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.      

Genomic organization and mutation screening of the human ortholog of Pkdr1 associated with polycystic kidney disease in the rat

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited disorders in humans. Although disease-causing mutations have been found in two genes, PKD1 and PKD2, a small number of ADPKD families exist that are unlinked to either of these genes, suggesting involvement of a third, as yet unidentified PKD3 gene. Susceptibility to renal cyst formation in the (cy/+) rat is caused by a missense mutation in Pkdr1 encoding the novel protein SamCystin. To initiate studies of the human orthologous gene, we determined the location and the organization of human PKDR1. We genotyped microsatellite markers flanking the human ortholog in PKD families that either are unlinked to known PKD genes, or in which mutations have not yet been identified and carried out mutation analysis in PKD patients. We identified eight novel single nucleotide polymorphisms, including three leading to amino acid changes. These variants are unlikely to account for PKD in these patients, yet the screening of other affected populations may provide information about the involvement of PKDR1 as a modifier gene in cystic kidney disease.     

Screening of the PKD1 duplicated region reveals multiple single nucleotide polymorphisms and a de novo mutation in Hellenic polycystic kidney disease families

Mutations in the PKD1 gene account for approximately 85% of cases with autosomal dominant polycystic kidney disease (ADPKD1; MIM# 601313), which is considered one of the most frequent monogenic disorders, with a frequency of approximately 1:1000. The main symptom is the formation of fluid-filled cysts in the kidneys and less often in other organs, such as the liver and pancreas. Since the cloning of the gene many mutations have been identified, although the screening is hampered by several unique features of this gene, the most significant one being that approximately 70% of the sequence at the 5'-end, is reiterated elsewhere on chromosome 16 with homology approaching 95%. Here, we used an oligonucleotide primer anchored in the unique part in exon 34, paired with a forward primer in exon 23 for specifically amplifying PKD1 sequences. We screened for mutations in samples from 32 Hellenic ADPKD families. We detected seven sequence variants, five of which most probably are single nucleotide polymorphisms (SNPs), especially useful for linkage analysis and disease association studies. One is a missense change, segregating with ADPKD in one family. The last one is a missense non-conservative change, H2921P, which appeared de novo in the proband, concurrently with the disease phenotype, and was passed on to another two generations. Two siblings who inherited the same haplotype as the proband, but not the de novo mutation, were not affected. This is only the fourth case of a molecularly documented de novo mutation in ADPKD. Somatic mosaicism in peripheral blood leukocytes of the proband was tested and excluded. Hum Mutat 16:176, 2000.     

Mutation analysis in PKD1 of Japanese autosomal dominant polycystic kidney disease patients

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic renal disorder (incidence, 1:1,000). The mutation of PKD1 is thought to account for 85% of ADPKD. Although a considerable number of studies on PKD1 mutation have been published recently, most of them concern Caucasian ADPKD patients. In the present study, we examined PKD1 mutations in Japanese ADPKD patients. Long-range polymerase chain reaction (LR-PCR) with PKD1-specific primers followed by nested PCR was used to analyze the duplicated region of PKD1. Six novel chain-terminating mutations were detected: three nonsense mutations (Q2014X transition in exon 15, Q2969X in exon 24, and E2810X in exon 23), two deletions (2132del29 in exon10 and 7024delAC in exon 15), and one splicing mutation (IVS21-2delAG). There was also one nonconservative missense mutation (T2083I). Two other potentially pathogenic missense mutations (G2814R and L2816P) were on the downstream site of one nonsense mutation. These three mutations and a following polymorphism (8662C>T) were probably the result of gene conversion from one of the homologous genes to PKD1. Six other polymorphisms were found. Most PKD1 mutations in Japanese ADPKD patients were novel and definitely pathogenic. One pedigree did not link to either PKD1 or PKD2.     

Novel mutations in the duplicated region of PKD1 gene

Autosomal dominant polycystic kidney disease (ADPKD) exhibits a genetically heterogeneous transmission involving at least three different genes. PKD1 gene linked mutations are responsible for the disease in about 85% of ADPKD cases. The search for mutations is a very important step in understanding the molecular mechanisms underlying ADPKD. We undertook this study using denaturing gradient gel electrophoresis (DGGE), after a stage of long range PCR, to scan for mutations in the duplicated region of the PKD1 gene in French ADPKD families. This allowed us to identify eight novel mutations and several polymorphisms: among the mutations, three are nonsense mutations, two are deletions in the coding sequence leading to frameshift mutations, one is a splice mutation and two are highly probable missense mutations. In this paper, we also provide a review of the mutations reported so far which are widespread throughout the gene. Although no clear hot spot for mutation is apparent, we will focus on some clustering observed.     

