Parentally imprinted allele (PIA) typing in the differentially methylated region upstream of the human H19 gene.
The H19 gene is a paternally imprinted gene located on chromosome 11p15.5. In this study, the H19FR1 and H19FR2 haplotype polymorphisms including four and three SNPs, respectively, upstream of the H19 gene according to the GenBank sequence (accession no. AF125183) were investigated. 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 genotyes 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 SNPs, a8097g, was detected. Based on the methylation status of the genomic DNA, selective detection of the parental alleles for H19FRs 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, as a result of cleavage of unmethylated recognition sites on the maternal allele. On the contrary, 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.
Huang
Daixin
D
Faculty of Forensic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China. huangdaixin@hotmail.com
Lin
Xiaoyan
X
Chen
Hui
H
Yang
Qingen
Q
Jie
Ya
Y
Zhai
Xiandun
X
Yin
Hui
H
D002874:Chromosome Mapping; D002880:Chromosomes, Human, Pair 11; D017931:DNA Primers; D053803:Forensic Genetics / Q000379:methods*; D018392:Genomic Imprinting; D005838:Genotype; D006239:Haplotypes / Q000235:genetics; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics; D018598:Minisatellite Repeats / Q000235:genetics; D010290:Parents; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated