Validating Laboratory Accuracy of the Next Generation Sequencing (NGS) Technology As a Newborn Screening Tool for Hemoglobinopathies and Thalassemia in Southeast Asian Population

Background

Thalassemia and hemoglobinopathies are one of the most important health burden to Thailand and several Southeast Asian countries due to a very high frequency of carriers of these globin disorders in the region. Although, Thailand as one of the leading countries on thalassemia research has established a national program for prevent and control of severe thalassemia syndrome at ante-natal screening follows by prenatal diagnosis in pregnant women. However, such program has several limitations due to a late antenatal care and a lack of public awareness. Early screening at neonatal levels together with data incorporated within our Thailand's national health care database (eGovernment) can improve the national thalassemia control program in a long-term run.

Aims

To evaluate the efficacy and accuracy of using next generation sequencing (NGS) technology as a fast, high through-put and scalable method for thalassemia screening in Southeast Asian setting.

Methods

Newborn peripheral blood (heel prick) were collected for hematologic testing including complete blood count and hemoglobin (Hb) typing. Also, genomic DNA (gDNA) was extracted and then analyzed using conventional multiplex PCR methods including α-GAP multiplex PCR, α-ARMS multiplex PCR and β-ARMS multiplex PCR, which covered > 95% of common mutations of globin genes found in the Thai population. All samples were then analyzed for sequences of α-globin (HBA1 and HBA2) and β-globin (HBB) genes using NGS technology and the combinatorial probe-anchor synthesis sequencing method (BGI VISTA^TM Thalassemia-Seq panel) and detection by software provided by BGI Biotechnology, Wuhan.

Results

Based on the comparative analysis of α-globin and β-globin gene mutations. The 352 neonatal gDNA samples by multiplex PCR methods and combinatorial probe-anchor synthesis sequencing methods, 335 samples (95%) showed identical results on globin gene mutations. However, the NGS approach has a superior on identification several rare but possible clinical significance variants such as Hb Westmead (2.6%), α-triplication (ααα^anti3.7, 0.3%) and ααα^anti3.7/–^SEA (0.3%) that were missed using the conventional methods.

Conclusion

Application of NGS technology to detect globin gene mutations that causes thalassemia and hemoglobinopathies showed a promising accurate technology for the future newborn screening program in Thailand and other Southeast Asian countries.

No relevant conflicts of interest to declare.