Loss of balancing selection in the βS globin locus

<p>Abstract</p> <p>Background</p> <p>Probably the best example of the rise and maintenance of balancing selection as an evolutionary trend is the role of S-haemoglobin (HbS - rs334) in protecting from malaria. Yet, the dynamics of such a process remains poorly understood, particularly in relation to different malaria transmission rates and the genetic background of the affected populations.</p> <p>Methods</p> <p>We investigated the association of haemoglobin HbS in protection from clinical episodes of malaria in two populations/villages where malaria is endemic, but mostly presenting in mild clinical forms. Five-hundred and forty-six individuals comprising 65 and 82 families from the Hausa and Massalit villages respectively were genotyped for HbS. Allele and genotype frequencies as well as departure from Hardy-Weinberg Equilibrium were estimated from four-hundred and seventy independent genotypes across different age groups. Age-group frequencies were used to calculate the coefficient-of-fitness and to simulate the expected frequencies in future generations.</p> <p>Results</p> <p>Genotype frequencies were within Hardy-Weinberg expectations in Hausa and Massalit in the total sample set but not within the different age groups. There was a trend for a decrease of the HbS allele frequency in Hausa and an increase of frequency in Massalit. Although the HbS allele was able to confer significant protection from the clinical episodes of malaria in the two populations, as suggested by the odds ratios, the overall relative fitness of the HbS allele seems to have declined in Hausa.</p> <p>Conclusions</p> <p>Such loss of balancing selection could be due to a combined effect of preponderance of non-clinical malaria in Hausa, and the deleterious effect of the homozygous HbS under circumstances of endogamy.</p

The molecular basis of thalassaemia in Sri Lanka

Blood samples from over 1600 schoolchildren revealed a prevalence of both trait and HbE trait, averaging 2.3% and 0.8% respectively. With the present popUlation estimate at 18.6 million, it is predicted that approximately 2650 patients with l3-thalassaemia major and 956 with HbE/I3-thalassaemia should consume >5% of the current health budget for treatment of these patients. Patients with the phenotype of severe thalassaemia, from seven centres, were analysed to understand the molecular basis of the disease. Twenty-four l3-globin gene mutations were identified with three mutations accounting for 84.4% of the 1234 alleles: IVSI-5 (G--7C) 56.1 %; IVSI-l (G--7A) 15.2% and HbE 13.1 %. Sixteen mutations have previously been described on the Indian subcontinent and demonstrate close regional links between the popUlations, especially North East India, and were supported by haplotype association. The IVSI-l (G--7A) mutation occurs commonly in countries around the Mediterranean but very rarely on the Indian subcontinent. In Sri Lanka it is associated with a different 3' haplotype, suggesting a multicentric origin. Three new mutations were found; a frameshift codon 6-10 IVSI-129 (A--7C) in the consensus splice site and a second frame shift, CD55 (-A). Four a-gene arrangements were demonstrated. An allele frequency of the a+-thalassaemia gene deletions were 6.5% and 1.1% for the _a3.7 deletion and the _a4.2 deletion, respectively. Cord blood analysis of Hb Bart's levels confirmed an estimated level of 7.1 % for a single a-gene nonexpression. Extra a-genes are common and the first instance of homozygosity for the aaaa arrangement was observed. No aOthalassaemia was revealed and only a single polymorphism was associated with a possible extremely mild a-thalassaemia allele. The phenotype associated with the various l3-globin genotypes was found to be extremely variable. Within the many factors that modify this phenotype the Xmn-I polymorphism was significantly associated with the level of haemoglobin expressed. A +1+ genotype appeared to be a good indicator of a milder outcome. The incidence of a-gene deletions was low in patients with HhElI3-thalassaemia and thus proposed this as a strong modifier for a mild phenotype. Other factors were associated with specific complications

Association Analysis of Additive Effects and Epistasis Between Human Candidate Malaria Protective Genes

Malaria is a major cause of childhood death in Africa and host genetic factors play a key role in determining survival from this disease. Although many candidate loci have been identified, there have been difficulties in confirming the significance of some of these loci. To some extent this might be explained by the added complexity of epistasis, or gene-gene interactions. Through this thesis I aimed: (1) to re-appraise a range of candidate malaria-association genes using a large-scale case-control study of severe malaria (SM) in Kilifi, Kenya; (2) to compare different approaches to detecting epistatic interactions; (3) to look for evidence of epistasis between candidate genes in my data set; (4) to examine the haplotype structure and linkage disequilibrium (LD) patterns for two such implicated variants (HbS and α+thalassaemia) and their gene regions, that coexist in the Kilifi population, and (5) to use these exemplars as a starting point for investigating the process of detecting epistasis in SM in a genome-wide association study (GWAS). Out of 71 candidate genes investigated, I observed that polymorphisms affecting various aspects of red blood cells (including HBB, HBA, G6PD, FREM3, INPP4B, ATP2B4 and ABO) were among those associated with the strongest signals of differential susceptibility to SM. Because of their prominence in malaria, HbS and α+thalassaemia were used to illustrate interaction analysis at the GWAS level. This included looking at the structure of the genomic regions surrounding the genes. As expected, a single haplotype of approximately 200kb was seen surrounding HbS, which then diverged into 2 major haplotypes spanning a further 1Mb either side, an observation that was largely explained by ethnicity. In contrast, no marked LD/haplotype structure was observed in the genomic region surrounding the α+thalassaemia deletion, suggesting that this is a very old polymorphism. Through this study, I confirmed the negative epistasis seen between HbS and α+thalassaemia using a study design (case-control) that was different to that used previously (cohort), although this was not among the most significant of the interactions I detected. I searched for pairwise interactions between these two polymorphisms at a genome wide level using heterozygous and additive models for HbS and α+thalassaemia respectively. For each scan a single region reaching a significance level of -7 was found (STX18 for HbS and MYEOV for α+thalassaemia), plus several other novel signals were identified in the 10-6 to 10-7 significance region. Further work will be required to validate these signals and the challenge will be to try and understand their biological relevance. This is now becoming possible with datasets in many diseases, including malaria, being released into the public domain. But, as this Kenyan study has shown, having large group sizes, high quality clinical and genetic data, it is possible to begin to explore genetic interactions in a disease setting

Programmierter Zelltod in Plasmodium infizierten HbA/A und HbA/S Erythrozyten

In the first part of my thesis, I investigated the activation of erythrocyte nonselective cation (NSC) permeability and of sphingomyelinase by Plasmodium falciparum infection. Both are involved in programmed cell death in erythrocytes, also referred to as eryptosis [Lang et al., 2006; Lang et al., 2005], and meet four requirements for parasite growth at the same time: i) Development of an inwardly directed Na+ and an outwardly directed K+ ion gradient across the parasite plasma membrane, this in turn making the parasite dependent on high Na+ and low K+ ion concentration extracellularly [Brand et al., 2003]. ii) The presence of Ca2+ ions in the culture medium and a Ca2+ permeability conferred by a NSC conductance [Brand et al., 2003]. iii) Increased ceramide formation (detected in the blood of P. berghei ANKA infected mice). iv) Activity of parasitic neutral sphingomyelinase and host acid sphingomyelinase, probably to provide ceramide for membrane synthesis [Hanada et al., 2000]. In the second part of my doctoral thesis I investigated differences in the induction of eryptosis in normal (HbA/A) and sickle trait (HbA/S) P. falciparum infected red blood cells (RBCs). The results were as follows: i) P. falciparum did not develop differently in HbA/A and HbA/S RBCs, but infected HbA/S RBCs showed enhanced PS exposure. ii) The host ceramide pathway played only a minor role in causing PS exposure in infected RBCs under normal culturing conditions. iii) Although parasite growth was dependent on ceramide, extragenously applied ceramide inhibited P. falciparum in vitro growth, and more so in HbA/S RBCs than in HbA/A RBCs. iv) Infected HbA/S RBCs secreted more prostaglandin E2 (PGE2) than did infected HbA/A RBCs, probably significantly contributing to the selective advantage of P. falciparum infected sickle cell trait carriers. Increased PGE2 levels are associated with a mild course of malaria infection [Perkins et al., 2001]. v) The increased PGE2 levels enhanced the infection-induced Ca2+ influx into ring-stage infected HbA/S RBCs. This promoted the observed increase in PS exposure on the outer membrane leaflet of infected HbA/S RBCs as compared to infected HbA/A RBCs. These observations show that the accelerated eryptosis of P. falciparum infected HbA/S RBCs is caused by enhanced activation of a NSC permeability during ring stage. Enhanced PS exposure acts as an 'eat-me' signal for macrophages. Together with other signals [Ayi et al., 2004], it triggers the early recognition and removal of ring-stage infected HbA/S RBCs [Kasinathan, 2007].Im ersten Teil meiner Doktorarbeit untersuchte ich die Aktivierung einer nichtselektiven Kationenkanalaktivität und von Sphingomyelinase in Plasmodium falciparum infizierten Erythrozyten. Beide Prozesse sind am programmierten Zelltod in Erythrozyten beteiligt, der auch Eryptose genannt wird [Lang et al., 2006; Lang et al., 2005], und erfüllen gleichzeitig vier Bedingungen für das Parasitenwachstum: i) Entwicklung eines einwärts gerichteteten Natrium- und eines auswärts gerichteten Kalium-Ionen-Gradienten über die Parasitenplasmamembran, deshalb war der Parasit abhängig von einer hohen Natrium- und einer niedrigen Kalium- Konzentration im Extrazellulärraum [Brand et al., 2003]. ii) Die Anwesenheit von Calcium-Ionen im Kulturmedium und einer Permeabilität für Calcium-Ionen durch eine nichtselektive Kationenleitfähigkeit [Brand et al., 2003]. iii) Erhöhte Ceramid-Bildung (im Blut von P. berghei ANKA-infizierten Mäusen nachgewiesen). iv) Aktivität einer parasitischen neutralen Sphingomyelinase und einer sauren Sphingomyelinase des Wirtes, wahrscheinlich um Ceramid zur Membransynthese bereitzustellen [Hanada et al., 2000]. Im zweiten Teil meiner Doktorarbeit untersuchte ich Unterschiede in der Induktion der Eryptose in normalen (HbA/A) P. falciparum infizierten Erythrozyten und in infizierten Erythrozyten von heterozygoten Trägern der Sichelzellen-Gens (HbA/S). Ich erzielte folgende Ergebnisse: i) P. falciparum entwickelte sich nicht unterschiedlich in HbA/A- und HbA/S-Erythrozyten, aber infizierte HbA/S-Erythrozyten zeigten erhöhte Phosphatidylserin-Exposition. ii) Der Stoffwechselweg in der Wirtszelle zur Bildung von Ceramid trug unter normalen Kulturbedingungen nur geringfügig zur Phosphatidylserin-Exposition bei. iii) Obwohl das Parasitenwachstum Ceramid-abhängig war, inhibierte von außen hinzugefügtes Ceramid das Wachstum von P. falciparum in vitro, und zwar mehr in HbA/S- als in HbA/A- Erythrozyten. iv) Infizierte HbA/S Erythrozyten sezernierten mehr Prostaglandin E2 (PGE2) als infizierte HbA/A Erythrozyten, und tragen deshalb wahrscheinlich wesentlich zum Selektionsvorteil von P. falciparum infizierten heterozygoten Trägern des Sichelzellen-Gens bei. Erhöhte PGE2-Plasmaspiegel stehen im Zusammenhang mit einem milden Verlauf einer Malariainfektion [Perkins et al., 2001]. v) Der erhöhte PGE2-Spiegel verstärkte den infektions-induzierten Calcium-Ionen-Einstrom in HbA/S-Erythrozyten, die von P. falciparum im Ringstadium infiziert sind. Dies förderte die beobachtete Erhöhung der Phosphatidylserin-Exposition in der äußeren Membranschicht der infizierten HbA/S-Erythrozyten im Vergleich zu infizierten HbA/A-Erythrozyten. Diese Beobachtungen zeigen, daß die beschleunigte Eryptose von P. falciparum-infizierten HbA/S-Erythrozyten durch erhöhte Aktivierung einer nichtselektiven Kationenpermeabilität während des Ringstadiums verursacht wird. Erhöhte Phosphatidylserin-Exposition wirkt als Freßsignal für Makrophagen. Zusammen mit anderen Signalen [Ayi et al., 2004] bewirkt sie die frühe Erkennung und Beseitigung von im Ringstadium infizierten HbA/S-Erythrozyten [Kasinathan, 2007]. Ayi K, Turrini F, Piga A, Arese P (2004). Enhanced phagocytosis of ring-parasitized mutant erythrocytes: a common mechanism that may explain protection against falciparum malaria in sickle trait and beta-thalassemia trait. Brand VB, Sandu CD, Duranton C, Tanneur V, Lang KS, Huber SM, Lang F (2003). Dependence of Plasmodium falciparum in vitro growth on the cation permeability of the human host erythrocyte. Cell Physiol Biochem. 13, 347-356. Hanada K, Mitamura T, Fukasawa M, Magistrado PA, Kurokawa K, Rai G, Sakata D, Hara T, Horri T, Nishijima M (2000). Neutral sphingomyelinase activity dependent on Mg2+ and anionic phospholipids in the intraerythrocytic malaria parasite Plasmodium falciparum. Biochem. J. 346 Pt3, 671-677. Kasinathan RS (2007). Cation channels in erythrocyte apoptosis and malaria. Dissertation. Fakultät für Biologie, Eberhard-Karls-Universität, Tübingen, Germany. Lang F, Lang KS, Lang PA, Huber SM, Wieder T (2006). Mechanisms and significance of eryptosis. Antioxid. Redox. Signal. 8, 1183-1192. Lang KS, Lang PA, Bauer C, Duranton C, Wieder T, Huber SM, Lang F (2005). Mechanisms of suicidal erythrocyte death. Cell Physiol Biochem. 15, 195-202. Perkins DJ, Kremsner PG, Weinberg JB (2001). Inverse relationship of plasma prostaglandin E2 and blood mononuclear cell cyclooxygenase-2 with disease severity in children with Plasmodium falciparum malaria. J. Infect. Dis. 183, 113-118

Sickle Cell Disease Erythrocyte Stiffness and Cytoadhesion Investigated via Atomic Force Microscopy

The biomechanical properties of red blood cells (RBCs), including increased stiffness and abnormal cytoadherence, are integral components in the cascade of events resulting to vasoocclusive episodes (VOEs) in sickle cell disease (SCD). VOEs are the main cause of morbidity in SCD and sickle cell trait (SCT). Using experimental techniques based on atomic force microscopy (AFM), we studied the stiffness and adhesion of RBCs from SCD patients and from subjects with SCT. We found that SCD and SCT RBCs are three-fold stiffer than normal RBCs. Further, a ten-fold increase in the stiffness of sickled RBCs was measured upon deoxygenation. In an effort to rectify the increased stiffness of sickle RBCs, mice were fed a diet supplemented with docosahexanoic acid (DHA), an omega-3 fatty acid. A decrease in RBC stiffness was measured suggesting therapeutic benefits of DHA. Cytoadherence of RBCs to subendothelial laminin via the basal cell adhesion molecule/Lutheran (BCAM/Lu) is implicated in vasculopathy, a common condition in SCD patients. We established the in vitro technique of single-molecule force spectroscopy (SMFS) which enables detection of single BCAM/Lu proteins on the RBC surface via measurement of the unbinding force with laminin. It was shown that epinephrine, acting through the cyclic adenosine monophosphate (cAMP) signaling pathway, increases the population of active BCAM/Lu receptors on SCT RBCs, suggesting a role in exercise-induced VOEs. The sensitivity of the SMFS system was validated in a neuronal system to quantitatively map SK channels and then employed to investigate the effects of cAMP pathway targeting on BCAM/Lu receptor expression on normal and SCD RBCs. We illustrated that A-kinase anchoring proteins are crucial for BCAM/Lu receptor activation. To examine the relevance of results based on SMFS in the cytoadhesion of entire RBCs, single-cell force spectroscopy (SCFS) was established to measure the adhesion of whole cells with a functionalized substrate. We established a correspondence between the SMFS and SCFS results. Both techniques were able to detect significant changes in the adhesive response of RBCs to cAMP pathway modulation and variability was measured amongst human subjects, suggesting that RBCs maintain diverse intracellular levels of tonic protein kinase A

Copy number variations (CNVs) in the assessment of stroke susceptibility in patients with sickle cell anemia

Orientador: Mônica Barbosa de MeloTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Embora a anemia falciforme (AF) resulte da homozigose de uma única mutação, na posição 6 do locus da ?-hemoglobina, fenotipicamente, é uma doença muito heterogênea, de modo que diferentes pacientes apresentam evoluções clínicas significativamente distintas. Praticamente todos os órgãos podem ser afetados pela oclusão vascular, merecendo destaque o Sistema Nervoso Central (SNC), onde são observados acidentes isquêmicos transitórios, infartos e hemorragia cerebral, os quais acometem aproximadamente 25% dos pacientes com AF. As complicações neurológicas são graves, podendo ser fatais em até 15% dos casos. A identificação precoce de pacientes com anemia falciforme suscetíveis ao Acidente Vascular Cerebral (AVC) poderia diminuir os riscos, possivelmente prevenir a recorrência de infartos e potencialmente reduzir sua incidência. Portanto, estudos que visem identificar novos grupos de risco para o desenvolvimento de AVC em pacientes com anemia falciforme seriam fundamentais para otimizar o controle clínico desta enfermidade, sendo uma dessas vias a abordagem molecular deste grupo de pacientes. Desta forma, propusemos a investigação da presença de variação no número de cópias alélicas ou "Copy Number Variation" (CNV), utilizando-se lâminas de alta densidade (microarrays), com o objetivo de identificar regiões do genoma, potencialmente envolvidas com o risco aumentado de AVC em pacientes com anemia falciforme. Este trabalho propôs ainda avaliar os aspectos populacionais relativos à população brasileira. É de grande interesse nos estudos de genética humana entender a manifestação de dado fenótipo em populações distintas, e se a associação de variantes em uma população pode ser extrapolada para outras. A anemia falciforme fornece um modelo nesse intento, passível de ser extrapolado para doenças complexas dado ser um grupo de origem bem conhecida, exemplo clássico de seleção balanceada em humanos, primeira doença a ter base molecular desvendada e ser de herança mendeliana. Observou-se marcante diferença na ancestralidade de indivíduos afetados por AF brasileiros em relação a norte-americanos, tanto no genoma geral, quanto no cromossomo 11. Chegou-se a uma listagem de regiões com variações no número de cópias potencialmente associadas ao AVC em ambas as populaçõesAbstract: Although sickle cell anemia results from homozygosity for a single mutation at position 6 of gamma-hemoglobin locus, this disease presents high heterogenity in phenotype, so that different patients may have significantly different clinical outcomes. Virtually all organs may be affected by vascular occlusion, with emphasis on the Central Nervous System (CNS), where are observed transient ischemic attacks, stroke and cerebral hemorrhage, which affect approximately 25% of patients with SCA. Neurological complications are serious and can be fatal in up to 15% of cases. Early identification of patients with sickle cell anemia, susceptible to stroke (CVA) could reduce the risk, possibly preventing the recurrence of heart attacks and potentially reduce their incidence. Therefore, studies aimed at identifying new risk groups for development of stroke in sickle cell patients would be essential to optimize the clinical management of this disease, one of these pathways the molecular approach of this group of patients. Thus, we propose to investigate the presence of copy number variation in allele or "Copy Number Variation" (CNV), using high density microarray in order to identify genomic regions potentially involved in the increased risk of stroke in sickle cell patients. The present work also proposes to assess the Brazilian population genetic aspects. It is highly desirable in human genetics to unveil the manifestation of a given phenotype in distinct populations, as well as whether an association identified on a specific population may hold to other populations. Sickle cell anemia provides a model for this purpose, and may be extrapolated to complex diseases given its well known geographical region, and for being a classic case of balanced selection in humans, the first disease to have its molecular mechanism unraveled, and a Mendelian trait. Ancestral components markedly differ in Brazilian SCA patients compared to the United States subjects on both the genomic landscape and the local ancestry on chromosome 11. Here we present a list of CNV events potentially associated to stroke on both populationsDoutoradoGenetica Animal e EvoluçãoDoutor em Genetica e Biologia Molecular140967/2012-52012/06438-5FAPESPCNP

Genomic, patterns of selection and differentiation in African populations and implications for mapping disease association

The main objective of this thesis is to gain a better understanding of genomic patterns of natural selection and population differentiation in Africa, where there is great genetic diversity, and of the implications for genetic mapping of complex diseases. I began by studying two neighbouring villages in eastern Sudan that are of different ethnicity, Hausa and Masalit, and that appear to have different susceptibility to malaria and visceral leishmaniasis (VL). Specifically, I investigated patterns of linkage disequilibrium (LD) and haplotypic signals of positive selection in the 5q31 genomic region which contains immune genes that have been implicated in susceptibility to malaria and VL. In my first analysis, by genotyping 34 single nucleotide polymorphisms (SNPs) in the 5q31 region, I did not find signals of selection or population differentiation between the Hausa and Masalit using available statistical methods. I conceived the idea that patterns of LD might provide a more sensitive test of population differentiation, and I developed an approach for this using permutation analysis. This method revealed differentiation between the Hausa, the Masalit and other African ethnic groups. To better understand signals of selection, I next studied a region of the genome associated with a known malaria resistance factor, the haemoglobin S (HbS) variant of the HBB gene. By genotyping 26 SNPs in the region of the HBB gene, I observed a haplotype that extended in excess of 1 Mb, despite being at high frequency and spanning several recombinational hotspots. This long haplotype carried the HbS allele but, importantly, it could be readily detected without typing the HbS variant. Building on this observation, I designed a new method to screen the whole genome for long haplotypes that might be signals of selection, and developed a software programme to implement this method. I validated this method using haplotypic data for the Yoruba generated by the HapMap project and complemented by additional SNP data that I generated on HapMap cell lines, and found that the HbS allele resides on a haplotype that extends to 1.2 Mb, and is at strikingly high frequency compared to other haplotypes of similar length on the same chromosome. Next I applied this method to a large family-based association study of severe malaria in The Gambia, and identified several novel genomic regions with unusually long haplotypes of high frequency. These included a number of regions that may be associated with resistance to severe malaria, and which merit further investigation

Human Blood Group Systems and Haemoglobinopathies

The past decade has seen remarkable improvements and advances in the fields of blood transfusion and hematology, particularly with regards to advances in science, technology, method development, quality, standardization, and governance. This book provides more evidenced-based insight into the field of blood transfusion and the management of hemoglobinopathies

Dissecting the genetic bases of severe malaria resistance using genome-wide and post genomewide study approaches

