A P-Value for an Exact Two-Sided Test of Hardy-Weinberg Equilibrium

A two-tailed P-value is proposed for testing two-sided departures from Hardy-Weinberg equilibrium at a diallelic locus. The calculation of P uses the exact conditional distribution of the test statistic P, the observed number of heterozygotes in the sample. The proposed P-value is always two-tailed, unlike other P-values proposed in the literature.     

A Two-Tailed P-Value for Fisher's Exact Test

A two-tailed P-value is presented for a significance test in two by two contingency tables. There is no extraneous quasi-observation such as is needed in the exact randomized uniformly most powerful unbiased (UMPU) test of the hypothesis of independence. The proposed P-value can never exceed unity and is always two-tailed, unlike other P-values proposed in the literature     

False discovery rate control for multiple testing based on discrete p-values

For multiple testing based on discrete p-values, we propose a false discovery rate (FDR) procedure “BH+” with proven conservativeness. BH+ is at least as powerful as the BH (i.e., Benjamini-Hochberg) procedure when they are applied to superuniform p-values. Further, when applied to mid-p-values, BH+ can be more powerful than it is applied to conventional p-values. An easily verifiable necessary and sufficient condition for this is provided. BH+ is perhaps the first conservative FDR procedure applicable to mid-p-values and to p-values with general distributions. It is applied to multiple testing based on discrete p-values in a methylation study, an HIV study and a clinical safety study, where it makes considerably more discoveries than the BH procedure. In addition, we propose an adaptive version of the BH+ procedure, prove its conservativeness under certain conditions, and provide evidence on its excellent performance via simulation studies.     

MAX-rank: a simple and robust genome-wide scan for case-control association studies

In genome-wide association studies (GWAS), single-marker analysis is usually employed to identify the most significant single nucleotide polymorphisms (SNPs). The trend test has been proposed for analysis of case-control association. Three trend tests, optimal for the recessive, additive and dominant models respectively, are available. When the underlying genetic model is unknown, the maximum of the three trend test results (MAX) has been shown to be robust against genetic model misspecification. Since the asymptotic distribution of MAX depends on the allele frequency of the SNP, using the P-value of MAX for ranking may be different from using the MAX statistic. Calculating the P-value of MAX for 300,000 (300 K) or more SNPs is computationally intensive and the software and program to obtain the P-value of MAX are not widely available. On the other hand, the MAX statistic is very easy to calculate without complex computer programs. Thus, we study whether or not one could use the MAX statistic instead of its P-value to rank SNPs in GWAS. The approaches using the MAX and its P-value to rank SNPs are referred to as MAX-rank and P-rank. By applying MAX-rank and P-rank to simulated and four real datasets from GWAS, we found the ranks of SNPs with true association are very similar using both approaches. Thus, we recommend to use MAX-rank for genome-wide scans. After the top-ranked SNPs are identified, their P-values based on MAX can be calculated and compared with the significance level. The work of Q. Li was partially supported by the Knowledge Innovation Program of the Chinese Academy of Sciences, No. 30465W0 and 30475V0. The research of Z Li was partially sponsored by NIH grant EY014478.     

The influence of radioactive phosphate level on the absorption of phosphate by plants and on the determination of labile soil phosphate

Summary Previous work has suggested that the presence of P³² in fertilizers inhibits the uptake of the applied phosphate from the soil by plants, and also that if the applied phosphate is not incorporated uniformly in the soil there will be preferential uptake from regions of low specific activity. This made it desirable to determine the effect of P³²-level on phosphate uptake and the determination ofL-values in pot experiments in which the labelled phosphate source is added as discrete particles of the phosphate form of an anion-exchange resin.Increasing the level of P³² from 0.05 to 1.25 mo per gram of phosphorus in the added phosphate did not have a significant effect on the fresh weight, dry weight or total phosphorus uptake of the ryegrass crop. The measuredL-value showed a significant increase, about 15 per cent for a five-fold increase in P³² level, on each of the four soil types used, as would be expected if P³² depressed the uptake of labelled fertilizer phosphate.Although a significant effect of P³² was observed this does not invalidate a comparison of soils with respect toL-value.     

Fine scale mapping of a genetic locus for conditioned fear

Abstract Fear conditioning is one of a number of models for investigating the genetic basis of individual variation in emotion and learning. Genetic mapping using crosses between strains of laboratory mice has identified a locus on chromosome one that appears to influence not only variation in conditioned fear, but also in other validated tests of fear-related behaviour, (including the open-field and the elevated-plus maze), suggesting that the rodent locus may act in ways consistent with how a locus influencing susceptibility to anxiety in humans is believed to operate. Here we use high-resolution mapping in genetically heterogeneous mice to show that a quantitative trait locus influencing conditioned fear can be separated from loci influencing open-field activity. Mapping in two different heterogeneous stocks, the Boulder and Northport HS, gave similar map locations for open-field activity at two positions on the current mouse physical map, one at 162 Mb on chromosome one (negative log P-value 5.4) the other at 173 Mb (negative log P-value 4.8), while mapping of contextual conditioned fear in the Boulder HS identified a locus at 170 Mb (negative log P-value 5.4). Estimates of the 95% confidence intervals show that the locations do not overlap. The region containing a gene or genes that influence variation in conditioned fear is approximately 1 megabase in size and contains only one gene of known function, a pre-B cell leukaemia factor.     

Small Sample Properties of the Mantel-Haenszel Test Statistic for Density Follow-Up Studies

For the analysis of combinations of 2×2 non-contingency tables as obtained from density follow-up studies (relating a number of events to a number of person-years of follow-up) an analogue of the Mantel-Haenszel test for 2×2 contingency tables is widely used. In this paper the small sample properties of this test, both with and without continuity correction, are evaluated. Also the improvement of the test-statistic by using the first four cumulants via the Edgeworth expansion was studied. Results on continuity correction agree with similar studies on the Mantel-Haenszel statistic for 2×2 contingency tables: Continuity correction gives a p-value which approximates the exact p-value better than the p-value obtained without this correction; both the exact test and its approximations show considerable conservatism in small samples; the uncorrected Mantel-Haenszel test statistic gives a p-value that agrees more with the nominal significance level, but can be anti-conservative. The p-value based on the first four cumulants gives a better approximation of the exact p-value than the continuity corrected test, especially when the distribution has marked skewness.     

Expression of insulin-like growth factor-II,matrix metalloproteinases,and their tissue inhibitors as predictive markers in the peripheral blood of HCC patients

Background/aim: Elevated relative expression of insulin-like growth factor-II (IGF-II) was observed in hepatocellular carcinoma (HCC) liver tissues with a role in neovascularization and associated with poor prognosis. IGF-II is influenced by the proteolytic cleavage of IGF-binding protein 3 and by matrix metalloproteinases (MMP), which are further regulated by their tissue inhibitors tissue inhibitor of metalloprotienase-1 (TIMP-1). Our aim is to study new molecular markers for HCC.

Patients/methods: RNA was extracted from the peripheral blood for evaluating the relative expression of IGF-II, MMP-9, and TIMP-1 in correlation with clinical staging of 39 HCC patients and 15 healthy controls using TaqMan real-time PCR.

Results: The relative expression of IGF-II and MMP-9 mRNA were significantly elevated in HCC patients compared with healthy controls; P-value <0.0001 for both. There was a significant correlation between MMP-9 and different HCC stages. On the other hand, TIMP-1 was significantly down-regulated in HCC patients; P?=?0.0003 with the elevation of the IGF-II/TIMP-1 ratio. Significant correlation between TIMP-1 and HCC Stage III and Stage IV was found; P-value?=?0.0138.

Conclusion: These results highlight the importance of profiling the expression of IGF-II, MMP-9, and TIMP-1 in the peripheral blood as prognostic molecular biomarkers in HCC.     

Identification of a combination of SNPs associated with Graves’ disease using swarm intelligence

Graves’ disease, the production of thyroid-stimulating hormone receptor-stimulating antibodies leading to hyperthyroidism, is one of the most common forms of human autoimmune disease. It is widely agreed that complex diseases are not controlled simply by an individual gene or DNA variation but by their combination. Single nucleotide polymorphisms (SNPs), which are the most common form of DNA variation, have great potential as a medical diagnostic tool. In this paper, the P-value is used as a SNP pre-selection criterion, and a wrapper algorithm with binary particle swarm optimization is used to find the rule for discriminating between affected and control subjects. We analyzed the association between combinations of SNPs and Graves’ disease by investigating 108 SNPs in 384 cases and 652 controls. We evaluated our method by differentiating between cases and controls in a five-fold cross validation test, and it achieved a 72.9% prediction accuracy with a combination of 17 SNPs. The experimental results showed that SNPs, even those with a high P-value, have a greater effect on Graves’ disease when acting in a combination.     

A genomic scan of porcine reproductive traits reveals possible quantitative trait loci (QTLs) for number of corpora lutea

Reproductive traits have low heritabilities, are expressed in only one sex, and are not measurable until sexual maturity (Avalos and Smith, Anim Prod 44:153, 1987). Using traditional methods, selection for reproductive traits is relatively less effective than selecting for growth or carcass traits. Traits most affected by a small number of genes with major effects rather than many genes with small effects are most amenable to MAS. As part of our porcine genome scan to identify quantitative trait loci (QTLs) of economic importance in marker-assisted selective (MAS) breeding programs, we examined 8 reproductive and farrowing traits in the University of Illinois (UI) Meishan × Yorkshire Resource Family. Gilts were genotyped with 119 microsatellite markers (MS) with intervals averaging 24 cM over all 18 porcine autosomes. F-ratios supporting QTL location were calculated by the least squares regression method. Results suggestive of linkage at the 5% genome-wide level were observed for the number of stillborn piglets on Chromosome (Chr) 4 (SSC4) (p-value = 0.0001), corpora lutea on SSC8 (p-value = 0.00027), and gestation length on SSC9 (p-value = 0.00019). Results for additional loci relevant to litter size, number of corpora lutea on SSC15 and 7 (p-value = 0.0029 and 0.0028 at 107 and 150 cM, respectively), gestation length on SSC15 and 1 (p-value = 0.0017 and 0.0069 at 96 and 166 cM, respectively), uterine length on SSC7 and 5 (p-value = 0.0044 and 0.0075 at 148 and 1 cM, respectively) and piglets born per litter on SSC6 (p-value = 0.0075 at 102 cM), were not statistically significant at the 5% genome-wide level. Thus, the use of a linked marker to facilitate selection for reproductive traits has considerable potential. By using linked markers, selection can be applied to both sexes before sexual maturity, making genetic selection considerably more efficient and less costly. Received: 24 June 1998 / Accepted 29 January 1999     

Transmission disequilibrium and sequence variants at the leptin receptor gene in extremely obese German children and adolescents

Genetic determinants of the degree of obesity and body fat distribution have been demonstrated by family studies. The heritability has been estimated to be in the range 0.2–0.7. Mutation leading to obesity in humans has been described for only two genes, one of them the leptin gene. The leptin gene codes for a cytokine secreted by fat cells that binds to the leptin receptor (Lep-R), which exerts some of its biological functions by expression in the brain. Hence, the Lep-R gene appears to be a promising candidate for the determination of obesity in humans. We isolated genomic DNA clones from the Lep-R gene region and identified a new polymorphic microsatellite marker (OBR-CA) within 80 kb of the translation start of Lep-R. We genotyped this and a second, intragenic microsatellite marker (D1S2852) in 130 nuclear families consisting of extremely obese children and adolescents and both parents. Using the most frequent parental allele of both markers, our analysis revealed a significant transmission disequilibrium for the 266-bp allele of D1S2852 (corrected P-value=0.042). No significant result was obtained with the most frequent allele of OBR-CA (corrected P-value=1.0). However, two rare alleles showed transmission disequilibrium and were subsequently used for constructing a haplotype with the 266-bp allele. This haplotype had a transmission rate of 80% (nominal P-value=0.02). In order to identify the underlying mutation, we sequenced all coding exons of Lep-R and the partially overlapping gene encoding the obese receptor gene-related protein (ob-rgrp) in individuals carrying this haplotype. We found one new mutation (Ser675Thr) in the Lep-R gene in one proband and several other mutations known to be not associated with obesity in other study groups. As this new mutation cannot explain our positive linkage result, the transmission disequilibrium of the 266-bp allele and the high transmission rate of the identified haplotype point towards a mutation in close proximity to marker D1S2852. Received: 9 March 1998 / Accepted: 17 July 1998     

Interval mapping of growth in divergent swine cross

A genomic scan of 18 swine autosomal chromosomes was constructed with 119 polymorphic microsatellite (ms) markers to identify quantitative trait loci (QTL) for 11 growth traits in the University of Illinois Meishan × Yorkshire Swine Resource Family. A significant QTL effect was found for post-weaning average daily gain (ADG) between 5.5 and 56 kg of body weight that mapped between markers SW373 and SW1301 near the telomere of Chromosome (Chr) 1 q (SSC1). This QTL effect had a nominal (pointwise) p-value of 0.000007, a genome wide p-value of 0.012, and accounted for 26% of the F₂ phenotypic variance. The same chromosome region also had significant effects on ADG between birth and 56 kg body weight (p-value = .000227), and on ADG between 35 and 56 kg (p-value = .00077). These observations suggest that a significant QTL for post-weaning growth resides on SSC1. Received: 18 May 1998 / Accepted: 6 October 1998     

Recurrence prediction using microRNA expression in hormone receptor positive breast cancer during tamoxifen treatment

Abstract

Purpose: To identify miRNAs associated with distant recurrence during tamoxifen treatment and build a recurrence prediction model.

Materials and methods: We measured the expression of five miRNAs (miR-134, miR-125b-5P, miRNA-30a, miR-10a-5p and miR-222). A total of 176 tumour tissues from 176 patients who had hormone receptor positive breast cancer with tamoxifen treatment were used to measure miRNA expression using quantitative real-time PCR (qRT-PCR).

Results: The five miRNAs were all up-regulated in distant recurrence cases within 5?years after surgery and during tamoxifen treatment. Kaplan-Meier survival analyses based on expression cut-offs determined by receiver characteristics curves (ROC) showed that high expression of miR-134, miR-125b-5P, miRNA-30a, miR-10a-5p and miR-222 were significantly (log-rank p-value =0.006, p-value <0.0001, p-value <0.0001, p-value <0.0001 and p-value <0.0001, respectively) associated with short relapse-free time. Our results were used to build a combined 3 miRNAs expression model. It could be used to categorize high-risk subset of patients with short relapse-free survival (AUC =0.891, p-value <0.0001).

Conclusions: Distant recurrence during tamoxifen treatment of hormone positive breast cancer might be affected by tamoxifen resistance related miRNAs. Such distant recurrence can be predicted using miRNA measurement.     

The generalized Fisher's combination and accurate p-value calculation under dependence

Combining dependent tests of significance has broad applications but the related p-value calculation is challenging. For Fisher's combination test, current p-value calculation methods (eg, Brown's approximation) tend to inflate the type I error rate when the desired significance level is substantially less than 0.05. The problem could lead to significant false discoveries in big data analyses. This paper provides two main contributions. First, it presents a general family of Fisher type statistics, referred to as the GFisher, which covers many classic statistics, such as Fisher's combination, Good's statistic, Lancaster's statistic, weighted Z-score combination, and so forth. The GFisher allows a flexible weighting scheme, as well as an omnibus procedure that automatically adapts proper weights and the statistic-defining parameters to a given data. Second, the paper presents several new p-value calculation methods based on two novel ideas: moment-ratio matching and joint-distribution surrogating. Systematic simulations show that the new calculation methods are more accurate under multivariate Gaussian, and more robust under the generalized linear model and the multivariate t-distribution. The applications of the GFisher and the new p-value calculation methods are demonstrated by a gene-based single nucleotide polymorphism (SNP)-set association study. Relevant computation has been implemented to an R package GFisher available on the Comprehensive R Archive Network.     

