Development of the Next Generation Sequencing-Based Diagnostic Test for β-Thalassemia and its Validation in a Pashtun Family

Abstract

β-Thalassemia (β-thal) is a common monogenic disease with ethnic-specific mutations on the HBB gene throughout the world. The reported mutations either reduce the expression or completely inactivate the HBB gene. In Pakistan, the prevalence of β-thal is high due to consanguineous marriages. Accurate identification of mutations in carriers is imperative for prevention of β-thal in subsequent generations. To overcome the limitations of traditional testing methods for β-thal, a next-generation sequencing (NGS)-based diagnostic test was designed and validated by sequencing the entire HBB gene. The primer set covering the entire HBB gene was designed and validated in a Pashtun β-thalassemic family. The polymerase chain reaction (PCR) product was sequenced using an Illumina MiSeq platform. A homozygous pathogenic insertion of A>AC/AC (rs35699606) was detected in an affected member of the family, while unaffected members were heterozygous for it. In addition, all family members were homozygous for the synonymous variant, A>G/G (rs713040), except the father who was heterozygous for it. We sequenced the entire HBB gene using the NGS-based test, which is highly sensitive, robust and specific for the diagnosis and screening of β-thal in Pakistan, especially for families practicing consanguineous marriages.

Keywords:

• β-Thalassemia (β-thal)
• genetic variation
• HBB
• next-generation sequencing (NGS)

Acknowledgments

The authors thank the β-thalassemic family for their participation in this study.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

We thank the Rehman Medical Institute, Peshawar, KP, Pakistan for their financial support.