Unidad de Gestión Clínica de Genética, Reproducción y Medicina Fetal, Instituto de Biomedicina de Sevilla (IBIS), Hospitales Universitarios Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain.
BMC Med Genet. 2010 May 11;11:71. doi: 10.1186/1471-2350-11-71.
Hirschsprung disease is characterized by the absence of intramural ganglion cells in the enteric plexuses, due to a fail during enteric nervous system formation. Hirschsprung has a complex genetic aetiology and mutations in several genes have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. In this study, we have looked for CNVs in some of the genes related to Hirschsprung (EDNRB, GFRA1, NRTN and PHOX2B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach.
CNVs screening was performed in 208 HSCR patients using a self-designed set of MLPA probes, covering the coding region of those genes.
A deletion comprising the first 4 exons in GFRA1 gene was detected in 2 sporadic HSCR patients and in silico approaches have shown that the critical translation initiation signal in the mutant gene was abolished. In this study, we have been able to validate the reliability of this technique for CNVs screening in HSCR.
The implemented MLPA based technique presented here allows CNV analysis of genes involved in HSCR that have not been not previously evaluated. Our results indicate that CNVs could be implicated in the pathogenesis of HSCR, although they seem to be an uncommon molecular cause of HSCR.
先天性巨结肠症的特征是肠神经系统形成失败，导致肠丛内缺少神经节细胞。先天性巨结肠具有复杂的遗传病因，已有多个基因的突变与该疾病相关。这些基因中的错义/无义突变明显占主导地位，而拷贝数变异（CNVs）则很少被描述，这可能是由于通常用于突变分析的常规技术的局限性所致。在这项研究中，我们使用多重连接依赖性探针扩增（MLPA）方法，针对与先天性巨结肠相关的一些基因（EDNRB、GFRA1、NRTN 和 PHOX2B）寻找 CNVs。
使用我们自行设计的 MLPA 探针集，对 208 例先天性巨结肠症患者进行了 CNV 筛查，涵盖了这些基因的编码区。
在 2 例散发性先天性巨结肠症患者中检测到 GFRA1 基因的第 1 至 4 个外显子缺失，计算机模拟表明突变基因中的关键翻译起始信号被破坏。在本研究中，我们已经能够验证该技术用于先天性巨结肠症 CNV 筛查的可靠性。
本研究中实施的基于 MLPA 的技术允许对先前未评估的先天性巨结肠症相关基因进行 CNV 分析。我们的研究结果表明，CNVs 可能与先天性巨结肠症的发病机制有关，尽管它们似乎是先天性巨结肠症的罕见分子病因。
