Chromoanagenesis, the mechanisms of a genomic chaos.

Designated under the name of chromoanagenesis, the phenomena of chromothripsis, chromanasynthesis and chromoplexy constitute new types of complex rearrangements, including many genomic alterations localized on a few chromosomal regions, and whose discovery over the last decade has changed our perception about the formation of chromosomal abnormalities and their etiology. Although exhibiting specific features, these new catastrophic mechanisms generally occur within a single cell cycle and their emergence is closely linked to genomic instability. Various non-exclusive exogenous or cellular mechanisms capable of generating chromoanagenesis have been evoked. However, recent experimental data shed light on 2 major processes, which following a defect in the mitotic segregation of chromosomes, can generate a cascade of cellular events leading to chromoanagenesis. These mechanisms are the formation of micronuclei integrating isolated chromosomal material, and the occurrence of chromatin bridges around chromosomal material resulting from telomeric fusions. In both cases, the cellular and molecular mechanisms of fragmentation, repair and transmission of damaged chromosomal material are consistent with the features of chromoanagenesis-related complex chromosomal rearrangements. In this review, we introduce each type of chromoanagenesis, and describe the experimental models that have allowed to validate the existence of chromoanagenesis events and to better understand their cellular mechanisms of formation and transmission, as well as their impact on the stability and the plasticity of the genome.

Non-invasive prenatal diagnosis (NIPD) of cystic fibrosis: an optimized protocol using MEMO fluorescent PCR to detect the p.Phe508del mutation.

BACKGROUND: Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. METHODS: To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. RESULTS: We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. CONCLUSIONS: This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.

Discordant sex in monozygotic XXY/XX twins: a case report.

We report a case of discordant phenotypic sex in monozygotic twins mosaic 47,XXY/46,XX: monozygotic heterokaryotypic twins. The twins presented with cognitive and comprehension delay, behavioural and language disorders, all symptoms frequently reported in Klinefelter syndrome. Molecular zygosity analysis with several markers confirmed that the twins are in effect monozygotic (MZ). Array comparative genomic hybridization found no evidence for the implication of copy number variation in the phenotypes. Ultrasound scans of the reproductive organs revealed no abnormalities. Endocrine tests showed a low testosterone level in Twin 1 (male phenotype) and a low gonadotrophin level in Twin 2 (female phenotype) which, combined with the results from ultrasound examination, provided useful information for potentially predicting the future fertility potential of the twins. Blood karyotypes revealed the presence of a normal 46,XX cell line and an aneuploïd 47,XXY cell line in both patients. Examination of the chromosome constitutions of various tissues such as blood, buccal smear and urinary sediment not surprisingly showed different proportions for the 46,XX and 47,XXY cell lines, which most likely explains the discordant phenotypic sex and mild Klinefelter features. The most plausible underlying biological mechanism is a post-zygotic loss of the Y chromosome in an initially 47,XXY zygote. This would result in an embryo with both 46,XX and 47,XXY cells lines which could subsequently divide into two monozygotic embryos through a twinning process. The two cell lines would then be distributed differently between tissues which could result in phenotypic discordances in the twins. These observations emphasize the importance of regular paediatric evaluations to determine the optimal timing for fertility preservation measures and to detect new Klinefelter features which could appear throughout childhood in the two subjects.

Molecular characterization of 39 de novo sSMC: contribution to prognosis and genetic counselling, a prospective study.

Small supernumerary marker chromosomes (sSMCs) are structurally abnormal chromosomes that cannot be characterized by karyotype. In many prenatal cases of de novo sSMC, the outcome of pregnancy is difficult to predict because the euchromatin content is unclear. This study aimed to determine the presence or absence of euchromatin material of 39 de novo prenatally ascertained sSMC by array-comparative genomic hybridization (array-CGH) or single nucleotide polymorphism (SNP) array. Cases were prospectively ascertained from the study of 65,000 prenatal samples [0.060%; 95% confidence interval (CI), 0.042-0.082]. Array-CGH showed that 22 markers were derived from non-acrocentric markers (56.4%) and 7 from acrocentic markers (18%). The 10 additional cases remained unidentified (25.6%), but 7 of 10 could be further identified using fluorescence in situ hybridization; 69% of de novo sSMC contained euchromatin material, 95.4% of which for non-acrocentric markers. Some sSMC containing euchromatin had a normal phenotype (31% for non-acrocentric and 75% for acrocentric markers). Statistical differences between normal and abnormal phenotypes were shown for the size of the euchromatin material (more or less than 1 Mb, p = 0.0006) and number of genes (more or less than 10, p = 0.0009). This study is the largest to date and shows the utility of array-CGH or SNP array in the detection and characterization of de novo sSMC in a prenatal context.

