- Methodology article
- Open Access

# Using the longest significance run to estimate region-specific p-values in genetic association mapping studies

- Ie-Bin Lian
^{1}, - Yi-Hsien Lin
^{2}, - Ying-Chao Lin
^{3}, - Hsin-Chou Yang
^{4}, - Chee-Jang Chang
^{5}and - Cathy SJ Fann
^{2}Email author

**9**:246

https://doi.org/10.1186/1471-2105-9-246

© Lian et al; licensee BioMed Central Ltd. 2008

**Received:**09 August 2007**Accepted:**27 May 2008**Published:**27 May 2008

## Abstract

### Background

Association testing is a powerful tool for identifying disease susceptibility genes underlying complex diseases. Technological advances have yielded a dramatic increase in the density of available genetic markers, necessitating an increase in the number of association tests required for the analysis of disease susceptibility genes. As such, multiple-tests corrections have become a critical issue. However the conventional statistical corrections on locus-specific multiple tests usually result in lower power as the number of markers increases. Alternatively, we propose here the application of the longest significant run (*LSR*) method to estimate a region-specific p-value to provide an index for the most likely candidate region.

### Results

An advantage of the *LSR* method relative to procedures based on genotypic data is that only p-value data are needed and hence can be applied extensively to different study designs. In this study the proposed *LSR* method was compared with commonly used methods such as Bonferroni's method and FDR controlling method. We found that while all methods provide good control over false positive rate, *LSR* has much better power and false discovery rate. In the authentic analysis on psoriasis and asthma disease data, the *LSR* method successfully identified important candidate regions and replicated the results of previous association studies.

### Conclusion

The proposed *LSR* method provides an efficient exploratory tool for the analysis of sequences of dense genetic markers. Our results show that the *LSR* method has better power and lower false discovery rate comparing with the locus-specific multiple tests.

## Keywords

- False Discovery Rate
- Tail Probability
- Test Size
- Linkage Disequilibrium Block
- Transmission Disequilibrium Test

## Background

Recently whole genome association studies (WGA) with high density SNP data are becoming popular due to new technology in genotyping (e.g., Affymetrix and Illumina) [1–4]. Optimal study design in whole genome association remains unresolved although the two-stage association test design has gained popularity [5, 6].

In order to improve power, many samples have been used in WGA studies; however the cost for such studies is still expensive even though the expense of genotyping has dropped significantly. One problem common of WGA studies is a high false positive rate if the direct method, based on the simple chi-square test for association, is performed without any correction for multiple testing. By far, the most commonly used remedy is the Bonferroni approach. However, its overly conservative correction might result in power reduction. There have been several methods proposed to circumvent the problem of the over correction of multiple testing procedures, e.g. false discovery rate (FDR) methods [7–9] that control the expected proportion of false rejections, or False Positive Report Probability (FPRP) [10] which requires explicit consideration of the prior probability for each hypothesis under association. Although the FPRP method aims to provide a higher power than traditional association test, the need for priors is not always possible. The FDR controlling method was originally designed for multiple comparisons of independent tests [7], and was then later extended to dependent cases under certain conditions, e.g. assuming the test statistics are either equally positively correlated and normally distributed, or having satisfied so-called the positive regression dependency [11]. As for the high density SNP data that were considered in this study, the aforementioned assumptions need to be justified in advance.

Most direct association tests are locus-specific and therefore seldom account for the association of different markers. Different markers usually have heterogeneous genetic backgrounds, such as allele frequency and marker characteristics. Single-point inference sometimes yields a misleading conclusion for an overall phenomenon. Methods considered multiple markers simultaneously and included logistic regression analysis, haplotype analysis [12, 13], global significance method [14], multivariate association analysis [15], and the consideration of gene-gene interactions [16].

Instead of testing the significance of a single marker, our novel method tests the significance of all markers within a defined region, and therefore can be regarded as a simultaneous test for multiple markers that account for the dependence of close genetic markers.

The proposed "longest significant run" (*LSR*) method is a two-stage procedure. The first stage conducts conventional association tests, such as the chi-square test for the case-control design or the transmission disequilibrium test [17] for the family-based design. Based on the pre-specified size of a given test, the p-value of each test is converted into a zero/one indicator (1 for significance or 0 otherwise). In the second stage, this binary sequence is scanned for the longest region of consecutive 1s (hence "longest significant run") and the results determine whether or not the run is inordinately long or simply a random pattern.

This region-specific testing procedure is motivated by the dependence of association tests on dense markers: if a disease susceptibility gene lies in a specific region, then the disease gene and the nearby markers will show a relatively positive trend of association (i.e., linkage disequilibrium (LD)). A special non-random pattern (i.e., a cluster of positive signals) indicates that disease genes may be included in the candidate region. The evidence supporting such a non-random pattern is then evaluated with the magnitude of the longest run of consecutive significance.

There is a long list of applications for longest run statistics [18, 19], one of which concerns the alignment and testing of the homology among DNA sequences. Considering two aligned sequences with length n, a match of locus between the sequences is assigned to be "1", and a mismatch is "0". More homologous sequences should have larger longest matching subsequences (run) than others [20], and its corresponding probability is used as an important reference to the homology between two sequences.

Given a similar concept, we use the longest significance run to find a region that is most likely to harbour a disease susceptibility gene in association studies. The dependent structure of the binary sequence is obtained from association tests by considering it as a discrete-time Markov chain model. Using extensive simulations, we demonstrate that the LSR approach provides a reasonable model of dependence for association test results whereby the false-positive and false-negative rates are all controlled effectively. The program in *R* code for *LSR* method is available (Additional File 1).

