Mutation and recombination in the human genome

Lead Research Organisation: University of Leicester
Department Name: Genetics

Abstract

All normal and pathological variation in human DNA arises from two processes that operate when DNA is transmitted from parent to child. The first is mutation, causing changes in the sequence of DNA either at a single base or at the larger scale of deletion or insertion of pieces of DNA. The second is recombination between chromosomes, a process operating during egg and sperm production that reshuffles DNA diversity into the endless new combinations seen in human populations. Recombination can also cause mutation when chromosomes pair up incorrectly, leading to duplications and deletions which often have disease consequences. Thanks to genomics programmes, we know a lot about patterns of human DNA diversity. In contrast, we still understand little about the dynamics and processes of mutation and recombination, and how resulting genetic changes are moulded, by for example natural selection, into the patterns of diversity seen in human populations. We are addressing this problem by analysing single DNA molecules, particularly in sperm, to gain direct insights into the types and rates of change going on within our DNA. We will look at DNA as it goes through the recombination process to learn more about why recombination events occur where they do in human chromosomes, what happens to DNA during recombination, and whether there is a link between recombination and mutation. We will also look at how recombination can lead to changes in gene copy number in human DNA, with particular focus on very common types of mutation that result in inherited anaemia. We are also developing new methods for investigating how pieces of DNA can be spontaneously lost, how jumping ?parasitic? DNA moves around the human genome, and how fundamental changes to the DNA sequence itself can arise. This ambitious research will help to understand the basis of human genetic variation and how pathological changes arise in our DNA, with potential applied aspects ranging from assisting in genetic counselling, through identifying factors that predispose to infertility and chromosome abnormalities, to producing new methods for identifying environmental factors that might influence human mutation. This work will interface with other research on human DNA instability in the Leicester MRC Co-operative Group on Human Genetics, and will be presented to lay audiences through a programme of talks to the media, schools and the general public, where we use aspects of our work to inform on basic issues in human genetics.

Technical Summary

Current descriptive databases of sequence and structural variation in the human genome are of major importance in facilitating genetic analysis, and provide a view of human genetic diversity complementary to that obtained through descriptions of pathological changes in human DNA. What is lacking is an understanding of the underlying dynamic processes operating in the human germline that create this variation. We will address this by direct mechanistic analyses, using single DNA molecule methods largely pioneered in our laboratory to access rare events. In a continuation of work currently funded by the MRC, we will extend our exploration of meiotic recombination. Specific goals include the characterisation of the most intense recombination hotspots in the human genome and their use to investigate recombination initiation and processing, to test whether recombination is mutagenic, and to identify DNA sequence and epigenetic factors that control hotspot activity and influence recombination outcome. This work will be extended to investigating ectopic recombination between repeated DNA sequences, with particular focus on unequal exchange operating within gene families, a major driver of pathological DNA rearrangements. Specific issues include the balance between mitotic and meiotic recombination, the rules governing duplication and deletion, the identification of elements that control exchange and the relationship between allelic and ectopic recombination. Information on germline dynamics will be used to explore levels of selection operating within human populations on loci that show copy number variation. We will also develop new approaches to investigate directly the dynamics and processes of spontaneous deletion in the human germline, to characterise de novo transposition events in human sperm, and most challengingly to detect de novo base substitutions and to characterise factors such as germline lineage structure and germcell selection that can influence mutation load. This work will generate new methods for monitoring de novo heritable mutation and will yield fundamental insights into sequence turnover in the human germline and the origin of pathological variants. Without this knowledge, we will never fully understand human DNA variation nor forces such as selection that operate at the population level to create patterns of contemporary human DNA diversity. This fundamental research programme lies at the heart of our current MRC Co-operative Group on Variability, Instability and Pathology of the Human Genome, and heavily interfaces with other research at Leicester involved in exploring human genome dynamics, diversity and disease.

Publications

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Berg IL (2011) Variants of the protein PRDM9 differentially regulate a set of human meiotic recombination hotspots highly active in African populations. in Proceedings of the National Academy of Sciences of the United States of America

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Jeffreys AJ (2013) Recombination regulator PRDM9 influences the instability of its own coding sequence in humans. in Proceedings of the National Academy of Sciences of the United States of America

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Jeffreys AJ (2009) The rise and fall of a human recombination hot spot. in Nature genetics

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Kauppi L (2009) Analysis of meiotic recombination products from human sperm. in Methods in molecular biology (Clifton, N.J.)

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Neumann R (2010) Dynamics and processes of copy number instability in human  -globin genes in Proceedings of the National Academy of Sciences

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Webb AJ (2008) Sperm cross-over activity in regions of the human genome showing extreme breakdown of marker association. in Proceedings of the National Academy of Sciences of the United States of America

 
Description Home Affairs Committee Review on Forensic DNA
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
Impact Highlighted issues concerning the National DNA Database, and in particular the retention of information from innocent individuals. Government has introduced new legislation to address this problem, with the result that innocents are now being removed from NDNAD.
 
Description House of Commons Science and Technology Committee, review of FSS closure
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
Impact Provided oral and written evidence concerning the likely impact of closure of the Forensic Science Service on UK forensic research and the provision of forensic services. The Committee's report highlighted my concerns about the parlous state of forensic research funding in the UK and made recommendations for addressing this issue.
 
