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目的通过2个巨轴索神经病(GAN)家系进行基因突变位点筛查,找到致病位点。方法采用盐析法提取GAN家系成员基因组DNA,采用PCR方法扩增GAN基因的编码外显子,纯化后直接测序。结果在1号先证者GAN基因上发现2个杂合错义突变。1.第2外显子核苷酸序列第224位T>A突变(c.224T>A),该错义突变引起编码的第75位氨基酸由亮氨酸(L)变为组氨酸(H)(L75H);先证者母亲是该突变的杂合携带者,具有正常表型,先证者父亲在该位点为正常基因型。2.第10外显子核苷酸序列第1634位G>A突变(c.1634G>A),该错义突变引起编码的第545位氨基酸从精氨酸(R)变为组氨酸(H)(R545H);先证者父亲是该突变的杂合携带者,具有正常表型,先证者母亲在该位点为正常基因型。这2个错义突变均能引起GAN基因编码氨基酸的改变,从而引起蛋白质结构和功能异常。2号先证者GAN基因未发现突变位点。结论在第1个家系中,c.224T>A和c.1634G>A错义突变导致先证者出现GAN的表型,其父母分别是2个突变的携带者,均具有正常表型。第2个家系可能存在其他遗传方式。
OBJECTIVE: To screen the genetic mutation sites by two GAN pedigrees and find the pathogenic sites. Methods The genomic DNA of GAN pedigree was extracted by salting-out method. The coding exon of GAN gene was amplified by PCR and sequenced directly after purification. Results Two heterozygous missense mutations were found on the GAN gene of proband 1. 1. T> A mutation at position 224 of the second exon nucleotide sequence (c.224T> A), which resulted in a change from leucine (L) to histidine H) (L75H); proband’s mother is a heterozygous carrier of the mutation, with a normal phenotype, proband’s father at this locus for the normal genotype. 2. The 1634th G> A mutation (c.1634G> A) in nucleotide 10 of the exon 10 resulted in a change of amino acid 544 from arginine (R) to histidine H) (R545H); proband’s father is a heterozygous carrier of the mutation, with a normal phenotype, proband’s mother at this locus for the normal genotype. These two missense mutations can cause amino acid changes in the GAN gene, causing protein structure and dysfunction. No. 2 proband GAN gene found no mutation sites. Conclusion In the first pedigree, the c.224T> A and c.1634G> A missense mutations lead to the phenotype of GAN in probands, and their parents are carriers of two mutations, all with normal phenotypes. There may be other ways of inheritance in the second family.