BACKGROUND
recently whole genome association studies  with high density snp data are becoming popular due to new technology in genotyping   <cit> .
optimal study design in whole genome association remains unresolved although the two-stage association test design has gained popularity  <cit> .
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  methods  <cit>  that control the expected proportion of false rejections, or false positive report probability   <cit>  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  <cit> , 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  <cit> .
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  <cit> , global significance method  <cit> , multivariate association analysis  <cit> , and the consideration of gene-gene interactions  <cit> .
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"  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  <cit>  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 .
in the second stage, this binary sequence is scanned for the longest region of consecutive 1s  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 ).
a special non-random pattern  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  <cit> , 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  than others  <cit> , 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 .
RESULTS
the program simla version  <dig>   <cit>  was used to simulate family-structure genetic data.
the program is available  <cit> .
in the process, we generated  <dig> trio families with genotypic data for snp  markers.
to determine the effects of the number of snp markers, we considered two cases for  <dig> and  <dig> snps in a candidate region.
to mimic the scenario of dependent markers, we considered a dense intermarker distance with a mean of  <dig> kb and a standard deviation of  <dig>  kb.
note that in  <dig> k affymetrix data, marker distance has an average of  <dig>  kb and a standard deviation of  <dig>  kb.
in the simulation, the intermarker distance is generated from a left-truncated normal distribution with mean  <dig> kb and standard deviation  <dig>  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  <dig> , and genetic relative risk was set to be  <dig> ,  <dig>  and  <dig> .
the allele frequencies were  <dig>  and  <dig> , 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 .
under a looser definition, the relative risk can be lowered to  <dig>  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  <dig> 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:  three common haplotypes each with a frequency of 30%,  two common haplotypes each with a frequency of 45%, and  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  <dig> .
<dig>  <cit>  was used to test the locus-specific association for each marker.
the output p-values {p^j, j =  <dig>  Λ n} were transformed into a sequence of significance indicators {xj, j =  <dig> Λ,n} based on a test size α <dig>  where xj is  <dig> if the test of the jth marker is significant and  <dig> otherwise.
in the second stage, we scanned this binary sequence to identify the longest perfect run of  <dig>  l <dig>  the nearly perfect run of  <dig> allowing one zero within, namely the l <dig> and the run of  <dig> allowing two zeros within, namely the l <dig>  for each of these three lsr statistics, the corresponding tail probabilities were calculated using the method of chang et al.
<cit> .
testing statistics
although allowing more interruptions  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 li with minimal tail probability, for i from  <dig> to k and considered only kϕ <dig>  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:  <dig> target markers where the disease gene lain between them,  <dig> nearby markers in linkage disequilibrium  with the  <dig> target markers, and other markers that are in linkage equilibrium .
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 < α2/n.
for the fdr controlling method, benjamini and hochberg  <cit>  proposed the following criterion.
the n single marker p-values were sorted from smallest to largest: p ⋯ p. starting from p, we compared p with i·α2/n.
this process was continued as long as p > i·α2/n.
if k is the turn around point, then significance is declared if a target marker corresponds to the kth smallest p-value.
for our lsr method, the criterion was that a target marker is covered by a significant lsr .
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.
the false-positive rate is the probability that a test mistakenly rejects the null hypothesis.
in our case, it is the rate at which a test falsely detects disease susceptibility genes where none exists.
in our first part of the simulation, no disease gene was assumed.
table  <dig> lists the corresponding false-positive rates for the two lsr approaches and bonferroni approach based on  <dig> replications for each of the eight scenarios .
the test size in the first stage was α <dig> =  <dig> , and the test size was α <dig> =  <dig>  in the second stage.
we found that all the false-positive rates were under or similar to the pre-specified nominal test size α <dig>  indicating that all the methods adequately controlled the false-positive rate.
simulation was based on  <dig> replications with sample size  <dig>  under additive disease model.
screening criteria for lsr:α <dig> =  <dig> , and level of significance for all methods:α <dig> =  <dig>
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  <dig> target markers with statistical significance.
