Semi-nested Taqman real-time quantitative PCR for noninvasive prenatal diagnosis of Bart's hydrops fetalis.

Abstract

BACKGROUND:

Non-invasive prenatal diagnosis based on detection of fetal cell-free DNA is limited when mother and father are both carriers for the same autosomal recessive mutation.

OBJECTIVE:

Develop the semi-nested Taqman real-time PCR for quantification of alpha-thalassemia-1 SEA type deletion allele in plasma of alpha-thalassemia-1 SEA carriage pregnancies.

MATERIAL AND METHOD:

Plasma DNA was extracted from six women who carried fetuses with normal, 11 with heterozygote alpha-thalassemia-1 SEA type deletion and seven with Bart's hydrops fetalis. DNA was amplified using conventional PCR with the primary specific primer set for alpha-thalassemia-1 SEA type deletion. PCR product was then subjected to the semi-nested real-time PCR using the secondary specific primer and Taqman probe set for alpha-thalassemia-1 SEA type deletion. The standard curve was constructed using ten-fold serial dilutions of conventional PCR product of the heterozygote alpha-thalassemia-1 SEA type deletion.

RESULTS:

Women who carried fetuses with Bart's hydrops fetalis displayed a trend toward higher mean copy number of alpha-thalassemia-1 SEA type deletion allele vs. women who carried fetuses with normal and heterozygote, albeit not reaching statistical significance.

CONCLUSION:

The maternally inheritedfetal allele present in maternal plasma is difficult to discern the fetal cell-free DNA from a higher background DNA of the mother Thus, further investigation is needed to improve the diagnosis ofBart's hydrops fetalis using this technique.

• Authors: Sakorn Pornprasert, Kanyakan Sukunthamala, Naowarat Kunyanone, Sririchai Sittiprasert, Khanungnit Thungkham, Sumeth Junorse, Khachonsilp Pongsawatkul, Wisut Pattanaporn, Chantip Jitwong
• Journal: Journal of the Medical Association of Thailand = Chotmaihet thangphaet (J Med Assoc Thai) Vol. 95 Issue 1 Pg. 6-9 (Jan 2012) ISSN: 0125-2208 [Print] Thailand
• PMID: 22379734 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Topics

• Adult
• Alleles
• Diagnosis, Differential
• Female
• Humans
• Hydrops Fetalis (diagnosis, genetics)
• Pregnancy
• Prenatal Diagnosis (methods)
• Real-Time Polymerase Chain Reaction (methods)
• alpha-Thalassemia (blood, genetics)