Accurate determination of copy number variations (CNVs): Application to the α- and β-defensin CNVs

Nuytten, Hilde; Włodarska, Iwona; Nackaerts, Kristiaan; Vermeire, Séverine; Vermeesch, Joris Robert; Cassiman, Jean‐Jacques; Cuppens, Harry

doi:10.1016/j.jim.2009.03.002

Cited by 13 publications

(17 citation statements)

References 21 publications

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“…Although it is possible that different population origins may influence the outcome, even the relatively small sample analysed by Linzmeier and Ganz seems incompatible with the values determined here, and may reflect limitations of real-time PCR typing for this locus. The study of Nuytten et al [52] used real-time PCR calibrated against concatemeric constructs, but reports a copy number distribution that is also very significantly different from the one reported here (P = 1.1 × 10 -10 ), with a much lower frequency of samples with copy numbers above 8. Nuytten et al do not use reference genomic DNA standards, and despite their careful and ingenious method to calibrate real-time PCR measurements, it is possible that in this case their cloned constructs do not produce the same calibration as would be obtained from genomic DNA samples of the same copy number.…”

Section: Discussioncontrasting

confidence: 85%

“…Distributions of diploid copy numbers in the 589 European samples typed in this work, and comparison with data taken or inferred from the previous studies of Aldred et al [30], Linzmeier and Ganz [7], and Nuytten et al [52]. The comparison is also made between the observed frequencies of copy number classes and those predicted from the haplotype frequencies determined in this study, assuming Hardy-Weinberg equilibrium (“Predicted frequency (HWE)”).…”

Section: Resultsmentioning

confidence: 52%

“…The comparison is also made between the observed frequencies of copy number classes and those predicted from the haplotype frequencies determined in this study, assuming Hardy-Weinberg equilibrium (“Predicted frequency (HWE)”). The frequencies of copy number classes were not given explicitly by Nuytten et al [52], but are reconstructed here from the data in their Figure seven (a). Jespersgaard et al [47] do not give details of individual copy number counts, but instead give counts above or below a copy number of 6.…”

Section: Resultsmentioning

confidence: 99%

“…Larger-scale typing then produced data that were internally consistent between PRT and ratio measurements and conformed well to the predictions of Hardy-Weinberg equilibrium using haplotype frequencies determined in three-generation families. Reassurance of the correct calibration of our typing methods is particularly important given the apparent differences with the population copy-number distributions discovered by other approaches [7, 47, 52]. …”

Section: Discussionmentioning

confidence: 99%

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Accurate measurement of gene copy number for human alpha-defensin DEFA1A3

et al. 2013

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BackgroundMulti-allelic copy number variants include examples of extensive variation between individuals in the copy number of important genes, most notably genes involved in immune function. The definition of this variation, and analysis of its impact on function, has been hampered by the technical difficulty of large-scale but accurate typing of genomic copy number. The copy-variable alpha-defensin locus DEFA1A3 on human chromosome 8 commonly varies between 4 and 10 copies per diploid genome, and presents considerable challenges for accurate high-throughput typing.ResultsIn this study, we developed two paralogue ratio tests and three allelic ratio measurements that, in combination, provide an accurate and scalable method for measurement of DEFA1A3 gene number. We combined information from different measurements in a maximum-likelihood framework which suggests that most samples can be assigned to an integer copy number with high confidence, and applied it to typing 589 unrelated European DNA samples. Typing the members of three-generation pedigrees provided further reassurance that correct integer copy numbers had been assigned. Our results have allowed us to discover that the SNP rs4300027 is strongly associated with DEFA1A3 gene copy number in European samples.ConclusionsWe have developed an accurate and robust method for measurement of DEFA1A3 copy number. Interrogation of rs4300027 and associated SNPs in Genome-Wide Association Study SNP data provides no evidence that alpha-defensin copy number is a strong risk factor for phenotypes such as Crohn’s disease, type I diabetes, HIV progression and multiple sclerosis.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-719) contains supplementary material, which is available to authorized users.

