Human defensin gene copy number polymorphisms: comprehensive analysis of independent variation in alpha- and beta-defensin regions at 8p22-p23

To investigate defensin gene copy number polymorphisms, a quantitative real-time PCR assay was developed and used to study DNA from 27 unrelated individuals of diverse ethnic and racial backgrounds. The DEFB4 and DEFB103A genes varied in tandem, with copy numbers 2 to 8, with a mode of 6 per diploid genome (PDG). The combined copy numbers of the DEFA1 and DEFA3 genes ranged from 5 to 14, with a mode of 10 copies PDG. The copy numbers of the DEFA1/3 genes varied independently of those of the DEFB4 and DEFB103A genes. The amount of HNP-1 and HNP-3 peptides expressed in neutrophils was found to be proportional to the combined copy number of DEFA1 and DEFA3. The DEFA3 allele was absent in 7/27 subjects. The highly copy-number-variable DEFA1 and DEFA3 genes are flanked by other defensin genes present uniformly at 2 copies PDG. The remarkable variability in defensin gene copy numbers could contribute to differences in individual resistance to infections.     

Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains

It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1 × 244 k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to contribute to the alcohol-related phenotypic differences associated with these strains.     

A 450-kb contig of defensin genes on human chromosome 8p23.

Defensins are a large family of host defense peptides expressed in leukocytes and epithelia. Using P1 and BAC clones, we have determined the organization of the human alpha-defensin genes and the beta-defensin gene HDEFB1 on chromosome 8p23. From the telomere, the order of the genes (with encoded peptides in parentheses) is HDEFA5 (HD-5), HDEFA1/1A (HNP-1/3), HDEFA4 (HNP-4), HDEFA6 (HD-6), and HDEFB1 (HBD-1). These genes span a region of approximately 450kb. Genes encoding intestinal Paneth cell defensins (HDEFA5 and HDEFA6) flank the myeloid defensin gene cluster (HDEFA1, HDEFA1A, HDEFA4). Based on our previous studies, the remaining known defensin gene, HDEFB2 (HBD-2), is about 400kb centromeric to HDEFB1. This map supports the hypothesis, originally proposed because of sequence similarities, that myeloid alpha-defensin genes evolved by reduplication and divergence from Paneth cell defensin genes, and identifies regions and clones, which should be useful in the search for new defensin genes.     

Genome-wide patterns of copy number variation in the diversified chicken genomes using next-generation sequencing

Background

Copy number variation (CNV) is important and widespread in the genome, and is a major cause of disease and phenotypic diversity. Herein, we performed a genome-wide CNV analysis in 12 diversified chicken genomes based on whole genome sequencing.

Results

A total of 8,840 CNV regions (CNVRs) covering 98.2 Mb and representing 9.4% of the chicken genome were identified, ranging in size from 1.1 to 268.8 kb with an average of 11.1 kb. Sequencing-based predictions were confirmed at a high validation rate by two independent approaches, including array comparative genomic hybridization (aCGH) and quantitative PCR (qPCR). The Pearson’s correlation coefficients between sequencing and aCGH results ranged from 0.435 to 0.755, and qPCR experiments revealed a positive validation rate of 91.71% and a false negative rate of 22.43%. In total, 2,214 (25.0%) predicted CNVRs span 2,216 (36.4%) RefSeq genes associated with specific biological functions. Besides two previously reported copy number variable genes EDN3 and PRLR, we also found some promising genes with potential in phenotypic variation. Two genes, FZD6 and LIMS1, related to disease susceptibility/resistance are covered by CNVRs. The highly duplicated SOCS2 may lead to higher bone mineral density. Entire or partial duplication of some genes like POPDC3 may have great economic importance in poultry breeding.

Conclusions

Our results based on extensive genetic diversity provide a more refined chicken CNV map and genome-wide gene copy number estimates, and warrant future CNV association studies for important traits in chickens.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-962) contains supplementary material, which is available to authorized users.     

A distance-type measure approach to the analysis of copy number variation in DNA sequencing data

Background

The next generation sequencing technology allows us to obtain a large amount of short DNA sequence (DNA-seq) reads at a genome-wide level. DNA-seq data have been increasingly collected during the recent years. Count-type data analysis is a widely used approach for DNA-seq data. However, the related data pre-processing is based on the moving window method, in which a window size need to be defined in order to obtain count-type data. Furthermore, useful information can be reduced after data pre-processing for count-type data.

