A 1-Mb BAC/PAC-based physical map of the autosomal recessive polycystic kidney disease gene (PKHD1) region on chromosome 6

The PKHD1 (polycystic kidney and hepatic disease 1) gene responsible for autosomal recessive polycystic kidney disease has been mapped to 6p21.1-p12 to an approximately 1-cM interval flanked by the markers D6S1714/D6S243 and D6S1024. We have developed a sequence-ready BAC/PAC-based contig map of this region as the next step for the positional cloning of PKHD1. This contig comprising 52 clones spanning approximately 1 Mb was established by content mapping of 44 BAC/PAC-end-derived STSs, 3 known genetic markers, 5 YAC-end-derived STSs, 3 random STSs, 1 previously mapped gene, and 1 EST. The average depth per marker is 6.3 clones, and the average STS density is 20 kb. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. A high-resolution BAC/PAC-based contig map is essential to the ultimate goal of identifying the PKHD1 gene.     

The Severe Perinatal Form of Autosomal Recessive Polycystic Kidney Disease Maps to Chromosome 6p21.1-p12: Implications for Genetic Counseling

Autosomal recessive polycystic kidney disease (ARPKD) is a one of the most common hereditary renal cystic diseases in children. Its clinical spectrum is widely variable with most cases presenting in infancy. Most affected neonates die within the first few hours of life. At present, prenatal diagnosis relies on fetal sonography, which is often imprecise in detecting even the severe form of the disease. Recently, in a cohort of families with mostly milder ARPKD phenotypes, an ARPKD locus was mapped to a 13-cM region of chromosome 6p21-cen. To determine whether severe perinatal ARPKD also maps to chromosome 6p, we have analyzed the segregation of seven microsatellite markers from the ARPKD interval in 22 families with the severe phenotype. In the majority of the affected infants, ARPKD was documented by histopathology. Our data confirm linkage and refine the ARPKD region to a 3.8-cM interval, delimited by the markers D6S465/D6S427/D6S436/D6S272 and D6S466. Taken together, these results suggest that, despite the wide variability in clinical phenotypes, there is a single ARPKD gene. These linkage data and the absence of genetic heterogeneity in all families tested to date have important implications for DNA-based prenatal diagnoses as well as for the isolation of the ARPKD gene.     

PKHD1基因缺陷与常染色体隐性遗传性多囊肾病的研究进展

PKHD1是目前所知人类常染色体隐性遗传多囊肾病(autosomal recessive polycystic kidney disease,ARPKD)的惟一致病基因。ARPKD临床病变以双肾多发性进行性充液囊泡为主要特征。目前对PKHDl基因在ARPKD发病中的作用了解并不多。该文对ARPKD的发病机制和PKHD1基因功能最新研究进展进行综述。     

The mouse polycystic kidney disease mutation (cpk) is located on proximal chromosome 12

The mouse congenital polycystic kidney (cpk) mutation produces a condition that resembles human autosomal recessive polycystic kidney disease (ARPKD) in its pattern of inheritance, clinical progression, and histopathology. Inheritance of this mouse mutation in crosses segregating the Rb(12.14)8Rma translocation chromosome and various DNA markers of Chromosome 12 have localized cpk to a site near D12Nyu2, approximately 7 cM from the centromere of Chromosome 12. This result suggests that the homologous PKD2 gene should be localized to either human chromosome 2p23-p25 or chromosome 7q22-q31.     

The mouse polycystic kidney disease mutation (cpk) is located on proximal chromosome 12

The mouse congenital polycystic kidney (cpk) mutation produces a condition that resembles human autosomal recessive polycystic kidney disease (ARPKD) in its pattern of inheritance, clinical progression, and histopathology. Inheritance of this mouse mutation in crosses segregating the Rb(12.14)8Rma translocation chromosome and various DNA markers of Chromosome 12 have localized cpk to a site near D12Nyu2, approximately 7 cM from the centromere of Chromosome 12. This result suggests that the homologous PKD2 gene should be localized to either human chromosome 2p23-p25 or chromosome 7q22-q31.     

The gene for autosomal dominant polycystic kidney disease lies in a 750-kb CpG-rich region.

