Genetic Analysis in the Tanzania Sickle Surveillance Study (TS3): Modifiers of Sickle Cell Disease and Identification of Hemoglobin Variants
Authors: Smart Luke R. , Ambrose Emmanuela E. , Charles Mwesige , Hernandez Arielle G. , Latham Teresa S. , Hokororo Adolfine , Beyanga Medard , Kamugisha Erasmus , Tebuka Erius , Howard Thad A. , Ware Russell E. 
Published: February 2020

Introduction. Based on sparse historical data, Tanzania ranks fourth globally for the estimated number of annual births with sickle cell disease. Northwest Tanzania is projected to have an especially high burden of sickle cell disease, but no contemporary surveillance data exist to verify this projection. We designed a large prospective study to determine the prevalence of both sickle cell trait and sickle cell disease in northwest Tanzania. Additional objectives included an analysis of two known genetic modifiers of sickle cell disease [alpha thalassemia trait and glucose-6-phosphate dehydrogenase (G6PD) deficiency], as well as genetic variants affecting fetal hemoglobin (HbF) expression and characterization of hemoglobin variants.
Methods. The Tanzania Sickle Surveillance Study (TS3) is a prospective cross-sectional study to determine the prevalence of sickle cell trait and disease in an area projected to be at high risk. Dried blood spots (DBS) from all children born to HIV-infected mothers in the 9 regions across northwest Tanzania were collected by the HIV Early Infant Diagnosis (EID) program and transported to a central laboratory at Bugando Medical Centre, a teaching and consultancy hospital in Mwanza. DBS were tested by isoelectric focusing and scored as normal, trait, disease, or variant. Samples scored as disease or variant were retested for confirmation, and then frozen for subsequent genetic analysis. In the US, genomic DNA was extracted from each DBS, and sickle cell disease status was confirmed by PCR and TaqMan genotyping. All confirmed samples were then analyzed for alpha thalassemia trait (rightward -3.7 gene deletion) using quantitative real time PCR, and for G6PD deficiency using three real time PCR probes to distinguish A and B G6PD isoforms, to identify the G6PD A- variant, and to confirm the sex, respectively. Single nucleotide polymorphisms within 3 quantitative trait loci [BCL11A, the HBS1L-MYB intergenic polymorphism (HMIP) region, and the gamma-globin promoter XmnI site] that modify baseline HbF were analyzed using TaqMan genotyping. DBS with hemoglobin variants were investigated for the presence of common variants found in East Africa.
Results. Between February 2017 and May 2018, a total of 232 IEF gels were completed by local staff in Tanzania. Among EID samples from children &lt;24 months of age, the median age was 52 days (IQR 41-93 days), and 17,278 unique DBS samples were scored. With 20 samples uninterpretable and 54 samples with missing results, the primary analysis was performed on 17,204 samples. The overall prevalence of sickle cell trait and disease in the entire cohort was 20.3% and 1.2%, respectively, for a sickle allele frequency of 0.114 with Hardy-Weinberg equilibrium. There was some geographical variation between individual districts in both sickle trait (15.2-27.8%) and disease (0.0-4.3%). Hemoglobin variants were rare (0.1%) and included 4 Hb G-Pest, 2 Hb Kenya, and 1 Hb P-Nilotic. Of 143 DBS confirmed to have sickle cell disease and available for further testing, 61 (43%) had one-gene deletion alpha thalassemia trait and 21 (15%) had two-gene deletion. A- G6PD deficiency was detected in 19.2% of males, and 25.7% of females were heterozygous carriers. The minor allelic frequency for known modifiers of HbF were 32.2% C at rs11886868 (BCL11A), 26.6% T at rs1427407 (BCL11A), 32.5% A at rs4671393 (BCL11A), 23.8% G at rs28384513 (HMIP), 4.5% C at rs9399137 (HMIP), and 0% A at rs7482144 (XmnI).
Conclusion. The prevalence of sickle cell trait and disease among infants in northwest Tanzania is very high, exceeding 20% trait and 1.2% disease. Regional prevalence data paired with region-specific crude birth rates predict 10,055 births annually in the northwest regions, more than doubling previous estimates. Concomitant alpha thalassemia trait and G6PD deficiency are frequently co-inherited and may affect the phenotype, as well as common genetic modifiers of HbF expression. Our detailed genetic analysis of a geographically representative surveillance cohort provides a foundation for future targeted screening and the introduction of hydroxyurea for treatment of sickle cell disease in northwest Tanzania.

Disclosures
Ware: Bristol Myers Squibb: Other: Research Drug Donation; Addmedica: Other: Research Drug Donation; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Nova Laboratories: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: DSMB.