P. falciparum malaria remains one of the leading public health problems worldwide. The global tally of malaria in 2018 was estimated at 228 million cases and 405, 000 deaths worldwide. African countries disproportionately carry the global burden of malaria accounting for 93% and 94% of cases and deaths, respectively. Even though most infected children recover from P. falciparum malaria, a small subset (~1%) of cases progresses to severe disease and death. Over the last decade, several genome-wide association studies (GWASs) have been conducted in diverse malaria endemic populations to understand the natural host protective immunity against severe malaria that can provide clues for the development of new vaccines and therapeutics. However, beyond identifying association variants, conventional GWAS approaches can't inform the underpinning biological functions. To bridge this gap, we applied various contemporary statistical genetic analytic approaches to malaria GWAS datasets of diverse malaria endemic populations. First, we accessed malaria resistance GWAS datasets of three African populations (N=~11,000) including Kenya, Gambia and Malawi from European Genome Phenome Archive (EGA) through MalariaGEN consortium standard data accession procedures. We explored the challenges of GWAS approaches in the genetically diverse Africa populations and figured out how various advanced statistical genetic methods can be implemented to address these challenges. We investigated single nucleotide polymorphism (SNP) heritability (h2 g) of malaria resistance in the Gambian populations and determined appropriate quality (QC) thresholds to accurately estimate the h2 g in our dataset. Second, we estimated h2 g in the three populations and partitioned the h2 g into chromosomes, allele frequencies and annotations using the genetic relationship-matrix restricted maximum likelihood approaches. We further created African specific reference panel from African population datasets obtained from 1000 Genomes Project and African Genome Variation Project dataset and computed linkage disequilibrium (LD). We used LD information obtained from these reference panels to compute cell-type specific and none cell-type specific enrichments for GWAS-summary statistics meta-analyzed across the three populations. Our results showed for the first time that malaria resistance is polygenic trait with h2 g of ~20% and that the causal variants are overrepresented around protein coding regions of the genome. We further showed that the h2 g is disproportionately concentrated on three chromosomes (chr 5, 11 and 20), suggesting cost-effectiveness of targeting these chromosomes in future malaria genomic sequencing studies. Third, we systematically predicted plausible candidate genes and pathways from functional analysis of severe malaria resistance GWAS summary statistics (N = 17,000) meta-analyzed across eleven populations in malaria endemic regions in Africa, Asia and Oceania. We applied positional mapping, expression quantitative trait locus (eQTL), chromatin interaction mapping and gene-based association analyses to identify candidate severe malaria resistance genes. We performed network and pathway analyses to investigate their shared biological functions. We further applied rare variant analysis to raw GWAS datasets of three malaria endemic populations including Kenya, Malawi and Gambia and performed various population genetic structures of the identified genes in the three endemic populations and 20 world-wide ethnics. Our functional mapping analysis identified 57 genes located in the known malaria genomic loci while our gene-based GWAS analysis identified additional 125 genes across the genome. The identified genes were significantly enriched in malaria pathogenic pathways including multiple overlapping pathways in erythrocyte-related functions, blood coagulations, ion channels, adhesion molecules, membrane signaling elements and neuronal systems. Furthermore, our population genetic analysis revealed that the minor allele frequencies (MAF) of the SNPs residing in the identified genes are generally higher in the three malaria endemic populations compared to global populations. Overall, our results suggest that severe malaria resistance trait is attributed to multiple genes that are enriched in pathways linked to severe malaria pathogenesis. This highlights the possibility of harnessing new malaria therapeutics that can simultaneously target multiple malaria protective host molecular pathways. In conclusions, this project showed that malaria resistance trait is mainly a polygenic trait which is influenced by genes and pathways linked to blood stage lifecycle of P. falciparum. These findings constitute the foundations for future experimental studies that can potentially lead to translational medicine including development of new vaccines and therapeutics. However, ‘-omics' studies including those implemented in this study, are limited to single datatype analysis and lack adequate power to explain the complexity of molecular processes and usually lead to identification of correlations than causations. Thus, beyond singe locus analysis, the future direction of malaria resistance requires a paradigm shift from single-omics to multi-stage and multi-dimensional integrative multi-omics studies that combines multiple data types from the human host, the parasite, and the environment. The current biotechnological and statistical advances may eventually lead to the feasibility of systems biology studies and revolutionize malaria research

Adaptation to Mediterranea

The Mediterranean region encompasses countries that surround Mediterranean Sea. Due to its position at the intersection of Eurasia and Africa it has often been a route of human migrations during history, which contributed to its high biodiversity. People living in this area had been exposed to the episodes of natural selection that led to the establishment of specific genetic variations, for which is thought to carry a certain adaptation. Some recent studies have shown that genetic adaptations are probably related to the immune defense against infectious pathogens. One of the most recognizable disease of the region is familial Mediterranean fever (FMF), a prototype of a monogenic autoinflammatory disease. FMF is predisposed by the mutations in the Mediterranean fever (MEFV) gene that encodes inflammasome regulatory protein - pyrin. Specific variations of several other genes have been proposed to confer a protection against Plasmodium malariae parasite. Some of these are hemoglobin S (HbS), thalassemia, glucose-6-phosphate dehydrogenase deficiency, ovalocytosis, and mutation in the Duffy antigen (FY). In this chapter we will summarize important genetics and pathogenesis features of diseases commonly encountered in the Mediterranean region with a short discussion of potential adaptations that they may carry