Comprehensive PKD1 and PKD2 Mutation Analysis in Prenatal Autosomal Dominant Polycystic Kidney Disease

Prenatal forms of autosomal dominant polycystic kidney disease (ADPKD) are rare but can be recurrent in some families, suggesting a common genetic modifying background. Few patients have been reported carrying, in addition to the familial mutation, variation(s) in polycystic kidney disease 1 (PKD1) or HNF1 homeobox B (HNF1B), inherited from the unaffected parent, or biallelic polycystic kidney and hepatic disease 1 (PKHD1) mutations. To assess the frequency of additional variations in PKD1, PKD2, HNF1B, and PKHD1 associated with the familial PKD mutation in early ADPKD, these four genes were screened in 42 patients with early ADPKD in 41 families. Two patients were associated with de novo PKD1 mutations. Forty patients occurred in 39 families with known ADPKD and were associated with PKD1 mutation in 36 families and with PKD2 mutation in two families (no mutation identified in one family). Additional PKD variation(s) (inherited from the unaffected parent when tested) were identified in 15 of 42 patients (37.2%), whereas these variations were observed in 25 of 174 (14.4%, P=0.001) patients with adult ADPKD. No HNF1B variations or PKHD1 biallelic mutations were identified. These results suggest that, at least in some patients, the severity of the cystic disease is inversely correlated with the level of polycystin 1 function.     

Seven novel mutations of the PKD2 gene in families with autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is genetically heterogeneous, with at least three chromosomal loci accounting for the disease. Mutations in the PKD2 gene on the long arm of chromosome 4 are expected to be responsible for approximately 15% of cases of ADPKD. METHODS: We report a systematic screening for mutations covering the 15 exons of the PKD2 gene in eight unrelated families with ADPKD type 2, using the heteroduplex technique. RESULTS: Seven novel mutations were identified and characterized that, together with the previously described changes, amount to a detection rate of 85% in the population studied. The newly described mutations are two nonsense mutations, a 1 bp deletion, a 1 bp insertion, a mutation that involves both a substitution and a deletion (2511AG-->C), a complex mutation in exon 6 consisting of a simultaneous 7 bp inversion and a 4 bp deletion, and the last one is a G-->C transversion that may be a missense mutation. Most of these mutations are expected to lead to the formation of shorter truncated proteins lacking the carboxyl terminus of PKD2. We have also characterized a frequent polymorphism, Arg-Pro, at codon 28 in this gene. The clinical features of these PKD2 patients are similar to the previously described, with the mean age of end-stage renal disease being 75.5 years (SE +/- 3.8 years). CONCLUSIONS: Our results confirm that many different mutations are likely to be responsible for the disease and that most pathogenic defects probably are point or small changes in the coding region of the gene.     

Mutations of the PKD2 gene in Taiwanese patients with autosomal dominant polycystic kidney disease

BACKGROUND: Mutation analysis in the context of clinical phenotypes helps clarify the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). Over 78 PKD2 gene mutations have been reported in the literature, but few have been described from an Asian population. This study attempted to characterize PKD2 mutations and their clinical implications among Taiwanese. METHODS: Twenty unrelated ADPKD patients with uncharacterized genotypes were screened for mutations in the PKD2 gene via single-strand conformation polymorphism (SSCP) of PCR products from genomic DNA, using previously reported PCR conditions and primers. RESULTS: This study identified two novel mutations (C681A and 2136-2137delG) and one mutation (C2407T) previously reported in a Cypriot family. Overall, we found PKD2 mutations in 15% (three out of 20) of the ADPKD patients screened. The mutations included two nonsense mutations (Y227X and R803X) and one frameshift mutation (712-715X) that could all lead to premature termination of translation. The locations of mutations in this study spanned the entire PKD2 gene on exons 2, 11, and 13 without clustering and did not influence the renal disease severity. CONCLUSIONS: The study identified two novel mutations and one recurrent mutation of the PKD2 gene in 20 Taiwanese patients. The characteristics of the mutations in this study resemble those reported among Western populations.     

