权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Mutations in beta-hexosaminidase beta-subunit gene and the clinical phenotypes

β-己糖胺酶β亚基基因突变与临床表型

基本信息

批准号：
08670905
负责人：
TANAKA Akemi
金额：
$ 1.22万
依托单位：
Osaka city University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Four unrelated japanese Patients with infantile Sandhoff disease (beta-hexosaminidase beta-subunit deficiency) have been studied for their molecular basis of their severe phenotype. Two patients had complex base substitutions ; one patient was homoallelic for a triple mutation (P417L,K121R and S255R) and the other was a compound heterozygote of a double (P417L and K121R) mutation and the triple mutation. K121R has been known to be a functional polymorphisms, while P417L (exon 11, +8 C*T) generates predominantly an abnormally spliced mRNA at +112 base of exon 11 and has been described in two patients with a juvenile form of the disease. The mild phenotype is attributed to the presence of a small amount of normally spliced mRNA.S255R is novel without prior description in the literature. An expression study of the normally spliced cDNA with the double and the triple mutations gave 78% and 28% of normal activity, respectively. This finding suggested that S255R further reduces the catalytic … More activity of the already below-the threshold amount of normally spliced mRNA and accounts for the more severe phenotype in our patients. In the other two patients a novel disease-causing base transition was found within intron 10, away from the intron/exon junction (-17 a*g). This mutation caused abnormal 3' splicing at-37 of intron 10, and normally spliced product was detectable in RT-PCR analysis. We noted an unusually low splice site score (61.8) for the exon 10-intron 11 junction and suspected that this might be partially responsible for these abberant splicing in these mutations. To test this hypothesis, we constructed four chimeric cDNAs all with additinal intron 10 inserted and evaluated their splicing efficiency. They respectively had the normal sequence, P417L (exon 11, +8 C*T), the intronic mutation (-17 a*g), and an artificially engineered intron 10-exon 11 junction with a higher splice site score (85.1). Fifty-eight % and 31% of total transcript were correctly spliced in the normal chimeric construct and P417L,respectively, while no normally spliced product was generated either in the chimeric construct with -17 a*g or in that with a high splice site score. The sequence around the adenosine 17 residues upstream of the normal acceptor splice site in this report UUGCAAU (-22 to -16) matches the consensus branchpoint sequence YNYRAY reported in the literature. The mutation in this study is most likely to abolish the lariat formation because the artificial site of high splice acore did not improve splicing efficiency. Less

研究了4例无亲缘关系的日本婴儿山德霍夫病（β-氨基己糖苷酶β-亚单位缺乏症），以了解其严重表型的分子基础。两名患者有复杂的碱基替换;一名患者是纯等位基因的三重突变（P417 L，K121 R和S255 R），另一个是复合杂合子的双重（P417 L和K121 R）突变和三重突变。已知K121 R是一种功能性多态性，而P417 L（外显子11，+8 C*T）主要在外显子11的+112碱基处产生异常剪接的mRNA，并已在两名患有幼年型疾病的患者中描述。S255 R是一种新的突变体，在文献中未见报道。正常剪接的cDNA与双和三重突变的表达研究给出了78%和28%的正常活动，分别。这一发现表明，S255 R进一步降低了 ...更多信息已经低于正常剪接的mRNA的阈值量的活性，并解释了我们患者中更严重的表型。在另外两名患者中，在内含子10内发现了一种新的致病碱基转换，远离内含子/外显子连接（-17 a*g）。该突变导致内含子10的-37位点发生异常3'剪接，RT-PCR检测到正常剪接产物。我们注意到外显子10-内含子11连接处的剪接位点评分异常低（61.8），并怀疑这可能是这些突变中异常剪接的部分原因。为了验证这一假设，我们构建了四个嵌合cDNA，它们都插入了额外的内含子10，并评估了它们的剪接效率。它们分别具有正常序列、P417 L（外显子11，+8 C*T）、内含子突变（-17 a*g）和具有较高剪接位点评分（85.1）的人工工程化内含子10-外显子11连接。在正常嵌合构建体和P417 L中分别有58%和31%的总转录物被正确剪接，而在具有-17 a*g的嵌合构建体或具有高剪接位点评分的嵌合构建体中没有产生正常剪接的产物。本报告中正常受体剪接位点上游腺苷17个残基周围的序列UUGCAAU（-22至-16）与文献中报道的共有分支点序列YNYRAY匹配。本研究中的突变最有可能消除lactone的形成，因为高剪接acore的人工位点并没有提高剪接效率。少