Hum Genet (2006) 120:126–138

DOI 10.1007/s00439-006-0209-y

ORIGINAL INVESTIGATION

Characterization of the human lineage-specific pericentric inversion that distinguishes human chromosome 1 from the homologous chromosomes of the great apes

Justyna M. Szamalek · Violaine Goidts · David N. Cooper · Horst Hameister · Hildegard Kehrer-Sawatzki

Received: 10 April 2006 / Accepted: 16 May 2006 / Published online: 15 June 2006© Springer-Verlag 2006

Abstract The human and chimpanzee genomes aredistinguishable in terms of ten gross karyotypic diVer-ences including nine pericentric inversions and a chro-mosomal fusion. Seven of these large pericentricinversions are chimpanzee-speciWc whereas two ofthem, involving human chromosomes 1 and 18, wereWxed in the human lineage after the divergence ofhumans and chimpanzees. We have performed detailedmolecular and computational characterization of thebreakpoint regions of the human-speciWc inversion ofchromosome 1. FISH analysis and sequence compari-sons together revealed that the pericentromeric regionof HSA 1 contains numerous segmental duplicationsthat display a high degree of sequence similaritybetween both chromosomal arms. Detailed analysis ofthese regions has allowed us to reWne the p-arm break-point region to a 154.2 kb interval at 1p11.2 and the q-arm breakpoint region to a 562.6 kb interval at 1q21.1.

Both breakpoint regions contain human-speciWc seg-mental duplications arranged in inverted orientation.We therefore propose that the pericentric inversion ofHSA 1 was mediated by intra-chromosomal non-homologous recombination between these highlyhomologous segmental duplications that had them-selves arisen only recently in the human lineage byduplicative transposition.

Introduction

The human and chimpanzee karyotypes are distin-guishable by virtue of nine pericentric inversions ofchromosomes homologous to HSA 1, 4, 5, 9, 12, 15,16, 17 and 18 as well as the fusion that gave rise tohuman chromosome 2 (Yunis and Prakash 1982;Gross et al. 2006). Seven of these inversions wereWxed in the chimpanzee lineage (reviewed in Kehrer-Sawatzki et al. 2005a). Comparative breakpoint anal-yses have shown that the two sister chimpanzee spe-cies, Pan troglodytes and Pan paniscus, share thesame pericentric inversions of these seven chromo-somes (Locke et al. 2003; Szamalek et al. 2006a).These inversions must therefore have predated theseparation of the two chimpanzee species »0.86 to2 Mya (Yoder and Yang 2000; Won and Hey 2005).Given that these seven inversions occurred speciW-cally in the chimpanzee lineage, it may be presumedthat the corresponding human and orangutan chro-mosomes resemble the respective ancestral hominoidchromosomes (Dutrillaux 1979; Yunis and Prakash1982; Müller and Wienberg 2001). By contrast, theinversions that distinguish human chromosomes 1 and18 from their respective chimpanzee homologues (as

Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at http://dx.doi.org/10.1007/s00439-006-0209-y.

Justyna M. Szamalek and Violaine Goidts are contributed equally to the paper.

J. M. Szamalek · V. Goidts · H. Hameister · H. Kehrer-Sawatzki (&)Department of Human Genetics, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germanye-mail: hildegard.kehrer-sawatzki@medizin.uni-ulm.de

D. N. CooperInstitute of Medical Genetics, CardiV University, Heath Park, CardiV, CF14 4XN, UK

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Hum Genet (2006) 120:126–138 127

well as those of the gorilla and orangutan) representhuman lineage-speciWc rearrangements (Yunis andPrakash 1982; McConkey 1997; Dennehey et al. 2004;Goidts et al. 2004; Weise et al. 2005).

Most of the nine pericentric inversions have nowbeen subjected to detailed molecular characterization(Nickerson and Nelson 1998; Marzella et al. 2000; Keh-rer-Sawatzki et al. 2002, 2005a, b, c; Locke et al. 2003;Dennehey et al. 2004; Goidts et al. 2004, 2005; Shimadaet al. 2005; Szamalek et al. 2005) and only the break-points of the relatively small chromosome 1 inversionremain to be reWned. As may be deduced from thecomparative ideogram proposed by Yunis and Prakash(1982), this inversion encompasses the pericentromericregion of HSA 1. This has recently been conWrmed byWeise et al. (2005) who showed by comparative FISHanalysis, using BAC RP11-35B4, that the pericentricinversion occurred between contigs NT 004754 and NT032962, corresponding to the region between positions119,538 and 148,790 Mb of human chromosome 1. ThisBAC clone detects numerous intra-chromosomalduplications but the observed FISH pattern suggeststhat the segmental duplications detected by this BACon q-arm are located within the inverted region onHSA 1 (Weise et al. 2005).

We report here the detailed molecular and compu-tational characterization of the breakpoint regions ofthe HSA 1 inversion. The proximal breakpoint wasnarrowed down to a 154.2 kb region on HSA 1p11.2and the distal breakpoint to a 562.6 kb region on HSA1q21.1. Both these regions are rich in segmental dupli-cations and hence, unsurprisingly, also contain unse-quenced gaps. We propose that this human-speciWcpericentric inversion was mediated by non-homolo-gous recombination between two 91 kb inverted dupli-cations, both of which represent human-speciWc gains.