The effects of human tear electrolytes on the viability and hydrogel colonization of Pseudomonas aeruginosa and Staphylococcus aureus

This study was designed to evaluate the effects of the inorganic electrolytes present in human tear film on the viability and colonization of bacteria to hydrogels. Pseudomonas aeruginosa and Staphylococcus aureus were used in these experiments. A D-value test was performed to investigate any bacteriostatic effect by measuring the reduction of viable test microorganisms over time when exposed to the inorganic electrolyte solution. No D-value was calculable for S. aureus in electrolyte solution whereas a D-value of 8.1 h was obtained for P. aeruginosa in electrolyte solution. The D-value data indicate that staphylococci have a greater survivability potential in a hypertonic environment than do pseudomonads. Bacterial adhesion to high water, ionic hydrogels was studied using the Modified Robbins Device (MRD). The data for P. aeruginosa recovered from the lenses showed an 82% decrease in bacterial counts in electrolyte solution as compared to bacteria incubated in control solution. In contrast there were slight increases in S. aureus counts recovered from the lenses. Journal of Industrial Microbiology & Biotechnology (2000) 25, 17–19. Received 12 August 1999/ Accepted in revised form 05 January 2000     

Genetic association analysis of KCNQ3 and juvenile myoclonic epilepsy in a South Indian population

Juvenile myoclonic epilepsy (JME) is a common subtype of idiopathic generalized epilepsy that shows a complex pattern of inheritance. We have tested the association between JME phenotype and an intragenic marker in KCNQ3 by using the transmission disequilibrium test in 119 probands and their parents. Mutations in KCNQ3 are known to cause benign familial neonatal convulsions and are involved in the physiologically important M current in neurons. Our results provide suggestive evidence of allelic association between JME and KCNQ3 (P-value=0.008) and raise an interesting possibility of a genetic contribution to JME, viz., of a gene that causes a monogenic form of human epilepsy.     

Regression Trees for Survival Data — an Approach to Select Discontinuous Split Points by Rank Statistics

Regression trees allow to search for meaningful explanatory variables that have a non linear impact on the dependent variable. Often they are used when there are many covariates and one does not want to restrict attention to only few of them. To grow a tree at each stage one has to select a cut point for splitting a group into two subgroups. The basis for this are the maxima of the test statistics related to the possible splits due to every covariate. They or the resulting P-values are compared as measure of importance. If covariates have different numbers of missing values, ties, or even different measurement scales the covariates lead to different numbers of tests. Those with a higher number of tests have a greater chance to achieve a smaller P-value if they are not adjusted. This can lead to erroneous splits even if the P-values are looked at informally. There is some theoretical work by Miller and Siegmund (1982) and Lausen and Schumacher (1992) to give an adjustment rule. But the asymptotic is based on a continuum of split points and may not lead to a fair splitting rule when applied to smaller data sets or to covariates with only few different values. Here we develop an approach that allows determination of P-values for any number of splits. The only approximation that is used is the normal approximation of the test statistics. The starting point for this investigation has been a prospective study on the development of AIDS. This is presented here as the main application.     

DNA methylation of loci within ABCG1 and PHOSPHO1 in blood DNA is associated with future type 2 diabetes risk

Identification of subjects with a high risk of developing type 2 diabetes (T2D) is fundamental for prevention of the disease. Consequently, it is essential to search for new biomarkers that can improve the prediction of T2D. The aim of this study was to examine whether 5 DNA methylation loci in blood DNA (ABCG1, PHOSPHO1, SOCS3, SREBF1, and TXNIP), recently reported to be associated with T2D, might predict future T2D in subjects from the Botnia prospective study. We also tested if these CpG sites exhibit altered DNA methylation in human pancreatic islets, liver, adipose tissue, and skeletal muscle from diabetic vs. non-diabetic subjects. DNA methylation at the ABCG1 locus cg06500161 in blood DNA was associated with an increased risk for future T2D (OR = 1.09, 95% CI = 1.02–1.16, P-value = 0.007, Q-value = 0.018), while DNA methylation at the PHOSPHO1 locus cg02650017 in blood DNA was associated with a decreased risk for future T2D (OR = 0.85, 95% CI = 0.75–0.95, P-value = 0.006, Q-value = 0.018) after adjustment for age, gender, fasting glucose, and family relation. Furthermore, the level of DNA methylation at the ABCG1 locus cg06500161 in blood DNA correlated positively with BMI, HbA1c, fasting insulin, and triglyceride levels, and was increased in adipose tissue and blood from the diabetic twin among monozygotic twin pairs discordant for T2D. DNA methylation at the PHOSPHO1 locus cg02650017 in blood correlated positively with HDL levels, and was decreased in skeletal muscle from diabetic vs. non-diabetic monozygotic twins. DNA methylation of cg18181703 (SOCS3), cg11024682 (SREBF1), and cg19693031 (TXNIP) was not associated with future T2D risk in subjects from the Botnia prospective study.     

