Dispersed repeat elements contribute to genome instability by de novo insertion and unequal recombination between repeats. To study the dynamics of these processes, we have developed single DNA molecule approaches to detect de novo insertions at a single locus and Alu-mediated deletions at two different loci in human genomic DNA. Validation experiments showed these approaches could detect insertions and deletions at frequencies below 10(-6) per cell. However, bulk analysis of germline (sperm) and somatic DNA showed no evidence for genuine mutant molecules, placing an upper limit of insertion and deletion rates of 2 x 10(-7) and 3 x 10(-7), respectively, in the individuals tested. Such re-arrangements at these loci therefore occur at a rate lower than that detectable by the most sensitive methods currently available

AJ Jeffreys

AR Jeffs

BF Koop

D Stoppa-Lyonnet

F Rouyer

HH Kazazian

HH: Kazazian

J Murray

J: Jurka

M: Kimura

PE Carter

PL Deininger

Y Miki

Z Wong

English

Dispersed repeat elements contribute to
								genome instability by de novo insertion
								and unequal recombination between
								repeats. To study the dynamics of these
								processes, we have developed single DNA
								molecule approaches to detect de novo
								insertions at a single locus and
								Alu-mediated deletions at two different
								loci in human genomic DNA. Validation
								experiments showed these approaches
								could detect insertions and deletions at
								frequencies below 10(-6) per cell.
								However, bulk analysis of germline
								(sperm) and somatic DNA showed no
								evidence for genuine mutant molecules,
								placing an upper limit of insertion and
								deletion rates of 2 x 10(-7) and 3 x
								10(-7), respectively, in the individuals
								tested. Such re-arrangements at these
								loci therefore occur at a rate lower
								than that detectable by the most
								sensitive methods currently available.Peer Reviewe

Hollies, C.R.

Monckton, D.G.

Jeffreys, A.J.

Repositorio da Universidade da Coruña

Attempts to detect retrotransposition
								and de novo deletion of Alus and other
								dispersed repeats at specific loci in
								the human genome

