一例散发1型神经纤维瘤病患者的NF1基因嵌合突变分析

Identification of a mosaic mutation ofNF1 gene in a sporadic case of neurofibromatosis type 1

目的对1例散发1型神经纤维瘤病患者的NF1基因进行嵌合突变分析。方法提取先证者外周血基因组RNA，PCR扩增NF1基因编码区序列并进行序列测定；找到突变后，基因组DNA途径证实突变，并对先证者儿子的NFI基因相应外显子也进行序列分析；针对NF1基因第51外显子已发现的突变，取先证者全血淋巴细胞、口腔上皮细胞和尿路上皮细胞基因组DNA进行PCR扩增，PcR产物T克隆及测序。结果先证者的临床表现符合1型神经纤维瘤病。先证者外周血RNA途径检测出无义突变C．7911C〉T（P．Q2510X）；基因组DNA途径证实患者外周血淋巴细胞、口腔上皮细胞和尿路上皮细胞中均有该突变，尿路上皮细胞中该突变测序信号较弱；在PCR产物的T克隆-测序中，来自先证者的血液、口腔上皮、尿液上皮细胞无义突变c．7911c〉T（P．Q2510X）突变体的重组菌分别占总数的42％、36％、12％。其儿子、正常对照不存在上述突变。结论先证者在胚胎早期发生了NF1基因突变，使其体内部分细胞带有NF1基因突变，导致形成全身嵌合的1型神经纤维瘤病。

Objective To detect NF1 gene mutation in a patient with neurofibromatosis type 1. Methods Five fragments encompassing the entire coding sequence of the NF1 gene were amplified with reverse transcription PCR. PCR products were directly sequenced. Suspected mutations were verified by sequencing of DNA amplified by PCR using genomic DNA as template. Corresponding exon of family members was also sequenced. Furthermore, the PCR products were inserted into a pGEM-T cloning vector to quantify cells carrying the mutation in different samples derived from the three embryonic layers. Results The proband＇s clinical manifestation was consistent with neurofibromatosis type 1. Sequence analysis has identified a novel heterozygous mutation c. 7911C〉T （p. Q2510X） in exon 51 of the NF1 gene in the proband. The same mutation was also detected in peripheral blood cells, uroepithelial cells and oral mucosal cells of the proband, though the signals of uroepithelial cells were significantly weaker. By T cloningsequencing, recombinants carrying the NF1 gene mutation respectively accounted for 42 %, 36 M and 12 % of all peripheral blood cells, oral mucosal cells and uroepithelial cells. Conclusion It is likely that a mutation of NF1 gene has occurred in early embryogenesis of the proband, which in turn has led to generalized mosaicism of neurofibromatosis type 1.