Results

In this study, we propose to analyze DNA-seq data based on the related distance-type measure. Distances are measured in base pairs (bps) between two adjacent alignments of short reads mapped to a reference genome. Our experimental data based simulation study confirms the advantages of distance-type measure approach in both detection power and detection accuracy. Furthermore, we propose artificial censoring for the distance data so that distances larger than a given value are considered potential outliers. Our purpose is to simplify the pre-processing of DNA-seq data. Statistically, we consider a mixture of right censored geometric distributions to model the distance data. Additionally, to reduce the GC-content bias, we extend the mixture model to a mixture of generalized linear models (GLMs). The estimation of model can be achieved by the Newton-Raphson algorithm as well as the Expectation-Maximization (E-M) algorithm. We have conducted simulations to evaluate the performance of our approach. Based on the rank based inverse normal transformation of distance data, we can obtain the related z-values for a follow-up analysis. For an illustration, an application to the DNA-seq data from a pair of normal and tumor cell lines is presented with a change-point analysis of z-values to detect DNA copy number alterations.

Conclusion

Our distance-type measure approach is novel. It does not require either a fixed or a sliding window procedure for generating count-type data. Its advantages have been demonstrated by our simulation studies and its practical usefulness has been illustrated by an experimental data application.

     

猪13号染色体上拷贝数变异区内基因信息发掘及遗传规律分析

拷贝数变异(Copy number variation, CNV)是染色体上发生的一种微结构变异, 已引起越来越多研究者的关注。本课题组前期已获得猪13号染色体上的32个CNV区域(CNV region, CNVR), 为了发掘CNVR内的基因信息, 文章在线检索了上述CNVR内的基因并进行基因本体(Gene Ontology)分析。结果共发现236个基因, 其中有注释基因169个, 主要参与蛋白质水解、细胞粘附、大分子降解等生物过程。为了探索这些基因拷贝数变异的遗传规律, 文章选择RCAN1(Regulators of calcineurin 1)基因为候选基因, 利用QPCR方法在莱芜猪群中检测了该基因的拷贝数, 并分析了CNV在莱芜猪3个家系中的遗传规律。结果表明, RCAN1基因在莱芜猪群体中存在拷贝数的缺失、重复现象, 其拷贝数变异的遗传规律符合孟德尔遗传方式。     

Proliferation and copy number variation of BEL-like long terminal repeat retrotransposons within the Diabrotica virgifera virgifera genome

The proliferation of retrotransposons within a genome can contribute to increased size and affect the function of eukaryotic genes. BEL/Pao-like long-terminal repeat (LTR) retrotransposons were annotated from the highly adaptable insect species Diabrotica virgifera virgifera, the Western corn rootworm, using survey sequences from bacterial artificial chromosome (BAC) inserts and contigs derived from a low coverage next-generation genome sequence assembly. Eleven unique D. v. virgifera BEL elements were identified that contained full-length gag–pol coding sequences, whereas 88 different partial coding regions were characterized from partially assembled elements. Estimated genome copy number for full and partial BEL-like elements ranged from ~ 8 to 1582 among individual contigs using a normalized depth of coverage (DOC) among Illumina HiSeq reads (total genome copy number ~ 8821). BEL element copy number was correlated among different D. v. virgifera populations (R² = 0.9846), but individual element numbers varied ≤ 1.68-fold and the total number varied by ~ 527 copies. These data indicate that BEL element proliferation likely contributed to a large genome size, and suggest that differences in copy number are a source of genetic variability among D. v. virgifera.     

General assessment of copy number variation in normal and tumor tissues of the domestic dog (Canis lupus familiaris)

In recent years, characterization of a copy number variation (CNV) of the genomic DNA has provided evidence for the relationship of this type of genetic variation with the occurrence of a broad spectrum of diseases, including cancer lesions. Copy number variants (CNVs) also occur in the genomes of healthy individuals as a result of abnormal recombination processes in germ cells and have a hereditary character contributing to the natural genetic diversity. Recent image analysis methods and advanced computational techniques allow for identification of CNVs using SNPs genotyping microarrays based on the analysis of signal intensity observed for markers located in the specific genomic regions. In this study we used CanineHD BeadChip assay (Illumina) to identify both natural and cancer-induced CNVs in the genomes of different dog breeds and in different cancer types occurring in this species. The obtained results showed that structural aberrations are a common phenomenon arising during a tumor progression and are more complex and widespread in tumors of mesenchymal tissue origin than in epithelial tissue originating tumors. The tumor derived CNVs, in comparison to healthy samples, were characterized by larger sizes of regions, higher number of amplifications, and in some cases encompassed genes with potential effect on tumor progression.     

