10Med Genet 1998;35:130-136 The mechanisms involved in formation of deletions and duplications of 15 q 11 -q 13 W P Robinson, F Dutly, R D Nicholls, F Bernasconi, M Pefiaherrera, R C Michaelis, D Abeliovich, AA Schinzel Department of Medical Genetics, University of British Columbia, Vancouver, Canada W P Robinson F Bernasconi M Pefiaherrera Institut fur Medizinische Genetik der Universitat Zurich, Switzerland F Dutly AA Schinzel Department of Genetics, Case Western Reserve University School of Medicine and Center for Human Genetics and University Hospital of Cleveland, Cleveland, USA R D Nicholls Greenwood Genetic Center, Greenwood, USA R C Michaelis Hadassah Hebrew University Hospital, Jerusalem, Israel D Abeliovich Correspondence to: Dr Robinson, BC Research Institute for Child and Family Health, Room 170-950, West 28th Avenue, Vancouver, BC, Canada V5Z 4H4. Received 8 July 1997 Revised version accepted for publication 15 August 1997 Abstract Haplotype analysis was undertaken in 20 cases of 15q11-q13 deletion associated with Prader-Willi syndrome (PWS) or Angelman syndrome (AS) to determine if these deletions arose through unequal meiotic crossing over between homolo- gous chromosomes. Of these, six cases of PWS and three of AS were informative for markers on both sides of the deletion. For four of six cases of paternal 15q11-q13 deletion (PWS), markers on both sides of the deletion breakpoints were inferred to be of the same grandparental origin, implying an intrachromosomal origin of the deletion. Although the remaining two PWS cases showed evidence of crossing over between markers flanking the dele- tion, this was not more frequent than expected by chance given the genetic distance between proximal and distal markers. It is therefore possible that all PWS deletions were intrachromosomal in origin with the deletion event occurring after normal meiosis I recombination. Alternatively, both sister chromatid and homologous chromosome unequal ex- change during meiosis may contribute to these deletions. In contrast, all three cases of maternal 15ql1-q13 deletion (AS) were associated with crossing over between flanking markers, which suggests signifi- candy more recombination than expected by chance (p=0.002). Therefore, there appears to be more than one mechanism which may lead to PWS/AS deletions or the resolution of recombination interme- diates may differ depending on the paren- tal origin of the deletion. Furthermore, 13 of 15 cases of 15qll-ql3 duplication, trip- lication, or inversion duplication had a distal duplication breakpoint which dif- fered from the common distal deletion breakpoint. The presence of at least four distal breakpoint sites in duplications indicates that the mechanisms of rear- rangement may be complex and multiple repeat sequences may be involved. (7Med Genet 1998;35:130-136) Keywords: Prader-Willi syndrome; Angelman syn- drome; deletion; duplication 1 5q 1 1 -q 13 Both Prader-Willi syndrome (PWS) and An- gelman syndrome (AS) result from large inter- stitial deletions of chromosome 1 5q 1l-ql 3 in 70-75% of cases.'` PWS results when the dele- tion occurs on the paternal chromosome and AS when the same deletion is on the maternal chromosome.5 Both syndromes occur at a frequency of about 1/15 000-1/20 0006 8 and therefore maternal and paternal deletions each occur in approximately 1/20 000-1/28 000 livebirths. Although there are a few exceptions ob- served, the vast majority of both maternal and paternal deletions of this region are of similar size (-4 Mb) and have tightly clustered breakpoints.59-1l There are two common proxi- mal breakpoints with similar frequencies (-50% of each) in both maternally and paternally derived deletions.'2 '1 It has been suggested that most distal breakpoints are included in the region covered by a single YAC.9 The common deletion is much larger than necessary to cause the PWS or AS pheno- type, as deletions of less than 7 kb including the imprinting control centre can also result in the clinically typical PWS or AS phenotype4 15 (unpublished results). The frequent occur- rence and high clustering of breakpoints in the large deletions imply an instability at these sites. It is commonly suggested that microdele- tions arise through mispairing of large dupli- cated sequences. Repetitive DNA is abundant in the human genome, but the frequency and distribution of microdeletions