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

Ho, S.; Huan, Pei Tee; Leow, Gek Har; Ching, Leng Kee; Chiu, Lily; Law, Hai Yang; Koay, Evelyn Siew Chuan

doi:10.1002/pd.4183

Cited by 2 publications

(2 citation statements)

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“…However, these techniques are labor intensive and time consuming. The use of pyrosequencing allele quantification for non-invasive prenatal diagnosis might be a solution in areas where simple, quick, and inexpensive techniques are needed, especially in areas where thalassemia is prevalent ( 22 ).…”

Section: Discussionmentioning

confidence: 99%

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

Maskoen

Rahayu²,

Laksono³

et al. 2022

Front. Pediatr.

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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.

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Section: Discussionmentioning

confidence: 99%

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

Maskoen

Rahayu²,

Laksono³

et al. 2022

Front. Pediatr.

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“…However, pyrosequencing has strict requirements on the concentration of DNA template, and the presence of maternal cell contamination can interfere with interpretation of results, particularly when the fetus carries the same mutation as the mother. [24] Murad et al used molecular screening and direct DNA sequencing to evaluate a prenatal diagnosis of β-thalassemia by collecting 55 amniotic fluid samples in Syria. [25] In another study fetal cells were isolated using micromanipulators at 8 weeks of gestation in women with high-risk pregnancies and nested polymerase chain reactions were performed.…”

Section: Discussionmentioning

confidence: 99%

Whole-exome sequencing analysis of amniotic fluid cells in 5 pregnant women with thalassemia: Case report

Zhang

Li²,

et al. 2022

Medicine

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Rationale: While thalassemia is a monogenic disease that is relatively common worldwide, there is no recognized radical cure for thalassemia in current medical practice. Prenatal diagnosis is the most important contribution to thalassemia prevention, but due to its technical limitations, rare thalassemia mutations cannot be detected; and the birth of thalassemic babies cannot be completely circumvented. Whole-exome sequencing can, however, compensate for this shortcoming. Patient concerns: We report the results of whole exon sequencing of amniotic cells in 5 pregnant women with thalassemia. Diagnosis: Prenatal diagnosis revealed that 4 of them were α thalassemia carriers and 1 of them was β thalassemia carrier. Interventions and Outcomes: We collected amniotic fluid of 5 pregnant women (age range: 25–27 years, Mean ± SD: 28 ± 1.8) with thalassemia. The gestational ages ranged between 16 and 19 weeks. The cells were separated from the amniotic fluid and passaged until a sufficient number of cells were obtained for exome sequencing. We therefore employed whole-exome sequencing of amniotic fluid cells from thalassemic carriers to validate prenatal diagnostic results and to identify novel mutation sites. We found that 4 of 5 samples are SEA which is consistent with the clinical prenatal diagnosis. However, 2 of 5 samples were point mutations in the HBB gene, and were thus different from the clinical prenatal diagnosis. Conclusion: The identifications from this study showed that prenatal diagnosis has limitations. Whole-exome sequencing can compensate for this shortcoming. And this study would add new insights into understanding of molecular mechanisms in thalassemia.

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

Abstract: Pyrosequencing is a rapid and sensitive method to detect common beta-thalassemia mutations without the need for cell culture, thus reducing the assay turnaround time.

Cited by 2 publications

References 20 publications

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

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

Whole-exome sequencing analysis of amniotic fluid cells in 5 pregnant women with thalassemia: Case report

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