[Approaches to detect the gene mutations in autosomal recessive Alport's syndrome: analysis of a family].
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).


CONCLUSIONS
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.
Hou
Ping
P
Department of Nephrology, Institute of Nephrology, First Hospital of Peking University, Beijing 100034, China.
Lü
Ji-Cheng
JC
Chen
Yu-Qing
YQ
Ding
Jia-Xiang
JX
Li
Guang-Tao
GT
Zhang
Hong
H
D000328:Adult; D000368:Aged; D001483:Base Sequence; D024141:Collagen Type IV / Q000235:genetics*; D004252:DNA Mutational Analysis; D005091:Exons; D005192:Family Health; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009394:Nephritis, Hereditary / Q000235:genetics*; D010375:Pedigree; D055815:Young Adult
D024141:Collagen Type IV