- Email: [email protected]
Preimplantation genetic diagnosis for polycystic kidney disease
FERTILITY AND STERILITY威 VOL. 82, NO. 4, OCTOBER 2004 Copyright ©2004 American Society for Reproductive Medicine Published by Elsevier Inc. Printed on acid-free paper in U.S.A.
Preimplantation genetic diagnosis for polycystic kidney disease Yury Verlinsky, Ph.D., Svetlana Rechitsky, Ph.D., Oleg Verlinsky, M.Sc., Seckin Ozen, M.D., Robin Beck, M.Sc., and Anver Kuliev, M.D., Ph.D. Reproductive Genetics Institute, Chicago, Illinois
Objective: To use preimplantation genetic diagnosis for achieving a polycystic kidney disease (PKD)-free pregnancy for a couple in which the female partner was affected by PKD but whose PKD1 or PKD2 carrier status was not established. Design: Case report. Setting: The IVF program of Reproductive Genetics Institute, Chicago, Illinois. Patient(s): An at-risk couple with the female partner affected by PKD, whose PKD1 or PKD2 carrier status was not established. Intervention(s): Removal of PB1 and PB2 and testing for three closely linked markers to PKD1 (Kg8, D16S664, and SM7) and four closely linked markers to PKD2 (D4S2922, D4S2458, D4S423, and D4S1557) after standard IVF. Main Outcome Measure(s): Deoxyribonucleic acid analysis of PB1 and PB2 indicating whether corresponding oocytes were PKD1 or PKD2 allele free, for the purpose of transferring only embryos resulting from mutation-free oocytes. Result(s): Of 11 oocytes tested by PB1 and PB2 DNA analysis, 7 were predicted to contain PKD1 or PKD2, with the remaining 4 free of both mutations. Three embryos resulting from these oocytes were transferred, yielding a twin pregnancy and the birth of two unaffected children. Conclusion(s): This is the first preimplantation genetic diagnosis for PKD, which resulted in the birth of healthy twins confirmed to be free of PKD1 and PKD2. Preimplantation genetic diagnosis based on linked marker analysis provides an alternative for avoiding the pregnancy and birth of children with PKD, even in at-risk couples without exact PKD1 or PKD2 carrier information. (Fertil Steril威 2004;82:926 –9. ©2004 by American Society for Reproductive Medicine.) Key Words: Preimplantation genetic diagnosis, polycystic kidney disease, PKD1, PKD2, polar body sampling, linkage analysis
Received September 17, 2003; revised and accepted March 2, 2004. Reprint requests: Anver Kuliev, M.D., Ph.D., Reproductive Genetics Institute, Chicago, 2825 North Halsted, Chicago, Illinois 60657 (FAX: 773-8715221; E-mail: anverkuliev@ hotmail.com). 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2004. 03.041
926
Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder, present in 1 of 1,000 people worldwide, which causes progressive cyst formation and which can lead to renal failure by late middle age, requiring renal transplantation or dialysis (1). The overall health implications of ADPKD are obvious, given that approximately 10% of all patients at need for renal transplantation or dialysis have this disease. Autosomal dominant polycystic kidney disease is caused by the genes PKD1 or PKD2, for which no direct testing is yet available, making linkage analysis a method of choice. Because of the extremely variable expression and age of onset (much later in PKD2 carriers), only half of the patients carrying these genes
might present with severe clinical manifestation and renal failure. This makes prenatal diagnosis and pregnancy termination highly controversial. Preimplantation genetic diagnosis might, therefore, be more attractive for at-risk couples, because an ADPKD-free pregnancy might be established from the onset, without risk for pregnancy termination after prenatal diagnosis. The majority (85%) of ADPKD is caused by PKD1, assigned to chromosome 16p13.3; the rest is attributed to PKD2, located on chromosome 4q21-q23 (1). Because both PKD1- and PKD2-related disease are characterized by the enlargement of kidneys due to the formation of bilateral or multiple unilateral fluid-filled cysts, presymptomatic diagnosis is available by abdominal ultrasound examination of young
