Cell-free fetal DNA as a non-invasive method using pyrosequencing in detecting beta-globin gene mutation: A pilot study from area with limited facilities in Indonesia

Abstract

BackgroundThalassemia is a monogenic, autosomal recessive, inherited disorder of the red blood cells caused by mutations or deletions in the globin gene. Approximately 6–10% of the Indonesian population carries the β-globin gene mutation; however, premarital screening is rarely conducted, and antenatal screening is optional. We explored the use of cell-free fetal DNA (cffDNA) as a potential non-invasive method of detecting the fetal β-globin gene mutation prenatally in pregnant women.Materials and methodsPregnant mothers (n = 10), who were known carriers of thalassemia and who had a history of having borne a baby with thalassemia major, and their carrier husbands (n = 4) were recruited after providing consent. EDTA blood was drawn, and maternal DNA, including cffDNA, and paternal DNA were isolated. Maternal contamination tests were conducted using the variable number tandem repeat test for ApoB and D1S80 loci. Allele quantification was performed by pyrosequencing. Known mutations from the bio-archived DNA of patients with thalassemia major (n = 16) were run alongside as a control.ResultsIn total, 7 out of 10 cffDNA successfully passed the maternal contamination test. The results of the allele quantification showed that six fetuses were predictive carriers of IVS1nt5 and one was predictive normal, in line with the allele quantification for the bio-archived DNA from patients with thalassemia major. The minimum threshold percentage for mutant A allele at cd26 was 32%, mutant T allele at IVS1nt1 was 23%, and mutant C allele at IVS1nt5 was 39%.ConclusionTaking cffDNA from the mother’s blood proved useful as a non-invasive means of detecting the β-globin gene mutation using pyrosequencing allele quantification. This non-invasive method is of great interest for prenatal diagnosis in settings with limited facilities, as it minimizes the risk of abortion. Further study of other mutations of the β-globin gene is needed.

Pharmacogenomics of Drugs Used in β-Thalassemia and Sickle-Cell Disease: From Basic Research to Clinical Applications

“…In this respect, the discovery of cell-free fetal DNA (cffDNA) in the peripheral blood of pregnant women has encouraged the research of innovative molecular protocols providing the possibility of NIPD [215]. For instance, the possibility of NIPD has been demonstrated using multiplex PCR/NGS [216], pyrosequencing [217], and droplet digital PCR [218][219][220]. In the near future, we expect that these protocols will be applied to the non-invasive prenatal identification of DNA polymorphisms associated with better outcomes of clinical treatments in β-thalassemia and SCD.…”

Noninvasive Prenatal Genetic Screening of Cell-Free Fetal DNA for Early Prediction of β-Thalassemia Using Fiber Optic Nanogold-Linked Sorbent Assay

“…7−11 Besides β-thalassemia, 12,13 the uses of NIPS for other genetic diseases, such as cystic fibrosis 14 and sickle cell disease 15,16 have also been employed. Currently, common techniques such as real-time PCR, 17,18 next-generation sequencing, 19−21 digital PCR, 22−24 and pyrosequencing 25 are applied in NIPS. Although these approaches have numerous advantages, such as high sensitivity and quantitative data, they are complicated, costly, and time-consuming.…”

“…However, it is important to note that these invasive procedures carry some risks, including a chance of miscarriage, the time-intensive nature of testing, or other complications. , Aiming to address the drawbacks associated with invasive procedures, noninvasive prenatal screening (NIPS), also known as noninvasive prenatal testing, involving the analysis of cell-free DNA (cfDNA) circulating in the pregnant mother’s blood, has been utilized in recent years. − Besides β-thalassemia, , the uses of NIPS for other genetic diseases, such as cystic fibrosis and sickle cell disease , have also been employed. Currently, common techniques such as real-time PCR, , next-generation sequencing, − digital PCR, − and pyrosequencing are applied in NIPS. Although these approaches have numerous advantages, such as high sensitivity and quantitative data, they are complicated, costly, and time-consuming.…”