Genome-Wide DNA Methylation and Gene Expression Analyses of Monozygotic Twins Discordant for Intelligence Levels

Human intelligence, as measured by intelligence quotient (IQ) tests, demonstrates one of the highest heritabilities among human quantitative traits. Nevertheless, studies to identify quantitative trait loci responsible for intelligence face challenges because of the small effect sizes of individual genes. Phenotypically discordant monozygotic (MZ) twins provide a feasible way to minimize the effects of irrelevant genetic and environmental factors, and should yield more interpretable results by finding epigenetic or gene expression differences between twins. Here we conducted array-based genome-wide DNA methylation and gene expression analyses using 17 pairs of healthy MZ twins discordant intelligently. ARHGAP18, related to Rho GTPase, was identified in pair-wise methylation status analysis and validated via direct bisulfite sequencing and quantitative RT-PCR. To perform expression profile analysis, gene set enrichment analysis (GSEA) between the groups of twins with higher IQ and their co-twins revealed up-regulated expression of several ribosome-related genes and DNA replication-related genes in the group with higher IQ. To focus more on individual pairs, we conducted pair-wise GSEA and leading edge analysis, which indicated up-regulated expression of several ion channel-related genes in twins with lower IQ. Our findings implied that these groups of genes may be related to IQ and should shed light on the mechanism underlying human intelligence.     

A unified sampling approach for multipoint analysis of qualitative and quantitative traits in sib pairs

Recent advances in molecular biology have enhanced the opportunity to conduct multipoint mapping for complex diseases. Concurrently, one sees a growing interest in the use of quantitative traits in linkage studies. Here, we present a multipoint sib-pair approach to locate the map position (tau) of a trait locus that controls the observed phenotype (qualitative or quantitative), along with a measure of statistical uncertainty. This method builds on a parametric representation for the expected identical-by-descent statistic at an arbitrary locus, conditional on an event reflecting the sampling scheme, such as affected sib pairs, for qualitative traits, or extreme discordant (ED) sib pairs, for quantitative traits. Our results suggest that the variance about tau&d4;, the estimator of tau, can be reduced by as much as 60%-70% by reducing the length of intervals between markers by one half. For quantitative traits, we examine the precision gain (measured by the variance reduction in tau&d4;) by genotyping extremely concordant (EC) sib pairs and including them along with ED sib pairs in the statistical analysis. The precision gain depends heavily on the residual correlation of the quantitative trait for sib pairs but considerably less on the allele frequency and exact genetic mechanism. Since complex traits involve multiple loci and, hence, the residual correlation cannot be ignored, our finding strongly suggests that one should incorporate EC sib pairs along with ED sib pairs, in both design and analysis. Finally, we empirically establish a simple linear relationship between the magnitude of precision gain and the ratio of the number of ED pairs to the number of EC pairs. This relationship allows investigators to address issues of cost effectiveness that are due to the need for phenotyping and genotyping subjects.     

Genes in the vicinity of <Emphasis Type="Italic">CFTR </Emphasis>modulate the cystic fibrosis phenotype in highly concordant or discordant F508del homozygous sib pairs

Cystic fibrosis (CF) is the most common severe autosomal recessive disease among Caucasians and is caused by lesions within the cystic fibrosis transmembrane conductance regulator ( CFTR) gene. The variability of CF disease severity suggests the effect of modifying factors. Thirty-four highly concordant and highly discordant F508del homozygous sib pairs, who have been selected out of a group of 114 pairs for extreme disease phenotypes by nutritional and pulmonary status, were typed at single nucleotide polymorphisms (SNPs) and short tandem repeat polymorphisms (STRPs) in the 24-cM CFTR-spanning region between D7S525 and D7S495. Allele frequencies differed significantly at D7S495, located within a 21-cM distance 3' of CFTR, comparing concordant mildly affected, concordant severely affected and discordant sib pairs, as judged by hypothesis-free permutation analysis by Monte Carlo simulation. A rare haplotype of two SNPs within the leptin gene promotor was found exclusively among the concordant mildly affected pairs. All concordant sib pairs shared the paternal F508del chromosome between CFTR and D7S495, whilst the cohort of discordant sib pairs inherited equal proportions of recombined and non-recombined parental chromosomes. We conclude that disease manifestation in CF is modulated by loci in the partially imprinted region 3' of CFTR that determine stature, food intake and energy homeostasis, such as the Silver-Russel-Syndrome candidate gene region and LEP.     