on the other hand, the false discovery rate is the fraction of false positives among all tests declared significant.
figures  <dig>   <dig>   <dig>   <dig> illustrate the performance of bonferroni and lsr methods given different nominal test sizes.
the test size in the first stage for lsr was α <dig> =  <dig>  and  <dig>  and the test size in the second stage was α <dig> =  <dig> .
higher level of significance  at screening stage resulted in better power than that of α <dig> =  <dig> , in trade of a slightly higher false discovery rate.
lsr <dig> had better performance in both power and false discovery rate than lsr <dig>  the stringent definition of a "run" for lsr <dig> yields a relatively narrow candidate region that was more likely to miss the target markers.
both lsr <dig> and lsr <dig> had better power and false discovery rate than those from the bonferroni approach among all the scenarios that we tried.
lsr <dig> has similar power to that of the fdr controlling method, but better false discover rate.
in summary, in terms of power, lsr <dig> > lsr1~ fdr controlling method > bonferroni; and in terms of false discovery rate, lsr2~lsr <dig> ~<fdr controlling method < bonferroni.
the detailed results are available in tables  <dig> and  <dig>
simulation was based on  <dig> replications with sample size  <dig>  and snp data were generated under additive disease model with  <dig> markers in linkage disequilibrium.
screening criteria for lsr:α <dig> =  <dig> , and level of significance for all methods:α <dig> =  <dig>
simulation was based on  <dig> replications with sample size  <dig>  and snp data were generated under additive disease model with  <dig> markers in linkage disequilibrium.
screening criteria for lsr:α <dig> =  <dig> , and level of significance for all methods:α <dig> =  <dig>
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 <dig>  although better than lsr <dig> 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 <dig> =  <dig>  over α <dig> =  <dig>  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  <cit> .
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  <dig>  <cit> .
helms et al.
<cit>  collected  <dig> european nuclear families comprising  <dig> psoriasis cases and genotyped  <dig> genetic markers on chromosome 17q <dig>  the family-based association test tdt-ae  <cit>  was applied to locate the psoriasis susceptibility genes.
in the first stage of lsr procedure, we used α <dig> =  <dig>  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 < <dig> , and the region of  <dig> consecutive markers identified by l <dig> captured two functional genes slc9a3r <dig> and dkfzpp564c <dig>  which are known to be psoriasis-related.
the results are consistent with the finding of helms et al.
<cit> .
however, when we proceeded with the sequential goodness-of-fit tests proposed by anderson and goodman  <cit> , the hypothetical assumption of {xj} 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  <dig> markers and sliding distance of  <dig> markers to estimates of the transition matrix under the hypothesis of markov dependency.
the same region was identified by lsr with p-value  <dig>  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 <dig> and l <dig> did not further extend the region of l <dig>  therefore, l <dig> 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 5th to the 86th loci among the  <dig> markers.
moreover, we also excluded results from marker loci with less than  <dig> trios in the analysis to preclude possibly unreliable association results.
it resulted in  <dig> loci included in the final analysis.
the consequent analysis also came out with the same significant region with a p-value < <dig> .
of particular note, all of the  <dig> markers had p-value > <dig> , 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.
<cit> , there were  <dig> families containing  <dig> asthma children and  <dig> markers covering a region of  <dig> kb on chromosome 2q <dig>  immunoglobulin e often causes asthmatic inflammation and has been recognized to be an important concomitant factor of children asthma.
allen et al.
<cit>  conducted the transmission disequilibrium tests to assess the relationship between marker loci with asthma and immunoglobulin e.
using test size α <dig> =  <dig> , the sequence of p-values was converted into a sequence of twenty-three 1s and fifty-six 0s:
x =  <cit> .
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 <dig> and η <dig> to be  <dig>  and  <dig> , respectively, and identified l <dig>  with length  <dig> and yielded the corresponding p-value  <dig> .
note that l <dig> contains a significant candidate region of  <dig> bp closed to an asthma-suspected gene dpp <dig> with functions of catalytic activity and dipetidyl-peptidase iv activity.
the conclusion is consistent with allen et al.
<cit>  who found the highly significant strp marker d2s <dig> at  <dig> in the vicinity of our identified region.
discussion
as discussed by rosenthal  <cit>  and zaykin et al.
<cit> , a series of border-line significant results may together suggest significance.
therefore, it is likely that two consecutive p-values of  <dig>  may suggest evidence against the null than one isolated p-value of  <dig> .
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  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  <cit>  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  <dig> % of the time .
on the other hand, in the simulations assuming a disease gene with allele frequency of  <dig>  and rr =  <dig>  imbedded in a region flanking with dense markers, the test rejected the null hypothesis of markov independence  <dig> % of the time  but did not reject the null hypothesis of first order of markov chain  <dig> % of the time .
likewise, the test did not reject the null hypothesis of first order markov chain  <dig> % of the time , assuming a disease susceptibility gene with allele frequency of  <dig> .
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  <dig> , therefore we chose to use disease-causing allele frequencies to be  <dig>  and  <dig> .
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  <cit> , 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  are allowed within the run.
our simulation results suggest that the lsrs 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  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.
to explore the genetic structure of human genome, we downloaded the first  <dig>  markers of chromosomes  <dig>   <dig>   <dig>   <dig> and  <dig>  respectively, from the hapmap website  <cit> .
according to gabriel's criterion   <cit> , there are  <dig> ld blocks among the  <dig>  markers.
the mean frequency and standard deviation of the top haplotype are  <dig>  and  <dig> .
they are  <dig>  and  <dig>  for the second haplotype and  <dig>  and  <dig>  for the third one.
if the top two or three haplotypes are defined as the "common haplotypes", about 62% of the  <dig> ld blocks have a cumulative frequency of 90% and about  <dig> % of the blocks have a cumulative frequency of 50%.
to compare the powers of our proposed method between optimal and modest ld scenarios, we used two settings of haplotype frequencies,  and  for the former and a setting of , where the frequencies were roughly those mean frequencies minus one respective standard deviation, for the latter.
as expected, the former scenario with optimal ld block resulted in better power for all methods in our simulation than the latter with modest ld block.
the power of lsr <dig> for this modest ld scenario is about 77% assuming  <dig>  disease allele frequency, rr =  <dig> and α <dig> =  <dig> .
the power is about 23% for the bonferroni procedure.
the results for the different ld scenarios comprised of  <dig> common haplotypes are also presented in figure  <dig>
we further compared the power of our proposed methods with those of haplotype analyses carried out by the fbat program  <cit>  assuming rr =  <dig> and disease allele frequency of  <dig> .
for the optimal ld scenarios, the multiple-degree of freedom  and single-degree of freedom  haplotype tests yield powers of  <dig>  and  <dig> , respectively, for the setting of .
the powers for m-df and 1-df are  <dig>  and  <dig> , respectively, for the setting of .
for the modest ld scenario of , the powers for m-df and 1-df are  <dig>  and  <dig> , respectively.
the power of lsr <dig> is  <dig>  with α <dig> =  <dig>  and the power of bonferroni procedure is  <dig> .
therefore, the power of lsr <dig> 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 <dig>  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  <dig> k assay of affymetrix  <cit>  is  <dig> kb.
the intermarker distance is only  <dig>  kb for the  <dig> 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.
CONCLUSIONS
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.

