Genetic Analyses in the Uganda Sickle Surveillance Study: Modifiers of Sickle Cell Anemia and Identification of Hemoglobin Variants

Introduction: The Uganda Sickle Surveillance Study (US3) analyzed almost 100,000 dried blood spots (DBS) collected from infants and toddlers, with the primary objective of determining the distribution and burden of sickle cell disease and trait in the Republic of Uganda. The recently published results document a high prevalence of both sickle trait (13.3%) and disease (0.7%) with non-uniform distribution across the country (Ndeezi et al, Lancet Global Health 2016). The prevalence of established genetic modifiers of sickle cell anemia in Uganda including α-thalassemia, β-globin haplotype, and G6PD deficiency is currently unknown. In addition, isoelectric focusing electrophoresis (IEF) revealed numerous hemoglobin variants of uncertain significance, with an overall prevalence of 0.5% warranting further investigation.

Methods: De-identified DBS samples identified as Disease or Variant on IEF were transported to Cincinnati Children's Hospital for analysis, with oversight from an IRB-approved protocol at both CCHMC and Makerere University. Using genomic DNA isolated from two 3mm punches, Disease samples were tested for genetic modifiers using a combination of PCR-based techniques: HbS allele (rs334) by TaqMan RT-PCR analysis; G6PD A^- by 3 TaqMan RT-PCR analyses for G202A (rs1050829) to distinguish A and B isoforms followed by A376G (rs1050828) to identify the A^- variant, and gender (SRY_VIC, ABCD1_CCHS0H-FAM); β-globin haplotype by PCR, restriction fragment length polymorphism (RFLP), and Taq Man RT-PCR for the classic DNA polymorphisms [XmnI (HBG2), rs7482144; HindIII ^Gγ (HBG2), rs2070972; HindIII ^Aγ (HBG1), rs28440105; HincII ψβ (HBBP1), rs10128556; HincII δ (HBD), rs968857; AvaII (HBB), rs10768683; HincII ε (ΗΒΕ1), rs3834466]; and deletional -α^3.7-thalassemia using a copy-number variant RT-PCR assay with probes inside and outside the α-globin region (Hs03947236_cn, HbA_Tri_CCPAD0C). Hemoglobin variants were identified using a combination of PCR amplification of alpha, beta, gamma, and delta globin exons followed by Sanger sequencing; after missense mutations and fusion genes were documented, rapid and specific PCR-based diagnostic techniques were developed using RFLP and RT-PCR.

Results: A total of 164 DBS samples with confirmed sickle cell anemia were found to have the following prevalence of genetic modifiers: G6PD A^- deficiency (N=160) in 6.6% of males and 1.2% of females for an overall A^-allele frequency of 15.7%; β-globin haplotypes (N=156) were Central African Republic (CAR) in 91.6% of alleles, along with a small number of Cameroon (5.1%), Benin (2.2%), or Atypical (1.0%) haplotypes; deletional α-thalassemia (N=150) had an overall prevalence of 44.6% with one-gene deletion in 38.6% and two gene-deletion in 6.0% of samples.

Subsequent DNA-based investigation of 190 DBS samples with hemoglobin variants identified five common patterns: two α-globin variants (Hb Stanleyville II, Asn78Lys and Hb G-Pest, Asp74Asn), one β-globin variant (Hb O-Arab, Glu121Lys), and two fusion globin variants (Hb P-Nilotic, β31-δ50 and Hb Kenya, ^Aγ81Leu-β86Ala). Hb Stanleyville II was identified in 30% of the variant samples, Hb Kenya in 27%, Hb P-Nilotic in 21%, Hb G-Pest in 10% and Hb O-Arab in 1%; an additional 6% remain unidentified by sequence or IEF pattern. Compound heterozygotes with Hb S were identified with Hb O-Arab and Hb Kenya. Geospatial mapping of Hb variants document a non-uniform distribution with geographic clustering: for example Hb Kenya was concentrated in the Eastern Regions and around Lake Victoria, Hb P-Nilotic was found primarily in Northern Regions, while Hb Stanleyville II and Hb G-Pest were identified across the country.

Conclusions: Genetic analyses of DBS collected in US3 reveal important findings that are relevant to the country's nascent newborn screening efforts. First, there is a high prevalence of known genetic modifiers, including >40% alpha thalassemia trait, >90% CAR haplotype, and >6% of males with G6PD deficiency. Second, analysis of five common IEF hemoglobin variants identified three missense mutations and two fusion proteins; recognition of their IEF patterns and development of PCR-based diagnostic algorithms will allow their correct identification. As newborn screening programs expand in Uganda and across sub-Saharan Africa, these results will be valuable adjuncts to standard IEF documentation of sickle cell trait and disease.