1 Center for Human Genetics and 2 Leuven University Fertility Center, University Hospital Leuven, Herestraat 49, B-3000 Leuven, Belgium
3 To whom correspondence should be addressed. Email: joris.vermeesch{at}uz.kuleuven.ac.be
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Abstract |
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Key words: insertional translocation/meiosis/preimplantation genetic diagnosis (PGD)/quadrivalents/reciprocal translocation
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Introduction |
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Preimplantation genetic diagnosis (PGD) can be offered to carriers of balanced translocations to reduce the risk of the unbalanced transmission of aberrant chromosomes. PGD can thus reduce the risk of chromosomally abnormal offspring, reduce the frequency of miscarriages and, as a result, increase the baby take home rate. In 2001, the ESHRE consortium reported 51 PGD cycles for carriers of Robertsonian translocations and 96 PGD cycles for carriers of balanced reciprocal translocations (ESHRE PGD Consortium Steering Committee, 2002). The large majority of reciprocal translocations are terminal translocations. It is well established that reciprocal translocations form quadrivalents during meiosis which, in turn, lead to a higher incidence of chromosomal anomalies (Goldman and Hulten, 1993
; Martin and Hulten, 1993
; Gersen and Keagle, 1999
). Not only can the 2:2 segregants be unbalanced for the translocation chromosomes, but also a higher incidence of 3:1 segregants resulting in aneuploidy for the abnormal chromosomes is observed. PGD of such translocations is performed by fluorescence in situ hybridization (FISH), using a variety of approaches (Munné et al., 1998
; Scriven et al., 1998
). The most common approach is by performing interphase FISH on blastomeres. One, time-consuming, approach has been to develop specific probes spanning the breakpoints of each translocation (Munné et al., 1998
; Weier et al., 1999
). Whilst it may be ideal to use a different enumerator probe for each of the four translocation segments (both translocated segments and both centric segments), suitable probes labelled with appropriate fluorochromes or haptens may not be readily available. However, only enumerator probes for three of the four segments are required to detect all the possible segregation products with chromosome imbalance (Scriven et al., 1998
). The use of three enumerator probes, one for each translocated segment (typically subtelomere probes) and one of the centric segments (typically a centromere probe), is recommended for single-cell biopsy because there are then two probes informative for imbalance consistent with adjacent1 segregation and, providing the centromere probe has been selected appropriately, for potentially viable imbalance associated with 3:1 segregation (Munné et al., 1998
, 2000
; Pierce et al., 1998
; Van Assche et al., 1999
). However, successful strategies in clinical practice have also used enumeration probes for both centric segments and one translocated segment in conjunction with 2-cell embryo biopsy (Simopoulou et al., 2003
).
A small minority of translocations, however, are not terminal but interstitial. Both intra- and interchromosomal insertional translocations have been reported (for a review see Van Hemel and Eussen, 2000). In the former case, only a single chromosome is involved. In the latter, an intrachromosomal fragment from one chromosome is inserted into another chromosome, thus generating one chromosome with an intrachromosomal deletion and another chromosome with an insertion. PGD for an intrachromosomal insertional translocation has been reported (Simopoulo et al., 2003
). However, PGD for interchromosomal insertional translocations has, to our knowledge, not yet been reported. Here we demonstrate that in the case of an interchromosomal insertional translocation, a minimum of four probes are needed to identify all possible unbalanced events and we apply PGD on a patient with an insertion of chromosome (2)(q31
q35) in chromosome 14q.
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Materials and methods |
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Fluorescence in situ hybridization
BAC/PAC DNA was isolated by Nucleobond AX (Machery-Nagel, Düren, Germany) and the DNA was directly labelled by the Random Prime Labeling System (Invitrogen, Carlsbad, CA). Different fluorochromes used were Spectrum orange, Spectrum green, Spectrum red (Vysis, Abbott laboratories, IL) and diethylaminocoumarin-5-dUTP (DEAC) (Perkin Elmer Life Sciences). Two clones of the same chromosomal regions were mixed to obtain strong fluorescent signals. A probe within the translocated region was obtained by Spectrum green labelling RP11-315D12 (2q33) (clone derived from the VGC library, Invitrogen Co.). Probes proximal and distal to the break on chromosome 2 were obtained by combining Spectrum orange-labelled PAC 278P6 and PAC 146O2 (2q12.13) (Nothwang et al., 1998) and combining DEAC-labelled RP11-367B19 and RP11-680O16 (2q37) (CHORI, Oakland, CA). For the detection of chromosome 14, the telomeric clone CTC-820M16 was labelled with Spectrum red.
To prepare the final probe mixture, the four different probes were combined and dissolved in hybridization mix containing 50% formamide, 2x SSC and 10% SDS. Then 1 µl of probe was applied to the slide, covered with a coverslip (diameter of 10 mm) and sealed with rubber cement. Nuclei and probe were denatured simultaneously on a hot plate at 75°C for 5 min. Hybridization was allowed to take place overnight in a humid chamber at 37°C. After hybridization, excess or non-specific bound probe was removed by subsequent washes in 0.4x SSC/0.3% NP-40 (73°C for 2 min), 2xSSC/0.1% NP-40 (room temperature for 1 min) and 2xSSC (room temperature for 1 min) followed by dehydration through ethanol series. After drying, the slides were mounted in Vectashield anti-fading medium (Vector Laboratories, Peterborough, UK) containing 2.5 ng/ml 4',6-diamidino-2-phenylindole (DAPI; Boehringer Ingelheim GmbH, Germany). Nuclei were examined using an Axioplan 2 microscope (Zeiss NV, Zaventem).
