Population genetics of CAPN10 and GPR35: implications for the evolution of type 2 diabetes variants.

A positional cloning study of type 2 diabetes in Mexican Americans identified a region, termed "NIDDM1," on chromosome 2q37 with significant linkage evidence. Haplotype combinations at the calpain-10 gene (CAPN10) within this region were shown to increase diabetes risk in several populations. On the basis of the thrifty genotype hypothesis, variants that increase susceptibility to type 2 diabetes under modern lifestyle conditions provided a survival advantage in past environments by increasing the efficiency of energy use and storage. Here, our goal is to make inferences about the evolutionary forces shaping variation in genes in the NIDDM1 region and to investigate the population genetics models that may underlie the thrifty genotype hypothesis. To this end, we surveyed sequence variation in CAPN10 and in an adjacent gene, G-protein-coupled receptor 35 (GPR35), in four population samples from different ethnic groups. These data revealed two distinct deviations from the standard neutral model in CAPN10, whereas GPR35 variation was largely consistent with neutrality. CAPN10 showed a significant deficit of variation in the haplotype class defined by the derived allele at SNP44, a polymorphism that is significantly associated with diabetes in meta-analysis studies. This suggests that this haplotype class was quickly driven to high frequency by positive natural selection. Interestingly, the derived allele at SNP44 is protective against diabetes. CAPN10 also showed a local excess of polymorphism and linkage disequilibrium decay in intron 13. Simulations show that this pattern may be explained by long-standing balancing selection that maintains multiple selected alleles. Alternatively, it is possible that the local mutation and recombination rates changed since the divergence of human and chimpanzee; this scenario does not require the action of natural selection on intron 13 variation.

Gene conversion and different population histories may explain the contrast between polymorphism and linkage disequilibrium levels.

To characterize linkage disequilibrium (LD) levels in human populations, we have analyzed 10 independent noncoding segments in three population samples from the major ethnic groups--that is, Africans, Asians, and Europeans. Descriptive statistics show that LD decays much faster in the African samples than in the non-African ones. With the assumption of an equilibrium model, we estimated the population crossing-over parameter (4N(e)r(bp), where N(e) is the effective population size and r(bp) is the crossing-over rate per generation between adjacent base pairs) in the presence of gene conversion. In the African sample, LD and polymorphism levels lead to similar estimates of effective population size, as expected under an equilibrium model. Conversely, in both non-African samples, LD levels suggest a smaller effective population size than that implied by polymorphism levels. This observation is paralleled by significant departures from an equilibrium model in the spectrum of allele frequencies of the non-African samples. Besides ruling out the possibility that non-African populations are at equilibrium, these results suggest different demographic history (temporal and spatial) of these groups. Interestingly, the African sample fits the expectations of an equilibrium model based on polymorphism and divergence levels and on frequency spectrum. For this sample, the estimated ratio of gene conversion to crossing-over rates is 7.3 for a mean tract length of 500 bp, suggesting that gene conversion may be more frequent than previously thought. These findings imply that disease-association studies will require a much denser map of polymorphic sites in African than in non-African populations.

Two-locus sampling distributions and their application.

Methods of estimating two-locus sample probabilities under a neutral model are extended in several ways. Estimation of sample probabilities is described when the ancestral or derived status of each allele is specified. In addition, probabilities for two-locus diploid samples are provided. A method for using these two-locus probabilities to test whether an observed level of linkage disequilibrium is unusually large or small is described. In addition, properties of a maximum-likelihood estimator of the recombination parameter based on independent linked pairs of sites are obtained. A composite-likelihood estimator, for more than two linked sites, is also examined and found to work as well, or better, than other available ad hoc estimators. Linkage disequilibrium in the Xq28 and Xq25 region of humans is analyzed in a sample of Europeans (CEPH). The estimated recombination parameter is about five times smaller than one would expect under an equilibrium neutral model.

LIAN 3.0: detecting linkage disequilibrium in multilocus data. Linkage Analysis.

SUMMARY: LIAN is a program to test the null hypothesis of linkage equilibrium for multilocus data. LIAN incorporates both a Monte Carlo method as well as a novel algebraic method to carry out the hypothesis test. The program further returns the genetic diversity of the sample and the pairwise distances between its members.

