Analysis of chromosome segregation during preimplantation genetic diagnosis in both male and female translocation heterozygotes

Individuals carrying translocations suffer from reduced fertility or spontaneous abortions and seek help in form of assisted reproductive technology (ART) and preimplantation genetic diagnosis (PGD). While most translocations are relatively easy to detect in metaphase cells, the majority of embryonic cells biopsied in the course of in vitro fertilization (IVF) procedures are in interphase. These nuclei are thus unsuitable for analysis by chromosome banding or painting using fluorescence in situ hybridization (FISH). Thus several methods have been devised to detect translocation imbalance through FISH in single cells for purpose of PGD, among them polar body chromosome painting, interphase FISH with combination of subtelomeric and centromeric probes, breakpoint spanning probes, and cell conversion. Results with PGD indicate a significant decrease in spontaneous abortions, from 81% before PGD to 13% after PGD. They also indicate very high rates of chromosome abnormalities in embryos from translocation carriers, 72% for Robertsonian translocations and 82% for reciprocal translocations. Sperm analysis was found to be a good predictor of IVF and PGD outcome, with samples with more than 60% abnormal forms indicating poor prognosis. Similarly, the predictability from first PGD cycle results for future cycles was 90%. In summary, PGD can help translocation carriers to achieve viable pregnancies, but the success of the process is conversely related to the baseline of unbalanced gametes.     

Precise localization of NF1 to 17q11.2 by balanced translocation.

A female patient is described with von Recklinghausen neurofibromatosis (NF1) in association with a balanced translocation between chromosome 17 and 22 [46,XX,t(17;22)(q11.2;q11.2)]. The breakpoint in chromosome 17 is cytogenetically identical to a previously reported case of NF1 associated with a 1;17 balanced translocation and suggests that the translocation events disrupt the NF1 gene. This precisely maps the NF1 gene to 17q11.2 and provides a physical reference point for strategies to clone the breakpoint and therefore the NF1 gene. A human-mouse somatic cell hybrid was constructed from patient lymphoblasts which retained the derivative chromosome 22 (22pter----22q11.2::17q11.2----17qter) but not the derivative 17q or normal 17. Southern blot analysis with genes and anonymous probes known to be in proximal 17q showed ErbA1, ErbB2, and granulocyte colony-stimulating factor (CSF3) to be present in the hybrid and therefore distal to the breakpoint, while pHHH202 (D17S33) and beta crystallin (CRYB1) were absent in the hybrid and therefore proximal to the breakpoint. The gene cluster including ErbA1 is known to be flanked by the constitutional 15;17 translocation breakpoint in hybrid SP3 and by the acute promyelocytic leukemia (APL) breakpoint, which provides the following gene and breakpoint order: cen-SP3-(D17S33,CRYB1)-NF1-(CSF3,ERBA1, ERBB2)-APL-tel. The flanking breakpoints of SP3 and API are therefore useful for rapidly localizing new markers to the neurofibromatosis critical region, while the breakpoints of the two translocation patients provide unique opportunities for reverse genetic strategies to clone the NF1 gene.     

Localization of the rhabdomyosarcoma t(2;13) breakpoint on a physical map of chromosome 13.

Previous investigations of the pediatric soft tissue tumor alveolar rhabdomyosarcoma have identified a characteristic translocation t(2;13)(q35;q14). We have employed a physical mapping strategy to localize the site of this translocation breakpoint on chromosome 13. Using a panel of somatic cell hybrid and lymphoblast cell lines with deletions and unbalanced translocations involving chromosome 13, we have mapped numerous probes from the 13q12-q14 region and demonstrate that this region is divisible into five physical intervals. These probes were then mapped with respect to the t(2;13) rhabdomyosarcoma breakpoint by quantitative Southern blot analysis of an alveolar rhabdomyosarcoma cell line with two copies of the derivative chromosome 13 and one copy of the derivative chromosome 2. Our findings demonstrate that the t(2;13) breakpoint is localized within a map interval delimited by the proximal deletion breakpoints in lymphoblast lines GM01484 and GM07312. Furthermore, the breakpoint is most closely flanked by loci D13S29 and TUBBP2 within this map interval. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the alveolar rhabdomyosarcoma translocation. In addition, this physical map will permit rapid determination of the proximity of new cloned sequences to the translocation breakpoint.     

Molecular mapping and cloning of the breakpoints of a chromosome 11p14.1-p13 deletion associated with the AGR syndrome

Chromosome 11p13 is frequently rearranged in individuals with the WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) or parts of this syndrome. To map the cytogenetic aberrations molecularly, we screened DNA from cell lines with known WAGR-related chromosome abnormalities for rearrangements with pulsed field gel (PFG) analysis using probes deleted from one chromosome 11 homolog of a WAGR patient. The first alteration was detected in a cell line from an individual with aniridia, genitourinary anomalies, mental retardation, and a deletion described as 11p14.1-p13. We have located one breakpoint close to probe HU11-164B and we have cloned both breakpoint sites as well as the junctional fragment. The breakpoints subdivide current intervals on the genetic map, and the probes for both sides will serve as important additional markers for a long-range restriction map of this region. Further characterization and sequencing of the breakpoints may yield insight into the mechanisms by which these deletions occur.     

Chromosomal distributions of breakpoints in cancer,infertility, and evolution

We extract 11 genome-wide sets of breakpoint positions from databases on reciprocal translocations, inversions and deletions in neoplasms, reciprocal translocations and inversions in families carrying rearrangements and the human-mouse comparative map, and for each set of positions construct breakpoint distributions for the 44 autosomal arms. We identify and interpret four main types of distribution: (i) a uniform distribution associated both with families carrying translocations or inversions, and with the comparative map, (ii) telomerically skewed distributions of translocations or inversions detected consequent to births with malformations, (iii) medially clustered distributions of translocation and deletion breakpoints in tumor karyotypes, and (iv) bimodal translocation breakpoint distributions for chromosome arms containing telomeric proto-oncogenes.     

Mapping of balanced chromosome translocation breakpoints to the basepair level from microdissected chromosomes

The analysis of structural variants associated with specific phenotypic features is promising for the elucidation of the function of involved genes. There is, however, at present no approach allowing the rapid mapping of chromosomal translocation breakpoints to the basepair level from a single chromosome. Here we demonstrate that we have advanced both the microdissection and the subsequent unbiased amplification to an extent that breakpoint mapping to the basepair level has become possible. As a case in point we analysed the two breakpoints of a t(7;13) translocation observed in a patient with split hand/foot malformation (SHFM1). The amplification products of the der(7) and of the der(13) were hybridized to custom‐made arrays, enabling us to define primers at flanking breakpoint regions and thus to fine‐map the breakpoints to the basepair level. Consequently, our results will also contribute to a further delineation of causative mechanisms underlying SHFM1 which are currently unknown.     

Molecular analysis of both translocation products of a Philadelphia-positive CML patient.

The breakpoint regions of both translocation products of the (9;22) Philadelphia translocation of CML patient 83-H84 and their normal chromosome 9 and 22 counterparts have been cloned and analysed. Southern blotting with bcr probes and DNA sequencing revealed that the breaks on chromosome 22 occurred 3' of bcr exon b3 and that the 88 nucleotides between the breakpoints in the chromosome 22 bcr region were deleted. Besides this small deletion of chromosome 22 sequences a large deletion of chromosome 9 sequences (greater than 70 kb) was observed. The chromosome 9 sequences remaining on the 9q+ chromosome (9q+ breakpoint) are located at least 100 kb upstream of the v-abl homologous c-abl exons whereas the translocated chromosome 9 sequences (22q-breakpoint) could be mapped 30 kb upstream of these c-abl sequences. The breakpoints were situated in Alu-repetitive sequences either on chromosome 22 or on chromosome 9, strengthening the hypothesis that Alu-repetitive sequences can be hot spots for recombination.     

Chromosome Segregation Analysis in Human Embryos Obtained from Couples Involving Male Carriers of Reciprocal or Robertsonian Translocation

The objective of this study was to investigate the frequency and type of chromosome segregation patterns in cleavage stage embryos obtained from male carriers of Robertsonian (ROB) and reciprocal (REC) translocations undergoing preimplantation genetic diagnosis (PGD) at our reproductive center. We used FISH to analyze chromosome segregation in 308 day 3 cleavage stage embryos obtained from 26 patients. The percentage of embryos consistent with normal or balanced segregation (55.1% vs. 27.1%) and clinical pregnancy (62.5% vs. 19.2%) rates were higher in ROB than the REC translocation carriers. Involvement of non-acrocentric chromosome(s) or terminal breakpoint(s) in reciprocal translocations was associated with an increase in the percent of embryos consistent with adjacent 1 but with a decrease in 3∶1 segregation. Similar results were obtained in the analysis of nontransferred embryos donated for research. 3∶1 segregation was the most frequent segregation type in both day 3 (31%) and spare (35%) embryos obtained from carriers of t(11;22)(q23;q11), the only non-random REC with the same breakpoint reported in a large number of unrelated families mainly identified by the birth of a child with derivative chromosome 22. These results suggest that chromosome segregation patterns in day 3 and nontransferred embryos obtained from male translocation carriers vary with the type of translocation and involvement of acrocentric chromosome(s) or terminal breakpoint(s). These results should be helpful in estimating reproductive success in translocation carriers undergoing PGD.     

Fine structure DNA mapping studies of the chromosomal region harboring the genetic defect in neurofibromatosis type 1

To better map the location of the von Recklinghausen neurofibromatosis (NF1) gene, we have characterized a somatic cell hybrid designated 7AE-11. This microcell-mediated, chromosome-transfer construct harbors a centromeric segment and a neo-marked segment from the distal long arm of human chromosome 17. We have identified 269 cosmid clones with human sequences from a 7AE-11 library and, using a panel of somatic cell hybrids with a total of six chromosome 17q breakpoints, have mapped 240 of these clones on chromosome 17q. The panel included a hybrid (NF13) carrying a der(22) chromosome that was isolated from an NF1 patient with a balanced translocation, t(17;22) (q11.2;q11.2). Fifty-three of the cosmids map into a region spanning the NF13 breakpoint, as defined by the two closest flanking breakpoints (17q11.2 and 17q11.2-q12). RFLP clones from a subset of these cosmids have been mapped by linkage analysis in normal reference families, to localize the NF1 gene more precisely and to enhance the potential for genetic diagnosis of this disorder. The cosmids in the NF1 region will be an important resource for testing DNA blots of large-fragment restriction-enzyme digests from NF1 patient cell lines, to detect rearrangements in patients' DNA and to identify the 17;22 NF1 translocation breakpoint.     

Induced reciprocal translocation in transgenic mice near sites of transgene integration

Transgenic mice (JCP₀ #18), heterozygous for an insertion of approximately 50 copies of the rat peripheral myelin (P₀) protein cDNA, displayed a pattern of reduced litter size that suggested a chromosome rearrangement. Chromosome banding studies of fetal cells disclosed the presence of an apparently balanced translocation between a Chromosome (Chr) 1 and 14 with breakpoints at bands 1H3 and 14C3. In situ hybridization of biotin-labeled P₀ rat cDNA probe to chromosome spreads and detection of specific signal with fluorescein isothiocyanate-conjugated avidin revealed a strong signal on the 1¹⁴ translocation chromosome at the site of the breakpoint. A weaker signal was present near the breakpoint on the 14¹ derivative chromosome. These results suggest an etiologic relationship between the insertion of the transgene and the origin of the translocation. To further elucidate possible mechanisms, we first mapped the endogenous P₀ gene (gene symbol Mpp). As previously reported (You et al., Genomics 9: 751, 1991), we found that Mpp is located on Chr 1 in the region of the translocation breakpoint in JCP₀ mice. Subsequently, we have carried out pulsed-field gel and standard Southern analyses with P₀ gene probes, but found no evidence for a direct involvement of the endogenous P₀ gene in the process that generated the balanced reciprocal translocation. Thus, we favor the hypothesis that, during repair of DNA strand breakage—possibly induced by the microinjection procedure—the transgene copies were ligated to broken ends of Chrs 1 and 14. According to convention, this translocation is designated T(1;14) 1Po. Homozygotes are phenotypically normal and breed well; they will be useful for genetic and physical mapping of Chrs 1 and 14.     

The establishment and application of preimplantation genetic haplotyping in embryo diagnosis for reciprocal and Robertsonian translocation carriers

Background

Preimplantation genetic diagnosis (PGD) is now widely used to select embryos free of chromosomal copy number variations (CNV) from chromosome balanced translocation carriers. However, it remains a difficulty to distinguish in embryos between balanced and structurally normal chromosomes efficiently.

Methods

For this purpose, genome wide preimplantation genetic haplotyping (PGH) analysis was utilized based on single nucleotide polymorphism (SNP) microarray. SNPs that are heterozygous in the carrier and, homozygous in the carrier’s partner and carrier’s family member are defined as informative SNPs. The haplotypes including the breakpoint regions, the whole chromosomes involved in the translocation and the corresponding homologous chromosomes are established with these informative SNPs in the couple, reference and embryos. In order to perform this analysis, a reference either a translocation carrier’s family member or one unbalanced embryo is required. The positions of translocation breakpoints are identified by molecular karyotypes of unbalanced embryos. The recombination of breakpoint regions in embryos could be identified.

Results

Eleven translocation families were enrolled. 68 blastocysts were analyzed, in which 42 were unbalanced or aneuploid and the other 26 were balanced or normal chromosomes. Thirteen embryos were transferred back to patients. Prenatal cytogenetic analysis of amniotic fluid cells was performed. The results predicted by PGH and karyotypes were totally consistent.

Conclusions

With the successful clinical application, we demonstrate that PGH was a simple, efficient, and popularized method to distinguish between balanced and structurally normal chromosome embryos.

     

Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22

We have identified and molecularly cloned 46 kb of human DNA from chromosome 22 using a probe specific for the Philadelphia (Ph') translocation breakpoint domain of one chronic myelocytic leukemia (CML) patient. The DNAs of 19 CML patients were examined for rearrangements on chromosome 22 with probes isolated from this cloned region. In 17 patients, chromosomal breakpoints were found within a limited region of up to 5.8 kb, for which we propose the term "breakpoint cluster region" (bcr). The two patients having no rearrangements within bcr lacked the Ph' chromosome. The highly specific presence of a chromosomal breakpoint within bcr in Ph'-positive CML patients strongly suggests the involvement of bcr in this type of leukemia.     

Molecular and cytogenetic characterization of a de novo t(5p;21q) in a patient previously diagnosed as monosomy 21.

Genomic single-copy DNA fragments were used to characterize an undetected chromosome translocation in an individual whose metaphase chromosome analysis revealed apparent monosomy 21. Eight RFLPs detected by six probes were used to identify homologous sequences from chromosome 21 in DNA digests from the proband and her parents. These family studies showed that the proband was disomic for the distal region of 21q. Reverse banding and in situ hybridization of chromosome 21-specific probes to metaphase chromosomes from the proband revealed a de novo translocation with breakpoints at 5p13 or 14 and 21q11 or 21. In situ hybridization permitted orientation of the translocated portion of chromosome 21 on the derivative chromosome 5 and, in conjunction with molecular analysis and previous mapping studies, refined the physical map for the long arm of chromosome 21.     

Identification and molecular cytogenetic characterization of a novel complex Y chromosome rearrangement in a boy with disorder of sexual development

Ambiguous genitalia or disorder of the sexual development is a birth defect where the external genitals do not have the typical appearance of either a male or female. Here we report a boy with ambiguous genitalia and short stature. The cytogenetic analysis by G-banding revealed a small Y chromosome and an additional material on the 15p arm. Further, molecular cytogenetic analysis by Fluorescence in situ hybridization (FISH) using whole chromosome paint probes showed the presence of Y sequences on the 15p arm, confirming that it is a Y;15 translocation. Subsequent, FISH with centromere probe Y showed two signals depicting the presence of two centromeres and differing with a balanced translocation. The dicentric nature of the derivative 15 chromosome was confirmed by FISH with both 15 and Y centromeric probes. Further, the delineation of the Y chromosomal DNA was also done by quantitative real time PCR. Additional Y-short tandem repeat typing was performed to find out the extent of deletion on small Y chromosome. Fine mapping was carried out with 8 Y specific BAC clones which helped in defining the breakpoint regions. MLPA was performed to check the presence or absence of subtelomeric regions and SHOX regions on Y. Finally array CGH helped us in confirming the breakpoint regions. In our study we identified and characterized a novel complex Y chromosomal rearrangement with a complete deletion of the Yq region and duplication of the Yp region with one copy being translocated onto the15p arm. This is the first report of novel and unique Y complex rearrangement showing a deletion, duplication and a translocation in the same patient. The possible mechanism of the rearrangement and the phenotype–genotype correlation are discussed.     

Mapping of Xp21 translocation breakpoints in and around the DMD gene by pulsed field gel electrophoresis

Balanced translocations with a breakpoint in the Xp21 region are likely to disrupt the giant Duchenne muscular dystrophy (DMD) locus and can be demonstrated in females suffering from the disease. Pulsed field gel electrophoresis allows the positioning of these breakpoints by detecting junction fragments on the derived chromosomes; DNA probes hybridizing to these fragments may be located as many as several hundred kilobases away from the breakpoints. By using this approach, 11 translocation breakpoints from the Xp21 region have been analyzed. The localization of three previously examined breakpoints was confirmed. Six other breakpoints, including a breakpoint flanking the DMD gene and not associated with the DMD phenotype, could be positioned relative to SfiI sites on a 3.5-Mb restriction map of the region.     

