Origin and mechanisms of formation of fetus-in-fetu: two cases with genotype and methylation analyses.

Fetus-in-fetu (FIF) is a condition in which a host infant has a fetus-like mass(es) within its body. We describe here results of molecular genetic analysis in two cases of FIF. In FIF-1, a male host had two retroperitoneal fetiform masses each with a vertebral column, and in FIF-2, a fetiform mass with vertebral column was present in the cranial cavity of a male host. Genotyping of each case using microsatellite markers showed that the host infant and its fetus(es) inherited one copy each of parental alleles and shared identical genotypes. These findings were confirmed by single nucleotide polymorphism (SNP) analysis using Affymetrix GeneChip Human Mapping 50K Array, and supported a monozygotic twin theory of FIF. Analysis of the methylation status was done in both cases at the differentially methylated region (DMR) within the human IGF2-H19 locus after bisulfite treatment, methylation-specific PCR, and cloning of PCR products. Normally, only the paternal allele is methylated and the maternal allele unmethylated in DMR. However, in FIF-1, 7 (46.7%) of 15 clones from a fetiform mass and 6 (66.7%) of 9 clones from the other mass showed an unmethylated paternal allele, while the methylation status of a host infant and its fetiform mass in FIF-2 was the same in all clones examined with normal patterns. These data suggest that in FIF-1, two isolated blastocysts originated from one zygote, one of the two was implanted into (or included by) the other blastocyst during the process of methylation, and such abnormal implantation may have occurred in FIF-2 after the establishment of methylation. This is the first case of FIF showing different methylation patterns between a host infant and fetiform mass.