Prenatal Diagnosis of Hemoglobinopathies by Pyrosequencing: A More Sensitive and Rapid Approach to Fetal Genotyping

Abstract

Prenatal diagnosis of the hemoglobinopathies by fetal DNA analysis is currently performed in most countries, either by DNA sequencing, restriction enzyme polymerase chain reaction (RE-PCR) or the amplification refractory mutation system (ARMS). These methods are time consuming and prolong the turnaround time for diagnosis. We here describe a method utilizing pyrosequencing for the prenatal diagnosis of 12 common nondeletional α- and β-globin gene mutations in the UK population. In particular, it replaced the diagnosis of sickle cell disease by RE-PCR and for the diagnosis of β-thalassemia (β-thal) by Sanger DNA sequencing. We have genotyped 148 chorionic villi and 29 uncultured amniotic fluid DNA samples by pyrosequencing and found 100% concordance with the fetal diagnosis result obtained by ARMS-PCR or DNA sequencing. Pyrosequencing was more robust, revealing an 83% decrease in diagnostic failures using uncultured amniocyte DNA samples, and also quantitative, revealing one case of allelic imbalance due to maternal DNA contamination. Overall, we found pyrosequencing to be simpler, more robust, quicker, and less expensive than conventional sequencing and RE-PCR, making it a good choice for rapid and cost-effective prenatal diagnosis of thalassemia and sickle cell disease.

Prenatal Molecular Diagnosis of β-Thalassemia and Sickle Cell Anemia in the Syrian Population

“…It has been actively pursued in many countries, such as Cyprus (23), China (24), Taiwan (25), Iran (26), Turkey (2,27), USA (28), UK (29) and Australia (30). Prenatal diagnosis had a great success in decreasing the number of live births with b-thal major (b-TM) in these countries.…”

Rapid prenatal diagnosis of common beta‐thalassemia mutations in Southeast Asia using pyrosequencing

“…Several molecular diagnostic methods have been developed to detect rare mutations as well as beta‐thalassemia mutations that are prevalent in specific ethnic groups. These include single strand conformation polymorphism, denaturing gradient gel electrophoresis, denaturing high performance liquid chromatography, restriction fragment length polymorphism, amplification refractory mutation system, direct Sanger sequencing, reverse dot blot hybridization, melting curve analysis and more recently, pyrosequencing . Pyrosequencing is a real‐time sequencing‐by‐synthesis method that enables rapid single nucleotide polymorphisms (SNPs) analysis of short DNA sequences.…”

“…These include single strand conformation polymorphism, 7 denaturing gradient gel electrophoresis, 8 denaturing high performance liquid chromatography, 9 restriction fragment length polymorphism, 10 amplification refractory mutation system, 11,12 direct Sanger sequencing, 13,14 reverse dot blot hybridization, 15,16 melting curve analysis 17,18 and more recently, pyrosequencing. 19,20 Pyrosequencing is a real-time sequencing-by-synthesis method that enables rapid single nucleotide polymorphisms (SNPs) analysis of short DNA sequences. In pyrosequencing, the annealing of a dispensed nucleotide with the template during synthesis will cause the release of a pyrophosphate, which is converted to fluorescence emission through a series of enzymatic reactions.…”

“…The fluorescence emission is measured to determine the amounts of synthesized nucleotide molecules, hence, determining the sequence of the template. Timbs et al 19 recently described the use of pyrosequencing in the prenatal diagnosis of 12 common nondeletional alpha-globin and beta-globin gene mutations in the UK population. 19 We aim to develop a rapid and sensitive prenatal diagnostic assay for the detection of 27 most common nondeletional and deletional beta-thalassemia mutations in Singapore by using pyrosequencing.…”

“…The fluorescence emission is measured to determine the amounts of synthesized nucleotide molecules, hence, determining the sequence of the template. Timbs et al . recently described the use of pyrosequencing in the prenatal diagnosis of 12 common nondeletional alpha‐globin and beta‐globin gene mutations in the UK population .…”