摘要目的 通过一例常染色体隐性遗传型Alport综合征患者家系的基因突变分析,探讨常染色体隐性遗传Alport综合征的基因诊断方法.方法 提取该患者外周血细胞的基因组DNA,应用基因组DNA PCR测序法对COL4A3和COL4A4基因各外显子进行突变筛查.对于基因组DNA序列异常者,从外周血细胞和EB病毒转染的细胞中提取总RNA,应用cDNA RT-PCR测序方法分析突变.同时在家系和正常人群中进行验证.结果 使用基因组DNA样本经PCR直接测序法检测到COL4A3基因上两个致病性新突变(IVS 39+1 G＞A剪接突变和c.1729-1737 de19bp缺失突变);经RT-PCR测序方法验证其与基因组DNA样本中检测到的突变一致,并证实剪接突变产生异常的转录产物.结论 基因组DNA PCR测序法和cDNA RT-PCR-测序法均检测到Alport综合征患者COL4A3的致病突变,两种方法结果一致;cDNA RT-PCR测序法因为其相对快捷,并且可以了解突变转录情况而优于经典的基因组筛查突变方法.

abstractsObjective To explore the gene diagnostic method for autosomal recessive Alport syndrome (AR-AS).Methotis Genomic DNA was extracted from the peripheral leukocytes of the proband of an AR-AS family.All the exons of COL4A3 and COL4A4 introns were amplified by PCR,and then the PCR products were sequenced by direct sequencing.Meanwhile,the mRNA of the coding region of type IV collagen α3 and α4 chain was extracted from the PBL and EB virus transfected cell and analyzed by using RT-PCR and sequencing to confoYm the genomic DNA analysis results.Results PCR-sequencing analysis identified two novel COL4A3 mutations.OBe was a 5' donor splice site mutation (C.3418+1 G to A) in exon 39,leading to the deletion of exon 39 in mRNA level by RT-PCR analysis.The other was a deletion mutation of 9 bp at exon 25 (C.1729-1737 del9).Conclusion Both genomic-DNA-PCR-sequencing and mRNA-RT-PCR-sequencing methods can be carried out to detect tlle pathogenic mutations.In particulr,mRNA-based approach Can identify the changes in transcript level,therefore it iS better than the genomic DNA-based method.