Combined Gap-Polymerase Chain Reaction and Targeted Next-Generation Sequencing Improve α- and β-Thalassemia Carrier Screening in Pregnant Women in Vietnam

Abstract

Vietnam has a high thalassemia burden. We collected blood samples from 5880 pregnant Vietnamese women during prenatal health checks to assess thalassemia carrier frequency using combined gap-polymerase chain reaction (gap-PCR) and targeted next-generation sequencing (NGS). Thalassemia carriers were identified with prevalence of 13.13% (772), including 7.82% (460) carriers of α-thalassemia (α-thal), 5.31% (312) carriers of β-thalassemia (β-thal), and 0.63% (37) concurrent α-/β-thal carriers. Deletional mutations (368) accounted for 80.0% of α-thal carriers, of which, ––^SEA (Southeast Asian) (n = 254; 55.0%) was most prevalent, followed by the –α^3.7 (rightward) (n = 66; 14.0%) and –α^4.2 (leftward) (n = 45; 9.8%) deletions. Hb Westmead (HBA2: c.369C>G) (n = 53) and Hb Constant Spring (Hb CS or HBA2: c.427T>C) (in 28) are the two most common nondeletional α-globin variants, accounting for 11.5 and 6.0% of α-thal carriers. We detected 11 different β-thal genotypes. Hb E (HBB: c.79G>A) (in 211) accounted for 67.6% of β-thal carriers. The most common β-thal genotypes were associated with mutations at codon 17 (A>T) (HBB: c.52A>T), codons 41/42 (–TTCT) (HBB: c.126_129delCTTT), and codon 71/72 (+A) (HBB: c.217_218insA) (prevalence 0.70%, 0.68%, and 0.2%, respectively). Based on mutation frequencies calculated in this study, estimates of 5021 babies in Vietnam are affected with clinically severe thalassemia annually. Our data suggest a higher thalassemia carrier frequency in Vietnam than previously reported. We established that combining NGS with gap-PCR creates an effective large-scale thalassemia screening method that can detect a broad range of mutations.

Keywords:

• α- and β-Globin gene
• gap-polymerase chain reaction (gap-PCR)
• next-generation sequencing (NGS)
• thalassemia

Acknowledgements

The authors thank all participants and their families who agreed to take part in this study, all the clinics and hospitals that assisted inpatient consultation and sample collection, and the laboratory technicians at the Medical Genetics Institute at Ho Chi Minh City, Vietnam, for performing the assays. They thank Angela Jansen, PhD, MHS of Angela Jansen and Associates (Laguna Beach, CA, USA), for her editorial services in preparing the manuscript for publication.

Author contributions

Doan-Tu Nguyen, Quang Thanh Le, Duy-Anh Nguyen, Diem-Tuyet Thi Hoang, Huu Du Nguyen, Canh Chuong Nguyen, Kim Phuong Thi Doan, Nhat Thang Tran, Thi-Minh-Thi Ha, Thu Huong Nhat Trinh, Van Thong Nguyen, Duc Tam Lam, Minh Tam Le, Xuan Thao Nguyen, Thu-Hang Thi Ho, Trung Hoanh Tran, Viet Thang Ho, Thanh Van Bui, Van Trong Nguyen, Phuoc Ba Hoang, Hoai Thanh Nguyen, Manh Hoan Nguyen, Thanh-Thanh Thi Nguyen, Bich-Ngoc Thi Huynh, Thanh-Phuong Thi Nguyen, Kim-Van Thi Tran, and Cong-Trai Nguyen recruited patients, performed clinical analysis and provided critical appraisal of the manuscript. Thanh-Binh Vo, Duy-Khang Nguyen Le, Thao Ngoc Truong, Hong-Thuy Thi Dao, Phuong-Anh Ngoc Vo, Thien-Chi Van Nguyen, Ngoc-Nhu Thi Tran, Quynh-Nhu Thi Tran, Yen-Linh Thi Van, Tuan-Thanh Lam, Phuoc-Loc Doan, Thanh-Dat Nguyen, Thanh-Thuy Thi Do, Dinh-Kiet Truong, Hung Sang Tang, Ngoc-Phuong Thi Cao, Minh-Duy Phan, Hoa Giang, and Hoai-Nghia Nguyen conceptualized study design, designed experiments. Ngoc-Phuong Thi Cao, Minh-Duy Phan, Hoa Giang, Phuoc-Loc Doan, and Thanh-Dat Nguyen developed bioinformatics and simulation algorithms. Tuan-Thanh Lam performed data clean-up, formal analysis and wrote the manuscript. Hoai Nghia Nguyen supervised the project.

Disclosure statement

Thanh-Thanh Thi Nguyen, Bich-Ngoc Thi Huynh, Thanh-Phuong Thi Nguyen, Kim-Van Thi Tran, Cong-Trai Nguyen, Thanh-Binh Vo, Duy-Khang Nguyen Le, Thao Ngoc Truong, Hong-Thuy Thi Dao, Phuong-Anh Ngoc Vo, Thien-Chi Van Nguyen, Ngoc-Nhu Thi Tran, Quynh-Nhu Thi Tran, Yen-Linh Thi Van, Tuan-Thanh Lam, Phuoc-Loc Doan, Thanh-Dat Nguyen, Hung Sang Tang, Ngoc-Phuong Thi Cao, Minh-Duy Phan, and Hoa Giang are currently employees of Gene Solutions Vietnam. The other authors declare no conflict of interests. The authors alone are responsible for the content and writing of this article.

Data availability statement

The data that support the findings of this study are available from the corresponding author, Hoai-Nghia Nguyen, upon reasonable request.

Additional information

Funding

This study was funded by Gene Solutions, Vietnam [Grant ID: GS004]. They confirm that the funder Gene Solutions provided support in the form of salaries for authors Thanh-Thanh Thi Nguyen, Bich-Ngoc Thi Huynh, Thanh-Phuong Thi Nguyen, Kim-Van Thi Tran, Cong-Trai Nguyen, Thanh-Binh Vo, Duy-Khang Nguyen Le, Thao Ngoc Truong, Hong-Thuy Thi Dao, Phuong-Anh Ngoc Vo, Thien-Chi Van Nguyen, Ngoc-Nhu Thi Tran, Quynh-Nhu Thi Tran, Yen-Linh Thi Van, Tuan-Thanh Lam, Phuoc-Loc Doan, Thanh-Dat Nguyen, Hung Sang Tang, Ngoc-Phuong Thi Cao, Minh-Duy Phan, and Hoa Giang but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are in the ‘author contributions’ section.