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Analysis of copy number variation using quantitative interspecies competitive PCR.

Williams NM, Williams H, Majounie E, Norton N, Glaser B, Morris HR, Owen MJ, O'Donovan MC - Nucleic Acids Res. (2008)

Bottom Line: Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility.Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts.In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychological Medicine, Wales School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. williamsnm@cf.ac.uk

ABSTRACT
Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

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Comparison of the copy number at the PARK2 locus as determined by MPLA and qicPCR. Copy number estimates of PARK2 exons 3, 4, 5, 6, 8, 9, 10 and 11 as determined by qicPCR and MLPA in two patients with previously characterized PARK2 mutations. The assays of exons 3 (A and B) and exon 6, which detect the deletion and duplication in our test samples PD-patient1 and PD-patient2, respectively, are highlighted.
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Figure 2: Comparison of the copy number at the PARK2 locus as determined by MPLA and qicPCR. Copy number estimates of PARK2 exons 3, 4, 5, 6, 8, 9, 10 and 11 as determined by qicPCR and MLPA in two patients with previously characterized PARK2 mutations. The assays of exons 3 (A and B) and exon 6, which detect the deletion and duplication in our test samples PD-patient1 and PD-patient2, respectively, are highlighted.

Mentions: To initially evaluate qicPCR we analysed a total of 11 probes designed to assay exons at the PARK2 locus and compared the results to data generated by the established technique of MPLA (23). The standard curves of each qicPCR assay being presented in Supplementary Table 3. All probes were analysed in 10 healthy controls as well as two PD individuals who had previously been characterized to carry PARK2 mutations. Analysis of the human DNA copy number (hsCN) determined for the assays targeting PARK2 exons 3 and 6 established that qicPCR was capable of detecting both the heterozygous deletion at exon 3 (mean hsCN = 1.08) and the heterozygous duplication at exon 6 (mean hsCN = 2.88) in the respective PD samples (Table 1). Neither assay detected any evidence for a change in copy number in any of the 10 control samples [exon 3a: mean hsCN = 1.92 (1.8–2.0); exon 3b: mean hsCN = 1.76 (1.66–1.88); exon 6: mean hsCN = 2.16 (2.04–2.26)], Table 1. Direct comparison of the hsCN estimates of qicPCR to those generated by MPLA showed that the results of both methods were highly correlated, r = 0.82, P = 0.005 (Table 1 and Figure 2). No evidence for change in copy number was observed in either the PD patients or the 10 control samples at the qicPCR assays that were not targeting PARK2 exons 3 or 6 (mean hsCN = 1.98, 95% CI 1.9–2.06), Table 1.Figure 2.


Analysis of copy number variation using quantitative interspecies competitive PCR.

Williams NM, Williams H, Majounie E, Norton N, Glaser B, Morris HR, Owen MJ, O'Donovan MC - Nucleic Acids Res. (2008)

Comparison of the copy number at the PARK2 locus as determined by MPLA and qicPCR. Copy number estimates of PARK2 exons 3, 4, 5, 6, 8, 9, 10 and 11 as determined by qicPCR and MLPA in two patients with previously characterized PARK2 mutations. The assays of exons 3 (A and B) and exon 6, which detect the deletion and duplication in our test samples PD-patient1 and PD-patient2, respectively, are highlighted.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553599&req=5

Figure 2: Comparison of the copy number at the PARK2 locus as determined by MPLA and qicPCR. Copy number estimates of PARK2 exons 3, 4, 5, 6, 8, 9, 10 and 11 as determined by qicPCR and MLPA in two patients with previously characterized PARK2 mutations. The assays of exons 3 (A and B) and exon 6, which detect the deletion and duplication in our test samples PD-patient1 and PD-patient2, respectively, are highlighted.
Mentions: To initially evaluate qicPCR we analysed a total of 11 probes designed to assay exons at the PARK2 locus and compared the results to data generated by the established technique of MPLA (23). The standard curves of each qicPCR assay being presented in Supplementary Table 3. All probes were analysed in 10 healthy controls as well as two PD individuals who had previously been characterized to carry PARK2 mutations. Analysis of the human DNA copy number (hsCN) determined for the assays targeting PARK2 exons 3 and 6 established that qicPCR was capable of detecting both the heterozygous deletion at exon 3 (mean hsCN = 1.08) and the heterozygous duplication at exon 6 (mean hsCN = 2.88) in the respective PD samples (Table 1). Neither assay detected any evidence for a change in copy number in any of the 10 control samples [exon 3a: mean hsCN = 1.92 (1.8–2.0); exon 3b: mean hsCN = 1.76 (1.66–1.88); exon 6: mean hsCN = 2.16 (2.04–2.26)], Table 1. Direct comparison of the hsCN estimates of qicPCR to those generated by MPLA showed that the results of both methods were highly correlated, r = 0.82, P = 0.005 (Table 1 and Figure 2). No evidence for change in copy number was observed in either the PD patients or the 10 control samples at the qicPCR assays that were not targeting PARK2 exons 3 or 6 (mean hsCN = 1.98, 95% CI 1.9–2.06), Table 1.Figure 2.

Bottom Line: Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility.Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts.In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychological Medicine, Wales School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. williamsnm@cf.ac.uk

ABSTRACT
Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

Show MeSH
Related in: MedlinePlus