Enlighten

SHORT REPORTAttempts to detect retrotransposition and de novodeletion of Alus and other dispersed repeats at specificloci in the human genomeCaroline R Hollies1, Darren G Monckton2 and Alec J Jeffreys*,31Department of Pathology, University of Wales College of Medicine, Wales, UK; 2Division of Molecular Genetics,University of Glasgow, Glasgow, Scotland, UK; 3Department of Genetics, University of Leicester, Leicester, UKDispersed repeat elements contribute to genome instability by de novo insertion and unequal recombinationbetween repeats. To study the dynamics of these processes, we have developed single DNA moleculeapproaches to detect de novo insertions at a single locus and Alu-mediated deletions at two different loci inhuman genomic DNA. Validation experiments showed these approaches could detect insertions anddeletions at frequencies below 1076 per cell. However, bulk analysis of germline (sperm) and somatic DNAshowed no evidence for genuine mutant molecules, placing an upper limit of insertion and deletion rates of261077 and 361077, respectively, in the individuals tested. Such re-arrangements at these loci thereforeoccur at a rate lower than that detectable by the most sensitive methods currently available. European Journalof Human Genetics (2001) 9, 143 – 146.Keywords: Alu; deletion; dispersed repeats; insertion; recombination; retropositionIntroductionHuman retroposons, in particular Alu and L1 elements, play amajor role in genome remodelling, both by de novo insertionand by promoting deletions and re-duplications as a result ofunequal recombination between dispersed repeat elements.1Mechanisms of re-arrangement, particularly in retroposition,are fairly well established;2 in contrast, little is known aboutthe dynamics of these events. De novo insertions are usuallyof complete Alu and partial L1 elements,1,2 while unequalexchanges occur mainly between Alu elements.3 – 5 Bothtypes of event are thought to occur mainly in the germline,2though examples of both have been observed in somatictissues.3,6 There is evidence that at least some unequalexchange events may be due to meiotic recombination.4 Todate, such insertions and re-arrangements have only beencharacterised in patients, resulting in considerable uncer-tainties about the nature of these processes. We havetherefore developed highly sensitive methods for mutationdetection, analogous to methods devised for studying de novomutation at highly unstable minisatellites,7 and have usedthese in an attempt to detect dispersed repeat-mediatedinstability in somatic and germline DNA at three differentloci.MethodsPCR amplificationPrimers used were: MS1-1, 5’-GCT-GCT-GTG-CCT-TCC-CCG-GTT-C-3’; MS1-2, 5’-ACC-CAC-TGA-CTC-ACA-GCA-AGG-G-3’; MS1-3 5’-ggc-gga-tcc-cgg-gaa-TTC-AGT-CTC-CTA-GTG-AAG-GTG-C-3’; MS1-4, 5’-gcg-aat-tcg-gat-ccc-GGT-GAT-TTC-TCA-ATT-TGC-TGG-3’ (lowercase – extensions incorpor-ating restriction endonuclease sites for subsequent cloning):32-5F, 5’-CAG-TGC-TTG-GCA-CAT-AAT-GAG-CAC-3’; 32-5NF, 5’-CTC-TTT-CTA-GAA-GCC-GTT-AGA-GGA-G-3’; 32-5R, 5’-GAA-TCC-TAC-ATG-TAG-GCG-AGC-AGT-3’; 32-1.6F,5’-GCC-AAC-AGT-GTA-CTT-TGA-AGA-GCA-3’; 32-1.3NR, 5’-GCA-GGT-AGA-TAG-TGG-CCC-AGA-G-3’; 32-1.3R, 5’-CCA-GGT-TCT-GGG-GTG-ACT-AG-3’: C1+2.4F, 5’-CCC-AAC-AGA-TTC-TCC-TAC-CC-3’; C1+2.4NF 5’-GCC-CAC-TAC-European Journal of Human Genetics (2001) 9, 143 – 146ª 2001 Nature Publishing Group All rights reserved 1018-4813/01 $15.00www.nature.com/ejhg*Correspondence: AJ Jeffreys, Department of Genetics, University ofLeicester, Leicester, LE1 7RH, UK. Tel: +44 116 252 3435; fax: +44 116 2523378; E-mail: ajj@leicester.ac.ukReceived 27 July 2000; revised 26 September 2000; accepted 20 October2000TGG-GTC-CTT-CTG-CCC-AG-3’; C1+2.8R, 5’ATA-CAT-CCC-TCT-ACC-CCA-CC-3’; C1+6.5F, 5’-TGG-GTC-AAA-GGA-GTC-TTG-G-3’; C1+6.8NR, 5’-ATG-GAC-CCT-GGC-TGA-GGC-TGG-GTG-3’; C1+6.9R, 5’GCA-GCA-GAA-CTG-CTT-TCA-AAC-3’. MS32 and C1NH outside and nested PCRreactions employed a long PCR protocol.7 Locus-specificprobes (c. 300 bp long) were synthesised at MS32 byamplification of 1 ng MS32 cosmid DNA8 using primer pairs32-5NF and 32-5R (probe 1), and 32-1.6F and 32-1.3NR(probe 2) for 20 cycles, denaturing at 948C for 15 s, annealingat 658C for 30 s and 1 min extension at 708C; and at C1NH byamplification of 200 ng genomic DNA using primersC1+2.4NF and C1+2.8R (probe 1), and C1+6.5F andC1+6.8NR (probe 2) for 30 cycles, denaturing at 968C for1 min, annealing at 628C for 30 s and 1 min extension at708C. At MS32 semi-nested PCR was carried out using primers32-5F and 32-1.3NR for 26 cycles, denaturing at 968C for 15 s,annealing at 688C for 30 s and 6 min extension at 708C.Nested PCR at C1NH was carried out using primers C1+2.4NFand C1+6.8NR for 28 cycles, denaturing at 948C for 15 s, andannealing and extending at 708C for 6 min.Size fractionation of genomic DNA to detect insertioneventsA total of 25 mg sperm DNA was amplified in five 100-mlreactions for 12 cycles, denaturing at 968C for 1 min,annealing at 638C for 30 s and 4 min extension at 708C,using the primers MS1-1 and MS1-2 to give a progenitor PCRproduct of 1.2 kb. Following amplification, 500 ng ofpAT153-DdeI/BamHI marker DNA was added to each sampleas an internal control and separated by agarose gelelectrophoresis. DNA between 1.4 and 9.5 kb was size-fractionated7 to enrich for alleles containing insertions ofbetween 200 and 7000 bp (consensus Alu and L1 elementshave lengths of c. 300 and 6000 bp respectively). Fifty percent of the purified DNA was re-amplified for between 12 – 16cycles such that the residual progenitor allele was amplifiedto levels no greater than 1 ng, and again size fractionated.These PCR and size fractionation steps were repeated twicemore using the outside primers, and four times with thenested primers. A sample of each reaction was analysed byagarose gel electrophoresis and ethidium bromide staining,followed by Southern hybridisation with 32P-labelled totalhuman DNA. Reactions were scaled down for subsequentanalysis of 10-mg samples.Size fractionation and screening of genomic