 recently whole genome association studies with high density snp data are becoming popular due to new technology in genotyping. optimal study design in whole genome association remains unresolved although the two-stage association test design has gained popularity. 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 methods that control the expected proportion of false rejections, or false positive report probability 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, 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. 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, global significance method, multivariate association analysis, and the consideration of gene-gene interactions. 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" 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 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. in the second stage, this binary sequence is scanned for the longest region of consecutive 1s 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 ). a special non-random pattern 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, 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 than others, 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.  the program simla version was used to simulate family-structure genetic data. the program is available. in the process, we generated trio families with genotypic data for snp markers. to determine the effects of the number of snp markers, we considered two cases for and snps in a candidate region. to mimic the scenario of dependent markers, we considered a dense intermarker distance with a mean of kb and a standard deviation of kb. note that in k affymetrix data, marker distance has an average of kb and a standard deviation of kb. in the simulation, the intermarker distance is generated from a left-truncated normal distribution with mean kb and standard deviation 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, and genetic relative risk was set to be, and. the allele frequencies were and, 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. under a looser definition, the relative risk can be lowered to 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 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: three common haplotypes each with a frequency of 30%, two common haplotypes each with a frequency of 45%, and 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. was used to test the locus-specific association for each marker. the output p-values {p^j, j = Λ n} were transformed into a sequence of significance indicators {xj, j = Λ,n} based on a test size α where xj is if the test of the jth marker is significant and otherwise. in the second stage, we scanned this binary sequence to identify the longest perfect run of l the nearly perfect run of allowing one zero within, namely the l and the run of allowing two zeros within, namely the l for each of these three lsr statistics, the corresponding tail probabilities were calculated using the method of chang et al.. testing statistics although allowing more interruptions 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 li with minimal tail probability, for i from to k and considered only kϕ 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: target markers where the disease gene lain between them, nearby markers in linkage disequilibrium with the target markers, and other markers that are in linkage equilibrium. 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 < α2/n. for the fdr controlling method, benjamini and hochberg proposed the following criterion. the n single marker p-values were sorted from smallest to largest: p ⋯ p. starting from p, we compared p with i·α2/n. this process was continued as long as p > i·α2/n. if k is the turn around point, then significance is declared if a target marker corresponds to the kth smallest p-value. for our lsr method, the criterion was that a target marker is covered by a significant lsr. 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. the false-positive rate is the probability that a test mistakenly rejects the null hypothesis. in our case, it is the rate at which a test falsely detects disease susceptibility genes where none exists. in our first part of the simulation, no disease gene was assumed. table lists the corresponding false-positive rates for the two lsr approaches and bonferroni approach based on replications for each of the eight scenarios. the test size in the first stage was α =, and the test size was α = in the second stage. we found that all the false-positive rates were under or similar to the pre-specified nominal test size α indicating that all the methods adequately controlled the false-positive rate. simulation was based on replications with sample size under additive disease model. screening criteria for lsr:α =, and level of significance for all methods:α = 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 target markers with statistical significance. on the other hand, the false discovery rate is the fraction of false positives among all tests declared significant. figures illustrate the performance of bonferroni and lsr methods given different nominal test sizes. the test size in the first stage for lsr was α = and and the test size in the second stage was α =. higher level of significance at screening stage resulted in better power than that of α =, in trade of a slightly higher false discovery rate. lsr had better performance in both power and false discovery rate than lsr the stringent definition of a "run" for lsr yields a relatively narrow candidate region that was more likely to miss the target markers. both lsr and lsr had better power and false discovery rate than those from the bonferroni approach among all the scenarios that we tried. lsr has similar power to that of the fdr controlling method, but better false discover rate. in summary, in terms of power, lsr > lsr1~ fdr controlling method > bonferroni; and in terms of false discovery rate, lsr2~lsr ~<fdr controlling method < bonferroni. the detailed results are available in tables and simulation was based on replications with sample size and snp data were generated under additive disease model with markers in linkage disequilibrium. screening criteria for lsr:α =, and level of significance for all methods:α = simulation was based on replications with sample size and snp data were generated under additive disease model with markers in linkage disequilibrium. screening criteria for lsr:α =, and level of significance for all methods:α = 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 although better than lsr 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 = over α = 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. 