羅素–西弗氏症與人類印記基因甲基化的關聯性研究

Abstract

羅素–西弗氏症(Russell–Silver syndrome; RSS)是一種十分罕見的遺傳疾病，病患於出生前，具有子宮內生長遲緩(intrauterine growth retardation; IUGR)的現象，因此，被認為與出生後的語言障礙(delayed speech)、發育遲緩(developmental delay)與第五根手指彎斜向內(clinodactyly)有關。病童主要臨床表徵包括面部形狀不正常(dysmorphic facial)與身體不對稱(asymmetry)。目前，導致RSS的病因仍不明瞭，但近來研究顯示可能與部分基因的甲基化程度有關。本實驗針對二十七位具有RSS類似臨床表徵的研究對象，進行人類印記基因(imprinting genes)甲基化程度的檢測，並嘗試與RSS進行相關性分析。 在以Hpa II/ Pst I PCR方法對十七位正常人所進行'基因甲基化程度測定'的前置實驗中，測得MEST、UBE3A、SNRPN、GRB10、H19、IGF2與NESP55等七個人類印記基因的甲基化指標正常參考值(平均值 ± 二個標準差)，分別為0.09 ± 0.16，0.11 ± 0.18，0.25 ± 0.40，0.59 ± 0.26，0.58 ± 0.48，0.77 ± 0.36與0.82 ± 0.24 (UBE3A、SNRPN兩個基因作為對照組)。以此參考值為標準，在二十七位RSS研究對象中，發現有十三位(48%)在GRB10基因的甲基化程度上明顯偏離正常值，其中，九位為基因高度甲基化(hypermethylation)，四位為基因低度甲基化(hypomethylation)。而在MEST、H19、IGF2與NESP55基因上，則分別有四位(15%)、一位(4%)、四位(15%)與四位(15%)的甲基化程度明顯偏離正常值，其中，一位H19、四位IGF2與四位NESP55研究對象皆為基因低度甲基化，而四位MEST研究對象皆為基因高度甲基化。在對照組UBE3A基因，所有二十七位研究對象的甲基化測定值皆落於正常參考值範圍內。就異常甲基化基因數目而言，在二十七位RSS研究對象中，分別有一位(男性)、六位(五男一女)、十位(七男三女)各有四個、二個及一個基因偏離甲基化正常值，然而有十位(七男三女)研究對象在五個檢測的基因中都未發現任何異常。 在RSS研究對象臨床表徵檢視方面，本次評估除了包括子宮內生長遲緩(IUGR)、生長遲滯(failure to thrive; FTT)、典型臉部特徵(不對稱倒三角形臉型)(typical face；TFA)、身體不對稱(包括四肢)、第五手指彎斜向內(clinodactyly；CLIN)之外，還包括生殖器異常(genital abormality；GAB)、餵食困難(feeding difficulty；FDIF)、語言學習延遲(delayed speech；DSP)、咖啡斑(Café’au lait spot；CLA)、發育遲緩(developmental delay；DDE)與其他項異常表徵(others anomaly；OAM)。 在具有臨床表徵資料的十九位病患中，有十七位除了RSS臨床診斷表徵之要項外還伴隨有其他方面的異常表徵，其次最多的臨床表徵依序為，十七位具生長遲滯，十六位具有子宮內生長遲緩，十四位具有典型臉部特徵，十五位具有身體不對稱(包括四肢)，十四位具第五根手指彎斜向內，各有九位具餵食困難與發育遲緩，四位有生殖器異常，四位具有語言學習延遲，但所有研究對象皆不具咖啡斑。 檢視六位(編號2、5、8、9、16、26)具有全部或部份臨床資料的研究對象，其在五個基因檢測中均無異常甲基化表現，可發現有67% (4/6)有子宮內生長遲緩，83% (5/6)有生長遲滯，典型臉部特徵佔50% (3/6)，身體不對稱(包括四肢)佔100% (6/6)，第五手指彎斜向內佔80% (4/5)，餵食困難佔100% (1/1)，發育遲緩佔100% (2/2)與其他異常佔100% (4/4)。除了咖啡斑、語言學習延遲與生殖器異常之外(因為無臨床資料)，幾乎所有臨床病徵皆存在於這六位病患，由此顯示，除受檢測印記基因外，應仍有其它基因參與RSS症狀的表現。再例如編號2 號，其五個基因皆正常，但所得的性狀表現總分卻高達8分，更可暗示存在某些未列於本次研究的檢測基因。 GRB10轉譯adapter protein，因此GRB10的異常甲基化將直接影響tyrosine kinases的細胞表面接受體的形狀與功能，造成分子訊息傳遞異常，另一方面也影響到細胞分裂、胚胎細胞的複製、繁殖與凋零。在GRB10高度甲基化且具有臨床資料的六位研究對象中，全部都具有子宮內生長遲緩現象，但儘管如此，研究對象中也有許多人的GRB10雖有正常的甲基化表現，但也具有子宮內生長遲緩的性狀。因此在本研究中，GRB10高度甲基化與生長遲滯、典型臉部特徵、身體不對稱(包括四肢)、第五手指彎斜向內、生殖器異常、餵食困難、語言學習延遲、咖啡斑、發育遲緩等性狀皆無直接相關聯性。 綜合以上結果顯示，基因甲基化的程度可能與RSS的病因有關但尚無法証實，且與RSS臨床表徵相關的基因應該不只本研究中所涉及之基因。

