目的:对2例散发性1型神经纤维瘤患者进行致病基因NF1编码序列的突变筛查以及拷贝数变异(copy number variation,CNV)研究,寻找致病性突变。方法:PCR扩增NF1基因的编码区及外显子-内含子交界区,对产物进行直接测序。在50例正常对照中进...
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目的:对2例散发性1型神经纤维瘤患者进行致病基因NF1编码序列的突变筛查以及拷贝数变异(copy number variation,CNV)研究,寻找致病性突变。方法:PCR扩增NF1基因的编码区及外显子-内含子交界区,对产物进行直接测序。在50例正常对照中进行新发现突变位点的测序分析,以排除多态性。用多重连接探针扩增技术(MLPA)对患者进行NF1基因CNV的检测,并对证实有NF1拷贝缺失的患者进行长片段PCR,以找寻断裂点。结果:患者S736于NF1基因检测到一个国际上尚未报道的新突变:c.6345_6346 ins G(***2116Alafs*4),患者父母均不携带此突变,故此突变为一个de novo突变。该突变使开放阅读框移位,提前引入终止密码子,导致蛋白质分子的截断,以致部分犰狳式褶皱(ARM-type fold)结构域丢失。在另一患者S743中检测到一个包含整个NF1基因的大片段缺失,缺失区域为1.3～1.9 Mb,但断裂点尚不明,此为我国首例NF1基因拷贝数目变异的报道。结论:患者NF1基因的de novo突变及CNV是引起这2例1型神经纤维瘤发病的分子机制,这些发现可用于临床1型神经纤维瘤的分子诊断。
ObjectiveThis study aimed to construct plant expression vector for recombinant human epidermal growth factor （hEGF） and further to provide a basis for the expression of hEGF in peanut hairy root system. MethodAccord...
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ObjectiveThis study aimed to construct plant expression vector for recombinant human epidermal growth factor （hEGF） and further to provide a basis for the expression of hEGF in peanut hairy root system. MethodAccording to the hEGF sequence in GenBank, hEGF was synthesized artificially; subsequently, hEGF gene was ligated with green fluorescent protein （GFP） gene, and their ligation product was then amplified with primers flanked with corresponding endonuclease cleavage sites, followed by double digestion by Sal I and EcoR I of the amplified products; next, pRI 101 AN DNA was extracted and digested by both Sal I and EcoR I; susequently, the digestion products of hEGF and GFP ligation fragment by Sal I and EcoR I and the digestion products of pRI 101 AN plasmid DNA by Sal I and EcoR I were ligated, and their ligation product was transformed into Escherichia coli XL10-Gold, followed by extraction of DNA from the recombinants exhibiting green fluorescence, which was then identified by enzymatic digestion and PCR, and the verified recombinant plasmid DNA was named pBZG101. ResultHuman epidermal growth factor gene （hEGF） and green fluorescent protein gene （GFP） were successfully ligated, and their ligation fragment was successfully ligated to pRI 101 AN DNA, finally with the acquirement of the plant expression vector for recombinant human epidermal growth factor-（pBZG101）. ConclusionThe plant expression vector for recombinant human epidermal growth factor-（pBZG101）- was successfully constructed in this study.
目的 揭示一疑似糖原贮积症（GSD）Ⅲ型家系发病的分子遗传学机制,并对其致病的AGL基因的新突变做致病性鉴定.方法 先证者于2012年初在福建医科大学附属二院诊断为疑似GSD患者,并转诊至中山大学中山医学院医学遗传学教研室.首先对先证者的AGL基因的所有编码区及各编码区与内含子交界部位的序列进行直接测序.在检出突变后,再对其父母的相应位点进行检测并确认患儿突变的来源.对所发现的杂合新突变再通过克隆测序予以确证,并对新突变的致病性进行一系列鉴定,包括对100名健康对照（近2年来人群中随机取样获得）进行变性高效液相色谱（DHPLC）筛检以统计突变的频率；在11个跨物种间对突变所在氨基酸序列的保守性进行分析；比对并分析突变蛋白和正常蛋白的高级结构的差异程度.结果 先证者为“c.100C＞ T（p.R34X）”无义突变和“c.1176_1178 del TCA（p.392delHis）”缺失突变的复合杂合子.其中,p.R34X国外已报道为致病性突变,而c.1176_1178 del TCA为新发现的缺失突变.其父为p.R34X无义突变的携带者；其母为c.1176_1178 del TCA缺失突变的携带者.dbSNP数据库、HGMD数据库及近几年最新文献的检索确认c.1176_1178 delTCA为一新突变,并排除多态性变异的可能.11个跨物种的保守性分析结果表明：该突变位点所在氨基酸在进化上具有高度保守性.突变蛋白和正常蛋白预测的高级结构的比对结果显示：此缺失突变造成了AGL蛋白空间立体构象的显著改变.结论 所发现的c.1176_1178 del TCA（p.392delHis）缺失突变是一新的致病性突变,它和“c.100C＞ T（p.R34X）”无义突变是先证者患GSDⅢa病的根本原因,这两种突变分别是由母亲和父亲各遗传一个而来.
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