- Methodology article
- Open Access
Inferring linkage disequilibrium from non-random samples^{†}
- Minghui Wang^{1},
- Tianye Jia^{1},
- Ning Jiang^{1},
- Lin Wang^{2},
- Xiaohua Hu^{2} and
- Zewei Luo^{1, 2}Email author
https://doi.org/10.1186/1471-2164-11-328
© Wang et al; licensee BioMed Central Ltd. 2010
- Received: 9 October 2009
- Accepted: 26 May 2010
- Published: 26 May 2010
Abstract
Background
Linkage disequilibrium (LD) plays a fundamental role in population genetics and in the current surge of studies to screen for subtle genetic variants affecting complex traits. Methods widely implemented in LD analyses require samples to be randomly collected, which, however, are usually ignored and thus raise the general question to the LD community of how the non-random sampling affects statistical inference of genetic association. Here we propose a new approach for inferring LD using a sample un-randomly collected from the population of interest.
Results
Simulation study was conducted to mimic generation of samples with various degrees of non-randomness from the simulated populations of interest. The method developed in the paper outperformed its rivals in adequately estimating the disequilibrium parameters in such sampling schemes. In analyzing a 'case and control' sample with β-thalassemia, the current method presented robustness to non-random sampling in contrast to two commonly used methods.
Conclusions
Through an intensive simulation study and analysis of a real dataset, we demonstrate the robustness of the proposed method to non-randomness in sampling schemes and the significant improvement of the method to provide accurate estimates of the disequilibrium parameter. This method provides a route to improve statistical reliability in association studies.
Keywords
- Linkage Disequilibrium
- Simulated Population
- Conditional Probability Distribution
- Linkage Disequilibrium Analysis
- Marker Allele Frequency
Background
Linkage disequilibrium (LD) has long been one of the central topics in evolutionary and population genetics. Linkage disequilibrium refers to non-random association of alleles at different linked or unlinked loci in a population. Inference about LD provides useful information for distinguishing between alternative evolutionary models of genetic polymorphisms within or divergence between populations [1]. The current surge of population based association studies has reported identification of causal genetic variants of disease susceptibilities in humans [2] and complex genetic variation in plants and animals [3, 4]. The kernel of these studies is inference of LD between the genetic variants and functional loci that are closely genetically linked. Thus, adequate prediction of LD is obviously crucial for reliability and accuracy of these studies.
The coefficient of LD between two biallelic loci is defined as D = f_{ AB }- f_{ A }f_{ B }in a randomly mating population, where f_{ AB }, f_{ A }and f_{ B }are frequencies of gametes AB, alleles A and B in the population. The genetic parameter has been re-parameterized into different forms for various purposes of LD analysis [5]. Hill proposed the well known "chromosome counting" method to estimate the parameter by using data of genotypes at the two loci from a random sample [6]. It has been widely used in population genetic analyses and studies on linkage disequilibrium based mapping [7, 8]. The principle of the analysis has also been employed to develop widely used methods for predicting haplotypes of DNA markers in natural populations [9, 10]. In practice, however, it is very rare that the samples for LD analyses are truly randomly collected from the population under study. For example, the samples used in many association studies or population genomics analyses were so collected that the frequencies of some genotypes are artificially inflated to ensure that genotypes involving a rare allele are well represented [11, 12]. Weir and Cockerham explored the consequences of implementing the method to estimate LD by using the samples in which some genotypes are missing and stressed that the method should not be used to estimate the parameter from non-random samples [13], but neither appropriate theory nor method has been developed for inferring population disequilibrium from non-random samples.
In this paper, we develop a new method to calculate the maximum likelihood estimate of the coefficient of linkage disequilibrium between genes at any pair of polymorphic loci in any randomly matting population by making use of samples of genotype data, which are non-randomly collected from the population of interest. On the basis of simulation studies, we demonstrate that bias in estimates of the disequilibrium parameter from Hill's method arising from the use of non-random samples can be substantially reduced by implementing the new method. We compared analyses of a 'case and control' dataset of β- thalassemia using three different methods: Hill's, haplotype prediction by the computer software PHASE2.1.1 and the method developed in the present study.
Results
We developed a likelihood-based statistical approach to estimate the coefficient of linkage disequilibrium between a pair of polymorphic loci in a natural population and to test for significance of the disequilibrium by making use of the samples that are not randomly collected from the population. The method uses information from the conditional distribution of genotypes at one locus given genotypes at the other in formulating the statistical analysis with non-random samples. This is in contrast to the approach proposed by Hill [6], which relies on information of a joint distribution of genotypes at the polymorphic loci and estimates the disequilibrium parameter from using the samples randomly collected from the population under question. Hill's method has been extended or converted into various forms/approaches that are widely used in the current surge of genetic association studies and population genetic analyses [11, 12, 14–18]. Here, our analysis is focused on Hill's method (H) and the method (L) developed in the present study.
To explore the adequacy of the methods H and L in estimating LD and their statistical power in detecting LD, we first conducted a simulation study to generate samples collected from the simulated population by sampling with various degrees of non-randomness. We then implemented the methods to analyze both the simulated datasets and the real data of 20.693 kb DNA sequence surrounding the β-globin gene from a 'case and control' study with β-thalassemia [19].
