| Summary: | <p>Abstract</p> <p>Background</p> <p>We have previously developed a long RT-PCR method for selective amplification of full-length <it>PKD1 </it>transcripts (13.6 kb) and a long-range PCR for amplification in the reiterated region (18 kb) covering exons 14 and 34 of the <it>PKD1 </it>gene. These have provided us with an opportunity to study <it>PKD1 </it>mutations especially in its reiterated region which is difficult to examine. In this report, we have further developed the method of multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP) for analysis of <it>PKD1 </it>mutations in the patients with autosomal dominant polycystic kidney disease (ADPKD). Novel and <it>de novo PKD1 </it>mutations are identified and reported.</p> <p>Methods</p> <p>Full-length <it>PKD1 </it>cDNA isolated from the patients with ADPKD was fractionated into nine overlapping segments by nested-PCR. Each segment was digested with sets of combined restriction endonucleases before the SSCP analysis. The fragments with aberrant migration were mapped, isolated, and sequenced. The presence of mutation was confirmed by the long-range genomic DNA amplification in the <it>PKD1 </it>region, sequencing, direct mutation detection, and segregation analysis in the affected family.</p> <p>Results</p> <p>Five <it>PKD1 </it>mutations identified are two frameshift mutations caused by two di-nucleotide (c. 5225_5226delAG and c.9451_9452delAT) deletions, a nonsense (Q1828X, c.5693C>T) mutation, a splicing defect attributable to 31 nucleotide deletion (g.33184_33214del31), and an in-frame deletion (L3287del, c.10070_10072delCTC). All mutations occurred within the reiterated region of the gene involving exons 15, 26, 15, 19 and 29, respectively. Three mutations (one frameshift, splicing defect, and in-frame deletion) are novel and two (one frameshift and nonsense) known. In addition, two mutations (nonsense and splicing defect) are possibly <it>de novo</it>.</p> <p>Conclusion</p> <p>The MRF-SSCP method has been developed to analyze PCR products generated by the long RT-PCR and nested-PCR technique for screening <it>PKD1 </it>mutations in the full-length cDNA. Five mutations identified were all in the reiterated region of this gene, three of which were novel. The presence of <it>de novo PKD1 </it>mutations indicates that this gene is prone to mutations.</p>
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