title: Diagnosis of Russell-Silver syndrome by the combined bisulfite restriction analysis-denaturing high-performance liquid chromatography assay.
abstract: Russell-Silver syndrome (RSS) is characterized by prenatal and postnatal growth retardation, triangular facies, and fifth-finger clinodactyly. Half of all patients with RSS have hypomethylation of the differentially methylated region of the H19 gene on chromosome 11p15.5. Hence, a quantitative methylation analysis of this region can be useful for the molecular diagnosis of RSS. However, conventional assays based on bisulfite clone sequencing are rather time and labor consuming and are not suitable for clinical use. In the present study, we investigated a possible method of quantitatively determining H19 hypomethylation in RSS patients using a combined bisulfite restriction analysis (COBRA)-denaturing high-performance liquid chromatography (DHPLC) assay; in this combined assay, polymerase chain reaction products amplified from the H19 differentially methylated region of bisulfite-treated genomic DNA were analyzed using a COBRA assay, which detects methylation-dependent sequence differences in the bisulfite-treated genomic DNA using a restriction enzyme analysis. We designed the assay so that a restriction enzyme (HinfI) would cut the methylated, but not the unmethylated, template. The molar ratio between the cut and uncut fragments was measured using DHPLC, and the construction of a calibration curve enabled the methylation index for the original genomic DNA to be estimated. An analysis of seven RSS patients using the COBRA-DHPLC assay demonstrated that three of the seven RSS patients had a low methylation index of around 10%. A comparison of the methylation indices obtained using COBRA-DHPLC and conventional bisulfite clone sequencing revealed an excellent intermethod agreement. In summary, we have developed a robust, rapid, and cost-effective COBRA-DHPLC-based screening system for RSS.
authors.affiliation: Division of Maxillofacial/Neck Reconstruction, Maxillofacial Orthognathics, Department of Maxillofacial Reconstruction and Function, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Mitsu
authors.initials: M
authors.lastname: Hattori
authors.forename: Chiharu
authors.initials: C
authors.lastname: Torii
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Yagihashi
authors.forename: Kosuke
authors.initials: K
authors.lastname: Izumi
authors.forename: Naoto
authors.initials: N
authors.lastname: Suda
authors.forename: Kimie
authors.initials: K
authors.lastname: Ohyama
authors.forename: Takao
authors.initials: T
authors.lastname: Takahashi
authors.forename: Keiji
authors.initials: K
authors.lastname: Moriyama
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Kosaki
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D017931:DNA Primers; D006801:Humans; D016133:Polymerase Chain Reaction; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism; D056730:Silver-Russell Syndrome / Q000175:diagnosis* / Q000235:genetics; D013447:Sulfites / Q000737:chemistry
chemical_list: D017931:DNA Primers; C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite