Genetic Heterogeneity And Gene Diagnosis Of Autosomal Dominant Polycystic Kidney Disease Using Microsatellite DNA And Single Nucleotide Polymorphisms

Objective The study aims are to study the genetic polymorphisms of seven (CA)n microsatellite DNA tightly linked to PKD1 and PKD2 in Shanghai Han nationality and to evaluate its value in genetic heterogeneity study and gene diagnosis. To search the genetic heterogeneity of ADPKD and to compare the clinical presentation between ADPKD type 1 and 2 in Shanghai Han population, develop a method of gene diagnosis by linkage analysis with microsatellite DNA tightly linked to PKD1 and PKD2 and make gene diagnosis of mutation carriers before cystogenesis, detect single nucleotide polymorphism of PKD2 and its frequence in Han population.Methods Informed consent was obtained before investigation. 80 unrelated healthy volunteers and 43 unrelated ADPKD families were included in this study. Each ADPKD family included at least three affected members or two affected and two unaffected members. All of the patients of the ADPKD families were physicially and ultrasonographically examined at hospitals. The clinical history and presentation of all of the patients were collected. Two milliliters of peripheral blood was removed from each member. DNA was extracted according to phenol-chloroform method. Genetic polymorphisms of four microsatellites linked to PKD1(KG8, SM6, CW2 and SM7) and three microsatellites linked to PKD2(D4S1534, D4S1542 and D4S423) were detected in 80 volunteers. ADPKD families were typed using five microsatellites for PKD1(KD8, SM6, 16AC2.5, CW2 and SM7) and five microsatellites for PKD2 (D4S423 , D4S1534, D4S1563, D4S2460 and D4S1542). Polymerase chain reation amplification was performed and the PCR products at each microsatellite DNA locus were determined on capillary electrophoresis and analyzed by GeneScan and Genotyper software. Genetic polymorphisms of seven microsatellites were analyzed by population genetic statistics. Genetic heterogeneity of ADPKD families were studied by linkage analysis. The comparison of pheotypes between ADPKD type 1 and 2 were performed by medical statistics. Gene diagnosis wasmade in nine individuals in seven ADPKD families by linkage analysis and haplotype analysis. Otherwise, single nucleotide polymorphisms of PKD2 were detected in 80 volunteers using restriction digestion and sequencing method.Results In Shanghai Han people, KG8, SM6, CW2, SM7 loci included 4, 1CK 11 and9 alleles, the sizes were 112~122bp, 86~110bp,111~131bpand 83~99bp, the heterozygosities and PIC were 0.29 and 0.275, 0.825 and 0.819, 0.786 and 0.779, 0.85 and 0.827, respectively. 9, 4 and 12 alleles were observed at D4S1534, D4S1542 and D4S423 loci, whose sizes are 144~160bp, 211~ 217bp , 103~ 125bp and whose heterozygosities and PIC are 0.862 and 0.849, 0.462 and 0.448 , 0.900 and 0.898, respectively. The allele distribution frequencies of seven microsatellite DNA was consistent with the Hardy-Weinberg equilibrium. In 43 ADPKD families, 36 families showed to be linked to PKD1, which accounted to 84% of all families, 7 families linked to PKD2, which accounted to 16%. The diagnosis age of patients with ADPKD type 1 was 36.8 (8.5-59.2) year, the frequence and diagnosis age of hypertension was 62% and 37.4 (22.5-62.6) year, and the frequence of hepatic cysts, urinary-tract infection, macrohaematuria, urinary-tract calculi, cerebrovascular accident were 59%, 20%, 33%, 24%, 13%, repectively, the onset age of ESRD was 51.2 (35.4-75.2) year. The diagnosis age of patients with type 2 was 46.3 (13.7-61.4) year, the frequence and diagnosis age of hypertension was 54% and 49.1 (35.2-64.5) year, and the frequence of hepatic cysts, urinary-tract infection, macrohaematuria, urinary-tract, calculi, cerebrovascular accident were 60%, 26%, 20% , 14%, 7%, respectively, the onset age of ESRD was 64.7(57.3-70.4) year. In 7 ADPKD families which required to be made gene diagnosis, 1 PKD1 mutation carrier was found in 5 individuals in 4 families linked to PKD1, and 2 PKD2 mutation carriers were found in 4 individuals in 3 families linked to PKD2. 4 SNPs were detectd, which were 420G/A and 568G/A of exon 1, and 1420A/G and 1546G/...