PKD1, the locus most commonly affected by mutations that produce autosomal dominant polycystic kidney disease (ADPKD), has previously been localized to chromosome 16p13.3. Since no cytogenetic abnormalities have been found in association with ADPKD, flanking genetic markers have been required to define an interval--the PKD1 region--that contains the PKD1 gene. In this report we demonstrate, through the construction of a long-range restriction map that links the flanking genetic markers GGG1 (D16S84) and 26.6PROX (D16S125), that the PKD1 gene lies within an extremely CpG-rich 750-kb segment of chromosome 16p13.3. Approximately 90% of this region has been cloned in three extensive cosmid/bacteriophage contigs. The cloned DNA is a valuable resource for identifying new closer flanking genetic markers and for isolating candidate genes from the region.     

Molecular and cellular pathophysiology of autosomal recessive polycystic kidney disease (ARPKD)

Autosomal recessive polycystic kidney disease (ARPKD) belongs to a group of congenital hepatorenal fibrocystic syndromes characterized by dual renal and hepatic involvement of variable severity. Despite the wide clinical spectrum of ARPKD (MIM 263200), genetic linkage studies indicate that mutations at a single locus, PKHD1 (polycystic kidney and hepatic disease 1), located on human chromosome region 6p21.1–p12, are responsible for all phenotypes of ARPKD. Identification of cystic disease genes and their encoded proteins has provided investigators with critical tools to begin to unravel the molecular and cellular mechanisms of PKD. PKD cystic epithelia share common phenotypic abnormalities despite the different genetic mutations that underlie the disease. Recent studies have shown that many cyst-causing proteins are expressed in multimeric complexes at distinct subcellular locations within epithelia. This co-expression of cystoproteins suggests that cyst formation, regardless of the underlying disease gene, results from perturbations in convergent and/or integrated signal transduction pathways. To date, no specific therapies are in clinical use for ameliorating cyst growth in ARPKD. However, studies noted in this review suggest that therapeutic targeting of the cAMP and epidermal growth factor receptor (EGFR)-axis abnormalities in cystic epithelia may translate into effective therapies for ARPKD and, by analogy, autosomal dominant polycystic kidney disease (ADPKD). A particularly promising approach appears to be the targeting of downstream intermediates of both the cAMP and EGFR axis. This review focuses on ARPKD and presents a concise summary of the current understanding of the molecular genetics and cellular pathophysiology of this disease. It also highlights phenotypic and mechanistic similarities between ARPKD and ADPKD.The authors are supported by the National Institutes of Health (grant no. 1-P50-DK57306), the PKD Foundation (grant no. 76a2r), and the Children’s Research Institute, Children’s Hospital of Wisconsin.     

An Integrated Genetic and Physical Map of the Autosomal Recessive Polycystic Kidney Disease Region

Autosomal recessive polycystic kidney disease is one of the most common hereditary renal cystic diseases in children. Genetic studies have recently assigned the only known locus for this disorder, PKHD1, to chromosome 6p21–p12. We have generated a YAC contig that spans 5 cM of this region, defined by the markers D6S1253–D6S295, and have mapped 43 sequence-tagged sites (STS) within this interval. This set includes 20 novel STSs, which define 12 unique positions in the region, and three ESTs. A minimal set of two YACs spans the segment D6S465–D6S466, which contains PKHD1, and estimates of their sizes based on information in public databases suggest that the size of the critical region is <3.1 Mb. Twenty-eight STSs map to this interval, giving an average STS density of <1/150 kb. These resources will be useful for establishing a complete trancription map of the PKHD1 region.     

Sequence analysis of the human hTg737 gene and its polymorphic sites in patients with autosomal recessive polycystic kidney disease

DNA sequence analysis of the human Tg737 gene was performed in 36 patients with the autosomal recessive form of polycystic kidney disease (ARPKD). Coding exons and their adjacent splice sites were screened for mutations. Pathogenic exon or splice region mutations were not identified although one exonic and two intronic polymorphic sites were discovered. These results are in agreement with another study that has recently reported linkage to Chromosome (Chr) 6p21-cen in a set of 16 ARPKD families. STS mapping has localized the gene to a YAC contig that includes D13S175 on chromosome 13q12.1. The polymorphisms found in the hTg737 gene will permit its future evaluation as a candidate gene for other recessive cystic renal diseases and as a modifier gene in human PKD.     

Mutations in PRKCSH cause isolated autosomal dominant polycystic liver disease

Autosomal dominant polycystic liver disease (ADPLD) is a distinct clinical and genetic entity that can occur independently from autosomal dominant polycystic kidney disease (ADPKD). We previously studied two large kindreds and reported localization of a gene for ADPLD to an approximately 8-Mb region, flanked by markers D19S586/D19S583 and D19S593/D19S579, on chromosome 19p13.2-13.1. Expansion of these kindreds and identification of an additional family allowed us to define flanking markers CA267 and CA048 in an approximately 3-Mb region containing >70 candidate genes. We used a combination of denaturing high-performance liquid chromatography (DHPLC) heteroduplex analysis and direct sequencing to screen a panel of 15 unrelated affected individuals for mutations in genes from this interval. We found sequence variations in a known gene, PRKCSH, that were not observed in control individuals, that segregated with the disease haplotype, and that were predicted to be chain-terminating mutations. In contrast to PKD1, PKD2, and PKHD1, PRKCSH encodes a previously described human protein termed "protein kinase C substrate 80K-H" or "noncatalytic beta-subunit of glucosidase II." This protein is highly conserved, is expressed in all tissues tested, and contains a leader sequence, an LDLa domain, two EF-hand domains, and a conserved C-terminal HDEL sequence. Its function may be dependent on calcium binding, and its putative actions include the regulation of N-glycosylation of proteins and signal transduction via fibroblast growth-factor receptor. In light of the focal nature of liver cysts in ADPLD, the apparent loss-of-function mutations in PRKCSH, and the two-hit mechanism operational in dominant polycystic kidney disease, ADPLD may also occur by a two-hit mechanism.     

Down-regulation of PKHD1 induces cell apoptosis through PI3K and NF-κB pathways

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). Fibrocystin/polyductin (FPC), encoded by PKHD1, is a membrane-associated receptor-like protein. Although it is widely accepted that cystogenesis is mostly due to aberrant cell proliferation and apoptosis, it is still unclear how apoptosis is regulated. The aim of this study is to analyze the relationship among apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor κB (NF-κB) in FPC knockdown kidney cells. We show that PKHD1-silenced HEK293 cells demonstrate a higher PI3K/Akt activity. Selective inhibition of PI3K/Akt using LY294002 or wortmannin in these cells increases serum starvation-induced HEK293 cell apoptosis with a concomitant decrease in cell proliferation and higher caspase-3 activity. PI3K/Akt inhibition also leads to increased NF-κB activity in these cells. We conclude that the PI3K/Akt pathway is involved in apoptotic function in PKHD1-silenced cells, and PI3K/Akt inhibition correlates with upregulation of NF-κB activity. These observations provide a potential platform for determining FPC function and therapeutic investigation of ARPKD.     

Germline PKHD1 mutations are protective against colorectal cancer

The autosomal recessive polycystic kidney disease (ARPKD) gene, PKHD1, has been implicated in the genesis or growth of colorectal adenocarcinoma, as a high level of somatic mutations was found in colorectal tumor tissue. To determine whether carriers of a single PKHD1 mutation are at increased risk of colorectal carcinoma, we assessed the prevalence of the commonest European mutation, T36M. First, we assayed a European cohort of ARPKD patients and found T36M was responsible for 13.1% of mutations. We then investigated two European cohorts with colorectal adenocarcinoma versus two control cohorts of similar age and gender. Screening for the most common PKHD1 mutation, T36M, we detected 15:3,603 (0.42%) controls versus 1:3,767 (0.027%) colorectal cancer individuals, indicating that heterozygous PKHD1 mutations are not a risk factor and are protective (p=0.0002). We also show that the carriage rate for PKHD1 mutations in the European population is higher than previous accepted at 3.2% (1:31 genomes).