Genome architecture, rearrangements and genomic disorders

An increasing number of human diseases are recognized to result from recurrent DNA rearrangements involving unstable genomic regions. These are termed genomic disorders, in which the clinical phenotype is a consequence of abnormal dosage of gene(s) located within the rearranged genomic fragments. Both inter- and intrachromosomal rearrangements are facilitated by the presence of region-specific low-copy repeats (LCRs) and result from nonallelic homologous recombination (NAHR) between paralogous genomic segments. LCRs usually span approximately 10-400 kb of genomic DNA, share >or= 97% sequence identity, and provide the substrates for homologous recombination, thus predisposing the region to rearrangements. Moreover, it has been suggested that higher order genomic architecture involving LCRs plays a significant role in karyotypic evolution accompanying primate speciation.     

Genome organization influences partner selection for chromosomal rearrangements

Chromosomal rearrangements occur as a consequence of the erroneous repair of DNA double-stranded breaks, and often underlie disease. The recurrent detection of specific tumorigenic rearrangements suggests that there is a mechanism behind chromosomal partner selection involving the shape of the genome. With the advent of novel high-throughput approaches, detailed genome integrity and folding maps are becoming available. Integrating these data with knowledge of experimentally induced DNA recombination strongly suggests that partner choice in chromosomal rearrangement primarily follows the three-dimensional conformation of the genome. Local rearrangements are favored over distal and interchromosomal rearrangements. This is seen for neutral rearrangements, but not necessarily for rearrangements that drive oncogenesis. The recurrent detection of tumorigenic rearrangements probably reflects their exceptional capacity to confer growth advantage to the rare cells that contain them. The abundant presence of neutral rearrangements suggests that somatic genome variation is also common in healthy tissue.     

Complex chromosomal rearrangements

Complex Chromosomal Rearrangements (CCRs) are constitutional structural rearrangements involving three or more chromosomes or having more than two breakpoints. CCRs preferentially occur during spermatogenesis and are transmitted in families through oogenesis. Recent investigation showed that CCRs are more complex and more common than initially appreciated. Here 1 present an overview of CCRs, including the important impact of CCRs in fertility, the mechanism of their development, the various meiotic errors that can occur and their consequences. The review also discusses the differential transmission of CCRs in males and females, the incidence of pregnancy outcomes of CCR carriers, genetic counseling and prenatal diagnosis.     

Engineering chromosomal rearrangements in mice

The combination of gene-targeting techniques in mouse embryonic stem cells and the Cre/loxP site-specific recombination system has resulted in the emergence of chromosomal-engineering technology in mice. This advance has opened up new opportunities for modelling human diseases that are associated with chromosomal rearrangements. It has also led to the generation of visibly marked deletions and balancer chromosomes in mice, which provide essential reagents for maximizing the efficiency of large-scale mutagenesis efforts and which will accelerate the functional annotation of mammalian genomes, including the human genome.     

A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders

The prevailing mechanism for recurrent and some nonrecurrent rearrangements causing genomic disorders is nonallelic homologous recombination (NAHR) between region-specific low-copy repeats (LCRs). For other nonrecurrent rearrangements, nonhomologous end joining (NHEJ) is implicated. Pelizaeus-Merzbacher disease (PMD) is an X-linked dysmyelinating disorder caused most frequently (60%-70%) by nonrecurrent duplication of the dosage-sensitive proteolipid protein 1 (PLP1) gene but also by nonrecurrent deletion or point mutations. Many PLP1 duplication junctions are refractory to breakpoint sequence analysis, an observation inconsistent with a simple recombination mechanism. Our current analysis of junction sequences in PMD patients confirms the occurrence of simple tandem PLP1 duplications but also uncovers evidence for sequence complexity at some junctions. These data are consistent with a replication-based mechanism that we term FoSTeS, for replication Fork Stalling and Template Switching. We propose that complex duplication and deletion rearrangements associated with PMD, and potentially other nonrecurrent rearrangements, may be explained by this replication-based mechanism.     

Genome rearrangements: mother knows best!

In Paramecium, developmentally programmed genome rearrangements can be altered by the presence of homologous sequences within the maternal somatic nucleus. Newly identified RNA-binding proteins appear to mediate the transfer of homologous sequence information from the maternal to the developing somatic nucleus, facilitating epigenetic regulation of this large-scale genome reorganization.     

Tissue specificity of chromosomal rearrangements in ataxia-telangiectasia

Summary Cytogenetic studies of lymphocytes and fibroblasts from individuals with ataxia-telangiectasia (AT) demonstrate spontaneous chromosomal breakage. In the AT lymphocytes, this damage results in a high frequency of balanced rearrangements involving chromosome bands 7p14, 7q35, 14q12, and 14q32. The T-cell receptor , , and chain gene complexes and the immunoglobulin heavy chain gene complex, all of which may be functional in lymphocytes, have been localized to these bands. To assess the relationship between genes at these breakpoints and the entirety of the AT phenotype, we undertook a detailed cytogenetic analysis of fibroblasts and lymphocytes from seven AT homozygotes. Our findings indicate that the rearrangements present in the lymphocytes are not commonly observed in the fibroblasts, despite the increased instability of chromosomes from these cells relative to lymphocytes. Furthermore, the changes in the fibroblasts are neither consistent within nor between patients, suggesting that chromosome rearrangement occurs more randomly in this tissue. Therefore, differential site-specific damage in separate tissues may generate the distinct features of the disease in those tissues and may account for the pleiotrophic effects of the AT gene.     