Molecular analysis of chromosome 1 abnormalities in neuroblastoma

Tumor cells from 70% of neuroblastoma patients contain a deletion of part of the short arm of chromosome 1, indicating that this chromosomal region includes a gene involved in tumor formation. To more precisely evaluate the boundaries and mechanisms involved in generating these deletions, we have examined four neuroblastoma cell lines using a combination of somatic cell hybridization, isozyme analysis, and nucleic acid hybridization employing both standard and restriction fragment length polymorphic probes. The data suggest that the truncation of chromosome 1 in these neuroblastomas was most likely due to a complex translocation and deletion mechanism rather than a simple unbalanced translocation or terminal or interstitial deletion. This conclusion is supported by the frequent removal of MYCL from the altered chromosome 1 to another chromosome. Furthermore, the data suggest that the frequency of breakpoints previously assigned by karyotypic analysis to bands other than 1p32 in neuroblastomas may be overestimated. Finally, this study identified a breakpoint at 1p32 that was localized between the genes JUN and MYCL for one neuroblastoma thus establishing the order of these genes as centromere, JUN, MYCL, telomere. We conclude that the observed breakpoints within chromosome 1p in human neuroblastoma are not as variable as previously described and suggest the results of this study provide evidence for the involvement of specific DNA sequences within 1p32 in the generation of neuroblastoma.     

Preimplantation genetic analysis of translocations: case-specific probes for interphase cell analysis

Carriers of balanced translocations show an increased risk of infertility and spontaneous abortions, because of errors in gametogenesis, and constitute a significant fraction of patients seeking assisted reproduction. The objective of this study was to design approaches for preimplantation diagnosis of chromosome translocations and to apply such techniques to the selection of chromosomally normal or balanced embryos prior to their transfer to the mother’s womb. Three slightly different approaches were assessed by means of chromosome-specific, non-isotopically labeled DNA probes and an assay based on fluorescence in situ hybridization- to score and characterize chromosomes in single blastomeres biopsied from embryos on their third day of development. The three approaches were used for preimplantation genetic diagnosis involving four couples who had enrolled in our IVF program and in which one of the partners was a carrier of one of the following translocations: 46,XX,t(12;20)(p13.1;q13.3), 46,XY,t(3;4) (p24;p15), 45,XY,der(14;15)(10q;10q), and 46,XY,t(6;11) (p22.1;p15.3). A total of 33 embryos were analyzed, of which 25 (75.8%) were found to be either unbalanced or otherwise chromosomally abnormal. Only a single embryo could be transferred to patients A and D, whereas three embryos were transferred to patient B in a total of two IVF cycles. Transfer of two embryos to patient C resulted in an ongoing pregnancy. Re-analysis of non-transferred embryos with additional probes confirmed the initial results in 95% (20/21) of the cases. In conclusion, case-specific translocation tests can be applied to any translocation carrier for the selection of normal or chromosomally balanced embryos prior to embryo transfer. This is expected significantly to increase the success rates in IVF cycles of translocation carriers, while preventing the spontaneous abortion or birth of abnormal offspring. Received: 13 January 1998 / Accepted: 24 March 1998     

Physical mapping of translocation breakpoints in rye by means of synaptonemal complex analysis

A physical map including 40 translocation breakpoints has been constructed in rye by means of synaptonemal complex (SC) analysis of well-paired pachytene quadrivalents. The chromosome arms involved in such translocations were previously identified either from mitotic C-banding analysis or from the meiotic configurations observed in the progenies of crosses with a rye line having multiple chromosome rearrangements. The synaptonemal complexes formed by some translocation homozygotes were also analyzed, the relative pachytene SC length of their translocated chromosomes being compared to that observed in the corresponding translocation heterozygotes. In the translocations in which the position of the breakpoint could be well defined from mitotic C-banding analysis, a good correspondence between the relative position of the point showing partner exchange in the pachytene quadrivalents and the actual location of the breakpoint was established. It is concluded that the mapping of translocation breakpoints by SC analysis of pachytene quadrivalents provides a more accurate estimate of the position of the breakpoints than that obtained from mitotic C-banding analysis, due to the lack of evenly-distributed interstitial C-bands in most rye chromosomes. The distribution of the breakpoints along the chromosomes in relation to their spontaneous or induced origin is also discussed.