Linkage analysis of quantitative trait loci: sib pairs or sibships?

Sib pair linkage studies are now widely used to investigate the genetic factors implicated in complex quantitative traits. To increase the power of these approaches, it has been proposed to select extremely discordant (ED) sib pairs which are expected to contain the highest linkage information. However, it is known that sibships of larger size contain more linkage information than independent sib pairs. In this paper we compare, in terms of power and cost considerations, the ED strategy, which uses information on sib pairs only, to the recently developed 'Maximum Likelihood Binomial' sibship-oriented method performed on the whole sibships from which the ED sib pairs have been extracted. We show that the use of these whole sibships is an efficient alternative to approaches focusing on ED sib pairs only.     

Optimal designs for linkage disequilibrium mapping and candidate gene association tests in livestock populations

Simulation was used to evaluate the performance of different selective genotyping strategies when using linkage disequilibrium across large half-sib families to position a QTL within a previously defined genomic region. Strategies examined included standard selective genotyping and different approaches of discordant and concordant sib selection applied to arbitrary or selected families. Strategies were compared as a function of effect and frequency of QTL alleles, heritability, and phenotypic expression of the trait. Large half-sib families were simulated for 100 generations and 2% of the population was genotyped in the final generation. Simple ANOVA was applied and the marker with the greatest F-value was considered the most likely QTL position. For traits with continuous phenotypes, genotyping the most divergent pairs of half-sibs from all families was the best strategy in general, but standard selective genotyping was somewhat more precise when heritability was low. When the phenotype was distributed in ordered categories, discordant sib selection was the optimal approach for positioning QTL for traits with high heritability and concordant sib selection was the best approach when genetic effects were small. Genotyping of a few selected sibs from many families was generally more efficient than genotyping many individuals from a few highly selected sires.     

Mapping quantitative-trait loci in humans by use of extreme concordant sib pairs: selected sampling by parental phenotypes.

In two previous articles, we have considered sample sizes required to detect linkage for mapping quantitative-trait loci in humans, using extreme discordant sib pairs. Here, we examine further the use of extreme concordant sib pairs but consider the effect of parents' phenotypes. Sample sizes necessary to obtain a power of 80% with concordant sib pairs at a significance level of .0001 are given, stratified by parental phenotypes. When there is no residual correlation between sibs, the parental phenotypes have little impact on the sample sizes. When residual correlations between sibs exist, we show, however, that power can be considerably reduced by including extreme sib pairs when the parents also have similarly extreme values. Thus, we recommend the exclusion of such pairs from linkage studies. This recommendation reduces the required sample sizes by 3- to 28-fold. The degree of saving in the required sample sizes varies among different models and allele frequencies. The reduction is most dramatic (a 28-fold reduction) for a rare recessive gene.     

A novel framework for sib pair linkage analysis

Sib pair linkage analysis of a dichotomous trait is a popular method for narrowing the search for genes that influence complex diseases. Although the pedigree structures are uncomplicated and the underlying genetic principles straightforward, a surprising degree of complexity is involved in implementing a sib pair study and interpreting the results. Ascertainment may be based on affected, discordant, or unaffected sib pairs, as well as on pairs defined by threshold values for quantitative traits, such as extreme discordant sib pairs. To optimize power, various domain restrictions and null hypotheses have been proposed for each of these designs, yielding a wide array of choices for the analyst. To begin, we systematically classify the major sources of discretion in sib pair linkage analysis. Then, we extend the work of Kruglyak and Lander (1995), to bring the various forms into a unified framework and to facilitate a more general approach to the analysis. Finally, we describe a new, freely available computer program, Splat (Sib Pair Linkage Analysis Testing), that can perform any sib pair statistical test currently in use, as well as any user-defined test yet to be proposed. Splat uses the expectation maximization algorithm to calculate maximum-likelihood estimates of sharing (subject to user-specified conditions) and then plots LOD scores versus chromosomal position. It includes a novel grid-scanning capability that enables simultaneous visualization of multiple test statistics. This can lead to further insight into the genetic basis of the disease process under consideration. In addition, phenotype definitions can be modified without the recalculation of inheritance vectors, thereby providing considerable flexibility for exploratory analysis. The application of Splat will be illustrated with data from studies on the genetics of diabetic nephropathy.     

