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One-step multiplex polymerase chain reaction for preimplantation genetic diagnosis of Huntington disease
One-step multiplex polymerase chain reaction for preimplantation genetic diagnosis of Huntington disease Ana Peci~ na, Ph.D.,a,b Marıa Dolores Lozano Arana, M.D.,a Juan C. Garcıa-Lozano, M.D.,a Salud Borrego, M.D., Ph.D.,a,b and Guillermo Anti~ nolo, M.D., Ph.D.a,b a b
Unidad de Gestion Clınica de Genetica, Reproduccion y Medicina Fetal, Hospitales Universitarios Virgen del Rocıo, and Centro de Investigaciones Biomedicas en Red de Enfermedades Raras, Instituto de Salud Carlos III (ISCIII), Seville, Spain
Objective: To develop a multiplex polymerase chain reaction (PCR) method for Huntington disease (HD) preimplantation genetic diagnosis (PGD) based on the coamplification of CAG repeats and three different polymorphic microsatellites in a single step of PCR. Design: Techniques and instrumentation. Setting: Tertiary clinical and academic medical center. Patient(s): Thirty-six embryos from seven clinical PGD cycles. Intervention(s): Patients underwent a PGD cycle with transfer of two unaffected embryos on day 5. Main Outcome Measure(s): PGD based on mutation identification or exclusion testing for at-risk HD carriers. Result(s): Thirty-six embryos from seven clinical PGD cycles were analyzed with the new method here developed, and results were obtained for 34 of them. Two embryos were transferred on day 5, resulting in two singleton pregnancies. Conclusion(s): An interesting application of this approach can be considered for PGD cycles in which numerous markers must be used. We have also used this one-step multiplex method for PGD for other pathological conditions. (Fertil Steril 2010;93:2411–2. 2010 by American Society for Reproductive Medicine.) Key Words: Multiplex fluorescent PCR, preimplantation genetic diagnosis, Huntington disease, single-cell PCR, exclusion testing
Huntington disease (HD) is a late-onset neurodegenerative disorder transmitted as an autosomal dominant trait caused by a so-called dynamic mutation. This mutation is caused by an expansion of a CAG repeat array in the first exon of the IT15 gene on chromosome 4 (4p16) (1). For at-risk HD carriers, preimplantation genetic diagnosis (PGD) based on mutation identification is available. Other potential HD carriers, however, prefer not to know their status but still wish to prevent the birth of a carrier child. For these patients, embryos obtained from a PGD cycle can then be selected, either by the amplification of the CAG repeats in the embryos without communicating results to the patients (nondisclosure testing) (2) or by exclusion testing (3). We present here a fluorescent multiplex polymerase chain reaction (PCR) technique based on the coamplification of CAG repeats and three different polymorphic microsatellites
Received September 22, 2008; revised November 28, 2008; accepted January 20, 2009; published online May 6, 2009. A.P. has nothing to disclose. M.D.L.A. has nothing to disclose. J.C.G.-L. has nothing to disclose. S.B. has nothing to disclose. G.A. has nothing to disclose. ~olo, M.D., Ph.D., Unidad de Gestio n Reprint requests: Guillermo Antin tica, Reproduccio n y Medicina Fetal, Hospitales UniverClınica de Gene sitarios Virgen del Rocıo, Avenida Manuel Siurot s/n, 41013 Sevilla, Spain (FAX: +34 9 55013473; E-mail: guillermo.antinolo.sspa@ juntadeandalucia.es).
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2009.01.120
commonly used for HD diagnosis (4) in a single step of PCR applied at the single-cell level for PGD. MATERIALS AND METHODS Genomic DNA was obtained from whole blood. Patient ovarian stimulation, oocyte retrieval, and intracytoplasmic sperm injection technique were carried out with standard protocols (3). Blastomeres were biopsied using a noncontact, 1.48-mm diode laser system (OCTAX; Olympus, Tokyo, Japan). Each blastomere was transferred into a reaction tube containing 2.5 mL lysis buffer (200 mM NaOH, 50 mM DTT) and frozen at 80 C before cell lysis. Cells were lysed by incubation at 65 C for 10 minutes before adding the PCR mix. A multiplex single-cell PCR for PGD of HD was performed. CAG repeats, an intragenic marker (I1CAHD), and two HD gene-flanking markers (D4S412 and D4S127) were amplified in a single PCR reaction using the QIAGEN Multiplex PCR kit (QIAGEN GmbH; Hilden, Germany). The reaction mix contained 200 mM Tricine, pH 4.93, 2 mM each primer, 5 Sol Q and 2 QIAGEN Multiplex PCR Master Mix, for a final volume of 25 mL. The PCR program was as follows: 15 minutes at 95 C, 10 cycles of 30 seconds at 96 C, 1 minute at 63 C, 90 seconds at 72 C, followed by 30 cycles of 30 seconds at 94 C, 1 minute at 63 C, 90 seconds at 72 C, and a final extension of
Fertility and Sterility Vol. 93, No. 7, May 1, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
2411
FIGURE 1 Examples of profiles obtained for four different markers. Amplified loci are indicated on the top right of the figure. Profiles were obtained after PCR on a single blastomere of an affected embryo (A) and an unaffected embryo (B).
