

  Ray A, Rahbari R and Badge RM

  Department of Genetics, University of Leicester, University Road, Leicester LE17RH, UK.

  Intracisternal A-type particle (IAP) elements are high copy number long terminal repeat (LTR) rodent retrotransposons. Some IAP elements can transpose, and are responsible for ~12% of spontaneous mouse mutations. Inbred mouse strains show variation in genomic IAP distribution, contributing to inter-strain genetic variability. Additionally IAP elements can influence the transcriptional regulation of neighbouring genes through their strong LTR promoter, effecting phenotypic variation. This genetic and phenotypic variability can translate into experimental variability between mouse strains. For example, it has been demonstrated that strain-specific genetic/epigenetic factors can interact to yield variable responses to drugs. Therefore, in experimental contexts it is essential to unequivocally identify mouse strains. Recently it was estimated that any two inbred strains share only ~40% of their IAP insertions. Of the remaining 60%, some insertions will be strain specific, fixed during inbreeding. These fixed insertions can be exploited as genetic markers to identify inbred strains, if they can be identified simply and efficiently. Here, we report the development of a PCR-based system allowing direct acquisition of strain-specific IAP insertions. In a pilot study, we identified 21 IAP loci, genotyped IAP insertions at 9 loci, and discovered two strain-specific insertions that could reliably identify these strains.

  Funded by: Wellcome Trust: 075163, 075163/Z/04/Z

  Molecular biotechnology 2011;47;3;243-52

  PUBMED: 20872285; PMC: 3032225; DOI: 10.1007/s12033-010-9338-6

• A novel L1 retrotransposon marker for HeLa cell line identification.

  Rahbari R, Sheahan T, Modes V, Collier P, Macfarlane C and Badge RM

  Department of Genetics, University of Leicester, Leicester, UK.

  The HeLa cell line is the oldest, most widely distributed, permanent human cell line. As a nearly ubiquitous inhabitant of laboratories using tissue culture techniques, its aggressive growth characteristics make it a problematic contaminant that can overgrow less robust cell lines. Consequently, HeLa contamination is common in both the research laboratory and cell line repository contexts, and its detection is hampered by the lack of a rapid, sensitive and robust assay. Here we report the development of a HeLa-specific DNA diagnostic test: a single duplex detection PCR assay targeting an L1 retrotransposon insertion. All HeLa clones from a geographically diverse panel were positive by this assay, and the particular L1 insertion we identified appears to be unique to the HeLa cell line. The assay can detect very low levels of HeLa contamination (<1%), and can be performed on un-purified cell pellets, allowing rapid routine screening.

  Funded by: Wellcome Trust: 075163, 075163/Z/04/Z

  BioTechniques 2009;46;4;277-84

  PUBMED: 19450234; PMC: 2696096; DOI: 10.2144/000113089

Art Wuster

- Postdoctoral Fellow

Art has a PhD in Computational Biology from the MRC Laboratory of Molecular Biology in Cambridge and a BSc in Biology from Sussex University. Before joining the Sanger Institute, Art worked at an international firm of management consultants.

Research

Art develops methods to identify mutations that cause genetic disorders.

References

• Cross-species chemogenomic profiling reveals evolutionarily conserved drug mode of action.

  Kapitzky L, Beltrao P, Berens TJ, Gassner N, Zhou C, Wüster A, Wu J, Babu MM, Elledge SJ, Toczyski D, Lokey RS and Krogan NJ

  Department of Cellular and Molecular Pharmacology, QB3 Institute, University of California, San Francisco, CA 94158, USA.

  We present a cross-species chemogenomic screening platform using libraries of haploid deletion mutants from two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. We screened a set of compounds of known and unknown mode of action (MoA) and derived quantitative drug scores (or D-scores), identifying mutants that are either sensitive or resistant to particular compounds. We found that compound-functional module relationships are more conserved than individual compound-gene interactions between these two species. Furthermore, we observed that combining data from both species allows for more accurate prediction of MoA. Finally, using this platform, we identified a novel small molecule that acts as a DNA damaging agent and demonstrate that its MoA is conserved in human cells.