Novel mutations of PKD1 gene in Chinese patients with autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common disease in China. The major gene responsible for ADPKD, PKD1, has been fully characterized and shown to encode an integral membrane protein, polycystin 1, which is thought to be involved in cell-cell and cell-matrix interaction. Until now, 82 mutations of PKD1 gene have been reported in European, American, and Asian populations. However, there has been no report on mutations of the PKD1 gene in a Chinese population. METHODS: Eighty Chinese patients in 60 families with ADPKD were screened for mutations in the 3' region of the PKD1 gene using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA-sequencing techniques. RESULTS: Three mutations were found. The first mutation is a 12593delA frameshift mutation in exon 45, and the polycystin change is 4129WfsX4197, 107 amino acids shorter than the normal polycystin (4302aa). The second mutation is a 12470InsA frameshift mutation in exon 45, producing 4088DfsX4156, and the predicted protein is 148 amino acids shorter than the normal. The third one is a 11151C-->T transition in exon 37 converting Pro3648 to Leu. In addition, nine DNA variants, including IVS44delG, were identified. CONCLUSIONS: Three mutations in Chinese ADPKD patients are described and all of them are de novo mutations. Data obtained from mutation analysis also suggests that the mutation rate of the 3' single-copy region of PKD1 in Chinese ADPKD patients is very low, and there are no mutation hot spots in the PKD1 gene. Mutations found in Chinese ADPKD patients, including nucleotide substitution and minor frameshift, are similar to the findings reported by other researchers. Many mutations of the PKD1 gene probably exist in the duplicated region, promoter region, and the introns of PKD1.     

Thirteen novel mutations of the replicated region of PKD1 in an Asian population

BACKGROUND: Mutations of PKD1 are thought to account for approximately 85% of all mutations in autosomal dominant polycystic kidney disease (ADPKD). The search for PKD1 mutations has been hindered by both its large size and complicated genomic structure. To date, few mutations that affect the replicated segment of PKD1 have been described, and virtually all have been reported in Caucasian patients. METHODS: In the present study, we have used a long-range polymerase chain reaction (PCR)-based strategy previously developed by our laboratory to analyze exons in the replicated region of PKD1 in a population of 41 unrelated Thai and 6 unrelated Korean families with ADPKD. We have amplified approximately 3.5 and approximately 5 kb PKD1 gene-specific fragments (5'MR and 5'LR) containing exons 13 to 15 and 15 to 21 and performed single-stand conformation analysis (SSCA) on nested PCR products. RESULTS: Nine novel pathogenic mutations were detected, including six nonsense and three frameshift mutations. One of the deletions was shown to be a de novo mutation. Four potentially pathogenic variants, including one 3 bp insertion and three missense mutations, were also discovered. Two of the nonconservative amino acid substitutions were predicted to disrupt the three-dimensional structure of the PKD repeats. In addition, six polymorphisms, including two missense and four silent nucleotide substitutions, were identified. Approximately 25% of both the pathogenic and normal variants were found to be present in at least one of the homologous loci. CONCLUSION: To our knowledge, this is the first report of mutation analysis of the replicated region of PKD1 in a non-Caucasian population. The methods used in this study are widely applicable and can be used to characterize PKD1 in a number of ethnic groups using DNA samples prepared using standard techniques. Our data suggest that gene conversion may play a significant role in producing variability of the PKD1 sequence in this population. The identification of additional mutations will help guide the study of polycystin-1 and better help us to understand the pathophysiology of this common disease.     

A novel frameshift mutation (2436insT) produces an immediate stop codon in the autosomal dominant polycystic kidney disease 2 (PKD2) gene.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder that can be caused by mutations in at least three different genes. Several mutations have been identified in PKD1 and PKD2 genes. Most of the mutations found in PKD2 gene are predicted to cause premature termination of the protein. METHODS: We analysed an Argentinian family characterized previously as PKD2. The PKD2 gene was amplified from genomic DNA using 17 primer pairs and the products were analysed by heteroduplex analysis. PCR products that showed a variation by heteroduplex analysis were sequenced directly. The mutation was confirmed by sequencing relatives. The segregation of the mutation in this family was verified by restriction endonuclease digestion of PCR products obtained from genomic DNA of all family members. Results and conclusions. Here, we report a novel mutation present in an Argentinian family characterized as PKD2 by linkage analysis. The mutation, shared by all affected members of the family, is a thymidine insertion at position 2436 of the gene, which results in a translation frameshift and creates an immediate stop codon. This mutation is expected to lead to a truncated protein that lacks the interacting domain with the PKD1 gene product. The thymidine insertion abolished a Ddel restriction site, allowing a rapid test for detection of PKD2 carriers in the family.     

Genotype-renal function correlation in type 2 autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a common Mendelian disorder that affects approximately 1 in 1000 live births. Mutations of two genes, PKD1 and PKD2, account for the disease in approximately 80 to 85% and 10 to 15% of the cases, respectively. Significant interfamilial and intrafamilial renal disease variability in ADPKD has been well documented. Locus heterogeneity is a major determinant for interfamilial disease variability (i.e., patients from PKD1-linked families have a significantly earlier onset of ESRD compared with patients from PKD2-linked families). More recently, two studies have suggested that allelic heterogeneity might influence renal disease severity. The current study examined the genotype-renal function correlation in 461 affected individuals from 71 ADPKD families with known PKD2 mutations. Fifty different mutations were identified in these families, spanning between exon 1 and 14 of PKD2. Most (94%) of these mutations were predicted to be inactivating. The renal outcomes of these patients, including the age of onset of end-stage renal disease (ESRD) and chronic renal failure (CRF; defined as creatinine clearance < or = 50 ml/min, calculated using the Cockroft and Gault formula), were analyzed. Of all the affected individuals clinically assessed, 117 (25.4%) had ESRD, 47 (10.2%) died without ESRD, 65 (14.0%) had CRF, and 232 (50.3%) had neither CRF nor ESRD at the last follow-up. Female patients, compared with male patients, had a later mean age of onset of ESRD (76.0 [95% CI, 73.8 to 78.1] versus 68.1 [95% CI, 66.0 to 70.2] yr) and CRF (72.5 [95% CI, 70.1 to 74.9] versus 63.7 [95% CI, 61.4 to 66.0] yr). Linear regression and renal survival analyses revealed that the location of PKD2 mutations did not influence the age of onset of ESRD. However, patients with splice site mutations appeared to have milder renal disease compared with patients with other mutation types (P < 0.04 by log rank test; adjusted for the gender effect). Considerable renal disease variability was also found among affected individuals with the same PKD2 mutations. This variability can confound the determination of allelic effects and supports the notion that additional genetic and/or environmental factors may modulate the renal disease severity in ADPKD.     

Somatic PKD2 mutations in individual kidney and liver cysts support a "two-hit" model of cystogenesis in type 2 autosomal dominant polycystic kidney disease.

An intriguing feature of autosomal dominant polycystic kidney disease (ADPKD) is the focal and sporadic formation of renal and extrarenal cysts. Recent documentation of somatic PKD1 mutations in cystic epithelia of patients with germ-line PKD1 mutations suggests a "two-hit" model for cystogenesis in type 1 ADPKD. This study tests whether the same mechanism for cystogenesis might also occur in type 2 ADPKD. Genomic DNA was obtained from 54 kidney and liver cysts from three patients with known germ-line PKD2 mutations, using procedures that minimize contamination of cells from noncystic tissue. Using intragenic and microsatellite markers, these cyst samples were screened for loss of heterozygosity. The same samples were also screened for somatic mutations in five of the 15 exons in PKD2 by single-stranded conformational polymorphism analysis. Loss of heterozygosity was found in five cysts, and unique intragenic mutations were found in seven other cysts. In 11 of these 12 cysts, it was also determined that the somatic mutation occurred nonrandomly in the copy of PKD2 inherited from the unaffected parent. These findings support the "two-hit" model as a unified mechanism for cystogenesis in ADPKD. In this model, the requirement of a somatic mutation as the rate-limiting step for individual cyst formation has potential therapeutic implications.     

Autosomal dominant polycystic kidney disease coexisting with cystic fibrosis

Autosomal dominant polycystic kidney disease (ADPKD) is a common, inherited disorder characterized by the progressive enlargement of fluid-filled cysts in the kidneys and liver. Since the cystic fibrosis transmembrane conductance regulator (CFTR) Cl--channel may mediate the secretion of Cl--and fluid into the cysts, it is conceivable that coexisting cystic fibrosis (CF) in patients with ADPKD could attenuate their development. We previously reported that two patients with ADPKD coexisting with cystic fibrosis (CF) had a milder cystic phenotype than that of kindred without CFTR mutations. A subsequent report failed to confirm this protective effect. We now have identified another family with coexisting type 1 ADPKD and CF. The kidney volumes and the number and size of renal and hepatic cysts were markedly less in a member of this family with ADPKD (PKD1 mutation C508R) and CF (homozygous DeltaF508 mutation) than in her sister with ADPKD alone at comparable ages.     

