10 Genome Plasticity in Evolution

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From: Genomic Disorders: The Genomic Basis of Disease Edited by: J. R. Lupski and P. Stankiewicz © Humana Press, Totowa, NJ

10 Genome Plasticity in Evolution

The Centromere Repositioning

Mariano Rocchi, ^{P h D} and Nicoletta Archidiacono, ^{P h D}

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ONTENTS

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ACKGROUND

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NTRODUCTION

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ENTROMERE

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EPOSITIONING

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VOLUTION OF

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ECURRENT

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EEDING

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EPOSITIONING

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ROGRESS

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EOCENTROMERES IN

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BACKGROUND

The centromere repositioning phenomenon consists in the move of the centromere along the chromosome during evolution. This phenomenon is relatively frequent, and has been doc- umented in primates, nonprimate mammals, and birds. It implies the inactivation of the old centromere and the rapid progression of the newly seeded centromere toward the complex organization that probably stabilizes its activity. Both events have a huge impact on chromo- somal architecture. The segmental duplicon clusters at 6p22.1 and 15q24-26 are clear examples of remains of inactivated ancestral centromeres. These duplicons are dispersed in a relatively large area (approx 10 Mb), and contribute to the bulk of nonpericentromeric segmental dupli- cations that constitute approx 5% of the human genome.

Human neocentromeres are rare newly created centromeres formed in a previously

noncentromeric location and devoid of alphoid sequences. Two human neocentromeres

emerged in the same 15q24-26 chromosomal region where an ancestral centromere inacti-

vated, establishing an intriguing connection between neocentromeres and inactivated ances-

tral centromeres. An other human neocentromere was found to be located in the same 3q26.1

region where an evolutionary centromere was seeded and fixed in Old World monkey (OWM)

ancestor, indicating that the 3q26.1 region conserved a centromeric competence that was

present before hominoids/OWM splitting. These findings further support the idea that some

chromosomal domains have inherent centromeric capability.

sis and meiosis, and is conserved throughout evolution. Unexpectedly, sequences underlying the centromere function are not evolutionary conserved, and show variations even among closely related species (1,2). Pericentromeric regions also show a high degree of evolutionary plasticity. They accumulate large amount of segmental duplications, which appear intrinsi- cally more unstable than the bulk of euchromatic DNA (3–7). A detailed analysis of pericentromeric segmental duplications and their role in evolution can be found in Chapter 5.

Evolutionary studies aimed at tracking chromosomal marker order conservation in primates unveiled an additional peculiar feature of centromeres: the centromere repositioning, consist- ing in the evolutionary movement of the centromere along the chromosome in the absence of any pericentric inversion that would account for the discrepancy in centromere location between two species. In this chapter, we focus on this biological phenomenon that is relatively frequent during the evolution of mammals.

CENTROMERE REPOSITIONING

We discovered this phenomenon while studying the evolutionary history of human chromo- some 9 in primates (8). Figure 1 summarizes the results obtained using a panel of yeast artificial chromosome clones used to establish marker order along this chromosome in 9 different primate species. If the position of the centromere is not taken into account, a relatively small number of rearrangements can be invoked to reconstruct the marker order in the studied species. Conversely, if the centromere is included in the analysis, a paradox emerges: the centromere appears to have undergone an independent evolutionary history. We hypothesized that the movement of the centromere toward the new location was not owing to classical chromosomal rearrangements (essentially inversions), but, rather, to the inactivation of the old centromere and the simultaneous appearance of a new centromere in a different location. The complexity of the evolutionary history of chromosome 9 did not allow certain alternative hypotheses to be discarded, such as two successive inversions, the first involving the centromere and the second restoring the previous marker order with the exception of the centromere.

For these reasons we undertook similar studies on other chromosomes. According to Ohno’s

law, the gene content of the chromosome X has barely changed throughout mammalian devel-

opment in the last 125 million years (9). The X chromosome was, therefore, an ideal chromo-

some in this respect. The position of the centromere of this chromosome is substantially

unchanged in primates, with two exceptions in prosimians: the black lemur (Eulemur macaco

[EMA]), and the ring-tailed lemur (Lemur catta [LCA]). The chromosome X appears acrocen-

tric in black lemur and almost metacentric in ring-tailed lemur (Fig. 2). We investigated the

marker order organization using a variety of different molecular cytogenetic tools, but no

variation in marker order was found (10). We concluded that the move of the centromere in

EMA and LCA was because of the centromere repositioning event.

Chapter 10 / Genome Plasticity in Evolution 155

A centromere repositioning implies the inactivation of an ancestral centromere and the seeding of a new centromere. Neocentromere appearance is not a rare event in human. Human neocentromeres are defined as “rare human chromosomal aberrations where a new centromere has formed in a previously non-centromeric location” (11). The first well-documented case of a human neocentromere was reported by du Sart et al. (12). Since then, more than 50 neo- centromere cases have been described (13). Some neocentromeres are clustered in “hotspots”

for neocentromere formation, which include 3q26-qter, 8p, 13q21-q32, and 15q24-q26 regions.

Most of the neocentromeres arise in acentric fragments because of a cytogenetic rearrange- ment. Acentric chromosomal fragments are usually lost. However, in some cases, their mitotic survival is rescued by neocentromere appearance, which is always an accidental event with respect to the rearrangement. In two cases, the neocentromere that emerged on chromosome

Fig. 1. Summary of fluorescence in situ hybridization results delineating the evolutionary history of human chromosome 9 in primates. The hypothesized pericentric or paracentric inversions are indicated by brackets spanning the inverted segment. The brackets at the bottom of the figure groups species in which the difference in centromere position cannot be explained on the basic of classical rearrangements.

Species studied: common chimpanzee (Pan troglodytes, PTR), gorilla (Gorilla gorilla, GGO), and orangutan (Pongo pygmaeus, PPY). Old World monkeys, silvered leaf-monkey (Presbytis cristata, PCR). New World monkeys, dusky titi (Callicebus molloch, CMO), spider monkey (Ateles geoffroyi);

common marmoset (Callithrix jacchus, CJA), and squirrel monkey (Saimiri sciureus, SSC). (Adapted with permission from ref.8.)

Fig. 2. DAPI-banded chromosome X in humans (HSA), ring-tailed lemur (LCA), and black lemur (EMA), showing a completely different position of the centromere, indicated by arrows. EMA chromo- some X is upside down to match marker order in humans and LCA.

Y was not accompanied by a rearrangement (14,15). In one of these cases, the chromosome was segregating in the family (15).

Remains of Inactivated Centromeres

Human chromosome 2 is the result of a telomere–telomere fusion between two ancestral acrocentric chromosomes that occurred after human–chimpanzee divergence (16), with one of the two centromeres becoming inactive. Indeed, stretches of alphoid sequences and clusters of segmental duplications, as remains of the inactivated centromere, are apparent at 2q21 (17,18).

We have hypothesized that the evolutionary appearance of new centromeres, as part of a centromere-repositioning event, implies the inactivation of a functioning centromere. Studies on the evolutionary history of chromosome 6 provided evidence that remains of an inactivated centromere, which resulted from a centromere repositioning, are present in the human genome (19). Figure 3 summarizes the evolutionary history of chromosome 6, which is substantially conserved in evolution. Marker order analysis strongly suggested that the arrangement of the two New World monkeys (NWM), common marmoset (Callithrix jacchus [CJA]), and wooly monkey (Lagothrix lagothricha [LLA]) maintained the primate ancestral form. The marker arrangement of cat, chosen as the outgroup, supported this hypothesis. The centromeres of OWM and great apes, therefore, are evolutionary new centromeres. The major point of this analysis was the finding, in humans, of a duplicon cluster at 6p22.1 exactly corresponding to the region where the ancestral centromere was inactivated (Fig. 4). No remains of alphoid centromeric sequences, however, were detected. It can be hypothesized that, following the centromere inactivation, the pericentromeric duplicons dispersed in a relatively wide area (approx 10 Mb), whereas the large alphoid block, typical of active primate centromeres, completely disappeared. A second example of remains of an ancestral inactivated centromere is presented next.

Ancestral Centromeres and Neocentromeres

Centromeres are visible cytogenetically as the primary constriction and in primates are always associated with the presence of an array of α-satellite DNA ( 20). α-satellite, however, it is not an absolute requirement for centromere function, as evident in neocentromeres, which are devoid of satellite DNA.