DNA todetect deletion mutantsGenomic DNA was digested with restriction enzymes whichreleased the target region (NcoI+KpnI for MS32, DraI forC1NH; Figure 2) and size-fractionated by agarose gelelectrophoresis7 to eliminate most or all progenitor mole-cules (c. 4.4 kb) and collect all possible Alu-Alu deletionmutant molecules (1.4 – 4.3 kb long). Multiple aliquots ofeach fraction, each containing DNA derived from 8 – 306104haploid genomes, were screened for deletion mutants in 7-mlreactions by long PCR. MS32 amplifications were carried outusing primers 32-5F and 32-1.3R for 26 cycles, denaturing at948C for 30 s, annealing at 688C for 30 s and 6 min extensionat 708C; C1NH amplifications were carried out using primersC1+2.4F and C1+6.9R for 32 cycles, denaturing at 948C for15 s, and annealing and extending at 688C for 5 min. Half ofeach reaction was analysed by agarose gel electrophoresis andSouthern hybridisation with both 32P-labelled end probes.ResultsAnalysis of de novo insertionsThe 1.2 kb target flanking the human minisatellite MS1(D1S7) contains a number of short AT-rich potentialintegration sites9 (Figure 1). To detect transposition eventsinto this locus, size enrichment for target molecules longerthan the progenitor (Methods) was carried out on two spermDNA samples (1625 mg plus 1610 mg) and 10-mg samples oftwo blood DNAs, two colon tumour DNAs and two placentalDNAs. No evidence for abnormal length mutants wasdetected. This technique was validated by amplifying two100-ml reactions each containing 10 mg sperm DNA plus 1 mlof a 2 molecules/ml dilution of the pseudoinsertion allele(Figure 1) with the nested primers MS1-3 and MS1-4, thenperforming two cycles of size fractionation and re-amplifica-tion. Analysis of the final PCR products by hybridisation witha pseudoinsertion specific probe showed that a singlemolecule of the synthetic mutant could be recovered froma background of c. 3.36106 normal molecules.Figure 1 The MS1 target region for insertion analysis. PCRprimers are shown as arrows. The 657 bp Tet fragment (shadedbox) was isolated from plasmid pBR322 vector by NarIdigestion. This fragment was ligated to the target PCR product,amplified from human DNA with primers MS1-3 plus MS1-4 anddigested with TaqI (compatible with NarI sticky ends). The1.9 kb pseudoinsertion allele was recovered by amplifying theligation products with primers MS1-3 plus MS1-4, using thesame PCR conditions as for primers MS-1 and MS-2 (Methods).Transposition and deletion in human DNACR Hollies et al144European Journal of Human GeneticsAnalysis of Alu-mediated deletionsThe first target lies 1.3 – 5 kb upstream of the end of humanminisatellite MS32 (D1S8),8 known to be active in meioticrecombination10 (Figure 2A). The second target was a 4.4 kbregion around exon 4 of the C1-inhibitor gene (C1NH, Figure2B). Twelve out of 18 deletion breakpoints identified in type1 hereditary angioedema (HAE) patients have been localisedto Alu elements within this region.5Evolutionary and population genetic estimates ofinstabilityTo investigate instability over evolutionary time, both lociwere PCR amplified from human, chimpanzee, gorilla andorang-utan DNA using semi-nested (MS32) or nested (C1NH)primers and analysed by restriction enzyme digestion (Figure3). The MS32 target showed no length changes in any of thespecies, and all changes in DNA fragment profiles could beattributed to restriction site gains and losses by basesubstitution. Similar analysis of the C1NH target showed noevidence for length variation except for a 200-bp sizedifference between orang-utan and the other three primates.Sequencing showed that this was the result of a 200-bpdeletion entirely within Alu 2. Neither target has thereforealtered its Alu content over approximately 34 million years ofprimate evolution (7 million years since the divergence ofhuman, chimpanzee and gorilla, and 10 million years sincethe divergence of orang-utan from this lineage).11 Assuminga generation time of 10 years, this implies that selectively-neutral Alu-mediated re-arrangements in this region arise at arate of 50.961076 per generation (upper 95% C.I.).12To investigate instability in human populations, bothtargets were PCR amplified from 20 unrelated Caucasiansfor MS32 and 39 unrelated Caucasians for C1NH. Both lociwere monomorphic in size in all individuals tested (data notshown). Assuming an effective population size of 10 000 forhumans, this suggests a rate in Caucasians for selectivelyneutral Alu-mediated re-arrangements of 54.461076 pergeneration at MS32 and 52.161076 at C1NH (upper 95%C.I.’s).Analysis of deletion mutants in sperm and blood DNASize enrichment methods were then used in an attempt todetect de novo mutant molecules in sperm and blood DNAFigure 2 Alu-rich regions of the human genome analysed forinstability. The locations and orientation of Alu elements areshown, together with their assigned number and Alu subfamilyclassification. The positions of PCR primers, end probes andrestriction sites used in size enrichment are indicated above. (A)The MS32 target region upstream of minisatellite MS3215. (B)The C1NH gene target spanning intron 3, exon 4 and intron416. The location and extent of different deletions (brackets) anda duplication (hatched bar) in HAE patients5 are shown below.Figure 3 Comparison of the Alu-rich MS32 and C1NH targetsin humans and apes. Target regions were amplified from human(H), chimpanzee (C), gorilla (G) and orang-utan (O) DNA usingprimers 32-5F & 32-1.3NR and C1+2.4NF & C1+6.8NRrespectively and analysed by agarose gel electrophoresis eitherbefore (uppercase) or after (lowercase) digestion with therestriction enzymes indicated. M, markers = lDNA6 HindIII +fX1746 HaeIII. (A) Comparative digestion of the MS32 region.