Abstract: FR-PO300

Validating Whole Genome Sequencing (WGS) as a Diagnostic Technique for Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Session Information

Category: Genetic Diseases of the Kidney

  • 801 Cystic Kidney Diseases

Authors

  • Mallawaarachchi, Amali, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  • Furlong, Tim, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  • Hort, Yvonne, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  • Senum, Sarah R., Mayo Clinic, Rochester, Minnesota, United States
  • Lundie, Ben, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  • Tao, Jiang, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  • Minoche, Andre E, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  • Cowley, Mark J, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  • Shine, John, Garvan Institute of Medical Research, Sydney, New South Wales, Australia
  • Harris, Peter C., Mayo Clinic, Rochester, Minnesota, United States
Background

ADPKD is the most common monogenic renal disease. There are many benefits of genetic diagnosis (early diagnosis, family planning, cascade testing, living-donor selection and to predict disease severity). However genetic diagnostics is not routinely pursued. Diagnostic sequencing is challenged because 6 pseudogenes share 97% sequence homology with PKD1 and confound standard sequencing techniques. WGS has the potential to overcome sequence homology, but has not been validated as a diagnostic test.

Methods

We studied 42 unrelated patients with an ADPKD phenotype. Thirty patients initially underwent long-range PCR/Sanger sequencing/MLPA (LR-PCR/SS/MLPA) of PKD1 and PKD2 in the Mayo Clinic (Grp1). Blinded WGS was then performed after PCR-free library preparation (Kappa Hyper kit, HiSeqX; 150bp paired-end sequencing) in the Garvan Institute. Concurrently, 12 patients were initially sequenced via WGS and then blinded LR-PCR/SS/MLPA of PKD1 and PKD2 (Grp2). Raw WGS data was analysed for single nucleotide and copy number variation via customized bioinformatics pipelines. WGS variant analysis was focussed on PKD1 and PKD2.

Results

WGS provided uniform coverage (mean 36x; range 24-47), including in homologous and GC-rich regions. The same results as LR-PCR/SS/MLPA were obtained in 40/42 patients (37 disease-causing variants; 3 patients unknown with both methods). In 2/42 patients, using standard filters, WGS did not detect mosaic variants, however in 1 mosaic patient, reanalysis of WGS data showed the variant in 8% of reads. WGS defined the breakpoints of 2 multi-exon deletions, which was not possible with prior methods. On initial analysis of Grp1, the disease-causing variant was confirmed in 24/30 patients. After adjusting the variant filtering stringency, this was improved to 28/30. There were no false positive or false negative results with WGS.

Conclusion

WGS provides the basis of a new diagnostic test for ADPKD. It avoids laborious sample preparation and overcomes pseudogene homology. Unlike targeted sequencing, WGS allows scope for broadened genomic analysis if no PKD1 or PKD2 variants are identified. This study highlights the value of validating next generation sequencing against a gold-standard cohort prior to diagnostic application.

Funding

  • Private Foundation Support