  Funded by: NIGMS NIH HHS: P50 GM081879, R01 GM084279-04, R01 GM084448-04, R01 GM084448-05

  Molecular systems biology 2010;6;451

  PUBMED: 21179023; PMC: 3018166; DOI: 10.1038/msb.2010.107

• Spial: analysis of subtype-specific features in multiple sequence alignments of proteins.

  Wuster A, Venkatakrishnan AJ, Schertler GF and Babu MM

  Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge CB2 1TP, UK.

  MOTIVATION: Spial (Specificity in alignments) is a tool for the comparative analysis of two alignments of evolutionarily related sequences that differ in their function, such as two receptor subtypes. It highlights functionally important residues that are either specific to one of the two alignments or conserved across both alignments. It permits visualization of this information in three complementary ways: by colour-coding alignment positions, by sequence logos and optionally by colour-coding the residues of a protein structure provided by the user. This can aid in the detection of residues that are involved in the subtype-specific interaction with a ligand, other proteins or nucleic acids. Spial may also be used to detect residues that may be post-translationally modified in one of the two sets of sequences. AVAILABILITY: http://www.mrc-lmb.cam.ac.uk/genomes/spial/; supplementary information is available at http://www.mrc-lmb.cam.ac.uk/genomes/spial/help.html.

  Funded by: Medical Research Council

  Bioinformatics (Oxford, England) 2010;26;22;2906-7

  PUBMED: 20880955; PMC: 2971580; DOI: 10.1093/bioinformatics/btq552

• Transcriptional control of the quorum sensing response in yeast.

  Wuster A and Babu MM

  MRC Laboratory of Molecular Biology, Cambridge, UK. awuster@mrc-lmb.cam.ac.uk

  Quorum sensing is a process of intercellular communication. It allows individual cells to assess population density and to co-ordinate behaviour by secreting and sensing communication molecules. In the yeast Saccharomyces cerevisiae, the communication molecules are the aromatic alcohols tryptophol and phenylethanol, and quorum sensing regulates the transition between the solitary yeast form and the filamentous form. Though it is known that addition of these communication molecules to yeast cultures causes large changes in gene expression, how these changes are orchestrated and whether this system is conserved in related fungal species is still unknown. In this work, by employing an integrated computational approach that makes use of large-scale genomics datasets, such as ChIP-ChIP and expression analysis upon deletion and over-expression of transcriptional factors, we predict CAT8 and MIG1 as key transcriptional regulators that control the differential expression of the genes affected by aromatic alcohol communication. In addition, through a comparative genomic analysis involving 31 fungal species, we show that the S. cerevisiae quorum sensing system is a recent evolutionary innovation and that the genes which are differentially expressed upon treatment with these molecules are distributed across the genome in a highly non-random manner. The identified transcription factors will aid in further unravelling the molecular mechanisms of S. cerevisiae quorum sensing and may facilitate the engineering of regulatory circuits for applications such as the expression of heterologous proteins via aromatic alcohols.

  Funded by: Medical Research Council

  Molecular bioSystems 2010;6;1;134-41

  PUBMED: 20024075; DOI: 10.1039/b913579k

• Genomic analysis reveals a tight link between transcription factor dynamics and regulatory network architecture.

  Jothi R, Balaji S, Wuster A, Grochow JA, Gsponer J, Przytycka TM, Aravind L and Babu MM

  Biostatistics Branch, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC 27709, USA. jothi@mail.nih.gov

  Although several studies have provided important insights into the general principles of biological networks, the link between network organization and the genome-scale dynamics of the underlying entities (genes, mRNAs, and proteins) and its role in systems behavior remain unclear. Here we show that transcription factor (TF) dynamics and regulatory network organization are tightly linked. By classifying TFs in the yeast regulatory network into three hierarchical layers (top, core, and bottom) and integrating diverse genome-scale datasets, we find that the TFs have static and dynamic properties that are similar within a layer and different across layers. At the protein level, the top-layer TFs are relatively abundant, long-lived, and noisy compared with the core- and bottom-layer TFs. Although variability in expression of top-layer TFs might confer a selective advantage, as this permits at least some members in a clonal cell population to initiate a response to changing conditions, tight regulation of the core- and bottom-layer TFs may minimize noise propagation and ensure fidelity in regulation. We propose that the interplay between network organization and TF dynamics could permit differential utilization of the same underlying network by distinct members of a clonal cell population.

