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Copy number deletion has little impact on gene expression levels in racehorses.

Park KD, Kim H, Hwang JY, Lee CK, Do KT, Kim HS, Yang YM, Kwon YJ, Kim J, Kim HJ, Song KD, Oh JD, Kim H, Cho BW, Cho S, Lee HK - Asian-australas. J. Anim. Sci. (2014)

Bottom Line: Indeed, it is widely accepted that CNVs are associated with differential disease susceptibility.We did not observe a clear and consistent relationship between the deletion status of CNVs and gene expression levels before and after exercise in blood and muscle.However, we found some pairs of CNVs and associated genes that indicated relationships with gene expression levels: a positive relationship with genes responsible for membrane structure or cytoskeleton and a negative relationship with genes involved in disease.

View Article: PubMed Central - PubMed

Affiliation: Genomic Informatics Center, Hankyong National University, Anseong 456-749, Korea.

ABSTRACT
Copy number variations (CNVs), important genetic factors for study of human diseases, may have as large of an effect on phenotype as do single nucleotide polymorphisms. Indeed, it is widely accepted that CNVs are associated with differential disease susceptibility. However, the relationships between CNVs and gene expression have not been characterized in the horse. In this study, we investigated the effects of copy number deletion in the blood and muscle transcriptomes of Thoroughbred racing horses. We identified a total of 1,246 CNVs of deletion polymorphisms using DNA re-sequencing data from 18 Thoroughbred racing horses. To discover the tendencies between CNV status and gene expression levels, we extracted CNVs of four Thoroughbred racing horses of which RNA sequencing was available. We found that 252 pairs of CNVs and genes were associated in the four horse samples. We did not observe a clear and consistent relationship between the deletion status of CNVs and gene expression levels before and after exercise in blood and muscle. However, we found some pairs of CNVs and associated genes that indicated relationships with gene expression levels: a positive relationship with genes responsible for membrane structure or cytoskeleton and a negative relationship with genes involved in disease. This study will lead to conceptual advances in understanding the relationship between CNVs and global gene expression in the horse.

No MeSH data available.


Related in: MedlinePlus

Copy number variation (CNV) region validation by genomic polymerase chain reaction (PCR) analysis. (a) Genomic DNA amplification model. To validate the reliability of predicted CNV regions using the CNV extraction tool, we performed PCR amplification using the model represented. The primer pairs were designed to contain the CNV region (empty box in CNV del-model and graded box in CNV in-model), and the amplicon is 150 to 350 bp longer than the CNV region. The CNV region deletion type in each individual was confirmed by comparison of the amplicon size. Diagram is not to scale. (b) Amplicon patterns of CNV-deleted alleles by PCR analysis. The PCR amplicons of individuals with CNV deleted and non-deleted alleles are shown (solid triangle for expected non-deleted and arrow for expected deleted). Compared to the predicted size, the following four categories of CNV-deleted allele amplicon size were identified (lane 1–4, red line): similar to predicted size (lane 1), large (lane 2), or small (lane 3) amplicon size, and absence of amplicon from the CNV deleted allele (lane 4, dashed red line). M indicates size marker. (c) CNV patterns of each individual summarized as a heat map. Randomly selected CNV regions obtained using the extraction tool were assessed by PCR analysis. Dark gray, gray, and white boxes indicate two, one, and non-CNV deletions, respectively. By comparing the CNV allele distribution in each individual and amplicon size in CNV-deleted alleles between the predicted and experimental results, we evaluated the suitability of each extracted CNV region by classifying the PCR results into five categories, as described in the Results section (green box to red box).
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f3-ajas-27-9-1345: Copy number variation (CNV) region validation by genomic polymerase chain reaction (PCR) analysis. (a) Genomic DNA amplification model. To validate the reliability of predicted CNV regions using the CNV extraction tool, we performed PCR amplification using the model represented. The primer pairs were designed to contain the CNV region (empty box in CNV del-model and graded box in CNV in-model), and the amplicon is 150 to 350 bp longer than the CNV region. The CNV region deletion type in each individual was confirmed by comparison of the amplicon size. Diagram is not to scale. (b) Amplicon patterns of CNV-deleted alleles by PCR analysis. The PCR amplicons of individuals with CNV deleted and non-deleted alleles are shown (solid triangle for expected non-deleted and arrow for expected deleted). Compared to the predicted size, the following four categories of CNV-deleted allele amplicon size were identified (lane 1–4, red line): similar to predicted size (lane 1), large (lane 2), or small (lane 3) amplicon size, and absence of amplicon from the CNV deleted allele (lane 4, dashed red line). M indicates size marker. (c) CNV patterns of each individual summarized as a heat map. Randomly selected CNV regions obtained using the extraction tool were assessed by PCR analysis. Dark gray, gray, and white boxes indicate two, one, and non-CNV deletions, respectively. By comparing the CNV allele distribution in each individual and amplicon size in CNV-deleted alleles between the predicted and experimental results, we evaluated the suitability of each extracted CNV region by classifying the PCR results into five categories, as described in the Results section (green box to red box).

Mentions: To confirm that the extracted CNV region is genuine, we performed genomic DNA PCR with CNV region-spanning primer sets (Figure 3a and Supplementary Table 4). Among the extracted CNV regions that were of appropriate size for PCR (<2.5 kb), 26 CNV regions were selected randomly and the CNV deletion type and length in each was examined (Supplementary Figure 5). The amplicons in the deleted allele in the CNV region were of three sizes in comparison to the prediction by GenomeSTRiP: i) similar size of amplicon in the deleted allele in the CNV region (Figure 3b, lane 1), ii) 100 to 200 bp difference in the deleted allele (Figure 3b, lanes 2 and 3), and iii) absence of amplicon (Figure 3b, lane 4). The amplicon size differences might be due to the genuine CNV region length being shorter (second case) or longer (third case) than that predicted by GenomeSTRiP. Then, the distribution of CNV-deleted or -non-deleted alleles in each individual by PCR was compared to the expected model. By combining the criteria of ‘deleted amplicon size’ and ‘distribution of CNV in each individual’, we evaluated the extracted CNV region as proper or non-proper according to five classifications (Figure 3c): same pattern and CNV length difference <100 bp (green box) or 100 to 200 bp difference (yellow-green box); different pattern but <100 bp CNV length difference (yellow box); same pattern but the CNV was larger than the amplifying region (orange box, CNV8), or both the CNV distribution and amplicon size did not match those expected (red box). We considered that the PCR result showing the same CNV distribution pattern and <200 bp difference in the amplicon of the deleted allele (green and yellow-green boxes) was the proper CNV region. According to this standard, the validation process indicated that the extracting CNV region had 76.92% accuracy (Figure 3c, 20 proper CNV regions of 26 CNV regions examined).


Copy number deletion has little impact on gene expression levels in racehorses.

Park KD, Kim H, Hwang JY, Lee CK, Do KT, Kim HS, Yang YM, Kwon YJ, Kim J, Kim HJ, Song KD, Oh JD, Kim H, Cho BW, Cho S, Lee HK - Asian-australas. J. Anim. Sci. (2014)

Copy number variation (CNV) region validation by genomic polymerase chain reaction (PCR) analysis. (a) Genomic DNA amplification model. To validate the reliability of predicted CNV regions using the CNV extraction tool, we performed PCR amplification using the model represented. The primer pairs were designed to contain the CNV region (empty box in CNV del-model and graded box in CNV in-model), and the amplicon is 150 to 350 bp longer than the CNV region. The CNV region deletion type in each individual was confirmed by comparison of the amplicon size. Diagram is not to scale. (b) Amplicon patterns of CNV-deleted alleles by PCR analysis. The PCR amplicons of individuals with CNV deleted and non-deleted alleles are shown (solid triangle for expected non-deleted and arrow for expected deleted). Compared to the predicted size, the following four categories of CNV-deleted allele amplicon size were identified (lane 1–4, red line): similar to predicted size (lane 1), large (lane 2), or small (lane 3) amplicon size, and absence of amplicon from the CNV deleted allele (lane 4, dashed red line). M indicates size marker. (c) CNV patterns of each individual summarized as a heat map. Randomly selected CNV regions obtained using the extraction tool were assessed by PCR analysis. Dark gray, gray, and white boxes indicate two, one, and non-CNV deletions, respectively. By comparing the CNV allele distribution in each individual and amplicon size in CNV-deleted alleles between the predicted and experimental results, we evaluated the suitability of each extracted CNV region by classifying the PCR results into five categories, as described in the Results section (green box to red box).
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Related In: Results  -  Collection