The quality of the probe mixture was tested on nuclei derived from stimulated blood lymphocytes from both parents. In 100 nuclei, the number of signals for each of the four probes was counted. Individual probe analysis showed 98, 99, 97 and 95% efficiencies for probes for 2q12, 2q33, 2q37 and 14qter, respectively. In combination, two signals were detected for all four probes in 91% of the nuclei.
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Results |
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Discussion |
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In total, we analysed 10 embryos that reached the 6-cell stage at day 3. Two cells were biopsied. For six out of 10 embryos, only one nucleus was present and available for analysis. Four embryos appeared balanced for the chromosome aberration and five embryos were unbalanced. One embryo was without diagnosis. From the unbalanced embryos, two (embryos 2-E1 and 2-E2) were consistent with the fertilization of a 3:1 segregant, one (embryo E-E3) was consistent with the fertilization of the meiotic product of a bivalent or a 2:2 segregant, one is mosaic (embryo 2-E4) and one (embryo 2-E8) was not consistent with any of the potential segregants of our scheme (Table II). Embryo 2-E4 is a mosaic embryo with one cell with three signals for 2q33 and two signals for 14qter and a second blastomere presenting with two signals for 2q33 and a single signal for 14qter. The simplest explanation would be that the embryo contained the der(14) which is lost by a non-disjunction event in the other cell. Embryo 2-E8 presented with two similar cells containing a single signal for both 2q33 and 2qter. Such a chromosomal constitution cannot be explained by non-disjunction events, but may reflect the presence of a terminal 2q deletion. Equally, we cannot exclude that the abnormal blastomeres with a chromosome constitution consistent with 3:1 segregation are actually more complex karyotypes resulting from additional aneuploidies for chromosomes 2 and 14 in the sperm or oocyte and/or, considering that the combined probe efficiency is 91%, that some apparent imbalances are actually due to technical artefacts. In addition, the two affected boys and single fetus carrying the der(14) chromosome provide additional insights in the meiotic process. Their karyogram is most probably the result of a bivalent or quadrivalent 2:2 segregation during maternal meiosis with transmission of the der(14), but without crossovers in the translocated segment.
While the meiotic intermediates and the resulting segregants of the meiosis of terminal translocation carriers is well documented (Goldman and Hulten, 1993; Martin and Hulten, 1993
), little is known about the meiotic intermediates of insertional translocations. It is generally assumed that such chromosomes can form quadrivalents (Gersen and Keagle, 1999
). However, no direct visualization of such quadrivalents during meiosis has been documented. Goldman et al. (1992)
have investigated both the meiotic process and the sperm composition in a male insertional translocation carrier. Electron microscopic investigations of the meiotic pairing did not show the expected hairpin loops in any of the 54 pachytene spermatocytes investigated, nor was any quadrivalent visible. Upon sperm karyotyping, approximately half were balanced and half were unbalanced, indicating a normal transmission ratio. Based on current knowledge of the meiotic process, it is not obvious that such quadrivalents will form. The meiotic homologue pairing appears to start at the telomeres during the so-called bouquet phase (Scherthan, 2001
). Subsequently, the two homologues synapse along their lengths. While it is obvious that such a process will result in quadrivalents for terminal translocations, the pairing of insertional translocations may not be necessary for the meiotic process to proceed. However, recently it was shown that telomere-independent pairing of chromosomes does occur. Artificial ring chromosomeswhich do not carry any telomerescan pair during meiosis and cross over (Voet et al., 2003
). Although quadrivalents have not been visualized, evidence that recombination does occur is provided by the recombination pattern in families transmitting insertional translocations. Three different case reports describe insertional translocation carriers with offspring carrying unbalanced chromosomes apparently as a result of recombination within an insertional translocation (Jalbert et al., 1975
; Romain et al., 1990
; Boyd et al., 1995
). The ability of quadrivalents to form is likely to be dependent on the size of the translocated segment. In an overview of all reported cases of interchromosomal insertions, it was noted that in all three case reports of likely recombination, the segmental translocation is relatively large with a HAL >1.5% (Van Hemel and Eussen, 2000
) Also in the present case, the HAL is
1.5%.
Whether or not quadrivalents are formed and, if so, whether size constraints of the insertion on the formation of quadrivalents exist and whether there is an increased incidence of 3:1 segregations are more than theoretical questions for the PGD community. With the increased acceptance of PGD, it is likely that more carriers of complex translocations will enter PGD programmes. The results presented here suggest that quadrivalents do form and, hence, considerably more care should be taken and a larger effort to generate the relevant FISH probes is needed. If there would be no increased incidence of 3:1 segregation of the quadrivalents, three probes would be sufficient to detect all possible segregation products. If, however, 3:1 segregation without crossover in the insertional translocated segment is also taken into account, four probes are required to detect all possible segregants. Therefore, more research to understand the behaviour of such rare complex chromosomal anomalies during meiosis and the outcome of meiosis is warranted.
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References |
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Submitted on May 6, 2004; resubmitted on July 2, 2004; accepted on September 3, 2004.
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