Novel method for genomic analysis ofPKD1andPKD2mutations in autosomal dominant polycystic kidney disease

Abstract

Genetic testing of PKD1 and PKD2 is useful for diagnosis and prognosis of autosomal dominant polycystic kidney disease (ADPKD), particularly in asymptomatic individuals or those without a family history. PKD1 testing is complicated by the large transcript size, complexity of the gene region, and the extent of gene variations. A molecular assay was developed using Transgenomic's SURVEYOR Nuclease and WAVE Nucleic Acid High Sensitivity Fragment Analysis System to screen for PKD1 and PKD2 variants, followed by sequencing of variant gene segments, thereby reducing the sequencing reactions by 80%. This method was compared to complete DNA sequencing performed by a reference laboratory for 25 ADPKD patients from 22 families. The pathogenic potential of gene variations of unknown significance was examined by evolutionary comparison, effects of amino acid substitutions on protein structure, and effects of splice-site alterations. A total of 90 variations were identified, including all 82 reported by the reference laboratory (100% sensitivity). A total of 76 variations (84.4%) were in PKD1 and 14 (15.6%) in PKD2. Definite pathogenic mutations (seven nonsense, four truncation, and three splicing defects) were detected in 64% (14/22) of families. The remaining 76 variants included 26 missense, 33 silent, and 17 intronic changes. Two heterozygous nonsense mutations were incorrectly determined by the reference laboratory as homozygous. "Probably pathogenic" mutations were identified in an additional five families (overall detection rate 86%). In conclusion, the SURVEYOR nuclease method was comparable to direct sequencing for detecting ADPKD mutations, achieving high sensitivity with lower cost, providing an important tool for genetic analysis of complex genes.

Type of PKD1 Mutation Influences Renal Outcome in ADPKD

“…11,12 This improvement is mainly due to the following two reasons: (1) the thorough analysis of the entire coding sequences and all the exon/intron boundaries of PKD1 and PKD2 for both conventional mutations (i.e., single nucleotide substitutions, small insertions, and small deletions) and gross genomic rearrangements 4 and (2) the identification of a significant number of milder mutations. In addition, it may be possible that rare founder mutations have been considered as "recurrent" ones, thereby slightly increasing our mutation detection rate.…”

“…We describe here a high level of private mutations (70.2%), which is consistent with previous findings. [10][11][12] We cannot exclude the possibility that some recurrent mutations might in fact be due to the presence of a common ancestor. However, to date, we have not been able to establish any genealogic link between these families despite careful reanalysis of the pedigrees.…”

Pancreatic Cysts in Autosomal Dominant Polycystic Kidney Disease: Prevalence and Association withPKD2Gene Mutations

“…After excluding patients without a detectable mutation in PKD1 or PKD2 and patients for whom no matching control subject could be found, 110 patients with ADPKD were included in the current study (Fig 1). Pathogenic variants of PKD1 or PKD2 were determined by means of mutation analysis, as reported previously (19). Age, sex, race, height, estimated glomerular filtration rate (eGFR) attained by using the serum creatinine level obtained within 3 months of the MR imaging examination, and PKD genotype data were obtained from the Rogosin Institute ADPKD repository database.…”

“…PKD1 (reference sequence NM_000296.2) and PKD2 (reference sequence NM_000297.2) were analyzed by using Surveyor Nuclease Wave (Transgenomic, Omaha, Neb) denaturing high-performance liquid chromatography screening, followed by confirmatory sequencing (19) or directly by Sanger sequencing (20) by using an ABI 3130 genetic analyzer (Applied Biosystems, Foster City, Calif). Alternatively, long-range polymerase chain reaction products covering the entire exonic regions and most of the 59 and 39 untranslated regions of PKD1 and PKD2 were sequenced by using next-generation sequencing on an Illumina MiSeq system (San Diego, Univariate and multivariable logistic regression analyses helped determine whether variables were associated with presence of pancreatic cysts in patients with ADPKD.…”

Biology of Cilia and Ciliopathies

“…There are more than 500 mutations known in PKD1 and 120 in PKD2 [132,133]. These genes code for proteins polycystin-1 and polycystin-2, which both localize to renal cilia [134].…”