does not seem to be random with regard to these sequences. Additional factors are therefore necessary to explain why certain regions of the genome are predisposed to frequent mispairing and deletion.'6 In addition, these breakpoints are not sites of high homologous recombination and cannot therefore be explained simply by a tendency to double strand breaks at these sites during meiosis I.'7 18 Interstitial duplications1 521 and triplications22'24 of the PWS/AS region have also been reported. In most of these cases, maternal heterozygosity of duplicated genes is observed, indicating involvement of two differ- ent maternal chromosomes. It is therefore of interest to determine if these duplications/ triplications are the result of a mechanism related to deletion events. A meiotic unequal crossover between homologous chromosomes is expected to result in the formation of recip- rocal duplication and deletion products, which show recombination of flanking markers (fig 1A). Such a mechanism has been reported to be involved in duplications of 17pl 1.2-pl2, associated with Charcot-Marie Tooth disease IA (CMT1A), and deletions of the same region which are associated with hereditary neu- ropathy with liability to pressure palsies (HNPP).25 26 In contrast, an intrachromosomal 130 on 29 August 2018 by guest. Protected by copyright. http://jmg.bmj.com/ J Med Genet: first published as 10.1136/jmg.35.2.130 on 1 February 1998. Downloaded from
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10Med Genet 1998;35:130-136
The mechanisms involved in formation ofdeletions and duplications of 15q 11 -q13
W P Robinson, F Dutly, R D Nicholls, F Bernasconi, M Pefiaherrera, R C Michaelis,D Abeliovich, A A Schinzel
Department ofMedical Genetics,University of BritishColumbia, Vancouver,CanadaW P RobinsonF BernasconiM Pefiaherrera
Institut furMedizinische Genetikder Universitat Zurich,SwitzerlandF DutlyA A Schinzel
Department ofGenetics, CaseWestern ReserveUniversity School ofMedicine and Centerfor Human Geneticsand UniversityHospital of Cleveland,Cleveland, USAR D Nicholls
Greenwood GeneticCenter, Greenwood,USAR C Michaelis
Correspondence to:Dr Robinson, BC ResearchInstitute for Child andFamily Health, Room170-950, West 28th Avenue,Vancouver, BC, Canada V5Z4H4.
Received 8 July 1997Revised version accepted forpublication 15 August 1997
AbstractHaplotype analysis was undertaken in 20cases of 15q11-q13 deletion associatedwith Prader-Willi syndrome (PWS) orAngelman syndrome (AS) to determine ifthese deletions arose through unequalmeiotic crossing over between homolo-gous chromosomes. Of these, six cases ofPWS and three ofAS were informative formarkers on both sides of the deletion. Forfour of six cases of paternal 15q11-q13deletion (PWS), markers on both sides ofthe deletion breakpoints were inferred tobe of the same grandparental origin,implying an intrachromosomal origin ofthe deletion. Although the remaining twoPWS cases showed evidence of crossingover between markers flanking the dele-tion, this was not more frequent thanexpected by chance given the geneticdistance between proximal and distalmarkers. It is therefore possible that allPWS deletions were intrachromosomal inorigin with the deletion event occurringafter normal meiosis I recombination.Alternatively, both sister chromatid andhomologous chromosome unequal ex-change during meiosis may contribute tothese deletions. In contrast, all three casesof maternal 15ql1-q13 deletion (AS) wereassociated with crossing over betweenflanking markers, which suggests signifi-candy more recombination than expectedby chance (p=0.002). Therefore, thereappears to be more than one mechanismwhich may lead to PWS/AS deletions orthe resolution of recombination interme-diates may differ depending on the paren-tal origin of the deletion. Furthermore, 13of 15 cases of 15qll-ql3 duplication, trip-lication, or inversion duplication had adistal duplication breakpoint which dif-fered from the common distal deletionbreakpoint. The presence of at least fourdistal breakpoint sites in duplicationsindicates that the mechanisms of rear-rangement may be complex and multiplerepeat sequences may be involved.(7Med Genet 1998;35:130-136)
Both Prader-Willi syndrome (PWS) and An-gelman syndrome (AS) result from large inter-stitial deletions of chromosome 1 5q 1l-ql 3 in70-75% of cases.'` PWS results when the dele-tion occurs on the paternal chromosome and
AS when the same deletion is on the maternalchromosome.5 Both syndromes occur at afrequency of about 1/15 000-1/20 0006 8 andtherefore maternal and paternal deletions eachoccur in approximately 1/20 000-1/28 000livebirths.Although there are a few exceptions ob-
served, the vast majority of both maternal andpaternal deletions of this region are of similarsize (-4 Mb) and have tightly clusteredbreakpoints.59-1l There are two common proxi-mal breakpoints with similar frequencies(-50% of each) in both maternally andpaternally derived deletions.'2 '1 It has beensuggested that most distal breakpoints areincluded in the region covered by a singleYAC.9 The common deletion is much largerthan necessary to cause the PWS or AS pheno-type, as deletions of less than 7 kb including theimprinting control centre can also result in theclinically typical PWS or AS phenotype4 15
(unpublished results). The frequent occur-rence and high clustering of breakpoints in thelarge deletions imply an instability at thesesites. It is commonly suggested that microdele-tions arise through mispairing of large dupli-cated sequences. Repetitive DNA is abundantin the human genome, but the frequency anddistribution of microdeletions does not seem tobe random with regard to these sequences.Additional factors are therefore necessary toexplain why certain regions of the genome arepredisposed to frequent mispairing anddeletion.'6 In addition, these breakpoints arenot sites of high homologous recombinationand cannot therefore be explained simply by atendency to double strand breaks at these sitesduring meiosis I.'7 18
Interstitial duplications1521 andtriplications22'24 of the PWS/AS region havealso been reported. In most of these cases,maternal heterozygosity of duplicated genes isobserved, indicating involvement of two differ-ent maternal chromosomes. It is therefore ofinterest to determine if these duplications/triplications are the result of a mechanismrelated to deletion events. A meiotic unequalcrossover between homologous chromosomesis expected to result in the formation of recip-rocal duplication and deletion products, whichshow recombination of flanking markers (fig1A). Such a mechanism has been reported tobe involved in duplications of 17pl 1.2-pl2,associated with Charcot-Marie Tooth diseaseIA (CMT1A), and deletions of the same regionwhich are associated with hereditary neu-ropathy with liability to pressure palsies(HNPP).25 26 In contrast, an intrachromosomal
Mechanisms involved in formation of deletions and duplications of 15ql1-q13
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Figure I Models of rearrangements in chromosome 15ql1-q13. (A) Classical n
unequal crossing over leading to reciprocal duplication (dup) and deletion (del) zrecombination offlanking markers. (B) Unequal sister chromatid exchange (left)intrachromatid (right) recombination may lead to deletion with no recombinatioiflanking markers. (C) Recombination between inverted repeats leading to inversi,duplication (inv dup) derivedfrom two different chromosomes. The presence ofmdirect and inverted repeats can lead to variable breakpoints; those shown are for ipurposes.
event will not result in recombinationing markers and should not be associaa reciprocal duplication product, ur
crossing over is between sister chromIB). Interestingly, in one case of HNmaternal origin, the deletion was detto be intrachromosomal by haplotypstruction using data from the grandrand it was suggested that the intrasomal events may be specific to femaleIn addition, the rarity of CMT1A dupof maternal origin is not explained bylevel of female than male meiotic rec
tion in this region.28 The sex specific dwas suggested to be a result of malefactors which help to form or stabduplicated chromosome.29 Atransposon-like element within the re
been suggested to be involved inthese recombination events.'0The 1 5ql1 -qi 3 region is also notab]
frequent occurrence of inversion duplPrevious reports indicate that "small" smerary isodicentric inversion duplical
(inv dup 15) chromosomes share breakpointsin common with the two common proximalPWS/AS deletion breakpoints, while "large"inv dup 15 chromosomes tend to be of twosizes with only one breakpoint similar to thedistal deletion breakpoint.3'" The formation ofisodicentric chromosomes may also involverecombination between inverted repeats asshown in fig 1C. However, alternative mecha-nisms include a "U type" exchange,"4 or repairof a broken chromosome through replicationand end to end fusion. Although the proposedmechanisms differ, the similarity of break-points between inversion duplications anddeletions of this region, as well as the observa-
^ tion of patients carrying both a small isodicen-tV tric 15 plus a 15q11-q13 deletion35 orrcltrK duplication,2' suggests that at least some of the
causes may be related.In order to determine if PWS/AS deletions
are associated with meiotic recombinationbetween flanking markers and hence if dele-tions are likely to be intra- or interchromo-somal events, marker haplotypes were analysedin 20 families. Grandparental DNA wasavailable in 18 of these, and haplotypes were
-- inferred in the two additional families using- unaffected sibs. The results exclude an unequal
crossover between homologous chromosomesat meiosis as the deletion mechanism in four ofsix informative PWS cases. However, all three
N informative AS deletion cases were associatedwith a crossover event. A comparison of proxi-mal and distal breakpoints between commondeletions and duplications (or triplications andinversion duplications) of this same regionindicates that the observed maternal duplica-tions of this region are not the reciprocal prod-
nodel of ucts of deletion events, or at least that thewith breakpoints are much more variable. It isor hypothesised that multiple mechanisms for
onPif rearrangement in this region exist and maylultiple involve unequal sister chromatid exchange orilustrative intrachromatid recombination either during or
after meiosis in at least a portion of cases. Theof flank- greater number of breakpoint sites associatedited with with inversion duplications than deletions maynless the also reflect relative location of inverted versus
atids (fig direct repeat sequences.IPP withterminedoe recon- Methods
?arents Patients with 15ql 1-q13 deletions were ascer-chromo- tained through routine molecular investiga-icatonse tions of PWS and AS patients. Deletions werefcalorns diagnosed by virtue of lack of maternal ora lower paternal inheritance of the commonly deleted
,ombina- RFLP probes and dosage analysis"6 37 or,ilfference usually, by microsatellite analysis.20 Becausespecific there are no published markers more proximal
ilise the than D15S1035, D15S541, D15S542, andmariner D1 5S18 (order of these four polymorphisms ispeat has not known), and as these markers are deleted iniediating half the patients," only patients with the
smaller deletion will be informative in thisle for the analysis (for recent mapping data in this regionlications. see Robinson et al"). In addition, many3upernu- patients were uninformative even if they weretion 1 5q intact for these markers if the parent in
p=0.37 to observe two or more crossovers by chance from these six meioses.*Grandparental haplotypes inferred from sibs and cannot be designated grandmaternal or grandpaternal.tTwo additional cases with grandparents were informative distally but not for any proximal marker; one showed grandpaternalinheritance at D 15S 165 and one grandmaternal inheritance at D 15S 118.
Table 2 Summary of inheritance in informative AS deletion cases
p=0.002 to observe 3/3 crossovers by chance.*Seven additional cases with grandparents were informative distally but either uninformative or deleted for proximal markers; ofthese, five showed grandpaternal and two grandmaternal inheritance for markers flanking the distal deletion breakpoint.
question was homozygous at these loci. In one
case (PW-99), grandparents were not availablebut haplotypes were inferred from two sibs.Normal maternal and paternal inheritance of
microsatellite markers was observed outsidethe deleted region, thus excluding uniparentaldisomy in all cases. PWS-47 is identical topatient PWS-47 previously reported.'PWS-RN was previously published as HS2.37Cytogenetic analysis in all cases indicated thatthe deletions were interstitial and not associ-ated with a translocation or other chromosomalrearrangement. In addition, the fact that proxi-mal loci were intact excludes a deletion arisingfrom an unbalanced cryptic translocation.
