Region Of PKD1 Research Articles

Murine Pkd1 is a developmentally regulated gene from morula to adulthood: role in tissue condensation and patterning.

PKD1 is the most common genetically mutated gene involved in autosomal dominant polycystic kidney disease (ADPKD). Our previous studies have shown that the pathogenesis of human and murine polycystic kidney disease (PKD) involves failure to switch out of a renal developmental program, suggesting a role for PKD1 in development. To investigate this hypothesis, we have cloned a portion of the murine Pkd1 gene and characterized the fetal to adult tissue expression pattern of Pkd1. We chose to clone the transmembrane region of Pkd1, a region prone to mutations in ADPKD. The transmembrane coding region (2.6 kb) has 80.3% nucleotide homology with human PKD1 and 85.3% amino acid similarity. The cloned murine Pkd1 fragment closely resembles that of human PKD1 with respect to both genomic size and exon/intron position. We have demonstrated that this Pkd1 region is not conserved in lower organisms and is mammalian specific. A detailed expression analysis of Pkd1 revealed expression as early as the morula stage and in ES cells with differential expression levels in various tissues/organs throughout development. Highest expression levels were observed in the early condensing mesenchyme of primitive mesoderm and ectoderm. Pkd1 was also expressed at high levels in developing neural tube, neural crest derivatives, prechondrogenic tissue, metanephros, bladder, salivary glands, lung, and blood vessels with lower expression levels in other organs and tissues. Specific spatial and temporal patterns of Pkd1 expression were demonstrated in individual organs, such as lung, kidney, brain, indicating it is highly developmentally regulated. Particularly high levels persisted in mature derivatives of neural tube, neural crest, chondrogenic tissue, metanephros, and lung. In summary, our data suggest that Pkd1 has at least two cellular functions, one a basic function involved in early tissue condensation processes, and the other a mammalian-specific function, that evolved with tissue patterning and tubulogenesis in metanephric and pulmonary development.

Gene conversion is a likely cause of mutation in PKD1.

Approximately 70% of the gene responsible for the most common form of autosomal dominant polycystic kidney disease ( PKD1 ) is replicated in several highly homologous copies located more proximally on chromosome 16. We recently have described a novel technique for mutation detection in the duplicated region of PKD1 that circumvents the difficulties posed by these homologs. We have used this method to identify two patients with a nearly identical cluster of base pair substitutions in exon 23. Since pseudogenes are known to be reservoirs for mutation via gene conversion events for a number of other diseases, we decided to test whether these sequence differences in PKD1 could have arisen as a result of this mechanism. Using changes in restriction digest patterns, we were able to show that these sequence substitutions are also present in N23HA, a rodent-human somatic cell hybrid that contains only the PKD1 homologs. Moreover, these changes were also detected in total DNA from several affected and unaffected individuals that did not harbor this mutation in their PKD1 gene copy. This is the first example of gene conversion in PKD1 , and our findings highlight the importance of using gene-specific reagents in defining PKD1 mutations.

Facilitated diagnosis of the contiguous gene syndrome: Tuberous sclerosis and polycystic kidneys by means of haplotype studies

Tuberous sclerosis (TSC) and autosomal dominant polycystic kidney disease (ADPKD) are genetically heterogeneous diseases. The major gene for ADPKD (PKD1) lies adjacent to the TSC2 gene on chromosome 16p13. Some reports in the literature referred to an unusual presentation of TSC with enlarged cystic kidneys at birth, but it was not until the localization of the TSC2 and PKD1 genes that it was possible to analyze the interaction between both genes. We describe a case of a child with TSC and enlarged cystic kidneys. The study of genetic marker segregation in the family pointed to the presence of a deletion involving the 3' region of PKD1. A further study of the region showed a deletion of 40 kb involving both PKD1 and TSC2. We suggest that an additive or synergistic effect between PKD1 and TSC2 may cause this renal phenotype. A contiguous gene syndrome involving PKD1 and TSC2 should be suspected in children with TSC and enlarged polycystic kidneys at birth. The first approach to identify a deletion of both genes could be the analysis of the segregation of PKD1 and TSC2 markers in the family. (Am J Kidney Dis 1998 Jun;31(6):1038-43)

An unusual pattern of mutation in the duplicated portion of PKD1 is revealed by use of a novel strategy for mutation detection.

