Silver-Russell syndrome in a girl born after in vitro fertilization: partial hypermethylation at the differentially methylated region of PEG1/MEST - PubMed (original) (raw)

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Silver-Russell syndrome in a girl born after in vitro fertilization: partial hypermethylation at the differentially methylated region of PEG1/MEST

Masayo Kagami et al. J Assist Reprod Genet. 2007 Apr.

Abstract

Purpose: The prevalence of low birth weight (LBW) is increased in subjects born after assisted reproduction technology (ART), and defective imprinting has frequently been identified in patients with Beckwith-Wiedermann and Angelman syndromes conceived by ART. Thus, we examined methylation pattern in a girl born after ART who had Silver-Russell syndrome (SRS) which can be caused by maternal uniparental disomy for chromosome 7 and by hypomethylation of the differentially methylated region (DMR) of H19.

Methods: We examined methylation status of 31 cytosines at the CpG dinucleotides in the DMR of PEG1/MEST on 7q32.2 and 23 cytosines at the CpG dinucleotides in the DMR of H19 on 11p15, using leukocyte genomic DNA.

Results: Eight of the 31 cytosines in the patient and four of the 31 cytosines in the father were hypermethylated in the PEG1/MEST-DMR. In the H19-DMR, no abnormal methylation pattern was identified in the patient.

Conclusion: The results suggest that hypermethylation of paternally expressed genes including PEG1/MEST, which usually have growth-promoting effects, may be relevant to LBW in subjects conceived by ART.

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Figures

Fig. 1

Fig. 1

Growth charts depicted on the standard growth curves for the Japanese girls and photographs of the twin sisters at 4 11/12 of age. GH: growth hormone; and GnRH: gonadotropin releasing hormone

Fig. 2

Fig. 2

Methylation analysis of the _PEG1/MEST_-DMR (differentially methylated region). (A) Methylation pattern analysis employed in the present study. The upper part: a schematic representation indicating the analyzed region within the DMR encompassing E1 (exon 1) of PEG1/MEST. The positions of the primers utilized for the polymerase chain reaction (PCR) are also shown. The lower left part: genomic sequence of the analyzed region containing 31 CpG dinucleotides. The cytosine residues surrounded by squares are methylated on the maternally derived alleles and unmethylated on the paternally derived alleles. The shaded nucleotide sequence is shown in the electrochromatograms. The lower right part: primer sequences utilized in this study. MET-F and MET-R are designed to hybridize the sequence harboring cytosines, so that they specifically amplify the maternally derived alleles with methylated cytosines at the CpG dinucleotides after bisulphite treatment. UNMET-F and UNMET-R are designed to hybridize the sequence harboring thymines, so that they specifically amplify the paternally derived alleles with thymines converted from unmethylated cytosines via uracils after bisulphite treatment. The nucleotides specific to the methylated and unmethylated alleles are surrounded by squares. COMMON-F and COMMON-R are designed to hybridize the sequence lacking CpG dinucleotides, so that they amplify both paternally and maternally derived alleles after bisulphite treatment. (B) Representative electrochromatograms of a region containing two CpG dinucleotide sequences obtained after bisulphite treatment. The cytosines at the CpG nucleotides are delineated as cytosines (arrows) in the PCR products obtained with MET-F and MET-R (Methylated) in the mother and the patient. In the PCR products obtained with UNMET-F and UNMET-R (Unmethylated), the cytosines at the CpG nucleotides are detected as thymines in the mother and as cytosines in the patient (arrows). (C) Summary of the methylation status of the cytosine residues at the 31 CpG dinucleotides examined after bisulphite treatment. The black circles represent cytosine residues, and the open circles denote thymine residues. The sequence results obtained with MET-F and MET-R are shown on the upper array of circles, and those obtained with UNMET-F and UNMET-R are indicated on the lower array of circles. Hypermethylation, as indicated by the presence of cytosine residues in the PCR products obtained with UNMET-F and UNMET-R, has been identified in 4 of the 31 cytosines in the father and in 8 of the 31 cytosines in the patient. In a subject with maternal disomy for chromosome 7, the PCR products have been obtained only with MET-F and MET-R, and cytosines only have been delineated. The methylation pattern is normal in the mother and the sister, as well as in control subjects

Fig. 3

Fig. 3

Methylation analysis of the _H19_-DMR (differentially methylated region). (A) A schematic representation indicating the analyzed region. PCR was performed after bisulphite treatment with the primers that amplify both the methylated and unmethylated alleles. (B) The results of methylation analysis. Each lane indicates a single cloned allele, and each circle denotes a CpG island; filled and open circles represent methylated and unmethylated cytosines, respectively. Both of the methylated and unmethylated alleles are identified with a similar frequency in the patient

Fig. 4

Fig. 4

Summary of microsatellite analysis. The ideogram of chromosome 7 is shown with the cytogenetic localization of the microsatellite loci examined. The locus position is based on Ensembl Human Genome Browser (http://www.ensembl.org). N.E.: not examined

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