adults at risk; this also allows for improving the management of hypertension in these patients, which appears long before the actual manifestation of renal disease. Although the mutation rate is believed to be high, especially in PKD1, approximately half of cases are still ancestry related, thus allowing for diagnosis by linkage analysis. The PKD1 gene contains 46 exons, encoding the membrane protein polycystin 1, involved in cell-to-cell interaction, whereas PKD2 has at least 15 exons and encodes polycystin 2, which is a channel protein. Both of these proteins interact to produce new calcium-permeable, nonselective cation currents, contributing to fluid flow sensation by the primary cilium in renal epithelium. Therefore, loss or dysfunction of any of these proteins leads to PKD, owing to the inability of cells to sense mechanical cues that normally regulate renal tubular morphology and function. Because direct mutation testing is not presently available either for PKD1 or PKD2, and their clinical manifestations are almost identical, except for the severity and earlier onset of PKD1 disease, linkage analysis is usually applied for both of these genes to trace the disease gene from the affected person through the family to the patient. Deoxyribonucleic acid sequencing might in future be applied for PKD2, but this might not be useful for the PKD1 gene, because a large part of this gene is a duplication of multiple pseudogenes, which produce messenger RNA but are not translated. This article presents the first experience of preimplantation genetic diagnosis for ADPKD performed for a couple, for whom neither PKD1 nor PKD2 could be excluded as a cause of ADPKD in the family, resulting in a twin pregnancy and the birth of two unaffected children.
CASE REPORT The couple presented for preimplantation genetic diagnosis, with the female partner having a family history of ADPKD. Her father had severe ADPKD, and she also had a brother with clinical symptoms of ADPKD. Initial linkage analysis could not exclude either PKD1 or PKD2 as a cause of ADPKD in this family, so a set of linked markers were designed to trace both PKD1 and PKD2 in the same reaction.
MATERIALS AND METHODS A preimplantation genetic diagnosis cycle was performed according to a standard IVF protocol, together with micromanipulation procedures for polar body sampling and blastomere biopsy, as described elsewhere (2). The first and second polar bodies (PB1 and PB2) were removed in sequence after maturation and fertilization of the oocytes and tested by multiplex nested polymerase chain reaction analysis, involving the linked markers simultaneously in a multiplex, heminested system (3). The protocol of the study was approved by our institutional review board. FERTILITY & STERILITY威
The preselection of mutation-free oocytes was based on linked marker analysis, involving three closely linked markers to PKD1 (Kg8, D16S664, and SM7) and four closely linked markers to PKD2 (D4S2922, D4S2458, D4S423, and D4S1557) (4 – 6). The maternal haplotypes were 90 base pair (bp), 110 bp, and 133 bp repeats for Kg8, D16S664, and SM7 markers, respectively, to trace PKD1, and 123 bp, 91 bp, 133 bp, and 100 bp repeats for D4S2922, D4S2458, D4S23, and D4S1557 markers, respectively, to trace the PKD2 gene. As per the informed consent form, approved by our institutional review board, the embryos derived from the oocytes free of PKD1 and PKD2, as determined by information about polymorphic markers, were preselected for transfer back to the patient. Those oocytes predicted to be mutant or with inconclusive marker information were further tested by analysis of blastomeres, removed from the resulting embryos, either to confirm the diagnosis or to identify additional mutation-free embryos for transfer.
