SNPs and real-time quantitative PCR method for constitutional allelic copy number determination, the VPREB1 marker case

BMC Medical Genetics

Sabrina Corbetta⁷  Palma Finelli³  Luca Persani⁵  Elena Costa⁴  Anita Donnangelo⁴  Mario Carminati¹  Rea Valaperta⁴  Daniela Rusconi²  Tiziana de Filippis⁶  Elena Passeri⁷  Marcello Frigerio⁴ 

[1] Pediatric Cardiology and Adult with Congenital Heart Disease Department, IRCCS Policlinico San Donato, San Donato Milanese (MI), Italy;Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Cusano Milanino (MI), Italy;Department of Biology and Genetics for Medical Sciences, Università di Milano, Milan, Italy;Research Laboratories - Molecular Biology, IRCCS Policlinico San Donato, San Donato Milanese (MI), Italy;Department of Medical Sciences, Università di Milano, Milan, Italy;Laboratorio di Ricerche Endocrino-Metaboliche, IRCCS Istituto Auxologico Italiano, Milan, Italy;Department of Medical-Surgical Sciences, Università di Milano, San Donato Milanese (MI), Italy

关键词: DiGeorge Syndrome;    22q11.2 microdeletion;    allelic copy number;    qRT-PCR;   

Others  :  1178049
DOI  :  10.1186/1471-2350-12-61

 received in 2010-06-28, accepted in 2011-05-05,  发布年份 2011

【 摘 要 】

Background

22q11.2 microdeletion is responsible for the DiGeorge Syndrome, characterized by heart defects, psychiatric disorders, endocrine and immune alterations and a 1 in 4000 live birth prevalence. Real-time quantitative PCR (qPCR) approaches for allelic copy number determination have recently been investigated in 22q11.2 microdeletions detection. The qPCR method was performed for 22q11.2 microdeletions detection as a first-level screening approach in a genetically unknown series of patients with congenital heart defects. A technical issue related to the VPREB1 qPCR marker was pointed out.

Methods

A set of 100 unrelated Italian patients with congenital heart defects were tested for 22q11.2 microdeletions by a qPCR method using six different markers. Fluorescence In Situ Hybridization technique (FISH) was used for confirmation.

Results

qPCR identified six patients harbouring the 22q11.2 microdeletion, confirmed by FISH. The VPREB1 gene marker presented with a pattern consistent with hemideletion in one 3 Mb deleted patient, suggestive for a long distal deletion, and in additional five non-deleted patients. The long distal 22q11.2 deletion was not confirmed by Comparative Genomic Hybridization. Indeed, the VPREB1 gene marker generated false positive results in association with the rs1320 G/A SNP, a polymorphism localized within the VPREB1 marker reverse primer sequence. Patients heterozygous for rs1320 SNP, showed a qPCR profile consistent with the presence of a hemideletion.

Conclusions

Though the qPCR technique showed advantages as a screening approach in terms of cost and time, the VPREB1 marker case revealed that single nucleotide polymorphisms can interfere with qPCR data generating erroneous allelic copy number interpretations.

【 授权许可】

   
2011 Frigerio et al; licensee BioMed Central Ltd.

【 图 表 】

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