Parentally Imprinted Allele (PIA) Typing in the Differentially Methylated Region Upstream of the Human H19 Gene

• NCJ Number: 225045
• Journal: Forensic Science International: Genetics Volume: 2 Issue: 4 Dated: September 2008 Pages: 286-291
• Author(s): Daixin Huang; Xiaoyan Lin; Hui Chen; Qingen Yang; Ya Jie; Xiandun Zhai; Hui Yin
• Date Published: September 2008
• Length: 6 pages
• Publication Format: Article
• Publication Type: Report (Study/Research)
• Language: English
• Country: Ireland

Annotation

This paper describes four closely related single nucleotide polymorphisms (SNPs) upstream of the imprinted H19 gene (H19FR1 and H19FR2, respectively), a paternally imprinted gene located on chromosome 11p15.5, as well as a selective discrimination of either parental allele by using an enzymatic digestion on differentially methylated genomic DNA.

Abstract

Five haplotypes and nine genotypes were detected for H19FR1 in the Chinese Han population by means of PCR and subsequent denaturing gradient gel electrophoresis (DGGE). The power of discrimination (Dp), polymorphism information content (PIC), and probability of paternity exclusion (PE) were estimated to be 0.803, 0.58, and 0.322, respectively. For the H19FR2, two haplotypes and three genotypes were observed, and the Dp, PIC, and PE were 0.626, 0.37, and 0.162, respectively. Sequencing results showed that only two of the four reported SNPs, a7342g and g7547a, were detected in H19FR1 in the Chinese Han population; and two new SNPs, g7351c and a7357g, were found. In the H19FR2 region, only one of the three reported DNAPs, a8097g, was detected. Based on the methylation status of the genomic DNA, selective detection of the parental alleles for H19FR was examined by using two types of enzymes, the methylation-sensitive restriction enzyme (msRE) HpaII or HhaI, and McrBC. Genomic DNA digested by either HpaII or HhaI, revealed a single band derived from the paternal allele, due to cleavage of unmethylated recognition sites on the maternal allele. The use of McrBC, which can digest a methylated paternal sequence, resulted in exclusively amplifying the maternal allele. This parentally imprinted allele (PIA) typing method could be one of the useful techniques for discriminating the parental origin of alleles. The descriptions of materials and methods address sample preparation, primers and PCR amplification, denaturing gradient gel electrophoresis, PIA typing, DNA sequencing, and statistical analysis. 4 tables, 4 figures, and 29 references