Pathway analysis of complex diseases for GWAS, extending to consider rare variants, multi-omics and interactions.

BACKGROUND: Genome-wide association studies (GWAS) is a major method for studying the genetics of complex diseases. Finding all sequence variants to explain fully the aetiology of a disease is difficult because of their small effect sizes. To better explain disease mechanisms, pathway analysis is used to consolidate the effects of multiple variants, and hence increase the power of the study. While pathway analysis has previously been performed within GWAS only, it can now be extended to examining rare variants, other "-omics" and interaction data. SCOPE OF REVIEW: 1. Factors to consider in the choice of software for GWAS pathway analysis. 2. Examples of how pathway analysis is used to analyse rare variants, other "-omics" and interaction data. MAJOR CONCLUSIONS: To choose appropriate software tools, factors for consideration include covariate compatibility, null hypothesis, one- or two-step analysis required, curation method of gene sets, size of pathways, and size of flanking regions to define gene boundaries. For rare variants, analysis performance depends on consistency between assumed and actual effect distribution of variants. Integration of other "-omics" data and interaction can better explain gene functions. GENERAL SIGNIFICANCE: Pathway analysis methods will be more readily used for integration of multiple sources of data, and enable more accurate prediction of phenotypes.

How does family functioning contribute to academic-related outcomes of Chinese adolescents: the mediating role of spirituality.

While family functioning is crucial to adolescent developmental outcomes, the mediating role of spirituality in the relationship between family functioning and academic-related outcomes of adolescents has been sparsely explored, particularly in non-Western contexts. To address this gap, based on a short-term longitudinal study, we examined the influence of family functioning on the academic values and academic anxiety of 4,981 Chinese adolescents in Sichuan, China, with spirituality as the mediator. We gathered data from students aged 11 and above at Wave 1 and at six months later (Wave 2). Analysis utilizing structural equation modeling indicated that prior family functioning positively and negatively predicted subsequent academic values and academic anxiety respectively, with spirituality as a significant mediator. Theoretically, this study helps to build up a conceptual model on how family functioning and spirituality of adolescents shape academic values and academic anxiety of adolescents. Practically, the present findings highlight the significance of enhancing family functioning and adolescent spirituality to help adolescents strive for academic success.

Psychometric properties of the Chinese Family Assessment Instrument: evidence from mainland China.

Regarding the assessment of family functioning in Chinese people, there are several research gaps. First, although there are some instruments in the field, there are very few validated instruments. Second, while some translated measures have been developed, there are very few assessment tools based on indigenous Chinese concepts. Third, compared to Hong Kong, research on family assessment is relatively inactive in mainland China. Fourth, there are very few family assessment tools to assess perceived family functioning in older children and early adolescents. Fifth, few studies used large samples to validate family assessment tools. Sixth, researchers seldom utilized longitudinal data to examine the psychometric properties of family assessment tools. Finally, few studies have examined factorial validity across samples and time to demonstrate the stability of Chinese family assessment measures. In Hong Kong, based on focus group data (i.e., indigenous concepts of family functioning) and an integration with the family science literature, we have developed the Chinese Family Assessment Instrument (C-FAI) to assess perceived family functioning according to the perception of adolescents. Results showed that the C-FAI possessed good reliability and validity. Specifically, five dimensions of the measure (mutuality, communication, conflict, parental concern and parental control) were supported via exploratory factor analysis and confirmatory factor analysis. Convergent validity and reliability of the C-FAI were illustrated. To understand the psychometric properties of the C-FAI in mainland China, we collected three waves of data from students in the period of preadolescence and early adolescence in mainland China (N = 3,732). Based on the data, we examined the psychometric properties of the measure, particularly factor invariance in different samples and at different times. Confirmatory factor analysis provided support for the five dimensions in C-FAI, including factorial invariance in terms of configuration, factor loading, intercepts, and over time. There was evidence for convergent validity and discriminant validity of the measure. Finally, reliability analyses showed that the total C-FAI scale and its subscales are internally consistent. The present findings suggest that family researchers and practitioners can use the C-FAI to objectively assess perceived family functioning in preadolescence and early adolescence in different Chinese communities.

