https://scholars.lib.ntu.edu.tw/handle/123456789/144766
標題: | 對蕾特氏症候群及類蕾特氏症候群患兒進行臨床以及MECP2基因的突變分析 Mutation Analysis of the MECP2 Gene in Patients with Rett Syndrome and Rett-like Features |
作者: | 蔡文心 Tsai, Wen-Hsin |
關鍵字: | 蕾特氏症候群;MECP2基因;MLPA;突變分析;Rett syndrome;MECP2;Mutation analysis | 公開日期: | 2009 | 摘要: | 一、研究目的:台灣地區典型或非典型蕾特氏症候群患者的基因診斷率向來偏低。我們首先藉由臨床診斷分群,再運用兩階段的篩檢方式,先進行MECP2基因編碼區直接定序,再加上MLPA(Multiplex Ligation-dependent Probe Amplification)的方式進行大片斷缺失突變分析,期能提高台灣地區有關蕾特氏症候群的基因突變篩檢率。我們針對大台北地區典型或非典型蕾特氏症候群以及類蕾特氏症候群患兒轉介至台大醫院做MECP2基因突變篩檢者,分析其MECP2基因突變的結果。我們希望建立台灣本土關於典型或非典型蕾特氏症候群或類蕾特氏症候群患兒MECP2基因突變型的資料庫。、研究背景:蕾特氏症候群是一種罕見的進行性神經系統退化及發展遲緩的疾病,通常好發於小女孩。蕾特氏症候群罹患率約1/10,000-15,000女孩左右。西元1999年蕾特氏症的致病基因被發現是位於染色體Xq28的MECP2基因突變所導致。若男性發生突變,則會死產或在出生後兩年內死亡。約有80~90%的典型蕾特氏症候群患兒在MECP2基因的編碼區(coding region)發生點突變,然而剩下的10~20%的患兒可能屬於基因的缺失突變,突變在其他基因的部分,或基因尚未確定。一些研究結果顯示有一部份MECP2基因的編碼區沒有點突變的病人,事實上有大片段的缺失突變。因此,我們決定使用兩階段的篩檢方式,除了直接定序外再加上MLPA進行MECP2基因大片斷缺失突變分析,期能提高台灣地區關於蕾特氏症候群患者MECP2基因突變的檢出率。、研究方法:我們收集自2003年12月至2008年11月期間由大台北地區各級醫院因臨床症狀疑似典型、非典型、或類蕾特氏症候群患兒而轉介至台大醫院來做MECP2基因突變分析的個案。研究個案依據Hagberg et al於西元2002發表的典型蕾特氏症候群診斷標準修改版,以及變異型蕾特氏症候群診斷標準來分類。若符合典型或非典型蕾特氏症候群診斷或類蕾特氏症候群患兒,則直接做MECP2基因直接定序來偵測其中基因編碼區是否存在突變。而未檢出突變者,再利用MLPA來偵測是否存在大片段的缺失突變。另一群病人未能符合典型或非典型蕾特氏症候群診斷標準,則歸類於未明原因身心發展遲緩患者。我們假設當中部分個案有可能疑似MECP2基因相關疾病患者,因此藉由MECP2-MLPA的試驗去探討此族群中偵測出MECP2雙倍數(duplication)突變的可能性。、研究結果:總共收案97位,其中有23符合典型蕾特氏症候群,37位為非典型或類蕾特氏症候群;其他有37位為歸類為未明原因身心發展遲緩疑似MECP2基因相關疾病患者。在23符合典型蕾特氏症候群族群中突變偵測率為60.1% (14/23)。在非典型或類蕾特氏症候群患者中突變偵測率為8.1% (3/37)。在37位未明原因身心發展遲緩個案中,我們找到1位有涵蓋MECP2基因的雙倍數突變,突變偵測率佔男性未明原因智障的4.0% (1/25)。我們總共從17個符合典型或非典型蕾特氏症候群患者的個案找到14種不同的突變,1種unclassified variant or mutation。14種不同的突變中,11個是點突變,3個是缺失突變。大部分的突變是missense mutations ,佔57.1% (8/14),其次是nonsense mutations,佔21.4% (3/14)以及frame shift mutations,佔21.4% (3/14)。 14種不同的突變當中有5種突變是本研究的新發現。其一是nonsense mutation,是C>T 的取代發生在cDNA 844 (c.844C>T),造成TGA (stop codon) 取代CGA (arginine) (p.R282X);另外三個是missense mutation (p.S149Y, p.V207M, p.Q437H);以及一個是有83個鹼基在exon4發生缺失突變(c.1117_1199del83)造成frameshift(p.S373fs)。另外有一個文獻中有報導過被歸類為unclassified variant or mutations的突變點(p.R344W),也在本研究中一位其臨床表現為典型蕾特氏症候群的女孩找到。此外,我們由MLPA篩檢找到台灣首例MECP2基因雙倍數突變的男性患者。、結論: 在台灣大部份針對MECP2基因突變的篩檢是PCR-based以及只侷限在基因的編碼區,因此很大的可能會遺漏掉此基因的大片段缺失突變。據我們了解,在台灣地區我們的研究是第一個用MLPA的方式去針對蕾特氏症候群及類蕾特氏症候群患兒進行MECP2基因的突變分析,也是第一個研究用MLPA的方式去找出MECP2雙倍數突變是造成男性智能障礙的可能原因之一。雖然,我們並未如預期地在蕾特氏症候群及類蕾特氏症候群患兒身上找到MECP2基因的大片段缺失突變,我們相信用MLPA的方式去針對定序結果陰性的蕾特氏症候群患者做進一步的篩檢,仍是非常重要的工作。 Background: Mutations in the methy-CpG-binding protein 2 (MECP2) gene are well known to cause Rett syndrome (RTT), a severe neurodevelopmental disorder, occurs almost exclusively in females, with an estimated prevalence of approximately 1 in 10,000-15,000 females. RTT is often considered an X-linked dominant condition with male lethality (Hagberg 1985) characterized by a progressive loss of intellectual function, fine gross motor skills, and communicative abilities; deceleration of head growth; and the development of stereotypic hand movements, all occurring after a period of normal development. To date, mutations have been identified in the MECP2 sequences of approximately 80-90% of all RTT; the remaining 10-20% may possess noncoding regions of this gene, or they may harbor a second RTT inducing gene or locus. Several reports have identified large gene deletions in RTT patients that escaped detection by PCR-based screening strategies (Laccone et al. 2004; Schollen et al. 2003). his study reports the result of the mutation analysis of MECP2 in 97 patients with classic or atypical RTT or RTT-like, conducted to obtain a genotypeic representation of the mutational spectrum in Taipei, Taiwan.atients and Methods: A total of 97 clinically suspicious RTT or RTT-like patients, referred to National Taiwan University Hospital for MECP2 gene mutation analysis, were involved in this study. Patients were grouping according to the clinical diagnostic criteria for the classic or variant Rett syndrome proposed by The Rett Syndrome Diagnostic Criteria Work Group (Hagberg et al. 2002), consisting of 23 classic, 37 atypical or RTT-like, and 37 grouped into idiopathic psychomotor retardation patients.The first group composed of RTT or RTT-like patients who were fulfilled the diagnostic criteria, and this group underwent DNA direct sequencing. In the mutation-negative patient, we used multiplex ligation-dependent probe amplification (MLPA) kit P015C to screen for large deletions. The other group who weren’t fulfill the classic or atypical RTT diagnostic criteria, were considered as idiopathic psychomotor retardation, but who may be related to the MECP2-related disorders were underwent MECP2-MLPA test directly to study the possible role of MECP2 duplication in this group.esults: Mutations in MECP2 were detected in 60.1% (14/23) of the patients presenting with classic RTT, and in 8.1% (3/37) of those with atypical RTT or RTT-like features. In total, 14 different MECP2 mutations, and 1 unclassified variant or mutation were identified in 17 of the 60 diagnosed as classical or atypical RTT or RTT-like patients. Most of the variants were missense mutations, accounting for 57.1% (8/14), followed by nonsense mutations 21.4% (3/14), and frame shift mutations 21.4% (3/14). We identified five novel mutations, four of which were point mutations, and one was deletion. The first, a nonsense mutation, C>T transition at the cDNA 844 (c.844C>T), lead to CGA change to TGA and became a stop codon (p.R282X). The other three were missense mutations (p.S149Y, p.V207M, p.Q437H), and one was a 83bp deletion (c.1117_1199del83) happened in exon 4 and lead to frameshift (p.S373fs). No mutations were found in exon 1 or 2 based on our study. However, we did not pinpoint a significant relationship between genotype and phenotype in these cases. In the group of 37 patients with idiopathic psychomotor retardation, we identified one case with Xq28 duplication including MECP2 gene.onclusion: In Taiwan, most of the screenings are PCR-based and restricted to the coding part of the gene and therefore prone to miss gene dosage changes. To our knowledge, this is the first study in Taiwan to determine the role of large deletions of the MECP2 in RTT or RTT-like patients using MLPA method. Also, it is the first study in Taiwan to determine the possible role of duplications of the MECP2 region in children with psychomotor retardation using MLPA method. Although there was no large deletion of MECP2 found in our patients with Rett or Rett-like syndrome, it remains important to screen large deletions in these patients. Because duplication of MECP2 gene remains one of the possibilities in patients with psychomotor retardation of unknown etiology, we recommend screening MECP2 gene duplications in males with moderate to severe developmental delay, especially when a history of recurrent infections has been documented. We will continue our work to screen the large deletion of MECP2 gene in RTT or RTT-like patients, and the duplication of MECP2 gene in children with psychomotor retardation of unknown etiology to clarify the role of such changes in these patients. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/181643 |
顯示於: | 臨床醫學研究所 |
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