台灣地區先天性巨腸症與其相關腸道蠕動異常遺傳因子研究

Study of the genetic factors of Hirschsprung disease and motility disturbances of the gut in Taiwan

巫堂鎥(Trang-Tiau Wu)

指導教授 ： 李娟

中山醫學大學/醫學院/醫學研究所/博士(2006年)

https://doi.org/10.6834/CSMU.2006.00013

摘要

先天性巨腸症 (HSCR) 或稱為先天性腸管無神經節細胞症係因結腸遠端的腸壁內神經節細胞缺少，而造成腸道功能的阻塞現象，也是新生兒腸阻塞最常見的原因之一。先天性巨腸症的基因變異主要來自於二種不同的receptor-ligand 機制，包括RET-GDNF-NTN和 EDNRB-EDN-3-ECE1及SOX10基因的發現。RET (receptor tyrosine kinase) 和EDNRB (endothelin receptor B) 基因已經被報導與先天性巨腸症有相關性。我們在中台灣地區收集了55位典型先天性巨腸症病人的DNA樣本及這些病患中部份雙親的DNA，針對RET 與EDNRB基因進行聚合酵素連鎖反應後定序DNA。在55位中發現有兩位同卵雙生兄弟在RET基因的exon 19 codon 1062的位置 A 轉換為G (Y1062C)，這位置已被報導與信息傳遞有相關性。同時在病人與正常族群中在exon 2,7, 11及13皆有發現多型性基因(SNPs)，而exon 15在正常族群中也有多型性的現象。在這些位置病人與正常族群的對偶基因分布在統計學上有顯著差異。在exon 2和13中基因型的分布情形病人有較多的比率為AA/GG，與正常族群有較多的比率為AG/GT有顯著的不同。又發現在exon2,7及13這三個多型性位置對偶基因的傳遞有不均的現象。此實驗是目前台灣針對先天性巨腸症病患基因研究的首例。我們更進一步在小兒外科收集具有嚴重腸道蠕動功能異常之病患如無肛症或是無肛併有假性巨腸症以及小兒腸道假性腸阻塞病人作為典型先天性巨腸症之對照組。在上述RET基因的五個多型性位置對偶基因的分布在無肛併有假性巨腸症與正常族群統計上並無明顯不同。在假性腸阻塞病人與典型先天性巨腸症病人間對偶基因分布在這幾個多型性位置有顯著差異。有趣的是在RET基因中exon2及13等多型性位置對偶基因分布假性腸阻塞病人與正常族群統計上也有顯著差異。在c135 (exon 2) 中對偶基因G及c2307 (exon 13) 對偶基因T 由典型先天性巨腸症病人至正常族群再到假性腸阻塞病人有逐漸遞增的現象。在這些多型性位置無肛併有假性巨腸症與正常族群間基因型的分布非常相似反映出兩者間對偶基因的分布也相似。從另一方面，假性腸阻塞病人與典型先天性巨腸症病人及正常族群基因型的分布在這五個多型性位置卻有顯著區別，顯示假性腸阻塞病人其對偶基因分布在這些病患中具有獨特性。更進一步我們也發現在exon 2 及13對偶基因的傳遞不論是假性腸阻塞病人或是無肛併有假性巨腸症皆無不均的現象。結果明顯顯示在RET 基因中這五個多型性位置為台灣典型先天性巨腸症所特見。此外我們也利用組織切片染色和組織免疫染色法，分析出先天性巨腸症病人受侵犯的結腸段缺乏神經節細胞，在無肛症併有假性巨腸的病人異常組織部位發現有神經節細胞，但較正常組織部位少。最後更進一步收集先天性巨腸症與無肛症併有假性巨腸病人，經手術切除病變部位的組織，利用二維電泳分析蛋白在正常與病變結腸組織中的差異表現，並且以MALDI-TOF 質譜儀鑑定蛋白。分析發現HSP27在先天性巨腸症部份病人與無肛症併有假性巨腸病人的病變部位組織與正常組織表現量有差異。但HSP27的表現量我們並未得到一致的結果。因此HSP 是否與先天性巨腸症的病因相關，需進一步探討。

關鍵字

先天性巨腸症 ； 無肛症併有假性巨腸 ； 假性腸阻塞

並列摘要

Hirschsprung disease (HSCR), or congenital intestinal aganglionosis, is a relatively common disorder characterized by the absence of ganglion cells in the nerve plexuses of the lower digestive tract, resulting in intestinal obstruction in neonate. Mutations in genes of the RET receptor tyrosine kinase and endothelin receptor B (EDNRB) signaling pathways have been shown to be associated in HSCR patients. In this study we collected genomic DNA samples from 55 HSCR patients in central Taiwan and analyzed the coding regions of the RET and EDNRB gene by PCR amplification and DNA sequencing. We detected an A to G transition in two patients (identical twin brothers) at the end of RET exon 19 at codon 1062 (Y1062C), a reported critical site for the signaling pathways. No sequence alteration was detected in other patients. Single nucleotide polymorphisms (SNP) in exon 2, 7, 11, 13 and 15 of RET, and exon 4 of EDNRB in the HSCR patients or controls were detected. The differences between patients and controls in allele distribution of the five RET polymorphic sites are statistically significant. The most frequent genotype encompassing the exon 2 and 13 SNP (the polymorphic sites with the highest percentage of heterozygotes) was AA/GG in patients, which was different from the AG/GT in the normal controls. Transmission disequilibrium was observed in exon 2, 7 and 13, indicating non-random association of the susceptibility alleles with the disease in the patients. This study represents the first comprehensive genetic analysis of HSCR disease in Taiwan. We also further collected patients encountered in department of pediatric surgery with severe gastrointestinal disorders such as anorectal malformations or anorectal malformations with co-existing syndromic Hirschsprung disease (syndromic HSCR) and pediatric intestinal pseudo-obstruction (IPO) as the “control” group for the typical HSCR patients. The allele distributions of all five RET SNPs of the syndromic HSCR patients do not statistically deviate from those of the normal population. The allele distributions of these RET SNPs of the IPO patients are all significantly different from those of the HSCR patients. It is interesting that for these IPO patients, allele distribution at the polymorphic sites of exon 2 and 13 are also statistically different from those of normal control population. The frequencies of the G allele of c135 (at exon 2) as well as the T allele of c2307 (exon 13) increased from HSCR patient to normal population to IPO patients. The genotype distribution of the syndromic HSCR patients were very close to that of the normal control population reflecting the similarity of allele distributions in these SNPs. On the other hand, the genotype distribution of the IPO patients is distinct from that of HSCR or normal population indicating the unique allele distribution in these patients. Furthermore, no transmission preference was detected in the alleles of exon 2 and 13 in either IPO or syndromic HSCR parents by the transmission disequilibrium test. The results in this study strengthen the association of specific RET SNP alleles with typical HSCR in Taiwan. We also performed immunohistochemical staining on tissues from related patients. No ganglion cells were detected in aganglionic colonic segments of HSCR patients. We detected fewer ganglion cells in the abnormal tissues than normal tissues from syndromic HSCR. At last we took the “proteomic” approach to 'differential display' proteins in the abnormal and normal colon tissues we collected by two dimensional gel electrophoresis and then MALDI-TOF mass spectrometry. We found the heat shock protein 27 (HSP27) differentially expressed between the normal and abnormal tissues in most of our HSCR and syndromic HSCR patients. Nevertheless, we could not obtain consistent results in all patients for the differential HSP27 expression, whether HSP involves in the etiology of HSCR requires more investigation.

並列關鍵字

Hirschsprung disease ； anorectal malformations with co-existing syndromic Hirschsprung disease ； pediatric intestinal pseudo-obstruction

參考文獻

Aldridge, R.T., and P.E. Campbell. 1968. Ganglion cell distribution in the normal rectum and anal canal. A basis for the diagnosis of Hirschsprung's disease by anorectal biopsy. J Pediatr Surg. 3:475-90.

Angrist, M., S. Bolk, M. Halushka, P.A. Lapchak, and A. Chakravarti. 1996. Germline mutations in glial cell line-derived neurotrophic factor (GDNF) and RET in a Hirschsprung disease patient. Nat Genet. 14:341-4.

Angrist, M., S. Bolk, B. Thiel, E.G. Puffenberger, R.M. Hofstra, C.H. Buys, D.T. Cass, and A. Chakravarti. 1995. Mutation analysis of the RET receptor tyrosine kinase in Hirschsprung disease. Hum Mol Genet. 4:821-30.

Angrist, M., E. Kauffman, S.A. Slaugenhaupt, T.C. Matise, E.G. Puffenberger, S.S. Washington, A. Lipson, D.T. Cass, T. Reyna, D.E. Weeks, and et al. 1993. A gene for Hirschsprung disease (megacolon) in the pericentromeric region of human chromosome 10. Nat Genet. 4:351-6.

Attie, T., A. Pelet, P. Edery, C. Eng, L.M. Mulligan, J. Amiel, L. Boutrand, C. Beldjord, C. Nihoul-Fekete, A. Munnich, and et al. 1995. Diversity of RET proto-oncogene mutations in familial and sporadic Hirschsprung disease. Hum Mol Genet. 4:1381-6.