Russell-Silver syndrome (RSS) is a rare genetic disorder characterized by growth delays before birth (intrauterine growth retardation; IUGR) and is associated with postnatal delayed speech, developmental delay and clinodactyly. Affected children exhibit unusual characteristic facial features and may have asymmetry or overgrowth of one side of the body. The aetiology of RSS is still unclear but it has been correlated with the degrees of gene methylation. To get more insights into the roles of gene methylation in RSS, we measure and analyze the degrees of methylations in a number of human imprinting genes for 27 patients with suspected RSS. The level of DNA methylation is determined by Hpa II/ PstⅠPCR. We first examined the methylation levels of MEST, UBE3A, SNRPN, GRB10, H19, IGF2 and NESP55 for 17 normal controls in order to set up the normal reference ranges. The normal methylation indices (mean±2 SD) for the 7 genes mentioned above were estimated as 0.09±0.16, 0.11±0.18, 0.25±0.40, 0.59±0.26, 0.58±0.48, 0.77±0.36 and 0.82±0.24, respectively. Among the 27 RSS patients, there were 13 patients (48%) with aberrant methylation levels in GRB10 (9 hypermethylations and 4 hypomethylations), and 4 patients (15%), one patients (4%), 4 patients (15%), 4 patients (15%) with aberrant methylation levels in MEST, H19, IGF2 and NESP55, separately. All of the one patient aberrant in H19, 4 patients aberrant in IGF2,and 4 patients aberrant in NESP55 were hypomethylated, while all 4 patients aberrant in MEST were hypermethylated. In addition, all the 27 RSS patients had normal methylation levels in UBE3A. As a whole, there were 1 male patient with aberrant methylation levels in 4 genes, 6 patients (5 males and 1 female) with aberrant levels in 2 genes, 10 patients (7 males and 3 females) with aberrant levels in 1 genes and 10 patients (7 males and 3 females) without any aberration in all of the 6 genes (not inclusive of SNRPN). The clinical findings complied with a very broad definition of RSS inclusive of IUGR, FTT (Failure to Thrive), typical face, asymmetry and clinodactyly. Notable additional findings include genital abnormalities, feeding difficulties, delayed speech, caf’e au lait spots, developmental delay and other anomaly. In 19 examined patients (not inclusive of the other 8 patients on account of unavailable clinical data), other anomaly were seen in 17 patients, FTT in 17, IUGR in 15, typical face in 14, asymmetry in 15, clinodactyly in 14, feeding difficulty in 9, developmental delay in 9, genital abnormality in 4, and delayed speech in 4. None of the patients had Café au lait spots. Among the 6 patients (no. 2, 5, 8, 9, 16, 26) who have no abberant methylation in any examined genes but with clinical data, there are 67% (4/6) with IUGR, 83% (5/6) with FTT, 50% (3/6) with typical face, 100% (6/6) with Asymmetry, 80% (4/5) with clinodactyly, 100% (1/1) with feed difficulty, 0% (0/1) with delay speech, 100% (2/2) with developmental delay, and 100% (4/4) with other anomaly. This finding suggests that there are other genes involved in RSS. For example, patient no. 2 has 8 scores, but all the 6 genes are normal. GRB10 known to interact with a number of receptor tyrosine kinases and signalling molecules belongs to a small family of adapter proteins in cytoplasma. GRB10 involves in signal transduction in developing embryos and regulates cell proliferation and apoptosis. Although 6 patients (exclude 3 patients without clinical syndrome data) with high methylation in GRB10 are all with IUGR, we can not jump to the conclusion that GRB10 is directly related to IUGR because there are patients with normal GRB10 who also have IUGR. Statistically, GRB10 hypermethylation are not related to FTT, TFA, ASY, CLIN, GAB, FDIF, DSP, CLA, DDE. In sum, our study indicated that the level of methylation in GRB10 might be related to RSS, but current data still don’t have enough power to prove it. There should be other genes not studied in the current study that are involved in the etiology of RSS.