Peci~ na. One-step multiplex PCR for PGD of Huntington. Fertil Steril 2010.
15 minutes at 60 C. One primer of each pair was fluorescently labeled. Primer sequences were described elsewhere (4). PCR products were analyzed on an ABI3730 automated sequencer (Applied Biosystems, Foster City, CA).
rate, the time required, and the cost of each PGD cycle. The assay allows the detection of contamination and indirect genetic diagnosis, thereby increasing the reliability of the results.
RESULTS Seven clinical PGD cycles were then performed using the method. Two blastomeres were removed from each of 36 embryos and analyzed, and results were obtained for 34 of these (94.4%). The allele drop out (ADO) rate was 2.7%. A typical PGD result of the one-step multiplex PCR is shown in Figure 1. Two embryos were transferred on day 5 in five out of seven cycles, resulting in two singleton pregnancies. One of the pregnant woman asked for confirmatory prenatal diagnosis; a chorionic villus sample was performed, and PGD results were confirmed.
REFERENCES
DISCUSSION The coamplification of CAG repeat mutations and three different microsatellite markers provides more information than previously published protocols (3–5), and it can also be used for at-risk persons who do not want to undergo presymptomatic mutation testing diagnosis. This method involves only a round of single PCR, thus decreasing the contamination
2412
Peci~na et al.
An interesting application of this approach can be considered for PGD cycles in which numerous markers must be used. We have also used this one-step multiplex method for PGD for other pathological conditions such as cystic fibrosis, spinal muscular atrophy, and Duchenne muscular dystrophy.
1. The Huntington’s Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expended and unstable on Huntington’s disease chromosomes. Cell 1993;72:971–83. 2. Stern HJ, Harton GL, Sisson ME, Jones SL, Fallon LA, Thorsell LP, et al. Non-disclosing preimplantation genetic diagnosis for Huntington disease. Prenat Diagn 2002;22:503–7. 3. Sermon K, De Rijcke M, Lissens M, De Vos A, Platteau P, Bonduelle M, et al. Preimplantation genetic diagnosis for Huntington’s disease with exclusion testing. Eur J Hum Genet 2002;10:591–8. 4. Moutou C, Gardes N, Viville S. New tools for preimplantation genetic diagnosis of Huntington’s disease and their clinical applications. Eur J Hum Genet 2004;12:1007–14. 5. Jasper MJ, Hu DG, Liebelt J, Sherrin D, Watson R, Tremellen KP, et al. Singleton births after routine preimplantation genetic diagnosis using exclusion testing (D4S43 and D4S 126) for Huntington’s disease. Fertil Steril 2006;85:597–2.
One-step multiplex PCR for PGD of Huntington
Vol. 93, No. 7, May 1, 2010
Unidad de Gestion Clınica de Genetica, Reproduccion y Medicina Fetal, Hospitales Universitarios Virgen del Rocıo, and Centro de Investigaciones Biomedicas en Red de Enfermedades Raras, Instituto de Salud Carlos III (ISCIII), Seville, Spain
Objective: To develop a multiplex polymerase chain reaction (PCR) method for Huntington disease (HD) preimplantation genetic diagnosis (PGD) based on the coamplification of CAG repeats and three different polymorphic microsatellites in a single step of PCR. Design: Techniques and instrumentation. Setting: Tertiary clinical and academic medical center. Patient(s): Thirty-six embryos from seven clinical PGD cycles. Intervention(s): Patients underwent a PGD cycle with transfer of two unaffected embryos on day 5. Main Outcome Measure(s): PGD based on mutation identification or exclusion testing for at-risk HD carriers. Result(s): Thirty-six embryos from seven clinical PGD cycles were analyzed with the new method here developed, and results were obtained for 34 of them. Two embryos were transferred on day 5, resulting in two singleton pregnancies. Conclusion(s): An interesting application of this approach can be considered for PGD cycles in which numerous markers must be used. We have also used this one-step multiplex method for PGD for other pathological conditions. (Fertil Steril 2010;93:2411–2. 2010 by American Society for Reproductive Medicine.) Key Words: Multiplex fluorescent PCR, preimplantation genetic diagnosis, Huntington disease, single-cell PCR, exclusion testing
Huntington disease (HD) is a late-onset neurodegenerative disorder transmitted as an autosomal dominant trait caused by a so-called dynamic mutation. This mutation is caused by an expansion of a CAG repeat array in the first exon of the IT15 gene on chromosome 4 (4p16) (1). For at-risk HD carriers, preimplantation genetic diagnosis (PGD) based on mutation identification is available. Other potential HD carriers, however, prefer not to know their status but still wish to prevent the birth of a carrier child. For these patients, embryos obtained from a PGD cycle can then be selected, either by the amplification of the CAG repeats in the embryos without communicating results to the patients (nondisclosure testing) (2) or by exclusion testing (3). We present here a fluorescent multiplex polymerase chain reaction (PCR) technique based on the coamplification of CAG repeats and three different polymorphic microsatellites
Received September 22, 2008; revised November 28, 2008; accepted January 20, 2009; published online May 6, 2009. A.P. has nothing to disclose. M.D.L.A. has nothing to disclose. J.C.G.-L. has nothing to disclose. S.B. has nothing to disclose. G.A. has nothing to disclose. ~olo, M.D., Ph.D., Unidad de Gestio n Reprint requests: Guillermo Antin tica, Reproduccio n y Medicina Fetal, Hospitales UniverClınica de Gene sitarios Virgen del Rocıo, Avenida Manuel Siurot s/n, 41013 Sevilla, Spain (FAX: +34 9 55013473; E-mail: guillermo.antinolo.sspa@ juntadeandalucia.es).