Simulation Study
Conditional probability distribution of disease genotypes for a given marker genotype
MM | Mm | mm | ||||||
---|---|---|---|---|---|---|---|---|
AA | Aa | aa | AA | Aa | aa | AA | Aa | aa |
Q ^{2} | 2Q(1 - Q) | (1 - Q)^{2} | QR | Q + R - 2QR | (1 - Q)(1 - R) | R ^{2} | 2R(1 - R) | (1 - R)^{2} |
n _{11} | n _{12} | n _{13} | n _{21} | n _{22} | n _{23} | n _{31} | n _{32} | n _{33} |
Prediction of Sampling Scheme I
Pop. | p | q | (D_{ min }, D_{ max }) | D | ||
---|---|---|---|---|---|---|
1 | 0.5 | 0.5 | (-0.25, 0.25) | 0.20 | 0.1999 ± 0.0078 | 0.2004 ± 0.0078 |
2 | 0.5 | 0.5 | (-0.25, 0.25) | 0.10 | 0.1002 ± 0.0145 | 0.1003 ± 0.0145 |
3 | 0.3 | 0.3 | (-0.09, 0.21) | 0.09 | 0.0898 ± 0.0133 | 0.0899 ± 0.0125 |
4 | 0.7 | 0.7 | (-0.09, 0.21) | 0.09 | 0.0895 ± 50.0133 | 0.0896 ± 0.0126 |
5 | 0.3 | 0.5 | (-0.15 0.15) | 0.10 | 0.0997 ± 0.0120 | 0.0998 ± 0.0111 |
6 | 0.5 | 0.3 | (-0.15, 0.15) | 0.10 | 0.0995 ± 0.0121 | 0.0993 ± 0.0109 |
7 | 0.5 | 0.5 | (-0.25, 0.25) | -0.20 | -0.1995 ± 0.0081 | -0.1998 ± 0.0081 |
8 | 0.5 | 0.5 | (-0.25, 0.25) | -0.10 | -0.0996 ± 0.0146 | -0.0997 ± 0.01460 |
9 | 0.3 | 0.3 | (-0.09, 0.21) | -0.09 | -0.0896 ± 0.0074 | -0.0899 ± 0.0068 |
10 | 0.7 | 0.7 | (-0.09, 0.21) | -0.09 | -0.0897 ± 0.0073 | -0.0899 ± 0.0065 |
11 | 0.3 | 0.5 | (-0.15 0.15) | -0.10 | -0.1000 ± 0.0124 | -0.1000 ± 0.0117 |
12 | 0.5 | 0.3 | (-0.15, 0.15) | -0.10 | -0.0995 ± 0.0120 | -0.0993 ± 0.0111 |
Prediction of Sampling Scheme II
Pop. | D | n_{1•} = 0 | n_{2•} = 0 | n_{3•} = 0 | n_{11} = 0 | n_{22} = 0 | n_{33} = 0 | |
---|---|---|---|---|---|---|---|---|
1 | 0.20 | 0.18 ± 0.01 0.20 ± 0.03 | 0.20 ± 0.01 0.20 ± 0.01 | 0.18 ± 0.01 0.20 ± 0.03 | 0.17 ± 0.01 0.18 ± 0.04 | 0.17 ± 0.01 0.17 ± 0.01 | 0.17 ± 0.01 0.18 ± 0.04 | |
2 | 0.10 | 0.09 ± 0.02 0.10 ± 0.02 | 0.10 ± 0.01 0.10 ± 0.01 | 0.09 ± 0.02 0.10 ± 0.02 | 0.05 ± 0.02 0.06 ± 0.02 | 0.07 ± 0.01 0.07 ± 0.01 | 0.05 ± 0.02 0.06 ± 0.02 | |
3 | 0.09 | 0.08 ± 0.02 0.09 ± 0.01 | 0.06 ± 0.01 0.10 ± 0.04 | 0.10 ± 0.02 0.09 ± 0.01 | 0.07 ± 0.01 0.08 ± 0.01 | 0.04 ± 0.01 0.09 ± 0.06 | 0.00 ± 0.02 0.04 ± 0.01 | |
4 | 0.09 | 0.10 ± 0.02 0.09 ± 0.01 | 0.06 ± 0.01 0.09 ± 0.01 | 0.08 ± 0.01 0.09 ± 0.01 | 0.00 ± 0.02 0.04 ± 0.02 | 0.04 ± 0.01 0.06 ± 0.01 | 0.07 ± 0.01 0.08 ± 0.01 | |
5 | 0.10 | 0.08 ± 0.01 0.10 ± 0.01 | 0.06 ± 0.01 0.10 ± 0.01 | 0.12 ± 0.01 0.10 ± 0.01 | 0.07 ± 0.01 0.08 ± 0.01 | 0.07 ± 0.01 0.08 ± 0.01 | 0.05 ± 0.02 0.06 ± 0.02 | |
6 | 0.10 | 0.09 ± 0.01 0.10 ± 0.01 | 0.10 ± 0.01 0.10 ± 0.01 | 0.09 ± 0.01 0.10 ± 0.01 | 0.07 ± 0.01 0.08 ± 0.01 | 0.07 ± 0.01 0.08 ± 0.01 | 0.05 ± 0.02 0.06 ± 0.02 |
Sampling scheme III considered the scenario that the disease allele had a low frequency but generated an equal number of the case individuals (i.e. either a homozygote or a heterozygote of the disease allele) and the controls (i.e. a homozygote of the wild type allele). To mimic the sampling scheme, we randomly generated a given number of 'case' or 'control' individuals for each of seven simulated populations. From these individuals, 100 'case' and another 100 'control' individuals were randomly collected, making a constant sample size of 200. The samples so generated make a severely non-random collection of the cases but they present a typical example of the samples widely used in many current genetic association studies with a case-control design in which roughly an equal number of sporadic case individuals and control individuals were collected from the population under question [11, 14–19]. Use of the samples so collected raised a question of how the allele frequency parameters, p and q, can be calculated and used to estimate the disequilibrium parameter, D. We proposed to calculate p, the marker allele frequency, from the control sub-samples, and explored two alternative ways to obtain the value of q, the disease allele frequency. Firstly, q was obtained from an independent population survey such as a prior epidemiological study or population survey. To assimilate this scenario, we used the stimulated value of q in estimation of D. Secondly, we explored the use of q values directly estimated from the case-control sample in estimation of D.
