An MLPA-based approach for high-resolution genotyping of disease-related multi-allelic CNVs.

abstract Article history:Received 23 April 2014Accepted 31 May 2014Available online 2 June 2014Keywords:MLPAMulti-allelic CNVCCL3L1DEFBUGT2B17 Copy number variation has recently been recognized as an important type of genetic variation that modifieshumanphenotypes.Copynumbervariants(CNVs)arebeingincreasinglyassociatedwithvarioushumanpheno-types and diseases. However, the lack of an appropriate method that allows fast, inexpensive and, most impor-tantly, accurate CNVs genotyping significantly hampers CNV analysis. This limitation especially affects theanalysis of multi-allelic CNVs that frequently modify various phenotypes. Recently, we developed a multiplexligation-dependent probe amplification (MLPA)-based strategy for multiplex copy number genotyping and thevalidation of candidate CNV-miRNAs. Here we present the adaptation and optimization of this recently devel-oped method for high-resolution genotyping of individual disease-related multi-allelic CNVs. We developedappropriate assays for three well-known and extensively studied CNVs: CNV-CCL3L1, CNV-DEFB, and CNV-UGT2B17, which have been associated with various human phenotypes including inflammation-related andinfectious diseases. With the use of these assays we identified several general factors that allow to increase theresolutionofthecopynumbergenotyping.Performedexperimentsconfirmedthehighreproducibilityandaccu-racy of the obtained genotypingresults. The reliability of theresults and relatively low per-genotype cost makesthis strategy an attractive method for large-scale experiments such as genotype–phenotype association studies.© 2014 Elsevier B.V. All rights reserved.