The gene for the most common and severe form of autosomal dominant polycystic kidney disease, PKD1, encodes a 14 kb mRNA that is predicted to result in an integral membrane protein of 4302 amino acids. The major challenge faced by researchers attempting to complete mutation analysis of the PKD1 gene has been the presence of several homologous loci also located on chromosome 16. Because the sequence of PKD1 and its homologs is nearly identical in the 5' region of the gene, most traditional approaches to mutation analysis cannot distinguish sequence variants occurring uniquely in PKD1. Therefore, only a small number of mutations have been identified to date and these have all been found in the 3', unique portion of the gene. In order to begin analysis of the duplicated region of PKD1, we have devised a novel strategy that depends on long-range PCR and a single gene-specific primer from the unique region of the gene to amplify a PKD1-specific template that spans exons 23-34. This 10 kb template, amplified from genomic DNA, can be employed for mutation analysis using a wide variety of sequence-based approaches. We have used our long-range PCR strategy to begin screening for sequence variants with heteroduplex analysis, and several affected individuals were discovered to have clusters of base pair substitutions in exons 23 and 25. In two patients, these changes, identified in exon 23, would be predicted to result in multiple amino acid substitutions in a short stretch of the protein. This clustering of base pair substitutions is unusual and suggests that mutation may result from unique structural features of the PKD1 gene.

Identification of Mutations in the Duplicated Region of the Polycystic Kidney Disease 1 Gene ( PKD1) by a Novel Approach

Mutation screening of the major autosomal dominant polycystic kidney disease gene (PKD1) has been complicated by the large transcript size (> 14 kb) and by reiteration of the genomic area encoding 75% of the protein on the same chromosome (the HG loci). The sequence similarity between the PKD1 and HG regions has precluded specific analysis of the duplicated region of PKD1, and consequently all previously described mutations map to the unique 3' region of PKD1. We have now developed a novel anchored reverse-transcription-PCR (RT-PCR) approach to specifically amplify duplicated regions of PKD1, employing one primer situated within the single-copy region and one within the reiterated area. This strategy has been incorporated in a mutation screen of 100 patients for more than half of the PKD1 exons (exons 22-46; 37% of the coding region), including 11 (exons 22-32) within the duplicated gene region, by use of the protein-truncation test (PTT). Sixty of these patients also were screened for missense changes, by use of the nonisotopic RNase cleavage assay (NIRCA), in exons 23-36. Eleven mutations have been identified, six within the duplicated region, and these consist of three stop mutations, three frameshifting deletions of a single nucleotide, two splicing defects, and three possible missense changes. Each mutation was detected in just one family (although one has been described elsewhere); no mutation hot spot was identified. The nature and distribution of mutations, plus the lack of a clear phenotype/genotype correlation, suggest that they may inactivate the molecule. RT-PCR/PTT proved to be a rapid and efficient method to detect PKD1 mutations (differentiating pathogenic changes from polymorphisms), and we recommend this procedure as a firstpass mutation screen in this disorder.

Loss of the polycystic kidney disease (PKD1) region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis.

It is not known whether mutations in the PKD1 gene cause autosomal dominant polycystic kidney disease (PKD) by an activating (gain-of-function) or an inactivating (loss-of-function) model. We analyzed DNA from cyst epithelial cells for loss of heterozygosity (LOH) in the PKD1 region of chromosome 16p13 using microsatellite markers. 29 cysts from four patients were studied. Five cysts from three patients had chromosome 16p13 LOH. Four of the cysts had loss of two chromosome 16p13 markers that flank the PKD1 gene. In two patients, microsatellite analysis of family members was consistent with loss of the wild-type copy of PKD1 in the cysts. In the third patient, 16p13 LOH was detected in three separate cysts, all of which showed loss of the same alleles. Chromosome 3p21 LOH was detected in one cyst. No LOH was detected in four other genomic regions. These results demonstrate that some renal cyst epithelial cells exhibit clonal chromosomal abnormalities with loss of the wild-type copy of PKD1. This supports a loss-of-function model for autosomal dominant PKD, with a germline mutation inactivating one copy of PKD1 and somatic mutation or deletion inactivating the remaining wild-type copy.

