Diagnosis of Russell-Silver syndrome by the combined bisulfite restriction analysis-denaturing high-performance liquid chromatography assay.

Mitsu Hattori, Chiharu Torii, Tatsuhiko Yagihashi, Kosuke Izumi, Naoto Suda, Kimie Ohyama, Takao Takahashi, Keiji Moriyama, Kenjiro Kosaki

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)623-630
Number of pages8
JournalGenetic Testing and Molecular Biomarkers
Volume13
Issue number5
Publication statusPublished - 2009 Oct

Fingerprint

Silver-Russell Syndrome
High Pressure Liquid Chromatography
Methylation
DNA
Clone Cells
hydrogen sulfite
Restriction Mapping
Calibration
Fingers
Chromosomes

ASJC Scopus subject areas

  • Genetics(clinical)

Cite this

Diagnosis of Russell-Silver syndrome by the combined bisulfite restriction analysis-denaturing high-performance liquid chromatography assay. / Hattori, Mitsu; Torii, Chiharu; Yagihashi, Tatsuhiko; Izumi, Kosuke; Suda, Naoto; Ohyama, Kimie; Takahashi, Takao; Moriyama, Keiji; Kosaki, Kenjiro.

In: Genetic Testing and Molecular Biomarkers, Vol. 13, No. 5, 10.2009, p. 623-630.

Research output: Contribution to journalArticle

Hattori, Mitsu ; Torii, Chiharu ; Yagihashi, Tatsuhiko ; Izumi, Kosuke ; Suda, Naoto ; Ohyama, Kimie ; Takahashi, Takao ; Moriyama, Keiji ; Kosaki, Kenjiro. / Diagnosis of Russell-Silver syndrome by the combined bisulfite restriction analysis-denaturing high-performance liquid chromatography assay. In: Genetic Testing and Molecular Biomarkers. 2009 ; Vol. 13, No. 5. pp. 623-630.
@article{8fbdd8b9a33944428961fa72baf9845a,
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.",
author = "Mitsu Hattori and Chiharu Torii and Tatsuhiko Yagihashi and Kosuke Izumi and Naoto Suda and Kimie Ohyama and Takao Takahashi and Keiji Moriyama and Kenjiro Kosaki",
year = "2009",
month = "10",
language = "English",
volume = "13",
pages = "623--630",
journal = "Genetic Testing and Molecular Biomarkers",
issn = "1945-0265",
publisher = "Mary Ann Liebert Inc.",
number = "5",

}

TY - JOUR

T1 - Diagnosis of Russell-Silver syndrome by the combined bisulfite restriction analysis-denaturing high-performance liquid chromatography assay.

AU - Hattori, Mitsu

AU - Torii, Chiharu

AU - Yagihashi, Tatsuhiko

AU - Izumi, Kosuke

AU - Suda, Naoto

AU - Ohyama, Kimie

AU - Takahashi, Takao

AU - Moriyama, Keiji

AU - Kosaki, Kenjiro

PY - 2009/10

Y1 - 2009/10

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=73949161338&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=73949161338&partnerID=8YFLogxK

M3 - Article

C2 - 19814617

AN - SCOPUS:73949161338

VL - 13

SP - 623

EP - 630

JO - Genetic Testing and Molecular Biomarkers

JF - Genetic Testing and Molecular Biomarkers

SN - 1945-0265

IS - 5

ER -