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f3-ajas-27-9-1345: Copy number variation (CNV) region validation by genomic polymerase chain reaction (PCR) analysis. (a) Genomic DNA amplification model. To validate the reliability of predicted CNV regions using the CNV extraction tool, we performed PCR amplification using the model represented. The primer pairs were designed to contain the CNV region (empty box in CNV del-model and graded box in CNV in-model), and the amplicon is 150 to 350 bp longer than the CNV region. The CNV region deletion type in each individual was confirmed by comparison of the amplicon size. Diagram is not to scale. (b) Amplicon patterns of CNV-deleted alleles by PCR analysis. The PCR amplicons of individuals with CNV deleted and non-deleted alleles are shown (solid triangle for expected non-deleted and arrow for expected deleted). Compared to the predicted size, the following four categories of CNV-deleted allele amplicon size were identified (lane 1–4, red line): similar to predicted size (lane 1), large (lane 2), or small (lane 3) amplicon size, and absence of amplicon from the CNV deleted allele (lane 4, dashed red line). M indicates size marker. (c) CNV patterns of each individual summarized as a heat map. Randomly selected CNV regions obtained using the extraction tool were assessed by PCR analysis. Dark gray, gray, and white boxes indicate two, one, and non-CNV deletions, respectively. By comparing the CNV allele distribution in each individual and amplicon size in CNV-deleted alleles between the predicted and experimental results, we evaluated the suitability of each extracted CNV region by classifying the PCR results into five categories, as described in the Results section (green box to red box).
Mentions: To confirm that the extracted CNV region is genuine, we performed genomic DNA PCR with CNV region-spanning primer sets (Figure 3a and Supplementary Table 4). Among the extracted CNV regions that were of appropriate size for PCR (<2.5 kb), 26 CNV regions were selected randomly and the CNV deletion type and length in each was examined (Supplementary Figure 5). The amplicons in the deleted allele in the CNV region were of three sizes in comparison to the prediction by GenomeSTRiP: i) similar size of amplicon in the deleted allele in the CNV region (Figure 3b, lane 1), ii) 100 to 200 bp difference in the deleted allele (Figure 3b, lanes 2 and 3), and iii) absence of amplicon (Figure 3b, lane 4). The amplicon size differences might be due to the genuine CNV region length being shorter (second case) or longer (third case) than that predicted by GenomeSTRiP. Then, the distribution of CNV-deleted or -non-deleted alleles in each individual by PCR was compared to the expected model. By combining the criteria of ‘deleted amplicon size’ and ‘distribution of CNV in each individual’, we evaluated the extracted CNV region as proper or non-proper according to five classifications (Figure 3c): same pattern and CNV length difference <100 bp (green box) or 100 to 200 bp difference (yellow-green box); different pattern but <100 bp CNV length difference (yellow box); same pattern but the CNV was larger than the amplifying region (orange box, CNV8), or both the CNV distribution and amplicon size did not match those expected (red box). We considered that the PCR result showing the same CNV distribution pattern and <200 bp difference in the amplicon of the deleted allele (green and yellow-green boxes) was the proper CNV region. According to this standard, the validation process indicated that the extracting CNV region had 76.92% accuracy (Figure 3c, 20 proper CNV regions of 26 CNV regions examined).

Bottom Line: Indeed, it is widely accepted that CNVs are associated with differential disease susceptibility.We did not observe a clear and consistent relationship between the deletion status of CNVs and gene expression levels before and after exercise in blood and muscle.However, we found some pairs of CNVs and associated genes that indicated relationships with gene expression levels: a positive relationship with genes responsible for membrane structure or cytoskeleton and a negative relationship with genes involved in disease.

View Article: PubMed Central - PubMed

Affiliation: Genomic Informatics Center, Hankyong National University, Anseong 456-749, Korea.

ABSTRACT
Copy number variations (CNVs), important genetic factors for study of human diseases, may have as large of an effect on phenotype as do single nucleotide polymorphisms. Indeed, it is widely accepted that CNVs are associated with differential disease susceptibility. However, the relationships between CNVs and gene expression have not been characterized in the horse. In this study, we investigated the effects of copy number deletion in the blood and muscle transcriptomes of Thoroughbred racing horses. We identified a total of 1,246 CNVs of deletion polymorphisms using DNA re-sequencing data from 18 Thoroughbred racing horses. To discover the tendencies between CNV status and gene expression levels, we extracted CNVs of four Thoroughbred racing horses of which RNA sequencing was available. We found that 252 pairs of CNVs and genes were associated in the four horse samples. We did not observe a clear and consistent relationship between the deletion status of CNVs and gene expression levels before and after exercise in blood and muscle. However, we found some pairs of CNVs and associated genes that indicated relationships with gene expression levels: a positive relationship with genes responsible for membrane structure or cytoskeleton and a negative relationship with genes involved in disease. This study will lead to conceptual advances in understanding the relationship between CNVs and global gene expression in the horse.

No MeSH data available.


Related in: MedlinePlus