A kindred exhibiting cosegregation of an overlap connective tissue disorder and the chromosome 16 linked form of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a disorder of adult onset manifested by bilaterally enlarged cystic kidneys frequently associated with progressive renal failure. The mutated gene (PKD1) responsible for 85 to 95% of cases has been localized to a small segment on the distal tip of the short arm of chromosome 16. A clinical spectrum of heritable connective tissue disorders that remain unclassifiable under the present nosology but that contain elements of the Marfan's syndrome have previously been described. The genetic localization and molecular basis of such overlap connective tissue disorders (OCTD) have not been elucidated. In this report, a kindred in which ADPKD and OCTD appear to cosegregate is described. The connective tissue phenotype in this family includes aortic root dilation, aortic and vertebral artery aneurysms with dissection, and aortic valve incompetence, as well as pectus abnormalities, pes planus, joint laxity, arachnodactyly, scoliosis, dolichostenomelia, and high arched palate. ADPKD was manifest primarily as bilateral renal cysts with or without renal failure. The DNA of all living family members was studied with markers recognizing polymorphic loci flanking the PKD1 region (3'HVR and O90a), as well as markers from the loci of chromosomes 15 and 5, associated with fibrillin genes FBN1 and FBN2, respectively. In this kindred of 20 family members traced through five generations, cosegregation of ADPKD and the OCTD phenotype was observed in 12 of 12 meioses and 3 of 3 phase known. Both markers for PKD1 were tightly linked to both ADPKD and OCTD, whereas there was no evidence for linkage with either fibrillin locus. In this family, the ADPKD and OCTD mutations are genetically linked. The presence of OCTD with ADPKD identifies a group of patients at significantly greater risk for sudden death from aortic root and other vascular aneurysmal dissection and rupture.

Icelandic families with autosomal dominant polycystic kidney disease: families unlinked to chromosome 16p13.3 revealed by linkage analysis

We have mainly used 3 highly polymorphic DNA markers, 3'HVR (D16S85), 16AC2.5 (D16S291) and SM7 (D16S283), flanking the PKD1 region on chromosome 16p13.3 to establish linkage status in seven Icelandic families with autosomal dominant polycystic kidney disease (ADPKD). In four families, the disease locus is in the PKD1 region, and three families are "unlinked" to chromosome 16p13.3. In one of the "unlinked" families, the disease locus is excluded from a part of the long arm of chromosome 2, and we support a theory of more than 2 loci being responsible for ADPKD. Our data confirm the location of the locus YNH24 (D2S44) to chromosome 2q13-q24.

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.

Cosmid walking and chromosome jumping in the region of PKD1 reveal a locus duplication and three CpG islands

The locus responsible for the most common form of autosomal dominant polycystic kidney disease (PKD1) is located on chromosome 16p13.3. Genetic mapping studies indicate that PKD1 is flanked on the proximal side by the DNA marker 26.6 (D16S125). Here we show that 26.6 has undergone a locus duplication and that the two loci are less than 150kb apart. One of the two loci contains a polymorphic TaqI site that has been used in genetic studies and represents the proximal boundary for the PKD1 locus. We demonstrate that the polymorphic locus is the more proximal of the two 26.6-hybridizing loci. Therefore, four cosmids isolated from the distal 26.6-hybridizing locus contain candidate sequences for the PKD1 gene. These cosmids were found to contain two CpG islands that are likely markers for transcribed regions. A third CpG island was detected and cloned by directional chromosome jumping.