Description SAIS committee, Royal Society
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in advisory committee
 
Description Wellcome Trust Technology Development Grant (A novel approach for the analysis of de novo point mutations in the human FGFR3 gene)
Amount £212,120 (GBP)
Funding ID 087707/Z/08/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2009 
End 10/2013
 
Title PRDM9 variants 
Description We have substantially extended the catalogue of zinc finger array coding variants found in the human PRDM9 gene. Also, ten different variants have been engineered for expression in E. coli and have been placed in long-term storage for future open use. 
Type Of Material Biological samples 
Year Produced 2010 
Provided To Others? Yes  
Impact Data on these variants are fully available through our open-access publications, and have already proved to be of great use to researchers exploring the extraordinary impact of PRDM9 variation on recombination and the generation of rearrangements in human chromosomes, and for defining those pathological genomic rearrangements for which PDRM9 variants are a major risk factor. 
 
Title Super-hotspots 
Description Details of extremely active human meiotic recombination hotspots identified by my group, with details of genotype data and sperm crossover assay conditions, all freely available on my website. 
Type Of Material Biological samples 
Year Produced 2008 
Provided To Others? Yes  
Impact These data are being used by bioinformatics experts interested in exploring the relationship between haplotype diversity and recombination. They also proved crucial in our analysis of the effects of PRDM9 variation on recombination hotspot activity, work we published in Nature Genetics in 2010 and in PNAS in 2011. 
 
Title de novo copy number variation 
Description New single molecule DNA techniques capable of recovering rare spontaneous duplications and deletions from bulk human genomic DNA, fully available in our open-access publications. 
Type Of Material Technology assay or reagent 
Year Produced 2007 
Provided To Others? Yes  
Impact Our unexpected direct detection of de novo base substitutions in human sperm; this was a major novel finding which we published in PNAS in 2010. 
 
Description Cis-regulation of hotspots 
Organisation University of Oxford
Department Department of Statistics
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration with bioinformatics expert to identify candidate recombination hotspots suitable for analysing cis-regulatory elements, with experimental work being conducted in my lab.
Collaborator Contribution Bioinformatics expertise.
Impact Strong evidence that hotspot-associated sequence motifs control recombination initiation.
Start Year 2007
 
Description Trans regulators of human meiotic recombination 
Organisation University of Montpellier
Country France 
Sector Academic/University 
PI Contribution This collaboration brought together my expertise in recombination and mutation detection at the single DNA molecule level with an expert in zinc finger proteins and minisatellite mutation, and was aimed at exploring mutation processes in a locus encoding a major trans regulator of meiotic recombination and genome instability in humans.
Collaborator Contribution This was initially a full collaboration involving the exchange of reagents and expertise. Sadly, our French collaborator became seriously ill and the collaboration had to be terminated, though the research was subsequently pursued successfully in my laboratory.
Impact This interdisciplinary collaboration laid the foundations for a subsequent study in my lab that revealed major new aspects of zinc finger evolution and how zinc finger proteins can influence the evolution of their own coding loci. This work has been submitted for publication.
Start Year 2010
 
Description Awards and award lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Ostler Lecture, Charter Lecture, Royal Society Croonian Prize Lecture, Jean Shanks Lecture to the Academy of Medical Sciences, Edinburgh Medal Lecture, ABRF Annual Award Plenary Lecture; all on various general issues of genome diversity, identification and DNA instability. Also awarded the Edward O. Wilson Biodiversity Technology Pioneer Award and the AstraZeneca Award from the Biochemical Society, and received the Excellence Award, Indian Congress of Forensic Medicine and Toxicology. Elected Fellow of the Royal Society of Chemistry and received Honorary Doctorates from the University of Hong Kong, De Montfort University and Wolverhampton University.

All attracted significant press coverage.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2012
 
Description Congress presentations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Keynote presentation at Promega DNA identification congress, Hollywood and 10th Indo-Pacific Congress on Legal Medicine and Forensic Science, Delhi. Presentations at ESHG meeting, Barcelona, and at WTSI meeting on genome dynamics. Participation in First British Meiosis Meeting, Sussex and keynote lecturer at the Second British Meiosis Meeting, Leicester. Plenary lecture to the European Society of Human Genetics, Gothenburg, and to the INPALMS Congress, Delhi.

Useful interactions at Barcelona meeting leading to collaboration.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2012
 
Description Desert Island Discs 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Guest on Radio 4's Desert Island Discs.

Received tremendous positive feedback from the listening public, generally along the lines of "good to hear about a scientist who is also a human being"!
Year(s) Of Engagement Activity 2007
 
Description Institutional/departmental seminars 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Thirteen seminars in UK, Germany, Hong Kong and India addressing our latest research on genome instability.

Useful press briefings after Hong Kong and India talks.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2012
 
Description Official openings 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Officially opened:

The Forensic Submissions Unit at Leicestershire Constabulary Headquarters

Gartree High School in Oadby, Leicester

Soar Valley College in Leicester

Luton 6th form College

New science labs at Leicester High School for Girls

The Sir Alec Jeffreys Scientific Support Building, West Yorkshire Police, Wakefield.

Considerable press interest in all events.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012
 
Description Public lectures 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact 54 public lectures in the UK, Cyprus, Finland, Germany, Hong Kong, India, Netherlands and Hungary, including to New Scotland Yard and the University of the Third Age. Audiences ranged from the general public to schoolchildren, postgraduates, police, lawyers, clergy, industrial research managers, and senior executives at Nokia Research, Helsinki.

Several of these lectures attracted media interest and were duly covered by the radio/TV/press.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011,2012