(B) Comparative digestion of the C1NH region. Brackethighlights the RsaI DNA fragments showing the 0.2 kb deletionin the orang-utan C1NH target region.European Journal of Human GeneticsTransposition and deletion in human DNACR Hollies et al145that had arisen by Alu-mediated deletion (Methods). A surveyof DNA from 107 sperm and 4.36106 sperm at MS32 andC1NH respectively revealed no deletion mutants at eitherlocus. Similar analysis of somatic (blood) DNA showed nodeletion mutants at either locus in DNA from 2.56106diploid cells. The enrichment technique was validated forC1NH by mixing 4 mg sperm DNA with 6 pg (equivalent toone diploid genome) genomic DNA from HAE patient F1(Figure 2B).5 Size fractionation and screening revealed asingle molecule of the deletion mutant of the correct size(1.2 kb), establishing that rare deletions can be detected in abackground of 1.36106 normal molecules (data not shown).DiscussionSystems have been developed for detecting very rareinsertion and deletion events using physical size-selectionof DNA and PCR recovery of single molecules. Despite thesensitivity of these methods, no de novo insertion or deletionmutants were detected. The absence of insertion events in50 mg of human sperm DNA indicates that the rate of de novotransposition into the 1.2-kb MS1 target is very low(5261077 per sperm (upper 95% C.I.) and 5161076 inthe other cell types investigated). Similarly, the lack ofdeletions in either of the Alu-rich loci investigated impliesdeletion rates in sperm of 5361077 at MS32 and 5761077per sperm at C1NH, with maximum frequencies of somaticmutation at both of these loci of 51076 per cell.The failure to detect any large insertions into the MS1target region may be because the target is nonoptimal forintegration of retroposons, or alternatively reflects anextremely low overall rate of transposition in the humangenome. The latter possibility is supported by indirectestimates13 of Alu and L1 insertion, calculated to beapproximately 3.3610711/kb target/generation, far lowerthan the upper limits obtained by sperm DNA analysis.The frequency of gross genomic re-arrangement within theC1NH gene, estimated from the rate of occurrence of HAE, isbetween 7.661075 and 1.561076 per gamete.14 These ratescontrast with the population estimate of selectively neutraldeletion rate of5161076 per gamete and the direct estimateof overall deletion rate in sperm of 5761077. Thisinconsistency suggests, first, that most or all Alu-mediatedre-arrangements in the C1NH gene are selectively disadvan-tageous, and second, that these re-arrangements may occurpreferentially in the female germline. The stability of the Alu-rich region associated with MS32 can be explained by recentevidence demonstrating that the target locus investigatedhere lies within a region of intense linkage disequilibrium,within which meiotic crossover is strongly suppressed (MPanayi and AJ Jeffreys, unpublished data).This work has demonstrated that the most sensitivegermline mutation detection systems currently available areunsuitable for the isolation of re-arrangements occurring at arate below 1077. These difficulties highlight the need formore powerful and flexible systems to detect extremely lowfrequency re-arrangements at the single molecule level. Untilsuch methods are developed, ascertainment of mutation ininherited disease will provide the only means of accessingsuch events in the human germline.AcknowledgementsWe are very grateful to volunteers for providing blood and semensamples, and to Mario Tosi from the Pasteur Institute for supplyingDNA from HAE patients. We thank Nicola Royle, John Armour andother colleagues for helpful discussions. This work was supported inpart by an International Research Scholars Award to AJ Jeffreys fromthe Howard Hughes Medical Institute, and in part by grants from theWellcome Trust, Medical Research Council and Royal Society.References1 Deininger PL, Batzer MA: Alu repeats and human disease. MolGenet Metab 1999; 67: 183 – 193.2 Kazazian HH, Moran JV: The impact of L1 retrotransposons onthe human genome. Nature Genet 1999; 19: 19 – 24.3 Jeffs AR, Benjes SM, Smith TL, Sowerby SJ, Morris CM: The BCRgene recombines preferentially with Alu elements in complexBCR-ABL translocations of chronic myeloid leukaemia. HumMol Genet 1998; 7: 767 – 776.4 Rouyer F, Simmler MC, Page DC, Weissenbach J: A sex-chromosome rearrangement in a human XX male caused byAlu-Alu recombination. Cell 1987; 51: 417 – 425.5 Stoppa-Lyonnet D, Duponchel C, Meo T et al: Recombinationalbiases in the rearranged C1-inhibitor genes of hereditaryangioedema patients. Am J Hum Genet 1991; 49: 1055 – 1062.6 Miki Y, Nishisho I, Horii A et al: Disruption of the APC gene by aretrotransposal insertion of L1 sequence in a colon cancer.Cancer Res 1992; 52: 643 – 645.7 Jeffreys AJ, Neil DL, Neumann R: Repeat instability at humanminisatellites arising from meiotic recombination. EMBO J1999; 17: 4147 – 4157.8 Wong Z, Wilson V, Patel I, Povey S, Jeffreys AJ: Characterisationof a panel of highly variable minisatellites cloned from humanDNA. Ann Hum Genet 1987; 51: 269 – 288.9 Jurka J: Sequence patterns indicate an enzymatic involvementin integration of mammalian retroposons. Proc Natl Acad Sci USA1997; 94: 1872 – 1877.10 Jeffreys AJ, Murray J, Neumann R: High-resolution mapping ofcrossovers in human sperm defines a minisatellite-associatedrecombination hotspot. Mol Cell 1998; 2: 267 – 273.11 Koop BF, Goodman M, Xu P, Chan K, Slightom JL: Primate Z-globin DNA sequences and man’s place among the great apes.Nature 1986; 319: 234 – 238.12 Kimura M: The neutral theory of molecular evolution. CambridgeUniversity Press, Cambridge, 1983.13 Kazazian HH: An estimated frequency of endogenous inser-tional mutations in human. Nature Genetics 1999; 2: 130.14 Hollies CR: The investigation of variation and de novo mutationin the human genome: PhD thesis. University of Leicester, 1999.15 Murray J, Buard J, Neil DL et al: Comparative sequence analysisof human minisatellites showing meiotic repeat instability.Genome Res 1999; 9: 130 – 136.16 Carter PE, Duponchel C, Tosi M et al: Complete nucleotidesequence of the gene for human C1-inhibitor with an unusuallyhigh density of Alu elements. Eur J Biochem 1991; 197: 301 – 308.Transposition and deletion in human DNACR Hollies et al146European Journal of Human Genetics

AJ: Characterisation of a panel of highly variable minisatellites cloned from human DNA. Ann Hum Genet

al: Recombinational biases in the rearranged C1-inhibitor genes of hereditary angioedema patients.

Alu repeats and human disease. Mol Genet Metab

An estimated frequency of endogenous insertional mutations in human.

CM: The BCR gene recombines preferentially with Alu elements in complex BCR-ABL translocations of chronic myeloid leukaemia. Hum Mol Genet

Complete nucleotide sequence of the gene for human C1-inhibitor with an unusually high density of Alu elements.

DL et al: Comparative sequence analysis of human minisatellites showing meiotic repeat instability. Genome Res

High-resolution mapping of crossovers in human sperm defines a minisatellite-associated recombination hotspot. Mol Cell

M i k iY ,N i s h i s h oI ,H o r i iAet al: Disruption of the APC gene by a retrotransposal insertion of L1 sequence in a colon cancer. Cancer Res

R o u y e rF ,S i m m l e rM C ,P a g eD C ,W e i s s e n b a c hJ :As e x -chromosome rearrangement in a human XX male caused by Alu-Alu recombination. Cell

Repeat instability at human minisatellites arising from meiotic recombination.

Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons.

Slightom JL: Primate Zglobin DNA sequences and man's place among the great apes. Nature

The impact of L1 retrotransposons on the human genome.

The investigation of variation and de novo mutation in the human genome: PhD thesis.

The neutral theory of molecular evolution.

Attempts to detect retrotransposition and de novo deletion of Alus and other dispersed repeats at specific loci in the human genome

MUCC (Crossref)