Materials and methods

Cell lines

The P. troglodytes lymphoblastoid cell line PTR-EB176 (ECACC No. 89072704) was purchased fromthe European Collection of Cell Cultures (http://www.ecacc.org.uk). The P. troglodytes lymphoblas-toid cell line PTR-L2008 was a generous gift fromDr W. Schempp (University of Freiburg, Germany).The lymphoblast cell line GM03446 from Macacafascicularis (crab-eating macaque) was obtained fromthe Coriell Cell Repository. Human chromosomalslides were prepared from cultured blood samples fromnormal human donors.

Fluorescence in situ hybridization

BAC and PAC clones listed in Tables 1 and 2 werepurchased from the BACPAC Resource Center(http://www.bacpac.chori.org). DNA was isolatedfrom the respective clones with the Qiagen-Midi-Kit(Qiagen, Hilden, Germany) and used as FISHprobes. At least 1 �g BAC/PAC-DNA was labelledeither with biotin-16-dUTP (Roche-Diagnostics,Mannheim, Germany) and detected with FITC-avi-din and biotinylated anti-avidin (Vector, Burlin-game, USA) or labelled with digoxygenin-11-dUTP(Roche-Diagnostics) and detected by mouse anti-digoxygenin. In a second step, rabbit anti-mouseantibodies coupled with Texas-Red were used, fol-lowed by anti-rabbit antibodies also conjugated withTexas-Red (Dianova, Hamburg, Germany). Slideswere counterstained with diamidinophenylindole(DAPI) and mounted with Vectashield antifade solu-tion (Vector).

Table 1 Fluorescence in situ hybridization analyses designed to narrow down the inversion breakpoint on HSA 1p

a Ensembl version 37.35j based on the NCBI Build 35 assembly; the most intensive signals of the respective probes are indicated in boldb According to the combined results of FISH analyses and sequence alignmentsc On the basis of the »91 kb sequence homology of BAC RP11-439A17 to the q-arm PAC RP5-998N21, it was concluded that non-allelichomologous recombination between these inverted repeats probably mediated the inversion

BAC/PAC Chromosomal position

Map position in bpa FISH signals on Position with respect to the inversionb

HSA PTR

RP11-192J8 1p12 118,079,902–118,243,631 p12 p12 Not invertedRP4-712E4 1p12 119,194,364–119,316,485 p12 p12 Not invertedRP4-683H9 1p12 119,979,355–120,045,236 p36, p12, q21 p36, p12 Not invertedRP5-1042I8 1p12 120,045,137–120,183,091 p36, p12, q21 p36, p12 Not invertedRP11-114O18 1p11.2-12 120,288,704–120,395,759 p11–12, q21 p11-12 Not invertedRP11-439A17c 1p11.2 120,459,199–120,648,737 p11, q12-21, q32 q21, q32 In breakpoint regionRP11-343N15 1p11.2 120,698,738–120,794,484 p11, q21, q32 p11, q21, q32 InvertedRP11-435B6 1p11.1–11.2 120,956,393–121,097,476 p11 Centromere Inverted

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128 Hum Genet (2006) 120:126–138

Sequence alignments and database analysis

Sequences alignments were performed using GenAlyzersoftware (Choudhuri et al. 2004), Miropeat software(Parsons 1995) and BLAST analyses (http://www.ncbi.nlm.nih.gov/). The Wisconsin Package, version 10.2(Genetics Computer Group) was also used forsequence comparisons. Breakpoint regions were analy-sed by reference to the Ensembl database v 37.35jbased on NCBI Build 35 and the chimpanzee draftsequence (Mikkelsen et al. 2005). BAC/PACs contain-ing segmental duplications were analysed using theHuman Genome Segmental Duplication Database(http://www.projects.tcag.ca/humandup/) based onNCBI Build 35. Repeat masking was carried out usingthe RepeatMasker server from EMBL, Heidelberg(http://www.woody.embl-heidelberg.de/repeatmask).

Results

Computational comparison of the pericentromeric region between HSA 1 and PTR I

The comparison of the pericentromeric regions of HSA 1and PTR I using the GenAlyzer sequence alignment toolindicated the presence of numerous duplicated sequenceswith homologues on both 1p and 1q (Fig. 1a). To distin-guish between inverted sequences and intra-chromosom-ally duplicated sequences, the latter were excluded fromthe alignments; only unduplicated sequences larger than1 kb were considered. According to these comparisons,

the inversion breakpoint on HSA 1p maps to a »538 kbregion between 120,240,975 and 120,778,956 bp at1p11.2-12 whereas the q-arm breakpoint is locatedwithin a »4 Mb region at 1q21.1-21.2 between positions142,657,563 and 146,690,980 bp (Fig. 1b).

