20 Inversion Chromosomes

Chapter 20 / Inversion Chromosomes 289 20 Inversion Chromosomes

Orsetta Zuffardi, PhD, Roberto Ciccone, PhD, Sabrina Giglio, MD, PhD, and Tiziano Pramparo, PhD

CONTENTS INTRODUCTION CLASSICAL CHROMOSOME INVERSIONS: EPIDEMIOLOGICAL DATA RECOMBINANT CHROMOSOMES FROM CARRIERS OF CLASSICAL HETEROZYGOUS INVERSIONS CRYPTIC INVERSIONS ASSOCIATED WITH SEGMENTAL DUPLICATIONS CRYPTIC INVERSION AT 8P23 CARRIERS OF THE 8P23 INVERSION: ARE THEY AT RISK FOR UNBALANCED OFFSPRING? OTHER CHROMOSOME REARRANGEMENTS MEDIATED BY CRYPTIC PARACENTRIC INVERSION CONCLUSIONS AND PROSPECTIVES REFERENCES

INTRODUCTION A number of findings revealed that chromosome inversions are more frequent than deduced from classical cytogenetic studies. Indeed, some paracentric cryptic inversions have been found to be flanked by segmental duplications, either causing a Mendelian disease owing to the interruption of specific genes at inversion breakpoints or being present in the normal population as a polymorphism. In the latter case, in the heterozygous state they predispose to further unbalanced rearrangements such as inv dup rearrangements or simple deletions and duplications. The importance of this susceptibility factor has been well clarified with respect to some genomic disorders involving chromosome 8p and it is now emerging as a possible model that may explain the genetic basis of other recurrent chromosome rearrangements.

CLASSICAL CHROMOSOME INVERSIONS: EPIDEMIOLOGICAL DATA Chromosome inversions are the most common rearrangement differentiating humans and the great ape species at the karyotypic level (1–4). Inversions in which a breakpoint is in heterochromatic regions (1qh, 9qh, 16qh, and Yq) are relatively frequent and are regarded as variants. The most common inversion not involving centromeric heterochromatin is the

From: Genomic Disorders: The Genomic Basis of Disease Edited by: J. R. Lupski and P. Stankiewicz © Humana Press, Totowa, NJ 289 290 Part IV / Genomic Rearrangements and Disease Traits inv(2)(p11q13) that is also considered a benign variant. Other polymorphic inversions are also frequent and include inv(5)(p13q13) and inv(10)(p11q21.2) (5). Apart from these cases, inversions are approx 10 times more rare than the other balanced rearrangements (Robertsonian translocations: 1 in 1000; reciprocal translocations: 1 in 625; [6]) having a frequency ranging from approx 0.012 to 0.07% for the pericentric inversions and approx 0.01 to 0.05% for the paracentric inversions (7). In contrast to translocations for which the most frequent cause of ascertainment is the presence of reproductive difficulties, the majority of inversions are ascertained at prenatal diagnosis or because of an abnormal phenotype (5). The risk of unbalanced offspring from an inversion carrier, putting together the data of pericentric and the paracentric inversions, is approx 1%, much lower than that of reciprocal translocation carriers (2.7% in families ascertained through a balanced proband and 19.2% among the families ascertained through an unbalanced proband) (5). Accordingly, the reproductive fitness of inversion carriers is 0.926 ± 0.085, higher than that of reciprocal translocation carriers (0.70 ± 0.048; [8]). Reproductive fitness in Robertsonian translocation carriers varies between 0.768 ± 0.056 for the D/D carriers and 0.921 ± 0.123 for the D/G carriers (8).

RECOMBINANT CHROMOSOMES FROM CARRIERS OF CLASSICAL HETEROZYGOUS INVERSIONS Studies on inversion heterozygotes in man and in other species have reported crossover suppression in the inverted region (9) and increased recombination elsewhere on the same chromosome (10). When recombination occurs within the inverted region between the normal and the inverted chromosomes two complementary recombinant chromosomes arise. Each of them is duplicated for the distal region of one arm and deleted for the opposite one. In the case of paracentric inversions the two recombinants are dicentric and acentric. The size of the inversion seems to be the main factor influencing the type of synapsis between the inverted and the normal chromosome. For a more complete discussion on this topic, see ref. 7.