## Results

The program SIMLA version 3.1 (SIMulation of pedigree data for Linkage and Association studies)[21] was used to simulate family-structure genetic data. The program is available [22]. In the process, we generated 1000 trio families with genotypic data for SNP (single nucleotide polymorphism) markers. To determine the effects of the number of SNP markers, we considered two cases for 50 and 100 SNPs in a candidate region. To mimic the scenario of dependent markers, we considered a dense intermarker distance with a mean of 6 kb and a standard deviation of 10.7 kb. Note that in 500 K Affymetrix data, marker distance has an average of 5.8 kb and a standard deviation of 10.7 kb. In the simulation, the intermarker distance is generated from a left-truncated normal distribution with mean 6 kb and standard deviation 10.7 kb. Under the setup, the distribution of intermarker distance is similar to that of real Affymetrix data in the aspects of mean and standard deviation. Furthermore, the prevalence of the disease in the simulation was set as 0.1, and genetic relative risk was set to be 2.0, 2.5 and 3.0. The allele frequencies were 0.075 and 0.1, and an additive disease model was assumed. Note that the use of high relative risk is for observing contrasting powers between the methods under the stringent definition we used for detecting true positive markers (described in next sections). Under a looser definition, the relative risk can be lowered to 1.5 or less.

In the simulation, the location of disease gene was assigned to be within the two central markers, and the disease haplotype was comprised by 15 markers clustering around the disease gene. We had also tried randomly assigning the location of disease genes to be within any non-boundary markers, and found similar results. We considered three haplotype scenarios: (1) three common haplotypes each with a frequency of 30%, (2) two common haplotypes each with a frequency of 45%, and (3) two haplotypes with frequencies of 85% and 5%. Since the results of the three scenarios were similar, we present the scenario of three-common-haplotype.

During the first stage of the *LSR* method, the transmission disequilibrium test from the TDT procedure of SAS/GENETIC package release 8.2.39 [23] was used to test the locus-specific association for each marker. The output p-values {${\widehat{p}}_{j}$, *j* = 1, Λ *n*} were transformed into a sequence of significance indicators {*X*_{
j
}, *j* = 1,Λ,*n*} based on a test size *α*_{1}, where *X*_{
j
}is 1 if the test of the *j*^{th} marker is significant and 0 otherwise. In the second stage, we scanned this binary sequence to identify the longest perfect run of 1, *L*_{0}, the nearly perfect run of 1 allowing one zero within, namely the *L*_{1} and the run of 1 allowing two zeros within, namely the *L*_{2}. For each of these three *LSR* statistics, the corresponding tail probabilities were calculated using the method of Chang *et al*. [20].

### Testing statistics

Although allowing more interruptions (larger k) seems to make the *LSR* approach more plausible, it is helpful only if it can bridge up nearby but separated clusters of "1's". On the other hand, containing irrelevant markers may complicate further analysis to identify the target gene. To compromise the situations we defined the testing statistics, *LSR*_{
{k}
}, as *L*_{
i
}with minimal tail probability, for i from 1 to k and considered only kϕ2. In the simulation, we compared our method with the Bonferroni method, the popular correction used for multiple tests, by evaluating three quantities, namely the false-positive rate, power, and false discovery rate in the following three sections.

### False-positive rate

According to our setup in the simulation, markers in a sequence can be divided into three categories: 2 target markers where the disease gene lain between them, 13 nearby markers in linkage disequilibrium (LD) with the 2 target markers, and other markers that are in linkage equilibrium (LE). For the purpose of stringency, a true positive detection was considered only if at least one of the two target markers was significant under the criterion of each method. For Bonferroni's method, the criterion is that a target marker's p-value < α_{2}/n. For the FDR controlling method, Benjamini and Hochberg [7] proposed the following criterion. The *n* single marker p-values were sorted from smallest to largest: *P*_{(1)} ⋯ *P*_{
(n)
}. Starting from *P*_{(n)}, we compared *P*_{(i)}with i·α_{2}/n. This process was continued as long as *P*_{(i)}> i·α_{2}/n. If k is the turn around point, then significance is declared if a target marker corresponds to the k^{th} smallest p-value. For our *LSR* method, the criterion was that a target marker is covered by a significant *LSR* (p-value< α_{2}). On the other hand, a false positive case was considered if a LE marker was detected under each of the three criteria described above. If a non-target LD marker was selected, neither a false positive nor a true positive would be counted.

*LSR*approaches and Bonferroni approach based on 200 replications for each of the eight scenarios (2 disease gene allele frequencies, 2 relative risk, 2 marker numbers). The test size in the first stage was

*α*

_{1}= 0.1, and the test size was

*α*

_{2}= 0.05 in the second stage. We found that all the false-positive rates were under or similar to the pre-specified nominal test size

*α*

_{2}, indicating that all the methods adequately controlled the false-positive rate.

Comparisons of false positive rate while the SNP data are in linkage disequilibrium.

Marker number | Relative risk | Allele frequency | False positive rate | ||
---|---|---|---|---|---|

Bonferroni | LSR | LSR | |||

50 | 2.0 | 0.075 | 0.035 | 0.01 | 0.015 |

2.0 | 0.1 | 0.04 | 0.01 | 0.01 | |

2.5 | 0.075 | 0.05 | 0.025 | 0.025 | |

2.5 | 0.1 | 0.04 | 0.015 | 0.015 | |

100 | 2.0 | 0.075 | 0.04 | 0.015 | 0.015 |

2.0 | 0.1 | 0.025 | 0.035 | 0.055 | |

2.5 | 0.075 | 0.045 | 0.025 | 0.035 | |

2.5 | 0.1 | 0.02 | 0.025 | 0.035 |

### Power and false discovery rate

Statistical power is the probability that a test correctly rejects the null hypothesis. In our case, it is the probability that a method correctly accesses at least one of 2 target markers with statistical significance. On the other hand, the false discovery rate is the fraction of false positives among all tests declared significant.

*LSR*methods given different nominal test sizes. The test size in the first stage for

*LSR*was

*α*

_{1}= 0.05 and 0.1 and the test size in the second stage was

*α*

_{2}= 0.05. Higher level of significance (0.1) at screening stage resulted in better power than that of

*α*

_{1}= 0.05, in trade of a slightly higher false discovery rate.

