Technical considerations for genotyping multi-allelic copy number variation (CNV), in regions of segmental duplication

BMC Genomics

Stefan J White¹  Paul N Baird²  Patrick S Western¹  Stuart Cantsilieris² 

[1] Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton 3168, Victoria, Australia;Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria, Australia

关键词: Paralogue ratio test;    Multiplex ligation-dependent probe amplification;    Real-time quantitative PCR;    CCL3L1;    Multi-allelic;    Copy number variation;   

Others  :  1217301
DOI  :  10.1186/1471-2164-15-329

 received in 2013-10-14, accepted in 2014-04-22,  发布年份 2014

【 摘 要 】

Background

Intrachromosomal segmental duplications provide the substrate for non-allelic homologous recombination, facilitating extensive copy number variation in the human genome. Many multi-copy gene families are embedded within genomic regions with high levels of sequence identity (>95%) and therefore pose considerable analytical challenges. In some cases, the complexity involved in analyzing such regions is largely underestimated. Rapid, cost effective analysis of multi-copy gene regions have typically implemented quantitative approaches, however quantitative data are not an absolute means of certainty. Therefore any technique prone to degrees of measurement error can produce ambiguous results that may lead to spurious associations with complex disease.

Results

In this study we have focused on testing the accuracy and reproducibility of quantitative analysis techniques. With reference to the C-C Chemokine Ligand-3-like-1 (CCL3L1) gene, we performed analysis using real-time Quantitative PCR (QPCR), Multiplex Ligation-dependent Probe Amplification (MLPA) and Paralogue Ratio Test (PRT). After controlling for potential outside variables on assay performance, including DNA concentration, quality, preparation and storage conditions, we find that real-time QPCR produces data that does not cluster tightly around copy number integer values, with variation substantially greater than that of the MLPA or PRT systems. We find that the method of rounding real-time QPCR measurements can potentially lead to mis-scoring of copy number genotypes and suggest caution should be exercised in interpreting QPCR data.

Conclusions

We conclude that real-time QPCR is inherently prone to measurement error, even under conditions that would seem favorable for association studies. Our results indicate that potential variability in the physicochemical properties of the DNA samples cannot solely explain the poor performance exhibited by the real-time QPCR systems. We recommend that more robust approaches such as PRT or MLPA should be used to genotype multi-allelic copy number variation in disease association studies and suggest several approaches which can be implemented to ensure the quality of the copy number typing using quantitative methods.