Mutation detection in the duplicated region of the polycystic kidney disease 1 (PKD1) gene in PKD1-linked Australian families

Screening for disease-causing mutations in the duplicated region of the PKD1 gene was performed in 17 unrelated Australian individuals with PKD1-linked autosomal dominant polycystic kidney disease. Exons 2-21 and 23-34 were assayed using PKD1-specific PCR amplification and direct sequencing. We have identified 12 novel probably pathogenic DNA variants, including five truncating mutations (Q563X, c.5105delAT, c.5159delG, S2269X, c.9847delC), two in-frame deletions (c.7472del3, c.9292del39), and two splice-site mutations (IVS14+1G>C, IVS16+1G>T). Three of the mutations (G381C, Y2185D, G2785D) were predicted to lead to the replacement of conserved amino acid residues, with ensuing changes in protein conformation. Defects in the duplicated region of PKD1 thus account for 63% of our patients. Together with the previously detected mutations (Q4041X, R4227P) in the 3 region of the gene, the study has achieved an overall mutation detection rate of 74%. In addition, we have detected 31 variants (nine novel and 22 previously published) that did not segregate with the disease and were considered to be neutral polymorphisms. Three of the nine novel polymorphisms were missense mutations with a predicted effect on protein conformation, emphasizing the problems of interpretation in PKD1 mutation screening.     

Large deletions in the polycystic kidney disease 1 (PKD1) gene

Since identification of the genes mutated in patients with Autosomal Dominant Polycystic Kidney Disease, PKD1 and PKD2, a large number of different germ line mutations in both genes have been found by conventional PCR-based mutation detection methods. Nevertheless, in approximately 40% of the PKD1 families the disease-causing mutation remains to be elucidated. Complex germ line rearrangements are often not detectable by these standard diagnostic techniques. To detect large deletions in the PKD1 gene we performed Field Inversion Gel Electrophoresis (FIGE) followed by Southern blot analysis with probes selected in the unique and in the reiterated region of this gene. Our analysis revealed 4 deletions in 125 patients, indicating that large deletions in PKD1 are rare. Likely, patients with a deletion that also affects the neighbouring Tuberous Sclerosis Complex 2 (TSC2) gene will be diagnosed as patients with tuberous sclerosis. It was speculated that the exceptional polypyrimidine tract located in intron 21 and the small tract in intron 22, might play a role in the pathogenesis of ADPKD. Since this region is extremely difficult to amplify by PCR, we analysed the 5.8 kb BamHI fragment that contains the polypyrimidine tracts. We did not observe a disease-linked alteration although we detected two different rare variants either in PKD1 or in one of its homologues.     

Four novel mutations of the PKD2 gene in Czech families with autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disease caused by mutations in at least three different loci. Mutations in the PKD2 gene are responsible for approximately 15% of the cases of the disease. We have screened 14 Czech families for mutation in the PKD2 gene. Clear evidence against linkage to the PKD1 gene was established by CA-repeat markers in five families. The disease could be linked to both genes according to linkage analysis in nine families but we have chosen these families because of the mild clinical course. An affected member from each family was analyzed by heteroduplex analysis (HA) and single strand conformation polymorphism (SSCP) for all 15 coding regions. Samples exhibiting shifted bands on HA or SSCP gels were sequenced. We detected five mutations (four new, and one which was previously described) and two polymorphisms. The four new mutations include one insertion, one deletion, one substitution (leading to premature translation stop), one amino acid substitution. Our results confirm that different point or small changes distributed throughout the PKD2 gene without clustering are responsible for the disease.     

Genetic evidence for a trans-heterozygous model for cystogenesis in autosomal dominant polycystic kidney disease

Polycystic kidney disease (ADPKD) is a condition with an autosomal dominant mode of inheritance and adult onset. Two forms of the disease, ADPKD1 and ADPKD2, caused by mutations in PKD1 and PKD2, respectively, are very similar, except that ADPKD1 patients run a more severe course. At the cellular level, ADPKD1 was first shown to be recessive, since somatic second hits are perhaps necessary for cyst formation. The near identical phenotype had suggested that ADPKD1 and ADPKD2 might have a similar pathogenesis and that the two gene products, poly- cystins 1 and 2, are part of a common developmental pathway. Work in transgenic mice showed that somatic loss of Pkd2 expression is necessary for renal cyst formation, and recently we showed that somatic mutations inactivating the inherited healthy allele were present in 9 of 23 cysts from a human ADPKD2 kidney, supporting a two-hit loss-of-function model for ADPKD2 cystogenesis. Here, we provide the first direct genetic evidence that polycystins 1 and 2 do interact, perhaps as part of a larger complex. In cystic DNA from a kidney of an ADPKD1 patient, we showed somatic mutations not only in the PKD1 gene of certain cysts, but also in the PKD2 gene of others, generating a trans -heterozygous state with mutations in both genes. One mutation in PKD1 is of germinal nature and the mutation in the PKD2 gene is of somatic nature. The implications of such a situation are enormous, not only for ADPKD, but also for many other conditions with phenotypic heterogeneity and age-dependent penetrance.     