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Section: Discussioncontrasting

confidence: 85%

Section: Resultsmentioning

confidence: 52%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Accurate measurement of gene copy number for human alpha-defensin DEFA1A3

et al. 2013

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“…Three PCR based methods, real-time PCR (qPCR) [8,15,17,18], paralog ratio tests (PRT) [14,16,19-21] and multiplex ligation-dependent probe amplification (MLPA) [7,20,22,23] have being extensively used to determine DEFB CN. qPCR was advantageous due to universal applicability and relative simplicity, but the reliability of this method was questioned [16,22,24].…”

Section: Introductionmentioning

confidence: 99%

8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations

et al. 2014

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BackgroundThe copy number variation (CNV) in beta-defensin genes (DEFB) on human chromosome 8p23 has been proposed to contribute to the phenotypic differences in inflammatory diseases. However, determination of exact DEFB CN is a major challenge in association studies. Quantitative real-time PCR (qPCR), paralog ratio tests (PRT) and multiplex ligation-dependent probe amplification (MLPA) have been extensively used to determine DEFB CN in different laboratories, but inter-method inconsistencies were observed frequently. In this study we asked which one is superior among the three methods for DEFB CN determination.ResultsWe developed a clustering approach for MLPA and PRT to statistically correlate data from a single experiment. Then we compared qPCR, a newly designed PRT and MLPA for DEFB CN determination in 285 DNA samples. We found MLPA had the best convergence and clustering results of the raw data and the highest call rate. In addition, the concordance rates between MLPA or PRT and qPCR (32.12% and 37.99%, respectively) were unacceptably low with underestimated CN by qPCR. Concordance rate between MLPA and PRT (90.52%) was high but PRT systematically underestimated CN by one in a subset of samples. In these samples a sequence variant which caused complete PCR dropout of the respective DEFB cluster copies was found in one primer binding site of one of the targeted paralogous pseudogenes.ConclusionMLPA is superior to PRT and even more to qPCR for DEFB CN determination. Although the applied PRT provides in most cases reliable results, such a test is particularly sensitive to low-frequency sequence variations preferably accumulating in loci like pseudogenes which are most likely not under selective pressure. In the light of the superior performance of multiplex assays, the drawbacks of such single PRTs could be overcome by combining more test markers.

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Targeted Screening and Validation of Copy Number Variations

Ceulemans

Ven

Del-Favero

2011

Methods in Molecular Biology

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The accessibility of genome-wide screening technologies considerably facilitated the identification and characterization of copy number variations (CNVs). The increasing amount of available data describing these variants, clearly demonstrates their abundance in the human genome. This observation shows that not only SNPs, but also CNVs and other structural variants strongly contribute to genetic variation. Even though not all structural variants have an obvious phenotypic effect, there is evidence that CNVs influence gene dosage and hence can have profound effects on human disease susceptibility, disease manifestation, and disease severity. Therefore, CNV screening and analysis methodologies, specifically focusing on disease-related CNVs are actively progressing. This chapter specifically describes different techniques currently available for the targeted screening and validation of CNVs. We not only provide an overview of all these CNV analysis methods, but also address their strong and weak points. Methods covered include fluorescence in situ hybridization (FISH), quantitative real-time PCR (qPCR), paralogue ratio test (PRT), molecular copy-number counting (MCC), and multiplex PCR-based approaches, such as multiplex amplifiable probe hybridization (MAPH), multiplex ligation-dependent probe amplification (MLPA), multiplex PCR-based real-time invader assay (mPCR-RETINA), quantitative multiplex PCR of short fluorescent fragments (QMPSF), and multiplex amplicon quantification (MAQ). We end with some general remarks and conclusions, furthermore briefly addressing the future perspectives.

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Accurate determination of copy number variations (CNVs): Application to the α- and β-defensin CNVs

Cited by 13 publications

References 21 publications

Accurate measurement of gene copy number for human alpha-defensin DEFA1A3

Accurate measurement of gene copy number for human alpha-defensin DEFA1A3

8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations

Targeted Screening and Validation of Copy Number Variations

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