914 J Med Genet 1999;36:914–918

Neocentromere formation in a stable ring J Med Genet: first published as 10.1136/jmg.36.12.914 on 1 December 1999. Downloaded from 1p32-p36.1

Howard R Slater, Sara Nouri, Elizabeth Earle, AnthonyWILo,Lyndon G Hale, K H Andy Choo

Abstract from apparently non-alphoid DNA sequences Neocentromeres are functional centro- on the arms of diVerent human .4 meres formed in chromosome regions We report here a new case of neocentromere outside the normal domains involving a formed from a and are found in an increasing number of previously undescribed region on the short arm mitotically stable human marker chromo- of . The ring chromosome was somes in both neoplastic and non- diagnosed in a 38 year old man who presented neoplastic cells. We describe here the clinically with oligospermia. We describe the formation of a neocentromere in a previ- detailed cytogenetic, immunofluorescence, and ously undescribed chromosomal region at FISH characterisation of this ring chromosome 1p32→p36.1 in an oligospermic patient. and discuss our results in light of the emerging Cytogenetic GTL banding analysis and the neocentromere phenomenon. absence of detectable fluorescence in situ hybridisation (FISH) signals using telo- Materials and methods meric probes indicate the marker to be a CYTOGENETIC STUDIES AND CELL LINES ring chromosome. The chromosome is Cultured peripheral blood lymphocytes were negative for CBG banding and is devoid of used for the preparation of metaphase chromo- detectable centromeric á satellite and its somes for GTL and CBG banding analyses associated centromere CENP-B, using standard procedures and for FISH suggesting activation of a neocentromere experiments. Skin fibroblast and Epstein-Barr within the 1p32-36.1 region. Functional virus transformed lymphoblast cell lines were activity of the neocentromere is shown by established from the patient for immunofluo- the retention of the ring chromosome in rescence studies using anti-centromere anti- 97% of the patient’s lymphocytes and 100% bodies or for further cytogenetic analysis. of his cultured fibroblasts, as well as by the presence of key centromere binding pro- MICROSATELLITE ANALYSIS teins CENP-E, CENP-F, and INCENP. DNA from blood and sperm was extracted These results indicate that in addition to with a commercial DNA extraction kit (Nu- http://jmg.bmj.com/ CENP-A, CENP-C, and CENP-E de- cleon, Amersham) and investigated using poly- morphic microsatellites (n=9) located between The Murdoch scribed in earlier studies, neocentromere Institute, Royal activity can further be defined by CENP-F chromosome bands 1p31.1 and 1p36.23. The Children’s Hospital, and INCENP binding. Our evidence proximal to distal order of these microsatellite Flemington Road, suggests that neocentromere formation loci were D1S230, D1S2890, D1S2797, Parkville 3052, constitutes a viable mechanism for the D1S255, D1S234, D1S199, D1S2697, Australia

D1S2667, and D1S450. The loci between on September 28, 2021 by guest. Protected copyright. H R Slater mitotic stabilisation of acentric ring chro- S Nouri mosomes. D1S2797 and D1S2697 were surmised to be E Earle (J Med Genet 1999;36:914–918) present on the rder(1) chromosome. PCR AWILo products were analysed on an Applied Biosys- K H A Choo Keywords: neocentromere; centromere ; á tems ABI Prism 377 DNA Sequencer. satellite DNA; chromosome 1 Victorian Clinical Genetics Service, FISH AND IMMUNOFLUORESCENCE DETECTIONS FISH was performed according to the manu- Royal Children’s Normal human contain an abun- Hospital, Flemington facturer’s instructions using the pan á satellite Road, Parkville 3052, dance of a tandemly repeated DNA known as á probe (designated “All Human Centromeres” Australia satellite. This DNA is made up of basic units of probe, ONCOR), probe (designated H R Slater 171 base pairs that are tandemly repeated into “All Human probe, ONCOR), and S Nouri arrays of up to 3-4 megabases on