Hans Ackerman

Detecting recent positive selection in the human genome from haplotype structure

The ability to detect recent natural selection in the human population would have profound implications for the study of human history and for medicine. Here, we introduce a framework for detecting the genetic imprint of recent positive selection by analysing long-range haplotypes in human populations. We first identify haplotypes at a locus of interest (core haplotypes). We then assess the age of each core haplotype by the decay of its association to alleles at various distances from the locus, as measured by extended haplotype homozygosity (EHH). Core haplotypes that have unusually high EHH and a high population frequency indicate the presence of a mutation that rose to prominence in the human gene pool faster than expected under neutral evolution. We applied this approach to investigate selection at two genes carrying common variants implicated in resistance to malaria: G6PD and CD40 ligand. At both loci, the core haplotypes carrying the proposed protective mutation stand out and show significant evidence of selection. More generally, the method could be used to scan the entire genome for evidence of recent positive selection.

Malaria biology and disease pathogenesis: insights for new treatments

Plasmodium falciparum malaria, an infectious disease caused by a parasitic protozoan, claims the lives of nearly a million children each year in Africa alone and is a top public health concern. Evidence is accumulating that resistance to artemisinin derivatives, the frontline therapy for the asexual blood stage of the infection, is developing in southeast Asia. Renewed initiatives to eliminate malaria will benefit from an expanded repertoire of antimalarials, including new drugs that kill circulating P. falciparum gametocytes, thereby preventing transmission. Our current understanding of the biology of asexual blood-stage parasites and gametocytes and the ability to culture them in vitro lends optimism that high-throughput screenings of large chemical libraries will produce a new generation of antimalarial drugs. There is also a need for new therapies to reduce the high mortality of severe malaria. An understanding of the pathophysiology of severe disease may identify rational targets for drugs that improve survival.

Functional consequences of a polymorphism affecting NF-kappaB p50-p50 binding to the TNF promoter region.

ABSTRACT Stimulation of the NF-κB pathway often causes p65-p50 and p50-p50 dimers to be simultaneously present in the cell nucleus. A natural polymorphism at nucleotide −863 in the human TNF promoter (encoding tumor necrosis factor [TNF]) region provides an opportunity to dissect the functional interaction of p65-p50 and p50-p50 at a single NF-κB binding site. We found that this site normally binds both p65-p50 and p50-p50, but a single base change specifically inhibits p50-p50 binding. Reporter gene analysis in COS-7 cells expressing both p65-p50 and p50-p50 shows that the ability to bind p50-p50 reduces the enhancer effect of this NF-κB site. Using an adenoviral reporter assay, we found that the variant which binds p50-p50 results in a reduction of lipopolysaccharide-inducible gene expression in primary human monocytes. This finding adds to a growing body of experimental evidence that p50-p50 can inhibit the transactivating effects of p65-p50 and illustrates the potential for genetic modulation of inflammatory gene regulation in humans by subtle nucleotide changes that alter the relative binding affinities of different forms of the NF-κB complex.

Unusual haplotypic structure of IL8, a susceptibility locus for a common respiratory virus

Interleukin-8 (IL8) is believed to play a role in the pathogenesis of bronchiolitis, a common viral disease of infancy, and a recent U.K. family study identified an association between this disease and the IL8-251A allele. In the present study we report data, from a different set of families, which replicate this finding; combined analysis of 194 nuclear families through use of the transmission/disequilibrium test gives P = .001. To explore the underlying genetic cause, we identified nine single-nucleotide polymorphisms (SNPs) in a 7.6-kb segment spanning the IL8 gene and its promoter region and used six of these SNPs to define the haplotypic structure of the IL8 locus. The IL8-251A allele resides on two haplotypes, only one of which is associated with disease, suggesting that this may not be the functional allele. Europeans show an unusual haplotype genealogy that is dominated by two common haplotypes differing at multiple sites, whereas Africans have much greater haplotypic diversity. These marked haplotype-frequency differences give an F(ST) of.25, and, in the European sample, both Tajimas D statistic (D = 2.58, P = .007) and the Hudson/Kreitman/Aguade test (chi(2) = 4.9, P = .03) reject neutral equilibrium, suggesting that selective pressure may have acted on this locus.

A Comparison of Case-Control and Family-Based Association Methods: The Example of Sickle-Cell and Malaria

There has been much debate about the relative merits of population‐ and family‐based strategies for testing genetic association, yet there is little empirical data that directly compare the two approaches. Here we compare case‐control and transmission/disequilibrium test (TDT) study designs using a well‐established genetic association, the protective effect of the sickle‐cell trait against severe malaria. We find that the two methods give similar estimates of the level of protection (case‐control odds ratio = 0.10, 95% confidence interval 0.03–0.23; family‐based estimate of the odds ratio = 0.11, 95% confidence interval 0.04–0.25) and similar statistical significance of the result (case‐control: χ2= 41.26, p= 10−10, TDT: χ2= 39.06, p= 10−10) when 315 TDT cases are compared to 583 controls. We propose a family plus population control study design, which allows both case‐control and TDT analysis of the cases. This combination is robust against the respective weaknesses of the case‐control and TDT study designs, namely population structure and segregation distortion. The combined study design is especially cost‐effective when cases are difficult to ascertain and, when the case‐control and TDT results agree, offers greater confidence in the result.

