Zhonghua yi xue za zhi · 2008
[Approaches to detect the gene mutations in autosomal recessive Alport's syndrome: analysis of a family].
Аннотация на английскомСтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Hou Ping, Lü Ji-Cheng, Chen Yu-Qing, Ding Jia-Xiang, Li Guang-Tao, Zhang Hong
Аннотация
OBJECTIVE: To explore the gene diagnostic method for autosomal recessive Alport syndrome (AR-AS).
METHODS: 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 alpha3 and alpha4 chain was extracted from the PBL and EB virus transfected cell and analyzed by using RT-PCR and sequencing to conform the genomic DNA analysis results.
RESULTS: PCR-sequencing analysis identified two novel COL4A3 mutations. One 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 the pathogenic mutations. In particular, mRNA-based approach can identify the changes in transcript level, therefore it is better than the genomic DNA-based method.
METHODS: 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 alpha3 and alpha4 chain was extracted from the PBL and EB virus transfected cell and analyzed by using RT-PCR and sequencing to conform the genomic DNA analysis results.
RESULTS: PCR-sequencing analysis identified two novel COL4A3 mutations. One 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 the pathogenic mutations. In particular, mRNA-based approach can identify the changes in transcript level, therefore it is better than the genomic DNA-based method.