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[Successful preimplantation genetic diagnosis for beta-thalassemia using multiplex nested polymerase chain reaction].
Zhonghua Yi Xue Za Zhi 2005; 85(12):811-5ZY

Abstract

OBJECTIVE

To develop single-cell multiplex nested polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples at risk of having child with beta-thalassemia.

METHODS

Primers were designed and synthesized according to the documented mutation sites common among Chinese. Venous blood was collected from 4 pairs of husband and wife, all heterozygotes for beta-thalassemia, and underwent multiple nested PCR. Intraooplasmic sperm injection and mechanical bio psy was used to obtain single blastomere. Multiplex nested PCR was used to detect the CD41-42 mutation and the closely linked polymorphic marker, HumTHO1 gene or CD41-42, CD41-28, IVSII654 mutation and HumTHO1 gene in the single blastomeres from four clinical PGD cycles. The normal embryos with high scores capable of continuing to divide were transplanted into the uteri. The process of gestation was observed.

RESULTS

200 lymphocytes were amplified by nested PCR. The average amplification rate of the most common 16 beta-thalassemia mutations in Chinese population was 91.3% and the average rate of allele drop out for different sites was 17.0% without differences between any 2 sites. During the 4 PGD cycles 33 embryos underwent bioassay with a success rate of 100%. 33 blastomeres were obtained to undergo PCR, of which 30 were successfully amplified with an amplification rate of 90.9%. Explicit diagnosis was obtained in 26 of the 30 embryos: 7 normal homozygotes, 11 heterozygotes, and 8 abnormal or complex heterozygotes. One or more embryos were transferred back into the uteri of the 4 women and clinical pregnancy occurred in one woman. Five weeks after the implantation B-mode ultrasonography showed monocyesis, and in the 17th week of gestational period paracentesis of cord blood showed normal homozygote. At last a normal female infant confirming the PGD result had been born, which was the first reported unaffected pregnancy resulting from PGD using multiplex nested PCR for couples as beta-thalassemia gene carriers. The results of diagnosis for embryo all corresponded to those for blastomere. The average ADO rate of blastomere was 13.3% (4/30).

CONCLUSION

PGD using multiplex nested PCR, as an alternative to prenatal diagnosis, is a reliable and effective way to help couples-carriers of pathogenetic genes to get a healthy baby.

Authors+Show Affiliations

Reproductive Medical Center, 1st Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

English Abstract
Journal Article

Language

chi

PubMed ID

15949396

Citation

Deng, Jie, et al. "[Successful Preimplantation Genetic Diagnosis for Beta-thalassemia Using Multiplex Nested Polymerase Chain Reaction]." Zhonghua Yi Xue Za Zhi, vol. 85, no. 12, 2005, pp. 811-5.
Deng J, Zhuang GL, Peng WL, et al. [Successful preimplantation genetic diagnosis for beta-thalassemia using multiplex nested polymerase chain reaction]. Zhonghua Yi Xue Za Zhi. 2005;85(12):811-5.
Deng, J., Zhuang, G. L., Peng, W. L., Zhou, C. Q., Li, J., Liang, X. Y., ... Sun, H. Y. (2005). [Successful preimplantation genetic diagnosis for beta-thalassemia using multiplex nested polymerase chain reaction]. Zhonghua Yi Xue Za Zhi, 85(12), pp. 811-5.
Deng J, et al. [Successful Preimplantation Genetic Diagnosis for Beta-thalassemia Using Multiplex Nested Polymerase Chain Reaction]. Zhonghua Yi Xue Za Zhi. 2005 Mar 30;85(12):811-5. PubMed PMID: 15949396.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Successful preimplantation genetic diagnosis for beta-thalassemia using multiplex nested polymerase chain reaction]. AU - Deng,Jie, AU - Zhuang,Guang-Lun, AU - Peng,Wen-Lin, AU - Zhou,Can-Quan, AU - Li,Jie, AU - Liang,Xiao-Yan, AU - Deng,Ming-Fen, AU - Zeng,Yan-Hong, AU - Sun,Hong-Yu, PY - 2005/6/14/pubmed PY - 2005/12/17/medline PY - 2005/6/14/entrez SP - 811 EP - 5 JF - Zhonghua yi xue za zhi JO - Zhonghua Yi Xue Za Zhi VL - 85 IS - 12 N2 - OBJECTIVE: To develop single-cell multiplex nested polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples at risk of having child with beta-thalassemia. METHODS: Primers were designed and synthesized according to the documented mutation sites common among Chinese. Venous blood was collected from 4 pairs of husband and wife, all heterozygotes for beta-thalassemia, and underwent multiple nested PCR. Intraooplasmic sperm injection and mechanical bio psy was used to obtain single blastomere. Multiplex nested PCR was used to detect the CD41-42 mutation and the closely linked polymorphic marker, HumTHO1 gene or CD41-42, CD41-28, IVSII654 mutation and HumTHO1 gene in the single blastomeres from four clinical PGD cycles. The normal embryos with high scores capable of continuing to divide were transplanted into the uteri. The process of gestation was observed. RESULTS: 200 lymphocytes were amplified by nested PCR. The average amplification rate of the most common 16 beta-thalassemia mutations in Chinese population was 91.3% and the average rate of allele drop out for different sites was 17.0% without differences between any 2 sites. During the 4 PGD cycles 33 embryos underwent bioassay with a success rate of 100%. 33 blastomeres were obtained to undergo PCR, of which 30 were successfully amplified with an amplification rate of 90.9%. Explicit diagnosis was obtained in 26 of the 30 embryos: 7 normal homozygotes, 11 heterozygotes, and 8 abnormal or complex heterozygotes. One or more embryos were transferred back into the uteri of the 4 women and clinical pregnancy occurred in one woman. Five weeks after the implantation B-mode ultrasonography showed monocyesis, and in the 17th week of gestational period paracentesis of cord blood showed normal homozygote. At last a normal female infant confirming the PGD result had been born, which was the first reported unaffected pregnancy resulting from PGD using multiplex nested PCR for couples as beta-thalassemia gene carriers. The results of diagnosis for embryo all corresponded to those for blastomere. The average ADO rate of blastomere was 13.3% (4/30). CONCLUSION: PGD using multiplex nested PCR, as an alternative to prenatal diagnosis, is a reliable and effective way to help couples-carriers of pathogenetic genes to get a healthy baby. SN - 0376-2491 UR - https://www.unboundmedicine.com/medline/citation/15949396/[Successful_preimplantation_genetic_diagnosis_for_beta_thalassemia_using_multiplex_nested_polymerase_chain_reaction]_ L2 - http://journal.yiigle.com/LinkIn.do?linkin_type=pubmed&issn=0376-2491&year=2005&vol=85&issue=12&fpage=811 DB - PRIME DP - Unbound Medicine ER -