Impact of Family Functioning on Adolescent Materialism and Egocentrism in Mainland China: Positive Youth Development Attributes as a Mediator.

Although adolescent materialism and egocentrism are growing problems in Chinese societies, there are very few studies investigating their predictors and related mediators. Longitudinal studies in this area are also sparse. Based on a short-term longitudinal study (n = 4981), we assessed the impact of family functioning on materialism and egocentrism of Chinese adolescents, with positive youth development attributes as a hypothesized mediating factor. Results showed that family functioning positively predicted positive youth development attributes but negatively predicted adolescent materialism and egocentrism; positive youth development attributes also negatively predicted adolescent materialism and egocentrism. Mediational analyses showed that positive youth development attributes mediated the impact of family functioning on adolescent materialism and egocentrism. The theoretical and methodological advances of the study are discussed.

Development and Evaluation of a Pioneer School-Based Gifted Education Program (Project GIFT) for Primary and Secondary Students in Hong Kong.

In this study, we used a quasi-experimental research design with pretest and post-test data collected from an experimental group and a control group to investigate changes in students after participating in a school-based gifted education program (Project GIFT) in Hong Kong. There were 3207 successfully matched students (3rd to 9th graders) joining the Level 1 program (for all students) alone or both the Level 1 program and Level 2 program (for gifted students). Participants of the experimental and control groups completed validated measures on creativity, multiple intelligences, gifted characteristics, self-efficacy, psychological well-being, and satisfaction with life before and after participating in the program(s). One-way ANCOVA results revealed that students in the experimental groups showed positive changes after joining the program(s), with a greater impact for students joining both Level 1 and Level 2 programs. Students participating in both Level 1 and Level 2 programs displayed significant improvement in creativity, academic performance, logical-mathematical intelligence, intrapersonal intelligence, self-efficacy, autonomy, environmental mastery, and personal growth compared to the control counterparts. This study illustrates the benefits of the Level 1 and Level 2 programs in promoting the holistic development of the program participants.

Professional Development for Teachers of Gifted Education in Hong Kong: Instrument Validation and Training Effectiveness.

Project GIFT is a pioneer research-based gifted education program which has been found to be effective in fostering holistic development of students in Hong Kong. Nevertheless, little is known whether the Project is beneficial to teachers. To investigate the changes in teachers after participating in the Project, we adopted a quasi-experimental design with pretest and posttest data collected from experimental and control groups in this study. A total of 2031 primary and secondary school teachers participated in the professional development program of the Project. They completed validated measures on teachers' knowledge of and attitudes toward gifted education, teaching behaviors, characteristics and competencies, in addition to well-being before and after participating in the program. Results of one-way ANCOVA showed that the program could promote teachers' knowledge of gifted education and specific teaching strategies to gifted learners. This study provides preliminary support for the program in promoting holistic professional development of participating teachers in gifted education. The theoretical and practical implications of the findings are discussed.

A DNA pooling-based case-control study of myopia candidate genes COL11A1, COL18A1, FBN1, and PLOD1 in a Chinese population.