*LSR*

_{2}had better performance in both power and false discovery rate than

*LSR*

_{1}. The stringent definition of a "run" for

*LSR*

_{1}yields a relatively narrow candidate region that was more likely to miss the target markers. Both

*LSR*

_{1}and

*LSR*

_{2}had better power and false discovery rate than those from the Bonferroni approach among all the scenarios that we tried.

*LSR*

_{1}has similar power to that of the FDR controlling method, but better false discover rate. In summary, in terms of power,

*LSR*

_{2}>

*LSR*

_{1}~ FDR controlling method > Bonferroni; and in terms of false discovery rate,

*LSR*

_{2}~

*LSR*

_{1}~<FDR controlling method < Bonferroni. The detailed results are available in Tables 2 and 3.

Comparisons of power and false discovery rate among the procedures of Bonferroni correction and two LSR methods.

Marker number | Relative risk | Allele frequency | Power | False discovery rate | Length* | |||||
---|---|---|---|---|---|---|---|---|---|---|

Bonferroni | LSR | LSR | Bonferroni | LSR | LSR | LSR | LSR | |||

50 | 2.0 | 0.075 | 0.15 | 0.16 | 0.255 | 0.14 | 0 | 0 | 4.27 | 5.72 |

2.0 | 0.1 | 0.285 | 0.3 | 0.48 | 0.07 | 0 | 0 | 5.765 | 7.645 | |

2.5 | 0.075 | 0.53 | 0.69 | 0.81 | 0.06 | 0 | 0 | 8.7 | 10.39 | |

2.5 | 0.1 | 0.68 | 0.815 | 0.92 | 0.03 | 0.005 | 0.005 | 9.695 | 11.74 | |

3.0 | 0.075 | 0.83 | 0.91 | 0.98 | 0.04 | 0 | 0 | 10.7 | 12.9 | |

3.0 | 0.1 | 0.925 | 0.99 | 1 | 0.03 | 0 | 0 | 11.5 | 14.0 | |

100 | 2.0 | 0.075 | 0.065 | 0.225 | 0.365 | 0.28 | 0.015 | 0.02 | 4.34 | 5.715 |

2.0 | 0.1 | 0.185 | 0.37 | 0.485 | 0.18 | 0.005 | 0.02 | 5.685 | 7.1 | |

2.5 | 0.075 | 0.47 | 0.72 | 0.87 | 0.11 | 0.005 | 0.025 | 8.435 | 10.26 | |

2.5 | 0.1 | 0.605 | 0.905 | 0.945 | 0.05 | 0.01 | 0.005 | 10.07 | 11.33 | |

3.0 | 0.075 | 0.78 | 0.94 | 0.985 | 0.02 | 0.005 | 0.005 | 10.9 | 13.0 | |

3.0 | 0.1 | 0.91 | 0.99 | 1 | 0.03 | 0.005 | 0.005 | 11.6 | 13.8 |

Comparison of power and false discovery rate among the procedures of Bonferroni correction and two LSR methods.

Marker number | Relative risk | Allele frequency | Power | False discovery rate | Length* | |||||
---|---|---|---|---|---|---|---|---|---|---|

Bonferroni | LSR | LSR | Bonferroni | LSR | LSR | LSR | LSR | |||

50 | 2.0 | 0.075 | 0.15 | 0.31 | 0.44 | 0.14 | 0 | 0.005 | 5.95 | 7.765 |

2.0 | 0.1 | 0.285 | 0.57 | 0.66 | 0.07 | 0 | 0 | 7.74 | 9.66 | |

2.5 | 0.075 | 0.53 | 0.855 | 0.895 | 0.06 | 0.005 | 0.005 | 10.22 | 12.05 | |

2.5 | 0.1 | 0.68 | 0.935 | 0.98 | 0.03 | 0 | 0 | 11.23 | 13.215 | |

3.0 | 0.075 | 0.83 | 0.965 | 0.99 | 0.04 | 0.005 | 0 | 11.9 | 13.9 | |

3.0 | 0.1 | 0.93 | 0.995 | 1 | 0.03 | 0 | 0 | 12.6 | 14.6 | |

100 | 2.0 | 0.075 | 0.065 | 0.33 | 0.5 | 0.28 | 0.03 | 0.04 | 5.91 | 7.51 |

2.0 | 0.1 | 0.185 | 0.55 | 0.635 | 0.18 | 0.02 | 0.055 | 7.28 | 9.055 | |

2.5 | 0.075 | 0.47 | 0.835 | 0.91 | 0.11 | 0.015 | 0.04 | 9.82 | 11.755 | |

2.5 | 0.1 | 0.605 | 0.945 | 0.98 | 0.05 | 0.005 | 0.005 | 11.33 | 13.475 | |

3.0 | 0.075 | 0.78 | 0.985 | 1 | 0.02 | 0.005 | 0 | 12.2 | 14.1 | |

3.0 | 0.1 | 0.91 | 1 | 1 | 0.03 | 0 | 0 | 12.8 | 14.6 |

The good performance of LSR does not indicate that it can completely replace the marker-specific test procedures like the Bonferroni approach. Rather, we consider LSR as a useful screening tool to find a smaller region that possibly contains the disease susceptibility gene. After the region is identified, each maker within the region still needs to be examined biologically. At this stage, *LSR*_{2}, although better than *LSR*_{1} with respect to both power and false discovery rate, pays a token for retaining more makers for further examining. Analogue can be inferred in comparing the performance of using *α*_{1} = 0.1 over *α*_{1} = 0.05 as the threshold in the first stage of LSR, where the former usually includes more markers.

The tail probabilities of *LSR* were calculated under the null hypothesis of no disease marker. In the simulation, Markov independence was assumed for the binary sequences under null hypothesis. Nonetheless, the method can also be applied to the scenario of first order Markov-dependency, if the corresponding transition matrix can be assumed or estimated. We provide a robust approach to estimate the dependency structure of the sequence using the concept of sliding window, which is described in the method section. The method was applied on the following authentic data as illustration.

