All ETDs from UAB

Advisory Committee Chair

Lisa M Guay-Woodford

Advisory Committee Members

Anupam Agarwal

Bradley K Yoder

Rosa A Serra

Zsuzsanna Bebok

Document Type


Date of Award


Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine


Mutations in PKHD1 gene cause autosomal recessive polycystic kidney disease. Our previous studies have shown that human PKHD1 and its mouse orthologue, Pkhd1, undergo an extensive pattern of alternative splicing. Pkhd1 is primarily expressed in renal and biliary tubular structures. Translation products of these alternative spliced transcripts are predicted to generate isoforms expressed in plasma membrane, primary cilium and cytoplasm. We have characterized the cyli mouse model of ARPKD and identified a frameshift mutation, c.7589delGGinsT, in Pkhd1 exon 48. In this thesis we have characterized the mouse Pkhd1cyli/cyli model (liver restricted phenotype) and evaluated the transcriptome in terms of alternative exon usage and alternative splicing events. We observed that the Pkhd1 transcriptional profiles were different in 18.5dpc embryonic kidney, liver and placenta as well as in multiple adult tissues. We have evaluated 41 kidney transcripts showing different patterns of Pkhd1 exons splicing for WT and MUT kidney transcripts. Majority of the splicing events involved exon 6. All the transcripts analyzed appeared to preserve the amino acid sequence of the Pkhd1 longest open reading frame, most of them using the original translation start site in exon 2, same as for the WT full length Pkhd1. All the novel exon junctions have been verified by RT-PCR. RNA-Seq studies in IMCD cells employing target enrichment strategy confirmed virtually all of the novel exon junctions observed in our catalogue. Analyses of exonic splice enhancers using bioinformatics and minigene experiments revealed consensus positions for serine/arginine-rich proteins (SR) that influence alternative exon usage. Site-directed mutagenesis and minigene experiments showed the importance of intronic splice enhancers and polypyrimidine tract in Pkhd1 splicing. Preparation of translationally active mRNA from membrane-bound polysomal fraction showed that Pkhd1 transcripts with novel exon junctions were polysome-bound and hence likely to be involved in the process of translation. Evaluation of human missense variants from Aachen mutation database for ARPKD revealed that R760H variant alters PKHD1 splicing by disrupting one of the SR protein motifs. Taken together, our data demonstrate PKHD1/Pkhd1 transcriptional processing is modulated in part by intragenic factors and dysregulated PKHD1 splicing represents an under-appreciated pathogenic mechanism in ARPKD.

Supplemental data 1.xlsx (12 kB)
Supplemental Data 1 - Primer Sequence

Supplemental data 2.xlsx (10 kB)
Supplemental Data 2 - RT Primer Sequence

Supplemental Data 3 - Sample 5