Comprehensive genomic analysis of PKHD1 mutations in ARPKD cohorts.

A utosomal recessive polycystic kidney disease (ARPKD; MIM 263200) is an important childhood nephropathy, occurring in 1 in 20 000 live births. The clinical phenotype is dominated by dilatation of the renal collecting ducts, biliary dysgenesis, and portal tract fibrosis. Affected children often present in utero with enlarged, echogenic kidneys, as well as oligohydramnios secondary to poor urine output. Approximately 30% of affected neonates die shortly after birth as a result of severe pulmonary hypoplasia and secondary respiratory insufficiency. Those who survive the perinatal period express widely variable disease phenotypes with systemic hypertension, renal insufficiency, and portal hypertension due to portal tract fibrosis as the most common clinical features. Linkage analysis indicates that mutations in a single locus on chromosome 6p12 are responsible for all typical forms of ARPKD. 4 Two groups working independently have identified PKHD1 (MIM 606702) as the locus responsible for ARPKD and have demonstrated that this novel gene is among the largest in the human genome, extending over at least 470 kb and including a minimum of 86 exons. 6 Both PKHD1 and its mouse orthologue (Pkhd1) encode a complex and extensive array of splice variants, with most abundant transcriptional expression in fetal and adult kidney and weaker expression in other tissues including liver and pancreas. 7 The longest PKHD1 transcript includes 67 exons with an open reading frame (ORF) composed of 66 exons that encode a 4074 amino acid protein, polyductin/fibrocystin. 6 The full length protein is predicted to have several immunoglobulin-like, plexin, transcription factor (IPT) domains and multiple parallel beta-helix 1 (PbH1) repeats in its approximately 3860 amino acid extracellular amino terminus; a single transmembrane (TM) spanning domain; and a short, cytoplasmic carboxyl terminus with potential phosphorylation sites. Alternatively spliced transcripts are predicted to fall into two broad groups. The first subset, polyductin-M, is comprised of polypeptides that contain the single TM element but vary with respect to inclusion of the other predicted domains. The second subset, polyductin-S, lacks the TM domain and thus its members may be secreted. The PKHD1 gene products share structural features with hepatocyte growth factor receptor and plexins, members of a superfamily of proteins involved in regulation of cellular adhesion and repulsion as well as cell proliferation. In addition, recent studies have demonstrated that like other cystoproteins, polyductin/fibrocystin is expressed in the primary apical cilium. 8–12 Based on the available data, ARPKD appears to result from partial or complete loss of polyductin/fibrocystin function. However, the mechanisms by which PKHD1 mutations cause clinical disease phenotypes are not well understood. Gene based analyses have been complicated by the large gene size and reported mutation detection rates have ranged from 47% to 61%. 6 13–15 The limited mutation detection rates and the absence of mutational hot spots in PKHD1 have confounded efforts to examine potential genotype-phenotype correlations. These methodological challenges must be overcome before such correlative analyses are revealing and gene based examination is robust enough for clinical diagnostic testing. In the current study, we have refined the mutation detection strategies and evaluated all 86 predicted exons, including the 67 exons in the longest ORF transcript as well the 19 alternative exons. Our mutation detection rate of 82.7% is the best reported to date in an ethnically diverse population with a wide range of ARPKD associated phenotypes. We have examined potential correlations between disease phenotypes and specific mutational mechanisms and/ or linear positions along PKHD1. Consistent with previous Key points