| Autosomal dominant polycystic kidney disease (ADPKD) is a very common hereditary disorder in human. Its clinical manifestation not only includes renal cyst formation, but also accompanied with other multi-organ disorders, such as hepatic cyst, cerebral aneurysms, mitral valve prolapse, and so on. At least 50 percent of patients have got end stage renal disease when 60 years old. As being a major gene responsible for the onset of 85% of ADPKD patients, PKD1 has a complicated structure with a length of 53 kb and its homologues co-existing in vivo , which greatly inhibited the study of its mutation detection. There are 1.5 millions ADPKD patients in China at present, however, there hasn't been no report about the entire PKD1mutation in ADPKD Hans yet. To explore the types of mutation, their actual position and the regular occurred, we selected 67 individuals in 19 pedigrees among 100 ADPKD Han families from different areas of China and screened the mutation of entire PKD1 exons using 85 healthy Hans as normal control. The research is supported by grants from National Science Foundation(No. 30170901) and informed consent was obtained from all the patients.This study consists of the following three parts:1. The specific amplification of the entire PKD1 exons: Based on the rare difference between PKD1 and its homologues, we designed 82 pairs of primers covering the entire PKD1 exons with 14.15 kb in length. As for the GC-rich areas and several repeats within in the multicopy region of PKD1, we firstly performed long-range PCR to overcome the homologues interferes and obtained 8 amplicons with an average of 3.7 kb in length, and then through nested PCR using the long-range PCR products as templates, we obtained 57 shorter amplicons(about 285 bp or so) which would be analyzed by single-strand conformation polymorphism (SSCP) further.2. The development of PCR-SSCP analysis for PKD1 mutation: Using 85 healthy persons as normal control, we searched the proper conditions for SSCP analysis by trying different protocols, such as the non-denaturing polyacrylamide gel in different thickness and with different components, SSCP electrophoresis under different voltages, different temperatures. Especially, weimproved the tradition method by using saccharose instead of formamide in loading buffer which could increase the sensitivity of mutation detection via greatly enhancing the single strand DNA concentration when SSCP. In the end, we developed a PCR-SSCP analysis system for mutation detection of PKD1 in ADPKD Hans, which has been applied for the National Inventor's Patent Right (No. 03115435.2).3. The mutation detection of PKD1 in ADPKD Han Chinese: Used the above system for mutation detection of PKD1 in ADPKD Hans, we performed the PKD1 mutation screen in 67 members from 19 ADPKD Han pedigrees. As a result, we found 9 normal DNA polymorphisms and 11 pathogensis mutations of PKD1 which included 7 missense , 2 nonsense , 1 insertion and 1 deletion. Among them, 7 polymorphisms and 10 pathogensis ones are firstly reported up to date. From the mutation data identified, we further found the following results: Firstly, the majority of mutations we identified was novel. Although the other three mutations in present study were once reported by others, the incidence of the DNA variants we detected was obviously different from theirs. We assumed it might be attributed to the different races of population involved in; Secondly, the majority of mutations was transition variants. Among them, the nucleotide changes from G to A and from C to T occupied about 50 percent and 35 percent, respectively; Thirdly, the majority of mutation occurred near the imperfect nucleotide repeats region; fourthly, in contrast to normal polymorphisms we identified, the pathogensis mutations often resulted in altering the charge or hydrophobicity of amino acids they coded. Taken together, it suggested that the right conformation of polypeptides formed by amino acids should play a key role in keeping a normal function...
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