Causes and Consequences of Copy Number Variation of the Human Glycophorin Gene Cluster

Human glycophorin proteins expressed on the surface of erythrocytes, and are receptors for invasion of the Plasmodium falciparum parasite, which causes malaria in sub-Saharan Africa. The proteins are encoded by the genes GYPA and GYPB which, together with GYPE, reside on a tandemly-duplicated repeat region on chromosome 4q31.21. Sequence read depth data of the 1000 Genome Project were used to determine the glycophorin variants. The positive control samples of eight variants were Sanger sequenced and the breakpoints of them were identified and analysed, DEL1, DEL2, DEL6, DEL7, DUP14, DUP29, DUP5 and the gene conversion. In this thesis, paralogue ratio test (PRT) assays were developed to type CNV of the glycophorin gene regions in the Benin malaria cohort (n=563), and an allele-specific PCR assay to genotype alleles of the novel SNP (rs186873296), which is related to resistance to severe malaria in the same malaria cohort. This showed that absent of the GYPB is not associated with the Benin malaria cohort phenotypes. In addition, rs186873296 SNP is not in strong linkage disequilibrium (LD) with the GYPB deletion in this malaria cohort. Previous genome-wide analysis has shown that a structural variant within this region (DUP4), is identical to the blood group antigen Dantu NE+, and confers a clinically-important protective effect, is common in East Africans and is strongly protective against severe malaria. DUP4 is a complex structural genomic variant that carries hybrid (GYPA/GYPB) fusion genes. Using fibre-FISH, we validate the structural arrangement of the glycophorin locus in the DUP4 variant, and provide evidence of somatic variation in the number of GYPA/GYPB fusion genes. Subsequently, we have developed a paralogue-specific junction fragment PCR to genotype DUP4. We demonstrate association of DUP4 variant with haemoglobin levels - a phenotype related to malaria - in 962 DNA samples from a Tanzanian village holoendemic for malaria using a family-based association test. Using the family-based association approach implemented in (QTDT), we have found a statistically significant association of the DUP4 variant with haemoglobin levels (p=0.0054). This thesis confirms the importance of the DUP4 variant in malaria protection, and raises the intriguing possibility of heightened somatic instability and somatic mosaicism at this locus in DUP4 carriers, which might confer added protection against malaria.