A Clinically Orientated Approach for Combining Discordant and Concordant Sib Pairs

The use of extreme discordant sib pairs (EDSP) or extreme concordant sib pairs (ECSP) has recently been proposed to increase power for mapping quantitative traits in humans (RISCH and ZHANG, 1995, 1996). In this paper we propose a test statistic to jointly analyze EDSP and ECSP based on a clinical sampling procedure. This test statistic does not fulfill any optimality criteria. However, this approach is useful for quantitative traits of clinical significance for which EDSP are rare and/or expensive to ascertain. We show how sample size calculations can be adjusted for recombination using single markers, multipoint analysis, incompletely polymorphic markers and varying proportions of ECSP. If the true genetic model is unknown, the combined approach appears to be more robust than sampling based on only EDSP or only ECSP. We discuss how to find the optimal proportion of EDSP and ECSP to be included in an analysis under power considerations.     

Gene-environment interaction and affected sib pair linkage analysis

OBJECTIVES: Gene-environment (GxE) interaction influences risk for many complex disease traits. However, genome screens using affected sib pair linkage techniques are typically conducted without regard for GxE interaction. We propose a simple extension of the commonly used mean test and evaluate its power for several forms of GxE interaction. METHODS: We compute expected IBD sharing by sibling exposure profile, that is by whether two sibs are exposed (EE), unexposed (UU), or are discordant for exposure (EU). We describe a simple extension of the mean test, the "mean-interaction" test that utilizes heterogeneity in IBD sharing across EE, EU, and UU sib pairs in a test for linkage. RESULTS: The mean-interaction test provides greater power than the mean test for detecting linkage in the presence of moderate or strong GxE interaction, typically when the interaction relative risk (R(ge)) exceeds 3 or is less than 1/3. In the presence of strong interaction (R(ge) = 10), the required number of affected sib pairs to achieve 80% power for detecting linkage is approximately 30% higher when the environmental factor is ignored in the mean test, than when it is utilized in the mean-interaction test. CONCLUSION: Linkage methods that incorporate environmental data and allow for interaction can lead to increased power for localizing a disease gene involved in a GxE interaction.     

ACE基因I/D多态与冠心病的遗传流行病学研究——表型不一致同胞对分析和传递不平衡检验

用表型不一致同胞对分析(DSP)和传递不平衡检验(TDT),在冠心病家系中探讨血管紧张素转换酶(ACE)基因内含子16中的插入/缺失(I/D)多态是否为冠心病的遗传易患因素。方法：1998年10月～1999年2月期间收集先证者一级亲属中至少有1例冠心病患者的家系45个,其中完整核心家系、父母一方、双方基因型缺失家系分别为21、2与22个,调查对象212人。PCR-RFLP方法鉴定ACE基因I/D多态性基因座基因型。条件Logistic回归进行DSP分析,TDT-STDT 1.1程序进行TDT、STDT检验。结果表明,45个冠心病家系共组成106对DSP,单变量条件Logistic回归及调整传统危险因素后的多变量条件logistic回归均未发现II、ID和DD基因型在表型不一致同胞对中的分布存在差别。对13个满足要求的核心家系进行TDT检验,杂合子父母传递给患病子代的D等位基因频率未显著偏离50％(P>0.05);24个满足要求的同胞组进行STDT检验亦未发现受累子代与非受累子代D等位基因分布有显著差异(P>0.05)。结论:在冠心病家系中未发现ACE基因I/D多态与冠心病存在关联或与疾病基因座存在连锁,说明该基因座可能不是国人冠心病的遗传易患基因。 Abstract:To investigate whether the insertion/deletion polymorphism of the human angiotensin I converting enzyme gene increased the risk of coronary heart disease (CHD) in CHD pedigrees,discordant sib pair analysis (DSP) and transmission/disequilibrium test (TDT) were used.Forty-five CHD pedigrees with at least one CHD patient in the first degree relatives of probands were recruited during Oct.1998 to Feb.1999,of which parental genotype known,one or both parental genotype missing was 21,2 and 22 respectively.ACE genotype was measured by PCR technique.Conditional Logistic regression was used to analyze the DSP,and TDT-STDT program 1.1 was used for TDT and STDT.Univariable conditional Logistic regression did not find significant difference of the distribution of three different ACE genotypes in the 106 discordant sib pairs obtained from the 45 pedigrees.After adjusting effects of traditional risk factors of CHD,no significant difference of the distribution was found by multiple Logistic regression model.Neither the TDT for 13 nuclear families or STDT(sib transmission/disequilibrium test) for 24 sibships showed significant difference between the transmitted and untransmitted ACE gene D allele distributions.Our results show that the insertion/deletion polymorphism of ACE gene is not associated or linked with CHD in Chinese population.     

