Preparation Of Tandem Repeat ASO Probes And Their Application In Diagnosis Of Wilson's Disease

Wilson's disease (WD) is an autosomal recessive inherited disorder of copper metabolism. Early diagnosis is important since it can be effectively treated if diagnosis can be made in early life. The WD gene codes for a copper transporting P-type ATPase. Molecular genetic analysis of patients with WD has revealed that there were over 400 distinct mutations, most of which are point mutations, including missense and nonsense mutations. There is a difference in the spectrum of mutations in Europeans and Asians, including the mutation hot spots.For effectively detection of the mutations in patients, a series of tandem repeats of the ASO probes for the most common mutations of WD in Chinese were synthesized by complementary ASO contigs with a short spacer of 5 bp. Reverse-dot hybridization is carried out to identify point mutations in patients with Wilson's disease using the labeled exon fragments as the probes.Eight pairs of probes were constructed. Twenty-three WD cases were tested, and the mutations identified in each case were validated by restrict endonuclease digestion and sequencing of the PCR products. Four known mutations were detected with reverse hybridization and 8 other changes were identified by direct sequencing of the 4 exons in this panel of patients. The detection rate was 58.7%. R778L, which was reported as the most common mutation in Chinese, have been validated to be the highest prevalence in this study. Further study is needed to characterize the novel mutations.Tandem-repeat-ASO-probes based reverse dot-blot hybridization is a novel rapid and effective method to identify known point mutations. It can also be applied to other genetic diseases which are characterized of point mutations besides WD.