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首页> 外文期刊>Genetic testing and molecular biomarkers >Diagnosis of Russell-Silver Syndrome by the Combined Bisulfite Restriction Analysis–Denaturing High-Performance Liquid Chromatography Assay
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Diagnosis of Russell-Silver Syndrome by the Combined Bisulfite Restriction Analysis–Denaturing High-Performance Liquid Chromatography Assay

机译：艾综合征的诊断亚硫酸氢结合限制Analysis-Denaturing高效液相色谱法测定

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摘要

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 (Hinfl) 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-DHPLCbased screening system for RSS.
机译：艾综合症(RSS)的特点是产前及产后生长迟缓,三角相,第五根手指指弯曲。一半的患者RSShypomethylation差异甲基化11 p15.5地区段H19基因的染色体上。因此,定量的甲基化分析这个区域可能是有用的分子RSS的诊断。基于亚硫酸氢克隆测序时间和劳动消耗和不适合临床使用。调查可能的定量方法确定段H19 hypomethylation RSS患者限制使用亚硫酸氢结合分析(眼镜蛇)变性高效液相色谱法(DHPLC)测定;试验、聚合酶链反应产品放大段H19差异甲基化地区bisulfite-treated基因组DNA使用眼镜蛇化验分析,检测methylation-dependent序列差异使用限制bisulfite-treated基因组DNA酶分析。限制性内切酶(Hinfl)会降低甲基化,但不是unmethylated模板。剪切和毛边的之间的摩尔比片段用DHPLC测量,建设使校准曲线原来的基因组DNA甲基化指数是估计的。使用COBRA-DHPLC试验证明三个RSS患者低甲基化指数约为10%。使用获得的甲基化指标COBRA-DHPLC亚硫酸氢和常规克隆测序揭示了一个很好的intermethod协议。健壮的、快速的和具有成本效益的RSS COBRA-DHPLCbased筛选系统。

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