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. helms et al. collected european nuclear families comprising psoriasis cases and genotyped genetic markers on chromosome 17q the family-based association test tdt-ae was applied to locate the psoriasis susceptibility genes. in the first stage of lsr procedure, we used α = 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 <, and the region of consecutive markers identified by l captured two functional genes slc9a3r and dkfzpp564c which are known to be psoriasis-related. the results are consistent with the finding of helms et al.. however, when we proceeded with the sequential goodness-of-fit tests proposed by anderson and goodman, the hypothetical assumption of {xj} 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 markers and sliding distance of markers to estimates of the transition matrix under the hypothesis of markov dependency. the same region was identified by lsr with p-value 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 and l did not further extend the region of l therefore, l 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 5th to the 86th loci among the markers. moreover, we also excluded results from marker loci with less than trios in the analysis to preclude possibly unreliable association results. it resulted in loci included in the final analysis. the consequent analysis also came out with the same significant region with a p-value <. of particular note, all of the markers had p-value >, 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., there were families containing asthma children and markers covering a region of kb on chromosome 2q immunoglobulin e often causes asthmatic inflammation and has been recognized to be an important concomitant factor of children asthma. allen et al. conducted the transmission disequilibrium tests to assess the relationship between marker loci with asthma and immunoglobulin e. using test size α =, the sequence of p-values was converted into a sequence of twenty-three 1s and fifty-six 0s: x =. 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 and η to be and, respectively, and identified l with length and yielded the corresponding p-value. note that l contains a significant candidate region of bp closed to an asthma-suspected gene dpp with functions of catalytic activity and dipetidyl-peptidase iv activity. the conclusion is consistent with allen et al. who found the highly significant strp marker d2s at in the vicinity of our identified region. discussion as discussed by rosenthal and zaykin et al., a series of border-line significant results may together suggest significance. therefore, it is likely that two consecutive p-values of may suggest evidence against the null than one isolated p-value of. 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 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 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 % of the time. on the other hand, in the simulations assuming a disease gene with allele frequency of and rr = imbedded in a region flanking with dense markers, the test rejected the null hypothesis of markov independence % of the time but did not reject the null hypothesis of first order of markov chain % of the time. likewise, the test did not reject the null hypothesis of first order markov chain % of the time, assuming a disease susceptibility gene with allele frequency of. 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, therefore we chose to use disease-causing allele frequencies to be and. 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, 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 are allowed within the run. our simulation results suggest that the lsrs 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 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. to explore the genetic structure of human genome, we downloaded the first markers of chromosomes and respectively, from the hapmap website. according to gabriel's criterion, there are ld blocks among the markers. the mean frequency and standard deviation of the top haplotype are and. they are and for the second haplotype and and for the third one. if the top two or three haplotypes are defined as the "common haplotypes", about 62% of the ld blocks have a cumulative frequency of 90% and about % of the blocks have a cumulative frequency of 50%. to compare the powers of our proposed method between optimal and modest ld scenarios, we used two settings of haplotype frequencies, and for the former and a setting of, where the frequencies were roughly those mean frequencies minus one respective standard deviation, for the latter. as expected, the former scenario with optimal ld block resulted in better power for all methods in our simulation than the latter with modest ld block. the power of lsr for this modest ld scenario is about 77% assuming disease allele frequency, rr = and α =. the power is about 23% for the bonferroni procedure. the results for the different ld scenarios comprised of common haplotypes are also presented in figure we further compared the power of our proposed methods with those of haplotype analyses carried out by the fbat program assuming rr = and disease allele frequency of. for the optimal ld scenarios, the multiple-degree of freedom and single-degree of freedom haplotype tests yield powers of and, respectively, for the setting of. the powers for m-df and 1-df are and, respectively, for the setting of. for the modest ld scenario of, the powers for m-df and 1-df are and, respectively. the power of lsr is with α = and the power of bonferroni procedure is. therefore, the power of lsr 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 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 k assay of affymetrix is kb. the intermarker distance is only kb for the 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.  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. 
the proposed lsr method provides an efficient exploratory tool for the analysis of sequences of dense genetic markers. 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 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. our results show that the lsr method has better power and lower false discovery rate comparing with the locus-specific multiple tests. 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. however the conventional statistical corrections on locus-specific multiple tests usually result in lower power as the number of markers increases. we found that while all methods provide good control over false positive rate, lsr has much better power and false discovery rate. in this study the proposed lsr method was compared with commonly used methods such as bonferroni's method and fdr controlling method.
 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 method to estimate a region-specific p-value to provide an index for the most likely candidate region.  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.  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. 