each chromosome 1 specific paint (CAMBIO Ltd). chromosome.1 Existing evidence based on the Freemason’s Hospital Four diVerent anti-centromere antibodies di- Medical Centre, East reintroduction of exogenous á satellite DNA rected against centromere proteins CENP-B, Melbourne, Victoria into mammalian cells to reconstitute active CENP-E, CENP-F, and INCENP were used 3002, Australia centromeres suggest that this DNA is likely to in immunofluorescence studies. Anti-CENP-B L G Hale have a direct role in centromere function.23 antibodies were prepared in our earlier study,5 However, in recent years, a novel class of Correspondence to: anti-CENP-E and anti-CENP-F antibodies Dr Choo mitotically stable human marker chromosomes were gifts fromTJYen,andanti-INCENP that are devoid of á satellite DNA has been antibody was a gift from W E Earnshaw. The Revised version received identified. These á satellite negative or anal- immunofluorescence detection procedure was 22 June 1999 6 Accepted for publication phoid marker chromosomes contain new as described in a previous study. Images for 2 August 1999 centromeres (neocentromeres) that are formed the pan á satellite, telomere, and chromosome Neocentromere on human ring 1p chromosome 915 J Med Genet: first published as 10.1136/jmg.36.12.914 on 1 December 1999. Downloaded from A otherwise appeared normal. GTL banding showed a deleted chromosome 1 (designated del(1)) in which a segment (band 32-36.1) on the p arm of chromosome 1 was missing in all the 100 cells examined (fig 1A). In addition, an apparently supernumerary, small, ring-like chromosome (designated rder(1)) was evident in 97% (97 out of 100) of the cells analysed. In these 97 ring chromosome containing cells, 10 were shown to carry an rder(1) chromosome that has doubled in size (fig 1A). Two reasons pointed to this rder(1) chromosome having a presumed origin from the deleted 1p32-36.1 segment: (1) its approximate banding mor- phology is consistent with that of 1p32-36.1, and (2) despite the patient’s infertility problem, the patient was phenotypically normal which Normal(1) del(1) rder(1) Double rder(1) suggested a more or less balanced since of the 1p32-36.1 region accompanied by a of an unknown but substantial genomic segment would probably have manifested in a more severe phenotype. B Confirmation of the chromosome 1 origin of the ring has come from FISH studies below. CBG banding showed positive staining of the centromeric/pericentric heterochromatins of all the chromosomes except the rder(1) chromosome (fig 1B). This result, together with the high level retention of the rder(1) chromosome, suggested the presence of a functional centromere that was devoid of centromeric heterochromatic DNA. When the chromosomes of the patient’s parents were cytogenetically analysed, these were shown to Normal(1) der(1) rder(1) be normal, suggesting a de novo occurrence of the del(1) and rder(1) chromosomes. The patient’s lymphocyte karyotype is therefore 47,XY,del(1)(p32p36.1)+r(1)(p32p36.1) de novo. http://jmg.bmj.com/ Cytogenetic analysis of the patient’s fibro- blast culture showed the presence of the del(1) C and rder(1) chromosomes in all the 100 cells analysed, although in five of these cells the rder(1) chromosome has doubled in size. In addition, a translocation involving the X chro- mosome and was observed in

60% of these fibroblast cells (fig 1C), suggest- on September 28, 2021 by guest. Protected copyright. ing that the patient may be for this rearrangement. The patient’s fibroblast karyo- Normal(4) der(4) der(X) type is therefore 47,Y,t(X;4)(q23;q13),del (1) (p32p36.1)+r(1) (p32p36.1)[60]/XY,del(1) Figure 1 Cytogenetic analyses. (A, B) GTL and CBG banding, respectively, of the (p32p36.1)+r(1)(p32p36.1) [40]. patient’s lymphocyte chromosomes showing a normal chromosome 1 and the deleted chromosome 1 (del(1)), ring chromosome (rder(1)), and the occasional double sized rder(1). (C) GTL banding of patient’s