### Demonstrating the applications of our method to two authentic data sets

#### Example of psoriasis data

We assessed the practical application of the *LSR* method using an authentic genetic data set collected for a psoriasis study [24]. Psoriasis is a common chronic skin disorder characterized by inflammation and scaling. Recent studies indicate a significant association of important psoriasis predisposing loci with chromosome 17 [24, 25]. Helms *et al*. [24] collected 242 European nuclear families comprising 572 psoriasis cases and genotyped 123 genetic markers on chromosome 17q25. The family-based association test TDT-AE [26] was applied to locate the psoriasis susceptibility genes.

In the first stage of *LSR* procedure, we used *α*_{1} = 0.05 as a test size to convert the sequence of p-values into a binary sequence. Under null hypothesis assuming Markov independency, the LSR obtained a significant p-value <0.001, and the region of 12 consecutive markers identified by *L*_{0} captured two functional genes *SLC9A3R1* and *DKFZPP564C103*, which are known to be psoriasis-related. The results are consistent with the finding of Helms *et al*. [24].

However, when we proceeded with the sequential goodness-of-fit tests proposed by Anderson and Goodman [27], the hypothetical assumption of {*X*_{
j
}} being an independent sequence was rejected thereby supporting the hypothesis of it being a first order Markov dependence. Therefore, a sliding windows approach was applied with size of 40 markers and sliding distance of 10 markers to estimates of the transition matrix under the hypothesis of Markov dependency. The same region was identified by *LSR* with p-value 0.007 under the dependency assumption of null hypothesis. Similar results were produced when minor variations in window size and sliding distance were used.

In this example, *L*_{1} and *L*_{2} did not further extend the region of *L*_{0}, therefore, *L*_{0} is sufficient to confirm this candidate region. On the other hand, in order to attain approximately evenly spaced intermarker distance for simple-count approach and avoid combining two distinct chromosome regions, we tried only using the results from the 5^{th} to the 86^{th} loci among the 123 markers. Moreover, we also excluded results from marker loci with less than 50 trios in the analysis to preclude possibly unreliable association results. It resulted in 78 loci included in the final analysis. The consequent analysis also came out with the same significant region with a p-value <0.001. Of particular note, all of the 123 markers had p-value >0.0001, therefore, none of them was significant after direct Bonferroni adjustment.

#### Example of asthma data

Asthma is a common chronic disease characterized by airway inflammation resulting in some symptoms such as the difficulty of breathing, attacks of wheezing and coughing, etc,. In the positional-cloning study of Allen *et al*. [28], there were 224 families containing 239 asthma children and 79 markers covering a region of 384 kb on chromosome 2q14. Immunoglobulin E often causes asthmatic inflammation and has been recognized to be an important concomitant factor of children asthma. Allen *et al*. [28] conducted the transmission disequilibrium tests to assess the relationship between marker loci with asthma and immunoglobulin E.

Using test size *α*_{1} = 0.05, the sequence of p-values was converted into a sequence of twenty-three 1s and fifty-six 0s:

**X** = [0000000011111110111110010011000100000101101011001000000000000000000000000000000].

The goodness-of-fit test suggested that the sequence follows a first order Markov chain. In the second stage of *LSR* method, we estimated the transition probabilities *η*_{00} and *η*_{11} to be 0.647 and 0.7, respectively, and identified *L*_{1} (from marker 543WTC21P at 191388 to 543WTC91P at 252279, details are listed in Additional File 2) with length 13 and yielded the corresponding p-value 0.0002. Note that *L*_{1} contains a significant candidate region of 60891 bp closed to an asthma-suspected gene *DPP10* with functions of catalytic activity and dipetidyl-peptidase IV activity. The conclusion is consistent with Allen *et al*. [28] who found the highly significant STRP marker D2S308 at 261056 in the vicinity of our identified region.

## Discussion

As discussed by Rosenthal [29] and Zaykin *et al*. [30], a series of border-line significant results may together suggest significance. Therefore, it is likely that two consecutive p-values of 0.06 may suggest evidence against the null than one isolated p-value of 0.05. This phenomenon is often observed in the identification of candidate regions of complex disorders if the marginal effect of a disease allele is modest or minor with a few adjacent loci that are in LD. In this study, we propose the longest significant run, *LSR*, to estimate region-specific p-values while searching for disease susceptibility genes in gene mapping studies. The method transforms the p-values from locus-specific association tests (e.g., the transmission disequilibrium test or any other association test) to a binary sequence with the value '1' representing significance and '0' otherwise, and determines the *LSR* to identify the location of the target disease susceptibility gene. The statistical significance of *LSR* method can be accessed by imbedding the sequence onto a Markov chain. A sequential goodness-of- fit-test [27] can be used to justify the assumption that a sequence of indicator of p-values is Markov chain, simulations assuming no disease gene were carried out, and the test did not reject the null hypothesis of Markov independence 99.8% of the time (998 out of 1000). On the other hand, in the simulations assuming a disease gene with allele frequency of 0.075 and RR = 2.5 imbedded in a region flanking with dense markers, the test rejected the null hypothesis of Markov independence 92.4% of the time (924 Out of 1000) but did not reject the null hypothesis of first order of Markov chain 74.4% of the time (744 out of 1000). Likewise, the test did not reject the null hypothesis of first order Markov chain 82.2% of the time (822 out of 1000), assuming a disease susceptibility gene with allele frequency of 0.1. The allele frequencies in our simulations were assumed for the disease-causing alleles. The allele frequency for most markers in genome-wide association study is higher than 0.05, therefore we chose to use disease-causing allele frequencies to be 0.075 and 0.1. Other frequencies were also tested and found that the power was positively correlated with the allele frequency. This disease related marker was removed in all our subsequent analyses. Only results from additive disease model were reported since it was more plausible for complex diseases; we also examined dominant model which showed higher power and recessive model which showed lower power.

The *LSR* method avoids the controversial multiple-test adjustment required for locus-specific association tests, and thus has potential applications in exploratory data analysis. For example, if there were a few significant markers in a genome-wide association found by using single locus test [31], our proposed methods can be applied to screen out the most likely region for further biological examination. However, for an isolated significant marker, it is likely that the *LSR* method will miss the signal. Therefore, single-maker method and *LSR* method should be considered complementary.

In addition to the perfect *LSR*, we also propose a "nearly perfect" *LSR* in which a few interruptions (insignificance) are allowed within the run. Our simulation results suggest that the *LSR* s which allow for one or two interruptions within the run are generally more flexible and have some gain of testing power. The approach can be easily extended to allow more interruptions by modifying the imbedded transition matrix in Equation (5) in the method section.

An advantage of our proposed method relative to testing procedures based on genotypic data is that only p-value data are needed. This method can be adapted extensively to different study designs and testing procedures if reliable p-values are provided. An important application is meta-analysis which combines p-values from different studies. Due to the high sensitivity of *LSR* method, many published results with summarized p-values originally reported as insignificant can be reanalyzed by this method for more convincing conclusions.

*LSR*

_{2}for this modest LD scenario is about 77% assuming 0.1 disease allele frequency, RR = 3 and α

_{1}= 0.1. The power is about 23% for the Bonferroni procedure. The results for the different LD scenarios comprised of 3 common haplotypes are also presented in Figure 5.

We further compared the power of our proposed methods with those of haplotype analyses carried out by the FBAT program [34] assuming RR = 2 and disease allele frequency of 0.075. For the optimal LD scenarios, the multiple-degree of freedom (m-df) and single-degree of freedom (1-df) haplotype tests yield powers of 0.46 and 0.232, respectively, for the setting of (85% and 5%). The powers for m-df and 1-df are 0.35 and 0.176, respectively, for the setting of (30%, 30%, 30%). For the modest LD scenario of (35%, 10%, 5%), the powers for m-df and 1-df are 0.18 and 0.126, respectively. The power of LSR_{2} is 0.44 with α_{1} = 0.1 and the power of Bonferroni procedure is 0.15. Therefore, the power of LSR_{2} is about the same as the power of haplotype analyses under optimal LD scenarios but outperforms under modest LD scenario. Powers using other values of RR were also calculated. In general, powers of both haplotype analyses and LSR increase assuming higher values of RR.

Our simulation study demonstrates that all the methods adequately control the false-positive rate, however, the *LSR* methods, in particular, *LSR*_{2}, had better performance in both power and false discovery rate than those for the Bonferroni and FDR controlling methods. Moreover, as the number of markers increases, the power of both the Bonferroni and FDR controlling method drops significantly, whereas the power of *LSR* remains high and may even increase slightly.

Further studies for potential extensions of the LSR method are currently under development. Firstly, the *LSR* method transforms continuous p-values to a dichotomous random sequence of 0s and 1s, which may cause a loss of information. This limitation implies that some isolated disease susceptibility loci cannot be identified by the *LSR* method. A model based on a more complex continuous-state stochastic process constitutes an alternative that would exploit more information. Secondly, in our simulation, we assumed that there is only one disease susceptibility gene in the region of interest. However, the *LSR* method will miss some isolated target genes when a region contains two or more. One remedy is to extend the *LSR* theory to incorporate the second or third longest significant run.

The remarkable advances in genotyping technology have facilitated the pursuit of genome-wide association mapping. The increased density of available SNP markers provides finer resolution and higher statistical power for gene mapping. For example, the average distance between two SNPs using the 100 K assay of Affymetrix [35] is 24 kb. The intermarker distance is only 5.8 kb for the 500 K chips. We foresee that the multiple tests correction will remain a crucial issue as the number of genetic markers becomes very large. Our method can be applied to such data in combination with the sliding window method to screen out candidate regions associated with the disease genes.

## Conclusion

In summary, the *LSR* method provides an efficient exploratory tool for the analysis of sequences of dense genetic markers thereby complementing current locus-specific methods. Our simulation study demonstrates that the *LSR* method has reasonable statistical power and avoids the over-correction problem that plagues most of the locus-specific methods. When applied to actual genetic data, the *LSR* method successfully confirmed the location of two important psoriasis-associated genes and an asthma-related gene. The application to genome-wide screening studies may further enhance the proposed *LSR* method.

## Methods

Suppose that there are a total of *n* genetic markers in a study. The *LSR* method contains two main stages, the first of which transforms the results from *n* locus-specific association tests into a sequence of significance indicators and the sequence is used in the second stage to locate the *LSR* and compute its tail probability.

### The first-stage procedure

Association tests are applied to examine the significance of associations between genetic markers and a disease susceptibility gene, and the corresponding p-values (significance probabilities) of the tests are denoted by {${\widehat{p}}_{j}$, *j* = 1, Λ *n*}. Under the pre-specified test size, *α*_{1}, we consider an indicator function of the test on marker *j*: *X*_{
j
}= *I*[${\widehat{p}}_{j}$, <*α*_{1}] where *I* [*A*] = 1 if event *A* holds, and *I* [*A*] = 0 otherwise.

In the following hypothetical example of 15 genotyped genetic markers (*n* = 15), the corresponding p-value vector of 15 association tests is $\widehat{P}=[{\widehat{p}}_{1},\cdots ,{\widehat{p}}_{15}]$ = [0.30, 0.04, 0.03, 0.04, 0.12, 0.04, 0.02, 0.01, 0.35, 0.50, 0.02, 0.03, 0.04, 0.45, 0.04] Based on the setting of *α*_{1} = 0.05, we obtain**X** = [*X*_{1}, L, *X*_{15}] = [0, 1, 1, 1, 0, 1, 1, 1, 0, 0, 1, 1, 1, 0, 1],

where markers 2, 3, 4, 6, 7, 8, 11, 12, 13, and 15 show significant association with the disease susceptibility gene.

### The second-stage procedure

Before applying the LSR method, the assumption of Markov chain for the sequence of the indicator of p-values can be checked by carrying out the goodness-of-fit test of Markov Chain [27]. Assuming that the binary random sequence {*X*_{
j
}, *j* = 1,Λ,*n* } to be a first order Markov chain stochastic process satisfyingPr{*X*_{
j
}= *s*_{
j
}| *X*_{j-1}= *s*_{j-1},Λ, *X*_{1} = *s*_{1} = Pr{*X*_{
j
}= *s*_{
j
}| *X*_{j-1}= *s*_{j-1}},

*s*

_{ k }∈ {0,1} = insignificance, significance},

*k*= 1,Λ,

*n*. Let the initial probability be Pr{

*X*

_{1}= 1} =

*η*

_{1}and Pr{

*X*

_{1}= 0} = 1 -

*η*

_{1}, then the transition probability matrix is

where *η*_{00j}+ *η*_{01j}= 1 and *η*_{10j}+ *η*_{11j}= 1, *j* = 2,Λ,*n*. This model assumes conditional independence and is well established in a variety of fields.

An inordinately long *LSR* indicates possible associations between markers and a disease susceptibility gene. Due to random error, low heterozygosity or other unknown reasons, not all markers linked to the disease gene show positive association. Hence, it is sensible to allow for a few interrupting 0s (insignificances) in a run. We denote the more flexible run as a "nearly perfect run" in contrast to a perfect run that does not contain 0s. A unifying notation of the length of an *LSR* is *L*_{
k
}, where the subscript *k* denotes the number of interrupting 0s. When *k* = 0, the case reduces to a perfect run. In the example in Equation (1), *L*_{0} = 3, *L*_{1} = 7, *L*_{2} = 8 and *L*_{3} = 12.

#### Consider the following hypotheses

H_{0}: "there is no disease susceptibility gene in the sequence of markers", versus

H_{1}:"there is at least one disease susceptibility gene in the sequence".

According to the argument above, an inordinately long *LSR* (i.e. large *L*_{
k
}) indicates a non-random cluster of significant associations with nearby markers, suggesting that disease susceptibility genes may exist in the candidate region. For a pre-chosen *k*, an intuitive rule to reject H_{0} is *L*_{
k
}> *m**, where the constant *m** is the minimum integer of *m* satisfying the following inequalityPr{*L*_{
k
}≥ *m* | H_{0}} ≤ *α*_{2}

for a test size *α*_{2}. To determine the critical region, we need to calculate the tail probability of testing statistic *L*_{
k
}.

### Distributions of LSR statistics

There has been a long history of studies on the distribution of the length of the longest run. Erdõs and Révész [36] considered a binary sequence from a coin-tossing game, with outcomes of head (coded as "1") and tail ("0"). The p-value of *L*_{0} of *LSR* is analogous to the tail probability of their longest head-run. Fu and Koutras [19] have provided an algorithm to calculate the exact probability of *L*_{0}. Arratial *et al*. [37] and Karlin *et al*. [38] have provided asymptotic results on the distribution *L*_{
k
}, however, the former method [37] is valid only for independent sequences, whereas the latter [38], although allowing dependency, yields a large bias for the estimation of tail probability when *n* is of moderate size (e.g., *n* = 200) [20].

*et al*. [20] provided an algorithm and software to calculate the exact probability of

*L*

_{ k }by the following formula:

where u = [1,0,Λ,0], *ν* = [1,Λ,1,0] and the exact form of the imbedded transition matrix Λ_{
i
}is a function of *η*_{1} and *η*_{
sti
}s in Equation (3). By considering a homogeneous Markov chain, the subscript *i* can be suppressed and Λ_{
i
}and *η*_{
sti
}can be reduced to Λ and *η*_{
st
}. We estimated the initial probability by the proportion of the occurrence of "1" in {*X*_{
j
}, *j* = 1,Λ,*n*}, i.e., ${\widehat{\eta}}_{1}$ = (number of "1")/n. Under null hypothesis *H*_{0} of independency, {*X*_{
j
}is assumed to be of no special pattern and the transition probabilities *η*_{1t}and *η*_{0t}can be replaced by ${\widehat{\eta}}_{1}$ and 1 - ${\widehat{\eta}}_{1}$. For dependent sequences, we propose the following approach.

### Estimation of dependency structure under null hypothesis

*X*

_{1}...

*X*

_{n}}, we used the following sequential goodness-of-fit tests proposed by Anderson and Goodman [27]:

- (i)
H

_{0}: independent sequence vs. H_{1}: first order Markov chain - (ii)
H

_{0}: first order Markov chain vs. H_{1}: second order Markov chain

If the first order Markov chain hypothesis is accepted, we suggest using the following approach to estimate the dependency structure and calculate the p-value.

Consider a set of sliding windows of fixed size *w* and sliding distance *d*, *d* <*w* <*n*, under proper choices of *d* and *w*, the windows {*X*_{1}...*X*_{w}}, {*X*_{1+d}...*X*_{w+d}}, ..., {*X*_{1+sd}...*X*_{w+sd}} will cover nearly the whole sequence, where *s* is the largest integer smaller than or equal to (*n-w*)/*d*. For the subsequence in the *i-* th window, the transition probabilities *η*_{
st
}(*i*) = *P*(*X*_{i+1}= *t* | *X*_{
i
}= *s*), where *s*, *t* = 0,1, are estimated by its maximal likelihood estimator, namely, ${\widehat{\eta}}_{11}$(*i*) = (number of consecutive (1,1) pairs)/(number of 1s), and ${\widehat{\eta}}_{00}$ (number of consecutive (0,0) pairs)/(number of 0s), for *i* = 1 to *d*+1. For a homogeneous first-order Markov chain, we estimate ${\widehat{\eta}}_{11}$ and ${\widehat{\eta}}_{00}$ by the medians of ${\widehat{\eta}}_{00}$(*i*)'s and ${\widehat{\eta}}_{11}$(*i*)'s, respectively, and ${\widehat{\eta}}_{10}=1-{\widehat{\eta}}_{11}$ and ${\widehat{\eta}}_{01}=1-{\widehat{\eta}}_{00}$.