Association Study of IL-12B Polymorphisms Susceptibility with Ankylosing Spondylitis in Mainland Han Population

Objective

This study aims to determine whether the genetic polymorphisms of IL-12B gene is a susceptibility factor to Ankylosing spondylitis (AS) in mainland Han Chinese population.

Method

Eight single-nucleotide polymorphisms (SNPs) (rs10045431, rs11167764, rs3212227, rs6556412, rs6556416, rs6871626, rs6887695 and rs7709212) in the IL-12B gene were genotyped by iMLDR Assay technology in 400 patients [96% (384/400) HLA-B27(+)] and 395 geographically and ethnically matched healthy controls in mainland Han Chinese population. The correlation between IL-12B genetic polymorphisms and AS activity index (BASDAI, BASFI) were tested.

Results

The significant difference was found in genotype distribution between AS and healthy controls (χ² = 6.942, P-value = 0.031) of the SNP rs6871626. Furthermore, significant evidence was also detected under the recessive model for minor allele A. The AA genotype carrier had 1.830 fold risk compared with C allele carrier (with CC and AC genotypes) [OR (95% CI) = 1.830 (1.131-2.961), P-value = 0.014]. Nevertheless, the difference was no longer significant after Bonferroni correction. Subset analysis on cases with HLA-B27(+) did find the same results. Three genotypic groups (AA, CC and CA) in rs6871626 site was highly associated with the BASDAI and BASFI (P-value = 0.012 and P-value = 0.023, respectively), after adjustment for effect of age, sex, and disease duration, the P-value was 0.031 and 0.041, respectively. The AA genotype of rs6871626 was also significantly correlated with an increased BASDAI and BASFI compared to the AC and CC genotypes in AS patients.

Conclusion

Our findings suggest that rs6871626 may be associated AS susceptibility and with disease activity (BASDAI, BASFI) in mainland Han Chinese population.     

Generation and exploration of a dense genetic map in a region of a QTL affecting corpora lutea in a Meishan × Yorkshire cross

Previously genomic scans revealed quantitative trait loci (QTL) on porcine Chromosome 8 (SSC8) as significantly affecting the number of corpora lutea (CL) in swine. In one study, statistical evidence for the putative QTL was found in the chromosomal region defined by the microsatellites (MS) SW205, SW444, SW206, and SW29. A Yeast Artificial Chromosome library was screened by using the corresponding primers for clones containing these MS by PCR. From five positive YAC clones, 10 additional MS were isolated and mapped to SSC8 with the INRA-University of Minnesota porcine Radiation Hybrid (IMpRH) panel. The genetic map position of the QTL has been refined by addition of these 10 markers. The QTL evaluation included pedigrees of F₂-intercross Meishan × Yorkshire design, with phenotypic data of 108 F₂ female offspring and genotypic data for 29 MS markers on SSC8. The analysis was performed by using the least squares regression method. The calculated QTL effect for CL obtained by the multilocus least squares method showed a maximum test statistic (F value = 13.98) at position 99 cM between three MS derived from YACs containing SW205 and SW1843 spanning an interval of 7.1 cM. The point-wise (nominal) P-value was 5.21 × 10⁻⁶ corresponding to a genome-wide P-value of 0.009. The additive QTL effect explained 17.4% of the phenotypic variance. Received: 23 December 2000 / Accepted: 07 May 2001