cultured fibroblast chromosomes showing the MICROSATELLITE ANALYSIS presence of a reciprocal 4;X translocation in 60% of the cells in addition to the del(1) and As spermatogenesis may be compromised by rder(1) chromosomes which are present in all the cells. ring chromosomes with consequent infertility 1 paint probes were analysed on a Zeiss for most heterozygous males,7 it was possible Axioskop microscope equipped with a Cytovi- that the presence of the rder(1) chromosome in sion Image Analysis System (Applied Imaging patient AS may explain his oligospermia. We Ltd), whereas those for anti-centromere anti- therefore used an array of chromosome 1p spe- bodies were analysed on a Zeiss Axioskop fluo- cific microsatellite markers to determine the rescence microscope equipped with a × 100 heterozygosity of the del(1) and rder(1) objective and a cooled CCD camera (Photo- chromosomes in the sperm DNA sample. As a metrics Image Point) controlled by a Power control, DNA from the patient’s blood was Mac computer. used and this indicated all the microsatellite loci (except D1S2697) to be heterozygous. Results Analysis of sperm DNA showed identical CYTOGENETIC STUDIES results (data not shown) and gave no evidence The patient was a 38 year old man who of selection against sperm containing the del(1) presented with infertility and oligospermia but or rder(1) or both. 916 Slater, Nouri, Earle, et al J Med Genet: first published as 10.1136/jmg.36.12.914 on 1 December 1999. Downloaded from FISH ANALYSES blast cells. In contrast to this, the second rear- The origin of the del(1) and rder(1) chromo- rangment, which involves a translocation be- somes was ascertained by performing FISH on tween chromosomes 4 and X, is only detected the patient’s metaphase preparations using a in the patient’s fibroblasts where 60% of the chromosome 1 paint probe. The result indi- cells carry this aberration in addition to the cated that in addition to the specific painting of del(1) and rder(1) chromosomes. It therefore the normal chromosome 1, both the del(1) and appears that the patient may be mosaic for the rder(1) chromosomes were painted (fig 2A), 4;X translocation although this awaits confir- thus confirming their chromosome 1 origin. mation using additional biopsy samples from FISH analysis of the patient’s chromosomes the patient. At this stage, it is diYcult to estab- using a pan á satellite probe under low lish which or whether both of the rearrange- stringency condition showed hybridisation sig- ments are contributing to oligospermia in the nals on all the chromosomes except rder(1) (fig patient since both types of rearrangements 2B). When the metaphase chromosomes were may, each on its own, cause such a phenotype. hybridised with a telomere probe, positive sig- For example, almost all instances of parent to nals were again observed on all the chromo- child transmission of ring chromosomes in- somes except for the rder(1) chromosome (fig volve the mother as the carrier parent,7 2C). These results were consistent with the suggesting that the presence of a ring chromo- formation of the rder(1) chromosome from an some in most male heterozygotes compromises interstitial portion of 1p, followed by rejoining spermatogenesis. Similarly, non-mosaic males of the sticky ends of the deleted fragments. with an X; translocation are almost They further indicated that the rder(1) chro- invariably infertile.9 mosome has not acquired detectable amounts The cytogenetic GTL and CBG banding of exogenous á satellite sequences. These patterns of the del(1) and rder(1) chromo- analyses raised the strong possibility that the somes, together with the FISH results using a rder(1) chromosome contained a neocentro- chromosome 1 specific painting probe, indicate mere that has been formed from analphoid the origin of rder(1) from the 1p32-36.1 DNA sequences within the 1p32-36.1 region. region. Two lines of evidence suggest that rder(1) is a ring