Enlighten: Publications

SHORT REPORT
Attempts to detect retrotransposition and de novo
deletion of Alus and other dispersed repeats at specific
loci in the human genome
Caroline R Hollies1, Darren G Monckton2 and Alec J Jeffreys*,3
1Department of Pathology, University of Wales College of Medicine, Wales, UK; 2Division of Molecular Genetics,
University of Glasgow, Glasgow, Scotland, UK; 3Department of Genetics, University of Leicester, Leicester, UK
Dispersed repeat elements contribute to genome instability by de novo insertion and unequal recombination
between repeats. To study the dynamics of these processes, we have developed single DNA molecule
approaches to detect de novo insertions at a single locus and Alu-mediated deletions at two different loci in
human genomic DNA. Validation experiments showed these approaches could detect insertions and
deletions at frequencies below 1076 per cell. However, bulk analysis of germline (sperm) and somatic DNA
showed no evidence for genuine mutant molecules, placing an upper limit of insertion and deletion rates of
261077 and 361077, respectively, in the individuals tested. Such re-arrangements at these loci therefore
occur at a rate lower than that detectable by the most sensitive methods currently available. European Journal
of Human Genetics (2001) 9, 143 – 146.
Keywords: Alu; deletion; dispersed repeats; insertion; recombination; retroposition
Introduction
Human retroposons, in particular Alu and L1 elements, play a
major role in genome remodelling, both by de novo insertion
and by promoting deletions and re-duplications as a result of
unequal recombination between dispersed repeat elements.1
Mechanisms of re-arrangement, particularly in retroposition,
are fairly well established;2 in contrast, little is known about
the dynamics of these events. De novo insertions are usually
of complete Alu and partial L1 elements,1,2 while unequal
exchanges occur mainly between Alu elements.3 – 5 Both
types of event are thought to occur mainly in the germline,2
though examples of both have been observed in somatic
tissues.3,6 There is evidence that at least some unequal
exchange events may be due to meiotic recombination.4 To
date, such insertions and re-arrangements have only been
characterised in patients, resulting in considerable uncer-
tainties about the nature of these processes. We have
therefore developed highly sensitive methods for mutation
detection, analogous to methods devised for studying de novo
mutation at highly unstable minisatellites,7 and have used
these in an attempt to detect dispersed repeat-mediated
instability in somatic and germline DNA at three different
loci.
Methods
PCR amplification
Primers used were: MS1-1, 5’-GCT-GCT-GTG-CCT-TCC-CCG-
GTT-C-3’; MS1-2, 5’-ACC-CAC-TGA-CTC-ACA-GCA-AGG-G-
3’; MS1-3 5’-ggc-gga-tcc-cgg-gaa-TTC-AGT-CTC-CTA-GTG-
AAG-GTG-C-3’; MS1-4, 5’-gcg-aat-tcg-gat-ccc-GGT-GAT-
TTC-TCA-ATT-TGC-TGG-3’ (lowercase – extensions incorpor-
ating restriction endonuclease sites for subsequent cloning):
32-5F, 5’-CAG-TGC-TTG-GCA-CAT-AAT-GAG-CAC-3’; 32-
5NF, 5’-CTC-TTT-CTA-GAA-GCC-GTT-AGA-GGA-G-3’; 32-
5R, 5’-GAA-TCC-TAC-ATG-TAG-GCG-AGC-AGT-3’; 32-1.6F,
5’-GCC-AAC-AGT-GTA-CTT-TGA-AGA-GCA-3’; 32-1.3NR, 5’-
GCA-GGT-AGA-TAG-TGG-CCC-AGA-G-3’; 32-1.3R, 5’-CCA-
GGT-TCT-GGG-GTG-ACT-AG-3’: C1+2.4F, 5’-CCC-AAC-
AGA-TTC-TCC-TAC-CC-3’; C1+2.4NF 5’-GCC-CAC-TAC-
European Journal of Human Genetics (2001) 9, 143 – 146
ª 2001 Nature Publishing Group All rights reserved 1018-4813/01 $15.00
www.nature.com/ejhg
*Correspondence: AJ Jeffreys, Department of Genetics, University of
Leicester, Leicester, LE1 7RH, UK. Tel: +44 116 252 3435; fax: +44 116 252
3378; E-mail: ajj@leicester.ac.uk
Received 27 July 2000; revised 26 September 2000; accepted 20 October
2000
TGG-GTC-CTT-CTG-CCC-AG-3’; C1+2.8R, 5’ATA-CAT-CCC-
TCT-ACC-CCA-CC-3’; C1+6.5F, 5’-TGG-GTC-AAA-GGA-
GTC-TTG-G-3’; C1+6.8NR, 5’-ATG-GAC-CCT-GGC-TGA-
GGC-TGG-GTG-3’; C1+6.9R, 5’GCA-GCA-GAA-CTG-CTT-
TCA-AAC-3’. MS32 and C1NH outside and nested PCR
reactions employed a long PCR protocol.7 Locus-specific
probes (c. 300 bp long) were synthesised at MS32 by
amplification of 1 ng MS32 cosmid DNA8 using primer pairs
32-5NF and 32-5R (probe 1), and 32-1.6F and 32-1.3NR
(probe 2) for 20 cycles, denaturing at 948C for 15 s, annealing
at 658C for 30 s and 1 min extension at 708C; and at C1NH by
amplification of 200 ng genomic DNA using primers
C1+2.4NF and C1+2.8R (probe 1), and C1+6.5F and
C1+6.8NR (probe 2) for 30 cycles, denaturing at 968C for
1 min, annealing at 628C for 30 s and 1 min extension at
708C. At MS32 semi-nested PCR was carried out using primers
32-5F and 32-1.3NR for 26 cycles, denaturing at 968C for 15 s,
annealing at 688C for 30 s and 6 min extension at 708C.
Nested PCR at C1NH was carried out using primers C1+2.4NF
and C1+6.8NR for 28 cycles, denaturing at 948C for 15 s, and
annealing and extending at 708C for 6 min.
Size fractionation of genomic DNA to detect insertion
events
A total of 25 mg sperm DNA was amplified in five 100-ml
reactions for 12 cycles, denaturing at 968C for 1 min,
annealing at 638C for 30 s and 4 min extension at 708C,
using the primers MS1-1 and MS1-2 to give a progenitor PCR
product of 1.2 kb. Following amplification, 500 ng of
pAT153-DdeI/BamHI marker DNA was added to each sample
as an internal control and separated by agarose gel
electrophoresis. DNA between 1.4 and 9.5 kb was size-
