Eggermann T, Schönherr N, Eggermann K, Buiting K, Ranke M B, Wollmann H A, Binder G
Institute of Human Genetics, RWTH Aachen, Aachen, Germany.
Clin Genet. 2008 Jan;73(1):79-84. doi: 10.1111/j.1399-0004.2007.00930.x. Epub 2007 Dec 7.
Silver-Russell syndrome (SRS) describes a malformation syndrome with severe intrauterine and postnatal growth retardation. Currently, two major (epi)mutations have been described: while approximately 10% of patients carry a maternal uniparental disomy of chromosome 7 (UPD7), 35-60% show a hypomethylation at the H19 differentially methylated regions (DMRs) in 11p15. Until recently, a Southern-blot based test was routinely used to identify epimutation carriers. Nevertheless, this test was time consuming and hampered by the huge amount of genomic DNA needed. With the methylation-specific multiplex ligation-dependent probe amplification assay (MLPA) for SRS, a PCR-based test is now available, allowing the analysis also of small amounts of DNA. Probes in this assay hybridize to the H19 DMRs but do not cover the genomic target of the Southern-blot probe. We now screened 72 patients with SRS by MLPA. Hypomethylation of the H19 DMRs was confirmed in all patients analyzed by Southern blot. In addition, we identified six individuals with hypomethylation of the H19 DMR who had previously normal blot results. This discrepancy can be explained by the observed generally lower degree of demethylation in this group, possibly not detectable by the less sensitive Southern-blot method but also with a varying degree of methylation at different DMRs in the same individual. Apart from hypomethylation in the H19 DMR, we observed a slight demethylation for one of the IGF2 probes. The total detection rate of 11p15 hypomethylation is now increased to >38%. Considering maternal UPD7 and chromosomal aberrations, (epi)genetic alterations now account for more than 50% of SRS patients. In summary, MLPA represents an easy, low cost and reliable system in the molecular diagnostics of SRS.
Silver-Russell综合征（SRS）是一种具有严重宫内和出生后生长迟缓的畸形综合征。目前，已发现两种主要的（表观）突变：约10%的患者携带7号染色体的母源单亲二倍体（UPD7），35 - 60%的患者在11p15的H19差异甲基化区域（DMR）表现为低甲基化。直到最近，基于Southern印迹的检测方法一直被常规用于鉴定表观突变携带者。然而，该检测方法耗时且需要大量基因组DNA，受到了限制。随着用于SRS的甲基化特异性多重连接依赖探针扩增分析（MLPA）的出现，现在有了一种基于PCR的检测方法，也能够分析少量DNA。该分析中的探针与H19 DMR杂交，但不覆盖Southern印迹探针的基因组靶点。我们现在通过MLPA对72例SRS患者进行了筛查。通过Southern印迹分析的所有患者中均证实了H19 DMR的低甲基化。此外，我们还鉴定出6例H19 DMR低甲基化但之前印迹结果正常的个体。这种差异可以通过该组中观察到的总体较低的去甲基化程度来解释，可能较不敏感的Southern印迹方法无法检测到，而且同一个体的不同DMR处甲基化程度也有所不同。除了H19 DMR的低甲基化外，我们还观察到胰岛素样生长因子2（IGF2）探针之一有轻微去甲基化。11p15低甲基化的总检出率现在提高到了>38%。考虑到母源UPD7和染色体畸变，（表观）遗传改变现在占SRS患者的比例超过50%。总之，MLPA在SRS的分子诊断中是一种简便、低成本且可靠的系统。
