Abstract
BackgroundThe human chromosome 14q32.2 imprinted region harbors the primary MEG3/DLK1:IG-differentially methylated region (DMR) and secondary MEG3:TSS-DMR. The MEG3:TSS-DMR can remain unmethylated only in the presence of unmethylated MEG3/DLK1:IG-DMR in somatic tissues, but not in the placenta, because of a hierarchical regulation of the methylation pattern between the two DMRs.MethodsWe performed molecular studies in a 4-year-old Japanese girl with Temple syndrome (TS14).ResultsPyrosequencing analysis showed extremely low methylation levels of five CpGs at the MEG3:TSS-DMR and grossly normal methylation levels of four CpGs at the MEG3/DLK1:IG-DMR in leukocytes. HumanMethylation450 BeadChip confirmed marked hypomethylation of the MEG3:TSS-DMR and revealed multilocus imprinting disturbance (MLID) including mild hypomethylation of the H19/IGF2:IG-DMR and mild hypermethylation of the GNAS A/B:TSS-DMR in leukocytes. Bisulfite sequencing showed markedly hypomethylated CpGs at the MEG3:TSS-DMR and irregularly and non-differentially methylated CpGs at the MEG3/DLK1:IG-DMR in leukocytes and apparently normal methylation patterns of the two DMRs in the placenta. Maternal uniparental disomy 14 and a deletion involving this imprinted region were excluded.ConclusionsSuch a methylation pattern of the MEG3/DLK1:IG-DMR has not been reported in patients with TS14. It may be possible that a certain degree of irregular hypomethylation at the MEG3/DLK1:IG-DMR has prevented methylation of the MEG3:TSS-DMR in somatic tissues and that a hypermethylation type MLID has occurred at the MEG3/DLK1:IG-DMR to yield the apparently normal methylation pattern in the placenta.
Highlights
The human chromosome 14q32.2 imprinted region harbors the primary MEG3/DLK1:IG-differentially methylated region (DMR) and secondary MEG3:TSS-differentially methylated regions (DMRs)
For the H19/IGF2:IG-DMR, the CpGs around CpG19–22 examined by pyrosequencing were shown to be normally methylated by HumanMethylation450 BeadChip analysis; for the GNAS A/B:TSS-DMR, the CpGs around CpG43– 49 examined by pyrosequencing were found to be hypermethylated by HumanMethylation450 BeadChip analysis. (Additional file 1: Figure S1)
Expression analysis Quantitative PCR analyses showed increased expression of MEG3 and miR134-5p regulated by the MEG3:TSS-DMR, increased expression of H19 controlled by the H19/ IGF2:IG-DMR, and decreased expression of GNAS A/B
Summary
Methylation analysis Pyrosequencing analysis showed extremely low methylation levels of five CpGs at the MEG3:TSS-DMR and variable but grossly normal methylation levels of four CpGs at the MEG3/DLK1:IG-DMR with a relatively hypermethylated CpG and a relatively hypomethylated CpG, as well as normal methylation levels of most CpGs at the remaining seven DMRs (Fig. 2a). HumanMethylation450 BeadChip analysis revealed marked hypomethylation of most CpGs at the MEG3:TSS-DMR accompanied by a hypermethylated CpG at the MEG8-DMR [12], together with mild hypomethylation of 30% of CpGs at the H19/ IGF2:IG-DMR and mild hypermethylation of 64% of CpGs at the GNAS A/B:TSS-DMR, in addition to a few of hypo- or hypermethylated CpGs at several DMRs (Fig. 2b) (for details, see Additional file 1: Table S2 and its footnotes). Bisulfite sequencing disclosed markedly hypomethylated CpGs at the MEG3:TSS-DMR and rather irregularly (non-differentially) methylated CpGs at the MEG3/DLK1:IG-DMR in leukocytes and apparently similar methylation patterns of the MEG3/DLK1:IG-DMR and MEG3:TSS-DMR between placentas of this patient and a control subject (Fig. 2c). Expression analysis Quantitative PCR analyses showed increased expression of MEG3 and miR134-5p regulated by the MEG3:TSS-DMR, increased expression of H19 controlled by the H19/ IGF2:IG-DMR, and decreased expression of GNAS A/B. Because no cSNP was identified in MEG3, miR134-5p, and H19, biparental expression of these genes was not demonstrated
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