fractionated7 to enrich for alleles containing insertions of
between 200 and 7000 bp (consensus Alu and L1 elements
have lengths of c. 300 and 6000 bp respectively). Fifty per
cent of the purified DNA was re-amplified for between 12 – 16
cycles such that the residual progenitor allele was amplified
to levels no greater than 1 ng, and again size fractionated.
These PCR and size fractionation steps were repeated twice
more using the outside primers, and four times with the
nested primers. A sample of each reaction was analysed by
agarose gel electrophoresis and ethidium bromide staining,
followed by Southern hybridisation with 32P-labelled total
human DNA. Reactions were scaled down for subsequent
analysis of 10-mg samples.
Size fractionation and screening of genomic DNA to
detect deletion mutants
Genomic DNA was digested with restriction enzymes which
released the target region (NcoI+KpnI for MS32, DraI for
C1NH; Figure 2) and size-fractionated by agarose gel
electrophoresis7 to eliminate most or all progenitor mole-
cules (c. 4.4 kb) and collect all possible Alu-Alu deletion
mutant molecules (1.4 – 4.3 kb long). Multiple aliquots of
each fraction, each containing DNA derived from 8 – 306104
haploid genomes, were screened for deletion mutants in 7-ml
reactions by long PCR. MS32 amplifications were carried out
using primers 32-5F and 32-1.3R for 26 cycles, denaturing at
948C for 30 s, annealing at 688C for 30 s and 6 min extension
at 708C; C1NH amplifications were carried out using primers
C1+2.4F and C1+6.9R for 32 cycles, denaturing at 948C for
15 s, and annealing and extending at 688C for 5 min. Half of
each reaction was analysed by agarose gel electrophoresis and
Southern hybridisation with both 32P-labelled end probes.
Results
Analysis of de novo insertions
The 1.2 kb target flanking the human minisatellite MS1
(D1S7) contains a number of short AT-rich potential
integration sites9 (Figure 1). To detect transposition events
into this locus, size enrichment for target molecules longer
than the progenitor (Methods) was carried out on two sperm
DNA samples (1625 mg plus 1610 mg) and 10-mg samples of
two blood DNAs, two colon tumour DNAs and two placental
DNAs. No evidence for abnormal length mutants was
detected. This technique was validated by amplifying two
100-ml reactions each containing 10 mg sperm DNA plus 1 ml
of a 2 molecules/ml dilution of the pseudoinsertion allele
(Figure 1) with the nested primers MS1-3 and MS1-4, then
performing two cycles of size fractionation and re-amplifica-
tion. Analysis of the final PCR products by hybridisation with
a pseudoinsertion specific probe showed that a single
molecule of the synthetic mutant could be recovered from
a background of c. 3.36106 normal molecules.
Figure 1 The MS1 target region for insertion analysis. PCR
primers are shown as arrows. The 657 bp Tet fragment (shaded
box) was isolated from plasmid pBR322 vector by NarI
digestion. This fragment was ligated to the target PCR product,
amplified from human DNA with primers MS1-3 plus MS1-4 and
digested with TaqI (compatible with NarI sticky ends). The
1.9 kb pseudoinsertion allele was recovered by amplifying the
ligation products with primers MS1-3 plus MS1-4, using the
same PCR conditions as for primers MS-1 and MS-2 (Methods).
Transposition and deletion in human DNA
CR Hollies et al
144
European Journal of Human Genetics
Analysis of Alu-mediated deletions
The first target lies 1.3 – 5 kb upstream of the end of human
minisatellite MS32 (D1S8),8 known to be active in meiotic
recombination10 (Figure 2A). The second target was a 4.4 kb
region around exon 4 of the C1-inhibitor gene (C1NH, Figure
2B). Twelve out of 18 deletion breakpoints identified in type
1 hereditary angioedema (HAE) patients have been localised
to Alu elements within this region.5
Evolutionary and population genetic estimates of
instability
To investigate instability over evolutionary time, both loci
were PCR amplified from human, chimpanzee, gorilla and
orang-utan DNA using semi-nested (MS32) or nested (C1NH)
primers and analysed by restriction enzyme digestion (Figure
3). The MS32 target showed no length changes in any of the
species, and all changes in DNA fragment profiles could be
attributed to restriction site gains and losses by base
substitution. Similar analysis of the C1NH target showed no
evidence for length variation except for a 200-bp size
difference between orang-utan and the other three primates.
Sequencing showed that this was the result of a 200-bp
deletion entirely within Alu 2. Neither target has therefore
altered its Alu content over approximately 34 million years of
primate evolution (7 million years since the divergence of
human, chimpanzee and gorilla, and 10 million years since
the divergence of orang-utan from this lineage).11 Assuming
a generation time of 10 years, this implies that selectively-
neutral Alu-mediated re-arrangements in this region arise at a
rate of 50.961076 per generation (upper 95% C.I.).12
To investigate instability in human populations, both
targets were PCR amplified from 20 unrelated Caucasians
for MS32 and 39 unrelated Caucasians for C1NH. Both loci
were monomorphic in size in all individuals tested (data not
shown). Assuming an effective population size of 10 000 for
humans, this suggests a rate in Caucasians for selectively
neutral Alu-mediated re-arrangements of 54.461076 per
generation at MS32 and 52.161076 at C1NH (upper 95%
