Standardization of the endpoint PCR for identification of β-globin gene haplotypes associated with sickle cell anemia in Neonatal Screening patients, Panama.

Abstract

Sickle cell anemia is an inherited hemoglobinopathy characterized by the presence of hemoglobin S (HbS), which is estimated to affect approximately 0.04% of the Panamanian population. Its clinical severity is modulated by the haplotypes of the β-globin gene—Benin, Bantu, Senegal, Cameroon, and Arab-Hindu—whose characterization is not possible using high-performance liquid chromatography (HPLC) as employed in the Neonatal Screening Program at Dr. José Renán Equivel Children’s Hospital. The objective of this study was to standardize the endpoint PCR technique for the identification of these haplotypes in 22 patients with confirmed HbSS, corresponding to the period January 2020–August 2025. Genomic DNA was extracted using the PureLink kit (Invitrogen), and six specific regions of the β-globin gene were amplified. The products were digested with the enzymes DdeI, HindIII, HincII, and HinfI, and visualized on 2% agarose gels. Three haplotypes were identified: Senegal (45.5%), Bantu (40.9%), and Benin (13.6%); the Cameroon and Arab-Hindu haplotypes were not detected. The standardized protocol proved to be reproducible and reliable, constituting a complementary molecular tool for the subclassification of patients with HbSS.