The mechanisms underpinning neocentromere emergence is obscure. du Sart et al. (12) have

hypothesized the existence of latent centromeres with a finite capacity for neocentromerization.

Chapter 10 / Genome Plasticity in Evolution 157

Our investigations of chromosome 15 evolution provided evidence of an obscure but intriguing relationship between neocentromeres and ancestral inactivated centromeres.

Chromosomes 14 and 15 in humans and in great apes are separate chromosomes. In OWM, they form a unique chromosome (chromosome 7 in macaque) (21). We used a panel of bacterial artificial chromosome (BAC) clones to track the evolutionary history of the 14/15 association in macaque (Macaca mulatta [MMU]), sacred baboon (Papio hamadryas [PHA]), long-tailed macaque (Macaca fascicularis [MFA]), and silvered-leaf monkey (Presbytis cristata [PCR]).

All these species were found to share the 14/15 association, corresponding to chromosomes MMU7, PHA7, MFA7, and PCR5 (22). The results are summarized in Fig. 5. In all species these chromosomes appeared consistent with human chromosome14 and 15 being fused head–tail and retaining colinearity. The 14/15 association has been reported as ancestral to mammals (23). It can be deduced that in the ancestor to great apes, a noncentromeric fission event occurred between markers F and G, disrupting the 14/15 association and generating the present-day human

Fig. 3. Evolutionary history of human chromosome 6 established on the bases of marker order compari- son among the orthologous chromosomes in great apes, Old World monkeys, New World monkeys, and prosimians. MFA, long-tailed macaque, Macaca fascicularis; PCR, silvered leaf-monkey, Presbytis cristata; CJA, common marmoset, Callithrix jacchus; LLA, wooly monkey, Lagothrix lagothricha;

EMA, black lemur, Eulemur macaco; FCA, cat, Felix catus. N in a gray circle stands for a new cen- tromere; A in the black circle stands for an ancestral centromere. PA, primate ancestor; CA, catarrhini ancestor; OA, OWM ancestor. (Adapted with permission from ref.19.)

chromosomes 14 and 15. The ancestral centromere at 15q25 was inactivated and two new cen- tromeres were generated in regions corresponding to their present-day locations in humans.

Segmental duplication analysis showed a large cluster of intrachromosomal segmental duplica- tions at 15q24-q26 (data not shown). This cluster very likely derives from the inactivated ances-

Fig. 4. Distribution of segmental duplications between chromosome 6 and other human chromosomes.

Lines represent interchromosomal (gray) and intrachromosomal (black) alignments. Arrow indicates the cluster of intrachromosomal duplications at 6p22.1, which are the remains of an inactivated ancestral centromere. Note also that the pericentromeric region is relatively devoid of segmental duplications.

This is in agreement with the finding that human the chromosome 6 centromere is an evolutionary neocentromere. (Courtesy of Dr. E. E. Eichler.)

Fig. 5. The diagram shows the organization of human chromosomes 14/15 association in macaque (MMU, Macaca mulatta). A fission event gave rise to chromosomes 14 and 15. The fission was followed by the emergence of two new centromeres and by the inactivation of the old centromere at 15q25.

(Adapted with permission from ref.22.)

Chapter 10 / Genome Plasticity in Evolution 159

tral centromere. Fluorescence in situ hybridization (FISH) experiments using BAC probes con- taining duplicons of this region flanked the centromere in macaque (Fig. 6), strongly indicating that they are dispersed remains of the ancestral inactivated centromere. The region encompassed by duplicons that flanked the ancestral centromere is approx 11 Mb in size. Similarly to the inactivated ancestral centromere at 6p22.1, no remains of alphoid sequences were found.

The structural organization of the centromeres of the present-day human chromosomes 14 and 15 are indistinguishable from any other human centromere. The conclusion is that the evolutionary new centromeres progressively acquired the complexity of a normal centromere, which very likely stabilize the centromere function.

Interestingly, one of the hotspot site of neocentromeres appearance in clinical cases is at 15q24-q26. We studied two of these neocentromeres that were found several megabases apart but inside the region encompassed by duplicons of the ancestral centromere.

