Mutation detection of ADPKD PKD1 gene in Hans by denaturing high-performance liquid chromatography / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a screening system for more rapid and sensitive mutation detection of autosomal dominant polycystic kidney disease (ADPKD) gene 1 (PKD1) by using denaturing high-performance liquid chromatography (DHPLC) protocol.</p><p><b>METHODS</b>Using genomic DNA as templates extracted from blood samples of 19 Han pedigrees with 67 family members, the complete codon areas were amplified by long-range PCR and nested PCR in succession, and then the PCR products were analyzed by DHPLC. The mutations from screened abnormal PCR products were confirmed by DNA sequencing, and then compared with the mutations identified by single strand conformation polymorphism (SSCP) before.</p><p><b>RESULTS</b>There were 14 mutations found in this study, including 10 missense, 1 insertion, 1 deletion and 2 nonsense mutations. Besides 12 mutations identified before, mutations nt32819G>A and nt37137T>C were the novel mutations found. The mutation detection ratio was 73.7%.</p><p><b>CONCLUSION</b>This developed system via DHPLC can be used as a more effective approach for mutation detection of autosomal dominant polycystic kidney disease PKD1 in Hans.</p>

Mutation detection of PKD2 gene in Chinese by denaturing high-performance liquid chromatograph / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect the mutations of autosomal dominant polycystic kidney disease gene 2(PKD2)in Chinese.</p><p><b>METHODS</b>The white blood cell genomic DNA from patients of 94 Chinese autosomal dominant polycystic kidney disease(ADPKD) pedigrees was isolated and amplified by polymerase chain reaction(PCR). The PCR products were analyzed by denaturing high-performance liquid chromatography(DHPLC). The samples with abnormal profiles were sequenced.</p><p><b>RESULTS</b>Eight mutations were identified, including 2 nonsense mutations, 2 deletion mutations,1 insertion mutation and 3 missense mutations. Two nonsense mutations occurred in exon 5(1249C-->T) and exon 13(2407C-->T),both resulted in a stop codon. The insertion was in exon 2(636-637 ins T),and the deletion mutations were in exons 12(2348-2351 del AGAA) and 13(2401 delete A),resulting in the reading frame shift. Three missense mutations were in exons 1(G568-->A),4(C964-->T),and 5(G1168-->A), which caused amino acid changes (190Ala-->Thr,322Arg-->Trp,390Gly-->Ser).</p><p><b>CONCLUSION</b>The method of DHPLC was used in detecting mutations successfully and 8 mutations in PKD2 were identified. It will be useful in the molecular diagnosis of ADPKD in advance of the cysts formation and birth.</p>

Gene diagnosis of autosomal dominant polycystic kidney disease type 2 using microsatellite DNA tightly linked to polycystic kidney disease gene 2 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To use microsatellite DNA tightly linked to polycystic kidney disease gene 2 in the gene diagnosis of autosomal dominant polycystic kidney disease type 2.</p><p><b>METHODS</b>Microsatellite DNA of D4S1534, D4S1542, D4S1563,D4S2460 and D4S423 were amplified with PCR and the fragments of products were analyzed by capillary electrophoresis and Genescan and Genotyper software, and then gene diagnosis of the pedigrees was made by linkage analysis.</p><p><b>RESULTS</b>Three families were found to be linked to PKD2 in 20 families. Two carriers of PKD2 mutation were revealed by linkage analysis.</p><p><b>CONCLUSION</b>Gene diagnosis can be done for PKD2 mutation carriers prior to cytogenesis. Linkage analysis is a rapid, simple method for studying the heterogeneity of polycystic kidney disease and for diagnosing the disease at the molecular level.</p>