RESULTS Of 14 oocytes available for testing, for only 11 was information available for both PB1 and PB2. Two of these oocytes were predicted to contain PKD1 (oocytes 1 and 4), three to contain PKD2 (oocytes 2, 8, and 9), and three to contain neither PKD1 nor PKD2 (oocytes 5, 7, and 10). Three oocytes were inconclusive for one or both genes due to failed amplification of PB2 (oocytes 3 and 6) or allele dropout in PB1 (oocyte 11) (Table 1). The latter oocyte, however, was shown to be normal after blastomere analysis, although there was still a probability of recombination between the Kg8 marker and the PKD1 gene. The other two embryos, resulting from oocytes 3 and 6 with inconclusive results, were left with insufficient information because of amplification failure (embryo 6) or shared parental markers for PKD2 (embryo 3). Blastomere biopsies from the embryos resulting from the oocytes predicted to be mutant have confirmed the embryos to be affected, including one (embryo 9) in which, according to linkage analysis, neither PKD1 nor PKD2 could be excluded. Three embryos, resulting from oocytes 5, 7, and 10, were transferred, yielding a twin pregnancy and birth of two children confirmed to be free of both PKD1 and PKD2. According to the pattern of markers used to exclude PKD1, these mutation-free children were dizygotic twins, because they inherited different normal chromosomes 16 from the father. Follow-up analysis of the embryos resulting from the mutant oocytes also confirmed the accuracy of sequential PB1 and PB2 by linked marker analysis, which is in accordance with previously reported data (3). In addition, in cases of inconclusive results by polar body analysis, blastomere biopsy of the resulting embryo might still allow for preselection of additional embryos for transfer, as with embryo 11. However, as for avoiding a potential recombination between PKD1 and Kg8 alleles, closer linked markers 927
TABLE 1 Results of polar body and blastomere testing for PKD1 and PKD2 by linked marker analysis. PKD1 Oocyte no.
PKD2
Cell type
Kg8
D16S664
SM7
D4S2922
D4S2458
D4S423
D4S1557
Predicted genotype
PB1 PB2 PB1 PB2 PB1 PB2 Blastomere
96 90a 90a/96 90a 90a/96 FA 96/96
112 110a 110a/112 110a 110a/112 110a 106/112
142 133a 133a/ADO 133a 133a/ADO 133a 133/142
123a/129 123a 123a/129 129 129 123a 127/123a
ADOa/93 91a 91a/93 93 91a/93 91a 91/93
133a/129 133a 129 129 133a/129 133a 129/129
100a/107 100a 100a 107 100a/107 100a 110/107
PB1 PB2 Blastomere
ADOa/96 96 96/90a
110a/112 112 ADO/110a
ADOa/142 142 133/133a
123a/129 123a 123/129
91a/93 91a 91/93
133a/129 133a 129/129
100a/107 100a 100/107
PB1 PB2 PB1 PB2 Blastomere PB1 PB2 PB1 PB2 Blastomere
90a/96 90a 96 FA FAa 90a/96 90a 96 FA 96/?
110a/112 110a 110a/112 110a FAa 110a/112 110a 110a/112 110a 106/112
133a/ADO 133a 133a/142 133a FAa 133a/142 133a 142 FAa 131/142
123a 129 123a 129 FAa 123a/129 123a 123a/129 129 127/123a
91a 93 91a/93 91a FAa 91a/93 91a 91a/93 93 91/91a
133a 129 133a/129 133a FAa 133a/129 133a 133a/129 129 129/133a
100a 107 100a/107 100a FAa 100a/107 100a 100a/107 107 110/100a
PB1 PB2 Blastomere
96 FA 96/90a
ADOa/112 112 106/110a
142 FAa 133/133a
123a/129 129 123/123a
91a/93 93 91/91a
133a/129 129 ADO/133a
100a/107 107 100/100a
PB1 PB2 PB1 PB2 Blastomere
90a/ADOa 90a 96/? 90a 96/?
110a/112 110a 110a/112 110a 106/112
133a/142 133a ADOa/142 133a 133/142
123a/129 123a 123a 129 123/129
91a/93 91a 91a 93 91/93
133a/129 133a 133a 129 129/129
100a/107 100a 100a 107 100/107
Oocyte: PKD1 affected PKD2 normal Oocyte: PKD1 normal PKD2 affected Oocyte: PKD1 normal PKD2 inconclusive Embryo: PKD1 normal PKD2 inconclusive Oocyte: PKD1 affected PKD2 normal Embryo: PKD1 affected PKD2 normal Oocyte: PKD1 normal PKD2 normal Oocyte: PKD1 inconclusive PKD2 inconclusive Embryo: inconclusive Oocyte: PKD1 normal PKD2 normal Oocyte: PKD1 inconclusive PKD2 affected Embryo: PKD1 inconclusive PKD2 affected Oocyte: PKD1 inconclusive PKD2 affected Embryo: PKD1 affected PKD2 affected Oocyte: PKD1 normal PKD2 normal Oocyte: PKD1 inconclusive PKD2 normal Embryo: PKD1 normal PKD2 normal
Affected mother
90a/96
110a/112
133a/142
123a/129
91a/93
133a/129
100a/107
Affected maternal brother Unaffected father
90a/96
110a/112
133a/142
131a/129
91a/93
133a/129
100a/107
96/96
106/106
131/133
123/127
91/91
129/129
100/110
1 2 3
4
5 6
7 8
9
10 11
ET No No
No
No
Yes No Yes
No
No
Yes
Yes
Note: FA ⫽ failed amplification; ADO ⫽ allele dropout. Question marks denote inconclusive results. a Number of repeats corresponds with expected mutant alleles. Verlinsky. Preimplantation testing for PKD. Fertil Steril 2004.
were required; this embryo was frozen for possible future use by the couple.
DISCUSSION In the present experience of preimplantation genetic diagnosis for Mendelian disorders, performed in more than 1,000 cases (7), the case presented here is the first known preimplantation genetic diagnosis for ADPKD. The clinical relevance of preimplantation genetic diagnosis for ADPKD is evident, given that prenatal diagnosis and termination of pregnancy might not be acceptable for a considerable pro928
Verlinsky et al.
Preimplantation testing for PKD
portion of ADPKD cases, which might be benign with no clinical manifestation during the entire lifetime. The practical implication of preimplantation genetic diagnosis for ADPKD is also obvious, given the high worldwide prevalence of this disease. Thus, preimplantation genetic diagnosis for ADPKD will be useful for practicing physicians, to provide at-risk couples with appropriate advice regarding the available options for avoiding the risk of producing a child with ADPKD. Because of the straightforward clinical manifestation of the disease and the availability of a simple ultrasound screening, with no need for mutation Vol. 82, No. 4, October 2004
identification, the preimplantation genetic diagnosis strategy described here can be applied without the extensive preparatory work that is usually required for preimplantation genetic diagnosis for many other conditions. The case presented here also demonstrates the feasibility of preimplantation genetic diagnosis in cases that lack exact information on the causative gene involved. Thus, this report might have practical implications for preimplantation genetic diagnosis of other conditions for which the mutation might not be known but for which tracing of the mutant chromosome is possible with highly variable linked markers. References 1. National Center for Biotechnology Information. Online mendelian in-
FERTILITY & STERILITY威
2. 3.
4. 5. 6. 7.
heritance in man [MIM 601313; MIM 173910]. Baltimore: Johns Hopkins University, 2001. Available at: http://www.ncbi.nlm.nih.gov/Omim. Accessed September 3, 2003. Verlinsky Y, Kuliev A. Atlas of preimplantation genetic diagnosis. London: Parthenon, 2000. Rechitsky S, Strom C, Verlinsky O, Amet T, Ivakhnenko V, Kukharenko V, et al. Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes. J Assist Reprod Genet 1999;16: 192– 8. Harris PC, Thomas S, Ratcliffe PJ, Breuning MH, Coto E, Lopez-Larrea C. Rapid genetic analysis of families with polycystic kidney disease 1 by means of a microsatellite marker. Lancet 1991;338:1484 –7. Peral B, Ward CJ, San Milan JL, Thomas S, Stalings RL, Moreno F, et al. Evidence of linkage disequilibrium in the Spanish polycystic kidney disease I population. Am J Hum Genet 1994;54:899 –908. Snarey A, Thomas S, Schneider MC, Pound SE, Barton N, Wright AF, et al. Linkage disequilibrium in the region of the autosomal dominant polycystic kidney disease gene (PKDI). Am J Hum Genet 1994;55:365–71. Kuliev A, Verlinsky Y. Thirteen-years experience of preimplantation diagnosis. Reprod Biomed Online 2004;8:229 –35.
