title: Novel and de novo PKD1 mutations identified by multiple restriction fragment-single strand conformation polymorphism (MRF-SSCP).
abstract: BACKGROUND
  We have previously developed a long RT-PCR method for selective amplification of full-length PKD1 transcripts (13.6 kb) and a long-range PCR for amplification in the reiterated region (18 kb) covering exons 14 and 34 of the PKD1 gene. These have provided us with an opportunity to study PKD1 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 PKD1 mutations in the patients with autosomal dominant polycystic kidney disease (ADPKD). Novel and de novo PKD1 mutations are identified and reported.
  
  
  METHODS
  Full-length PKD1 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 PKD1 region, sequencing, direct mutation detection, and segregation analysis in the affected family.
  
  
  RESULTS
  Five PKD1 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 de novo.
  
  
  CONCLUSIONS
  The MRF-SSCP method has been developed to analyze PCR products generated by the long RT-PCR and nested-PCR technique for screening PKD1 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 de novo PKD1 mutations indicates that this gene is prone to mutations.
authors.affiliation: Division of Molecular Genetics, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. siwtn@mucc.mahidol.ac.th
authors.forename: Wanna
authors.initials: W
authors.lastname: Thongnoppakhun
authors.forename: Chanin
authors.initials: C
authors.lastname: Limwongse
authors.forename: Kriengsak
authors.initials: K
authors.lastname: Vareesangthip
authors.forename: Chintana
authors.initials: C
authors.lastname: Sirinavin
authors.forename: Duangkamon
authors.initials: D
authors.lastname: Bunditworapoom
authors.forename: Nanyawan
authors.initials: N
authors.lastname: Rungroj
authors.forename: Pa-thai
authors.initials: PT
authors.lastname: Yenchitsomanus
mesh_terms: D018389:Codon, Nonsense; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D016368:Frameshift Mutation; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016891:Polycystic Kidney, Autosomal Dominant / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D018807:Polymorphism, Single-Stranded Conformational; D011506:Proteins / Q000235:genetics*; D012326:RNA Splicing; D017384:Sequence Deletion; D050396:TRPP Cation Channels
chemical_list: D018389:Codon, Nonsense; D011506:Proteins; D050396:TRPP Cation Channels