Prediction of Sampling Scheme III
p | q | (D_{ min }, D_{ max }) | D | q was from population survey | q was from sample estimation | ||
---|---|---|---|---|---|---|---|
0.6 | 0.005 | (-0.003,0.002) | -0.002 | -0.011 ± 0.149 | -0.002 ± 0.000 | -0.113 ± 0.015 | -0.071 ± 0.011 |
0.5 | 0.01 | (-0.005,0.005) | 0.004 | 0.280 ± 0.011 | 0.004 ± 0.001 | 0.108 ± 0.013 | 0.080 ± 0.013 |
0.5 | 0.02 | (-0.010, 0.010) | 0.008 | 0.273 ± 0.010 | 0.008 ± 0.001 | 0.110 ± 0.014 | 0.081 ± 0.013 |
0.3 | 0.03 | (-0.009, 0.021) | 0.010 | 0.191 ± 0.026 | 0.011 ± 0.002 | 0.104 ± 0.019 | 0.057 ± 0.018 |
0.7 | 0.04 | (-0.028, 0.012) | 0.010 | 0.309 ± 0.016 | 0.011 ± 0.002 | 0.071 ± 0.012 | 0.061 ± 0.017 |
0.3 | 0.05 | (-0.015, 0.035) | 0.020 | 0.192 ± 0.044 | 0.021 ± 0.004 | 0.122 ± 0.017 | 0.066 ± 0.017 |
0.5 | 0.10 | (-0.050, 0.050) | 0.040 | 0.227 ± 0.008 | 0.045 ± 0.006 | 0.124 ± 0.014 | 0.088 ± 0.012 |
Prediction from case-control samples with various sample sizes
p | q | D | n= 100 | n= 200 | n= 400 | n= 800 |
---|---|---|---|---|---|---|
0.6 | 0.005 | -0.002 | 0.002 ± 0.001 | -0.002 ± 0.002 | -0.002 ± 0.000 | -0.002 ± 0.000 |
0.5 | 0.010 | 0.004 | 0.004 ± 0.001 | 0.004 ± 0.001 | 0.004 ± 0.000 | 0.004 ± 0.000 |
0.5 | 0.020 | 0.008 | 0.008 ± 0.002 | 0.008 ± 0.001 | 0.008 ± 0.001 | 0.008 ± 0.001 |
0.3 | 0.030 | 0.010 | 0.010 ± 0.003 | 0.011 ± 0.002 | 0.010 ± 0.002 | 0.010 ± 0.001 |
0.7 | 0.040 | 0.010 | 0.010 ± 0.004 | 0.011 ± 0.002 | 0.010 ± 0.002 | 0.010 ± 0.001 |
0.3 | 0.050 | 0.020 | 0.021 ± 0.005 | 0.021 ± 0.004 | 0.021 ± 0.003 | 0.021 ± 0.002 |
0.5 | 0.100 | 0.040 | 0.044 ± 0.008 | 0.045 ± 0.006 | 0.046 ± 0.004 | 0.045 ± 0.003 |
LOD ± s.d. | ||||||
0.6 | 0.005 | -0.002 | 1.852 ± 1.047 | 3.619 ± 1.467 | 6.913 ± 2.112 | 13.774 ± 2.927 |
0.5 | 0.010 | 0.004 | 1.949 ± 1.036 | 3.692 ± 1.454 | 7.397 ± 1.959 | 14.390 ± 2.809 |
0.5 | 0.020 | 0.008 | 2.010 ± 1.045 | 3.843 ± 1.483 | 7.657 ± 2.070 | 14.955 ± 2.832 |
0.3 | 0.030 | 0.010 | 1.545 ± 1.047 | 2.957 ± 1.395 | 5.596 ± 1.977 | 11.085 ± 2.717 |
0.7 | 0.040 | 0.010 | 1.115 ± 0.707 | 2.093 ± 1.043 | 3.958 ± 1.377 | 7.682 ± 2.063 |
0.3 | 0.050 | 0.020 | 2.325 ± 1.278 | 4.393 ± 1.690 | 8.648 ± 2.452 | 17.191 ± 3.332 |
0.5 | 0.100 | 0.040 | 2.493 ± 1.100 | 4.860 ± 1.549 | 9.560 ± 2.165 | 18.948 ± 2.994 |
LD estimation from case and control samples with varying proportions
p | q | D | c:c= 3/4:1/4 | c:c= 2/3:1/3 | c:c= 1/3:2/3 | c:c= 1/4:3/4 |
---|---|---|---|---|---|---|
0.6 | 0.005 | -0.002 | 0.002 ± 0.000 | -0.002 ± 0.000 | -0.002 ± 0.000 | -0.002 ± 0.000 |
0.5 | 0.010 | 0.004 | 0.004 ± 0.001 | 0.004 ± 0.001 | 0.004 ± 0.001 | 0.004 ± 0.001 |
0.5 | 0.020 | 0.008 | 0.008 ± 0.001 | 0.008 ± 0.001 | 0.008 ± 0.001 | 0.008 ± 0.002 |
0.3 | 0.030 | 0.010 | 0.010 ± 0.003 | 0.010 ± 0.003 | 0.010 ± 0.002 | 0.010 ± 0.003 |
0.7 | 0.040 | 0.010 | 0.010 ± 0.003 | 0.011 ± 0.002 | 0.010 ± 0.002 | 0.010 ± 0.003 |
0.3 | 0.050 | 0.020 | 0.021 ± 0.004 | 0.021 ± 0.004 | 0.021 ± 0.004 | 0.021 ± 0.004 |
0.5 | 0.100 | 0.040 | 0.043 ± 0.007 | 0.044 ± 0.006 | 0.045 ± 0.006 | 0.045 ± 0.007 |
LOD ± s.d. | ||||||
0.6 | 0.005 | -0.002 | 1.814 ± 0.855 | 2.420 ± 1.063 | 4.707 ± 2.172 | 5.362 ± 2.695 |
0.5 | 0.010 | 0.004 | 1.906 ± 0.832 | 2.493 ± 0.997 | 4.915 ± 1.994 | 5.715 ± 2.502 |
0.5 | 0.020 | 0.008 | 1.969 ± 0.812 | 2.579 ± 1.029 | 5.112 ± 2.160 | 5.916 ± 2.653 |
0.3 | 0.030 | 0.010 | 1.481 ± 0.812 | 1.950 ± 1.025 | 3.888 ± 2.011 | 4.463 ± 2.519 |
0.7 | 0.040 | 0.010 | 1.063 ± 0.580 | 1.453 ± 0.724 | 2.714 ± 1.368 | 3.130 ± 1.783 |
0.3 | 0.050 | 0.020 | 2.162 ± 0.966 | 2.982 ± 1.161 | 5.876 ± 2.446 | 6.695 ± 3.029 |
0.5 | 0.100 | 0.040 | 2.275 ± 0.816 | 3.152 ± 1.071 | 6.657 ± 2.241 | 7.616 ± 2.772 |
Analysis of β-thalassemia dataset
β-thalassemia is an autosomal recessive hemoglobinopathy caused by mutations in the β-globin (HBB) gene (OMIM 141900). The disorder is one of the most common inherited hemoglobinopathies in the world, with estimates of carrier frequencies ranging from 3 to 10% in some areas of the tropics and subtropics including southern China [19, 21, 22]. A frame shift mutation in codons 41 and 42, a 4-bp deletion (-CTTT), of the human β-globin gene represents the most common β-thalassemia mutations in East and Southeast Asia. The population frequency of the deletion is as high as 3% in South China [19]. To survey the distribution of linkage disequilibrium among the polymorphic sites surrounding the β-globin gene, Zhang et al collected a sample of 40 Chinese individuals, including 16 β^{CD41/42} thalassemia heterozygotes and 24 normal individuals [19]. They directly sequenced a 15.933-kb DNA region spanning 20.693 kb of the β-globin cluster surrounding the deletion and detected 50 bi-allelic sites in the sequenced region. All individuals in the sample were genotyped at the polymorphic markers. This dataset represents a typical example of the selected sample in which disease carriers are deliberately enriched and no homozygote of the disease allele (i.e. at the deletion locus) is present in the sample.
Discussion
The past decade has witnessed great progress in high-throughput detection and genotyping of single nucleotide polymorphisms (SNPs) in the genomes of plants, animals and humans. This has stimulated tremendous interest in mapping and identifying subtle genetic variants contributing to phenotypic variation of complex traits in natural populations through detecting linkage disequilibrium maintained in the populations by close linkage between alleles at genetic polymorphic sites and at trait loci. However, a common concern of association studies is the high proportion of false positive or false negative tests of association of trait phenotype with causal genetic polymorphisms as well as the limited statistical power in detecting genuine associations [23, 24]. A rich pool of literature has been focused on exploring factors that cause these problems and seeking for solutions to them. The most prominent among these is population stratification that could cause both false positive and false negative inferences of association [25].
Although it has been well established that skewed sampling from a population with a linkage equilibrium distribution of multi-locus genotypes may result in spurious linkage disequilibrium [26], there has not been a comprehensive investigation of the consequences of non-random samples on statistical inference of LD from populations under disequilibrium. We demonstrate in the present study that the use of non-random samples could result in severely biased estimates of LD from the method proposed for random samples. The simulation study showed that the estimates could be so biased as to be outside the theoretical limits of the corresponding simulated values and the biases can be either up- or downwards. These results indicate that the non-randomness of the sample may result in considerable false positive or false negative inference of the disequilibrium parameter and, in turn, false positives or false negatives in association analyses in which significant degree of marker-disease association implies significant linkage disequilibrium. Instead of considering the joint probability distribution of genotypes at the marker and disease loci in estimating LD from random samples [6, 13], we propose the use of the conditional probability distribution of disease genotypes given any marker genotype in developing a new method to estimate the parameter. The method avoids or effectively alleviates the influence of non-random presentation of any marker-disease genotype in the samples on the parameter estimation. On the basis of simulation studies, we show that the method yields equally adequate estimates of LD to the method previously proposed [6] and currently widely cited in the literature when individual genotypes are randomly sampled from the populations. However, the method confers significant improvement over the current method when the parameter estimation is made from using artificially selected samples. Methodologically, the improvement in the parameter estimation of the method developed in the present study over the current methods [6, 13] can be explained by their difference in extracting information of linkage disequilibrium between the two loci of interest from the samples under study. The present method uses the conditional probability distribution of genotypes at one locus on any given genotype at the other locus as illustrated in Table 1. When the sample of study is collected in such a way that genotype(s) at one of the two loci undergo selection, for example, the case-control samples where genotypes at the disease locus are strongly selected, the joint distribution of genotypes at the two loci in the sample deviates greatly from the joint genotypic distribution in the population from which the sample is collected. However, the conditional distribution of genotypes at one locus given any genotype at the other selected or unselected locus in the sample remains approximately the same as that in the population. Hence the analysis based on the conditional genotypic distribution is more robust to non-randomness of the samples to infer linkage disequilibrium in populations of interest than that based on the joint genotypic distribution.
The 'case and control' design used in many association studies to screen for genetic polymorphisms in significant association with phenotypic variation probably illustrates the most popular example of LD analysis with non-random samples [11, 12, 14–18]. Although the base populations under investigation may be randomly mating with respect to genotypes at marker-disease loci, the samples used in the studies are collected so that case individuals are well represented and thus create severely non-random presentation of the populations. Given that the analysis of the samples is to infer the situation in the base populations, accurate inference of LD from the samples is obviously crucial for reliability of these analyses. The β-thalassemia data analysis in the present study represents a typical example of such studies. The analysis with the dataset, although very limited in sample size, highlights the importance of adequately tackling the non-randomness in the 'case and control' studies in order to achieve reliable assessment of LD. It clearly demonstrates the robustness of the method developed in the present study to the non-randomness. Previous methods, which are widely implemented in the current literature of 'case and control' studies are only suitable for analysis with random samples and so can result in seriously biased inference of the disequilibrium parameter.
Methodologically, the present study has been focused on the most prominent linkage disequilibrium measure, D as defined above. There are several other measures, such as D' or r^{2} etc, frequently used in the literature of genetic association study or population genetic analysis. These latter measures are either a scaled or standardized form of the basic disequilibrium parameter D. Bias in estimate of D will be inherent to that of transformed versions of the parameter. For example, we re-analyzed the β-thalassemia data by using r^{2}, which is defined as D^{2}/p(1-p)q(1-q) in the present notation, as the disequilibrium measure and showed the analysis in Additional file 3. The disequilibrium distribution shows an almost identical pattern to that demonstrated in Figure 1, in which D was the disequilibrium measure.
One of the key properties of 'case and control' designs is its statistical power in detecting association between a genetic polymorphic marker to the phenotype of a disease trait. It has been widely accepted that level of LD is a critical factor in determining the power of 'case and control' designs to detect significance of genetic association [27, 28]. Several studies have focused on investigating factors affecting statistical power of the 'case and control' design, including marker allele frequency and errors in marker genotype and trait phenotype [29–31]. An equal proportion of cases and controls were proposed in these studies and implemented in many real 'case and control' experimental analyses [11, 31]. The present study investigated the impact of using varying proportions of the cases and controls on the power of the 'case and control' design in detecting linkage disequilibrium and shows that, for a given size of the 'case and control' sample, increasing the proportion of the 'cases' decreases rather than increases the statistical power (Table 6). In fact, increasing the proportion of control individuals in the 'case and control' samples alleviates the effects of non-randomness of the samples. This result indicates a need of reconsideration of the commonly accepted sampling strategy for 'case and control' study designs. We re-examined the question by implementing the chi-square based test proposed to test for significance of difference in marker allele frequency between cases and controls. The analysis was summarized as an Additional file 4 and shows that use of an equal proportion of cases and controls in the case-control design is favoured for a higher statistical power to detect the genetic associations. However, it has been well established that the chi-square based association test is highly vulnerable to deviation of genotypic distribution from the Hardy-Weinberg equilibrium [32]. Any violation to the equilibrium may result in severe type I error. Moreover, it is clear from Additional file 2 that the equilibrium does not usually hold in these samples.
It is well established that LD based association analysis is effective only for the genes underlying Mendelian traits or the genes with major genetic effects on polygenic traits [23, 33]. In the present study we assumed that genotypes at the putative disease locus are observable as are those at the marker locus. The assumption holds for Mendelian traits but may be questionable for quantitative traits. In fact, the theoretical model and analysis developed in the present study can be incorporated into the statistical framework we previously developed for detecting and estimating linkage disequilibrium between a genetic marker and a locus affecting a quantitative trait showing continuous or dichotomous phenotypic variation [20, 34, 35]. The quantitative genetic model allows allelic frequencies at the marker and trait loci, the coefficient of linkage disequilibrium between the two loci, genetic effects at the trait locus and the residual variance component to be modelled and inferred. Integration of the two models enables the non-randomness of samples to be properly accounted for in the statistical inference on the genetic parameters.
Conclusions
We demonstrated that the non-randomness may cause seriously biased assessment of LD when using the current methods originally developed for random samples. We have developed a new approach for inferring LD from samples with various degrees of non-randomness, and showed the significantly improved robustness of the present approach over the current methods when non-random samples were to evaluate LD through intensive simulation studies and analysis of a case and control sample of β-thalasemia. As accurate estimation of the disequilibrium parameter is crucial for any association study, in which the case/control design represents a typical example of non-random samples, the present paper highlights the importance of tackling the problem of using non-random samples to the community of LD analysis, and in addition, provides a route to improve statistical reliability in association studies.
Methods
Inferring LD from Using Nonrandom Samples: we consider two biallelic loci M (marker locus) and A (disease locus). There are two alleles at the marker locus, M and m, with population frequencies p and 1-p respectively and two alleles at the disease locus, A and a, with population frequencies q and 1-q. For simplicity, but without loss of generality, we denote by D, the coefficient of linkage disequilibrium between genes at the two loci. The conditional probability (f_{ ij }) of genotypes at one of two loci given a genotype at the other locus can be expressed in term of these genetic parameters when assuming random mating in the population. For example, the disease genotype distribution given any marker genotype is presented in Table 1.
which asymptotically follows a chi-square distribution with 1 degree of freedom. It should be noticed that the above analysis is based on the conditional probability distribution of genotypes at one locus given any genotype at the other of the two loci.
where b_{0} = -(2n_{11} + n_{12} + n_{21})pq, b_{1} = 2npq - (2n_{11} + n_{12} + n_{21})(1 - 2p - 2q) - n_{22}(1 - p - q), b_{2} = 2n(1 - 2p - 2q) - 2(2n_{11} + n_{12} + n_{21}) - n_{22} and b_{3} = 2n. With , and the MLE of g_{11}, p and q respectively, the MLE of the coefficient of disequilibrium coefficient, D, can be calculated as . Weir and Cockerham stressed that calculation of by solving the cubic equation may avoid the scenario of the iterative procedure may be diverging or converging but to the wrong roots [13]. Because b_{3} = 2n < 0, the cubic must have at least one real root. The theoretical bounds for g_{11} are given by {max(0, p + q - 1), min(p,q)} [13]. Significance of the MLE of D can also be tested through a likelihood ratio test statistic with a form similar to equation (3). However, to evaluate the likelihood function, one needs to use the joint genotype probability distribution given by Hill [6]. It should be noticed that the above analysis is based on the joint probability distribution of genotypes at the two loci of interest.
The above analyses show that the disequilibrium parameter can be estimated and detected from the two methods. In the Results section, we explored the performance of these methods at inferring the linkage disequilibrium using a simulation study and by analysis of real data.
Declarations
Acknowledgements
We thank Dr. Xiang-Min Xu for allowing us to share the thalassemia data analysed in the present study. We are grateful to three anonymous reviewers for their constructive comments which have helped improve presentation and clarity of an earlier version of the paper. This study was supported by a research grant from Biology and Biotechnology Research council (BBSRC, UK) and by research grants from China's National Natural Science Foundation to ZWL.
^{†} We would like to dedicate this paper to Professor Michael J. Kearsey, our mentor, for his 70^{th} birthday.
Authors’ Affiliations
References
- Lewontin RC, Kojima K-I: The evolutionary dynamics of complex polymorphisms. Evolution. 1960, 4: 458-472. 10.2307/2405995.View ArticleGoogle Scholar
- Easton DF, Pooley KA, Dunning AM, Pharoah PDP, Thompson D, Ballinger DG, Struewing JP, Morrison J, Field H, Luben R, Wareham N, Ahmed S, Healey CS, Bowman R, Meyer KB, Haiman CA, Kolonel LK, Henderson BE, Le Marchand L, Brennan P, Sangrajrang S, Gaborieau V, Odefrey F, Shen C-Y, Wu P-E, Wang H-C, Eccles D, Evans DG, Peto J, Fletcher O: Genome-wide association study identifies novel breast cancer susceptibility loci. Nature. 2007, 447: 1087-93. 10.1038/nature05887.PubMed CentralPubMedView ArticleGoogle Scholar
- Remington DL, Thornsberry JM, Matsuoka Y, Wilson LM, Whitt SR, Doeblay J, Kresovich S, Goodman MM, Buckler ES: Structure of linkage disequilibrium and phenotypic associations in the maize genome. Proceedings of the National Academy of Sciences of the United States of America. 2001, 98: 11479-11484. 10.1073/pnas.201394398.PubMed CentralPubMedView ArticleGoogle Scholar
- Valdar W, Solberg LC, Gauguier D, Burnett S, Klenerman P, Cookson Wo, Taylor MS, Rawlins JNP, Mott R, Flint J: Genome-wide genetic association of complex traits in heterogeneous stock mice. Nature Genetics. 2006, 38: 879-887. 10.1038/ng1840.PubMedView ArticleGoogle Scholar
- Devlin B, Risch N: A comparison of linkage disequilibrium measures for fine-scale mapping. Genomics. 1995, 29: 311-322. 10.1006/geno.1995.9003.PubMedView ArticleGoogle Scholar
- Hill WG: Estimation of linkage disequilibrium in randomly mating populations. Heredity. 1974, 33: 229-239. 10.1038/hdy.1974.89.PubMedView ArticleGoogle Scholar
- Carlson CS, Eberle MA, Rieder MJ, Smith JD, Kruglyak L, Nickerson DA: Additional SNPs and linkage-disequilibrium analyses are necessary for whole-genome association studies in humans. Nature Genetics. 2003, 33: 518-521. 10.1038/ng1128.PubMedView ArticleGoogle Scholar
- Slatkin M: Linkage disequilibrium - understanding the evolutionary past and mapping the medical future. Nature Reviews Genetics. 2008, 9: 477-485. 10.1038/nrg2361.PubMedView ArticleGoogle Scholar
- Long JC, Williams RC, Urbanek M: An E-M algorithm and testing strategy for multiple-locus haplotypes. American Journal of Human Genetics. 1995, 56: 799-810.PubMed CentralPubMedGoogle Scholar
- Stephens M, Smith NJ, Donnelly P: A new statistical method for haplotype reconstruction from population data. American Journal of Human Genetics. 2001, 68: 978-989. 10.1086/319501.PubMed CentralPubMedView ArticleGoogle Scholar
- Fung H-C, Scholz S, Matarin M, Simon-Sanchez J, Hernandez D, Britton A, Gibbs JR, Langefeld C, Stiegert ML, Schymick J, Okun MS, Mandel RJ, Fernandez HH, Foote KD, Rodriguez RL, Peckham E, De Vrieze FW, Gwinn-Hardy K, Hardy JA, Singleton A: Genome-wide genotyping in Parkinson's disease and neurologically normal controls first stage analysis and public release of data. Lancet Neurobiology. 2006, 5: 911-16. 10.1016/S1474-4422(06)70578-6.View ArticleGoogle Scholar
- Dawson E, Abecasis GR, Bumpstead S, Chen Y, Hunt S, Beare DM, Pabial J, Dibling T, Tinsley E, Kirby S, Carter D, Papaspyridonos M, Livingstone S, Ganske R, Lohmussaar E, Zernant J, Tonisson N, Remm M, Magi R, Puurand T, Vilo J, Kurg A, Rice K, Deloukas P, Mott R, Metspalu A, Bentley DR, Cardon LR, Dunham I: A first-generation linkage disequilibrium map of human chromosome 22. Nature. 2002, 418: 544-48. 10.1038/nature00864.PubMedView ArticleGoogle Scholar
- Weir BS, Cockerham CC: Estimation of linkage disequilibrium in randomly mating populations. Heredity. 1979, 42: 105-111. 10.1038/hdy.1979.10.View ArticleGoogle Scholar
- Scott LJ, Bonnycastle LL, Willer CJ, Sprau AG, Jackson AU, Narisu N, Duren WL, Chines PS, Stringham HM, Erdos MR, Valle TT, Tuomilehto J, Bergman RN, Mohlke KL, Collins FS, Boehnke M: Association of transcription factor 7-like 2 (TCF7L2) variants with type 2 diabetes in a Finnish sample. Diabetes. 2006, 55: 2649-53. 10.2337/db06-0341.PubMedView ArticleGoogle Scholar
- The Welcome Trust Case Control Consortium: Genome-wide association studye of 14,000 cases of seven common diseases and 3,000 share controls. Nature. 2007, 447: 661-78. 10.1038/nature05911.View ArticleGoogle Scholar
- The International HapMap Consortium: A second generation human haplotype map of over 3.1 million SNPs. Nature. 2007, 449: 851-861. 10.1038/nature06258.PubMed CentralView ArticleGoogle Scholar
- Maris JM, Mosse YP, Bradfield JP, Hou CP, Monni S, Scott RH, Asgharzadeh S, Attiyeh EF, Diskin SJ, Laudenslager M, Winter C, Cole KA, Glessner JT, Kim C, Frackelton EC, Casalunovo T, Eckert AW, Capasso M, Rappaport EF, McConville C, London WB, Seeger RC, Rahman N, Devoto M, Grant SFA, Li HZ, Hakonarson H: Chromosome 6p22 locus associated with clinically aggressive neuroblastoma. New England Journal of Medicine. 2008, 358: 2585-93. 10.1056/NEJMoa0708698.PubMed CentralPubMedView ArticleGoogle Scholar
- Australia and New Zealand Multiple Sclerosis Genetics Consortium (ANZgene): Genome-wide association study identifies new multiple sclerosis susceptibility loci on chromosomes 12 and 20. Nature Genetics. 2009, 41: 824-28. 10.1038/ng.396.View ArticleGoogle Scholar
- Zhang W, Cai WW, Zhou WP, Li HP, Li L, Yan W, Deng QK, Zhang YP, Fu YX, Xu XM: Evidence of gene conversion in the evolutionary process of the codon 41/42 (-CTTT) mutation causing beta-thalassemia in southern China. Journal of Molecular Evolution. 2008, 66: 436-445. 10.1007/s00239-008-9096-2.PubMedView ArticleGoogle Scholar
- Luo ZW: Detecting linkage disequilibrium between a polymorphic marker locus and a trait locus in natural populations. Heredity. 1998, 80: 198-208. 10.1046/j.1365-2540.1998.00275.x.PubMedView ArticleGoogle Scholar
- Weatherall DJ, Clegg JB: Inherited haemoglobin disorders an increasing global health problem. Bulletin of the World Health Organization. 2001, 79: 704-712.PubMed CentralPubMedGoogle Scholar
- Xu XM, Zhou YQ, Luo GX, Liao C, Zhou M, Chen PY, Lu JP, Jia SQ, Xiao GF, Shen X, Li J, Chen HP, Xia YY, Wen YX, Mo QH, Li WD, Li YY, Zhuo LW, Wang ZQ, Chen YJ, Qin CH, Zhong M: The prevalence and spectrum of alpha and beta thalassaemia in Guangdong Province implications for the future health burden and population screening. Journal of Clinical Pathology. 2004, 57: 517-522. 10.1136/jcp.2003.014456.PubMed CentralPubMedView ArticleGoogle Scholar
- Cardon LR, Palmer LJ: Population stratification and spurious allelic association. Lancet. 2003, 361: 598-604. 10.1016/S0140-6736(03)12520-2.PubMedView ArticleGoogle Scholar
- Balding DJ: A tutorial on statistical methods for population association studies. Nature Reviews Genetics. 2006, 7: 781-791. 10.1038/nrg1916.PubMedView ArticleGoogle Scholar
- Pritchard JK, Stephens M, Rosenberg NA, Donnelly P: Inference of population structure using multilocus genotype data. Genetics. 2000, 155: 945-59.PubMed CentralPubMedGoogle Scholar
- Avery PJ, Hill WG: Distribution of linkage disequilibrium with selection and finite population size. Genetical Research. 1979, 33: 29-48. 10.1017/S0016672300018140.View ArticleGoogle Scholar
- Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, Higgins J, DeFelice M, Lochner A, Faggart M, Liu-Cordero SN, Rotimi C, Adeyemo A, Cooper R, Ward R, Lander ES, Daly MJ, Altshuler D: The structure of haplotype blocks in the human genome. Science. 2002, 296: 2225-2229. 10.1126/science.1069424.PubMedView ArticleGoogle Scholar
- Weir BS: Linkage disequilibrium and association mapping. Annual Review of Genomics and Human Genetics. 2008, 9: 129-142. 10.1146/annurev.genom.9.081307.164347.PubMedView ArticleGoogle Scholar
- Edwardes MD: Sample size requirements for case-control study designs. BMC Med Res Methodol. 2001, 1: 11-10.1186/1471-2288-1-11.PubMed CentralPubMedView ArticleGoogle Scholar
- Gordon D, Finch SJ: Factors affecting statistical power in the detection of genetic association. Journal of Clinical Investigation. 2005, 115: 1408-1418. 10.1172/JCI24756.PubMed CentralPubMedView ArticleGoogle Scholar
- Barral S, Haynes C, Stone M, Gordon D: LRTae: improving statistical power for genetic association with case/control data when phenotype and/or genotype misclassification errors are present. BMC Genetics. 2006, 7: 5-10.1186/1471-2156-7-24.View ArticleGoogle Scholar
- Sasieni PD: From genotypes to genes doubling the sample size. Biometrics. 1977, 53: 1253-1261. 10.2307/2533494.View ArticleGoogle Scholar
- Yu JM, Buckler ES: Genetic association mapping and genome organization of maize. Current Opinion in Biotechnology. 2006, 17: 155-160.PubMedView ArticleGoogle Scholar
- Luo ZW, Tao SH, Zeng ZB: Inferring linkage disequilibrium between a polymorphic marker locus and a trait locus in natural populations. Genetics. 2006, 156: 457-467.Google Scholar
- Luo ZW, Wu CI: Modeling linkage disequilibrium between a polymorphic marker locus and a locus affecting complex dichotomous traits in natural populations. Genetics. 2001, 158: 1785-1800.PubMed CentralPubMedGoogle Scholar
- Wolfram S: Mathematica. 1991, Addison-Wesley Publishing Co, Inc, 2Google Scholar
- Riley KF: Mathematical Methods for the Physical Sciences. 1978, Cambridge Univ. PressGoogle Scholar
- Mano S, Yasuda N, Katoh T, Tounai K, Inoko H, Imanishi T, Tamiya G, Gojobori T: Notes on the maximum likelihood estimation of haplotype frequencies. Ann Hum Genet. 2004, 68: 257-264. 10.1046/j.1529-8817.2003.00088.x.PubMedView ArticleGoogle Scholar
Copyright
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.