Alonso et al. (24) have reported that neocentromeres mapping at the 13q32 hotspot span a 6.5-Mb region. Both sets of data disproved the hypothesis that the neocentromere hotspots refer to a single specific locus. Interestingly, however, both 15q25 neocentromeres mapped to duplicons that are the remains of the ancestral centromere, therefore establishing an intriguing but unclear correlation between duplicon dispersal and neocentromere emergence. If this correlation is correct, one would expect the appearance of neocentromeres at 6p22.1 and at 2q21, where abundant duplicon remains of an inactivated centromere are present. We think, however, that other important factors are involved in neocentromere appearance. It is worth noting that most of the acentric fragments are small in size and associated with phenotypes compatible with embryo development. It could be hypothesized that aneuploidies generated by other latent cen- tromeres are incompatible with life and will never be detected among liveborn.

EVOLUTION OF CHROMOSOME 3:

RECURRENT SITES FOR NEW CENTROMERE SEEDING

Because the evolutionary history of the neocentromere hotspot at 15q25 revealed a connec-

tion to an ancestral inactivated centromere, we undertook a comprehensive study of the evo-

lutionary history of chromosome 3, harboring an additional neocentromere hotspot at 3q25-q26

Fig. 6. Partial metaphases showing fluorescence in situ hybridization experiments using the human bacterial artificial chromosome clone RP11-182J1 (chr15:82,764,242-82,935,727; 15q25.2) on human chromosome 15 (left) and on macaque chromosome X.

Fig. 7. Summary of fluorescence in situ hybridization results delineating the evolutionary history of human chromosome 3 in primates. The hypothesized pericentric or paracentric inversions are indicated by square parentheses spanning the inverted segment. Some chromosomes are upside down to facilitate comparison. PA, primate ancestor; CA, catarrhini ancestor; OWM-A, Old World monkeys ancestor; NWM-A, New World monkeys ancestor; GA-A, great ape ancestor; N (in a gray circle), new centromere. The number that identifies the chromosome in each species is reported on top of the chromosome. Arrows with hatched lines point to species for which an intermediate ancestor has not been drawn. A white N in a gray circle indicates the chromosomes where new centromeres appeared during evolution. The black circles (HSA and OWM-A) and the black squares (GA-A and NWM-A) are positioned at loci where seeding of an evolutionary new centromere was detected. Cat (FCA) probes were identified using the following strategy. The sequence encompassed by each human bacterial artificial chromosome (BAC) was searched for conservation against mouse and rat genome. “Overgo” probes were designed on the most conserved region of each BAC and were then used to screen the cat BAC library RPCI-86. Cat probes, therefore, map to loci orthologous the loci encompassed by the corresponding human BACs. Species analyzed: great ape: common chimpanzee (Pan troglodytes, PTR), gorilla (Gorilla gorilla, GGO), Borneo (Pongo pygmaeus pygmaeus, PPY-B) and Sumatra (Pongo pygmaeus abelii, PPY-S) orangu- tan; Old World monkeys (OWM): rhesus monkey (Macaca mulatta, MMU, Cercopithecinae), sacred baboon (Papio hamadryas, PHA, Cercopithecinae), African green monkey (Cercopithecus aethiops, CAE, Cercopithecinae), silvered leaf-monkey (Presbytis cristata, PCR, Colobinae); New World monkeys (NWM):

wooly monkey (Lagothrix lagothricha, LLA, Atelinae), common marmoset (Callithrix jacchus, CJA, Callitrichinae), dusky titi (Callicebus moloch, CMO, Callicebinae), squirrel monkey (Saimiri boliviensis, SBO);

prosimians: ring-tailed lemur (Lemur catta, LCA). (Adapted with permission from ref.25.)

(25). A panel of human BAC clones was used to reconstruct the order of chromosome 3 markers

in representative extant primates (Fig. 7). The analysis clearly showed the emergence of new

centromeres in OWM and great ape ancestors. Two human neocentromere cases at 3q25-26

were investigated. These two cases were found to have distinct but important implications