C.I.’s).
Analysis of deletion mutants in sperm and blood DNA
Size enrichment methods were then used in an attempt to
detect de novo mutant molecules in sperm and blood DNA
Figure 2 Alu-rich regions of the human genome analysed for
instability. The locations and orientation of Alu elements are
shown, together with their assigned number and Alu subfamily
classification. The positions of PCR primers, end probes and
restriction sites used in size enrichment are indicated above. (A)
The MS32 target region upstream of minisatellite MS3215. (B)
The C1NH gene target spanning intron 3, exon 4 and intron
416. The location and extent of different deletions (brackets) and
a duplication (hatched bar) in HAE patients5 are shown below.
Figure 3 Comparison of the Alu-rich MS32 and C1NH targets
in humans and apes. Target regions were amplified from human
(H), chimpanzee (C), gorilla (G) and orang-utan (O) DNA using
primers 32-5F & 32-1.3NR and C1+2.4NF & C1+6.8NR
respectively and analysed by agarose gel electrophoresis either
before (uppercase) or after (lowercase) digestion with the
restriction enzymes indicated. M, markers = lDNA6 HindIII +
fX1746 HaeIII. (A) Comparative digestion of the MS32 region.
(B) Comparative digestion of the C1NH region. Bracket
highlights the RsaI DNA fragments showing the 0.2 kb deletion
in the orang-utan C1NH target region.
European Journal of Human Genetics
Transposition and deletion in human DNA
CR Hollies et al
145
that had arisen by Alu-mediated deletion (Methods). A survey
of DNA from 107 sperm and 4.36106 sperm at MS32 and
C1NH respectively revealed no deletion mutants at either
locus. Similar analysis of somatic (blood) DNA showed no
deletion mutants at either locus in DNA from 2.56106
diploid cells. The enrichment technique was validated for
C1NH by mixing 4 mg sperm DNA with 6 pg (equivalent to
one diploid genome) genomic DNA from HAE patient F1
(Figure 2B).5 Size fractionation and screening revealed a
single molecule of the deletion mutant of the correct size
(1.2 kb), establishing that rare deletions can be detected in a
background of 1.36106 normal molecules (data not shown).
Discussion
Systems have been developed for detecting very rare
insertion and deletion events using physical size-selection
of DNA and PCR recovery of single molecules. Despite the
sensitivity of these methods, no de novo insertion or deletion
mutants were detected. The absence of insertion events in
50 mg of human sperm DNA indicates that the rate of de novo
transposition into the 1.2-kb MS1 target is very low
(5261077 per sperm (upper 95% C.I.) and 5161076 in
the other cell types investigated). Similarly, the lack of
deletions in either of the Alu-rich loci investigated implies
deletion rates in sperm of 5361077 at MS32 and 5761077
per sperm at C1NH, with maximum frequencies of somatic
mutation at both of these loci of 51076 per cell.
The failure to detect any large insertions into the MS1
target region may be because the target is nonoptimal for
integration of retroposons, or alternatively reflects an
extremely low overall rate of transposition in the human
genome. The latter possibility is supported by indirect
estimates13 of Alu and L1 insertion, calculated to be
approximately 3.3610711/kb target/generation, far lower
than the upper limits obtained by sperm DNA analysis.
The frequency of gross genomic re-arrangement within the
C1NH gene, estimated from the rate of occurrence of HAE, is
between 7.661075 and 1.561076 per gamete.14 These rates
contrast with the population estimate of selectively neutral
deletion rate of5161076 per gamete and the direct estimate
of overall deletion rate in sperm of 5761077. This
inconsistency suggests, first, that most or all Alu-mediated
re-arrangements in the C1NH gene are selectively disadvan-
tageous, and second, that these re-arrangements may occur
preferentially in the female germline. The stability of the Alu-
rich region associated with MS32 can be explained by recent
evidence demonstrating that the target locus investigated
here lies within a region of intense linkage disequilibrium,
within which meiotic crossover is strongly suppressed (M
Panayi and AJ Jeffreys, unpublished data).
This work has demonstrated that the most sensitive
germline mutation detection systems currently available are
unsuitable for the isolation of re-arrangements occurring at a
rate below 1077. These difficulties highlight the need for
more powerful and flexible systems to detect extremely low
frequency re-arrangements at the single molecule level. Until
such methods are developed, ascertainment of mutation in
inherited disease will provide the only means of accessing
such events in the human germline.
Acknowledgements
We are very grateful to volunteers for providing blood and semen
samples, and to Mario Tosi from the Pasteur Institute for supplying
DNA from HAE patients. We thank Nicola Royle, John Armour and
other colleagues for helpful discussions. This work was supported in
part by an International Research Scholars Award to AJ Jeffreys from
the Howard Hughes Medical Institute, and in part by grants from the
Wellcome Trust, Medical Research Council and Royal Society.
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Attempts to detect retrotransposition and de novo deletion of Alus and other dispersed repeats at specific loci in the human genome

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