Apolipoprotein E and H polymorphisms in Mongolian Buryat: allele frequencies and relationship with plasma lipid levels

A Buryat population consisting of seven tribal groups in eastern Mongolia has been screened to determine the frequency distribution of different apolipoprotein E and H alleles (APOE and APOH, genes) coding for common isoforms and their association with quantitative plasma lipid levels. Allele frequencies at the APOE locus in 125 healthy Buryat aged 17 to 73 years were highest for APOE*3 (0.804), followed by APOE*4 (0.164) and APOE*2 (0.032). The APOH locus had high frequencies of APOH*2 (0.912) and APOH*3 (0.088). APOH*1 was not detected. No significant differences were observed in the overall APOE allele frequencies between the Buryat and the Siberian Evenki, Inuits, and Indians in Asia, or with some European whites. The frequency distribution of the overall APOH alleles of the Buryat was similar to that of the Japanese in Asia. Overall plasma lipid levels of the Buryat (males aged 20 to 73 years, females aged 21 to 64 years) were considerably lower, comparable to those of the Evenki. The APOE*4/E*3 males had significantly high total- and LDL-cholesterol levels compared with the APOE*3/E*3 males (p < 0.025 and p < 0.01, respectively). No significant effects of the APOH genotypes on any of the plasma lipid levels were observed. In particular, our data regarding APOE suggest that the Buryat are genetically close in allele frequencies to the Evenki and Inuits, but differ from them in the association of genotype APOE*4/E*3 with cholesterol levels.     

Variations in genome-wide gene expression in identical twins – a study of primary osteoblast-like culture from female twins discordant for osteoporosis

Background  

Monozygotic twin pairs who are genetically identical would be potentially useful in gene expression study for specific traits as cases and controls, because there would be much less gene expression variation within pairs compared to two unrelated individuals. However the twin pair has to be discordant for the particular trait or phenotype excluding those resulting from known confounders. Such discordant monozygotic twin pairs are rare and very few studies have explored the potential usefulness of this approach.     

Comprehensive evaluation of apolipoprotein H gene (APOH) variation identifies novel associations with measures of lipid metabolism in GENOA

Apolipoprotein H (apoH, also named beta-2 glycoprotein I) is found on several classes of lipoproteins, and is involved in the activation of lipoprotein lipase in lipid metabolism. We have comprehensively investigated the association of variation in the apoH gene (APOH) with lipid traits in hepatic cholesterol transport, dietary cholesterol transport (DCT), and reverse cholesterol transport (RCT). Our study population consisted of families from the Genetic Epidemiology Network of Arteriopathy multicenter study that include African Americans, Mexican Americans, and European Americans. We individually tested 36 single-nucleotide polymorphisms (SNPs) that span the APOH locus, including nonsynonymous variants that result in known apoH charge isoforms. In addition, we constructed haplotypes from SNPs in the 5' promoter region that comprise cis-acting regulatory elements, as well as haplotypes for multiple amino acid substitutions. We found point-wise significant associations of APOH variants with various lipid measures in the three racial groups. The strongest associations were found for DCT traits (triglyceride and apoE levels) in Mexican Americans with a nonsynonymous variant (SNP 14917, Cys306Gly) that may alter apoH protein folding in a region involved in phospholipid binding. In conclusion, family-based analyses of APOH variants have identified associations with measures of lipid metabolism in three American racial groups.     

Chimpanzee apolipoprotein H (beta2-glycoprotein I): report on the gene structure, a common polymorphism, and a high prevalence of antiphospholipid antibodies

Apolipoprotein H (apoH, protein; APOH, gene) is a 50-kDa glycoprotein that binds to negatively charged substrates, including phospholipids. ApoH is a main target antigen for the binding of antiphospholipid antibodies that are associated with thrombotic events. We have previously characterized the structural organization of the human APOH gene. Because of the significant structural homology between the human and chimpanzee genomes, we have employed oligonucleotides from the human APOH gene sequence to amplify chimpanzee DNA covering the entire transcribed region together with flanking sequence in the 5' region. As in humans, the chimpanzee APOH gene consists of eight exons and seven introns and encodes for a 326-amino-acid protein. The deduced amino acid and nucleotide sequence show 99.4% and 99.6% similarity between human and chimpanzee APOH, respectively. Using isoelectric focusing (IEF) and immunoblotting, we screened 155 chimpanzees (128 unrelated captured parents and 27 captive-born offspring) for the apoH protein polymorphism. The most common IEF pattern in chimpanzees was identical to a previously described APOH*3 allele in humans. In addition, an anodally shifted pattern was observed in chimpanzees with an allele frequency of 0.168, and the corresponding allele was designated as APOH*4. DNA sequencing of APOH*4 carriers revealed a missense mutation in exon 6 (A-->G) at codon 210, which replaces the amino acid lysine by glutamic acid. This mutation does not affect the binding of apoH to cardiolipin as revealed by cardiolipin/enzyme-linked immunosorbent assay (ELISA). We also evaluated the prevalence of anti-apoH antibodies in chimpanzee plasma by using human-apoH-based ELISA and the association of the Lys210Glu mutation with the occurrence of anti-apoH antibodies. The prevalence of anti-apoH antibodies in chimpanzees (64%) was found to be unusually high compared with that found in humans. However, the Lys210Glu mutation showed no association with the occurrence of anti-apoH antibodies. The prevalence of anti-apoH antibodies in chimpanzees may serve as a useful animal model for the human antiphospholipid syndrome, where these antibodies are associated with clinical manifestations.     

A linkage strategy for detection of human quantitative-trait loci. I. Generalized relative risk ratios and power of sib pairs with extreme trait values.

We generalize the concept of the relative risk ratio (lambda) to the case of quantitative traits, to take into account the various trait outcomes of a relative pair. Formulas are derived to express the expected proportions of genes shared identical by descent by a sib pair, in terms of the generalized lambda's for sib pairs (lambda S), parent-offspring pairs (lambda O), and monozygotic twins (lambda M) and in terms of the recombination fraction, with the assumption of no residual correlations. If residual correlations are nonzero among relative pairs, we assume that they are the same among sib pairs, parent-offspring pairs, and monozygotic twins, and we employ a slightly different definition for the generalized lambda so that the same set of formulas still hold. The power (or, the sample size necessary) to detect quantitative-trait loci (QTLs) by use of extreme sib pairs (ESPs) is shown to be a function of the three generalized lambda's. Since lambda M can be derived by use of values of lambda S and lambda O, estimates of the latter two lambda's will suffice for the analysis of power and the necessary sample sizes of ESPs, for a QTL linkage study.     

Selection and subsequent analysis of sib pair data for QTL detection.