Chapter 10 / Genome Plasticity in Evolution 161

toward a better understanding of new centromere emergence phenomenon both in evolution and in clinical cases. In the first case, the neocentromere emergence occurred at 3q26.1 as part of a complex rearrangement. The functional centromere was excised, and a neocentromere seeded in the acentric derivative chromosome. Human BAC clone RP11-498P15 was identi- fied to be the closest to the neocentromere. This same clone (nc1 in Fig. 7) was found a few kilobases apart from the evolutionary new centromere that appeared in OWM ancestor. This coincidence suggested that the region at 3q26.1 maintained a latent centromere competence that was present before OWM/Hominidae divergence that occurred approx 25 million years ago (26). Furthermore, the new centromere that appeared in the great ape ancestor was seeded very close to marker G (Fig. 7). Interestingly, a new centromere was also seeded in this region in NWM ancestor (chromosome GF21 in Fig. 7) suggesting, again, that the same latent cen- tromeric region was activated twice, in great ape and NWM ancestors. Pevzner and Tesler (27), comparing mouse and human evolutionary breakpoints, have suggested the “reuse” of breakpoint regions in evolution. Similarly, the examples of neocentromeres we have discov- ered, both in evolution and in clinical cases, seem to extend the “reuse” concept to centromeres.

Evolution of Chromosome 3: Centromeres and Telomeres

In four NWM species examined, human chromosome 3 is split into three chromosomes annotated as (I), (II), and (III) in the Fig. 7. Chromosomes (I) and (II) have an identical marker order in common marmoset (CJA), wooly monkey (LLA), and dusky titi (Callicebus moloch [CMO]), if centromeres are not considered. The position of the centromeres, on the contrary, appears peculiar: the centromere is located at the opposite telomere in both (I) and (II) chro- mosomes in CMO with respect to the ancestral form of CJA and LLA. We have found similar examples of telomere–centromere functional exchanges in the evolution of other chromo- somes, for instance, chromosome 20 (28), and chromosomes 11 and 13 (Rocchi, unpublished results, 2005). Low-copy repeat-mediated exchanges may be responsible for centromere jump- ing between telomeres.

Evolutionary New Centromere Progression

A new centromere was seeded in OWM chromosome 6 during evolution (see Remains of

Inactivated Centromeres; Fig. 3). The human BAC clone RP11-474A9 (L2 in Fig. 3), mapping

at 6q24.3, yielded clear signals on both sides of the MFA6 centromere. Apparently, the new

centromere recruited the huge amount of centromeric/pericentromeric sequences without

affecting the displaced flanking sequences. The scenario accompanying the progression of the

OWM new centromere that appeared on OWM chromosome 3 was different. We found that

this centromere is encompassed by human K1 and K2 markers (BAC clones RP11-355I21 and

RP11-418B12, respectively), located 436 kb apart in human. BAC clones spanning this gap did

not yield any appreciable FISH signal in macaque, African green monkey (CAE), and PCR,

whereas giving normal FISH signals in all great apes. Thus the 436-kb region appears lost in

OWMs as a consequence of the neocentromerization event. This chromosomal trait was inves-

tigated for gene content. The human region (UCSC chr3:163,941,067-164,377,941) appears

devoid of genes, harboring only two transcribed but not translated spliced RNAs (UCSC, July

2003 release). It can be hypothesized that the absence of functional constraint made possible

the loss (or complete restructuring) of the region. On the contrary, the functional elements

around the new centromere insertion on chromosome 6 in OWM (see above) behaved as a

strong selective barrier to the degenerative activity of the evolving centromeric region.

chromatin, as if the centromere were at an intermediate stage between new centromere seeding and a final stage in which all genes are excluded from the completely heterochromatized centromere, as occurs in the remaining rice centromeres.

Additional Euchromatic Duplicon Clusters of Pericentromeric Origin Segmental duplications accumulate at greater density in pericentromeric and telomeric regions (6). However, large blocks of duplicons exist also in euchromatic locations. We have previously shown that duplicon clusters located, in humans, at euchromatic regions 6p22.1 and 15q25 are the remains of inactivated centromeres. Other chromosomal rearrangements can relocate pericentromeric duplicons in noncentromeric areas. One of the two breakpoints of both pericentric and paracentric evolutionary inversions falls, rather frequently, in pericentromeric regions (8,28,32). In these cases part of a pericentromeric duplicated region can be repositioned far apart from the centromere. Two nonpericentromeric olfactory receptor clusters lying on chromosome 3 are precisely located at domains identified by A2 and I1 markers (Fig. 7). The evolutionary history of chromosome 3 we have reconstructed indicated that these two clusters are examples of duplicons that were pericentromerically located during evolution (see marker organization in the primate ancestor [PA] in Fig. 7).