DNA ANALYSISIsolation of genomic DNA from peripheralblood, restriction enzyme analysis, electro-phoresis, and Southern blotting were per-formed using standard procedures as describedpreviously.36 37 Probes used include pIR39(D15S18), IR4-3R (DI5S1 1), p3-21(D15SSO), and pCMW-1 (D15S24). PCRamplification of microsatellite loci was per-formed using standard conditions (usually55°C annealing temperature). A total of0.5-3 gl
of reagent was then mixed with an equal volumeof urea loading buffer (42% urea, 0.1% xylenecyanol, 0.1% bromphenol blue, and 0.1% of 0.5mol/l EDTA) and directly loaded onto a 0.4mm thick 6% polyacrylamide/50% urea gel.Visualisation of bands was done by silver stain-ing of the gels. Information on microsatelliteloci tested can be obtained from the GenomeData Base. All primers were obtained fromResearch Genetics Inc (Huntsville, AL).The probe IR39 (D15S18) maps proximal to
the common PWS/AS deletion in about 50% ofpatients'2 (unpublished data). Similarly, themicrosatellite loci D15S541 and D15S542,which map to a YAC containing D 15S 18, are
deleted in roughly half ofPWS or AS patients.'3The deleted region is about 4 Mb9 and covers
more than 10 cM.18 D 15S 1l is located withinthe commonly deleted region. The probeCMW-1, detecting the D15S24 locus, maps
outside (distal to) the deletion region in almostall patients' 3 (R D Nicholls, M Mascariunpublished data). D15S165 maps distal toD15S24 (R D Nicholls, unpublished results)and less than 5 cM from the distal breakpoint.D1 5S1048 has also been localised near toD15S165 based on recombination in CEPH
Table 3 Extent of duplication in one tandem duplication, two interstitial triplications, and 12 inv dupl5ql 1-q13 cases. The genetic distance of eachmarker in cMfrom D15S541 is indicated
D15S12t -17 nt dup* nt nt nt dup* dup* nt dup* dup* dup* dup* dup* N* ND15S1019 20 ui ui dup* dup* nt ui nt nt ui nt nt N* ui ui ND15S1048 20 ui ui dup* ui ui ui ui ui dup dup ui N N N uiD15S165 22 dup* ui ui ui dup* dup* dup ui dup nt dup N N N ND15S24 - nt dup* nt nt ui dup* ui nt dup* dup* dup* N N N ntD15S1031 22 dup* nt nt dup dup* dup nt ui dup* dup* dup* ui nt nt ntD15S976 23 ui ui dup* ui nt nt dup* dup dup* ui nt ui N ui uiD15S1043 23 ui dup* ui dup dup ui dup dup* ui ui ui N ui ui ND15S100 24 N* ui N ui dup nt ui N* ui ui n* nt ui ui uiD15S144 27 N ui N dup ui nt nt nt N ui N nt nt nt nt
*dup or N inferred by dosage of alleles only.tThe common deletion breakpoint lies just distal to D15S12.nt=not tested; dup (bold)=three distinct alleles were visible.N (bold)=only one allele from heterozygous mother was transmitted.
haplotype; PWS-47 has inherited the grandmaternal alleleon the father's haplotype at both D15S541 and D15S976.
and UPD15 families (W P Robinson, unpub-lished results). Sex specific genetic distancesgiven in table 1 are taken from CEPH/Ge'nethon online data and Robinson andLalande."8 CEPH based genetic locations aregiven in tables 2 and 3 for additional markerstested in this region.
Microsatellite loci were also used to deline-ate further the extent of deletions and duplica-tions in this region. Lack of transmission of apaternal or maternal allele in deletion cases wasconsidered evidence that this locus was in-cluded in the deletion, whereas heterozygosityin the patient indicated normal biparentalinheritance. In cases of duplication, the pres-ence of three distinct alleles was consideredconclusive evidence that this locus was in-cluded in the duplication, whereas a dosagedifference between alleles was considered sug-gestive of this. Dosage was measured qualita-tively by comparing the intensities of bands inparents and controls. At least two independentobservers judged dosage. If it was not clear toboth observers whether one band was dupli-cated, the result was recorded as uninforma-tive. Although this method cannot be consid-ered I100% accurate, it is strongly supportive ofthe presence or absence of duplication. Lack ofinheritance of both maternal alleles and anequal dosage of two alleles present in thepatient was taken as definite evidence that thislocus was not included in the duplication.
ID189
a,) aaab aa bc abcc ab
D15S1043 D 15S1G43
~j* " +03149 D188
cc acC.c a3 bc' at) acD15S165 D15SI65
Figure 3 Sample molecular results in inv dup 15 cases.ID7 appears to have a duplication at D15S1043 by dosageonly, whereas a duplication of this locus in ID189 is clearlyindicated by three distinct alleles. ID149 appears to have amaternal duplication at DISS165 by dosage, whereasID188 has inherited only one maternal allele in a singledose (as compared to the paternal allele).
ResultsSample molecular results are given in figs 2 and3. Haplotype data for all informative familiesare summarised in table 1. In four of fiveinformative PWS deletion cases with grandpar-ents available, markers flanking the paternaldeletion both proximally (Dl 5S541,D 15S542, D 15S 1035, or D15S 18) and distally(D15S144, D15S24, or D15S118) show inher-itance from the same grandparent (table 1). Intwo cases, the flanking markers showed originfrom the grandmother and, in the other twocases, the deletion arose on the grandpaternalhaplotype. Specifically, there was no evidenceof crossing over between the grandmaternaland grandpaternal haplotypes within the de-leted region. In contrast, case PWS-340showed grandpaternal inheritance forD1 5S1035 but grandmaternal inheritance dis-tal to the deletion breakpoint at D15S165.Thus, a crossover event between two father'schromosomes must have occurred either be-fore or during the deletion event. In the case ofPWS-99, the grandparents were not availableand haplotypes were inferred from the sibs.Two possibilities exist in this family: either thepatient has inherited a recombinant chromo-some from the father, or both of the sibs haveinherited a paternal recombinant chromosomein this region. Given a male recombination dis-tance of 17 cM between D15S541 andD15S165, the odds are 6:1 in favour of theformer (only one rather than two recombinantoffspring in this region). Furthermore, as thetwo normal sibs share the same paternal alleleat the proximal marker D15S541 and atGABRB3, the probability of the latter hypoth-esis is reduced as it would require a recombina-tion specifically between GABRB3 andD15S165 in both sibs. This case has thereforebeen classified as a probable recombinant.Even though two cases appear to show a
recombination between the markers flankingthe deletion, this does not exclude a post-meiosis I intrachromosomal origin in thesecases. To calculate the probability of observingchance recombinants from cases presented intable 1, one needs to account for the geneticdistance between markers in each informativecase. Assuming that the probability of observ-ing recombination between two markers corre-sponds to genetic distance (that is, 1 cM=1%recombination), the probability of not observ-ing any crossovers between the flanking mark-ers in these six cases if in fact normal levels ofmeiotic recombination precede the deletionevent is simply:
p(0/6 recombinant) = 1i (1-0j),where Oi is the recombination fraction betweeninformative flanking markers for case i, and Nis the total number of cases. Thus, p(0/6recombinant)= (1-0.23)*(1-0.17)*(1-0.17)*(1-0.27)*(1-0.26)*(1-0. 17)=0.24. Similarly,one can consider the probability that one of thesix cases would be recombinant: p(1/6 recom-binant)=0.39, or that two or more crossoverswould be observed: p( , 2/6 crossovers)=1-p(0/6 recombinant)-p(1/6 recombinant)=0.37.Therefore observing two recombinant cases is
well within the expected number if recombina-tion is completely independent of the deletionevent.The result is different in the case of the three
fully informative AS cases carrying maternaldeletions of 1 5ql1 -qi 3. The female recombi-nation distance in this region is slightly shorterthan in males, and the expected probabilities ofobserving 0/3, 1/3, 2/3, or 3/3 crossovers bychance (as calculated above) in the three casespresented in table 2 are 0.659, 0.295, 0.044,and 0.002, respectively. However, markerresults showed different grandparental originof haplotypes on either side of the deletionbreakpoints in all three cases. Thus, for the ASdeletions it is unlikely that the recombinationevents were the result of chance and are notassociated with the deletion (p=0.002).
It appears from tables 1 and 2 that there is nosignificant bias in terms of grandparental originof the chromosomes involved in the exchangeevent in either the maternal or paternaldeletions. Including additional cases whichwere informative for distal but not proximalmarkers, grandpaternal inheritance was seenfor markers flanking the deletion distally inthree of seven paternal and seven of 10 mater-nal deletion cases. The lack of bias ininvolvement of grandparental chromosomeswould seem to exclude a mechanism ofdeletion which specifically occurred before orduring resetting of the imprint on one or theother parental chromosome, unless the reset-ting of a paternal imprint as maternal and viceversa somehow involved exchange or associ-ation between homologues as part of the proc-ess, as has been previously suggested.38 9
To refine the distal breakpoints further,D15S165, D15S1043, D15S1019, D15S976,D15S1031, D15S10, D15S144, andD15S1048 have been examined in a subset of15 PWS or AS patients for inclusion indeletions of this region. None showed evidencefor uniparental inheritance of any of thesemarkers, with the exception of one unusualdeletion patient (PW-93) previously known tobe deleted distally for D15S24, but also notcarrying the same proximal breakpoint as com-mon deletions' (data not shown). The distalbreakpoint in PW-93 occurred betweenD15S24, D15S1048 (both deleted) andD 15S 1031, D 15S 1043 (both heterozygous).
In contrast to the common PWS/AS dele-tion, duplications of the PWS/AS regionfrequently included markers distal to D15S12.One tandem duplication patient and two intra-chromosomal triplication patients showed un-equal intensities of the two amplified alleles atseveral loci, which is consistent with inclusionin the duplications (table 3). For many of the12 inv dup 15q1 1-q13 patients examined,three distinct alleles were observed at distalloci, providing definitive proof of inclusion inthe duplication. Interestingly, a minimum offour different breakpoints were observed in thisregion: (1) distal to D15S144, (2) betweenD15S1043 and D15S1010, (3) betweenD15S12 and D15S1019 (equivalent to thecommon deletion breakpoint), and (4) be-tween GABRB3 and D15S12. Thus although
inv dup 15 chromosomes can generally bedistinguished as "large" (including thePWS/AS region) or "small" (excluding thisregion), there appears to be much morevariability in breakpoint location than is seenamong PWS/AS deletion patients.
DiscussionUnequal crossing over between homologouschromosomes at meiosis was excluded as themechanism of 15ql1l-qi 3 deletion formationin four of six deletions of paternal origin. Incontrast, all three informative AS cases showedevidence of recombination between markersflanking the deletion breakpoints, indicating anassociation with the deletion event. Theseresults are opposite to those observed forHNPP deletions, where haplotype analysisshowed the single maternal deletion case to beintrachromosomal in origin in contrast torecombination associated paternal deletions ofthe same region.27 Furthermore, the CMT1Aduplications (which are duplications of thesame region deleted in HNPP) are almost allpaternal in origin, in contrast to 1Sqll-q13duplications which are all maternal in origin.As deletion breakpoints do not appear todepend on parental origin in either case, thesex and region specific differences may simplyreflect differences in the resolution of certainrecombination intermediates rather than repre-senting completely distinct mechanisms. Stud-ies on the origin of the 7ql 1.23 deletion asso-ciated with Williams syndrome (WBS) showedevidence for a meiotic interchromosomalexchange between markers flanking the dele-tion in 22 of 27 cases.40 41 However, there wasno apparent bias in parental origin of thoseWBS deletions which were associated withexchange.
Several hypotheses should be consideredwhen considering the mechanism of microde-letion formation: (1) a premeiotic germlineorigin; (2) a meiotic event; or (3) an earlysomatic event.
(1) A premeiotic germline origin. Recurrenceof an interstitial deletion from the same parenthas never been reported for either PWS or AS(or any other interstitial microdeletion), mak-ing an early germline origin unlikely. Agermline origin would also be likely to cause apaternal bias owing to increased cell divisionsin spermatogenesis. However, the populationfrequency of maternal deletions ascertainedthrough AS and paternal deletions ascertainedthrough PWS are roughly equal (see introduc-tion).
(2) A meiotic origin. Meiotic recombinationin yeast is initiated by double strand breaksforming early in prophase. A chromosomeinstability leading to a high rate of non-homologous recombination would probablyalso involve double strand breaks and beexpected to show a high rate of homologousrecombination at the same sites. Studies ofunequal recombination between repeatedgenes in yeast show that those sequencesinvolved in the highest rate ofnon-homologous(unequal) exchange also show high rates ofhomologous exchange.42 However, no excess of
Mechanisms involved in formation of deletions and duplications of 15qll-ql13
meiotic recombination is apparent at thePWS/AS deletion breakpoint sites,'7 18 al-though the exact physical distance betweenD15S12 and D15S144 is not yet known. Theobservation that maternal and paternal dele-tions occur with equal frequency and involvethe same breakpoint sites 13 is also apparentlyinconsistent with the striking sex specificdifferences in meiotic recombination in thisregion. 18The above arguments do not exclude a mei-
otic origin, only suggest that we may not com-pletely understand the mechanism yet. There isevidence in yeast that exchange can occur dur-ing meiosis which is independent of exchangeassociated with functional chiasmata (see, forexample, Hawley and Arbel43). Similarly, stud-ies in Drosophila show evidence of recombina-tion events owing to recombinatorial repair oftransposon induced breaks." Perhaps thePWS/AS deletions are instead the result of animperfect repair mechanism invoked to repairpost-recombinational breaks that tend to occurwithin this region. The paternal intrachromo-somal versus maternal interchromosomal dif-ference could be the result of sex specificdifferences in use of sister chromatids versushomologous chromosomes for repair or ofhowthe recombination-repair intermediate is re-solved. However, because of the small numberof informative cases, it is not yet possible todetermine if the ratio of intra- to interchromo-somal events is significantly different in PWSversus AS deletion cases.
