Improved Multiplex Ligation-dependent Probe Amplification (i-MLPA) for rapid copy number variant (CNV) detection

• Published in: Clinica chimica acta Vol. 450; pp. 19 - 24
• Main Authors: Saxena, Sonal, Gowdhaman, Kavitha, Kkani, Poornima, Vennapusa, Bhavyasri, Rama Subramanian, Chellamuthu, Ganesh Kumar, S., Mohan, Kommu Naga
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 23-10-2015
• Subjects: Chromosome Aberrations; Chromosomes, Human, Pair 15 - genetics; CNV detection; DGV; DNA Copy Number Variations - genetics; Humans; Intellectual Disability - genetics; MLPA; Multiplex Polymerase Chain Reaction; Polymorphism, Single Nucleotide - genetics; Time Factors; MLPA; CNV detection; DGV
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/j.cca.2015.07.028

In Multiplex Ligation-dependent Probe Amplification (MLPA), copy number variants (CNVs) for specific genes are identified after normalization of the amounts of PCR products from ligated reference probes hybridized to genomic regions that are ideally free from normal variation. However, we observed ambiguous calls for two reference probes in an investigation of the human 15q11.2 region by MLPA among 20 controls, due to the presence of single nucleotide polymorphisms (SNPs) in the probe-binding regions. Further in silico analysis revealed that 18 out of 19 reference probes hybridize to regions subject to variation, underlining the requirement for designing new reference probes against variation-free regions.
An improved MLPA (i-MLPA) method was developed by generating a new set of reference probes to reduce the chances of ambiguous calls and new reagents that reduce hybridization times to 30min from 16h to obtain MLPA ratio data within 6h. Using i-MLPA, we screened 240 schizophrenia patients for CNVs in 15q11.2 region.
Three deletions and two duplications were identified among the 240 schizophrenia patients. No variation was observed for the new reference probes. Taken together, i-MLPA procedure helps obtaining non-ambiguous CNV calls within 6h without compromising accuracy.
•The manuscript reports ambiguous CNV calls using the currently available reference probes.•Ambiguous calls can misdirect investigation of irrelevant regions.•New set of reference probes minimize ambiguous calls.•New reagents yield reliable MLPA data within 6h.•i-MLPA method provides a better platform for fast and accurate CNV detection.