Investigation of inversion polymorphisms in the human genome using principal components analysis

Despite the significant advances made over the last few years in mapping inversions with the advent of paired-end sequencing approaches, our understanding of the prevalence and spectrum of inversions in the human genome has lagged behind other types of structural variants, mainly due to the lack of a cost-efficient method applicable to large-scale samples. We propose a novel method based on principal components analysis (PCA) to characterize inversion polymorphisms using high-density SNP genotype data. Our method applies to non-recurrent inversions for which recombination between the inverted and non-inverted segments in inversion heterozygotes is suppressed due to the loss of unbalanced gametes. Inside such an inversion region, an effect similar to population substructure is thus created: two distinct "populations" of inversion homozygotes of different orientations and their 1:1 admixture, namely the inversion heterozygotes. This kind of substructure can be readily detected by performing PCA locally in the inversion regions. Using simulations, we demonstrated that the proposed method can be used to detect and genotype inversion polymorphisms using unphased genotype data. We applied our method to the phase III HapMap data and inferred the inversion genotypes of known inversion polymorphisms at 8p23.1 and 17q21.31. These inversion genotypes were validated by comparing with literature results and by checking Mendelian consistency using the family data whenever available. Based on the PCA-approach, we also performed a preliminary genome-wide scan for inversions using the HapMap data, which resulted in 2040 candidate inversions, 169 of which overlapped with previously reported inversions. Our method can be readily applied to the abundant SNP data, and is expected to play an important role in developing human genome maps of inversions and exploring associations between inversions and susceptibility of diseases.     

The Effect of Genomic Inversions on Estimation of Population Genetic Parameters from SNP Data

In recent years it has emerged that structural variants have a substantial impact on genomic variation. Inversion polymorphisms represent a significant class of structural variant, and despite the challenges in their detection, data on inversions in the human genome are increasing rapidly. Statistical methods for inferring parameters such as the recombination rate and the selection coefficient have generally been developed without accounting for the presence of inversions. Here we exploit new software for simulating inversions in population genetic data, invertFREGENE, to assess the potential impact of inversions on such methods. Using data simulated by invertFREGENE, as well as real data from several sources, we test whether large inversions have a disruptive effect on widely applied population genetics methods for inferring recombination rates, for detecting selection, and for controlling for population structure in genome-wide association studies (GWAS). We find that recombination rates estimated by LDhat are biased downward at inversion loci relative to the true contemporary recombination rates at the loci but that recombination hotspots are not falsely inferred at inversion breakpoints as may have been expected. We find that the integrated haplotype score (iHS) method for detecting selection appears robust to the presence of inversions. Finally, we observe a strong bias in the genome-wide results of principal components analysis (PCA), used to control for population structure in GWAS, in the presence of even a single large inversion, confirming the necessity to thin SNPs by linkage disequilibrium at large physical distances to obtain unbiased results.     

Cheap, accurate and rapid allele frequency estimation of single nucleotide polymorphisms by primer extension and DHPLC in DNA pools

At present, the cost of genotyping single nucleotide polymorphisms (SNPs) in large numbers of subjects poses a formidable problem for molecular genetic approaches to complex diseases. We have tested the possibility of using primer extension and denaturing high performance liquid chromatography to estimate allele frequencies of SNPs in pooled DNA samples. Our data show that this method should allow the accurate estimation of absolute allele frequencies in pooled samples of DNA and also of the difference in allele frequency between different pooled DNA samples. This technique therefore offers an efficient and cheap method for genotyping SNPs in large case-control and family-based association samples.     

A method of oligochip for single nucleotide polymorphism genotyping in the promoter region of the interleukin-1 beta gene and its clinical application

Single nucleotide polymorphisms (SNPs) can contribute to genetic predispositions or serve as genetic markers that are associated with complex diseases. So far, a few SNP arrays containing a limited number of SNPs have been used in routine genetic testing. This study described an oligochip-based method that genotypes two SNPs (-511 and -31) in the promoter region of the interleukin (IL)-1 beta gene. The sensitivity of this SNP genotyping method is derived from polymerase chain reaction (PCR)-amplified allele-specific primer-probes with a biotin label incorporated from the reverse primers. The amplified primer-probes can specifically hybridize with the oligonucleotides that are spotted on the oligochip. This oligochip-based method successfully discriminated the two biallelic SNPs with 9 different genotypes and all the genotyping results are in concordance with those from PCR restriction fragment length polymorphism (RFLP) analysis. Selective samples with various genotypes were also confirmed by direct sequencing. This method was applied in the genotyping of the patients with tuberculosis or gastric cancer and healthy controls. In the case control study, our genotyping data supported the reported association between gastric cancer and the genotypes of IL-1 beta -31 TT and -511 CC (p < 0.05). We also found that there is a significant difference of IL-1 beta -31 genotypes between 98 tuberculosis patients and healthy controls (p < 0.002). All of our results demonstrated that the oligochip can effectively and accurately identify SNP genotypes in the IL-1 beta promoter region.     

Powerful Identification of Cis-regulatory SNPs in Human Primary Monocytes Using Allele-Specific Gene Expression

A large number of genome-wide association studies have been performed during the past five years to identify associations between SNPs and human complex diseases and traits. The assignment of a functional role for the identified disease-associated SNP is not straight-forward. Genome-wide expression quantitative trait locus (eQTL) analysis is frequently used as the initial step to define a function while allele-specific gene expression (ASE) analysis has not yet gained a wide-spread use in disease mapping studies. We compared the power to identify cis-acting regulatory SNPs (cis-rSNPs) by genome-wide allele-specific gene expression (ASE) analysis with that of traditional expression quantitative trait locus (eQTL) mapping. Our study included 395 healthy blood donors for whom global gene expression profiles in circulating monocytes were determined by Illumina BeadArrays. ASE was assessed in a subset of these monocytes from 188 donors by quantitative genotyping of mRNA using a genome-wide panel of SNP markers. The performance of the two methods for detecting cis-rSNPs was evaluated by comparing associations between SNP genotypes and gene expression levels in sample sets of varying size. We found that up to 8-fold more samples are required for eQTL mapping to reach the same statistical power as that obtained by ASE analysis for the same rSNPs. The performance of ASE is insensitive to SNPs with low minor allele frequencies and detects a larger number of significantly associated rSNPs using the same sample size as eQTL mapping. An unequivocal conclusion from our comparison is that ASE analysis is more sensitive for detecting cis-rSNPs than standard eQTL mapping. Our study shows the potential of ASE mapping in tissue samples and primary cells which are difficult to obtain in large numbers.     

Estimation of relative allele frequencies of single-nucleotide polymorphisms in different populations by microarray hybridization of pooled DNA

Single-nucleotide polymorphisms (SNPs) are considered useful polymorphic markers for genetic studies of polygenic traits. A new practical approach to high-throughput genotyping of SNPs in a large number of individuals is needed in association study and other studies on relationships between genes and diseases. We have developed an accurate and high-throughput method for determining the allele frequencies by pooling the DNA samples and applying a DNA microarray hybridization analysis. In this method, the combination of the microarray, DNA pooling, probe pair hybridization, and fluorescent ratio analysis solves the dual problems of parallel multiple sample analysis, and parallel multiplex SNP genotyping for association study. Multiple DNA samples are immobilized on a slide and a single hybridization is performed with a pool of allele-specific oligonucleotide probes. The results of this study show that hybridization of microarray from pooled DNA samples can accurately obtain estimates of absolute allele frequencies in a sample pool. This method can also be used to identify differences in allele frequencies in distinct populations. It is amenable to automation and is suitable for immediate utilization for high-throughput genotyping of SNP.     

Finding haplotype tagging SNPs by use of principal components analysis

The immense volume and rapid growth of human genomic data, especially single nucleotide polymorphisms (SNPs), present special challenges for both biomedical researchers and automatic algorithms. One such challenge is to select an optimal subset of SNPs, commonly referred as "haplotype tagging SNPs" (htSNPs), to capture most of the haplotype diversity of each haplotype block or gene-specific region. This information-reduction process facilitates cost-effective genotyping and, subsequently, genotype-phenotype association studies. It also has implications for assessing the risk of identifying research subjects on the basis of SNP information deposited in public domain databases. We have investigated methods for selecting htSNPs by use of principal components analysis (PCA). These methods first identify eigenSNPs and then map them to actual SNPs. We evaluated two mapping strategies, greedy discard and varimax rotation, by assessing the ability of the selected htSNPs to reconstruct genotypes of non-htSNPs. We also compared these methods with two other htSNP finders, one of which is PCA based. We applied these methods to three experimental data sets and found that the PCA-based methods tend to select the smallest set of htSNPs to achieve a 90% reconstruction precision.     

Multiple chromosomal rearrangements in a hybrid zone between Littorina saxatilis ecotypes

Both classical and recent studies suggest that chromosomal inversion polymorphisms are important in adaptation and speciation. However, biases in discovery and reporting of inversions make it difficult to assess their prevalence and biological importance. Here, we use an approach based on linkage disequilibrium among markers genotyped for samples collected across a transect between contrasting habitats to detect chromosomal rearrangements de novo. We report 17 polymorphic rearrangements in a single locality for the coastal marine snail, Littorina saxatilis. Patterns of diversity in the field and of recombination in controlled crosses provide strong evidence that at least the majority of these rearrangements are inversions. Most show clinal changes in frequency between habitats, suggestive of divergent selection, but only one appears to be fixed for different arrangements in the two habitats. Consistent with widespread evidence for balancing selection on inversion polymorphisms, we argue that a combination of heterosis and divergent selection can explain the observed patterns and should be considered in other systems spanning environmental gradients.     

Polytene chromosome map and inversion polymorphism in Drosophila mediopunctata

Drosophila mediopunctata belongs to the tripunctata group, and is one of the commonest Drosophila species collected in some places in Brazil, especially in the winter. A standard map of the polytene chromosomes is presented. The breakpoints of the naturally occurring chromosomal rearrangements are marked on the map. The distribution of breaking points through the chromosomes of D. mediopunctata is apparently non-random. Chromosomes X, II and IV show inversion polymorphisms. Chromosome II is the most polymorphic, with 17 inversions, 8 inversions in the distal region and 9 in the proximal region. Chromosome X has four different gene arrangements, while chromosome IV has only two.     

Long-range, high-throughput haplotype determination via haplotype-fusion PCR and ligation haplotyping

Ligation Haplotyping is a robust, novel method for experimental determination of haplotypes over long distances, which can be applied to assaying both sequence and structural variation. The simplicity and efficacy of the method for genotyping large chromosomal rearrangements and haplotyping SNPs over long distances make it a valuable and powerful addition to the methodological repertoire, which will be beneficial to studies of population genetics and evolution, disease association and inheritance, and genomic variation. We illustrate the versatility of the method both by genotyping a Yp paracentric inversion, found in approximately 60% of Northwest European males, that strongly influences the germline rate of infertility-causing XY translocations and by haplotyping two autosomal SNPs that lie 16.4 kb apart on chromosome 7, and which influence an individual's susceptibility to systemic lupus erythematosus.     

Nucleotide,Cytogenetic and Expression Impact of the Human Chromosome 8p23.1 Inversion Polymorphism

Background

The human chromosome 8p23.1 region contains a 3.8–4.5 Mb segment which can be found in different orientations (defined as genomic inversion) among individuals. The identification of single nucleotide polymorphisms (SNPs) tightly linked to the genomic orientation of a given region should be useful to indirectly evaluate the genotypes of large genomic orientations in the individuals.

Results

We have identified 16 SNPs, which are in linkage disequilibrium (LD) with the 8p23.1 inversion as detected by fluorescent in situ hybridization (FISH). The variability of the 8p23.1 orientation in 150 HapMap samples was predicted using this set of SNPs and was verified by FISH in a subset of samples. Four genes (NEIL2, MSRA, CTSB and BLK) were found differentially expressed (p<0.0005) according to the orientation of the 8p23.1 region. Finally, we have found variable levels of mosaicism for the orientation of the 8p23.1 as determined by FISH.

Conclusion

By means of dense SNP genotyping of the region, haplotype-based computational analyses and FISH experiments we could infer and verify the orientation status of alleles in the 8p23.1 region by detecting two short haplotype stretches at both ends of the inverted region, which are likely the relic of the chromosome in which the original inversion occurred. Moreover, an impact of 8p23.1 inversion on gene expression levels cannot be ruled out, since four genes from this region have statistically significant different expression levels depending on the inversion status. FISH results in lymphoblastoid cell lines suggest the presence of mosaicism regarding the 8p23.1 inversion.     

Analysis of association at single nucleotide polymorphisms in the APOE region

The discussion of the prospects of using a dense map of single nucleotide polymorphisms (SNPs) to identify disease genes with association analysis has been extensive. However, there is little empiric evidence to support this strategy. To begin to examine the practical issues surrounding this methodology, we identified 10 SNPs in the region immediately surrounding the apolipoprotein E locus (APOE), an established susceptibility gene for Alzheimer disease. Our goal was to examine patterns of allelic association to begin to investigate the question of whether APOE could have been identified using SNPs. Our strongest evidence of association was at the 2 SNPs immediately flanking APOE.     

