Moore Sam W, Zaahl Monique G
Division of Paediatric Surgery, Faculty of Medicine, University of Stellenbosch, P.O. Box 19063, Tygerberg 7505, South Africa.
J Pediatr Surg. 2007 Jul;42(7):1266-70. doi: 10.1016/j.jpedsurg.2007.02.019.
Animal models have demonstrated the role of genetic influences in anorectal malformations (ARM), although the pathogenetic mechanism remains uncertain. A body of collateral evidence points to possible connection with the endothelin-beta receptor (EDNRB) gene and the endothelin system. This study investigates the EDNRB gene in patients with ARM. Resected surgical specimens of terminal colonic tissue were obtained from 14 children (6 males and 8 females) undergoing surgery for ARM correction with ethical permission. DNA samples were screened for mutations in EDNRB. Polymerase chain reaction amplification of 7 exons of EDNRB was followed by heteroduplex single-strand conformation polymorphism analysis. Heteroduplex single-strand conformation polymorphism variants were validated with automated sequencing techniques on polymerase chain reaction products showing conformational variants in acrylamide gel. All investigated patients with ARM showed mobility shift aberrations and polymorphisms in the EDNRB gene. These included one previously described polymorphism in exon 4 (831G/A) seen in association with Hirschsprung disease and 6 novel polymorphisms identified in exons 1 (178G/A), 2 (552C/T and 561C/T), and 3 (702C/T). No aberrant banding patterns were observed. The exon 1 (178 G/A) variation was identified in 2 (50%) of 4 low lesions compared with 1 (1%) of 84 control samples. The exon 3 (702C/T) single nucleotide polymorphism was present in 3 (60%) of 5 of the supralevator lesions being associated with exon 4 (831G/A). The patient with VATER associations including cardiac and limb anomalies had the 831G/A variation only. Analysis revealed statistically significant differences for the polymorphism 178G/A (P < .01, chi2 with Yates correction = 8.24) compared to controls. Potential disease-related mutations were identified in South African patients with ARM, raising the question of its potential role in the pathogenesis of this condition.
动物模型已证明基因影响在肛门直肠畸形（ARM）中的作用，尽管其发病机制仍不确定。一系列旁证表明可能与内皮素-β受体（EDNRB）基因及内皮素系统有关。本研究对ARM患者的EDNRB基因进行了调查。在获得伦理许可后，从14名接受ARM矫正手术的儿童（6名男性和8名女性）身上获取了终末结肠组织的手术切除标本。对DNA样本进行EDNRB突变筛查。对EDNRB的7个外显子进行聚合酶链反应扩增，随后进行异源双链单链构象多态性分析。对在丙烯酰胺凝胶中显示构象变异的聚合酶链反应产物，用自动测序技术验证异源双链单链构象多态性变异体。所有接受调查的ARM患者在EDNRB基因中均显示迁移率改变异常和多态性。这些包括先前在第4外显子中描述的一种多态性（831G/A），其与先天性巨结肠病相关，以及在第1外显子（178G/A）、第2外显子（552C/T和561C/T）和第3外显子（702C/T）中鉴定出的6种新的多态性。未观察到异常条带模式。在4例低位病变患者中有2例（50%）检测到第1外显子（178G/A）变异，而84例对照样本中只有1例（1%）检测到。第3外显子（702C/T）单核苷酸多态性存在于5例高位病变患者中的3例（60%），且与第4外显子（831G/A）相关。患有包括心脏和肢体异常的VATER综合征的患者仅存在831G/A变异。分析显示，与对照组相比，多态性178G/A具有统计学显著差异（P <.01，经Yates校正的卡方检验= 8.24）。在南非ARM患者中鉴定出潜在的疾病相关突变，这引发了其在该病发病机制中潜在作用的问题。
