Functional analysis of PKHD1 splicing in autosomal recessive polycystic kidney disease

Abstract

Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutations in the PKHD1 (polycystic kidney and hepatic disease 1) gene on chromosome 6p12. The longest continuous open reading frame comprises 66 exons encoding a novel 4,074 aa multidomain integral membrane protein (polyductin/fibrocystin) of unknown function. Various alternatively spliced transcripts may additionally result in different isoproteins. Overall, the large size of PKHD1, its complex pattern of splicing, multiple allelism and lack of knowledge of the encoded protein's/proteins' functions pose significant challenges to DNA-based diagnostic testing. Nucleotide substitutions, particularly if residing in regulatory elements or introns outside the splice consensus sites, are often difficult to assess without further functional analyses and cannot be unambiguously classified as diseaseassociated. Investigations on the transcript level, however, are hampered as PKHD1 is not widely expressed in blood lymphocytes. We thus determined the functional significance of the novel splice site mutation c.53-3C>A in intron 2 by RNA analyses by minigeneconstruction. The mutant allele was shown to cause skipping of exon 3. Thus, given the minigene results together with 400 control chromosomes negative for this change, segregation of the mutation with the phenotype, and a significant lowering of the strength of the splice site by bioinformatics, the mutant allele is most likely pathogenic. To the best of our knowledge, this is the first study that defines the consequences of a PKHD1 splice mutation and underlines the relevance of functional analyses in determining the pathogenicity of changes of unknown significance.

In vivo and in vitro splicing assay of SLC12A1 in an antenatal salt-losing tubulopathy patient with an intronic mutation

“…In vitro splicing assays have been conducted for several diseases to identify the splicing abnormalities derived from intronic and sometimes exonic mutations (Thi Tran et al 2005;Tran et al 2006;Takeshima et al 1995;Tran et al 2007). For inherited kidney diseases, Bergmann et al conducted a splicing analysis to conWrm whether the PKHD1 c.53-3C > A in intron 2 mutation leads to exon 3 skipping in the transcripts (Bergmann et al 2006). They used this analysis because they could not get mRNA from kidney specimens and proved that this mutation was disease causing.…”

Polycystic liver diseases

“…This gene consists of at least 86 exons spanning 470 kb on chromosome 6p12 and produces a 16-kb transcript. The longest open reading frame is predicted to include 66 exons and to encode the 4074–amino acid membrane-associated receptor-like protein FPC [13,38]. FPC has a signal peptide, and structural predictions indicate a large extracellular region with multiple copies of the TIG domain (an immunoglobulin-like fold), a single transmembrane region, and a short cytoplasmic tail [39].…”

Fibrocystin/Polyductin Modulates Renal Tubular Formation by Regulating Polycystin-2 Expression and Function

“…The longest open reading frame is predicted to include 66 exons and to encode the 4074 -amino acid membrane-associated receptor-like protein fibrocystin/polyductin (FPC). [23][24][25][26] It was shown that FPC is associated with the basal bodies/primary cilia of epithelial cells [27][28][29][30] and co-localizes with PC2 within the cell. 31 These observations suggest the possibility that FPC and PC2 may function in a common molecular pathway in vivo.…”