Tag SNP selection using particle swarm optimization

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variations amongst species. With the genome‐wide SNP discovery, many genome‐wide association studies are likely to identify multiple genetic variants that are associated with complex diseases. However, genotyping all existing SNPs for a large number of samples is still challenging even though SNP arrays have been developed to facilitate the task. Therefore, it is essential to select only informative SNPs representing the original SNP distributions in the genome (tag SNP selection) for genome‐wide association studies. These SNPs are usually chosen from haplotypes and called haplotype tag SNPs (htSNPs). Accordingly, the scale and cost of genotyping are expected to be largely reduced. We introduce binary particle swarm optimization (BPSO) with local search capability to improve the prediction accuracy of STAMPA. The proposed method does not rely on block partitioning of the genomic region, and consistently identified tag SNPs with higher prediction accuracy than either STAMPA or SVM/STSA. We compared the prediction accuracy and time complexity of BPSO to STAMPA and an SVM‐based (SVM/STSA) method using publicly available data sets. For STAMPA and SVM/STSA, BPSO effective improved prediction accuracy for smaller and larger scale data sets. These results demonstrate that the BPSO method selects tag SNP with higher accuracy no matter the scale of data sets is used. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Accumulation of P elements in minority inversions in natural populations of Drosophila melanogaster.

The accumulation of a transposable element inside chromosomal inversions is examined theoretically by a mathematical model, and empirically by counts of P elements associated with inversion polymorphisms in natural populations of Drosophila melanogaster. The model demonstrates that, if heterozygosity for an inversion effectively reduces element associated production of detrimental chromosome rearrangements, a differential accumulation of elements is expected, with increased copy number inside the minority inversion. Several-fold differential accumulations are possible with certain parameter values. We present data on P element counts for inversion polymorphisms on all five chromosome arms of 157 haploid genomes from two African populations. Our observations show significantly increased numbers of elements within the regions associated with the least common, or minority arrangements, in natural inversion polymorphisms.     

DTNBP1 (Dystrobrevin binding protein 1) and schizophrenia: association evidence in the 3' end of the gene

OBJECTIVES: Dysbindin (DTNBP1) has been identified as a susceptibility gene for schizophrenia (SZ) through a positional approach. However, a variety of single nucleotide polymorphisms (SNPs) and haplotypes, in different parts of the gene, have been reported to be associated in different samples, and a precise molecular mechanism of disease remains to be defined. We have performed an association study with two well-characterized family samples not previously investigated at the DTNBP1 locus. METHODS: We examined 646 subjects in 136 families with SZ, largely of European ancestry (EA), genotyping 26 SNPs in DTNBP1. RESULTS: Three correlated markers (rs875462, rs760666, and rs7758659) at the 3' region of DTNBP1 showed evidence for association to SZ (p = 0.004), observed in both the EA (p = 0.031) and the African American (AA) subset (p = 0.045) with the same over-transmitted allele. The most significant haplotype in our study was rs7758659-rs3213207 (global p = 0.0015), with rs3213207 being the most frequently reported associated marker in previous studies. A non-conservative missense variant (Pro272Ser) in the 3' region of DTNBP1 that may impair DTNBP1 function was more common in SZ probands (8.2%) than in founders (5%) and in dbSNP (2.1%), but did not reach statistical significance. CONCLUSION: Our results provide evidence for an association of SZ with SNPs at the 3' end of DTNBP1 in the samples studied.     

CSF1 gene associated with aggressive periodontitis in the Japanese population

Aggressive periodontitis (AgP) is characterized by the early onset of the rapid and progressive destruction of the alveolar bone. We investigated the correlation of single nucleotide polymorphisms (SNPs) in candidate genes with AgP in the Japanese population in order to determine the genetic risk factors for this complex disease. Among 11 genes related to bone formation and resorption, 43 known SNPs were tested in 98 case and 88 control samples for association with AgP by using SNP genotyping techniques. Among these, three polymorphisms located in the colony stimulating factor 1 (CSF1) gene showed a positive association with AgP. This is the first case of an association between a CSF1 polymorphism and a human disease.