FISH analysis of the inversion using BAC/PACs that do not span segmental duplications

In order to conWrm the results obtained by sequencealignment, comparative Xuorescence in situ hybridiza-tion (FISH) was performed on human and chimpanzeechromosomes (Tables 1, 2). In accordance with theresults of the in silico analyses, HSA PAC clone RP4-712E4 from 1p12 and BAC clone RP11-458I7 from1q21.2 were shown not to be inverted (Fig. 1C). The»22 Mb inverted region (between 120,778,956 and142,657,563 bp) contains numerous segmental duplica-tions. This notwithstanding, the respective regionswere screened to identify interspersed unduplicatedsequences that would facilitate unambiguous FISH-based breakpoint mapping. By these means, we identi-Wed BACs RP11-30I17, RP11-337C18 and RP11-314N2which form part of contigs NT_004434 and NT_034400in HSA 1q21.1. FISH analysis then revealed that theseBACs are inverted since they hybridize at HSA 1q21but at PTR Ip11-12 (RP11-314N2, Fig. 1C; Table 2).

Taken together, these FISH analyses allowed us tonarrow down the q-arm breakpoint to a »2 Mb regionbetween 144,630,713 bp (map position of BAC RP11-314N2) and 146,690,980 bp. All BAC/PAC cloneslocated within this »2 Mb region on HSA 1q21.1-21.2, as

Table 2 Fluorescence in situ hybridization analyses designed to narrow down the inversion breakpoint in HSA 1q

a Ensembl version 37.35j based on the NCBI build 35 assembly; the most intensive signals of the respective probes are indicated in boldb According to the combined results of FISH analyses and sequence alignmentc On the basis of the »91-kb sequence homology of PAC RP5-998N21 to the p-arm BAC RP11-439A17, it was concluded that non-allelichomologous recombination between these inverted repeats probably mediated the inversion

BAC/PAC Chromosomal position

Map position in bpa FISH signals on Position with respect to the inversionb

HSA PTR

RP11-30I17 1q21.1 142,951,739–143,139,578 q21 p11-12 InvertedRP11-337C18 1q21.1 143,826,832–144,029,786 q21 p11-12 InvertedRP11-314N2 1q21.1 144,490,622–144,630,713 q21 p11-12 InvertedRP3–328E19 1q21.1 145,043,332–145,140,950 p36, p11-12, q21 p36, p11-12 InvertedRP11-763B22 1q21.1 145,513,740–145,671,828 p36, p11-12, q21 p36, p11-12 InvertedRP11-14N7 1q21.1 145,669,829–145,727,426 p11-12, q21 p11-12, q21 In breakpoint regionRP11-744H18 1q21.1 145,784,053–145,890,590 p36, p11-12, q21 p36, p11-12, q21 In breakpoint regionRP11-403I13 1q21.1 145,888,591–146,093,292 p36, p11-12, q21 p36, p11-12, q21 In breakpoint regionRP5-998N21c 1q21.1 146,143,293–146,272,718 p11, q21, q32 q21, q32 In breakpoint regionRP11–277L2 1q21.1 146,322,719–146,401,747 p36, p11-12, q21 p36, p11-12, q21 In breakpoint regionRP11-353N4 1q21.1-2 146,399,748–146,524,913 p36, p11-12, q21 p36, p11-12, q21 In breakpoint regionRP11-196G18 1q21.2 146,522,914–146,711,869 p11-12, q21 q21 Not invertedRP11-21J4 1q21.2 146,606,778–146,787,659 p11-12, q21 q21 Not invertedRP11-458I7 1q21.2 146,785,068–146,973,278 q21 q21 Not invertedRP11-363I22 1q21.2 147,432,838–147,586,905 p36, q21 p36, q21 Not inverted

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well as within the »538 kb region between 120,240,975and 120,778,956 bp on HSA 1p11.2-12, within which theinversion breakpoints must have occurred, contain seg-mental duplications.

Inversion breakpoint mapping by FISH and sequence comparisons using BAC/PACs that span the segmental duplications

To reWne further the breakpoint regions, we investi-gated several BAC/PACs that map to the abovemen-tioned breakpoint intervals and which containsegmental duplications.

Breakpoint on HSA 1p

Clones RP4-683H9, RP5-1042I8 and RP11-114O18map to HSA 1p11-12 in Ensembl (v37.35j; Build 35)but FISH also revealed additional signals on chromo-some 1 (summarized in Table 1) due to segmentalduplications detected by these BAC/PACs. GenAlyzerand BLAST analyses indicated that the “original” loca-tions of these clones mapping to HSA 1p11-12 are onthe p-arm of PTR I and cannot therefore be inverted.Accordingly, all three clones displayed FISH signalsonly on the p-arm of PTR I. From the map position ofBAC RP11-114O18 on HSA 1p and the results of the

Fig. 1 GenAlyzer display of homologous sequences between hu-man and chimpanzee chromosome 1 in the pericentromeric re-gions (118–147 Mb according to the NCBI Build 35). The redhorizontal bars represent the centromeres and heterochromatin.Homologous sequences between PTR and HSA are indicated asvertical or diagonal lines. a Homologous sequences between PTRI and HSA 1 ranging from 30 to 2,029 bp according to the colorscale below which indicates the length of the respective segments.b Homologous sequences ranging from 1,000 to 2,029 bp after

exclusion of the duplicated sequences. According to these analy-ses, the p-arm inversion breakpoint maps to the region between120,240,975 and 120,778,956 bp, whereas the q-arm breakpoint islocated between 142,657,563 and 146,690,980 bp. c Clones RP4-712E4 and RP11-458I7 localize outside the putative inversion re-gion on HSA 1 and hybridized to the same chromosomal arm inHSA and PTR. BAC RP11-314N2 maps within the inverted re-gion since it hybridized at HSA 1q21 but in chimpanzee at Ip11-12. The white arrows indicate the positions of the centromeres