Haplotypic analysis of the TNF locus by association efficiency and entropy

BackgroundTo understand the causal basis of TNF associations with disease, it is necessary to understand the haplotypic structure of this locus. We genotyped 12 single-nucleotide polymorphisms (SNPs) distributed over 4.3 kilobases in 296 healthy, unrelated Gambian and Malawian adults. We generated 592 high-quality haplotypes by integrating family- and population-based reconstruction methods.ResultsWe found 32 different haplotypes, of which 13 were shared between the two populations. Both populations were haplotypically diverse (gene diversity = 0.80, Gambia; 0.85, Malawi) and significantly differentiated (p < 10-5 by exact test). More than a quarter of marker pairs showed evidence of intragenic recombination (29% Gambia; 27% Malawi). We applied two new methods of analyzing haplotypic data: association efficiency analysis (AEA), which describes the ability of each SNP to detect every other SNP in a case-control scenario; and the entropy maximization method (EMM), which selects the subset of SNPs that most effectively dissects the underlying haplotypic structure. AEA revealed that many SNPs in TNF are poor markers of each other. The EMM showed that 8 of 12 SNPs (Gambia) and 7 of 12 SNPs (Malawi) are required to describe 95% of the haplotypic diversity.ConclusionsThe TNF locus in the Gambian and Malawi sample is haplotypically diverse and has a rich history of intragenic recombination. As a consequence, a large proportion of TNF SNPs must be typed to detect a disease-modifying SNP at this locus. The most informative subset of SNPs to genotype differs between the two populations.

Interethnic studies of TNF polymorphisms confirm the likely presence of a second MHC susceptibility locus in ankylosing spondylitis.

The objective of this study was to investigate TNF promoter region polymorphisms for association with susceptibility to ankylosing spondylitis (AS). The TNF −238 and −308 polymorphisms were genotyped in 306 English AS cases and 204 ethnically matched healthy B27-positive controls, and 96 southern German AS cases, 58 B27-positive and 251 B27-negative ethnically matched controls. Additionally, the TNF −376 polymorphism was genotyped in the southern German cases and controls. In the southern German AS patients a significant reduction in TNF −308.2 alleles was seen, compared with B27 positive controls (odds ratio 0.4, P = 0.03, 95% confidence interval 0.2–0.9), but no difference in allele frequencies was observed at TNF −238. Significant association between AS and both TNF −238 and TNF −308 was excluded in the English cases. These results confirm previous observations in the southern German population of association between TNF promoter region polymorphisms and AS, but the lack of association in the English population suggests that these polymorphisms themselves are unlikely to be directly involved. More likely, a second, non-HLA-B, MHC locus is involved in susceptibility to AS in these two populations.

Haplotypic relationship between SNP and microsatellite markers at the NOS2A locus in two populations

The density of genetic markers required for successful association mapping of complex diseases depends on linkage disequilibrium (LD) between non-functional markers and functional variants. The haplotypic relationship between stable markers and potentially unstable but highly informative markers (e.g. microsatellites) indicates that LD might be maintained over considerable genetic distance in non-African populations, supporting the use of such ‘mixed marker haplotypes’ in LD-based mapping, and allowing inferences to be drawn about human origins. We investigated sequence variation in the proximal 2.6 kb of the inducible nitric oxide synthase (NOS2A) promoter and the relationship between SNP haplotypes and a pentanucleotide microsatellite (the ‘NOS2A−2.6 microsatellite’) in Gambians and UK Caucasians. UK Caucasians exhibited a subset of sequence diversity observed in Gambians, sharing four of 11 SNPs and a similar haplotypic structure. Five SNPs were found in the sequence of interspersed repetitive DNA elements. In both populations, there was dramatic loss of LD between SNP haplotypes and microsatellite alleles across a very short physical distance, suggesting a high intrinsic mutation rate of the NOS2A−2.6 microsatellite, the SNP haplotypes are relatively ancient, or that this was a region of frequent recombination. Understanding locus- and population-specific LD is essential when designing and interpreting genetic association studies.

Nucleotide diversity of the TNF gene region in an African village.

The wide variety of disease associations reported at the TNF locus raises the question of how much variation exists within a single population. To address this question, we sequenced the entire TNF gene in 72 chromosomes from healthy residents of a village in The Gambia, West Africa. We found 12 polymorphisms in 4393 nucleotides, of which five have not been previously described, giving an estimated nucleotide diversity (θ) of 5.6 × 10−4. A significantly higher frequency of polymorphisms was found in the promoter region than in the coding region (8/1256 vs 0/882 nucleotides, P = 0.02). All polymorphisms with the exception of one rare allele were found to be present in Malawi, which is both geographically and genetically distant from The Gambia. Genotyping of 424 Gambian and 121 Malawian adults showed a significant frequency difference between the two populations for eight of the 12 polymorphisms, but the average fixation index across the variable sites was relatively low (FST = 0.007). We conclude that, at the TNF locus, the nucleotide diversity found within a single African village is similar to the global value for human autosomal genes sampled across different continents.

Complex haplotypic structure of the central MHC region flanking TNF in a West African population.

TNF polymorphisms have been associated with susceptibility to malaria and other infectious and inflammatory conditions. We investigated a sample of 150 West African chromosomes to determine linkage disequilibrium (LD) between 25 SNP markers located in an 80 kb segment of the MHC Class III region encompassing TNF and eight neighbouring genes. We observed 45 haplotypes, and 22 of them comprise 80% of the sample. The pattern of LD is remarkably patchy, such that many markers show no LD with adjacent markers but high LD with markers that are much further away. We introduce a method of examining the implications of LD data for disease association studies based on sample size considerations: this shows that certain TNF polymorphisms would be likely to yield positive associations if the true disease allele resided in LTA or BAT1. We conclude that detailed marker maps are needed to resolve the causal origin of disease associations observed at the TNF locus.