Templated insertions in the rearranged chicken IgL V gene segment arise by intrachromosomal gene conversion

Chickens create a repertoire for their immunoglobulin light-chain gene by a novel process of sequence substitution within a unique rearranged V gene segment (VL1) during B-cell development in the bursa of Fabricius. Sequence analysis has shown that these nucleotide substitutions are not random. Potential donors for observed sequence substitutions are present within the 25 psi VL segments located 5' of the VL1 gene. In this report, we demonstrate that VL1 sequence substitutions: (1) are derived from the psi VL donor segment templates in cis, (2) do not result in reciprocal transfer of VL1 gene sequences to the psi VL segments, and (3) lead to the rapid disappearance of cells with nondiversified rearranged VL1 genes during B-cell development in the bursa of Fabricius. Together, these data provide evidence that VL1 sequence diversity arises as a result of intrachromosomal gene conversion.     

A stable, nonsense mutation associated with a case of infantile onset polycystic kidney disease 1 (PKD1)

Autosomal dominant polycystic kidney disease (ADPKD) is the most common single gene disorder resulting in renal failure. It is generally an adult onset disease, but rarely, cases of severe childhood polycystic disease arise in ADPKD families. The clear clinical anticipation in these pedigrees has led to the suggestion that the mutation may be an unstable trinucleotide repeat. We have now identified a nonsense mutation, Tyr3818Stop, in one such family (P117) within the major ADPKD gene, polycystic kidney disease 1 (PKD1). The mutation is shown to be a de novo change in the father, and of grandpaternal origin. PKD1 manifests as typical adult onset disease in the father, but is seen as severe disease, detected as enlarged polycystic kidneys in utero, in one of a pair of dizygotic twins; the other twin has the mutation but no evidence of cysts, consistent with an adult onset disease course. The finding of the same stable mutation associated with very different disease severity in this family indicates that phenotypic variation in PKD1 is not due to a dynamic mutation. It seems most likely that a small number of modifying factors may radically affect the course of disease in PKD1; identification of such factors will have important prognostic implications in this disorder.      

Mutational analysis of PKD1 gene in a Chinese family with autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disease and common renal disease. Mutations of PKD genes are responsible for this disease. We analyzed a large Chinese family with ADPKD using Sanger sequencing to identify the mutation responsible for this disease. The family comprised 27 individuals including 10 ADPKD patients. These ADPKD patients had severe renal disease and most of them died very young. We analyzed 6 survival patients gene and found they all had C10529T mutation in exon 35 of PKD1 gene. We did not found gene mutation in any unaffected relatives or 300 unrelated controls. These findings suggested that the C10529T mutation in PKD1 gene might be the pathogenic mutation responsible for the disease in this family.     

Gene conversion between functional trypsinogen genes PRSS1 and PRSS2 associated with chronic pancreatitis in a six-year-old girl

Gene conversion--the substitution of genetic material from another gene--is recognized as the underlying cause of a growing number of genetic diseases. While in most cases conversion takes place between a normal gene and its pseudogene, here we report an occurrence of disease-associated gene conversion between two functional genes. Chronic pancreatitis in childhood is frequently associated with mutations of the cationic trypsinogen gene (serine protease 1; PRSS1). We have analyzed PRSS1 in 1106 patients with chronic pancreatitis, and identified a novel conversion event affecting exon 2 and the subsequent intron. The recombination replaced at least 289 nucleotides with the paralogous sequence from the anionic trypsinogen gene (serine protease 2; PRSS2), and resulted in the PRSS1 mutations c.86A > T and c.161A > G, causing the amino acid substitutions N29I and N54S, respectively. Analysis of the recombinant N29I-N54S double mutant cationic trypsinogen revealed increased autocatalytic activation, which was solely due to the N29I mutation. In conclusion, we have demonstrated that gene conversion between two functional paralogous trypsinogen genes can occur and cause genetically determined chronic pancreatitis.     

