Hirschsprung’s Disease and the related genes in Taiwan

目的 先天性巨大結腸症為一腸道神經發育疾病。患者在腸道黏膜下及肌肉層無神經節細胞，使腸道無法擴張而造成功能性的阻塞。此疾病可影響不同長度的腸道，且在亞洲人，男性有較高的發生率，同時也有家族傾向，顯示其與基因相關。此外，基因型對應到多樣的表現型也暗示著此疾病可能牽涉多重基因的調控。越來越多的基因被發現相關，如RET , GDNF, EDNRB, SOX10等。其中RET被認為相關性最高，同時RET中和巨大結腸症相關的SNP也在不同的文獻中提及在亞洲人比率較高。更明確的基因變異與此疾病的關係需要被了解。 方法 仔細瀏覽文獻之後，有31個基因懷疑與先天性巨大結腸症相關，且部分突變點位於內含子的區段。我們針對此31個基因設計了次世代基因定序所需的晶片。其中除了NRG3只做外顯子，其餘的30個基因均為全基因定序。 凡經病理報告確診之先天性巨大結腸症患者及其父母與兄弟姊妹均納入為受試者。但若有伴隨其他症候群 (如唐氏症) 之患者，則予以排除。預計收集30個家庭，150名受試者。在向受試者解釋並取得同意後，採集受試者20毫升之周邊血，(若受試者小於一歲，採血量可降為10cc)，並從中萃取DNA。之後將病患的DNA利用次世代基因定序找尋是否有之前文獻所提及之三十一個可能的致病基因。在找到可能致病的基因變異點後，再進一步以Sanger’s 定序確認次世代定序所看到之變異點。除了病人本身之外，其家族中沒有患病之家人也會針對該變異點做Sanger’s 定序，以便確認該變異點為遺傳而來，或是由新的突變形成。並尋找各基因型與表現型之間的關係。 研究結果 目前收案的家族已有26個家族，一共29位病患，95位受試者。基於成本的考量，每15個家族會進行一輪的次世代基因定序。 在目前的收案分布中，26個家庭共29位病患。有6人為家族性，占了20.69%。然而我們收案的病人當中，短型巨大結腸症的病人只有14位，占了48.28%；而長型的患者有15位，占了51.72%，遠高於一般只有20%左右的比率；且其中全大腸沒有神經節的患者高達11位，同時有一位是跳躍式神經節缺失(skip type)，或許是因為病情較嚴重的患者其父母較有意願參與試驗而導致收案的偏差。男女比則為21比8，也較一般3~4：1來的略低。過去的研究認為病灶的腸段越長，其家族性的傾向越明顯，而男女比的差異也越接近1:1。這也比較可以解釋我們收案的狀況。 目前為止的結果我們在23個偶發性的病人身上，有19人找到此31個基因外顯子上的變異點，約占了82.61%。除了RET約占了所有變異的50%之外，剩下一半分散在各個基因上。相較於過去一個病患一次只能做少數基因分析的時代，明顯提高了基因變異的偵測率。這也是我們希望利用次世代基因定序能跟過往產生不一樣的結果。 在RET有變異的病患身上，有三人發生了frame shift的變異，分別位於exon7及exon11，且此三人均為長型甚至是全大腸型的先天性巨大結腸症。此外，有1人為NRG1在exon2產生stop codon，1人在L1CAM產生frame shift的變異，也同樣發生了長型的先天性巨大結腸症。而相對於短型的患者，只有一人有ENDRB exon 7 上有stop codon的變異點。 結論 次世代基因定序是一個研究多基因疾病的強大工具。這是一個病人數量很少的研究，但可以發現在RET，L1CAM，NRG1若產生會嚴重影響功能的變異，如frameshift或是stop coden，雖然是異型合子，都足以讓病患產生長型的先天性巨大結腸症。此外在台灣地區的病人，除了RET之外，SEMA3C上V337M的變異點有者異常高的比例，是否是人種差異或是因案例數太少所產生的誤差需進一步分析。可惜病人的數量太少，而可能致病的基因相對太多，以至於無法明確地找到基因型跟表現型之間的關係，後續需要收納更多的病人或許可以讓我們對這個疾病有更深的認識。Background Hirschsprung’s disease (HSCR) is a congenital disease when the enteric ganglion cells were absent in the variable lengths of gastrointestinal tract. Several genes have been found to be involved in the development of HSCR, such as, RET, GDNF, EDNRB, SOX10, etc. Although RET is considered as the main gene related to HSCR, the phenotypic variability and incomplete penetrance observed in HSCR might suggest the involvement of modifier genes. Thus, a more thorough analysis of these HSCR-related genes might help to understand the HSCR genetic background that remains shrouded in mystery. Patients and Methods Patients proved to be HSCR by pathology were included after obtaining their written consent under the ethical guidance of the Institutional Review Board of Chang Gung Memorial Hospital, so as their parents, siblings, and/or off-springs. Total 30 families and 150 participants were included. Patients with syndromic presentations (such as Down syndrome) were disqualified. Peripheral blood is drawn for 20cc in all participants if possible, for children younger than 1-year- old, the amount is 10cc. After reviewing previous articles thoroughly, we identified 31 genes related to HSCR. A next-generation sequence (NGS) panel was designed accordingly. The DNA samples of all the patients were sent for NGS to detect possible genetic mutations. Further Sanger’s sequencing was performed to verify the mutations detected by NGS in the patients as well as their family members to characterize the mutation. Results There are 29 patients in 26 families were enrolled for NGS. Mutation of 13 different genes were identified in 23 patients. The detection rate of a possibly pathogenic mutation is 82.61%, which was much higher than previous reported. Frame-shift in RET, L1CAM and a stop codon in NRG1 were only found in long segment HSCR patients, indicating a clear-cut functional loss of the 3 HSCR-related genes and a severe form of the disease. Besides, there was an extraordinarily high frequency of SEMA3C mutations in our patients. For most of the patients, the relation between phenotype and the genotype is unclear, and our case number is too small to made solid conclusion in penetrance. Conclusion NGS is a powerful tool to work up for a multigenic disease, such as HSCR. An obvious loss of function in RET, L1CAM and NRG1 contributed to long segment HSCR, so such mutations of these genes might be employed for genetic counseling of HSCR family. Keywords: Next generation sequencing，Congenital megacolon, Hirschsprung’s disease, multigenic diseas