Synaptonemal complex analysis of mouse chromosomal rearrangements

Two paracentric inversions in the mouse, In(1)1 Rk and In(2)5 Rk, have been studied in surface microspreads of spermatocytes from heterozygotes. At zytogene, synaptic initiation occurs independently in three regions: within the inversion, and without, on either side. Synaptonemal complex (SC) formation is restricted to homologous regions, resulting in inversion loops in all early pachytene spermatocytes. An adjusting phase then occurs during pachytene in which the inversion loop is reduced by desynapsis of homologously synapsed SC, followed immediately by non-homologous synapsis with the alternate pairing partner, progressing from the ends toward the middle. Adjustment occurs during the first half of pachytene, but is not closely synchronized with sub-stage. It is complete by late pachytene, the loop having been eliminated in all cases and replaced by straight SCs in which the inverted region is heterosynapsed. Synapsis in the adjustment phase is evidently permitted only after the homosynaptic phase, and is indifferent to homology. It may lead to heterosynapsis, as in the inversion region, or to synapsis of homologous regions not synapsed at zytogene. The anaphase bridge frequency, a measure of crossing over within the inversion, is about 34% for both inversions studied, indicating that such crossovers do not block adjustment, that crossing over probably occurs before or during the adjustment period, and that there is some crossover suppression. The last could be the consequence of blocking by desynapsis/heterosynapsis. Synaptic adjustment appears to be a general phenomenon that occurs to varying extents in different forms. A hypothetical scheme for two phases of synapsis is proposed: at zytogene, a basic propensity for indifferent SC formation is limited by a restricting condition to synapsis between homologous regions. Subsequently, the restriction is lifted, whereupon synaptic instability is resolved by desynapsis, followed by resynapsis that is indifferent to homology, but that results in a topologically more stable structure.     

Synaptonemal complex analysis of mouse chromosomal rearrangements

Synaptonemal complex (SC) analysis by electron microscopy of spermatocytes in surface microspreads was carried out in mice heterozygous for two paracentric inversions: either In(1)1RK or In(2)5Rk. Characteristic SC inversion loops are formed at synapsis in bivalents carrying the rearrangements. Although all loops were observed to be eliminated by late pachytene through synaptic adjustment, every spermatocyte at early pachytene contained a fully synapsed loop. Cells in the earliest stage of pachytene contained the longest loops and thus had undergone minimal adjustment. The SC estimates of inversion lengths and breakpoint positions in such cells corresponded well with those from mitotic chromosome banding and could be correlated with genetic maps of chromosomes # 1 and # 2, thus demonstrating the basis for the mapping of pachytene chromosomes. The regularity of loop formation and reproducibility of the SC analysis are reflected in the constant relative positions of the estimated breakpoints. The method is sensitive enough to reflect small, real, interstitial length differences between meiotic and mitotic chromosomes. The results demonstrate the feasibility and precision of detection and quantitative characterization of inversions at early meiotic prophase by SC analysis.This paper is warmly dedicated to Prof. Dr. Wolfgang Beermann, on the occasion of his 60th birthday     

Synaptonemal complex analysis of mouse chromosomal rearrangements

Electron microscopy of surface-spread spermatocytes from mice heterozygous for a tandem duplication shows the heteromorphic synaptonemal complex (SC) to comprise two lateral elements of unequal length, the longer of which is buckled out in a characteristic loop, representing the unsynapsed portion of the duplication. The loop is a regular feature of late zygotene-early pachytene nuclei; it is longest at these early stages, but, through equalization of the two axes as a consequence of synaptic adjustment, it is replaced by a normal appearing SC at late pachytene. Because equalization, as indicated by a decrease in the percent difference between axes, may begin shortly after completion of synapsis, estimates of duplication segment length are restricted to a sample selected for least adjustment. — Although the mean position of the loop is constant at various pachytene substages, individual positions vary widely from cell to cell, consistent with the behavior expected of a duplication, but not of a deletion or an inversion. The length of the segment that is duplicated is estimated to be 22% of the normal chromosome, the midpoint of the segment is mapped at 0.61 of the chromosome distal to the kinetochore, and the ends of the segment are mapped at 0.50 to 0.72. Measurements of G-banded mitotic chromosomes give comparable values: duplication length, 24%; midpoint, 0.60, and segment ends, 0.48 and 0.71. This agreement constitutes further validation of the SC/spreading method for detecting and analyzing chromosomal rearrangements at pachytene and substantiates the fidelity with which the axes and SCs represent the behavior of chromosomes in synapsis.     