929
Preimplantation genetic diagnosis for polycystic kidney disease Yury Verlinsky, Ph.D., Svetlana Rechitsky, Ph.D., Oleg Verlinsky, M.Sc., Seckin Ozen, M.D., Robin Beck, M.Sc., and Anver Kuliev, M.D., Ph.D. Reproductive Genetics Institute, Chicago, Illinois
Objective: To use preimplantation genetic diagnosis for achieving a polycystic kidney disease (PKD)-free pregnancy for a couple in which the female partner was affected by PKD but whose PKD1 or PKD2 carrier status was not established. Design: Case report. Setting: The IVF program of Reproductive Genetics Institute, Chicago, Illinois. Patient(s): An at-risk couple with the female partner affected by PKD, whose PKD1 or PKD2 carrier status was not established. Intervention(s): Removal of PB1 and PB2 and testing for three closely linked markers to PKD1 (Kg8, D16S664, and SM7) and four closely linked markers to PKD2 (D4S2922, D4S2458, D4S423, and D4S1557) after standard IVF. Main Outcome Measure(s): Deoxyribonucleic acid analysis of PB1 and PB2 indicating whether corresponding oocytes were PKD1 or PKD2 allele free, for the purpose of transferring only embryos resulting from mutation-free oocytes. Result(s): Of 11 oocytes tested by PB1 and PB2 DNA analysis, 7 were predicted to contain PKD1 or PKD2, with the remaining 4 free of both mutations. Three embryos resulting from these oocytes were transferred, yielding a twin pregnancy and the birth of two unaffected children. Conclusion(s): This is the first preimplantation genetic diagnosis for PKD, which resulted in the birth of healthy twins confirmed to be free of PKD1 and PKD2. Preimplantation genetic diagnosis based on linked marker analysis provides an alternative for avoiding the pregnancy and birth of children with PKD, even in at-risk couples without exact PKD1 or PKD2 carrier information. (Fertil Steril威 2004;82:926 –9. ©2004 by American Society for Reproductive Medicine.) Key Words: Preimplantation genetic diagnosis, polycystic kidney disease, PKD1, PKD2, polar body sampling, linkage analysis
Received September 17, 2003; revised and accepted March 2, 2004. Reprint requests: Anver Kuliev, M.D., Ph.D., Reproductive Genetics Institute, Chicago, 2825 North Halsted, Chicago, Illinois 60657 (FAX: 773-8715221; E-mail: anverkuliev@ hotmail.com). 0015-0282/04/$30.00 doi:10.1016/j.fertnstert.2004. 03.041
926
Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder, present in 1 of 1,000 people worldwide, which causes progressive cyst formation and which can lead to renal failure by late middle age, requiring renal transplantation or dialysis (1). The overall health implications of ADPKD are obvious, given that approximately 10% of all patients at need for renal transplantation or dialysis have this disease. Autosomal dominant polycystic kidney disease is caused by the genes PKD1 or PKD2, for which no direct testing is yet available, making linkage analysis a method of choice. Because of the extremely variable expression and age of onset (much later in PKD2 carriers), only half of the patients carrying these genes
might present with severe clinical manifestation and renal failure. This makes prenatal diagnosis and pregnancy termination highly controversial. Preimplantation genetic diagnosis might, therefore, be more attractive for at-risk couples, because an ADPKD-free pregnancy might be established from the onset, without risk for pregnancy termination after prenatal diagnosis. The majority (85%) of ADPKD is caused by PKD1, assigned to chromosome 16p13.3; the rest is attributed to PKD2, located on chromosome 4q21-q23 (1). Because both PKD1- and PKD2-related disease are characterized by the enlargement of kidneys due to the formation of bilateral or multiple unilateral fluid-filled cysts, presymptomatic diagnosis is available by abdominal ultrasound examination of young