chromosome. The first comes CENTROMERE PROTEINS from our failure to detect any telomeric signal In order to establish the structural and which is consistent with the circularisation of a functional integrity of the putative neocentro- deleted interstitial chromosomal fragment. mere on the rder(1) chromosome, the status of Secondly, the observation of the occasional various centromere specific proteins was deter- double sized ring chromosomes shows struc- mined by immunofluorescence staining. As tural instability typical of larger rings which shown in fig 2D, use of an anti-CENP-B form complex ring structures as a result of sis- monoclonal antibody showed variable but ter exchanges. detectable levels of CENP-B on the centro- The high stability of the rder(1) chromo- meres of all the chromosomes except for the some in both the patient’s lymphocytes and his http://jmg.bmj.com/ rder(1) and the Y chromosomes. The absence established fibroblast cell line strongly suggests of CENP-B-binding on rder(1) was consistent the presence of a functional centromere on the with the lack of detectable á satellite DNA on ring chromosome. Proof of this comes from this chromosome (see Discussion), whereas the immunofluorescence studies using antibodies centromeric á satellite of the human Y against three centromere proteins (CENP-E, chromosome is known to have little or no CENP-F, and INCENP) that are known to be CENP-B box motifs upon which the CENP-B important for centromere function. CENP-E is 8

proteins associate. a molecular motor that moves chromosomes on September 28, 2021 by guest. Protected copyright. The other three centromere proteins tested along microtubules.10 Although the specific were CENP-E, CENP-F, and INCENP. As role of CENP-F remains unclear, its apparent shown in figs 2E and F, CENP-E and CENP-F interaction with CENP-E suggests a possible were clearly detected on the rder(1) chromo- role in the proper functioning of a motor some, with the immunofluorescence signals complex.11 That both CENP-E and CENP-F being as strong as those seen on the other are intimately associated with centromere chromosomes. Compared to the signals for functions is suggested by the observation that these two proteins, the INCENP signals these proteins are found on active but not inac- showed slightly more variability on the diVer- tive centromeres in dicentric or multicentric ent centromeres, with the rder(1) chromosome chromosomes.12 13 In addition to these two generally giving the weakest signal (fig 2G). proteins, studies involving overexpression of The implications of these results are discussed INCENP mutant proteins in cell culture have below. shown this protein to be an essential compo- nent of the mitotic cell cycle, being involved in Discussion prometaphase chromosome congression and Cytogenetic analyses have identified two ap- cytokinesis.14 Thus, the demonstration of these parently unrelated chromosomal rearrange- three key centromere proteins on the rder(1) ment events in the patient. The first involves an chromosome clearly indicates the presence of interstitial within the short arm of an active centromere on this chromosome. chromosome 1, leading to the formation of the At present, it is unclear why the anti- del(1) and rder(1) chromosomes. This rear- INCENP antibody signals on the rder(1) chro- rangement is found in 97% of the patient’s mosome are greatly reduced in relation to those lymphocytes and 100% of his cultured fibro- on the other chromosomes. Earlier studies have Neocentromere on human ring 1p chromosome 917

shown a diVuse localisation of INCENP within on the rder(1) chromosome con- J Med Genet: first published as 10.1136/jmg.36.12.914 on 1 December 1999. Downloaded from the centromeric of normal tributes to the severe reduction of centromeri- centromeres.15 Whether the absence of hetero- cally bound INCENP and, if so, what the http://jmg.bmj.com/ on September 28, 2021 by guest. Protected copyright.

