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The causes of misdiagnosis and adverse outcomes in PGD - the case for chromosome rearrangements
- Paul N Scriven, Caroline Mackie Ogilvie (18 February 2009)
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Paul N Scriven, Clinical Scientist Centre for Preimplantation Genetic Diagnosis, Guy’s and St Thomas’ NHS Foundation Trust, London, UK, Caroline Mackie Ogilvie
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Paul.Scriven{at}gsts.com Paul N Scriven, et al.
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The recent paper on the causes of misdiagnosis and adverse outcomes in PGD (Wilton et al. 2009) is in general comprehensive and authoritative. However, the section on PGD for chromosome rearrangements is inadequate and in places, we believe, inaccurate. In order to ensure that Centres undertaking PGD are fully aware of the complexities of this area, we feel that it is important to expand on this issue. We agree that for two-way reciprocal translocations, which have two centric segments and two terminal translocated segments (this is not the case for every “translocation”), a minimum of three probes labelled with appropriate fluorochromes or haptens is needed in order to detect all the unbalanced segregation products. Unfortunately, the two strategies presented by the authors are poorly described and could be open to misinterpretation. In addition, the authors inappropriately quote a poster abstract (Conn et al. 1995), which anyway does not relate to the strategies described but only to experience testing research blastomeres with YAC probes for chromosomes 13, 14, 18 and 21, using dual colour FISH. The first strategy presented in the misdiagnosis paper is described as using two probes on “either side” of the breakpoints of one translocation chromosome. This is misleading and could be misinterpreted to mean that both probes could be designed on one side of the breakpoint or the other. It would have been better to describe this strategy as employing two locus-specific probes for target regions, one on each side of the breakpoint of one of the translocation chromosomes and a third locus-specific probe for any target region on the other translocation chromosome. The second strategy as described by the authors, and credited to us, is incorrect. The description should have indicated that this strategy employs three probes: a locus-specific probe for each of the translocated segment subtelomere regions (not “one sub-telomeric probe for each of the chromosomes”; the centric segments also have subtelomere regions) and a third locus-specific probe for one of the centric segments (which typically could be a centromere-region probe). This centric segment probe is described by the authors as a “control probe”; again, this is inaccurate and misleading, because the centric segment probe is actually diagnostic for chromosome imbalance consistent with adjacent-2 and 3:1 segregation products of reciprocal translocations. The theoretical proof and comprehensive discussion of this approach can be found elsewhere (Scriven et al, 1998; Scriven and Ogilvie, 2007). For testing two-way reciprocal translocations using one cell and a single hybridization reaction we recommend using at least a locus-specific probe for each of the translocated segments and a third probe for the centric segment of the smallest chromosome (typically we use commercial subtelomere and centromere-region probes), each labelled with a different fluorochrome and visualised with appropriate filters (three-colour FISH). However, for some two-way reciprocal translocations, a different strategy may be required, and this will depend upon the chromosomes involved and the position of the breakpoints. It is therefore critical to assess the nature of the chromosome rearrangement prior to designing preimplantation genetic FISH tests; failure to do so could result in a significant risk of an affected pregnancy. For example, a test that uses probes for both centric segments and only one of the translocated segments would have a significant single-cell risk of misdiagnosing unbalanced products with monosomy and trisomy for the translocated segments (consistent with adjacent-1, the most common abnormal segregation outcome); this risk would be significantly reduced by ensuring that the test included probes for both of the translocated segments, or by testing two cells and only recommending for transfer embryos with a concordant normal/balanced result from both cells. For other translocations where monosomy or trisomy for whole chromosomes is potentially viable (consistent with 3:1 