adults at risk; this also allows for improving the management of hypertension in these patients, which appears long before the actual manifestation of renal disease. Although the mutation rate is believed to be high, especially in PKD1, approximately half of cases are still ancestry related, thus allowing for diagnosis by linkage analysis. The PKD1 gene contains 46 exons, encoding the membrane protein polycystin 1, involved in cell-to-cell interaction, whereas PKD2 has at least 15 exons and encodes polycystin 2, which is a channel protein. Both of these proteins interact to produce new calcium-permeable, nonselective cation currents, contributing to fluid flow sensation by the primary cilium in renal epithelium. Therefore, loss or dysfunction of any of these proteins leads to PKD, owing to the inability of cells to sense mechanical cues that normally regulate renal tubular morphology and function. Because direct mutation testing is not presently available either for PKD1 or PKD2, and their clinical manifestations are almost identical, except for the severity and earlier onset of PKD1 disease, linkage analysis is usually applied for both of these genes to trace the disease gene from the affected person through the family to the patient. Deoxyribonucleic acid sequencing might in future be applied for PKD2, but this might not be useful for the PKD1 gene, because a large part of this gene is a duplication of multiple pseudogenes, which produce messenger RNA but are not translated. This article presents the first experience of preimplantation genetic diagnosis for ADPKD performed for a couple, for whom neither PKD1 nor PKD2 could be excluded as a cause of ADPKD in the family, resulting in a twin pregnancy and the birth of two unaffected children.
CASE REPORT The couple presented for preimplantation genetic diagnosis, with the female partner having a family history of ADPKD. Her father had severe ADPKD, and she also had a brother with clinical symptoms of ADPKD. Initial linkage analysis could not exclude either PKD1 or PKD2 as a cause of ADPKD in this family, so a set of linked markers were designed to trace both PKD1 and PKD2 in the same reaction.
MATERIALS AND METHODS A preimplantation genetic diagnosis cycle was performed according to a standard IVF protocol, together with micromanipulation procedures for polar body sampling and blastomere biopsy, as described elsewhere (2). The first and second polar bodies (PB1 and PB2) were removed in sequence after maturation and fertilization of the oocytes and tested by multiplex nested polymerase chain reaction analysis, involving the linked markers simultaneously in a multiplex, heminested system (3). The protocol of the study was approved by our institutional review board. FERTILITY & STERILITY威
The preselection of mutation-free oocytes was based on linked marker analysis, involving three closely linked markers to PKD1 (Kg8, D16S664, and SM7) and four closely linked markers to PKD2 (D4S2922, D4S2458, D4S423, and D4S1557) (4 – 6). The maternal haplotypes were 90 base pair (bp), 110 bp, and 133 bp repeats for Kg8, D16S664, and SM7 markers, respectively, to trace PKD1, and 123 bp, 91 bp, 133 bp, and 100 bp repeats for D4S2922, D4S2458, D4S23, and D4S1557 markers, respectively, to trace the PKD2 gene. As per the informed consent form, approved by our institutional review board, the embryos derived from the oocytes free of PKD1 and PKD2, as determined by information about polymorphic markers, were preselected for transfer back to the patient. Those oocytes predicted to be mutant or with inconclusive marker information were further tested by analysis of blastomeres, removed from the resulting embryos, either to confirm the diagnosis or to identify additional mutation-free embryos for transfer.