【 授权许可】

   
2014 Cantsilieris et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Gonzalez E, Kulkarni H, Bolivar H, Mangano A, Sanchez R, Catano G, Nibbs RJ, Freedman BI, Quinones MP, Bamshad MJ, Murthy KK, Rovin BH, Bradley W, Clark RA, Anderson SA, O'Connell RJ, Agan BK, Ahuja SS, Bologna R, Sen L, Dolan MJ, Ahuja SK: The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility. Science 2005, 307(5714):1434-1440.
• [2]Aldhous M, Abu Bakar S, Prescott N, Palla R, Soo K, Mansfield J, Mathew C, Satsangi J, Armour J: Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn’s disease. Hum Mol Genet 2010, 19(24):4930-4938.
• [3]Cantsilieris S, Baird PN, White SJ: Molecular methods for genotyping complex copy number polymorphisms. Genomics 2012, 101(2):86-93.
• [4]Hollox EJ: The challenges of studying complex and dynamic regions of the human genome. Methods Mol Biol 2012, 838:187-207.
• [5]Carpenter D, Walker S, Prescott N, Schalkwijk J, Armour J: Accuracy and differential bias in copy number measurement of CCL3L1 in association studies with three auto-immune disorders. BMC Genomics 2011, 12(1):418. BioMed Central Full Text
• [6]Notini AJ, Craig JM, White SJ: Copy number variation and mosaicism. Cytogenet Genome Res 2009, 123(1–4):270-277.
• [7]Cantsilieris S, White SJ: Correlating multi-allelic copy number polymorphisms with disease susceptibility. Hum Mutat 2013, 34(1):1-13.
• [8]Bhattacharya T, Stanton J, Kim EY, Kunstman KJ, Phair JP, Jacobson LP, Wolinsky SM: CCL3L1 and HIV/AIDS susceptibility. Nat Med 2009, 15(10):1112-1115.
• [9]Urban TJ, Weintrob AC, Fellay J, Colombo S, Shianna KV, Gumbs C, Rotger M, Pelak K, Dang KK, Detels R, Martinson JJ: CCL3L1 and HIV/AIDS susceptibility. Nat Med 2009, 15(10):1110-1112.
• [10]Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a Laboratory manual. Cold Spring Harbor Laboratory Press 1989, 1:E3-E7.
• [11]White SJ, Vink GR, Kriek M, Wuyts W, Schouten J, Bakker B, Breuning MH, den Dunnen JT: Two-color multiplex ligation-dependent probe amplification: detecting genomic rearrangements in hereditary multiple exostoses. Hum Mutat 2004, 24(1):86-92.
• [12]White SJ, Breuning MH, den Dunnen JT: Detecting copy number changes in genomic DNA: MAPH and MLPA. Methods Mol Biol 2004, 75:751-768.
• [13]Sudmant P, Kitzman JO, Antonacci F, Alkan C, Malig M, Tsalenko A, Sampas N, Bruhn L, Shendure J, Eichler EE: 1000 Genomes Project. Diversity of human copy number variation and multicopy genes. Science 2010, 330(6004):641-646.
• [14]Field SF, Howson JMM, Maier LM, Walker S, Walker NM, Smyth DJ, Armour JAL, Clayton DG, Todd JA: Experimental aspects of copy number variant assays at CCL3L1. Nat Med 2009, 15(10):1115-1117.
• [15]Fode P, Jespersgaard C, Hardwick R, Bogle H, Theisen M, Dodoo D, Lenicek M, Vitek L, Vieira A, Freitas J, Andersen PS, Hollox EJ: Determination of beta-defensin genomic copy number in different populations: a comparison of three methods. PLoS One 2011, 6(2):e16768.
• [16]Perne A, Zhang X, Lehmann LE, Groth M, Stuber F, Book M: Comparison of Multiplex Ligation-dependant Probe Amplification and real time PCR accuracy for gene copy number quantification using the Beta Defensin Locus. Biotechniques 2009, 47(6):1023-1026.
• [17]Cukier HN, Pericak-Vance MA, Gilbert JR, Hedges DJ: Sample degradation leads to false-positive copy number variation calls in multiplex real-time polymerase chain reaction assays. Anal Biochem 2009, 386(2):288-290.
• [18]Fernandez-Jimenez N, Castellanos-Rubio A, Plaza-Izurieta L, Gutierrez G, Irastorza I, Castan L, Vitoria J, Bilbao J: Accuracy in copy number calling by qPCR and PRT: a matter of DNA. PLoS One 2011, 6(12):e28910.
• [19]Weaver S, Dube S, Mir A, Qin J, Gang S, Ramakrishnan R, Jones RC, Livak K: Taking qPCR to a higher level: analysis of CNV reveals the power of high throughpout qPCR to enhance quantitative resolution. Methods 2010, 50:271-276.
• [20]Hollox EJ, Armour JA, Barber JCK: Extensive normal copy number variation of a β-defensin antimicrobial-gene cluster. A J Hum Genet 2003, 73(3):591-600.
• [21]Mamtani M, Rovin B, Brey R, Camargo JF, Kulkarni H, Herrera M, Correa P, Holliday S, Anaya JM, Ahuja SK: CCL3L1 gene-containing segmental duplications and polymorphisms in CCR5 affect risk of systemic lupus erythaematosus. Ann Rheum Dis 2008, 67(8):1076-1083.
• [22]Fellermann K, Stange DE, Schaeffeler E, Schmalzl H, Wehkamp J, Bevins CL, Reinisch W, Teml A, Schwab M, Lichter P, Radlwimmer B, Stange EF: A chromosome 8 gene-cluster polymorphism with Low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon. Am J Hum Genet 2006, 79(3):439-448.
• [23]Bentley RW, Pearson J, Gearry RB, Barclay ML, McKinney C, Merriman TR, Roberts RL: Association of higher DEFB4 genomic copy number with Crohn’s disease. Am J Gastroenterol 2010, 105(2):354-359.
• [24]Mehlotra R, Dazard J, John B, Zimmerman P, Weinberg A, Jurevic R: Copy number variation within human β-defensin gene cluster influences progression to AIDS in the multicenter AIDS cohort study. J Aids Clin Res 2012, 3(10):1000184.
• [25]Hollox EJ: Beta-Defensins and Crohn’s disease: confusion from counting copies. Am J Gastroenterol 2010, 105(2):360-362.
• [26]McKinney C, Broen JCA, Vonk MC, Beretta L, Hesselstrand R, Hunzelmann N, Riemekasten G, Scorza R, Simeon CP, Fonollosa V, Carreira PE, Ortego-Centeno N, Gonzalez-Gay MA, Airo P, Coenen M, Martin J, Radstake TRDJ, Merriman TR: Evidence that deletion at FCGR3B is a risk factor for systemic sclerosis. Genes Immun 2012, 13(6):458-460.
• [27]McKinney C, Merriman ME, Chapman PT, Gow PJ, Harrison AA, Highton J, Jones PBB, McLean L, O’Donnell JL, Pokorny V, Spellerberg M, Stamp LK, Willis J, Steer S, Merriman TR: Evidence for an influence of chemokine ligand 3-like 1 (CCL3L1) gene copy number on susceptibility to rheumatoid arthritis. Ann Rheum Dis 2008, 67(3):409-413.
• [28]Barnes C, Plagnol V, Fitzgerald T, Redon R, Marchini J, Clayton D, Hurles ME: A robust statistical method for case–control association testing with copy number variation. Nat Genet 2008, 40(10):1245-1252.
• [29]D’haene B, Vandesompele J, Hellemans J: Accurate and objective copy number profiling using real-time quantitative PCR. Methods 2010, 50:262-270.
• [30]Boettger L, Handsaker R, Zody M, McCarroll S: Structural haplotypes and recent evolution of the human 17q21.31 region. Nat Genet 2012, 44(8):881-886.
• [31]Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP, et al.: High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 2011, 83(22):8604-8610.
• [32]Nuttle X, Huddleston J, O’Roak BJ, Antonacci F, Fichera M, Romano C, Shendure J, Eichler EE: Rapid and accurate large-scale genotyping of duplicated genes and discovery of interlocus gene conversions. Nat Meth 2013, 10(9):903-909.