Complex chromosomal rearrangements: origin and meiotic behavior.

BACKGROUND: Complex chromosomal rearrangements (CCRs) describe structural rearrangements, essentially translocations, involving at least three breakpoints on two or more chromosomes. Although they are rare in humans, their clinical identification is important since CCR carriers can display various phenotypes which include phenotypically normal subjects, infertile males and patients with mental retardation and/or congenital abnormalities. The rearrangement can be de novo or familial. The use of fluorescent in situ hybridization assays and molecular techniques for the characterization of CCRs have indicated that the rearrangements could be more complex than initially assumed. Accumulating data have revealed that the mechanisms underlying the genesis of CCRs remain elusive. METHODS: We performed a large PubMed search in order to summarize the current knowledge in this field and address important aspects of CCR formation and meiotic behavior, highlighting the complexity of these rearrangements at the chromosomal and genomic level. RESULTS: The review of published data indicates that the complexity of CCRs is becoming increasingly known, thanks to the application of more and more efficient molecular techniques. These approaches have allowed the precise sequence analysis of breakpoints and the identification of insertions, deletions, inversions and recombination events. New models have been proposed for the formation of CCRs, based on replication-based mechanisms and specific sequence elements. Their meiotic behavior has been discussed in the light of these new molecular data. CONCLUSIONS: Despite the increasing understanding of the mechanisms involved in their genesis, CCRs arise as unique, complex events for which the genetic and reproductive counseling of carriers remains a challenge.

Breakpoint characterization: a new approach for segregation analysis of paracentric inversion in human sperm.

Paracentric inversions (PAI) are structural chromosomal rearrangements generally considered to be harmless. Nevertheless, cases of viable recombinants have been reported, indicating the interest of studying the meiotic behaviour of these chromosomal abnormalities. To date, the few studies reported have been performed using either the human-hamster fertilization system or fluorescence in situ hybridization with centromeric or telomeric DNA probes. In order to improve the assessment of meiotic segregation in PAI, we present a new strategy based on the use of bacterial artificial chromosome (BAC) probes which allow a precise localization of chromosome breakpoints and the identification of all meiotic products in human sperm. Sperm samples from carriers of an inv(5) and an inv(14) were used to test this new high-resolution procedure.

Rare Robertsonian translocations and meiotic behaviour: sperm FISH analysis of t(13;15) and t(14;15) translocations: a case report.

t(13;15) and t(14;15) are two rare Robertsonian translocations. Meiotic segregation was studied in four males heterozygous for the rare Robertsonian translocations t(13;15) and t(14;15). Both locus-specific probes (LSPs) and whole chromosome painting (WCP) probes, specific to chromosomes 13, 14 and 15, were used in this study. The number of spermatozoa scored for each carrier ranged from 891 to 5000. The frequencies of normal and balanced sperm resulting from the alternate mode of segregation ranged from 77.6 to 92.8%, confirming the prevalence of alternate segregation over other segregation modes in all Robertsonian translocations. The incidences of unbalanced complements ranged from 6.7 to 20.4%, with a significant excess of disomy rates over the complementary frequencies of nullisomy. This variability might reflect differences in the location of breakpoints in translocated chromosomes, leading to the variable production of unbalanced gametes and the variable alterations of semen parameters in Robertsonian translocation carriers.

Meiotic segregation of rare Robertsonian translocations: sperm analysis of three t(14q;22q) cases.