Effects of chromosomal rearrangements on human-chimpanzee molecular evolution

Many chromosomes are rearranged between humans and chimpanzees while others remain colinear. It was recently observed, based on over 100 genes, that the rates of protein evolution are substantially higher on rearranged than on colinear chromosomes during human-chimpanzee evolution. This finding led to the conclusion, since debated in the literature, that chromosomal rearrangements had played a key role in human-chimpanzee speciation. Here we re-examine this important conclusion by employing larger a data set (over 7000 genes), as well as alternative analyses. We show that the higher rates of protein evolution on rearranged chromosomes observed in the earlier study are not reproduced by our survey of the larger data set. We further show that the conclusion of the earlier study is likely confounded by two factors introduced by the relatively limited sample size: (1) nonuniform distribution of genes in the genome, and (2) stochastic noise in substitution rates inherent to short lineages such as the human-chimpanzee lineage. Our results offer a general cautionary note on the importance of controlling for hidden factors in studies involving bioinformatic surveys.     

Genome rearrangements by nonlinear transposons in maize.

Transposable elements have long been considered as potential agents of large-scale genome reorganization by virtue of their ability to induce chromosomal rearrangements such as deletions, duplications, inversions, and reciprocal translocations. Previous researchers have shown that particular configurations of transposon termini can induce chromosome rearrangements at high frequencies. Here, we have analyzed chromosomal rearrangements derived from an unstable allele of the maize P1 (pericarp color) gene. The progenitor allele contains both a full-length Ac (Activator) transposable element and an Ac terminal fragment termed fAc (fractured Ac) inserted in the second intron of the P1-rr gene. Two rearranged alleles were derived from a classical maize ear twinned sector and were found to contain a large inverted duplication and a corresponding deficiency. The sequences at the junctions of the rearrangement breakpoints indicate that the duplication and deletion structures were produced by a single transposition event involving Ac and fAc termini located on sister chromatids. Because the transposition process we describe involves transposon ends located on different DNA molecules, it is termed nonlinear transposition (NLT). NLT can rapidly break and rejoin chromosomes and thus could have played an important role in generating structural heterogeneity during genome evolution.     

Genome-wide detection of spontaneous chromosomal rearrangements in bacteria

Genome rearrangements have important effects on bacterial phenotypes and influence the evolution of bacterial genomes. Conventional strategies for characterizing rearrangements in bacterial genomes rely on comparisons of sequenced genomes from related species. However, the spectra of spontaneous rearrangements in supposedly homogenous and clonal bacterial populations are still poorly characterized. Here we used 454 pyrosequencing technology and a 'split mapping' computational method to identify unique junction sequences caused by spontaneous genome rearrangements in chemostat cultures of Salmonella enterica Var. Typhimurium LT2. We confirmed 22 unique junction sequences with a junction microhomology more than 10 bp and this led to an estimation of 51 true junction sequences, of which 28, 12 and 11 were likely to be formed by deletion, duplication and inversion events, respectively. All experimentally confirmed rearrangements had short inverted (inversions) or direct (deletions and duplications) homologous repeat sequences at the endpoints. This study demonstrates the feasibility of genome wide characterization of spontaneous genome rearrangements in bacteria and the very high steady-state frequency (20-40%) of rearrangements in bacterial populations.     

Spontaneous chromosomal rearrangements in cultivated and wild barleys

Summary Four of 1,240 cultivated barley lines collected from different regions of the world and 3 of 120 lines of wild barley, Hordeum spontaneum C. Koch, carry spontaneous reciprocal translocations. Break-point positions and rearrangements in the interchanged chromosomes have been examined by both test crosses and Giemsa banding techniques. The four translocation lines in cultivated barley were all of Ethiopian origin and have the same translocation involving chromosomes 2 and 4. The breakpoints are at the centromeres of both chromosomes, resulting in interchanged chromosomes 2S+4S and 2L+4L (S=short arm, L=long arm). A wild barley line, Spont.II, also has translocated chromosomes 2 and 4 which are broken at the centromeres. The resultant chromosomes are, however, 2S+4L and 2L+4S. Another wild barley line, Spont.S-4, has interchanged chromosomes with breakpoints in the short arm of chromosome 3 and the long arm of chromosome 7. In addition, this line has a paracentric inversion in the short arm of chromosome 7 that includes a part of nucleolar constriction, resulting in two tandemly arranged nucleolar constrictions. The third wild barley line, Spont.S-7, has interchanged chromosomes with breakpoints in the long arms of both chromosomes 3 and 6. The translocated chromosome 3 is metacentric and the translocated chromosome 6 has a long arm similar in length to the long arm of chromosome 7.     

Structural chromosomal rearrangements in HpaII-treated human lymphocytes.

Restriction endonucleases have been shown to induce chromosome damage in a variety of cultured cells. We recently reported the coincidence between MspI-induced breakage and the location of common fragile sites. We have extended our study to HpaII, which induced a 4.5-fold increase in total breakage compared to controls. It appeared that a major contribution was given by stable chromosome rearrangements, which were present at a 14-fold increased frequency in comparison to the spontaneous levels. Moreover, several chromosome bands were involved in rearrangements in different cultures from different donors. Notably, HpaII-induced breakage occurred in the same bands where breakpoints of constitutional and neoplastic rearrangements are located.