Haseman and Elston (1972) developed a robust regression method for the detection of linkage between a marker and a quantitative trait locus (QTL) using sib pair data. The principle underlying this method is that the difference in phenotypes between pairs of sibs becomes larger as they share a decreasing number of alleles at a particular QTL identical by descent (IBD) from their parents. In this case, phenotypically very different sibs will also on average share a proportion of alleles IBD at any marker linked to the QTL that is lower than the expected value of 0.5. Thus, the deviation of the proportion of marker alleles IBD from the expected value in pairs of sibs selected to be phenotypically different (i.e. discordant) can provide a test for the presence of a QTL. A simple regression method for QTL detection in sib pairs selected for high phenotypic differences is presented here. The power of the analytical method was found to be greater than the power obtained using the standard analysis when samples of sib pairs with high phenotypic differences were used. However, the use of discordant sib pairs was found to be less powerful for QTL detection than alternative selective genotyping schemes based on the phenotypic values of the sibs except with intense selection, when its advantage was only marginal. The most effective selection scheme overall was the use of sib pairs from entire families selected on the basis of high within-family variance for the trait in question. There is little effect of selection on QTL position estimates, which are in good agreement with the simulated values. However, QTL variance estimates are biased to a greater or lesser degree, depending on the selection method.     

Molecular Surveillance of Antiviral Drug Resistance of Influenza A/H3N2 Virus in Singapore, 2009-2013

Adamantanes and neuraminidase inhibitors (NAIs) are two classes of antiviral drugs available for the chemoprophylaxis and treatment of influenza infections. To determine the frequency of drug resistance in influenza A/H3N2 viruses in Singapore, large-scale sequencing of neuraminidase (NA) and matrix protein (MP) genes was performed directly without initial culture amplification. 241 laboratory-confirmed influenza A/H3N2 clinical samples, collected between May 2009 and November 2013 were included. In total, 229 NA (95%) and 241 MP (100%) complete sequences were obtained. Drug resistance mutations in the NA and MP genes were interpreted according to published studies. For the NAIs, a visual inspection of the aligned NA sequences revealed no known drug resistant genotypes (DRGs). For the adamantanes, the well-recognised S31N DRG was identified in all 241 MP genes. In addition, there was an increasing number of viruses carrying the combination of D93G+Y155F+D251V (since May 2013) or D93G (since March 2011) mutations in the NA gene. However, in-vitro NAI testing indicated that neither D93G+Y155F+D251V nor D93G alone conferred any changes in NAI susceptibility. Lastly, an I222T mutation in the NA gene that has previously been reported to cause oseltamivir-resistance in influenza A/H1N1/2009, B, and A/H5N1, was detected from a treatment-naïve patient. Further in-vitro NAI testing is required to confirm the effect of this mutation in A/H3N2 virus.     

High-resolution genetic mapping of complex traits.

Positional cloning requires high-resolution genetic mapping. To plan a positional cloning project, one needs to know how many informative meioses will be required to narrow the search for a disease gene to an acceptably small region. For a simple Mendelian trait studied with linkage analysis, the answer is straightforward. In this paper, we address the situation of a complex trait studied with affected-relative-pair methods. We derive mathematical formulas for the size of an appropriate confidence region, as a function of the relative risk attributable to the gene. Using these results, we provide graphs showing the number of relative pairs required to narrow the gene hunt to an interval of a given size. For example, we show that localizing a gene to 1 cM requires a median of 200 sib pairs for a locus causing a fivefold increased risk to an offspring and 700 sib pairs for a locus causing a twofold increased risk. We discuss the implications of these results for the positional cloning of genes underlying complex traits.     

Differential expression of genes in milk of dairy cattle during lactation

The milk fat globule (MFG) is one of the most representative of mammary gland tissues and can be utilized to study gene expression of lactating cows during lactation. In this study, RNA‐seq technology was employed to detect differential expression of genes in MFGs at day 10 and day 70 after calving between two groups of cows with extremely high (H group) and low (L group) 305‐day milk yield, milk fat yield and milk protein yield. In total, 1232, 81, 429 and 178 significantly differentially expressed genes (false discovery rate q < 0.05) were detected between H10 and L10, H70 and L70, H10 and H70, and L10 and L70 respectively. Gene Ontology enrichment and pathway analysis revealed that these differentially expressed genes were enriched in biological processes involved in mammary gland development, protein and lipid metabolism process, signal transduction, cellular process, differentiation and immune function. Among these differentially expressed genes, 178 (H10 vs. L10), 4 (H70 vs. L70), 68 (H10 vs. H70) and 22 (L10 vs. L70) were found to be located within previously reported QTL regions for milk production traits. Based on these results, some promising candidate genes for milk production traits in dairy cattle were suggested.