  Funded by: Medical Research Council

  Molecular systems biology 2009;5;294

  PUBMED: 19690563; PMC: 2736650; DOI: 10.1038/msb.2009.52

• Chemogenomics and biotechnology.

  Wuster A and Madan Babu M

  Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. awuster@mrc-lmb.cam.ac.uk

  A robust knowledge of the interactions between small molecules and specific proteins aids the development of new biotechnological tools and the identification of new drug targets, and can lead to specific biological insights. Such knowledge can be obtained through chemogenomic screens. In these screens, each small molecule from a chemical library is applied to each cell type from a library of cells, and the resulting phenotypes are recorded. Chemogenomic screens have recently become very common and will continue to generate large amounts of data. The interpretation of this data will occupy biologists and chemists alike for some time to come. This review discusses methods for the acquisition and interpretation of chemogenomic data, in addition to possible applications of chemogenomics in biotechnology.

  Trends in biotechnology 2008;26;5;252-8

  PUBMED: 18346803; DOI: 10.1016/j.tibtech.2008.01.004

• Conservation and evolutionary dynamics of the agr cell-to-cell communication system across firmicutes.

  Wuster A and Babu MM

  MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, United Kingdom. awuster@mrc-lmb.cam.ac.uk

  We present evidence that the agr cell-to-cell communication system is present across firmicutes, including the human pathogen Clostridium perfringens. Although we find that the agr system is evolutionarily conserved and that the general functions which it regulates are similar in different species, the individual regulated genes are not the same. This suggests that the regulatory network controlled by agr is dynamic and evolves rapidly.

  Journal of bacteriology 2008;190;2;743-6

  PUBMED: 17933897; PMC: 2223712; DOI: 10.1128/JB.01135-07

Margriet van Kogelenberg

mvk1@sanger.ac.uk Postdoctoral Fellow

In 2004 Margriet obtained a Bachelors degree in Molecular Biology from the Institute of Life Sciences and Chemistry, Utrecht, the Netherlands which was followed by a Masters degree in Oncology from the VU University Amsterdam, the Netherlands in 2006. Margriet undertook a PhD in clinical genetics under supervision of Professor Stephen Robertson at the University of Otago, New Zealand, which was successfully completed in 2010.

Research

In 2011 Margriet started as a Postdoctoral Fellow at the Wellcome Trust Sanger Institute where she works on the Deciphering Developmental Disorders (DDD) project. This study aims to delineate the genetic architecture in children with undiagnosed severe developmental disorders and employs both array and sequencing technologies to enhance the detection rate of rare causative mutations and to gain a holistic view of the genomic architecture in developmental disorders. Margriet is primarily focussed on the exome sequence analysis.

References

• A novel Xp22.11 deletion causing a syndrome of craniosynostosis and periventricular nodular heterotopia.

  van Kogelenberg M, Lerone M, De Toni T, Divizia MT, de Brouwer AP, Veltman JA, van Bokhoven H and Robertson SP

  Department of Paediatrics and Child Health, Dunedin School of Medicine, Otago University, Dunedin, New Zealand.

  We report on a follow-up evaluation of a male with a phenotype including craniosynostosis, periventricular nodular heterotopia, and neurodevelopmental delay. He was initially assigned a clinical diagnosis of Fontaine-Farriaux syndrome (FFS) as an infant although now, with improved delineation of this entity, it is evident that this diagnosis is not applicable to this individual. Array comparative genomic hybridization has demonstrated a 300  kb interstitial deletion on Xp22.11 affecting all or part of three annotated genes, ZFX, PDK3, and PCYT1B in this subject. The deletion was inherited from the phenotypically normal mother who also exhibited markedly skewed X-inactivation. These findings implicate hemizygosity for one or all three of these genes as the cause of this phenotype.

  American journal of medical genetics. Part A 2011;155A;12;3144-7

  PUBMED: 22052819; DOI: 10.1002/ajmg.a.34311

• Periventricular heterotopia in common microdeletion syndromes.

  van Kogelenberg M, Ghedia S, McGillivray G, Bruno D, Leventer R, Macdermot K, Nelson J, Nagarajan L, Veltman JA, de Brouwer AP, McKinlay Gardner RJ, van Bokhoven H, Kirk EP and Robertson SP

  Department of Paediatrics and Child Health, Dunedin School of Medicine, Otago University, Dunedin, New Zealand.

  Periventricular heterotopia (PH) is a brain malformation characterised by heterotopic nodules of neurons lining the walls of the cerebral ventricles. Mutations in FLNA account for 20-24% of instances but a majority have no identifiable genetic aetiology. Often the co-occurrence of PH with a chromosomal anomaly is used to infer a new locus for a Mendelian form of PH. This study reports four PH patients with three different microdeletion syndromes, each characterised by high-resolution genomic microarray. In three patients the deletions at 1p36 and 22q11 are conventional in size, whilst a fourth child had a deletion at 7q11.23 that was larger in extent than is typically seen in Williams syndrome. Although some instances of PH associated with chromosomal deletions could be attributed to the unmasking of a recessive allele or be indicative of more prevalent subclinical migrational anomalies, the rarity of PH in these three microdeletion syndromes and the description of other non-recurrent chromosomal defects do suggest that PH may be a manifestation of multiple different forms of chromosomal imbalance. In many, but possibly not all, instances the co-occurrence of PH with a chromosomal deletion is not necessarily indicative of uncharacterised underlying monogenic loci for this particular neuronal migrational anomaly.

  Molecular syndromology 2010;1;1;35-41

  PUBMED: 20648244; PMC: 2883850; DOI: 10.1159/000274491

• Germline mutations in WTX cause a sclerosing skeletal dysplasia but do not predispose to tumorigenesis.

  Jenkins ZA, van Kogelenberg M, Morgan T, Jeffs A, Fukuzawa R, Pearl E, Thaller C, Hing AV, Porteous ME, Garcia-Miñaur S, Bohring A, Lacombe D, Stewart F, Fiskerstrand T, Bindoff L, Berland S, Adès LC, Tchan M, David A, Wilson LC, Hennekam RC, Donnai D, Mansour S, Cormier-Daire V and Robertson SP

  Departments of Paediatrics, Dunedin School of Medicine, Otago University, Dunedin 9054, New Zealand.

  Abnormalities in WNT signaling are implicated in a broad range of developmental anomalies and also in tumorigenesis. Here we demonstrate that germline mutations in WTX (FAM123B), a gene that encodes a repressor of canonical WNT signaling, cause an X-linked sclerosing bone dysplasia, osteopathia striata congenita with cranial sclerosis (OSCS; MIM300373). This condition is typically characterized by increased bone density and craniofacial malformations in females and lethality in males. The mouse homolog of WTX is expressed in the fetal skeleton, and alternative splicing implicates plasma membrane localization of WTX as a factor associated with survival in males with OSCS. WTX has also been shown to be somatically inactivated in 11-29% of cases of Wilms tumor. Despite being germline for such mutations, individuals with OSCS are not predisposed to tumor development. The observed phenotypic discordance dependent upon whether a mutation is germline or occurs somatically suggests the existence of temporal or spatial constraints on the action of WTX during tumorigenesis.

  Nature genetics 2009;41;1;95-100

  PUBMED: 19079258; DOI: 10.1038/ng.270