PTR I

HSA 1

RP4-712E4 RP11-314N2 RP11-458I7

PTR I

HSA 1

A

B

120,778,956

PTR I

HSA 1

C

p

p

p

p

q

q

q

q

30 230 430 630 830 1030 1230 1430 1630 1830 2029

142,657,563 146,690,980120,240,975bp

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sequence alignments, the p-arm breakpoint was nar-rowed down to the 1p11.2 region, between positions120,395,759 and 120,778,956 bp.

BAC RP11-343N15 maps to the proximal boundaryof this 382 kb region and hybridizes both to HSA 1p11,1q21, 1q32 and to orthologous regions on PTR I. Owingto the segmental duplications detected by this BAC,FISH analyses per se would not have been informative.However, taken together with detailed sequence analy-sis, RP11-343N15 turned out to be informative withrespect to breakpoint mapping. In addition to the intra-chromosomally duplicated sequences, RP11-343N15also spans a unique 19 kb segment that has not beenduplicated on chromosome 1 (1,789–20,655 bp ofAL358175). Sequence alignments of this 19 kb regionindicated that it is inverted in chimpanzee as comparedto human. Thus, RP11-343N15 must be located withinthe boundaries of the HSA 1 inversion.

The completely sequenced BAC RP11-435B6 fromHSA 1p is located very close to the centromere in theEnsembl database and contains 92.94% alpha satelliteDNA. Employing FISH, only faint signals from thisBAC were noted in the vicinity of the centromere onPTR I, indicative of the weak conservation of chromo-some 1-speciWc alpha-satellite sequence betweenhumans and chimpanzees.

BAC RP11-439A17 played an important role withrespect to the mapping of the HSA 1p breakpoint, whichis localized between inverted and non-inverted clones(Table 1). Unfortunately, this BAC is unconnected toXanking sequence contigs and is instead surrounded bysequence gaps. RP11-439A17 detects a human-speciWcsegmental duplication as determined by FISH per-formed in this study (Fig. 2a, b) and array CGH as previ-ously described (Goidts et al. 2006). Three signals wereobserved on human chromosome 1, at p11, q12-21 andq32 but only two signals were noted on PTR Iq inregions orthologous to HSA 1q21 and 1q32. It was notpossible solely on the basis of this FISH result to catego-rize the BAC as mapping either inside or outside theinverted region. However, since this BAC is directlyXanked by inverted and non-inverted regions and exhib-its high sequence homology to PAC RP5-998N21 (whichmaps to the breakpoint region on HSA 1q), we mayassume that the inversion breakpoint on HSA 1p islocated either within BAC RP11-439A17 or within theadjacent unsequenced gaps.

Breakpoint on HSA 1q

Fluorescence in situ hybridization and sequence align-ments were employed to investigate several BAC/PACclones mapping to the »2 Mb breakpoint interval on

HSA 1q. Although PAC RP3-328E19 and BAC RP11-763B22 span segmental duplications, FISH was infor-mative since the signal patterns diVered betweenhuman and chimpanzee. Both clones displayed stronghybridization signals on PTR Ip11-12 but hybridizedon both chromosomal arms in human (1p12 and 1q21;RP11-763B22, Fig. 2c, d). We therefore conclude thatthe loci detected by these clones on 1q21 are invertedby comparison with PTR I.

BAC RP11-196G18 was also informative withrespect to breakpoint mapping, exhibiting 46 kb ofsequence homology with BAC RP11-439A17 fromHSA 1p11.2. This 46 kb region was speciWcally dupli-cated in the human lineage as evidenced by array CGH(Goidts et al. 2006). FISH analysis with RP11-196G18and the overlapping BAC RP11-21J4 conWrmed theduplication, since in addition to a strong signal on HSA1q21, a signal at 1p11-12 was noted (RP11-196G18,Fig. 2e). Since this duplication is absent from PTR,BACs RP11-196G18 and RP11-21J4 hybridized exclu-sively to PTR Iq21 (RP11-196G18, Fig. 2f). Since the‘original’ locus maps to the q-arm in both human andchimpanzee, we conclude that the q-arm inversionbreakpoint must be located proximal to the region cov-ered by BAC RP11-196G18 on HSA 1q21 (Table 2). Insummary, the FISH patterns of BACs RP11-196G18and RP11-763B22 delimit the inversion breakpoint onHSA 1q to the 851 kb interval between positions145,671,828 and 146,522,914 bp.

Subsequently, we investigated BAC/PAC clonesfrom this interval and noted that, with the exception ofPAC RP5-998N21, all of them hybridize to both p andq arms in HSA and PTR (Table 2). Surprisingly, PACRP5-998N21, which is not anchored on either side toany sequence contig, exhibits very similar hybridiza-tion pattern to BAC RP11-439A17 located within thep-arm breakpoint region. The similar FISH patternsdisplayed by RP11-439A17 and RP5-998N21 are indic-ative of a 91 kb segmental duplication. According tothe Human Genome Segmental Duplication Database,the duplication covered by RP5-998N21 is termedDC0215 and shares > 99% sequence homology withDC0154 which is covered by RP11-439A17.

