Detection of Feline Autosomal Dominant Polycystic Kidney Disease by a PCR-DGGE Assay

• Main Authors: Yu-Jong Weng, 翁郁中
• Other Authors: Yang-Tsung Chung
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/80433059609132285187

碩士 === 國立中興大學 === 生命科學院碩士在職專班 === 97 === The detection of mutations by DGGE is based on the sequence-dependent electrophoretic mobility of double-stranded DNA fragments in a polyacrylamide gel that contains a linear denaturing gradient. Initially, the migration rate of the fragment depends on its molecular weight. However, at a specific point in the gel, the combination of denaturant concentration and temperature equals the Tm of the lower melting domain, resulting in a partially single-stranded fragment. The mobility of these branched fragments in the polyacrylamide is abruptly retarded. The addition of an artificial GC-rich sequence, a so-called GC-clamp, to DNA fragments has been a major improvement in DGGE methodology. Such a GC-clamp serves as the most stable part of the fragment and allows the detection of sequence variation in the remainder of the fragment. In PKD affected cats, the disease was linked to the feline PKD1 gene, coding polycystin, a protein similar to the transmembrane receptor. A C/A transversion resulting in a premature stop codon was identified in exon 29 of the feline PKD1 gene. PKD is inherited as an autosomal dominant trait. Cysts are present from birth, but start out small, slowly increasing in size. Cysts can range from very small to several centimeters in diameter. The increasing size of the cysts damage the normal kidney tissue, eventually causing kidney failure. Feline PKD is the most prevalent inherited disease of cats. It affects 38% of Persian cats worldwide. Ultrasound diagnosis is 98% accurate after approximately 10 months of age. We collected the cats submitted to NCHU VMTH from December 2006 to December 2008. One hundred and twenty-three cats were involved, consisting of 78 Persians, 14 Chinchilla, 11 Domestic Shorthair, 5 Himalaya, 6 American Shorthair, 3 Ragdoll, 3 Bengal, 1 Irish Shorthair, 1 Exotic Shorthair and 1 Siamese. Ultrasonographic examination of urinary bladder, both kidneys and liver was performed in 56 cats. ADPKD was diagnosed in 12 of the 58 cats. The blood of the 12 cats with ADPKD was obtained. Restriction fragment length polymorphism (RFLP) analysis and polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE) assay were performed to compare the diagnostic accuracy between these two genetic biotechnology. Only 10 of the 12 ADPKD blood samples were positive of RFLP analysis and PCR-DGGE assay. Two ADPKD affected cats diagnosed by ultrasonography were absent of the point mutation in exon 29. Other mutations may contribute to feline ADPKD. The accuracy of RFLP and PCR-DGGE was the same. PCR-DGGE can detect other single nucleotide polymorphism than PKD1. This is the first study to apply PCR-DGGE to detect PKD1 gene. More cases are needed for further evaluation of this methodology to detect PKD1 gene.