Tags

Type your tag names separated by a space and hit enter

Detection of SEA-type α-thalassemia in embryo biopsies by digital PCR.
Taiwan J Obstet Gynecol. 2017 Aug; 56(4):487-494.TJ

Abstract

OBJECTIVE

Accurate and efficient pre-implantation genetic diagnosis (PGD) based on the analysis of single or oligo-cells is needed for timely identification of embryos that are affected by deleterious genetic traits in in vitro fertilization (IVF) clinics. Polymerase chain reaction (PCR) is the backbone of modern genetic diagnoses, and a spectrum of PCR-based techniques have been used to detect various thalassemia mutations in prenatal diagnosis (PND) and PGD. Among thalassemias, SEA-type α-thalassemia is the most common variety found in Asia, and can lead to Bart's hydrops fetalis and serious maternal complications.

MATERIALS AND METHODS

To formulate an efficient digital PCR for clinical diagnosis of SEA-type α-thalassemia in cultured embryos, we conducted a pilot study to detect the α-globin and SEA-type deletion alleles in blastomere biopsies with a highly sensitive microfluidics-based digital PCR method. Genomic DNA from embryo biopsy samples were extracted, and crude DNA extracts were first amplified by a conventional PCR procedure followed by a nested PCR reaction with primers and probes that are designed for digital PCR amplification.

RESULTS

Analysis of microfluidics-based PCR reactions showed that robust signals for normal α-globin and SEA-type deletion alleles, together with an internal control gene, can be routinely generated using crude embryo biopsies after a 106-fold dilution of primary PCR products.

CONCLUSION

The SEA-type deletion in cultured embryos can be sensitively diagnosed with the digital PCR procedure in clinics. The adoption of this robust PGD method could prevent the implantation of IVF embryos that are destined to develop Bart's hydrops fetalis in a timely manner. The results also help inform future development of a standard digital PCR procedure for cost-effective PGD of α-thalassemia in a standard IVF clinic.

Authors+Show Affiliations

Institute of Biomedical Science, Chang Gung University, No. 259, Wenhua 1st Road, Guishan Township, Taoyuan City 33302, Taiwan.Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital Linkou Medical Center, Chang Gung University, 5 Fu-Shin Street, Gueishan Township, Taoyuan County 333, Taiwan.Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital Linkou Medical Center, Chang Gung University, 5 Fu-Shin Street, Gueishan Township, Taoyuan County 333, Taiwan. Electronic address: amego@cgmh.org.tw.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28805606

Citation

Lee, Ta-Hsien, et al. "Detection of SEA-type Α-thalassemia in Embryo Biopsies By Digital PCR." Taiwanese Journal of Obstetrics & Gynecology, vol. 56, no. 4, 2017, pp. 487-494.
Lee TH, Hsu YC, Chang CL. Detection of SEA-type α-thalassemia in embryo biopsies by digital PCR. Taiwan J Obstet Gynecol. 2017;56(4):487-494.
Lee, T. H., Hsu, Y. C., & Chang, C. L. (2017). Detection of SEA-type α-thalassemia in embryo biopsies by digital PCR. Taiwanese Journal of Obstetrics & Gynecology, 56(4), 487-494. https://doi.org/10.1016/j.tjog.2017.03.002
Lee TH, Hsu YC, Chang CL. Detection of SEA-type Α-thalassemia in Embryo Biopsies By Digital PCR. Taiwan J Obstet Gynecol. 2017;56(4):487-494. PubMed PMID: 28805606.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Detection of SEA-type α-thalassemia in embryo biopsies by digital PCR. AU - Lee,Ta-Hsien, AU - Hsu,Ya-Chiung, AU - Chang,Chia Lin, PY - 2017/03/07/accepted PY - 2017/8/15/entrez PY - 2017/8/15/pubmed PY - 2018/5/10/medline KW - Digital PCR KW - Embryo biopsy KW - IVF KW - SEA-Type KW - α-thalassemia SP - 487 EP - 494 JF - Taiwanese journal of obstetrics & gynecology JO - Taiwan J Obstet Gynecol VL - 56 IS - 4 N2 - OBJECTIVE: Accurate and efficient pre-implantation genetic diagnosis (PGD) based on the analysis of single or oligo-cells is needed for timely identification of embryos that are affected by deleterious genetic traits in in vitro fertilization (IVF) clinics. Polymerase chain reaction (PCR) is the backbone of modern genetic diagnoses, and a spectrum of PCR-based techniques have been used to detect various thalassemia mutations in prenatal diagnosis (PND) and PGD. Among thalassemias, SEA-type α-thalassemia is the most common variety found in Asia, and can lead to Bart's hydrops fetalis and serious maternal complications. MATERIALS AND METHODS: To formulate an efficient digital PCR for clinical diagnosis of SEA-type α-thalassemia in cultured embryos, we conducted a pilot study to detect the α-globin and SEA-type deletion alleles in blastomere biopsies with a highly sensitive microfluidics-based digital PCR method. Genomic DNA from embryo biopsy samples were extracted, and crude DNA extracts were first amplified by a conventional PCR procedure followed by a nested PCR reaction with primers and probes that are designed for digital PCR amplification. RESULTS: Analysis of microfluidics-based PCR reactions showed that robust signals for normal α-globin and SEA-type deletion alleles, together with an internal control gene, can be routinely generated using crude embryo biopsies after a 106-fold dilution of primary PCR products. CONCLUSION: The SEA-type deletion in cultured embryos can be sensitively diagnosed with the digital PCR procedure in clinics. The adoption of this robust PGD method could prevent the implantation of IVF embryos that are destined to develop Bart's hydrops fetalis in a timely manner. The results also help inform future development of a standard digital PCR procedure for cost-effective PGD of α-thalassemia in a standard IVF clinic. SN - 1875-6263 UR - https://www.unboundmedicine.com/medline/citation/28805606/Detection_of_SEA_type_α_thalassemia_in_embryo_biopsies_by_digital_PCR_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1028-4559(17)30145-6 DB - PRIME DP - Unbound Medicine ER -