Genome‐wide copy number variation in the bovine genome detected using low coverage sequence of popular beef breeds,

Copy number variations (CNVs) are large insertions, deletions or duplications in the genome that vary between members of a species and are known to affect a wide variety of phenotypic traits. In this study, we identified CNVs in a population of bulls using low coverage next‐generation sequence data. First, in order to determine a suitable strategy for CNV detection in our data, we compared the performance of three distinct CNV detection algorithms on benchmark CNV datasets and concluded that using the multiple sample read depth approach was the best method for identifying CNVs in our sequences. Using this technique, we identified a total of 1341 copy number variable regions (CNVRs) from genome sequences of 154 purebred sires used in Cycle VII of the USMARC Germplasm Evaluation Project. These bulls represented the seven most popular beef breeds in the United States: Hereford, Charolais, Angus, Red Angus, Simmental, Gelbvieh and Limousin. The CNVRs covered 6.7% of the bovine genome and spanned 2465 protein‐coding genes and many known quantitative trait loci (QTL). Genes harbored in the CNVRs were further analyzed to determine their function as well as to find any breed‐specific differences that may shed light on breed differences in adaptation, health and production.     

Analysis of copy number variation in the normal human population within a region containing complex segmental duplications on 22q11 using high-resolution array-CGH

A previously detected copy number polymorphism (Ep CNP) in patients affected with neuroectodermal tumors led us to investigate its frequency and length in the normal population. For this purpose, a program called Sequence Allocator was developed and applied for the construction of an array that consisted of unique and duplicated fragments, allowing the assessment of copy number variation within regions of segmental duplications. The average resolution of this array was 11 kb and we determined the size of the Ep CNP to be 290 kb. Analysis of normal controls identified 7.7 and 7.1% gains in peripheral blood and lymphoblastoid cell line (LCL) DNA, respectively, while deletions were found only in the LCL group (7.1%). This array platform allows the detection of DNA copy number variation within regions of pronounced genomic complexity, which constitutes an improvement over available technologies.     

Direct evidence for the adaptive role of copy number variation on antifolate susceptibility in Plasmodium falciparum

Resistance to antimalarials targeting the folate pathway is widespread. GTP‐cyclohydrolase (gch1), the first enzyme in this pathway, exhibits extensive copy number variation (CN) in parasite isolates from areas with a history of longstanding antifolate use. Increased CN of gch1 is associated with a greater number of point mutations in enzymes targeted by the antifolates, pyrimethamine and sulphadoxine. While these observations suggest that increases in gch1 CN are an adaptation to drug pressure, changes in CN have not been experimentally demonstrated to directly alter drug susceptibility. To determine if changes in gch1 expression alone modify pyrimethamine sensitivity, we manipulated gch1 CN in several parasite lines to test the effect on drug sensitivity. We report that increases in gch1 CN alter pyrimethamine resistance in most parasites lines. However we find evidence of a detrimental effect of very high levels of gch1 overexpression in parasite lines with high endogenous levels of gch1 expression, revealing the importance of maintaining balance in the folate pathway and implicating changes in gch1 expression in preserving proper metabolic flux. This work expands our understanding of parasite adaptation to drug pressure and provides a possible mechanism for how specific mutations become fixed within parasite populations.     

Increased paternal age and the influence on burden of genomic copy number variation in the general population

Genomic copy number variations (CNVs) and increased parental age are both associated with the risk to develop a variety of clinical neuropsychiatric disorders such as autism, schizophrenia and bipolar disorder. At the same time, it has been shown that the rate of transmitted de novo single nucleotide mutations is increased with paternal age. To address whether paternal age also affects the burden of structural genomic deletions and duplications, we examined various types of CNV burden in a large population sample from the Netherlands. Healthy participants with parental age information (n = 6,773) were collected at different University Medical Centers. CNVs were called with the PennCNV algorithm using Illumina genome-wide SNP array data. We observed no evidence in support of a paternal age effect on CNV load in the offspring. Our results were negative for global measures as well as several proxies for de novo CNV events in this unique sample. While recent studies suggest de novo single nucleotide mutation rate to be dominated by the age of the father at conception, our results strongly suggest that at the level of global CNV burden there is no influence of increased paternal age. While it remains possible that local genomic effects may exist for specific phenotypes, this study indicates that global CNV burden and increased father’s age may be independent disease risk factors.     

Contemporary evolution of resistance at the major insecticide target site gene Ace‐1 by mutation and copy number variation in the malaria mosquito Anopheles gambiae

Functionally constrained genes are ideal insecticide targets because disruption is often fatal, and resistance mutations are typically costly. Synaptic acetylcholinesterase (AChE) is an essential neurotransmission enzyme targeted by insecticides used increasingly in malaria control. In Anopheles and Culex mosquitoes, a glycine–serine substitution at codon 119 of the Ace‐1 gene confers both resistance and fitness costs, especially for 119S/S homozygotes. G119S in Anopheles gambiae from Accra (Ghana) is strongly associated with resistance, and, despite expectations of cost, resistant 119S alleles are increasing significantly in frequency. Sequencing of Accra females detected only a single Ace‐1 119S haplotype, whereas 119G diversity was high overall but very low at non‐synonymous sites, evidence of strong purifying selection driven by functional constraint. Flanking microsatellites showed reduced diversity, elevated linkage disequilibrium and high differentiation of 119S, relative to 119G homozygotes across up to two megabases of the genome. Yet these signals of selection were inconsistent and sometimes weak tens of kilobases from Ace‐1. This unexpected finding is attributable to apparently ubiquitous amplification of 119S alleles as part of a large copy number variant (CNV) far exceeding the size of the Ace‐1 gene, whereas 119G alleles were unduplicated. Ace‐1 CNV was detectable in archived samples collected when the 119S allele was rare in Ghana. Multicopy amplification of resistant alleles has not been observed previously and is likely to underpin the recent increase in 119S frequency. The large CNV compromised localization of the strong selective sweep around Ace‐1, emphasizing the need to integrate CNV analysis into genome scans for selection.     

Effect of plasmid copy number and lac operator sequence on antibiotic-free plasmid selection by operator-repressor titration in Escherichia coli

The Escherichia coli strain DH1lacdapD enables plasmid selection and maintenance that is free from antibiotics and selectable marker genes. This is achieved by using only the lac operator sequence as a selectable element. This strain is currently used to generate high copy number plasmids with no antibiotic resistance genes for use as DNA vaccines and for expression of recombinant proteins. Until now these have been limited to pUC-based plasmids containing a high copy number pMB1-derived origin of replication, and the principle lacO(1) and auxiliary lacO(3) operators. In this study we have shown that this system can also be used to select and maintain pBR322-based plasmids with the lower copy number pMB1 origin of replication, and that lacO(1) alone or a palindromic version of lacO(1) can provide a sufficient level of repressor titration for plasmid selection. This is advantageous for recombinant protein production, where low copy number plasmids are often used and plasmid maintenance is important. The degree of repressor titration due to these plasmids was measured using the natural lactose operon in E. coli DH1 as a model.     

Evidence for duplication of the human defensin gene DEFB4 in chromosomal region 8p22-23 and implications for the analysis of SNP allele distribution

Defensins constitute a primary mechanism in the innate immune system of humans and all mammals. Defensins are short, processed peptide molecules that are classified by structure into three groups: alpha-defensins, beta-defensins and theta-defensins. In humans, four beta-defensins have been described so far, corresponding to the products of the genes DEFB1 (hBD1, NM_005218), DEFB4 (hBD2, NM_004942.2), DEFB103 (hBD3, NM_018661), and DEFB104 (hBD4, NM_080389), respectively. All these genes have been mapped to chromosome 8p22-23. Much interest has been shown in genetic variation in the population at defensin loci to understand individual differences in disease susceptibility and severity. In this study, we have used an electronic search and then fluorescence in situ hybridization (FISH) on elongated chromosomes to demonstrate that the region containing the DEFB4 gene is duplicated on human chromosome 8p, making difficult the discovery of new SNPs in this gene and compromising the assessment of their allelic distribution in various ethnic populations for disease association studies.