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Background Approximately 5% of the human genome shows common structural variation, which is enriched for genes involved in the immune response and cell-cell interactions. A well-established region of extensive structural variation is the glycophorin gene cluster, comprising three tandemly-repeated regions about 120 kb in length and carrying the highly homologous genes GYPA , GYPB and GYPE . Glycophorin A (encoded by GYPA ) and glycophorin B (encoded by GYPB ) are glycoproteins present at high levels on the surface of erythrocytes, and they have been suggested to act as decoy receptors for viral pathogens. They are receptors for the invasion of the protist parasite Plasmodium falciparum, a causative agent of malaria. A particular complex structural variant, called DUP4, creates a GYPB-GYPA fusion gene known to confer resistance to malaria. Many other structural variants exist across the glycophorin gene cluster, and they remain poorly characterised. Results Here, we analyse sequences from 3234 diploid genomes from across the world for structural variation at the glycophorin locus, confirming 15 variants in the 1000 Genomes project cohort, discovering 9 new variants, and characterising a selection of these variants using fibre-FISH and breakpoint mapping at the sequence level. We identify variants predicted to create novel fusion genes and a common inversion duplication variant at appreciable frequencies in West Africans. We show that almost all variants can be explained by non-allelic homologous recombination and by comparing the structural variant breakpoints with recombination hotspot maps, confirm the importance of a particular meiotic recombination hotspot on structural variant formation in this region. Conclusions We identify and validate large structural variants in the human glycophorin A-B-E gene cluster which may be associated with different clinical aspects of malaria.

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.

Approximately 5% of the human genome consists of structural variants, which are enriched for genes involved in the immune response and cell-cell interactions. A well-established region of extensive structural variation is the glycophorin gene cluster, comprising three tandemly-repeated regions about 120kb in length, carrying the highly homologous genes GYPA, GYPB and GYPE. Glycophorin A and glycophorin B are glycoproteins present at high levels on the surface of erythrocytes, and they have been suggested to act as decoy receptors for viral pathogens. They act as receptors for invasion of a causative agent of malaria, Plasmodium falciparum. A particular complex structural variant (DUP4) that creates a GYPB/GYPA fusion gene is known to confer resistance to malaria. Many other structural variants exist, and remain poorly characterised. Here, we analyse sequences from 6466 genomes from across the world for structural variation at the glycophorin locus, confirming 15 variants in the 1000 Genomes project cohort, discovering 9 new variants, and characterising a selection using fibre-FISH and breakpoint mapping. We identify variants predicted to create novel fusion genes and a common inversion duplication variant at appreciable frequencies in West Africans. We show that almost all variants can be explained by unequal cross over events (non-allelic homologous recombination, NAHR) and by comparing the structural variant breakpoints with recombination hotspot maps, show the importance of a particular meiotic recombination hotspot on structural variant formation in this region. Footnotes * Correction to institutional affiliation of Mayana Zatz

Structural variation in the human genome can affect risk of disease. An example is a complex structural variant of the human glycophorin gene cluster, called DUP4, which is associated with a clinically-significant level of protection against severe malaria. The human glycophorin gene cluster harbours at least 23 distinct structural variants and accurate genotyping of this complex structural variation remains a challenge. Here, we use a PCR-based strategy to genotype structural variation at the human glycophorin gene cluster. We validate our approach, based on a triplex paralogue ratio test (PRT) combined with junction-fragment specific PCR, on publically-available samples from the 1000 Genomes project. We then genotype a longitudinal birth cohort using small amounts of DNA at low cost. Our approach readily identifies known deletions and duplications, and can potentially identify novel variants for further analysis. It will allow exploration of genetic variation at the glycophorin locus, and investigation of its relationship with malaria, in large sample sets at minimal cost, using standard molecular biology equipment.

Glycophorin A and glycophorin B are red blood cell surface proteins that are both receptors for the parasite Plasmodium falciparum, which is the principal cause of malaria in sub-Saharan Africa. DUP4 is a complex structural genomic variant that carries extra copies of a glycophorin A - glycophorin B fusion gene, and has a dramatic effect on malaria risk by reducing the risk of severe malaria by up to 40%. Using fiber-FISH and Illumina sequencing, we validate the structural arrangement of the glycophorin locus in the DUP4 variant, and reveal somatic variation in copy number of the glycophorin A- glycophorin B fusion gene. By developing a simple, specific, PCR-based assay for DUP4 we show the DUP4 variant reaches a frequency of 13% in a village in south-eastern Tanzania. We genotype a substantial proportion of that village and demonstrate an association of DUP4 genotype with hemoglobin levels, a phenotype related to malaria, using a family-based association test. Taken together, we show that DUP4 is a complex structural variant that may be susceptible to somatic variation, and show that it is associated with a malarial-related phenotype in a non-hospitalized population. Footnotes * Some minor changes clarifying the origin of the HG02554 cells and DNA, references to previous work, and the previously known DUP4 allele frequency in Tanzania.