Figure 2 FISH and immunofluorescence studies. (A-C) FISH using chromosome 1 specific paint (green), pan á satellite probe (red), and telomere probe (red), respectively, on a DAPI stained (blue) chromosome background. (D-G) Immunofluorescence detection using antibodies against CENP-B, CENP-E, CENP-F,and INCENP,respectively. In the left hand panel, chromosomes are shown in red while the centromeric antigen specific signals are shown in green (or yellow on a red background). The right hand panel represents split images of the green immunofluorescence signals shown in the left hand panel. Solid arrows indicate the rder(1) chromosome. Open arrow in D points to the CENP-B negative . The detection of two areas of fluorescence on many of the chromosomes (including rder(1)) represents well resolved signals on sister , rather than implying two diVerent centromeres located on diVerent parts of a chromosome, especially in the case of rder(1). Note the presence of CENP-E, CENP-F,and INCENP,but not CENP-B signals on the rder(1) chromosome. 918 Slater, Nouri, Earle, et al

underlying mechanism is for INCENP binding have shown that neocentromere formation J Med Genet: first published as 10.1136/jmg.36.12.914 on 1 December 1999. Downloaded from on a “euchromatic neocentromere”, remain to constitutes a viable mechanism for the mitotic be determined. stabilisation of acentric ring chromosomes. Our failure to detect any á satellite DNA on Further molecular studies will be necessary to the rder(1) chromosome is consistent with the unveil the intricate mechanisms underlying absence of CENP-B-binding on the rder(1) neocentromere formation. chromosome since previous studies have estab- lished the specific requirement of this protein We are grateful to K Ewen for the microsatellite assay, D F in binding the CENP-B box sequence motifs Hudson for anti-CENP-B antibody, T J Yen for anti-CENP-E 16 and anti-CENP-F antibodies, andWEEarnshawfor on the á satellite DNA. These results indicate anti-INCENP antibody. This work was supported by the that the observed mitotic stability of this chro- National Health and Medical Research Council of Australia. KHAC is a Senior Associate of the Univeristy of Melbourne and mosome is not associated with the inherent a Principal Research Fellow of NH&MRC. presence or acquisition of any substantial amount of normal centromeric á satellite 1 Choo KHA. The centromere. Oxford: Oxford University DNA. Based on this and the above described Press, 1997:1-304. cytogenetic and immunofluorescence charac- 2 Harrington JJ, Van Bokkelen G, Mays RW, Gustashaw K, Willard HF. Formation of de novo centromeres and teristics, it can be concluded that the rder(1) construction of first-generation human artificial chromo- chromosome necessarily contains a functional somes. Nat Genet 1997;15:345-55. 3 Ikeno M, Grimes B, Okazaki T, et al. Construction of YAC- neocentromere formed from the apparently based mammalian artificial chromosomes. Nat Biotech euchromatic DNA of the chromosome 1p32- 1998;16:431-9. 4 Choo KHA. Centromere DNA dynamics: latent centro- 36.1 region. meres and neocentromeres. Am J Hum Genet 1997;61: The best characterised analphoid neocentro- 1225-33. 5 Hudson DF, Fowler KJ, Earle E, et al. mere to date has been described for a marker null mice are mitotically and meiotically normal but have chromosome derived from chromosome lower body and testis weights. J Cell Biol 1998;141:309-19. 6 17–19 6 Voullaire LE, Slater HR, Petrovic V, Choo KHA. A 10. This neocentromere forms a distinct functional marker centromere with no detectable alpha- primary constriction at the 10q25 region, binds satellite, satellite III or CENP-B protein: activation of a latent centromere? Am J Hum Genet 1993;52:1153-63. functionally critical centromere antigens 7 MacDermot KD, Jack E, Cooke A, et al. Investigation of CENP-A and CENP-C but not CENP-B, and three patients with the “ring syndrome”, including familial transmission of ring 5, and estimation of reproductive risks. confers 100% mitotic stability to the patient’s Hum Genet 1990;85:516-20. lymphocytes and cultured lymphoblasts and 8 Pluta