CRYPTIC INVERSIONS ASSOCIATED WITH SEGMENTAL DUPLICATIONS In the last 15 years, a series of submicroscopic paracentric inversions have been discovered while studying specific genes at a genomic level. Some of these inversions lead to the breakage of a dosage sensitive gene and, therefore, cause a Mendelian disease, other inversions break within either an extragenic region or disrupt nondosage sensitive genes and are, thus, considered benign. Most of these cryptic inversions are mediated by large (usually >10 kb), highly homologous low-copy repeat (LCR) structures (also called segmental duplications or duplicons) that can act as recombination substrates in nonallelic homologous recombination (NAHR). There are clear evidences that some of the benign inversions are not mere neutral polymorphisms but may predispose to other rearrangements. The story began in 1993 when Lakich et al. (11) discovered that nearly half of the patients with severe haemophilia A had a breakage of the factor VIII gene caused by an inversion mediated essentially by intrachromatid recombination between DNA sequences in the A gene in intron 22 and one or other of two inverted copies of this sequence (int22h-2, int22h-3) located, respectively, 500 and 600 kb more telomeric. A few years later a similar mechanism was shown to be responsible for 13% of Hunter disease patients. In this case the inversion occurs because of abnormal recombination between the IDS gene and its pseudogene located 90 kb away, resulting in the disruption of the Chapter 20 / Inversion Chromosomes 291 gene (12). Other recurrent cryptic inversions mediated by homologous LCRs have been reported in normal individuals and, at least some of them, are regarded as benign. The inversion at the Xq28 emerin/filamin region was found while trying to elucidate the discrepancies observed between the genetic and physical map distances (13). This inversion, present in the heterozygous state in 33% of females and in the hemizygous state in 19% of males, is mediated by two 99% sequence identical segmental duplications flanking the emerin and filamin genes. Although inversion carriers are completely normal, some of the emerin deletions associated with Emery-Dreifuss muscular dystrophy have been hypothesized to be the result of inversion- mediated rearrangements (14). Moreover, it is not impossible that some of the dicentric X chromosomes pter-qter::qter-pter (15) are formed as a consequence of unequal crossovers between the same LCRs that mediate the inversions. Another recurrent benign cryptic inversion has been detected at the region surrounding the NPHP1 locus at 2q13. Saunier et al. (16) discovered that homozygous deletion of 290 kb responsible for nephronophthisis 1 is mediated by two copies of segmental duplications with the same orientation. These LCRs are surrounded and partially embedded into two homologous segmental duplications having opposite orientation and mediating an approx 500-kb inversion present in 1.3% of the population in the homozygous state. No negative effect has been demonstrated in association with this inversion. It is obvious that embryos with any dicentric chromosome 2pter-2q13::2q13-2pter would not be viable. The paradigmatic situation is that of the Y chromosome. This chromosome has an abun- dance of LCR (amplicons), which render this chromosome susceptible to a multitude of rearrangements that, when involving the long arm, are often the cause of spermatogenic failure (see Chapter 19) (17). It has been assumed that the polymorphisms observed at loci on the Y chromosome and mtDNA are selectively neutral and, therefore, existing patterns of molecular variation could be used to deduce the histories of populations in terms of drift, population movements, and cultural practices. However, Jobling et al. (18) demonstrated that the 3-Mb Yp inversion present in the male population is the preferential background for the PRKX/PRKY translocation underlying most XX males and some XY females (Fig. 1). This is a clear dem- onstration that this Y inversion “polymorphism” is not neutral. It seems likely that other cryptic inversions are responsible for the complex series of deletions and duplications mediated by several types of LCRs associated with the AZFc locus (19,20).

CRYPTIC INVERSION AT 8p23 The finding that a Y chromosome inversion was the basis for a recurrent translocation (18) was at first considered peculiar to the sex chromosomes. Further relevance of these data emerged following our studies (21) reporting that some recurrent chromosome rearrangements at 8p occur as a consequence of an 8p submicroscopic paracentric inversion present in the parent transmitting the abnormal chromosome. At that time, we already knew that the recurrent inv dup(8p) rearrangement, an inverted duplication of the chromosome 8 short arm associated with deletion of the very distal 8p (8p23.2-pter), was not the primary product of an abnormal recombination but instead was produced by the breakage of a dicentric chromosome 8qter- 8p23.1::8p23.1-8qter (22) leading to the formation of an inv dup(8p) and, though formally not demonstrated, to a chromosome 8 deleted for part of its short arm (8p-) (Fig. 2). In the dicentric chromosome the two duplicated regions are separated by a small single copy region (Fig. 3). The fact that the original inv dup (8p) was a dicentric chromosome led us to hypothesize that 292 Part IV / Genomic Rearrangements and Disease Traits

Fig. 1. Mechanism of the Xp/Yp translocation at the basis of XX males and XY females. (Top) Recom- bination between the Xp and Yp chromosome normally occurs at the pseudo-autosomal region 1 (PAR). PRKX gene lies on the X chromosome approx 1 Mb centromeric to PAR. On the Y chromosome the SRY gene is just centromeric to PAR. PRKY, homologous to PRKX, lies about 4.5 Mb centromeric to PAR and is included within a polymorphic inversion region (yellow region) of 3 Mb. In noninverted subjects PRKX and PRKY have opposite orientation, thus preventing any possible recombination event. In subjects with inverted PRKX and PRKY acquire the same orientation (Bottom), thus, allowing an ectopic recombination. The result is the transposition of SRY on the Xp chromosome and the absence of SRY on the Y chromosome. the reciprocal analphoid chromosome might be present in some subjects who acquired of a neocentromere. From a literature review we were able to identify two of such cases where the analphoid chromosome was a supernumerary marker that was mosaic with a normal cell line. Molecular definition of this marker (+der[8p]) demonstrated that it was exactly the reciprocal of the dicentric chromosome 8 and that both rearrangements were mediated by a pair of olfactory receptor (OR)-gene clusters mapping to 8p23.1 (21) (Figs. 3 and 4). The refined distance between the two clusters (the single-copy region) is approx 3.5 Mb. We reasoned that, according to the classical cytogenetics, the production of two reciprocal rearrangements, one dicentric and the other acentric, could only result following a crossover within a paracentric inversion. We, thus, studied by fluorescent in situ hybridization the apparently normal chromosomes 8 in the parent-of-origin of several inv dup(8p)s (thus far 16 cases) and of one case of +der(8p) and discovered that a heterozygous paracentric inversion with the same breakpoints of the dicentric and the acentric chromosomes was present in all of them. The presence of the Chapter 20 / Inversion Chromosomes 293

Fig. 2. The dicentric chromosome resulting from nonallelic homologous recombination can undergo a breakage at the level of the second centromere or a more proximal breakage, leading to an inv dup(8)s and a del(8p). Note that the inv dup(8p)s may differ according to the duplication size.

Fig. 3. Cryptic paracentric inversion between the two pairs of olfactory receptor (OR) gene clusters mapping to 8p23.1. Non-allelic homologous recombination results in a dicentric and an acentric recombinant chromosome. The single copy region is delimited by the two OR gene clusters (red arrows).

inversion also explained a highly unlikely crossover pattern at 8p23 observed in members of some Centre d’Etude du Polymorphisme Humain families (23) that showed apparent triple recombination events in a very short region. Studies on normal populations revealed that the 8p23 inversion is present in the heterozygous state in 26% of the European population (21) and in 39% of the Japanese population (24). Thus, the inversion represents a genomic polymorphism that, 294 Part IV / Genomic Rearrangements and Disease Traits

Fig. 4. The inv dup(8p) and the analphoid supernumerary chromosome +der(8p) are the two reciprocal products of the same abnormal recombination. Both fluorescent in situ hybridization images show the results obtained with the bacterial artificial chromosome (BAC) clone RP11-287P18 specific to the distal 8p olfactory receptor genes cluster. This clone gives signals on different chromosomes owing to high sequence identity of some of the olfactory receptor family members. Normal chromosome 8 and the inv dup(8p) are indicated by an arrow and an arrowhead, respectively. On the right image, the +der(8) appears completely covered by the signals. In the small squares, the green signals correspond to a single copy BAC clone (RP11-5E15) in distal 8p. The inv dup(8) lacks this signal, whereas the +der(8p) shows two opposite signals. in the heterozygous state, renders misalignment and abnormal recombination more likely, just as it occurs in cytogenetically identifiable inversions (7). In other words, the inversion predis- poses the individual to a susceptibility to the formation of what were considered de novo chromosome rearrangements (inv dup[8p] and the + der[8p]) (Fig. 3).

CARRIERS OF THE 8P23 INVERSION: ARE THEY AT RISK FOR UNBALANCED OFFSPRING? An unexpected finding is that both subjects with the inv dup(8p) or with +der(8) are single cases in their families, although the parent who has transmitted the anomalous chromosome is a carrier of the inversion and, therefore, one would have anticipated more than one unbalanced child. However, if we consider the analphoid chromosome, the occurrence of a neocentromere is a rare event (25). Thus, most of the zygotes containing a +der(8p) will lose it very soon after acquiring a normal chromosome complement. As to the dicentric chromosome, we had assumed that it did undergo a breakage at the second meiotic division thus leading to a gamete having the inv dup(8p) and a gamete deleted for a portion of 8p. Floridia et al. (22) demonstrated that the size of the inv dup(8p) may differ according to the size of the 8p duplication which may involve even the centromere (from 8p22 to 8pcen) or may be much smaller (from 8p21.2 to 8p22) (Fig. 2). On the contrary, the inv dup(8p) size is remarkably constant as to the deletion region (8p23.2-pter) and the single copy region at 8p23.1 flanked by the two clusters of OR genes. These findings demonstrated that the dicentric breakage may occur in different positions between the Chapter 20 / Inversion Chromosomes 295 two centromeres leading to a dicentric inv dup(8p) and a reciprocal acentric 8q, or to inv dup (8p)s with smaller duplications and reciprocal 8p- with different degrees of deletion (Fig. 2). It seems very likely that most of the deleted chromosomes 8 are not compatible with embryonic development resulting in premature termination of the pregnancy. Moreover, we demonstrated that in some cases the dicentric does not break at meiosis II but is inherited as such leading to an almost completely trisomic 8 zygote. Because of its instability, the dicentric may undergo breakage generating different cell lines in the embryo (26). Should the inactivation of one centromere occur very early, thus stabilizing the dicentric 8, the resulting embryo will be trisomic for an almost entire chromosome 8. It has been clearly demonstrated that survival of trisomy 8 is possible when the aneuploid cell line arises relatively late in development (27). Thus, embryos with trisomy 8 owing to the presence of a dicentric chromosome are expected to be prematurely aborted. Because no evidence of increased spontaneous abortions has been found in mothers of inv dup(8p) subjects (manuscript in preparation) we postulate that the trisomy 8 fetuses result in preclinical abortions. In conclusion, as Madan (28) and Sutherland et al. (29) already observed with paracentric inversion carriers, also subjects carrying the 8p23 cryptic inversion have a very low risk for unbalanced progeny.