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2009.01.120
commonly used for HD diagnosis (4) in a single step of PCR applied at the single-cell level for PGD. MATERIALS AND METHODS Genomic DNA was obtained from whole blood. Patient ovarian stimulation, oocyte retrieval, and intracytoplasmic sperm injection technique were carried out with standard protocols (3). Blastomeres were biopsied using a noncontact, 1.48-mm diode laser system (OCTAX; Olympus, Tokyo, Japan). Each blastomere was transferred into a reaction tube containing 2.5 mL lysis buffer (200 mM NaOH, 50 mM DTT) and frozen at 80 C before cell lysis. Cells were lysed by incubation at 65 C for 10 minutes before adding the PCR mix. A multiplex single-cell PCR for PGD of HD was performed. CAG repeats, an intragenic marker (I1CAHD), and two HD gene-flanking markers (D4S412 and D4S127) were amplified in a single PCR reaction using the QIAGEN Multiplex PCR kit (QIAGEN GmbH; Hilden, Germany). The reaction mix contained 200 mM Tricine, pH 4.93, 2 mM each primer, 5 Sol Q and 2 QIAGEN Multiplex PCR Master Mix, for a final volume of 25 mL. The PCR program was as follows: 15 minutes at 95 C, 10 cycles of 30 seconds at 96 C, 1 minute at 63 C, 90 seconds at 72 C, followed by 30 cycles of 30 seconds at 94 C, 1 minute at 63 C, 90 seconds at 72 C, and a final extension of
Fertility and Sterility Vol. 93, No. 7, May 1, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
2411
FIGURE 1 Examples of profiles obtained for four different markers. Amplified loci are indicated on the top right of the figure. Profiles were obtained after PCR on a single blastomere of an affected embryo (A) and an unaffected embryo (B).
Peci~ na. One-step multiplex PCR for PGD of Huntington. Fertil Steril 2010.
15 minutes at 60 C. One primer of each pair was fluorescently labeled. Primer sequences were described elsewhere (4). PCR products were analyzed on an ABI3730 automated sequencer (Applied Biosystems, Foster City, CA).
rate, the time required, and the cost of each PGD cycle. The assay allows the detection of contamination and indirect genetic diagnosis, thereby increasing the reliability of the results.
RESULTS Seven clinical PGD cycles were then performed using the method. Two blastomeres were removed from each of 36 embryos and analyzed, and results were obtained for 34 of these (94.4%). The allele drop out (ADO) rate was 2.7%. A typical PGD result of the one-step multiplex PCR is shown in Figure 1. Two embryos were transferred on day 5 in five out of seven cycles, resulting in two singleton pregnancies. One of the pregnant woman asked for confirmatory prenatal diagnosis; a chorionic villus sample was performed, and PGD results were confirmed.
REFERENCES
DISCUSSION The coamplification of CAG repeat mutations and three different microsatellite markers provides more information than previously published protocols (3–5), and it can also be used for at-risk persons who do not want to undergo presymptomatic mutation testing diagnosis. This method involves only a round of single PCR, thus decreasing the contamination
2412
Peci~na et al.
An interesting application of this approach can be considered for PGD cycles in which numerous markers must be used. We have also used this one-step multiplex method for PGD for other pathological conditions such as cystic fibrosis, spinal muscular atrophy, and Duchenne muscular dystrophy.
1. The Huntington’s Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expended and unstable on Huntington’s disease chromosomes. Cell 1993;72:971–83. 2. Stern HJ, Harton GL, Sisson ME, Jones SL, Fallon LA, Thorsell LP, et al. Non-disclosing preimplantation genetic diagnosis for Huntington disease. Prenat Diagn 2002;22:503–7. 3. Sermon K, De Rijcke M, Lissens M, De Vos A, Platteau P, Bonduelle M, et al. Preimplantation genetic diagnosis for Huntington’s disease with exclusion testing. Eur J Hum Genet 2002;10:591–8. 4. Moutou C, Gardes N, Viville S. New tools for preimplantation genetic diagnosis of Huntington’s disease and their clinical applications. Eur J Hum Genet 2004;12:1007–14. 5. Jasper MJ, Hu DG, Liebelt J, Sherrin D, Watson R, Tremellen KP, et al. Singleton births after routine preimplantation genetic diagnosis using exclusion testing (D4S43 and D4S 126) for Huntington’s disease. Fertil Steril 2006;85:597–2.
One-step multiplex PCR for PGD of Huntington
Vol. 93, No. 7, May 1, 2010