BACKGROUND: The t(14;22) remains one of the rare Robertsonian translocations observed in human, with an occurrence estimated at 1.2%. Three cases of rare Robertsonian translocation t(14;22) were investigated for meiotic segregation in sperm samples from male carriers using the fluorescent in situ hybridization (FISH) procedure. The three carriers included two men with an abnormal semen analysis and one with normal semen parameters. METHODS: Both locus-specific probes and whole-chromosome painting probes, specific for chromosomes 14 and 22, were used in this study. The number of spermatozoa scored for each probe set ranged from 3279 to 10,024. RESULTS: In the three carriers, similar frequencies, ranging from 78.53 to 81.76%, were found for normal and balanced spermatozoa resulting from alternate segregation. The total proportion of unbalanced spermatozoa resulting from adjacent modes of segregation ranged from 17.59 to 20.94%. CONCLUSION: This finding confirmed the predominance of alternate segregation over other segregation types in all Robertsonian translocations and indicates a higher production of imbalances in the t(14;22) than in most of the Robertsonian translocations previously analysed. This could be related to the variable location of breakpoints in Robertsonian translocations. This breakpoint diversity could also play a role in the differences in reproductive status observed in male carriers of Robertsonian translocations.

Hypothesis: for the worst and for the best, L1Hs retrotransposons actively participate in the evolution of the human centromeric alphoid sequences.

A number of questions concerning the evolution and the function of the alpha satellite DNA sequences present at the centromere of all human chromosomes are still open. In this paper, we present data which could contribute to understanding these points. It is shown here that the alphoid sequences within which L1 elements are found are quite divergent from those of the homogeneous alphoid subsets present at each centromere where none has so far been detected. In addition, a number of L1s are detected close to the ends of the alpha satellite blocks. A fairly high proportion exhibit a polymorphism of presence/absence. Strikingly, several L1s localized at a distance from each other are always either present or absent simultaneously. This is interpreted as resulting from intrachromosomal recombination, through distant L1s, leading to deletion of several of them at once together with their surrounding alphoid sequences. The parameters determining which portion of the several megabases of alphoid sequences is actually involved in the centromeric function are not known. From the above data we suggest that the alpha satellite domain within which DNA sequences are recruited to form a centromere is both homogeneous in sequence and uninterrupted by L1s or any other retrotransposons. Conversely, non-centromere competent alphoid sequences would be both divergent and punctuated by scattered L1 elements, particularly at the borders of the alphoid blocks. On the grounds of these data and hypotheses, a model is presented in which it is postulated that accumulation of L1 insertions within a centromere competent alphoid domain is ruining this competence, the consequence being damage to or even loss of the centromere-forming capability of the chromosome. Restoration of fully centromere-forming competence is supposed to occur by two alternative means, either de-novo amplification of a homogeneous and uninterrupted alphoid domain or by unequal crossing over with a homologue harbouring a large competent one. If L1 retrotransposons are acting detrimentally to centromere integrity (for the worst), one must also consider them as having positive consequences on chromosomes by preventing their centromeres from swelling indefinitely by the addition of alphoid sequences (for the best). The data and ideas presented here fit well with those already put forward by Csink and Henikoff (1998) using the example of Drosophila.

Genetic and physical analyses of the centromeric and pericentromeric regions of human chromosome 5: recombination across 5cen.

Human centromeres are poorly understood at both the genetic and the physical level. In this paper, we have been able to distinguish the alphoid centromeric sequences of chromosome 5 from those of chromosome 19. This result was obtained by pulsed-field gel electrophoresis after cutting genomic DNA with restriction endonucleases NcoI (chromosome 5) and BamHI (chromosome 19). We could thus define a highly polymorphic marker, representing length variations of the D5Z1 domain located at the q arm boundary of the chromosome 5 centromere. The centromeric region of chromosome 5 was then analyzed in full detail. We established an approximately 4.6-Mb physical map of the whole region with five rare-cutting enzymes by using nonchimeric YACs, two of which were shown to contain the very ends of 5cen on both sides. The p-arm side of 5cen was shown to contain an alphoid subset (D5Z12) different from those described thus far. Two genes and several putative cDNAs could be precisely located close to the centromere. Several L1 elements were shown to be present within alpha satellites at the boundary between alphoid and nonalphoid sequences on both sides of 5cen. They were used to define STSs that could serve as physical anchor points at the junction of 5cen with the p and q arms. Some STSs were placed on a radiation hybrid map. One was polymorphic and could therefore be used as a second centromeric genetic marker at the p arm boundary of 5cen. We could thus estimate recombination rates within and around the centromeric region of chromosome 5. Recombination is highly reduced within 5cen, with zero recombinants in 58 meioses being detected between the two markers located at the two extremities of the centromere. In its immediate vicinity, 5cen indeed exerts a direct negative effect on meiotic recombination within the proximal chromosomal DNA. This effect is, however, less important than expected and is polarized, as different rates are observed on both arms if one compares the 0 cM/Mb of the p proximal first 5.5 Mb and the 0.64 cM/Mb of the q proximal first 5 Mb to the sex-average 1.02 cM/Mb found throughout the entire chromosome 5. Rates then become close to the average when one goes further within the arms. Finally, most recombinants (21/22), irrespective of the arm, are of female origin, thus showing that recombination around 5cen is essentially occurring in the female lineage.