Proposed mechanism underlying the chromosome 1 inversion

Miropeat analysis (Parsons 1995) indicated manyregions of high sequence homology between the peri-centromeric regions of HSA 1p and 1q (Fig. 3). As men-tioned above, segments of high homology (> 99%)between the p- and q-arm breakpoint-spanning regionsare covered by clones RP11-439A17 and RP5-998N21.

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It is highly likely that these 91 kb segmental duplicationsmediated, or at least facilitated, the inversion (Fig. 4).

These 91 kb segmental duplications are absent in thechimpanzee and we may therefore conclude that theyarose after the divergence of the human and chimpan-zee lineages.

A model to explain how these segmental duplica-tions could have arisen during human evolution is pre-sented in Fig. 4. In the chimpanzee, sequences withhomology to portions of human clones RP5-998N21and RP11-439A17 map to PTR Iq21 and Iq32, respec-tively (Fig. 4a). Two duplicative transposition eventsoccurred in the ancestral forms of HSA 1 (Fig. 4b, c)thereby giving rise to the highly homologous 91 kb seg-mental duplications located in inverted orientation onthe p- and q-arms of the ancestral chromosome 1(Fig. 4c). It would appear likely that the high degree of

sequence homology between these inverted repeatsfacilitated non-allelic pairing of chromosomal regions1p11 and 1q21, thereby mediating the intra-chromo-somal recombination event (Fig. 4d). According to thismodel, based both on FISH data and sequence com-parisons, the inversion breakpoints map between120,395,759 and 120,549,995 bp on HSA 1p andbetween 145,671,828 and 146,234,395 bp on HSA 1q.

In order to simplify the model, we have indicated onHSA 1q21 only the 91 kb duplicon, covered by BACRP11-439A17, that potentially mediated the inversion(Fig. 4). There are however three further duplicons of10, 46 and 73.5 kb covered by this BAC which displayhomology to sequences on HSA 1q12-21. It is theseadditional duplicons that were probably responsiblefor the intensity of the FISH signal generated by BACRP11-439A17 on HSA 1q (Fig. 2a).

Fig. 2 Fluorescence in situ hybridization (FISH) analysis performed with human BACs from the pericentromeric re-gion containing segmental duplications. a, b BAC RP11-439A17 from HSA 1p covers a human-speciWc segmental duplication located at HSA 1p11, and paralogous se-quences in 1q12-21 and 1q32. In PTR, no signal was ob-served on the p-arm. c, d BAC RP11-763B22 maps to 1q21 according to Ensembl (v37.35j based on NCBI Build 35) and detects segmental duplica-tions located at 1p36 and 1p11-12 in human (c) and chimpanzee (d). This BAC displayed signals only on PTR Ip. In humans, this BAC maps to within the inverted region since the main signal was lo-cated at 1q21 (e, f). The undu-plicated portion of BAC RP11-196G18 hybridized at 1q in humans and chimpan-zees and is thus not inverted in humans

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Evolutionary history of the HSA 1 pericentric inversion

In addition to the pericentric inversion of HSA 1, a fur-ther large pericentric inversion of this chromosomeoccurred during primate evolution. Comparative FISHanalyses indicated that the chromosome I homologueof the Old World monkeys diVers from the analogouschromosome in great apes by a second pericentricinversion (Buckland et al. 1992; Maresco et al. 1998;Ruiz-Herrera et al. 2002). To verify these Wndings andto investigate the question of breakpoint position simi-larity or heterogeneity, we performed FISH withBACs RP11-314N2 and RP11-458I17. In the macaque(MFA), an Old World monkey, both BACs map toMFA 1p (Fig. 5a). The Wrst pericentric inversionoccurred after the separation of the Old World mon-keys from the lineage leading to the great apes andhumans. As a consequence, BAC RP11-458I7 isinverted in PTR Iq (Fig. 5b). The second pericentricinversion is conWned to the human lineage and wasintroduced after the separation from the chimpanzeelineage. This second inversion includes the region cov-ered by BAC RP11-314N2 which maps to the q-arm inhumans (Fig. 5c). Thus, we may conclude that the twoinversions which have occurred during the evolution-ary history of chromosome 1 have diVerent break-points (Fig. 5d). As a consequence of the Wrstinversion, the FCGR1 gene became inverted in great

apes by comparison with Old World monkeys (Mare-sco et al. 1998). During human evolution, two duplica-tions then gave rise to the FCGR1A, FCGR1B andFCGR1C genes, which are separated by the secondinversion that occurred speciWcally in the human line-age. From these comparisons, it may be inferred thatthe breakpoints of both inversions are separated by aminimum of several hundred kilobases.

