Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the CAP-related domain
Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the CAP-related domain
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DOI：
10.1086/302809
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发表时间：
2000-03-01
影响因子：
9.8
通讯作者：
Nürnberg, P
中科院分区：
文献类型：
--
作者：
Fahsold, R;Hoffmeyer, S;Nürnberg, P
在NF1基因中筛选了500多名1型神经纤维瘤病（NF1）的非相关患者（NF1）。对于每个患者，通过蛋白质截断测试（PTT），基因组PCR产物的温度梯度凝胶电泳（TGGE）研究了整个编码序列和所有剪接位点的畸变，或者通常通过直接基因组测序（所有外显子的DGS）。确定了总共301个序列变体，包括278个真正的致病性突变。与Upadhyaya和Cooper的最新发现或NNFF突变数据库相比，包含179或161个不同的突变的216或183个，包括179或161个不同的突变。各种筛查方法的突变检测效率相似：MT的47.1％，TGGE为53.7％，DGS为54.9％。大约224个突变（80.2％）直接或间接过早终止密码子产生。这些突变显示从外显子1到外显子47的整个基因的分布均匀分布。在我们的研究中确定的所有序列变体中，<20％<20％代表c-> t或g-> a cpg二核苷酸内的转变，也仅发生六个不同的突变，并且也发生在NF1假基因中，其中五个是CpG中的典型C-> T转变。因此，5-甲基胞菌素的频繁脱氨基和染色体基因转化率都不能解释NF1基因的高突变命运。与截断突变相反，鉴定出在两个不同的区域（GAP相关结构域（GRD））和一个包含外显子11-17的上游基因段的28个（10.1％）错义或单氨基酸 - 缺失突变。后者形成了一个所谓的半胱氨酸/丝氨酸富含域，具有三个半胱氨酸对，暗示了ATP结合，以及三种潜在的依赖性cAMP依赖性蛋白激酶（PKA）识别位点，PKA明显磷酸化。突变的氨基酸与人类和果蝇之间的保守的一致性强烈表明该区域具有显着的功能相关性，外显子12A和15和Exon 16的一部分发挥了主要作用。
More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for MT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study < 20% represent C->T or G->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation fate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16.