Sourcetree login required1/30/2024 ![]() ![]() ![]() LR PCR was performed using the GeneAmp High Fidelity PCR System (Applied Biosystems, Foster City, CA), as previously described. The LR PCR primer sequences are shown in Table 1. The entire coding region, 5′ and 3′ untranslated regions, and exon-intron boundaries of PKD1 were amplified in nine distinct LR PCRs (Gene1, Gene2-7, Gene8-12, Gene13-15, Gene15-21, Gene22-26, Gene27-34, Gene35-41, and Gene42-46), using PCR primers (Sigma-Genosys Ltd, St Louis, MO) anchored in either the rare mismatched region with the HGs or the single-copy region of PKD1. Genomic DNA was extracted from peripheral blood lymphocytes using standard DNA extraction methods. In this study, the accuracy of the new assay was evaluated using a panel of DNA samples that were previously analyzed by two different methods that have been previously validated. To improve genetic analysis of ADPKD and to avoid the use of a nested PCR, we have developed a new method for PKD1 genetic testing by directly sequencing LR PCR fragments covering the entire PKD1 gene. The number of sequencing reactions can be substantially reduced by mutation screening procedures 12 however, these procedures do not eliminate the need for a nested PCR. 11 However, this process is highly labor intensive, susceptible to PCR contamination, and expensive. This step prevents amplification of the PKD1 HGs that potentially could confound the analysis. 11 The nested-PCR approach comprises two primary steps: a long-range (LR) PCR with primers located in the rare mismatch sites distinguishing PKD1 and the HGs and a nested PCR to amplify each individual exon. Although the entire PKD2 gene and the single-copy region of the PKD1 gene can be directly amplified by PCR from genomic DNA, the duplicated PKD1 region requires nested PCR to amplify the true gene among the highly identical HGs. ![]() The main strategies used for ADPKD genetic analysis are all PCR based, followed by sequencing of the corresponding exons. 8 However, genetic testing is complicated by the presence of PKD1 homologous genes (HGs), the large size of PKD1, and marked allelic heterogeneity of the disease-associated mutations. 7 Genetic testing plays an increasingly significant role in the diagnosis of patients with an unclear renal phenotype, particularly in the absence of a known family history, and in the evaluation of family members who are considering kidney donation to affected individuals. 5,6 ADPKD is caused by mutations in two genes, with the PKD1 gene accounting for 75% to 85% of the cases and the PKD2 gene responsible for the remainder of the cases. It is characterized by bilateral kidney cyst development 1–4 and progressive chronic kidney disease, leading to end-stage renal disease. In conclusion, LR PCR sequencing was superior to the direct sequencing and screening methods for detecting genetic variations, achieving high sensitivity and improved intronic coverage with a faster turnaround time and lower costs, and providing a reliable tool for complex genetic analyses.Īutosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, affecting approximately 1 in 500 individuals in the United States and 12.5 million patients worldwide. Compared with the reference laboratory, LR PCR sequencing had a sensitivity of 100%, a specificity of 98.5%, and an accuracy of 98.8% compared with the SURVEYOR-WAVE method, it had a sensitivity of 97.1%, a specificity of 100%, and an accuracy of 99.4%. A total of 53 heterozygous genetic changes were identified by LR PCR sequencing, including 41 (of 42) variations detected by SURVEYOR nuclease and all 32 variations reported by the reference laboratory, detecting an additional 12 intronic changes not identified by the other two methods. This method was compared with direct sequencing used by a reference laboratory and the SURVEYOR-WAVE Nucleic Acid High Sensitivity Fragment Analysis System (Transgenomic) screening method for five patients with autosomal dominant polycystic kidney disease. By using this method, the entire PKD1 coding region was amplified by nine reactions, generating product sizes from 2 to 6 kb, circumventing the need for specific PCR amplification of individual exons. We developed a novel mutation screening assay for PKD1 by directly sequencing long-range (LR) PCR products. Genetic testing of PKD1 and PKD2 is useful for the diagnosis and prognosis of autosomal dominant polycystic kidney disease however, analysis is complicated by the large transcript size, the complexity of the gene region, and the high level of gene variations. ![]()
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