title: Genotyping of common SIRPB1 copy number variant using Paralogue Ratio Test coupled to MALDI-MS quantification.
abstract: Copy number variant (CNV) regions have been proven to have a significant impact on gene expression. Some of them have been also found to be associated to different human diseases. CNV genotyping is often prone to error and cross-validation with independent methods is frequently required. The platform of choice depends on whether it is a genome-wide discovery screening or a candidate CNV study, the cohort size and the number of CNVs included in the assay and, finally, the budget available. Here we illustrate a affordable approach to determine the CNV genotype using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and based on the quantitative determination of single nucleotide duplicated mismatches (SNDM) mapping the CNV region and a paralogue genomic region that is used as a two-copy reference. We have genotyped nsv436327, a common CNV mapping SIRPB1 intron 1 that has been associated to human personality behavior. SIRP cluster region was subjected to several ancestral duplication events what makes SIRPB1 CNV genotyping technically challenging. We designed three sets of primer pairs that amplified paralogue regions inside and outside the CNV, containing three SNDMs. Post-PCR extension analyses of sequencing oligonucleotides mapping immediately upstream each SNDM allowed us to quantify using MALDI-MS the proportion of PCR products derived from the CNV region versus the external reference. In contrast to other approaches, setting up this genotyping method requires an affordable investment.
authors.affiliation: Department of Basic Medical Sciences, University of Lleida, Spain; Institute of Biomedical Research of Lleida, Spain. Electronic address: jlroyo@cmb.udl.cat.
authors.forename: Jose Luis
authors.initials: JL
authors.lastname: Royo
authors.affiliation: Department of Basic Medical Sciences, University of Lleida, Spain.
authors.forename: Mariona
authors.initials: M
authors.lastname: Pascual-Pons
authors.affiliation: Department of Basic Medical Sciences, University of Lleida, Spain.
authors.forename: Arantxa
authors.initials: A
authors.lastname: Lupiañez
authors.affiliation: Proteomics and Genomics Scientific Technical Service, University of Lleida, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Sanchez-López
authors.affiliation: Department of Basic Medical Sciences, University of Lleida, Spain; Institute of Biomedical Research of Lleida, Spain. Electronic address: joan.fibla@cmb.udl.cat.
authors.forename: Joan
authors.initials: J
authors.lastname: Fibla
mesh_terms: D056915:DNA Copy Number Variations; D017931:DNA Primers / Q000235:genetics; D019143:Evolution, Molecular; D060005:Genotyping Techniques; D006801:Humans; D010554:Personality Disorders / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D011956:Receptors, Cell Surface / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D017931:DNA Primers; D011956:Receptors, Cell Surface; C501921:SIRPB1 protein, human
keywords: Copy-number variant; Gene cluster; Genotyping; MALDI-TOF-MS; Paralogue; SIRPB1