segregation) it is important to ensure that the test includes probes for the centric and translocated segments of the “viable” chromosomes, and if a probe is only available for one of the segments, to test two cells. In addition, some translocations have potentially viable unbalanced products that have monosomy and trisomy for the centric segments (consistent with adjacent-2 segregation); for these translocations, the test should include probes for both centric segments for single-cell diagnosis, or, if only one centric segment probe is available, two cells should be tested. Three misdiagnoses following PGD for chromosome rearrangements are reported. As the authors indicate, the 11/22 reciprocal translocation case has been discussed extensively previously. However, no further information was apparently available concerning the tests used for the translocation trisomy 13 case following testing for a 13/14 Robertsonian translocation, or for the unbalanced translocation associated with a 13/15 (sic) reciprocal translocation. We recommend for Robertsonian translocations involving chromosome 13 using two different locus-specific probes for chromosome 13 (Scriven and Ogilvie, 2007). Where this is not possible, testing two cells would be appropriate. The karyotype given in Table IV for the unbalanced reciprocal translocation is 46,XY,der(15)t(13;15)(q25.1;q26.3)pat; however, chromosome 13 does not have a q25.1 band and we believe that the correct karyotype is 46,XY,der(15)t(3;15)(q25.1;q26.3)pat (Karen Sermon pers. com.). The typographical error appears to have been first made in the report for the ESHRE PGD consortium data collection VII (Harper et al. 2008). The chromosome imbalance would therefore be consistent with adjacent-1 segregation of the translocation resulting in trisomy for the translocated segment of chromosome 3 (3q25.1-->qter) and monosomy for the translocated segment of chromosome 15 (15q26.3-->qter). The chromosome 15 breakpoint is given as the terminal band of the long arm, and may have been distal to loci for available FISH probes (in which case there would technically be no evidence of a reciprocal exchange of material); if no probe had been available for the chromosome 15 translocated segment and only one cell tested, the misdiagnosis may have been due to mis-scoring of three chromosome 3 translocated segment probes as two signals. In this situation, two-cell biopsy would minimise the risk of this error, and significantly reduce the risk of misdiagnosis. The general principle and recommended practice for all chromosome rearrangements is that the test should include sufficient probes to detect all the unbalanced segregation products of the rearrangement and if testing only one cell to have two probes that are diagnostic for the chromosome imbalance associated with segregation products that are likely to be frequent or have potential to be viable (Thornhill et al. 2005). This ensures that two scoring errors would have to occur in order to diagnose an abnormal embryo as normal/balanced. We strongly agree with the authors that all preimplantation genetic testing must be undertaken by suitably qualified scientific staff in laboratories with appropriate medical laboratory accreditation. References Conn CM, Harper JC, Winston RML, Delhanty JDA. Preimplantation diagnosis of trisomies 13, 14, 18 and 21 in translocation carriers using multicolour fluorescent in situ hybridisation. Am J Hum Genet 1995;(Suppl 57):A1611. Harper JC, de Die-Smulders C, Goossens V, Harton G, Moutou C, Repping S, Scriven PN, SenGupta S, Traeger-Synodinos J, Van Rij MC, Viville S, Wilton L, Sermon KD. ESHRE PGD consortium data collection VII: cycles from January to December 2004 with pregnancy follow-up to October 2005. Hum Reprod 2008;23:741-755. Scriven P, Handyside A, Ogilvie C. Chromosome translocations: segregation modes and strategies for preimplantation genetic diagnosis. Prenat Diagn 1998;18:1437–1449. Scriven PN, Ogilvie CM. Fluorescence in situ hybridization on single cells. (Sex determination and chromosome rearrangements). Methods Mol Med 2007;132:19-30. Wilton L, Thornhill A, Traeger-Synodinos J, Sermon KD, Harper JC. The causes of misdiagnosis and adverse outcomes in PGD. Hum Reprod. 2009 Jan 20. [Epub ahead of print] Paul Scriven 1,2 Caroline Mackie Ogilvie 1,3 1 Centre for Preimplantation Genetic Diagnosis, Guy’s and St Thomas’ NHS Foundation Trust, London, UK 2 Department of Cytogenetics, GSTS Pathology, Guy’s Hospital, London, UK 3 Genetics Centre, Guy’s and St Thomas’ NHS Foundation Trust, London, UK Conflict of Interest:None declared |
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