Allelic Copy Number Variation in FSCN2 Detected using Allele-Specific Genotyping and Multiplex Real-Time PCRs

¹ Lab. for Retinal Regeneration, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan
² Laboratory for Retinal Regeneration, RIKEN Center for Developmental of Biology, Kobe, Hyogo, Japan
³ Department of Ophthalmology & Visual Science, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan

^* To whom correspondence should be addressed. E-mail: jinzb{at}cdb.riken.jp.

	Abstract

PURPOSE: Allelic copy number variation (CNV) may alter the functional effects of a heterozygous mutation. The underlying mechanisms and their roles in hereditary diseases, however, are largely unknown. We have examined a FSCN2 mutation which has been reported not only in patients with retinitis pigmentosa (RP), but also in the normal population. METHODS AND RESULTS: We carried experiments to investigate the gene and allele copy numbers of FSCN2 in RP patients carrying the c.72delG mutation as well as healthy subjects with or without the mutation. After establishing a real-time PCR-based genotyping approach, which employed a real-time PCR assay to qualify the copy numbers of both the wild-type and mutant alleles of the FSCN2 gene, we found that three RP patients and three normal subjects had an equal ratio of the alleles. Interestingly, another RP patient had an asymmetric allele ratio (4:1) of the copy number of the wild-type allele compared with that of the mutant allele. These findings were further verified using quantitative assays. Allele-specific methylation assay demonstrated a random methylation pattern in the FSCN2 gene. CONCLUSION: The copy numbers of the FSCN2 gene and of each allele in the mutant samples were quantified. The findings excluded the possibility that allelic CNV was associated with RP, suggesting that the c.72delG variant is not the primary cause of RP. It is not likely that the FSCN2 gene is imprinted differentially. The real-time PCR-based genotyping method developed in this study is useful for investigations of allelic asymmetries within genomic regions with CNVs.

Key Words: retinitis pigmentosa, genetic diseases, mutation screening, FSCN2 gene, Copy number variation, Imprinting