Evaluating the clinical utility of a molecular genetic test for polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is estimated to affect 1/600-1/1000 individuals worldwide. The disease is characterized by age dependent renal cyst formation that results in kidney failure during adulthood. Although ultrasound imaging may be an adequate diagnostic tool in at risk individuals older than 30, this modality may not be sufficiently sensitive in younger individuals or for those from PKD2 families who have milder disease. DNA based assays may be indicated in certain clinical situations where imaging cannot provide a definitive clinical diagnosis. The goal of this study was to evaluate the utility of direct DNA analysis in a test sample of 82 individuals who were judged to have polycystic kidney disease by standard clinical criteria. The samples were analyzed using a commercially available assay that employs sequencing of both genes responsible for the disorder. Definite disease causing mutations were identified in 34 (approximately 42%) study participants. An additional 30 (approximately 37%) subjects had either in frame insertions/deletions, non-canonical splice site alterations or a combination of missense changes that were also judged likely to be pathogenic. We noted striking sequence variability in the PKD1 gene, with a mean of 13.1 variants per participant (range 0-60). Our results and analysis highlight the complexity of assessing the pathogenicity of missense variants particularly when individuals have multiple amino acid substitutions. We conclude that a significant fraction of ADPKD mutations are caused by amino acid substitutions that need to be interpreted carefully when utilized in clinical decision-making.     

Characterization of 11 new mutations in COL3A1 of individuals with Ehlers-Danlos syndrome type IV: preliminary comparison of RNase cleavage, EMC and DHPLC assays

We report on 12 patients with EDS IV in whom clinical diagnosis was confirmed by biochemical analysis of collagen type III, and further proven by mutation analysis of the COL3A1 gene. Four overlapping RT-PCR products covering the coding sequence for the triple-helical domain of type III collagen were analyzed by direct sequencing. So far, we have identified, 4 base changes at donor splice junctions, and 1 base change at an acceptor splice site, which all affect mRNA splicing; 1 genomic deletion, which removes exon 45; and 6 nucleotide changes, which cause substitutions of glycine residues within the triple helix. Eleven of the 12 identified mutations are newly recognized. Furthermore, we report a preliminary comparison of RNase cleavage, EMC and DHPLC assays in mutation detection in the COL3A1 gene.     

Genetics and phenotypic characteristics of autosomal dominant polycystic kidney disease in Finns

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, leading to renal insufficiency and renal transplantation. Mutation screening in the major gene for ADPKD, the polycystic kidney disease type 1 (PKD1) gene, has often been incomplete because of multiple homologous copies of this gene elsewhere on chromosome 16. Furthermore, there are only a few studies investigating genotype–phenotype correlations in patients with ADPKD. In this study, we screened the entire coding region of the PKD1 and PKD2 genes in 17 Finnish families with ADPKD via long-range polymerase chain reaction, single-strand conformation polymorphism analysis, and direct sequencing. We were able to identify mutations co-segregating with ADPKD in all 16 families linked to PKD1 by haplotype analysis. Of these mutations, six were insertions/deletions, five nonsense mutations, and five missense mutations. In the only PKD2-linked family, we found a missense mutation, R322Q. With the exception of one mutation (L845S in PKD1), all mutations were novel. Mutations and their location did not have a strong correlation with the phenotype with the exception of subarachnoidal hemorrhage or brain aneurysm, where mutations were located more often at the 5 end of the PKD1 gene than at the 3 end of the PKD1 gene.Electronic Supplementary Material Supplementary material is available for this article at .     

Novel PKD1 deletions and missense variants in a cohort of Hellenic polycystic kidney disease families

The autosomal dominant form of polycystic kidney disease is a very frequent genetically heterogeneous inherited condition affecting approximately 1 : 1000 individuals of the Caucasian population. The main symptom is the formation of fluid-filled cysts in the kidneys, which grow progressively in size and number with age, and leading to end-stage renal failure in approximately 50% of patients by age 60. About 85% of cases are caused by mutations in the PKD1 gene on chromosome 16p13.3, which encodes for polycystin-1, a membranous glycoprotein with 4302 amino acids and multiple domains. Mutation detection is still a challenge owing to various sequence characteristics that prevent easy PCR amplification and sequencing. Here we attempted a systematic screening of part of the duplicated region of the gene in a large cohort of 53 Hellenic families with the use of single-strand conformation polymorphism analysis of exons 16-34. Our analysis revealed eight most probably disease causing mutations, five deletions and three single amino acid substitutions, in the REJ domain of the protein. In one family, a 3-bp and an 8-bp deletion in exons 20 and 21 respectively, were co-inherited on the same PKD1 chromosome, causing disease in the mother and three sons. Interestingly we did not find any termination codon defects, so common in the unique part of the PKD1 gene. In the same cohort we identified 11 polymorphic sequence variants, four of which resulted in amino acid variations. This supports the notion that the PKD1 gene may be prone to mutagenesis, justifying the relatively high prevalence of polycystic kidney disease.