OTHER CHROMOSOME REARRANGEMENTS MEDIATED BY CRYPTIC PARACENTRIC INVERSION Osborne et al. (30) observed a heterozygous inversion of the Williams-Beuren syndrome region in 4 of 12 parents transmitting the disease-related chromosome. Their data had been fully confirmed by Bayés et al. (31), who found that one-third of the transmitting progenitors were heterozygous for an inversion between the centromeric and the telomeric segmental duplications at 7q11.23, which are in opposite orientation. The mechanism by which the inversion generates an interstitial deletion is probably owing to the fact that the normal and the inverted chromosome synapse for all their length and “balloon out” at the inversion region, thus allowing NAHR between the segmental duplications lying in opposite orientation (Fig. 5). We also demonstrated that the t(4;8)(p16;p23) recurrent translocation, reported in several cases (32) either in the balanced or unbalanced form, is mediated by two pairs of homologous OR-gene clusters located at 4p16 and 8p23, respectively (33). In five de novo cases of unbalanced and balanced translocations, all of maternal origin, we could demonstrate a double heterozygous inversion between the two pairs of OR-gene clusters at 4p16 and 8p23 in all the mothers. It seems likely that the two pairs of homologous chromosomes cannot synapse in their distal short arm regions involved in the inversion and this allows the occurrence of a cross- ingover between homologous regions located into nonhomologous chromosomes (Fig. 6). 4p16 and 8p23 heterozygous inversion was detected in 12.5% (4/40) and 26% (13/50) of control subjects, respectively, whereas 2.5% (1/40) were scored as double heterozygous. Heterozygous cryptic inversions as the basis of unbalanced rearrangements have also been found in mothers having a 15q11-q13 deleted Angelman syndrome (AS) child (34). The inversion was detected in the mothers of four out six AS cases with the breakpoint 2–3 (BP2/3) 15q11–q13 deletion, but not in seven mothers of AS because of paternal uniparental disomy 15. The BP2–BP3 chromosome 15q11–q13 inversion was detected in 4 of 44 subjects (9%) of the general population. From these data it emerges that the heterozygous inversion between homologous segmental duplications is an important mechanism causing abnormal synapsis and NAHR and, thus, that 296 Part IV / Genomic Rearrangements and Disease Traits

Fig. 5. Possible mechanism explaining how the paracentric inversion may lead to a deleted recombinant chromosome. The abnormal synapsis created by the inversion allows recombination between identical or nearly identical segmental duplications (gray blocks, A and B). Arrows indicate the occurrence of recombination. Chess-patterned circles indicate the centromeres. these types of inversions constitute an important factor for susceptibility to the occurrence of unbalanced chromosome rearrangements. However, it is also clear that not all recurrent rearrangements can be explained by this mechanism: Saitta et al. (35) did not find evidence of inversion of 22q11.2 in the chromosome that becomes deleted in subjects with a DiGeorge/ velocardiofacial syndrome phenotype. Similarly, several researchers could not find any inversion at 15q11-q13 in the fathers of deletion Prader-Willi syndrome subjects. Thus, it seems obvious that other mechanisms beyond to the inversion may cause synapsis displacement between homologous segmental duplications, in turn, leading to NAHR. Recent studies (36,37) demonstrated the existence of large segments of the genome, ranging in size from 100 kb to 2 Mb, varying several folds in copy number in the human population and Chapter 20 / Inversion Chromosomes 297

Fig. 6. The recurrent translocation t(4;8)(p16;p23) is mediated by two pairs of olfactory receptor gene clusters at 4p16 and 8p23 (yellow and orange blocks). Double heterozygous inversions between these two segmental duplications prevent a normal synapsis to occur along each pair of homologous chromosomes and allows nonallelic homologous recombination between nonhomologous chromosomes.

denominated large-scale copy number variations (LCVs). A higher than expected association between LCVs and known segmental duplications has been noted both by Iafrate et al. and Sebat et al. (36,37). It has been demonstrated that the instability of some genomic regions is owing to the presence of segmental duplications (38). This suggests that LCVs and genomic rearrangements might have a common mechanistic basis (39).

CONCLUSIONS AND PROSPECTIVES Inversions and more specifically cryptic inversions flanked by inversely oriented segmental duplications could be much more frequent than previously estimated. Segmental duplications represent approx 5% of our genome. A systematic study of the genomic regions flanked by segmental duplications (40) might reveal other inversion genomic polymorphisms. Ours and other studies revealed that cryptic inversions flanked by LCRs are an important but not the only susceptibility factor for the occurrence of unbalanced constitutional chromosome rearrangements. After years of darkness on the molecular causes of structural chromosome abnormalities and on the possible susceptibility factors behind them, it seems that the tangle begins to be unravelled. Segmental duplications are the cause of genomic instability and heterozygous carriers of cryptic inversions are at enhanced risk to form gametes with chromosome imbal- ances. Investigations on the presence of heterozygous variants with respect to LCVs in the parents transmitting structural chromosome abnormalities will likely determine whether these variants represent another risk factor. 298 Part IV / Genomic Rearrangements and Disease Traits

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