Informative genetic polymorphic markers within the centromeric regions of human chromosomes 17 (D17S2205) and 11 (D11S4975).

We have taken advantage of the presence of retrotransposed L1 elements within the centromeric alphoid sequences of the human genome to characterize polymorphic markers at the centromeres of human chromosomes 17 and 11 (D17S2205 and D11S4975, respectively). They correspond to microsatellites found at the 3' ends of L1 elements inserted within the alpha satellite sequences of the two chromosomes. They were detected after PCR by direct analysis in sequencing gels. Eight and five alleles, respectively, were found with heterozygosities of 0.67 and 0.68. They were converted into STSs by designing primers specific for each. D17S2205 and D11S4975 can be used as genuine anchor-informative genetic points for chromosomes 17 and 11. Both markers have been placed on the available genetic maps of their centromeric regions. The alphoid domain within which D17S2205 is embedded is ancestral to the canonical ones on chromosome 17 that exhibit several haplotypes in present-day human populations.

Site-specific retrotransposition of L1 elements within human alphoid satellite sequences.

In the course of a search for microsatellites as centromeric polymorphic markers at the 3' ends of Alu or L1 elements, we observed a much higher frequency of L1 than Alu elements embedded within alpha satellite DNA. By sequence analysis of the L1 elements at their alphoid locus of insertion, we found that the insertion site was specific, with the consensus being (Py)2-10/ (Pu)3-7. All potential sites within the consensus alphoid 171-bp repeat are occupied by such elements. This confirms the finding by Feng et al. (1996; Human retrotransposon encodes a conserved endonuclease required for retrotransposition, Cell 87:905-916) that the progenitor L1 elements encode a site-specific endonuclease and that they generate copies that are inserted at these specific sites. The analysis of retrotransposed L1 elements within the alphoid domains of the acrocentric chromosomes showed that a number of loci are shared among all five acrocentrics. This sheds light on the manner in which centromeric regions of these chromosomes are exchanging information during evolution.

SINE and LINE within human centromeres.

A number of the Alu and L1 elements present within the centromeric regions of the human chromosomes have been analyzed by polymerase chain reaction amplification. The oligonucleotide primers were homologous to the 3' end consensus sequences of either Alu or L1 in conjunction with an oligonucleotide primer homologous to alphoid sequences specific to different chromosomes. This allowed one to detect an unusual number of Alu and L1 polymorphisms at different loci. It is proposed that this results from molecular rearrangements which occur within the alpha-satellite DNA in which they are embedded (Marçais et al. J. Mol. Evol. 33:42-48, 1991) and not because the centromeric regions are targets for new insertions of such elements. The same analyses were made on cosmids and YACs originating from the centromeric region of chromosome 21 as well as on a collection of somatic hybrids containing chromosome 21 centromere as unique common human genetic material. The results were consistent with the above hypothesis.

A 19-allele polymorphic marker within the centromere of human chromosome 5.

We have detected and characterized a highly polymorphic marker that maps to the centromere of human chromosome 5. The localization was established by both linkage analysis within the CEPH reference families and fluorescence in situ hybridization. The marker consists of a sequence of five nucleotides, (CCTTT)n. Nineteen alleles have been detected in 46 unrelated individuals from 12 CEPH families, with a calculated heterozygosity of 0.91. This is the first truly centromeric, highly polymorphic genetic marker described so far.