Analysis of the gene content in the inversion breakpoint regions

The inversion breakpoint regions were assessed in termsof their gene content (Supplementary Table 1). Owing tothe 91 kb sequence homology between BAC RP11-439A17 and PAC RP5-998N21, the genes encompassedby these clones represent duplicated, human-speciWc cop-ies. Among them are genes encoding the human high-aYnity receptors for immunoglobulin G (FCGR1B,FCGR1C), the histone 2 pseudogenes, as well as theFAM72A and FAM72C genes (family with sequencesimilarity 72, members A and C). In the region coveredby BAC clones RP11-14N7, RP11-744H18 and RP11-403I13 on the q-arm, mainly tRNA pseudogenes andgenes encoding spliceosomal RNA were noted. Since theexact nucleotide positions of the breakpoints areunknown, we cannot establish whether any codingsequence was disrupted by the inversion.

Fig. 3 Comparison of the pericentromeric region of HSA 1 usingMiropeat software (Parsons 1995). The input sequences fromHSA 1p and HSA 1q are represented as horizontal lines; »3 Mbfrom the p-arm were compared to »4 Mb from the q-arm. Thehomologies between the p- and q-arms are depicted as connectorsbetween both input sequences. Each connector symbolizes at

least 2 kb in length. BAC RP11-439A17 from HSA 1p exhibits se-quence homology with PAC RP5-998N21 and BAC RP11-196G18 of 91 and 46 kb, respectively. PAC RP5-998N21 is locat-ed within the q-arm breakpoint region (indicated with horizontalrectangle), whilst RP11-196G18 maps outside the inversion, asshown by FISH analyses

p

qCentromere

118 Mb 121 Mb

143 Mb – 147 Mb

RP11-439A17

RP5-998N21

91 kb

46 kb

RP11-196G18

91 kbCentromere

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Discussion

Human chromosome 1 constitutes the largest physicalunit of the human karyotype, accounting for approxi-mately 9% of the human genome (White and Matise2003). It encompasses 245.5 million base-pairs and con-tains 2,062 known protein coding genes (Ensembl, ver-sion 37.35j, NCBI 35 assembly). HSA 1 is rich insegmental duplications (She et al. 2004), particularlywithin the pericentromeric regions (Bailey et al. 2001,2002; Cheung et al. 2003). During human evolution,chromosome 1 acquired a block of pericentromericheterochromatin in the proximal q-arm (1qh), which isabsent in the chimpanzee homologue. The 1qh (alsotermed 1q12) heterochromatic region displays homol-ogy to 2p11 due to the presence of numerous paralo-gous sequences (Horvath et al. 2000).

HSA 1 constitutes an ancestral chromosomal entitysince a single chromosome 1 homologue has been identi-

Wed in phylogenetically quite distant taxa (Murphy et al.2003). Although the major portion of HSA 1 represents aconserved linkage group that was present in the mostrecent common ancestor of eutherian mammals, a 4 Mbregion with homology to mouse chromosome 11 wasadded after the separation of the primate lineage from itscommon ancestor with rodents (Haig 2005). Two largepericentric inversions of chromosome 1 then occurredduring primate evolution. The Wrst of these made itsappearance in the common ancestor of the great apesand now serves to distinguish chromosome 1 of OldWorld monkeys from its homologues in great apes andhuman (Dutrillaux 1979; Buckland 1992, Maresco et al.1998, Ruiz-Herrera et al. 2002). A second inversion ofchromosome 1 however occurred speciWcally in thehuman lineage, after the divergence of humans and chim-panzees (Yunis and Prakash 1982; Weise et al. 2005).

In this study, we have narrowed down the breakpointregions of the human-speciWc pericentric inversion of

Fig. 4 Model to explain the pericentric inversion of HSA 1.a Schematic representation of PTR I homologous to HSA 1. Thered and blue striped boxes indicate sequence homologous to thehuman BAC RP11-439A17 and PAC RP5-998N21, respectively.Their orientation is indicated by arrows. A duplication followedby a transposition from 1q32 to 1q21 occurred after the separa-tion of the human and chimpanzee lineages. b Subsequently, a91 kb segment has been duplicated and transposed in an invertedorientation to the p-arm of the ancestral form of HSA 1. c Thehigh degree of similarity (> 99%) conferred by the inverted re-peats may have brought the chromosomal regions 1p11.2 and1q21 together, thereby mediating an intra-chromosomal recombi-

nation event. d Extant human chromosome 1. The red and bluevertical lines represent the p-and q-arms respectively. Red andblue numbers indicate the positions within a BAC, in kb, whilstthe black numbers refer to the positions within the Human Ge-nome Assembly (NCBI Build 35). The dotted lines denote the re-gion of the putative position of the inversion breakpoints.According to our inversion model, clones RP11-439A17 andRP5-998N21 are located in inverted orientation by comparisonwith the data given by Ensembl Database (v 37.35j; based onNCBI Build 35), where neither clone is anchored to any contigwithin the chromosome 1 assembly. The position of FCGR1genes is schematically depicted

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134 Hum Genet (2006) 120:126–138

chromosome 1 by a combination of FISH and genomicalignments between the human reference sequence andthe chimpanzee draft sequence. According to oursequence alignment data and FISH analysis, this rear-rangement is likely to be a pericentric inversion ratherthan a shift in the position of the centromere. We haveshown that a 19 kb segment of BAC RP11-343N15 fromHSA 1p is located in inverted orientation on PTR 1q(123,919,082–123,937,415 according to the PanTro 1.0,Nov 2003 assembly). Furthermore, a 28 kb regionhomologous to BAC RP11-782C8 from HSA 1q wasdetected in inverted orientation on PTR I at(123,028,935–123,062,479), proximal to the 19 kb seg-ment of BAC RP11-343N15. These Wndings suggestthat an inversion rather than centromeric repositioninghas been responsible for the rearrangement that nowdistinguishes HSA 1 from the homologous chromosomein the great apes.

The p-arm inversion breakpoint maps to 1p11.2 withina 154.2 kb region covered by BAC RP11-439A17 whilstthe q-arm breakpoint maps to 1q21.1 within a 562.6 kb

region. Sequence comparisons of these breakpointregions indicated a very high level of sequence homology(> 99%) over a 91 kb-spanning region that was almostcertainly responsible for mediating the pericentric inver-sion by non-allelic homologous recombination. These91 kb duplications are absent from the orthologousregions of chimpanzee chromosome 1, and constitutehuman lineage-speciWc segmental duplications that origi-nated by duplicative transposition (Fig. 4).

Two other studies have reported the breakpoints ofthis chromosome 1 inversion at the DNA sequencelevel (Mikkelsen et al. 2005; Newman et al. 2005).However, as indicated in Table 3, the locations of thebreakpoint regions determined by these authors devi-ate markedly from those reported here. In both previ-ous studies, sequence alignments were performed butthese data were not supported by FISH analysis. As wehave shown, the combination of FISH and sequencealignment constitutes a very powerful approach to nar-rowing down the breakpoint regions within the dupli-cation-enriched pericentromeric region of HSA 1.

Fig. 5 Fluorescence in situ hybridization (FISH) with BACsRP11-314N2 (green) and RP11-458I7 (red) on the metaphasechromosomes of macaque (MFA) (a), chimpanzee (PTR) (b) andhuman (HSA) (c). As schematically indicated under (d) andaccording to the FISH pattern, both BACs are located on MFA1p. The Wrst pericentric inversion event transposed BAC RP11-458I7 to the q arm in PTR (b). The second pericentric inversion

occurred speciWcally in the human lineage and included the regioncovered by BAC RP11-314N2 which maps to the q-arm in humans(c). Dotted lines in d indicate breakpoint regions for the two inver-sions that are located at diVerent locations. e The time scale indi-cates the estimated times in millions of years (Myrs) for theseparations of the diVerent primate lineages from their commonancestor as well as the occurrence of the two pericentric inversions

12-13

0.86-2

7-8

5-7

PPYPPY GGOGGO HSAHSA

Myrs

PPAPPAPTRPTRMFAMFA

~25Inversion 1

Inversion 2

CA B

HSAPTRMFA

X

X

c

MFA

pericentricinversion 1

pericentricinversion 2

PTR

c cc

HSA

458I7314N2

D

E

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Hum Genet (2006) 120:126–138 135

According to Mikkelsen et al. (2005), the breakpointon HSA 1p maps to the region between 113,283,739and 113,894,129 bp. However, this region actually cor-responds to a diVerent chromosomal rearrangementbetween human and chimpanzee, viz. a previouslydetected paracentric micro-inversion (Newman et al.2005; Szamalek et al. 2006b). We have shown that WveBAC/PAC clones distal to the breakpoint suggested byMikkelsen et al. on HSA 1p, which ranges from118,079,902 to 120,395,759, are not inverted in chim-panzee. Furthermore the distal breakpoint region pro-posed by Mikkelsen et al. (142,920,927–145,140,950)encompasses more than 2 Mb of HSA 1 sequence thatis rich in segmental duplications. According to ourFISH results, BAC RP11-763B22 from 145,513,740 to145,671,828 still lies within the inverted region. Thus,our FISH data are clearly incompatible with the break-point positions as claimed by Mikkelson et al. (2005).

In support of the validity of our conclusions, boththe locations of the breakpoint regions reported here,and our proposed model of inversion formation, areconsistent with the analysis of Maresco et al. (1998).These authors performed FISH analyses in primateswith a probe designed to detect the FCGR1 genes andprovided molecular evidence for the occurrence of twopericentric inversions during the evolution of primate/human chromosome 1. In Old World monkeys, a soli-tary FCGR1 gene maps to the homologue of human1p. In great apes, however, FCGR1 maps to the q-arm,indicative of a pericentric inversion that occurred afterthe separation of the Old World monkeys from thelineage leading to the great apes. In the human lineage,local serial duplications of the FCGR1 gene gave riseto the highly homologous FCGR1A, FCGR1B andFCGR1C genes. These duplications were then fol-lowed by the human-speciWc pericentric inversionwhich was mediated by two 91 kb segmental duplica-tions of high sequence homology (> 99%) that containthe FCGR1B and FCGR1C genes, respectively(Fig. 4). The human lineage-speciWc copy number gainscharacteristic of the FCGR1 gene family have also

been demonstrated by aCGH using genomic clones(Goidts et al. 2006; Wilson et al. 2006) and cDNAs(Fortna et al. 2004).

