NRG1 表达和甲基化对多因素 Hirschsprung 病的影响。
The impact of NRG1 expressions and methylation on multifactorial Hirschsprung disease.
机构信息
Pediatric Surgery Division, Department of Surgery/Genetics Working Group/Translational Research Unit, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito Hospital, 55281, Yogyakarta, Indonesia.
Department of Child Health/Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/UGM Academic Hospital, Yogyakarta, 55291, Indonesia.
出版信息
BMC Pediatr. 2022 Apr 20;22(1):216. doi: 10.1186/s12887-022-03287-1.
BACKGROUND
Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the lack of ganglion cells in the intestines. A current study showed that the NRG1 rare variant frequency in Indonesian patients with HSCR is only 0.9%. Here, we investigated the impact of NRG1 expressions and methylation patterns on the pathogenesis of HSCR.
METHODS
This cross-sectional study determined NRG1 type I (HRGα, HRGβ1, HRGβ2, HRGβ3, HRGγ, and NDF43 isoforms), type II and type III expressions in both ganglionic and aganglionic colons of 20 patients with HSCR and 10 control colons by real-time polymerase chain reaction (qPCR). For methylation studies, we treated the extracted gDNA from 16 HSCR patients' and 17 control colons with sodium bisulfate and analyzed the methylation pattern of NRG1 exon 1 with methylation-specific PCR. The samples were collected and analyzed at our institution from December 2018 to December 2020.
RESULTS
NRG1 types I, II and III expressions were upregulated (17.2-, 3.2-, and 7.2-fold, respectively) in the ganglionic colons compared with control colons (type I: 13.32 ± 1.65 vs. 17.42 ± 1.51, p < 0.01; type II: 13.73 ± 2.02 vs. 16.29 ± 2.19, p < 0.01; type III: 13.47 ± 3.01 vs. 16.32 ± 2.58, p = 0.03; respectively); while only type I (7.7-fold) and HRGβ1/HRGβ2 (3.3-fold) isoforms were significantly upregulated in the aganglionic colons compared to the controls (type I: 14.47 ± 1.66 vs. 17.42 ± 1.51, p < 0.01; HRGβ1/HRGβ2: 13.62 ± 3.42 vs 14.75 ± 1.26, p = 0.01). Moreover, the frequency of partially methylated NRG1 was higher in the ganglionic (81%) and aganglionic (75%) colons than in the controls (59%).
CONCLUSIONS
Our study provides further insights into the aberrant NRG1 expression in the colons of patients with HSCR, both ganglionic and aganglionic bowel, which might contribute to the development of HSCR, particularly in Indonesia. Furthermore, these aberrant NRG1 expressions might be associated with its methylation pattern.
背景
先天性巨结肠（HSCR）是一种复杂的遗传疾病，其特征是肠道内缺乏神经节细胞。目前的研究表明，印度尼西亚 HSCR 患者中 NRG1 罕见变异的频率仅为 0.9%。在这里，我们研究了 NRG1 表达和甲基化模式对 HSCR 发病机制的影响。
方法
本横断面研究通过实时聚合酶链反应（qPCR）确定了 20 例 HSCR 患者和 10 例对照结肠的神经调节蛋白 1 型（HRGα、HRGβ1、HRGβ2、HRGβ3、HRGγ 和 NDF43 异构体）、II 型和 III 型在有神经节和无神经节结肠中的表达。为了进行甲基化研究，我们用亚硫酸氢钠处理从 16 例 HSCR 患者和 17 例对照结肠中提取的 gDNA，并通过甲基化特异性 PCR 分析 NRG1 外显子 1 的甲基化模式。样本于 2018 年 12 月至 2020 年 12 月在我们机构采集和分析。
结果
与对照组相比，神经节结肠中 NRG1 类型 I、II 和 III 的表达分别上调（分别上调 17.2、3.2 和 7.2 倍）（I 型：13.32±1.65 vs. 17.42±1.51，p<0.01；II 型：13.73±2.02 vs. 16.29±2.19，p<0.01；III 型：13.47±3.01 vs. 16.32±2.58，p=0.03；分别）；而仅 I 型（7.7 倍）和 HRGβ1/HRGβ2（3.3 倍）异构体在无神经节结肠中与对照组相比显著上调（I 型：14.47±1.66 vs. 17.42±1.51，p<0.01；HRGβ1/HRGβ2：13.62±3.42 vs 14.75±1.26，p=0.01）。此外，在神经节（81%）和无神经节（75%）结肠中，部分甲基化 NRG1 的频率高于对照组（59%）。
结论
我们的研究进一步深入了解了 HSCR 患者结肠中神经调节蛋白 1 的异常表达，包括神经节和无神经节肠段，这可能有助于 HSCR 的发生，特别是在印度尼西亚。此外，这些异常的 NRG1 表达可能与其甲基化模式有关。
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