The validity of the sliding window approach is based on the assumption that most of the widows contain no disease markers, and therefore the sequences within the set of sliding windows of fixed size *w* can be used to estimate the transition probabilities. After ${\widehat{\eta}}_{st}$(*i*)'s are estimated from each window, the robustness of median can diminish the influence from those few windows which might contain disease markers. With the estimated *η*_{
st
}'s, tail probabilities of *LSR* statistics can be calculated using (5), and then the null hypothesis of no disease gene is rejected if *L*_{
k
}≥ *m*_{
α
}, where *m*_{
α
}is a threshold such that Pr{*L*_{
k
}≥ *m*_{
α
}|*H*_{0}} = *α*.

To summarize, we performed simulation studies to investigate the false-positive rate, the power, and the false discovery rate of the proposed *LSR* method, and we compared the results with those from Bonferroni method.

## Declarations

### Acknowledgements

This research was supported by National Science Council grants (NSC 92-3112-B-001-014, 92-2320-B-018-001) and an Academia Sinica grant (93IBMS2PP-C) of Taiwan. We would like to thank three anonymous reviewers; their valuable comments have greatly improved this manuscript.

## Authors’ Affiliations

## References

- Barrett JC, Cardon LR: Evaluating coverage of genome-wide association studies.
*Nat Genet*2006, 38: 659–662. 10.1038/ng1801View ArticlePubMedGoogle Scholar - Scott LJ, Mohlke KL, Bonnycastle LL, Willer CJ, Li Y, Duren WL, Erdos MR, Stringham HM, Chines PS, Jackson AU, Olsson LP, Ding CJ, Swift AJ, Narisu N, Hu T, Pruim R, Xiao R, Li X-Y, Conneely KN, Riebow NL, Sprau AG, Tong M, White PP, Hetrick KN, Barnhart MW, Bark CW, Goldstein JL, Watkins L, Xiang F, Saramies J, Buchanan TA, Watanabe RM, Valle TT, Kinnunen L, Abecasis GR, Pugh EW, Doheny KF, Bergman RN, Tuomilehto J, Collins FS, Boehnke M: A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants.
*Science*2007, 316: 1336–1341. 10.1126/science.1142382View ArticleGoogle Scholar - Sladek R, Rocheleau G, Rung J, Dina C, Shen L, Serre D, Boutin P, Vincent D, Belisle A, Hadjadj S, Balkau B, Heude B, Charpentier G, Hudson TJ, Montpetit A, Pshezhetsky AV, Prentki M, Posner BI, Balding DJ, Meyre D, Polychronakos C, Frogue P: A genome-wide association study identifies novel risk loci for type 2 diabetes.
*Nature*2007, 445: 881–885. 10.1038/nature05616View ArticlePubMedGoogle Scholar - Zeggini E, Weedon MN, Lindgren CM, Frayling TM, Elliott KS, Lango H, Timpson NJ, Perry JRB, Rayner NW, Freathy RM, Barrett JC, Shields B, Morris AP, Ellard S, Groves CJ, Harries LW, Marchini JL, Owen KR, Knight B, Cardon LR, Walker M, Hitman GA, Morris AD, Doney ASF;, The Wellcome Trust Case Control Consortium (WTCCC), McCarthy MI, Hattersley AT: Replication of genome-wide association signals in U.K. samples reveals risk loci for type 2 diabetes.
*Science*2007, 316: 1336–1341. 10.1126/science.1142364PubMed CentralView ArticlePubMedGoogle Scholar - Herbert A, Gerry NP, McQueen MB, Heid IM, Pfeufer A, Illig T, Wichmann H-E, Meitinger T, Hunter D, Hu FB, Colditz G, Hinney A, Hebebrand J, Koberwitz K, Zhu X, Cooper R, Ardlie K, Lyon H, Hirschhorn JN, Laird NM, Lenburg ME, Lange C, Christman MF: A common genetic variant is associated with adult and childhood obesity.
*Science*2006, 312: 279. 10.1126/science.1124779View ArticlePubMedGoogle Scholar - Lin DY: Evaluating statistical significance in two-stage genomewide association studies.
*Am J Hum Genet*2006, 78: 505–509. 10.1086/500812PubMed CentralView ArticlePubMedGoogle Scholar - Benjamini Y, Hochberg Y: Controlling the false discovery rate – a practical and powerful approach to multiple testing.
*J Roy Stat Soc B Met*1995, 57: 289–300.Google Scholar - Benjamini Y, Yekutieli D: Quantitative trait loci analysis using the false discovery rate.
*Genetics*2005, 171: 783–790. 10.1534/genetics.104.036699PubMed CentralView ArticlePubMedGoogle Scholar - Benjamini Y, Yekutieli D: The control of the false discovery rate in multiple testing under dependency.
*Ann Stat*2001, 29: 1165–1188. 10.1214/aos/1013699998View ArticleGoogle Scholar - Wacholder S, Chanock S, Garcia-Closas M, El Ghormli L, Rothman N: Assessing the probability that a positive report is false: an approach for molecular epidemiology studies.
*Journal of the National Cancer Institute*2004, 96: 434–442.View ArticlePubMedGoogle Scholar - Cardon LR, Bell JI: Association study designs for complex diseases.
*Nat Rev Genet*2001, 2: 91–99. 10.1038/35052543View ArticlePubMedGoogle Scholar - Becker T, Schumacher J, Cichon S, Baur MP, Knapp M: Haplotype interaction analysis of unlinked regions.
*Genet Epidemiol*2005, 29: 313–322. 10.1002/gepi.20096View ArticlePubMedGoogle Scholar - Tzeng JY, Devlin B, Wasserman L, Roeder K: On the identification of disease mutations by the analysis of haplotype similarity and goodness of fit.
*Am J Hum Genet*2003, 72: 891–902. 10.1086/373881PubMed CentralView ArticlePubMedGoogle Scholar - McIntyre LM, Martin ER, Simonsen KL, Kaplan NL: Circumventing multiple testing: a multilocus Monte Carlo approach to testing for association.
*Genet Epidemiol*2000, 19: 18–29. 10.1002/1098-2272(200007)19:1<18::AID-GEPI2>3.0.CO;2-YView ArticlePubMedGoogle Scholar - Fan R, Knapp M: Genome association studies of complex diseases by case-control designs.
*Am J Hum Genet*2003, 72: 850–868. 10.1086/373966PubMed CentralView ArticlePubMedGoogle Scholar - Musani SK, Shriner D, Liu N, Feng R, Coffey CS, Yi N, Tiwari HK, Allison DB: Detection of gene × gene interactions in genome-wide association studies of human population data.
*Human Heredity*2007, 63: 67–84. 10.1159/000099179View ArticlePubMedGoogle Scholar - Spielman RS, McGinnis RE, Ewens WJ: Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM).
*Am J Hum Genet*1993, 52: 506–516.PubMed CentralPubMedGoogle Scholar - Ewens WJ, Grant GR:
*Statistical Methods in Bioinformatics.*New York: Springer; 2001:184–189.View ArticleGoogle Scholar - Fu JC, Koutras MV: Distribution theory of runs: A Markov chain approach.
*J Am Stat Assoc*1994, 89: 1050–1058. 10.2307/2290933View ArticleGoogle Scholar - Chang CJ, Fann CSJ, Chou WC, Lian IB: On the tail probability of the longest well-matching run.
*Stat and Probability Letters*2003, 63: 267–274. 10.1016/S0167-7152(03)00091-9View ArticleGoogle Scholar - Schmidt M, Hauser ER, Martin ER, Schmidt S: Extension of the SIMLA package for generating pedigrees with complex inheritance patterns: environmental covariates, gene-gene and gene-environment interaction.
*Stat Appl Genet Mol Biol*2005, 4: Article 15.Google Scholar - SIMLA Simulation Software Version 2.3[http://wwwchg.duhs.duke.edu/software/simla.html]
- SAS Institute:
*SAS/Genetics User's Guide.*Cary, North Carolina; 2002.Google Scholar - Helms C, Cao L, Krueger JG, Wijsman EM, Chamian F, Gordon D, Heffernan M, Daw JAW, Robarge J, Ott J, Kwok P-Y, Menter A, Bowcock AM: A putative RUNX1 binding site variant between SLC9A3R1 and NAT9 is associated with susceptibility to psoriasis.
*Nat Genet*2003, 35: 349–356. 10.1038/ng1268View ArticlePubMedGoogle Scholar - Hwu WL, Yang CF, Fann CSJ, Chen CL, Tsai TF, Chien YH, Chiang SC, Chen CH, Hung SI, Wu JY, Chen YT: Mapping of psoriasis to 17q terminus.
*J Med Genet*2005, 42: 152–158. 10.1136/jmg.2004.018564PubMed CentralView ArticlePubMedGoogle Scholar - Gordon D, Heath SC, Liu X, Ott J: A transmission disequilibrium test that allows for genotyping errors in the analysis of single nucleotide polymorphism data.
*Am J Hum Genet*2001, 69: 371–380. 10.1086/321981PubMed CentralView ArticlePubMedGoogle Scholar - Anderson TW, Goodman LA: Statistical inference about Markov chains.
*Ann Math Stat*1957, 28: 89–110. 10.1214/aoms/1177707039View ArticleGoogle Scholar - Allen M, Heinzmann A, Noguchi E, Abecasis G, Broxholme J, Ponting CP, Bhattacharyya S, Tinsley J, Zhang Y, Holt R, Jones EY, Lench N, Carey A, Jones H, Dickens NJ, Dimon C, Nicholls R, Baker C, Xue L, Townsend E, Kabesch M, Weiland SK, Carr D, von Mutius E, Adcock IM, Barnes PJ, Lathrop GM, Edwards M, Moffatt MF, Cookson WOCM: Positional cloning of a novel gene influencing asthma from Chromosome 2q14.
*Nat Genet*2003, 35: 258–263. 10.1038/ng1256View ArticlePubMedGoogle Scholar - Rosenthal R: Combining results of independent studies.
*Psychological Bulletin*1978, 85: 185–193. 10.1037/0033-2909.85.1.185View ArticleGoogle Scholar - Zaykin DV, Zhivotovsky LA, Westfall PH, Weir BS: Truncated product method for combining p-values.
*Genet Epidemiol*2002, 22: 170–185. 10.1002/gepi.0042View ArticlePubMedGoogle Scholar - Owen MJ, Williams NM, O'Donovan MC: The molecular genetics of schizophrenia: new findings promise new insights.
*Mol Psychiatry*2004, 9: 14–27. 10.1038/sj.mp.4001444View ArticlePubMedGoogle Scholar - International HapMap Project[http://www.hapmap.org/downloads/index.html.en]
- 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.1069424View ArticlePubMedGoogle Scholar - Horvath S, Xu X, Lake SL, Silverman EK, Weiss ST, Laird NM: Family-based tests for associating haplotypes with general phenotype data: application to asthma genetics.
*Genet Epidemiol*2004, 26: 61–69. 10.1002/gepi.10295View ArticlePubMedGoogle Scholar - Liu G, Loraine AE, Shigeta R, Cline M, Cheng J, Valmeekam V, Sun S, Kulp D, Siani-Rose MA: NetAffx: Affymetrix probesets and annotations.
*Nucleic Acids Res*2003, 31: 82–86. 10.1093/nar/gkg121PubMed CentralView ArticlePubMedGoogle Scholar - Erdõs P, Révész P: On the length of the longest head-run.
*Colloq Math Soc Janos Bolyai*1977, 16: 219–228.Google Scholar - Arratial R, Gordan L, Waterman MS: The Erdos-Renyi law in distribution for coin tossing and sequence matching.
*Ann Stat*1990, 18: 539–570. 10.1214/aos/1176347615View ArticleGoogle Scholar - Karlin S, Ost F, Blaisdell BE:
*Mathematical Methods for DNA Sequences.*Boca Raton: CRC Press; 1990.Google 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.