Most mutations of the CYP21 locus (steroid 21-hydroxylase gene) are owing to a close pseudogene (CYP21P) that triggers gene conversions during unequal crossovers (33). Both CYP21and CYP21P are located inside the duplicon cluster at 6p22.1 that represents the remains of an evolutionary inactivated centromere (see above). Gene conversion, therefore, is an addi- tional, indirect mechanism by which duplicon cluster contribute to human genome morbidity.

CENTROMERE REPOSITIONING “IN PROGRESS”

Most of human neocentromere cases arise as a consequence of a rearrangement that generated an acentric fragment. The presence of the acentric fragment as a supernumerary chromosome results in phenotypic abnormalities, which indirectly prevent the transmission of the neocentromere-bearing chromosome to successive generations. They, therefore, have no evolu- tionary relevance. Very recently, however, Amor et al. (34) and Ventura et al. (25) have doc- umented two unprecedented neocentromere cases, at 4q21.3 and 3q24, respectively, that arose in an otherwise normal and mitotically stable chromosomes that were transmitted to subsequent generations. These individuals had normal phenotypes. They can be regarded as present-day episodes equivalent to new centromere appearance that occurred during the course of evolution and that were fixed in the population. Individuals with chromosomal changes not accompanied by clinical signs or reproductive problems usually escape detection. It can be reasonably sup- posed that the “normal” cases reported so far are only a minimal fraction of the existing ones.

In individuals heterozygous for the neocentromere, an odd number of meiotic exchanges

internal to the chromosomal region encompassed by the two active centromeres leads to the

Chapter 10 / Genome Plasticity in Evolution 163

formation of dicentric and acentric chromosomes. These problems could affect the fitness of heterozygous individuals and act as a negative selective pressure. Meiotic drive in females in favor of the repositioned chromosome is among the possible mechanisms favoring the fixation of the neocentromere. Meiotic drive favoring Robertsonian translocations has been reported by Pardo-Manuel de Villenna and Sapienza (35).

NEOCENTROMERES IN NONPRIMATE MAMMALS

One of the first steps in a sequencing project is the construction of detailed genetic and physical maps obtained using linkage analysis and radiation hybrids studies, respectively.

Radiation hybrid maps have been constructed, i.e., for cattle (36), cat (37), horse (38), and pig (39). These studies could represent, in theory, an efficient tool to pinpoint centromere- repositioning events when maps of different species are compared. However, most of these studies were not focused on evolutionary aspect of genome organization. The paper on the bovine radiation hybrids map published by Band et al. (40), on the contrary, took into account the position of the cattle centromeres with respect to humans. The authors concluded that “the comparative map suggests that 41 translocation events, a minimum of 54 internal rearrange- ments, and repositioning of all but one centromere can account for the observed organizations of the cattle and human genomes.” Most likely, they have overestimated the centromere- repositioning phenomenon in cattle. However, it indicates that the repositioning phenomenon may be much more widespread than expected.

Horse and donkey (equidae) diverged about 2 million years ago (41). They produce hybrids (mule and hinny) that are mostly sterile owing to the several chromosomal rearrangements that differentiate horse and donkey chromosomes (42). Yang et al. (43), using a combination of classical G-banding and whole chromosome paints, have noticed the discordant position of the centromere in four orthologous chromosomes, i.e., four distinct centromere repositioning events occurred in a relatively short evolutionary time.

CONCLUDING REMARKS

Centromere repositioning is a newly discovered, unprecedented phenomenon that has a substantial impact on chromosomal architecture. Indeed, the newly seeded centromere rapidly acquires the large size and the complex organization of a normal centromere, whereas the inactivated one disperses in the area large blocks of segmental duplications that were recruited around it when active. In addition, these remainings can contribute to the instability of the human genome (44). Segmental duplicons, indeed, have been shown to play a crucial role in triggering genomic disorders (see Chapters 2–23).

It can be reasonably assumed that only a few new centromere events have been fixed in the population during evolution. Therefore, the baseline rate of new centromere occurrence could be relatively high. The two healthy human individuals with a repositioned but otherwise “normal”

chromosomes, recently reported in the literature, provide living examples of centromere reposition- ing events at the very early stages, further supporting the centromere repositioning hypothesis.

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Centro di Eccellenza Geni in campo Biosanitario e Agroalimentare (CEGBA), Ministero

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