title: 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 disease-associated. 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 minigene-construction. 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.
authors.affiliation: Department of Human Genetics, Aachen University, Pauwelsstrasse 30, 52074, Aachen, Germany. cbergmann@ukaachen.de.
authors.forename: Carsten
authors.initials: C
authors.lastname: Bergmann
authors.affiliation: Department of Human Genetics, Aachen University, Pauwelsstrasse 30, 52074, Aachen, Germany.
authors.forename: Valeska
authors.initials: V
authors.lastname: Frank
authors.affiliation: Department of Human Genetics, Aachen University, Pauwelsstrasse 30, 52074, Aachen, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Küpper
authors.affiliation: Department of Human Genetics, Aachen University, Pauwelsstrasse 30, 52074, Aachen, Germany.
authors.forename: Christa
authors.initials: C
authors.lastname: Schmidt
authors.affiliation: Department of Human Genetics, Aachen University, Pauwelsstrasse 30, 52074, Aachen, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Senderek
authors.affiliation: Department of Human Genetics, Aachen University, Pauwelsstrasse 30, 52074, Aachen, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Zerres
mesh_terms: D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D009154:Mutation; D017044:Polycystic Kidney, Autosomal Recessive / Q000235:genetics*; D022821:RNA Splice Sites; D011956:Receptors, Cell Surface / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; C451750:PKHD1 protein, human; D022821:RNA Splice Sites; D011956:Receptors, Cell Surface