The pericentromeric region of HSA 1 is character-ized both by its instability in human disease and by itsgenomic variability. Thus, a large number of supernu-merary marker or ring chromosomes derived from thischromosomal region have been reported to be associ-ated with varying phenotypes (reviewed in Barbi et al.2005). Proximal duplications in HSA 1q have beenfound in patients with multiple congenital abnormali-ties and/or mental retardation (Faas et al. 2003).Breakpoints in the pericentromeric region of chromo-some 1 have also been identiWed in numerous humancancers as listed in the Mitelman Database of Chromo-some Aberrations in Cancer (Mitelman et al. 2006).Further, structural polymorphisms identiWed in thepericentromeric region of chromosome 1 provide addi-tional evidence for the plasticity of this genomicregion. By mapping end sequences of fosmid clonesfrom a human genomic library against the humanreference sequence, Tuzun et al. (2005) identiWed sev-eral insertions/deletions and duplications as well as aparacentric inversion of 31.5 kb (146,634,712–146,666,180; NCBI Build 35) located »400 kb distal tothe q-arm breakpoint region of the pericentric inver-sion investigated here. Within the region spanned bythe pericentric inversion, Sebat et al. (2004) detected a38.6 kb insertion/deletion variant (142,364,023–142,402,592; NCBI Build 35). In the region of the q-armbreakpoint of the pericentric inversion, 120 kb proximalto the 91 kb segmental duplication, two regions of copynumber variation have been identiWed, spanning 117and 42.2 kb, respectively (Loci 1474 and 1475 accordingto the Human Genome Segmental Duplication Data-base; Conrad et al. 2006; McCarroll et al. 2005).

The abundance of duplicated sequences in the peri-centromeric region of HSA 1 renders these regions sus-ceptible to both pathological and evolutionaryrearrangements. Indeed, many of the duplicatedsequences in the pericentromeric region of HSA 1 rep-resent recent acquisitions during human evolution. The91 kb duplication identiWed here in the breakpointregions of the human-speciWc pericentric inversion isassumed to have mediated the rearrangement. How-ever, a pericentric inversion of the ancestral chromo-some 1 occurred at a much earlier stage of primateevolution, after the separation of the Old World mon-key and great ape lineages. However, using compara-tive FISH analysis, we have shown that the breakpointsof these two inversions occur at non-identical positions(Fig. 5). This is in accord with the hypothesis that thehuman-speciWc pericentric inversion was mediated by

Table 3 Comparison of the breakpoint regions suggested for thepericentric inversion of HSA 1

a According to the NCBI Build 35

Reference Breakpoint regiona

HSA 1p HSA 1q

This study 120,395,759–120,549,995 145,671,828–146,234,395Mikkelsen

et al. (2005)113,283,739–113,894,129 142,920,927–145,140,950

Newman et al. (2005)

87,577,874–87,617,874 144,800,023–144,832,722

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136 Hum Genet (2006) 120:126–138

segmentally duplicated sequences that arose after thedivergence of humans and chimpanzees.

The model we propose to explain the molecularmechanism underlying the inversion (Fig. 4) is in per-fect agreement with the results of the FISH analysisand sequence alignment data reported here. However,future improvements of the sequence contigs in thepericentromeric region of chromosome 1 in bothhumans and chimpanzees should eventually allowindependent veriWcation of the model.

The co-localization of lineage-speciWc segmentalduplications and the breakpoints of large-scale rear-rangements that distinguish the human and chimpan-zee chromosomes is not without precedent. Thehuman-speciWc inversion of HSA 18 is thought tohave been mediated by segmental duplications of19 kb that are also human-speciWc (Dennehey et al.2004; Goidts et al. 2004). Further, the breakpoints ofthe chimpanzee-speciWc inversion of PTR XII map tolineage-speciWc duplications (Kehrer-Sawatzki et al.2005b). The fusion of the two ancestral submetacen-tric chromosomes that gave rise to human chromo-some 2 also occurred within regions enriched with lowcopy repeats (Fan et al. 2002). Segmental duplicationsand in particular lineage-speciWc duplications havethus clearly played an important role during karyo-type evolution in hominoids. The potential of lineage-speciWc duplications to inXuence karyotypic evolutionand species-speciWc features of the karyotype is alsoreXected in the very signiWcant diVerences evidentbetween subtelomeric regions of human and chim-panzee chromosomes. In the chimpanzee, massiveexpansions of a speciWc segmental duplication havebeen identiWed in subtelomeric regions, associatedwith the addition of 16 Mb of DNA sequence that isabsent from humans (Cheng et al. 2005). Theseexpansions are directly associated with ‘telomericcaps’, structures which are observed at the ends ofchimpanzee chromosomes but not at those of humanchromosomes (Yunis and Prakash 1982). Further sig-niWcant structural diVerences in subtelomeric seg-ments between the human and chimpanzee genomesare due to copy number diVerences mediated by seg-mental duplications (Trask et al. 1998; Monfouillouxet al. 1998; Martin et al. 2002; Linardopoulou et al.2005). Taken together, these Wndings emphasize thatsegmental duplications in general, and lineage-spe-ciWc duplications in particular, have exerted a veryconsiderable inXuence on karyotypic evolution inboth humans and chimpanzees.

Acknowledgment This research was funded by the DeutscheForschungsgemeinschaft (DFG KE 724/2-1).

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