If a classical meiotic unequal crossing overfully explained the occurrence of the maternaldeletions, we might expect to observe recipro-cal duplications of this region. Althoughmaternal duplications of this region are com-monly observed, an examination of distalbreakpoints in interstitial duplications/triplications of the commonly deleted PWS/ASregion indicates a difference. The commondeletion breakpoint occurs immediately distalto D15S129 and rarely includes D15S241'
(present results). A YAC contig of the PWSregion shows that D 1 5S24 lies on a YAC whichdoes not overlap the YAC spanning the distaldeletion breakpoint.9 D15S24 was, however,shown by FISH or molecular dosage analysis tobe included in two duplication patients20 andthree tandem triplication patients.2223 In addi-tion, results for one tandem duplication andtwo tandem triplication patients (including thecase of Schinzel et al") presented here showedunequal intensities of the amplified alleles con-sistent with duplication of markers distal to thecommon deletion (table 2). Therefore, theduplicated region in all of seven duplication ortriplication cases most probably extendedfurther than the common deletion.
Furthermore, we were able to identify aminimum of four breakpoints among inv dup15(ql 1-ql 3) chromosomes which included thePWS/AS critical regions, one of which corre-sponds to the common distal deletion break-point. Previous studies using FISH had identi-fied only two of these breakpoints.33 Thus,although inv dup 15 chromosomes can gener-ally be distinguished as "large" (including the
PWS/AS region) or "small" (excluding thisregion), there appears to be much morevariability in breakpoint location than is seenamong deletion patients. Perhaps the recombi-nation events leading to duplication anddeletions are the result of the same initiatingmechanism, for example, a chromosome break-age, but the resolution as a duplication versus adeletion is influenced by which distal se-quences are used for repair. As a series ofrepeated sequences at the proximal and distalbreakpoint regions have been identified in thisregion,33 45 46 it may be that the location andorientation of specific repeats determineswhether the recombination event will result indeletion, duplication, or inversion duplication.
(3) A post-meiotic origin. A post-meioticorigin also remains possible for at least somedeletions and duplications. Mosaicism of15q1 1-q13 deletions has been observed inassociation with hypomelanosis of Ito.'7 48 Thepresence ofmosaicism for 1 5q 1 -q 1 3 deletionswithin PWS patients has also beensuggested,6 49 although these results suffer frommethodological errors and are not convincing.However, observable mosaicism is not neces-sary to postulate a post-meiotic origin. Asomatic origin of non-disjunction has beeninferred in multiple instances of non-mosaictrisomy and uniparental disomy.50 5' It has alsobeen proven that post-meiotic events are com-mon in the formation of homologous Robert-sonian translocations and isochromosomes,despite the lack of any observed mosaicism,52and has also been observed for de novorearrangements53 including PWS/AS imprint-ing centre deletions.'5 Furthermore, the mater-nal and paternal 1qll-qi3 homologues ap-pear to "pair" in late S phase of the mitotic cellcycle in lymphocytes."9 The mechanism andfunction of this somatic "pairing" is unknown.However, it may provide a means to bring thetwo chromosomes 15 near enough to facilitatea mitotic recombination event.Although there is an abundance of repetitive
sequences in the human genome, recombina-tion between repeats in non-homologous sitesis not common. The observation of recurrentde novo deletions therefore implies severalcharacteristics of chromatin at the breakpointsites in addition to assuming that there existssome degree of homology between breakpointsites. The proximal or distal deletion break-points or both must be sites prone to single ordouble strand breaks or otherwise susceptibleto recombination/repair at some point in thecell or developmental cycle. The high rate ofexchange between sequences 4-5 Mb apartalso implies some physical proximity of thesesequences in the nucleus, which can potentiallybe achieved simply by the nature of chromatinpackaging in the region. Clearly more familiesneed to be studied to determine if there is aparent of origin effect on the frequency ofintra- versus interchromosomal involvement.Nonetheless, the initial observations indicateheterogeneity in the specific mechanism in-volved in the origin of 15ql1-qi3 rearrange-ments, including maternal and paternal dele-tions and maternal duplications, triplications,
and inv dup 15q11-q13. Although a simpleunequal crossing over mechanism may beinvolved, it is not sufficient to explain all thedata. Further investigations including identifi-cation of repetitive sequences in the proximaland distal regions are needed to determine theexact cause of instability at the breakpoint sites.
We wish to thank the families and physicians for their coopera-tion in contributing blood samples to this study. This work wassupported primarily by Swiss NF grant No 32-37798.93 (AS);part of this work was performed in 1988 by RDN in the labora-tory of the late Professor S A Latt. RDN was supported by aresearch grant from March of Dimes Birth Defects Foundation.
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