A new statistic for detecting genetic differentiation.

A new statistic for detecting genetic differentiation of subpopulations is described. The statistic can be calculated when genetic data are collected on individuals sampled from two or more localities. It is assumed that haplotypic data are obtained, either in the form of DNA sequences or data on many tightly linked markers. Using a symmetric island model, and assuming an infinite-sites model of mutation, it is found that the new statistic is as powerful or more powerful than previously proposed statistics for a wide range of parameter values.

Adjusting the focus on human variation.

Studies of nuclear sequence variation are accumulating, such that we can expect a good description of the structure of human variation across populations and genomic regions in the near future. This description will help to elucidate the evolutionary forces that shape patterns of variability. Such an understanding will be of general biological interest, but could also facilitate the design and interpretation of disease-mapping studies. Here, we integrate the results from surveys of nuclear sequence variation. When nuclear sequences are considered together with mtDNA and microsatellites, it becomes clear that neither the standard neutral model, nor a simple long-term exponential growth model, can account for all the available human variation data. A possible explanation is that a subset of loci are not evolving neutrally; even so, more-complex models of effective population size and structure might be necessary to explain the data.

ATP enhances repair of hair bundles in sea anemones.

Hair bundle mechanoreceptors of sea anemones are similar to those of the acousticolateralis system of vertebrates (Watson, Mire and Hudson, 1997, Hear. Res. 107, 53-63). Anemone hair bundles are repaired by 'repair proteins' secreted following a complete loss of structural integrity and loss of function caused by 1 h exposure to calcium free seawater. Exogenously supplied repair proteins (RP) restore structural integrity to hair bundles and restore vibration sensitivity in 7-8 min (Watson, Mire and Hudson, 1998, Hear. Res. 115, 119-128). We here report that exogenously supplied ATP enhances the rate by which RP restore vibration sensitivity. A bimodal dose response to ATP indicates maximal enhancement at picomolar and micromolar concentrations of ATP. At these concentrations of ATP, vibration sensitivity is restored in 2 min. These data suggest that at least two ATPases exhibiting different binding affinities for ATP are involved in the repair process. Whereas the higher affinity site is specific for ATP, the lower affinity site does not discriminate between ATP and ADP. Nucleotidase cytochemistry localizes ATPase activity in isolated repair proteins. In the absence of exogenously added RP, sea anemones secrete and consume ATP during the 4 h recovery period after 1 h exposure to calcium free seawater. In the presence of exogenously added RP, ATP is secreted and then consumed within 10 min. Quinacrine cytochemistry localizes possible stores of ATP in the apical cytoplasm of sensory neurons located at the center of the hair bundle. According to our model, ATP is secreted by the sensory neuron after its hair bundle loses structural integrity. Hydrolysis of ATP by repair proteins is essential to the repair process.

Type I error and the power of the s-test: old lessons from a new, analytically justified statistical test for phylogenies.

We present a new procedure for assessing the statistical significance of the most likely unrooted dichotomous topology inferrable from four DNA sequences. The procedure calculates directly a P-value for the support given to this topology by the informative sites congruent with it, assuming the most likely star topology as the null hypothesis. Informative sites are crucial in the determination of the maximum likelihood dichotomous topology and are therefore an obvious target for a statistical test of phylogenies. Our P-value is the probability of producing through parallel substitutions on the branches of the star topology at least as much support as that given to the maximum likelihood dichotomous topology by the aforementioned informative sites, for any of the three possible dichotomous topologies. The degree of statistical significance is simply the complement of this P-value. Ours is therefore an a posteriori testing approach, in which no dichotomous topology is specified in advance. We implement the test for the case in which all sites behave identically and the substitution model has a single parameter. Under these conditions, the P-value can be easily calculated on the basis of the probabilities of change on the branches of the most likely star topology, because under these assumptions, each site can become informative independently from every other site; accordingly, the total number of informative sites of each kind is binomially distributed. We explore the test's type I error by applying it to data produced in star topologies having all branches equally long, or having two short and two long branches, and various degrees of homoplasy. The test is conservative but we demonstrate, by means of a discreteness correction and progressively assumption-free calculations of the P-values, that (1) the conservativeness is mostly due to the discrete nature of informative sites and (2) the P-values calculated empirically are moreover mostly quite accurate in absolute terms. Applying the test to data produced in dichotomous topologies with increasing internal branch length shows that, despite the test's "conservativeness," its power is much higher than that of the bootstrap, especially when the relevant informative sites are few.

Detecting linkage disequilibrium in bacterial populations.

The distribution of the number of pairwise differences calculated from comparisons between n haploid genomes has frequently been used as a starting point for testing the hypothesis of linkage equilibrium. For this purpose the variance of the pairwise differences, VD, is used as a test statistic to evaluate the null hypothesis that all loci are in linkage equilibrium. The problem is to determine the critical value of the distribution of VD. This critical value can be estimated either by Monte Carlo simulation or by assuming that VD is distributed normally and calculating a one-tailed 95% critical value for VD, L, L = EVD + 1.645 sqrt(VarVD), where E(VD) is the expectation of VD, and Var(VD) is the variance of VD. If VD (observed) > L, the null hypothesis of linkage equilibrium is rejected. Using Monte Carlo simulation we show that the formula currently available for Var(VD) is incorrect, especially for genetically highly diverse data. This has implications for hypothesis testing in bacterial populations, which are often genetically highly diverse. For this reason we derive a new, exact formula for Var(VD). The distribution of VD is examined and shown to approach normality as the sample size increases. This makes the new formula a useful tool in the investigation of large data sets, where testing for linkage using Monte Carlo simulation can be very time consuming. Application of the new formula, in conjunction with Monte Carlo simulation, to populations of Bradyrhizobium japonicum, Rhizobium leguminosarum, and Bacillus subtilis reveals linkage disequilibrium where linkage equilibrium has previously been reported.

Frequency specificity of vibration dependent discharge of nematocysts in sea anemones.

Hair bundles on tentacles of sea anemones are similar to vertebrate hair bundles in terms of structure and function. Anemone hair bundles are involved in regulating discharge of nematocysts, "stinging capsules," used to capture prey. N-acetylated sugars from the prey including N-acetylneuraminic acid (NANA) induce hair bundles to elongate while shifting vibration dependent discharge of nematocysts to lower frequencies matching prey movements. In the present study, we find that vibration dependent discharge of nematocysts exhibits sharp frequency discrimination to within one Hz. Testing at one-Hz intervals over the range of frequencies spanning 1-75 Hz, we find that seven of these are stimulatory in seawater alone. A total of twenty-six frequencies are stimulatory in the presence of NANA. Stimulatory frequencies in NANA are lower than those in seawater alone. We find that antagonists of ryanodine receptors including ryanodine, procaine and tetracaine shift discharge to lower frequencies. Fluorescently tagged ryanodine labels numerous small loci in the apical cytoplasm of supporting cells. We propose that calcium induced calcium release (CICR) via ryanodine receptors may sharpen frequency specificity and/or cause shortening of hair bundles to shift frequency specificity to higher frequencies.

Malaria's Eve: evidence of a recent population bottleneck throughout the world populations of Plasmodium falciparum.

We have analyzed DNA sequences from world-wide geographic strains of Plasmodium falciparum and found a complete absence of synonymous DNA polymorphism at 10 gene loci. We hypothesize that all extant world populations of the parasite have recently derived (within several thousand years) from a single ancestral strain. The upper limit of the 95% confidence interval for the time when this most recent common ancestor lived is between 24,500 and 57,500 years ago (depending on different estimates of the nucleotide substitution rate); the actual time is likely to be much more recent. The recent origin of the P. falciparum populations could have resulted from either a demographic sweep (P. falciparum has only recently spread throughout the world from a small geographically confined population) or a selective sweep (one strain favored by natural selection has recently replaced all others). The selective sweep hypothesis requires that populations of P. falciparum be effectively clonal, despite the obligate sexual stage of the parasite life cycle. A demographic sweep that started several thousand years ago is consistent with worldwide climatic changes ensuing the last glaciation, increased anthropophilia of the mosquito vectors, and the spread of agriculture. P. falciparum may have rapidly spread from its African tropical origins to the tropical and subtropical regions of the world only within the last 6,000 years. The recent origin of the world-wide P. falciparum populations may account for its virulence, as the most malignant of human malarial parasites.

Repair of hair bundles in sea anemones by secreted proteins.

Sea anemones are sessile invertebrates that detect movements of prey using numerous hair bundles located on tentacles surrounding their mouth. Previously we found that hair bundles of anemones are structurally and functionally similar to those of vertebrates. After 10-15 min exposure to calcium depleted buffers, hair bundles in chickens suffer moderate damage from which they recover in 12 h without requiring new protein synthesis [Zhao, Yamoah and Gillespie, Proc. Natl. Acad. Sci. USA 94 (1996) 15469-15474]. We find that after 1 h exposure to calcium free seawater, hair bundles of anemones suffer extensive damage from which they recover in 4 h, apparently because of newly synthesized, secretory proteins called 'repair proteins'. Recovery is delayed in a dose dependent fashion by cycloheximide. In the presence of exogenously added repair proteins, recovery occurs within 8 min and is cycloheximide insensitive. Recovery is ascertained by a bioassay performed on intact specimens, by electrophysiology, and by timelapse video microscopy. Fraction beta, a chromatographic fraction with bioactivity comparable to the complete mixture of repair proteins, consists of complexes having an estimated mass of 2000 kDa. Avidin based cytochemistry suggests that biotinylated fraction beta binds to damaged hair bundles. SDS-PAGE gel electrophoresis demonstrates that fraction beta contains 8-10 polypeptides of 90 kDa or smaller. At least four of these polypeptides apparently are consumed during the repair process. Negatively stained samples of fraction beta are shown by transmission electron microscopy to include filamentous structures similar in length (150 nm) and width (6 nm) to linkages between stereocilia. The filamentous structures can be associated with globular structures (20 nm in diameter). A model is presented wherein repair proteins comprise replacement linkages and enzymes that attach linkages to appropriate membrane proteins.

Plasmodium falciparum antigenic diversity: evidence of clonal population structure.

Plasmodium falciparum, the agent of malignant malaria, is one of mankind's most severe scourges. Efforts to develop preventive vaccines or remedial drugs are handicapped by the parasite's rapid evolution of drug resistance and protective antigens. We examine 25 DNA sequences of the gene coding for the highly polymorphic antigenic circumsporozoite protein. We observe total absence of silent nucleotide variation in the two nonrepeated regions of the gene. We propose that this absence reflects a recent origin (within several thousand years) of the world populations of P. falciparum from a single individual; the amino acid polymorphisms observed in these nonrepeat regions would result from strong natural selection. Analysis of these polymorphisms indicates that: (i) the incidence of recombination events does not increase with nucleotide distance; (ii) the strength of linkage disequilibrium between nucleotides is also independent of distance; and (iii) haplotypes in the two nonrepeat regions are correlated with one another, but not with the central repeat region they span. We propose two hypotheses: (i) variation in the highly polymorphic central repeat region arises by mitotic intragenic recombination, and (ii) the population structure of P. falciparum is clonal--a state of affairs that persists in spite of the necessary stage of physiological sexuality that the parasite must sustain in the mosquito vector to complete its life cycle.

Genetic tagging of humpback whales.

The ability to recognize individual animals has substantially increased our knowledge of the biology and behaviour of many taxa. However, not all species lend themselves to this approach, either because of insufficient phenotypic variation or because tag attachment is not feasible. The use of genetic markers ('tags') represents a viable alternative to traditional methods of individual recognition, as they are permanent and exist in all individuals. We tested the use of genetic markers as the primary means of identifying individuals in a study of humpback whales in the North Atlantic Ocean. Analysis of six microsatellite loci among 3,060 skin samples collected throughout this ocean allowed the unequivocal identification of individuals. Analysis of 692 'recaptures', identified by their genotype, revealed individual local and migratory movements of up to 10,000 km, limited exchange among summer feeding grounds, and mixing in winter breeding areas, and also allowed the first estimates of animal abundance based solely on genotypic data. Our study demonstrates that genetic tagging is not only feasible, but generates data (for example, on sex) that can be valuable when interpreting the results of tagging experiments.

Before crossing over: the advantages of eukaryotic sex in genomes lacking chiasmatic recombination.

Non-recombining populations should suffer from four classic population genetic disadvantages: (1) they cannot reverse Muller's Ratchet, the accumulation of deleterious mutations caused by genetic drift and mutation; (2) whenever the fix a favourable mutation they lose all unlinked favourable variants; (3) they tend to lose favourable mutations that are linked to deleterious mutations; and (4) their genetic loads can be quite high when deleterious mutations have synergistic effects. It is commonly assumed that inter-chromosomal recombination (independent assortment) can counter these phenomena, but this has been studied only for the genetic load case. In contrast, many studies have shown that recombination via crossing over can counter these phenomena. Here we first show that segregation alone can strongly decelerate Muller's Ratchet in diploids, i.e. that recombination is not the only way to do so. We then show that inter-chromosomal recombination can indeed deal with phenomena (1) to (3) above very effectively if the genome consists of a moderate number of chromosomes. Therefore, if the above advantages of genetic recombination played a large role in the initial success of eukaryotic sex, the crucial moment in the origin of sex might have been the evolution of inter-chromosomal recombination, i.e. the evolution of genome segmentation, segregation, and syngamy. Crossing over might have become established as a major recombinational device only later, eliminating the disadvantages of extensively segmented genomes.

DNA variation at the Sod locus of Drosophila melanogaster: an unfolding story of natural selection.

Patterns of variation at the Sod locus of Drosophila melanogaster suggest that the protein polymorphism at this locus has very recently arisen. In addition, it appears that a previously rare DNA variant has been recently and rapidly driven to intermediate frequency. From the size of the region (>20 kb) that has been swept along with this rare variant, and patterns of linkage disequilibrium in the region, it is inferred that strength of selection was large (s > 0.01) and that the sweep occurred more than 25,000 generations ago. In addition, there are striking similarities to patterns of variation observed at the Est6 and Est-P loci, which are located approximately 1,000 kb from Sod.

Hair bundles of sea anemones as a model system for vertebrate hair bundles.

Sea anemones are marine invertebrates that use hair bundles to detect swimming movements of prey. Prey are captured by nematocysts (stinging capsules) that discharge into the prey. To further characterize anemone hair bundles and to compare hair bundles in anemones with hair bundles in vertebrates, we investigated fine structure and cytochemistry of anemone hair bundles. In addition, using a biological assay based on counting nematocysts discharged into vibrating test probes, we examined sensitivity of vibration detection to aminoglycoside antibiotics, Ca(2+)-free seawater, and amiloride. Like vertebrate hair bundles, anemone hair bundles are composed of stereocilia, possess lateral linkages between stereocilia whose preservation for transmission electron microscopy is enhanced by ruthenium red, and possess tip links morphologically similar to vertebrate tip links. Furthermore, vibration-dependent discharge of nematocysts is reversibly inhibited by 10(-4) M streptomycin and abolished by brief exposure to Ca(2+)-free seawater. However, unlike vertebrate hair bundles, anemone hair bundles appear to be insensitive to amiloride since vibration-dependent discharge of nematocysts is unaffected by up to mM amiloride. Thus, anemone hair bundles may serve as a useful model system for vertebrate hair bundles with the interesting feature of being insensitive to amiloride.

Deleterious background selection with recombination.

An analytic expression for the expected nucleotide diversity is obtained for a neutral locus in a region with deleterious mutation and recombination. Our analytic results are used to predict levels of variation for the entire third chromosome of Drosophila melanogaster. The predictions are consistent with the low levels of variation that have been observed at loci near the centromeres of the third chromosome of D. melanogaster. However, the low levels of variation observed near the tips of this chromosome are not predicted using currently available estimates of the deleterious mutation rate and of selection coefficients. If considerably smaller selection coefficients are assumed, the low observed levels of variation at the tips of the third chromosome are consistent with the background selection model.

The coalescent process and background selection.

Some statistical properties of gene trees are described for a model with background deleterious mutations. It is argued that the history of a small sample of genes under this model is a continuous time Markov chain that quickly reaches stationarity. This observation leads to simple expressions for the expected nucleotide diversity and suggests that the frequency spectrum in small samples should be approximately the same as under a strict neutral model. The results concerning expected nucleotide diversity are compared with observed variation on the third chromosome of Drosophila melanogaster.