RESULTS Of 14 oocytes available for testing, for only 11 was information available for both PB1 and PB2. Two of these oocytes were predicted to contain PKD1 (oocytes 1 and 4), three to contain PKD2 (oocytes 2, 8, and 9), and three to contain neither PKD1 nor PKD2 (oocytes 5, 7, and 10). Three oocytes were inconclusive for one or both genes due to failed amplification of PB2 (oocytes 3 and 6) or allele dropout in PB1 (oocyte 11) (Table 1). The latter oocyte, however, was shown to be normal after blastomere analysis, although there was still a probability of recombination between the Kg8 marker and the PKD1 gene. The other two embryos, resulting from oocytes 3 and 6 with inconclusive results, were left with insufficient information because of amplification failure (embryo 6) or shared parental markers for PKD2 (embryo 3). Blastomere biopsies from the embryos resulting from the oocytes predicted to be mutant have confirmed the embryos to be affected, including one (embryo 9) in which, according to linkage analysis, neither PKD1 nor PKD2 could be excluded. Three embryos, resulting from oocytes 5, 7, and 10, were transferred, yielding a twin pregnancy and birth of two children confirmed to be free of both PKD1 and PKD2. According to the pattern of markers used to exclude PKD1, these mutation-free children were dizygotic twins, because they inherited different normal chromosomes 16 from the father. Follow-up analysis of the embryos resulting from the mutant oocytes also confirmed the accuracy of sequential PB1 and PB2 by linked marker analysis, which is in accordance with previously reported data (3). In addition, in cases of inconclusive results by polar body analysis, blastomere biopsy of the resulting embryo might still allow for preselection of additional embryos for transfer, as with embryo 11. However, as for avoiding a potential recombination between PKD1 and Kg8 alleles, closer linked markers 927
TABLE 1 Results of polar body and blastomere testing for PKD1 and PKD2 by linked marker analysis. PKD1 Oocyte no.
PKD2
Cell type
Kg8
D16S664
SM7
D4S2922
D4S2458
D4S423
D4S1557
Predicted genotype
PB1 PB2 PB1 PB2 PB1 PB2 Blastomere
96 90a 90a/96 90a 90a/96 FA 96/96
112 110a 110a/112 110a 110a/112 110a 106/112
142 133a 133a/ADO 133a 133a/ADO 133a 133/142
123a/129 123a 123a/129 129 129 123a 127/123a
ADOa/93 91a 91a/93 93 91a/93 91a 91/93
133a/129 133a 129 129 133a/129 133a 129/129
100a/107 100a 100a 107 100a/107 100a 110/107
PB1 PB2 Blastomere
ADOa/96 96 96/90a
110a/112 112 ADO/110a
ADOa/142 142 133/133a
123a/129 123a 123/129
91a/93 91a 91/93
133a/129 133a 129/129
100a/107 100a 100/107
PB1 PB2 PB1 PB2 Blastomere PB1 PB2 PB1 PB2 Blastomere
90a/96 90a 96 FA FAa 90a/96 90a 96 FA 96/?
110a/112 110a 110a/112 110a FAa 110a/112 110a 110a/112 110a 106/112
133a/ADO 133a 133a/142 133a FAa 133a/142 133a 142 FAa 131/142
123a 129 123a 129 FAa 123a/129 123a 123a/129 129 127/123a
91a 93 91a/93 91a FAa 91a/93 91a 91a/93 93 91/91a
133a 129 133a/129 133a FAa 133a/129 133a 133a/129 129 129/133a
100a 107 100a/107 100a FAa 100a/107 100a 100a/107 107 110/100a
PB1 PB2 Blastomere
96 FA 96/90a
ADOa/112 112 106/110a
142 FAa 133/133a
123a/129 129 123/123a
91a/93 93 91/91a
133a/129 129 ADO/133a
100a/107 107 100/100a
PB1 PB2 PB1 PB2 Blastomere
90a/ADOa 90a 96/? 90a 96/?
110a/112 110a 110a/112 110a 106/112
133a/142 133a ADOa/142 133a 133/142
123a/129 123a 123a 129 123/129
91a/93 91a 91a 93 91/93
133a/129 133a 133a 129 129/129
100a/107 100a 100a 107 100/107
Oocyte: PKD1 affected PKD2 normal Oocyte: PKD1 normal PKD2 affected Oocyte: PKD1 normal PKD2 inconclusive Embryo: PKD1 normal PKD2 inconclusive Oocyte: PKD1 affected PKD2 normal Embryo: PKD1 affected PKD2 normal Oocyte: PKD1 normal PKD2 normal Oocyte: PKD1 inconclusive PKD2 inconclusive Embryo: inconclusive Oocyte: PKD1 normal PKD2 normal Oocyte: PKD1 inconclusive PKD2 affected Embryo: PKD1 inconclusive PKD2 affected Oocyte: PKD1 inconclusive PKD2 affected Embryo: PKD1 affected PKD2 affected Oocyte: PKD1 normal PKD2 normal Oocyte: PKD1 inconclusive PKD2 normal Embryo: PKD1 normal PKD2 normal
Affected mother
90a/96
110a/112
133a/142
123a/129
91a/93
133a/129
100a/107
Affected maternal brother Unaffected father
90a/96
110a/112
133a/142
131a/129
91a/93
133a/129
100a/107
96/96
106/106
131/133
123/127
91/91
129/129
100/110
1 2 3
4
5 6
7 8
9
10 11
ET No No
No
No
Yes No Yes
No
No
Yes
Yes
Note: FA ⫽ failed amplification; ADO ⫽ allele dropout. Question marks denote inconclusive results. a Number of repeats corresponds with expected mutant alleles. Verlinsky. Preimplantation testing for PKD. Fertil Steril 2004.
were required; this embryo was frozen for possible future use by the couple.
DISCUSSION In the present experience of preimplantation genetic diagnosis for Mendelian disorders, performed in more than 1,000 cases (7), the case presented here is the first known preimplantation genetic diagnosis for ADPKD. The clinical relevance of preimplantation genetic diagnosis for ADPKD is evident, given that prenatal diagnosis and termination of pregnancy might not be acceptable for a considerable pro928
Verlinsky et al.
Preimplantation testing for PKD
portion of ADPKD cases, which might be benign with no clinical manifestation during the entire lifetime. The practical implication of preimplantation genetic diagnosis for ADPKD is also obvious, given the high worldwide prevalence of this disease. Thus, preimplantation genetic diagnosis for ADPKD will be useful for practicing physicians, to provide at-risk couples with appropriate advice regarding the available options for avoiding the risk of producing a child with ADPKD. Because of the straightforward clinical manifestation of the disease and the availability of a simple ultrasound screening, with no need for mutation Vol. 82, No. 4, October 2004
identification, the preimplantation genetic diagnosis strategy described here can be applied without the extensive preparatory work that is usually required for preimplantation genetic diagnosis for many other conditions. The case presented here also demonstrates the feasibility of preimplantation genetic diagnosis in cases that lack exact information on the causative gene involved. Thus, this report might have practical implications for preimplantation genetic diagnosis of other conditions for which the mutation might not be known but for which tracing of the mutant chromosome is possible with highly variable linked markers. References 1. National Center for Biotechnology Information. Online mendelian in-
FERTILITY & STERILITY威
2. 3.
4. 5. 6. 7.
heritance in man [MIM 601313; MIM 173910]. Baltimore: Johns Hopkins University, 2001. Available at: http://www.ncbi.nlm.nih.gov/Omim. Accessed September 3, 2003. Verlinsky Y, Kuliev A. Atlas of preimplantation genetic diagnosis. London: Parthenon, 2000. Rechitsky S, Strom C, Verlinsky O, Amet T, Ivakhnenko V, Kukharenko V, et al. Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes. J Assist Reprod Genet 1999;16: 192– 8. Harris PC, Thomas S, Ratcliffe PJ, Breuning MH, Coto E, Lopez-Larrea C. Rapid genetic analysis of families with polycystic kidney disease 1 by means of a microsatellite marker. Lancet 1991;338:1484 –7. Peral B, Ward CJ, San Milan JL, Thomas S, Stalings RL, Moreno F, et al. Evidence of linkage disequilibrium in the Spanish polycystic kidney disease I population. Am J Hum Genet 1994;54:899 –908. Snarey A, Thomas S, Schneider MC, Pound SE, Barton N, Wright AF, et al. Linkage disequilibrium in the region of the autosomal dominant polycystic kidney disease gene (PKDI). Am J Hum Genet 1994;55:365–71. Kuliev A, Verlinsky Y. Thirteen-years experience of preimplantation diagnosis. Reprod Biomed Online 2004;8:229 –35.
929