Kidney Week

Abstract: TH-PO670

Species-Specific Differences in Pkhd1 Transcriptional Regulation: The Role of Cystin, the Cys1 Gene Product

Session Information

• ADPKD: Genetic and Model Studies
  October 25, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidney

• 1001 Genetic Diseases of the Kidney: Cystic

Authors

• Harafuji, Naoe, Children's National Health System, Washington, District of Columbia, United States

Naoe Harafuji,

• Cuevas, Santiago, Children's National Health System, Washington, District of Columbia, United States

Santiago Cuevas,

• Yang, Chaozhe, Children's National Health System, Washington, District of Columbia, United States

Chaozhe Yang,

• Odinakachukwu, Maryanne, Children's National Health System, Washington, District of Columbia, United States

Maryanne Odinakachukwu,

• Majmundar, Amar J., Boston Children's Hospital, Boston, Massachusetts, United States

Amar J. Majmundar,

• Hildebrandt, Friedhelm, Boston Children's Hospital, Boston, Massachusetts, United States

Friedhelm Hildebrandt,

• Guay-Woodford, Lisa M., Children's National Health System, Washington, District of Columbia, United States

Lisa M. Guay-Woodford,

Background

ARPKD (MIM 263200) typically results from mutations in PKHD1, which encodes a set of secreted and membrane-bound isoforms, referred to as FPC (Onuchic, 2002). Our previous studies showed: 1) mouse Pkhd1 is transcriptionally complex (Boddu, 2014); 2) cystin, the protein disrupted in the Cys1^cpk mouse, interacts with Srsf5, a pre-mRNA splicing factor (Watts, 2016); 3) Pkhd1 exon 51 contains a functional Srsf5 binding site. In the current study, we compared Pkhd1/PKHD1 and cystin in mouse, rat and human; assessed whether cystin is a transcriptional regulator of Pkhd1; and examined consanguineous families with genetically unresolved hepato-renal fibrocystic disease (HRFD) for CYS1 mutations.

Methods

We performed RT-PCR and comparative informatic analyses for mouse, rat and human Pkhd1/PKHD1 and cystin, respectively. For minigene splicing assays, we transfected a mouse Pkhd1 exon 6, 7, 51 construct into TERT-immortalized Cys1^wt (wt) and Cys1^cpk (cpk) stable cell lines, and wt and cpk primary collecting duct cells. Whole exome sequencing was performed in 258 consanguineous HRFD families.

Results

Comparative RT-PCR showed that mouse Pkhd1 encodes multiple isoforms; rat Pkhd1 and human PKHD1 have minimal transcriptional complexity. There is limited inter-species PKHD1/Pkhd1 homology (72%, human-mouse; 71%, human-rat; 86%, mouse-rat). In contrast, cystin is identical in mouse and rat, but rodent-human identities are limited (58%, human-mouse; 59%, human-rat). Quantitative minigene assays revealed differential splicing of the Pkhd1 construct in wt and cpk cell lines. Preliminary Iso-Seq analyses of wt and cpk kidneys support differential Pkhd1 splicing. Finally, we identified a 5 yo HRFD boy with a homozygous CYS1 variant, c.318+5G>A, that is predicted to disrupt the exon 1 donor site.

Conclusion

Our data demonstrate that: 1) the transcriptional regulation of mouse Pkhd1 is distinct from its rat and human orthologues; 2) cystin is highly conserved among rodents, but there is limited rodent-human homology; 3) cystin is a transcriptional regulator of mouse Pkhd1; and 4) CYS1 mutations are a rare cause of human HRFD. Our data suggest that in Cys1^cpk/cpk mice, loss of cystin function may contribute to renal cystogenesis due in part to alterations in Pkhd1 transcriptional regulation.