PURPOSE: We examined the relationship between high myopia and common polymorphisms in four candidate genes: collagen, type XI, alpha 1 (COL11A1); collagen, type XVIII, alpha 1 (COL18A1); fibrillin 1 (FBN1); and procollagen-lysine 1,2-oxoglutarate 5-dioxygenase 1 (PLOD1). These genes were selected because rare pathogenic mutations in these genes cause disease syndromes that have myopia, usually high myopia, as one of the common presenting features. METHODS: This study recruited 600 unrelated Han Chinese subjects including 300 cases with high myopia (spherical equivalent or SE≤-8.00 diopters) and 300 controls (SE within ±1.00 diopter). A total of 66 tag single nucleotide polymorphisms (SNPs) were selected for study from these four candidate genes. The study adopted a DNA pooling strategy with an initial screen of DNA pools to identify putatively positive SNPs and then confirmed the "positive" SNPs by genotyping individual samples forming the original DNA pools. DNA pools were each constructed by mixing equal amounts of DNA from 50 individuals with the same phenotype status. Six case pools were prepared from 300 cases and six control pools from 300 controls. Allele frequencies of DNA pools were estimated by analyzing the primer-extended products with denaturing high performance liquid chromatography and compared between case pools and control pools with nested ANOVA. RESULTS: In the first stage, 60 SNPs from the 4 candidate genes were successfully screened using the DNA pooling approach. Of these, 6 SNPs showed a statistical significant difference in estimated allele frequencies between case pools and controls at p<0.10. In the second stage, these "positive" SNPs were followed up by individual genotyping, but failed to be confirmed via standard single-marker and haplotype analyses. CONCLUSIONS: Common polymorphisms in these four candidate genes (COL11A1, COL18A1, FBN1 and PLOD1) were unlikely to play important roles in the genetic susceptibility to high myopia.

The myopia susceptibility locus vasoactive intestinal peptide receptor 2 (VIPR2) contains variants with opposite effects.

Myopia is the commonest eye disorder in the world. High myopes are predisposed to ocular pathologies. The vasoactive intestinal peptide receptor 2 (VIPR2) gene was identified as a myopia susceptibility locus by our group and another group. We continued to fine-map this locus. A case-control study was performed in 4 sequential stages with a total of 941 highly myopic subjects and 846 control subjects, all unrelated Chinese. Stage 1 experimentally genotyped 64.4% of the entire cohort for 152 single-nucleotide polymorphisms (SNPs) and Stage 2 the remaining subjects for 21 SNPs. Stage 3 combined the genotypes for 21 SNPs for the entire cohort, and identified one group of high-risk haplotypes and one group of protective haplotypes significantly associated with high myopia. Stage 4 imputed genotypes for variants in the VIPR2 region and identified two independent groups of variants: one group with high-risk minor alleles and another with protective minor alleles. Variants within each group were generally in strong linkage disequilibrium among themselves while high-risk variants were in linkage equilibrium with protective variants. Therefore, the VIPR2 locus seems to contain variants with opposite effects. This is the first study that has examined the genetic architecture of a myopia susceptibility locus in detail.

Sex- and age-dependent association of SLC11A1 polymorphisms with tuberculosis in Chinese: a case control study.

BACKGROUND: Host genetic factors are important determinants in tuberculosis (TB). The SLC11A1 (or NRAMP1) gene has been studied extensively for genetic association with TB, but with inconsistent findings. In addition, no study has yet looked into the effect of sex and age on the relationship between SLC11A1 polymorphisms and TB. METHODS: A case-control study was conducted. In total, 278 pulmonary TB patients and 282 sex- and age-matched controls without TB were recruited. All subjects were ethnic Chinese. On the basis of linkage disequilibrium pattern, three genetic markers from SLC11A1 and one from the nearby IL8RB locus were selected and examined for association with TB susceptibility. These markers were genotyped using single strand conformation polymorphism analysis or fragment analysis of amplified products. RESULTS: Statistically significant differences in allele (P = 0.0165, OR = 1.51) and genotype (P = 0.0163, OR = 1.59) frequencies of the linked markers SLC6a/b (classically called D543N and 3'UTR) of the SLC11A1 locus were found between patients and controls. With stratification by sex, positive associations were identified in the female group for both allele (P = 0.0049, OR = 2.54) and genotype (P = 0.0075, OR = 2.74) frequencies. With stratification by age, positive associations were demonstrated in the young age group (age < or =65 years) for both allele (P = 0.0047, OR = 2.52) and genotype (P = 0.0031, OR = 2.92) frequencies. All positive findings remained significant even after correction for multiple comparisons. No significant differences were noted in either the male group or the older age group. No significant differences were found for the other markers (one SLC11A1 marker and one IL8RB marker) either. CONCLUSION: This study confirmed the association between SLC11A1 and TB susceptibility and demonstrated for the first time that the association was restricted to females and the young age group.

Genetic Association Study of KCNQ5 Polymorphisms with High Myopia.

Identification of genetic variations related to high myopia may advance our knowledge of the etiopathogenesis of refractive error. This study investigated the role of potassium channel gene (KCNQ5) polymorphisms in high myopia. We performed a case-control study of 1563 unrelated Han Chinese subjects (809 cases of high myopia and 754 emmetropic controls). Five tag single-nucleotide polymorphisms (SNPs) of KCNQ5 were genotyped, and association testing with high myopia was conducted using logistic regression analysis adjusted for sex and age to give Pasym values, and multiple comparisons were corrected by permutation test to give Pemp values. All five noncoding SNPs were associated with high myopia. The SNP rs7744813, previously shown to be associated with refractive error and myopia in two GWAS, showed an odds ratio of 0.75 (95% CI 0.63-0.90; Pemp = 0.0058) for the minor allele. The top SNP rs9342979 showed an odds ratio of 0.75 (95% CI 0.64-0.89; Pemp = 0.0045) for the minor allele. Both SNPs are located within enhancer histone marks and DNase-hypersensitive sites. Our data support the involvement of KCNQ5 gene polymorphisms in the genetic susceptibility to high myopia and further exploration of KCNQ5 as a risk factor for high myopia.

Identification of myopia-associated WNT7B polymorphisms provides insights into the mechanism underlying the development of myopia.

Myopia can cause severe visual impairment. Here, we report a two-stage genome-wide association study for three myopia-related traits in 9,804 Japanese individuals, which was extended with trans-ethnic replication in 2,674 Chinese and 2,690 Caucasian individuals. We identify WNT7B as a novel susceptibility gene for axial length (rs10453441, Pmeta=3.9 × 10(-13)) and corneal curvature (Pmeta=2.9 × 10(-40)) and confirm the previously reported association between GJD2 and myopia. WNT7B significantly associates with extreme myopia in a case-control study with 1,478 Asian patients and 4,689 controls (odds ratio (OR)meta=1.13, Pmeta=0.011). We also find in a mouse model of myopia downregulation of WNT7B expression in the cornea and upregulation in the retina, suggesting its possible role in the development of myopia.

Evaluation of proteoglycan gene polymorphisms as risk factors in the genetic susceptibility to high myopia.

PURPOSE. To investigate the relationship between high myopia and single nucleotide polymorphisms (SNPs) in six proteoglycan genes: aggrecan (ACAN), fibromodulin (FMOD), decorin (DCN), lumican (LUM), keratocan (KERA), and epiphycan (EPYC). These genes were selected for study because they are involved in induced myopia in animals and/or are within the human MYP3 locus identified by linkage analysis of families with high myopia. METHODS. Two groups of Chinese subjects were studied: group 1 (300 cases and 300 controls) and group 2 (356 cases and 354 controls). Cases were high myopes with spherical equivalent (SE) ≤ -8.00 D, and controls had SE between +1.0 and -1.0 D. From these candidate genes, 60 tagging SNPs were selected. First, 12 DNA pools were each constructed from 50 samples of the same phenotype from group 1 subjects and were tested for association with the SNPs. Second, putatively positive SNPs were confirmed by individual genotyping of group 1 subjects. Finally, positive results were replicated in group 2 subjects. RESULTS. Of the 58 SNPs successfully screened by DNA pooling, 8 ACAN SNPs passed the threshold of P ≤ 0.10 (nested ANOVA) and were then genotyped in the individual samples. Haplotypes rs3784757 and rs1516794 showed significant association with high myopia. However, the positive result could not be replicated in the second subject group. CONCLUSIONS. These six proteoglycan genes were not associated with high myopia in these Chinese subjects and hence are unlikely to be important in the genetic predisposition to high myopia.

Systematic investigation of the relationship between high myopia and polymorphisms of the MMP2, TIMP2, and TIMP3 genes by a DNA pooling approach.

PURPOSE: This study examined the relationship between high myopia and three myopia candidate genes--matrix metalloproteinase 2 (MMP2) and tissue inhibitor of metalloproteinase-2 and -3 (TIMP2 and TIMP3)--involved in scleral remodeling. METHODS: Recruited for the study were unrelated adult Han Chinese who were high myopes (spherical equivalent, ≤ -6.0 D in both eyes; cases) and emmetropes (within ±1.0 D in both eyes; controls). Sample set 1 had 300 cases and 300 controls, and sample set 2 had 356 cases and 354 controls. Forty-nine tag single-nucleotide polymorphisms (SNPs) were selected from these candidate genes. The first stage was an initial screen of six case pools and six control pools constructed from sample set 1, each pool consisting of 50 distinct subjects of the same affection status. In the second stage, positive SNPs from the first stage were confirmed by genotyping individual samples forming the DNA pools. In the third stage, positive SNPs from stage 2 were replicated, with sample set 2 genotyped individually. RESULTS: Of the 49 SNPs screened by DNA pooling, three passed the lenient threshold of P < 0.10 (nested ANOVA) and were followed up by individual genotyping. Of the three SNPs genotyped, two TIMP3 SNPs were found to be significantly associated with high myopia by single-marker or haplotype analysis. However, the initial positive results could not be replicated by sample set 2. CONCLUSIONS: MMP2, TIPM2, and TIMP3 genes were not associated with high myopia in this Chinese sample and hence are unlikely to play a major role in the genetic susceptibility to high myopia.

PAX6 haplotypes are associated with high myopia in Han chinese.

BACKGROUND: The paired box 6 (PAX6) gene is considered as a master gene for eye development. Linkage of myopia to the PAX6 region on chromosome 11p13 was shown in several studies, but the results for association between myopia and PAX6 were inconsistent so far. METHODOLOGY/PRINCIPAL FINDINGS: We genotyped 16 single nucleotide polymorphisms (SNPs) in the PAX6 gene and its regulatory regions in an initial study for 300 high myopia cases and 300 controls (Group 1), and successfully replicated the positive results with another independent group of 299 high myopia cases and 299 controls (Group 2). Five SNPs were genotyped in the replication study. The spherical equivalent of subjects with high myopia was ≤-8.0 dioptres. The PLINK package was used for genetic data analysis. No association was found between each of the SNPs and high myopia. However, exhaustive sliding-window haplotype analysis highlighted an important role for rs12421026 because haplotypes containing this SNP were found to be associated with high myopia. The most significant results were given by the 4-SNP haplotype window consisting of rs2071754, rs3026393, rs1506 and rs12421026 (P = 3.54×10(-10), 4.06×10(-11) and 1.56×10(-18) for Group 1, Group 2 and Combined Group, respectively) and the 3-SNP haplotype window composed of rs3026393, rs1506 and rs12421026 (P = 5.48×10(-10), 7.93×10(-12) and 6.28×10(-23) for the three respective groups). The results remained significant after correction for multiple comparisons by permutations. The associated haplotyes found in a previous study were also successfully replicated in this study. CONCLUSIONS/SIGNIFICANCE: PAX6 haplotypes are associated with susceptibility to the development of high myopia in Chinese. The PAX6 locus plays a role in high myopia.

Association of PAX6 polymorphisms with high myopia in Han Chinese nuclear families.

PURPOSE: The paired box 6 (PAX6) gene is critical to eye development. Based on prior linkage evidence, this study was conducted to investigate the association of PAX6 polymorphisms with high myopia in a Han Chinese population. METHODS: Tag single nucleotide polymorphisms (tSNPs) in the PAX6 locus were selected based on HapMap data, and other polymorphisms in its functional regions were also identified. Both tSNPs and identified variants were genotyped in 164 nuclear families with 170 highly myopic (spherical equivalent < -6.0 D in both eyes) offspring. The linkage disequilibrium pattern of SNPs was established in the parental group (n = 328). Family-based association tests were performed using family-based association testing (FBAT) and genetic association computer analyses. RESULTS: Single marker analysis of SNPs rs3026390 and rs3026393 showed significant association with high myopia as a qualitative trait in dominant and recessive models (P = 0.0014 and P = 0.0011, respectively). For rs3026393, the genotype relative risk was 2.57 for G/T and 2.22 for T/T with reference to G/G. Significantly increased transmission was demonstrated for the haplotypes carrying allele T of rs3026393 in the additive and dominant models (P < 0.0070), whereas significantly decreased transmission was found for haplotypes carrying allele G of rs3026393 in the recessive model (P = 0.0173). Preferential transmission of single alleles and haplotypes remained significant after correction for multiple comparisons. CONCLUSIONS: This study demonstrates the association of PAX6 variants with susceptibility to high myopia. The PAX6 locus may contain polymorphisms playing a role in high myopia in southern Han Chinese.

TGFB1 as a susceptibility gene for high myopia: a replication study with new findings.

OBJECTIVE: To investigate the genetic association between transforming growth factor beta1 (TGFB1) gene polymorphisms and high myopia in a Chinese population. METHODS: Six hundred adults were recruited for this case-control study, including 300 subjects with high myopia (-8.0 diopters or worse) and 300 control subjects (within +/-1.0 diopters). Seven tag single-nucleotide polymorphisms (SNPs) and 1 coding SNP were genotyped. Their frequencies were compared between cases and controls by statistical tests. RESULTS: Four SNPs in the 5' half of the gene showed significant differences in allele and genotype frequencies between cases and controls. The results remained significant after correction for multiple comparisons. The previously reported association of the coding SNP rs1800470 with high myopia was successfully replicated. The tag SNP rs4803455 in intron 2 was found to account for the positive results of the other 3 SNPs by stepwise logistic regression. The minor allele T of rs4803455 was protective against high myopia with an odds ratio of 0.67 (95% confidence interval, 0.53-0.86; P= .001). CONCLUSION: TGFB1 is a myopia susceptibility gene. CLINICAL RELEVANCE: TGFB1 is the first myopia susceptibility gene successfully replicated. The functional significance of rs4803455 or the genuine causative SNPs in linkage disequilibrium with it remains to be determined.

ABO blood group in Kuwaitis: detailed allele frequency distribution and identification of novel alleles.

BACKGROUND: The ABO blood group is clinically the most important blood group system and can now be genotyped easily by DNA-based methods without family studies. STUDY DESIGN AND METHODS: Samples (n = 166) from a Kuwaiti population were phenotyped by standard serologic techniques for the ABO blood group and genotyped for the ABO locus by an established multiplex polymerase chain reaction protocol followed by single-strand conformation polymorphism (SSCP) analysis. Nonstandard SSCP patterns were investigated by DNA sequencing of exons 6 and 7 and, if necessary intron 6. RESULTS: Standard SSCP patterns identified six classical alleles in this population: A101 (0.1115), A102 (0.0181), A201 (0.0301), B101 (0.1627), O101 (0.3103), and O201 (0.2500). One A, 1 B, and 8 O variant alleles were identified (total frequency, 0.1175). All variant alleles were each present in one or two chromosomes (< or =0.0060) in our samples except O109 (0.0813). Three of these 10 variant alleles were novel alleles defined by newly identified single-nucleotide polymorphisms in exon 7 (527G>A, 687C>T, and 1116G>A). One new base substitution result in amino acid change. CONCLUSIONS: This is the first study reporting the detailed distribution of ABO alleles and genotypes in Kuwaitis. Sixteen alleles were identified, including 3 novel alleles.