Experimental Pathology Division, Institute of Pathology, University of Lausanne, Lausanne, Switzerland.
PLoS One. 2009 Aug 13;4(8):e6631. doi: 10.1371/journal.pone.0006631.
Silver-Russell syndrome (SRS) is a genetically and clinically heterogeneous disease. Although no protein coding gene defects have been reported in SRS patients, approximately 50% of SRS patients carry epimutations (hypomethylation) at the IGF2/H19 imprinting control region 1 (ICR1). Proper methylation at ICR1 is crucial for the imprinted expression of IGF2, a fetal growth factor. CTCFL, a testis-specific protein, has recently been proposed to play a role in the establishment of DNA methylation at the murine equivalent of ICR1. A screen was undertaken to assess whether CTCFL is mutated in SRS patients with hypomethylation, to explore a link between the observed epimutations and a genetic cause of the disease.
METHODOLOGY/PRINCIPAL FINDINGS: DNA was obtained from 36 SRS patients with hypomethylation at ICR1. All CTCFL coding exons were sequenced and analyzed for duplications/deletions using both multiplex ligation-dependent probe amplification, with a custom CTCFL probe set, and genomic qPCR. Novel SNP alleles were analyzed for potential differential splicing in vitro utilizing a splicing assay. Neither mutations of CTCFL nor duplications/deletions were observed. Five novel SNPs were identified and have been submitted to dbSNP. In silico splice prediction suggested one novel SNP, IVS2-66A>C, activated a cryptic splice site, resulting in aberrant splicing and premature termination. In vitro splicing assays did not confirm predicted aberrant splicing.
CONCLUSIONS/SIGNIFICANCE: As no mutations were detected at CTCFL in the patients examined, we conclude that genetic alterations of CTCFL are not responsible for the SRS hypomethylation. We suggest that analysis of other genes involved in the establishment of DNA methylation at imprinted genes, such as DNMT3A and DNMT3L, may provide insight into the genetic cause of hypomethylation in SRS patients.
银-罗素综合征（SRS）是一种遗传和临床异质性疾病。尽管 SRS 患者中未报告有蛋白编码基因突变，但大约 50%的 SRS 患者携带 IGF2/H19 印迹控制区 1（ICR1）的表观遗传突变（低甲基化）。ICR1 处的适当甲基化对于 IGF2（一种胎儿生长因子）的印迹表达至关重要。CTCFL，一种睾丸特异性蛋白，最近被提出在鼠类 ICR1 等效物的 DNA 甲基化建立中发挥作用。进行了一项筛选，以评估 CTCFL 是否在 ICR1 低甲基化的 SRS 患者中发生突变，以探索观察到的表观遗传突变与疾病遗传原因之间的联系。
方法/主要发现：从 36 名 ICR1 低甲基化的 SRS 患者中获得 DNA。使用多重连接依赖性探针扩增，以及定制的 CTCFL 探针集和基因组 qPCR，对所有 CTCFL 编码外显子进行测序和分析，以检测缺失/重复。利用体外剪接测定，分析新 SNP 等位基因是否存在潜在的差异剪接。未观察到 CTCFL 的突变或缺失/重复。鉴定了五个新的 SNP 并已提交给 dbSNP。基于计算机的剪接预测提示一个新的 SNP，IVS2-66A>C，激活了一个隐蔽的剪接位点，导致异常剪接和过早终止。体外剪接测定未证实预测的异常剪接。
结论/意义：由于在检查的患者中未检测到 CTCFL 的突变，因此我们得出结论，CTCFL 的遗传改变不是 SRS 低甲基化的原因。我们建议，对其他参与印迹基因 DNA 甲基化建立的基因，如 DNMT3A 和 DNMT3L 的分析，可能有助于深入了解 SRS 患者低甲基化的遗传原因。
