Comparison of statistics for candidate-gene association studies using cases and parents.

Studies of association between candidate genes and disease can be designed to use cases with disease, and in place of nonrelated controls, their parents. The advantage of this design is the elimination of spurious differences due to ethnic differences between cases and nonrelated controls. However, several statistical methods of analysis have been proposed in the literature, and the choice of analysis is not always clear. We review some of the statistical methods currently developed and present two new statistical methods aimed at specific genetic hypotheses of dominance and recessivity of the candidate gene. These new methods can be more powerful than other current methods, as demonstrated by simulations. The basis of these new statistical methods is a likelihood approach. The advantage of the likelihood framework is that regression models can be developed to assess genotype-environment interactions, as well as the relative contribution that alleles at the candidate-gene locus make to the relative risk (RR) of disease. This latter development allows testing of (1) whether interactions between alleles exist, on the scale of log RR, and (2) whether alleles originating from the mother or father of a case impart different risks, i.e., genomic imprinting.     

Evidence of association between methylenetetrahydrofolate reductase gene and susceptibility to breast cancer: a candidate-gene association study in a South-eastern European population

The methylenetetrahydrofolate reductase (MTHFR) gene has been proposed as a candidate gene for breast cancer (BC). However, the specific role of MTHFR polymorphisms and haplotypes has not been fully clarified and replicated. We examined the association of two common MTHFR polymorphisms (C677T and A1298C) and their haplotypes in a candidate-gene association study, involving 300 female patients with BC and 283 healthy women. The single locus analysis for the two polymorphisms revealed an association only for the C677T polymorphism [odds ratio (95% confidence interval), OR=2.05 (1.21-3.48)], but adjustment for age diminished this association [OR=1.76 (0.92-3.42)]. The menopausal status showed no significant effect in the association between the MTHFR polymorphisms and BC. The analysis of haplotypes showed an association for the C677-A1298 haplotypes (p=0.04). The available evidence from our study may support a contributory role of MTHFR polymorphisms in BC development. Future larger studies may help in elucidating the genetics of BC further.     

Number of individuals and molecular markers to use in genetic differentiation studies

Molecular markers are frequently used to study genetic variation among individuals within or between populations. Differences in marker banding patterns can be used to verify if individuals do, or do not, represent distinct groups or populations. Only in 2005, more than 500 studies used molecular markers to group individuals in clusters. Such studies make use of an arbitrary number of molecular markers from each of an arbitrary number of individuals presumed to represent distinct genotypes. However, the greater the genetic variation, the more likely a larger number of individuals and markers will be needed to capture a population's genetic signature. The numbers of both, markers and individuals included thus affect the way in which individuals are organized through cluster analyses, thereby affecting the conclusions drawn. Here we present a method that provides statistical criteria to verify that individual and marker sample sizes are sufficient to accurately depict genetic differentiation among different populations. Our method uses a resampling technique to assess the reproducibility of obtaining a particular grouping pattern for specific data sets. It thus, allows to estimate the robustness of the results obtained without including additional individuals, or markers.     

The association of genetic markers in two related swine breeds]

The combinations of genetic markers (allotypes, blood-group systems, and polymorphic proteins) were analyzed in pigs of two phylogenetically related Siberian populations: the newly selected Siberian specialized meat breed SM-1 (daughter breed) and the reproductive core of the Kemerovo breed (maternal breed). Statistically significant interlocus associations were found between the genetic markers that are known not to be linked, being assigned to different linkage groups, and the main selected meat-productivity traits. Some of these associations were shared by both breeds; others were found to be specific to either maternal or daughter populations. Possible genetic mechanisms underlying these associations and their significance are discussed.     

Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing.

We have developed a new method for forensic identification of individuals, in which a panel of biallelic DNA markers are amplified by the PCR, and the variable nucleotides are detected in the amplified DNA fragments by the solid-phase minisequencing method. A panel of 12 common polymorphic nucleotides located on different chromosomes with reported allele frequencies close to .5 were chosen for the test. The allele frequencies for most of the markers were found to be similar in the Finnish and other Caucasian populations. We also introduce a novel approach for rapid determination of the population frequencies of biallelic markers. By this approach we were able to determine the allele frequencies of the markers in the Finnish population, by quantitative analysis of three pooled DNA samples representing 3,000 individuals. The power of discrimination and exclusion of the solid-phase minisequencing typing test with 12 markers was similar to that of three VNTR markers that are routinely used in forensic analyses at our institute. The solid-phase minisequencing method was successfully applied to type paternity and forensic case samples. We also show that the quantitative nature of our method allows typing of mixed samples.     

Testing association of statistically inferred haplotypes with discrete and continuous traits in samples of unrelated individuals

There have been increasing efforts to relate drug efficacy and disease predisposition with genetic polymorphisms. We present statistical tests for association of haplotype frequencies with discrete and continuous traits in samples of unrelated individuals. Haplotype frequencies are estimated through the expectation-maximization algorithm, and each individual in the sample is expanded into all possible haplotype configurations with corresponding probabilities, conditional on their genotype. A regression-based approach is then used to relate inferred haplotype probabilities to the response. The relationship of this technique to commonly used approaches developed for case-control data is discussed. We confirm the proper size of the test under H(0) and find an increase in power under the alternative by comparing test results using inferred haplotypes with single-marker tests using simulated data. More importantly, analysis of real data comprised of a dense map of single nucleotide polymorphisms spaced along a 12-cM chromosomal region allows us to confirm the utility of the haplotype approach as well as the validity and usefulness of the proposed statistical technique. The method appears to be successful in relating data from multiple, correlated markers to response.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

Phylogenetic relationships between Leymus and related diploid Triticeae species revealed by ISSR markers

To investigate the genetic diversity and phylogenetic relationships between polyploid Leymus and related diploid species of the Triticeae tribe, inter-simple sequence repeats (ISSR) markers was used to analyze 41 Leymus accessions representing 22 species and 2 subspecies, together with Pseudoroegneria stipifolia (St), Psathyrostachys fragilis (Ns), Australopyrum retrofractum (W), Hordeum bogdanii, H. chilense (H) and Lophopyrum elongatum (E^e). A total of 376 clear and reproducible DNA fragments were amplified by 29 ISSR primers, among which 368 (97.87%) fragments were found to be polymorphic. 8–18 polymorphic bands were amplified by each polymorphic primer, with an average of 12.69 bands. The data of 376 ISSR bands were used to generate Nei’s similarity coefficients and to construct a dendrogram by means of UPGMA. The similarity coefficients data suggested great genetic diversity in genus Leymus and related diploid Triticeae species, the genetic diversity among the different species more abundant than that of the different accessions. The dendrogram and principal coordinate analysis showed explicit interspecific relationships and demonstrated close phylogenetic relationships between Leymus species and Psathyrostachys.     

The relative efficiency of the Hardy-Weinberg equilibrium-likelihood and the conditional on parental genotype-likelihood methods for candidate-gene association studies.

Selecting a control group that is perfectly matched for ethnic ancestry with a group of affected individuals is a major problem in studying the association of a candidate gene with a disease. This problem can be avoided by a design that uses parental data in place of nonrelated controls. Schaid and Sommer presented two new methods for the statistical analysis using this approach: (1) a likelihood method (Hardy-Weinberg equilibrium [HWE] method), which rests on the assumption that HWE holds, and (2) a conditional likelihood method (conditional on parental genotype [CPG] method) appropriate when HWE is absent. Schaid and Sommer claimed that the CPG method can be more efficient than the HWE method, even when equilibrium holds. It can be shown, however that in the equilibrium situation the HWE method is always more efficient than the CPG method. For a dominant disease, the differences are slim. But for a recessive disease, the CPG method requires a much larger sample size to achieve a prescribed power than the HWE method. Additionally, we show how the relative risks for the various candidate-gene genotypes can be estimated without relying on iterative methods. For the CPG method, we represent an asymptotic power approximation that is sufficiently precise for planning the sample size of an association study.     

Assessing familial aggregation of age at onset, by using estimating equations, with application to breast cancer.

In genetic research of chronic diseases, age-at-onset outcomes within families are often correlated. The nature of correlation of age-at-onset outcomes is indicative of common genetic and/or shared environmental risk factors among family members. Understanding patterns of such correlation may shed light on the disease etiology and, hence, is an important step to take prior to further searching for the responsible genes via segregation and linkage studies. Age-at-onset outcomes are different from those familiar quantitative or qualitative traits for which many statistical methods have been developed. In comparison with the quantitative traits, age-at-onset outcomes are often censored, i.e., instead of actual age-at-onset outcomes, only the current ages or ages at death are observed. They are also different from qualitative traits because of their continuity. Because of the complexity of correlated censored outcomes, few methods have yet been developed. A traditional approach is to impose a parametric joint distribution for the correlated age-at-onset outcomes, which has been criticized for requiring a stringent assumption about the entire distribution of age at onset. The purpose of this paper is to describe a method for assessing familial aggregation of correlated age-at-onset outcomes semiparametrically, by use of estimating equations. This method does not require any parametric assumption for modeling the age at onset. The estimates of parameters, including those quantifying the correlation within families, are consistent and have an asymptotic normal distribution that can be used to make inferences. To illustrate this new method, we analyzed two age-at-onset data sets that were obtained from studies conducted in the States of Washington and Hawaii, with the objective of quantifying the familial aggregations of age at onset of breast cancer.     

Ethnic-affiliation estimation by use of population-specific DNA markers.

During the past 10 years, DNA analysis has revolutionized the determination of identity in a forensic context. Statements about the biological identity of two human DNA samples now can be made with complete confidence. Although DNA markers are very powerful for distinguishing among individuals, most offer little power to distinguish ethnicity or to support any statement about the physical characteristics of an individual. Through a search of the literature and of unpublished data on allele frequencies we have identified a panel of population-specific genetic markers that enable robust ethnic-affiliation estimation for major U.S. resident populations. In this report, we identify these loci and present their levels of allele-frequency differential between ethnically defined samples, and we demonstrate, using log-likelihood analysis, that this panel of markers provides significant statistical power for ethnic-affiliation estimation. In addition to their use in forensic ethnic-affiliation estimation, population-specific genetic markers are very useful in both population- and individual-level admixture estimation and in mapping genes by use of the linkage disequilibrium created when populations hybridize.     

Testing for association between disease and linked marker loci: a log-linear-model analysis.

One approach frequently used for identifying genetic factors involved in the process of a complex disease is the comparison of patients and controls for a number of genetic markers near a candidate gene. The analysis of such association studies raises some specific problems because of the fact that genotypic and not gametic data are generally available. We present a log-linear-model analysis providing a valid method for analyzing such studies. When studying the association of disease with one marker locus, the log-linear model allows one to test for the difference between allelic frequencies among affected and unaffected individuals, Hardy-Weinberg (H-W) equilibrium in both groups, and interaction between the association of alleles at the marker locus and disease. This interaction provides information about the dominance of the disease susceptibility locus, with dominance defined using the epidemiological notion of odds ratio. The degree of dominance measured at the marker locus depends on the strength of linkage disequilibrium between the marker locus and the disease locus. When studying the association of disease with several linked markers, the model becomes rapidly complex and uninterpretable unless it is assumed that affected and unaffected populations are in H-W equilibrium at each locus. This hypothesis must be tested before going ahead in the analysis. If it is not rejected, the log-linear model offers a stepwise method of identification of the parameters causing the difference between populations. This model can be extended to any number of loci, alleles, or populations.     

Testing for association based on excess allele sharing in a sample of related cases and controls

Samples consisting of a mix of unrelated cases and controls, small pedigrees, and much larger pedigrees present a unique challenge for association studies. Few methods are available for efficient analysis of such a broad spectrum of data structures. In this paper we introduce a new matching statistic that is well suited to complex data structures and compare it with frequency-based methods available in the literature. To investigate and compare the power of these methods we simulate datasets based on complex pedigrees. We examine the influence of various levels of linkage disequilibrium (LD) of the disease allele with a marker allele (or equivalently a haplotype). For low frequency marker alleles/haplotypes, frequency-based statistics are more powerful in detecting association. In contrast, for high frequency marker alleles, the matching statistic has greater power. The highest power for frequency-based statistics occurs when the disease allele frequency closely matches the frequency of the linked marker allele. In contrast maximum power of the matching statistic always occurs for intermediate marker allele frequency regardless of the disease allele frequency. Moreover, the matching and frequency-based statistics exhibit little correlation. We conclude that these two approaches can be viewed as complementary in finding possible association between a disease and a marker for many different situations.     

Extreme morphological variation between related individuals of Gyrodactylus pungitii Malmberg, 1964 (Monogenea)

An example of extreme morphological variation in a gyrodactylid monogenean is described. A specimen of Gyrodactylus pungitii collected from Pungitius pungitius at Crowland, Lincolnshire, UK, was found to have very short, slightly asymmetrical hamuli with roots only 12 and 15 µm long, compared to 16–28 µm in typical specimens of this species. When the specimen was cloned on G. aculeatus, the grand-daughter was found to be a typical specimen of G. pungitii. This extreme morphological variation, which was not inherited by the offspring, highlights the dangers of describing gyrodactylids from single small collections of parasites.     

Linkage map of phenotype and RFLP markers in rice

The results from linkage mapping activities at Kyushu University during the last 10 years are summarized in this paper. The present paper concisely reveals present situation on linkage map of phenotype markers, the integration linkage map of phenotype and RFLP markers and the genetic stocks available. Some of the problems in this field, in addition, are pointed out and discussed.     

Correlation between Waardenburg syndrome phenotype and genotype in a population of individuals with identified PAX3 mutations

Waardenburg syndrome (WS) type 1 is an autosomal dominant disorder characterized by sensorineural hearing loss, pigmentary abnormalities of the eye, hair, and skin, and dystopia canthorum. The phenotype is variable and affected individuals may exhibit only one or a combination of several of the associated features. To assess the relationship between phenotype and gene defect, clinical and genotype data on 48 families (271 WS individuals) collected by members of the Waardenburg Consortium were pooled. Forty-two unique mutations in the PAX3 gene, previously identified in these families, were grouped in five mutation categories: amino acid (AA) substitution in the paired domain, AA substitution in the homeodomain, deletion of the Ser-Thr-Pro-rich region, deletion of the homeodomain and the Ser-Thr-Pro-rich region, and deletion of the entire gene. These mutation classes are based on the structure of the PAX3 gene and were chosen to group mutations predicted to have similar defects in the gene product. Association between mutation class and the presence of hearing loss, eye pigment abnormality, skin hypopigmentation, or white forelock was evaluated using generalized estimating equations, which allowed for incorporation of a correlation structure that accounts for potential similarity among members of the same family. Odds for the presence of eye pigment abnormality, white forelock, and skin hypopigmentation were 2, 8, and 5 times greater, respectively, for individuals with deletions of the homeodomain and the Pro-Ser-Thr-rich region compared to individuals with an AA substitution in the homeodomain. Odds ratios that differ significantly from 1.0 for these traits may indicate that the gene products resulting from different classes of mutations act differently in the expression of WS. Although a suggestive association was detected for hearing loss with an odds ratio of 2.6 for AA substitution in the paired domain compared with AA substitution in the homeodomain, this odds ratio did not differ significantly from 1.0. Received: 27 July 1997 / Accepted: 9 December 1997     

Polymorphisms and haplotypes in MyD88 are associated with the development of sarcoidosis: a candidate-gene association study

Sarcoidosis is considered as a disorder of protracted immune response to an as yet unidentified causative agent that leads to granuloma formation. Material from M. tuberculosis and P. acne has been repeatedly detected in the sarcoidosis lesions, implying the involvement of the Toll-like receptor2 (TLR2) gene that responds to these intracellular pathogens. Since TLR2 association studies have produced controversial results, we sought to investigate whether the downstream signalling molecule MyD88 could be linked to disease susceptibility. We analyzed a total of 93 cases with sarcoidosis and of 89 controls for the most common MyD88 SNPs: ?938C>A (rs4988453) and 1944C>G (rs4988457). There is evidence that the genotype distributions of both variants are associated with the development of sarcoidosis (p = 0.038 for ?938C>A and p = 0.026 for 1944C>G). In particular, ?938A and 1944G carriers were associated with risk of sarcoidosis [OR = 2.48 (1.23–5.02) and OR = 0.33 (0.14–0.76)], respectively, indicating dominance of the mutant alleles; however, the adjustment of the effect size for age and sex diminished the significance. The haplotype analysis showed association for the ?938A/1944G haplotype (p < 0.001). Since genetic association studies have linked MyD88 to Hodgkin’s lymphoma it is tempting to speculate that MyD88 may contribute to the granuloma formation that characterizes sarcoidosis.     

Patterns of intermediate host use and levels of association between two conflicting manipulative parasites.

For many parasites with complex life cycles, manipulation of intermediate host phenotypes is often regarded as an adaptation to increase the probability of successful transmission. This phenomenon creates opportunities for either synergistic or conflicting interests between different parasite species sharing the same intermediate host. When more than one manipulative parasite infect the same intermediate host, but differ in their definitive host, selection should favour the establishment of a negative association between these manipulators. Both Polymorphus minutus and Pomphorhynchus laevis exploit the amphipod Gammarus pulex as intermediate host but differ markedly in their final host, a fish for P. laevis and a bird for P. minutus. The pattern of host use by these two conflicting manipulative parasites was studied. Their incidence and intensity of infection and their distribution among G. pulex were first examined by analysing three large samples of gammarids collected from the river Tille, Eastern France. Both parasites had low prevalence in the host population. However, temporal fluctuation in the level of parasitic infection was observed. Overall, prevalence of both parasite species was higher in male than in female G. pulex. We then assessed the degree of association between the two parasites among their intermediate hosts, using two different methods: a host-centred measure and a parasite-centred measure. Both measures gave similar results; showing random association between the two acanthocephalan species in their intermediate hosts. We discuss our results in relation to the selective forces and ecological constraints that may determine the pattern of association between conflicting manipulative parasites.     

Revisiting the association between alcohol drinking and oral cancer in nonsmoking and betel quid non-chewing individuals

Background: Alcohol drinking is an oral cancer (OC) risk factor; tobacco smoking (TS) and betel quid chewing (BQC) are oral carcinogens and effect modifiers of drinking. Although the assessment of the independent effect of drinking on OC must necessarily account for effect modifiers, no observational study has included interaction terms between drinking, TS, BQC in regression analyses. In order to assess the independent association between drinking and OC, this pooled analysis focused on subjects who were not exposed to such effect modifiers. Methods: Case-control studies on OC, which discriminated non TS/non BQC drinkers from multiexposed drinkers were searched. Exposed subjects (≥1 drink daily, ≥10 years) were compared to unexposed subjects (non/occasional drinkers). Unadjusted odds ratios (ORs) were extracted/calculated. Pooled ORs were assessed with the random-effect method, which assumed high between-study heterogeneity (assessed with Cochran's Q). Robustness of estimates was investigated through use of adjusted ORs, correction for publication bias, sensitivity analysis to inclusion criteria. The drinking–TS interaction was assessed with the Interaction Contrast Ratio (ICR) and the Attributable Proportion due to Interaction (AP). Results: Sixteen studies were used, with substantially high heterogeneity. The pooled OR was 0.787 (95CI, 0.677–0.914). Use of adjusted ORs, correction for publication bias, sensitivity analysis corroborated these results. ICR and AP were 2.444 and 54.6%. Conclusions: Consistent with stratified analyses reporting non significant/negative associations between alcohol drinking and OC in non multiexposed subjects, an OC preventive activity of drinking is inferable. However, given the high prevalence and the oral carcinogenicity of concomitant drinking and smoking, drinking control policies remain essential.