title: Identification of PKHD1 multiexon deletions using multiplex ligation-dependent probe amplification and quantitative polymerase chain reaction.
abstract: BACKGROUND
  Mutations in the PKHD1 gene are responsible for autosomal recessive polycystic kidney disease (ARPKD). Using exon scanning by denaturing high-performance liquid chromatography (dHPLC) or bidirectional sequencing of all exons constituting the longest open reading frame, the mutation detection rate reaches approximately 82% and minor lesion mutations include truncating, splice, and missense mutations.
  
  
  OBJECTIVE
  The main aim of this study was to screen ARPKD patients in whom only one pathogenic PKHD1 mutation was identified after bidirectional sequencing of the longest open reading frame, for gene copy number alterations by employing multiplex ligation-dependent probe amplification complemented with quantitative real-time polymerase chain reaction.
  
  
  RESULTS
  Sixteen ARPKD probands were studied in whom only one clearly pathogenic mutation was previously identified. One patient with a suspected homozygous deletion of the exons 1-37 was also included in this cohort. Three distinct PKHD1 germ-line deletions were identified. Two of these deletions encompassed multiple exons of PKHD1 extending far beyond the 5' and 3' untranslated regions of the gene, and spanning at least 170 and 470 kb, respectively. The third 3.7 kb intragenic deletion affected only exons 20-21 of the PKHD1 gene. Thus, this is the first report presenting analysis of the entire PKHD1 longest open reading frame for gene deletions/duplications in a select cohort of ARPKD patients, in whom previously only one mutation was identified after bidirectional sequencing of the entire longest open reading frame.
  
  
  CONCLUSIONS
  The data indicate that multiplex ligation-dependent probe amplification is a sensitive and rapid method to identify PKHD1 deletions. Our study demonstrates that dosage analysis will increase the PKHD1 mutation detection rate and should be performed as a complementary assay in patients suspected to have ARPKD in the absence of two clear pathogenic mutations.
authors.affiliation: Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA. zverefv@labcorp.com
authors.forename: Val
authors.initials: V
authors.lastname: Zvereff
authors.forename: Suxia
authors.initials: S
authors.lastname: Yao
authors.forename: Julia
authors.initials: J
authors.lastname: Ramsey
authors.forename: Fady M
authors.initials: FM
authors.lastname: Mikhail
authors.forename: Raymon
authors.initials: R
authors.lastname: Vijzelaar
authors.forename: Ludwine
authors.initials: L
authors.lastname: Messiaen
mesh_terms: D015331:Cohort Studies; D004252:DNA Mutational Analysis; D015342:DNA Probes / Q000235:genetics; D005091:Exons; D005260:Female; D018628:Gene Dosage; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D017044:Polycystic Kidney, Autosomal Recessive / Q000175:diagnosis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011956:Receptors, Cell Surface / Q000235:genetics*; D017384:Sequence Deletion; D013997:Time Factors
chemical_list: D015342:DNA Probes; C451750:PKHD1 protein, human; D011956:Receptors, Cell Surface