AF, Cooke CA, Earnshaw WC. Structure of the human centromere at metaphase. Trends Biochem 1990;15: fibroblasts. Using the chromosome walking 181-5. technique, the core functional antigen binding 9 Schmidt M, duSart D. Functional disomies of the influence the cell selection and hence the X domain of this neocentromere has been inactivation pattern in females with balanced X-autosome localised to an 80 kb DNA.17 Extensive restric- translocations: a review of 122 cases. Am J Med Genet 1992;42:161-9. tion mapping indicates that the neocentromere 10 Thrower DA, Jordan MA, Schaar BT, Yen TJ, Wilson L. DNA is not significantly diVerent from the Mitotic HeLa cells contain a CENP-E-associated minus end-directed microtubule motor. EMBO J 1995;14:918- corresponding DNA of the normal 10q25 26. region, suggesting a mechanism of neocentro- 11 Chan GK, Schaar BT, Yen TJ. Characterization of the kine- mere formation through the epigenetic activa- tochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1. J http://jmg.bmj.com/ tion of a normally non-centromeric genomic Cell Biol 1998;143:49-63. 17 18 12 Sullivan B, Schwartz S. Identification of centromeric sequence. Direct sequencing and detailed antigens in dicentric Robertsonian translocations: computational analyses of the sequence show CENP-C and CENP-E are necessary components of func- tional centromeres. Hum Mol Genet 1995;4:2189-97. no distinct homology or structural resemblance 13 Faulkner NE, Vig B, Echeverri CJ, Wordeman L, Vallee RB. to any known eukaryotic centromere.19 The Localization of motor-related proteins and associated com- plexes to active, but not inactive, centromeres. Hum Mol discovery of an increasing number of putative Genet 1998;7:671-7. neocentromeres on other chromosomal sites 14 Mackay AM, Ainsztein AM, Eckley DM, Earnshaw WC. A dominant mutant of inner centromere protein (INCENP), that carry unrelated, unique genomic a chromosomal protein, disrupts prometaphase congres- on September 28, 2021 by guest. Protected copyright. sequences4 provides further support for the sion and cytokinesis. J Cell Biol 1998;140:991-1002. 15 Earnshaw WC, Cooke CA. Analysis of the distribution of belief that there appears not to be a conserved the INCENPs throughout reveals the existence of a “magic” primary sequence for the eukaryotic pathway of structural changes in chromosomes during metaphase and early events in cleavage formation. J Cell Sci centromeres and that seemingly “ordinary” 1991;98:443-61. genomic sequences can be activated to become 16 Muro Y, Masumoto H, Yoda K, Nozaki N, Ohashi M, Oka- 419 zaki T. Centromere protein B assembles human centro- a functional neocentromere. The recent meric alpha-satellite DNA at the 17-bp sequence, CENP-B identification of a neocentromere in a Dro- box. J Cell Biol 1992;116:585-96. 20 17 du Sart D, Cancilla MR, Earle E, et al. A functional sophila minichromosome suggests that this neo-centromere formed through activation of a latent phenomenon may be widespread throughout human centromere and consisting of non-alpha-satellite DNA. Nat Genet 1997;16:144-53. evolution. 18 Cancilla MR, Tainton KM, Barry AE, et al. Direct cloning In the present study, we have investigated the of human 10q25 neocentromere DNA using transformation-associated recombination (TAR) in yeast. detailed cytogenetic and antigenic properties of Genomics 1998;47:399-404. a neocentromere on a ring Barry AE, Howman EV, Cancilla MR, SaVery R, Choo KHA. Sequence analysis of an 80-kb human neocentro- formed from a previously undescribed 1p32- mere DNA. Hum Mol Genet (in press). 36.1 region. We presented evidence that in 20 Williams BC, Murphy TD, Goldberg ML, Karpen GH. Neocentromere activity of structurally acentric minichro- addition to CENP-A, CENP-C, and CENP-E mosomes in Drosophila. 1998;18:30-7. 17 21 Nat Genet binding described in earlier studies, neo- 21 Depinet TW, Zackowski JL, Earnshaw WC, et al. Charac- centromere activity can further be defined by terizaton of neocentromeres in marker chromosomes lack- ing detectable alpha-satellite DNA. Hum Mol Genet CENP-F and INCENP binding. Finally, we 1997;6:1195-204.