Adult patients with sporadic polycystic kidney disease: the importance of screening for mutations in the PKD1 and PKD2 genes

Background

ADPKD is one of the most common inherited disorders, with high risk for end-stage renal disease. Numerous patients, however, have no relatives in whom this disorder is known and are unsure whether they may transmit the disease to their offsprings. The aim of this study was to evaluate whether germline mutation analysis adds substantial information to clinical symptoms for diagnosis of ADPKD in these patients.

Methods

Clinical data included renal function and presence of liver or pancreas cysts, heart valve insufficiency, intracranial aneurysms, colonic diverticles, and abdominal hernias. Family history was evaluated regarding ADPKD. Germline mutation screening of the PKD1 and PKD2 genes was performed for intragenic mutations and for large deletions.

Results

A total of 324 adult patients with ADPKD including 30 patients without a family history of ADPKD (sporadic cases) were included. PKD1 mutations were found in 24/30 and PKD2 mutations in 6 patients. Liver cysts were present in 14 patients and intracranial aneurysms in 2 patients. Fourteen patients (45%) had no extrarenal involvement. Compared to the 294 patients with familial ADPKD, the clinical characteristics and the age at the start of dialysis were similar in those with sporadic ADPKD.

Conclusion

The clinical characteristics of patients with sporadic and familial ADPKD are similar, but sporadic ADPKD is often overlooked because of the absence of a family history. Molecular genetic screening for germline mutations in both PKD1 and PKD2 genes is essential for the definitive diagnosis of ADPKD.     

Polycystic kidney disease genes and polycystins

Conclusion The past decade has seen extraordinary progress in the study of autosomal-dominant polycystic kidney disease. The 2 major genes for this disorder have been identified. Animal models of ADPKD have been produced. The molecular basis of the disease has been characterized. ADPKD is a “second-hit” disease, much like many cancer predisposition syndromes. This has profound implications for our understanding. The progression of ADPKD in individual patients is likely related more to their individual rate of acquisition of second hits at thePKD1 orPKD2 locus than to the inherited germ line mutation itself. Therapeutic approaches will perhaps now be considered, which will include interventions that may limit the rate at which somatic mutations occur in the kidney. The major focus of research at present is to elucidate the normal functions ofPKD1 andPKD2. Protein binding partners are being sought for both proteins. The possible calcium channel function ofPKD2 is being investigated. The downstream effects of cellular deficiency of either protein are likely to yield many clues. Modifying genetic factors that may independently affect disease progression are likely to be identified using the several mouse models. Perhaps the next decade will bring great strides in understanding and in potential therapy for this common disease. This paper was presented at the 2nd International Forum “The Frontiers of Nephrology,” Tokyo, May 10, 1998.     

Identification of gene mutations in autosomal dominant polycystic kidney disease through targeted resequencing

Mutations in two large multi-exon genes, PKD1 and PKD2, cause autosomal dominant polycystic kidney disease (ADPKD). The duplication of PKD1 exons 1-32 as six pseudogenes on chromosome 16, the high level of allelic heterogeneity, and the cost of Sanger sequencing complicate mutation analysis, which can aid diagnostics of ADPKD. We developed and validated a strategy to analyze both the PKD1 and PKD2 genes using next-generation sequencing by pooling long-range PCR amplicons and multiplexing bar-coded libraries. We used this approach to characterize a cohort of 230 patients with ADPKD. This process detected definitely and likely pathogenic variants in 115 (63%) of 183 patients with typical ADPKD. In addition, we identified atypical mutations, a gene conversion, and one missed mutation resulting from allele dropout, and we characterized the pattern of deep intronic variation for both genes. In summary, this strategy involving next-generation sequencing is a model for future genetic characterization of large ADPKD populations.     

Genotype-phenotype correlations in autosomal dominant and autosomal recessive polycystic kidney disease

The phenotypes that are associated with the common forms of polycystic kidney disease (PKD)--autosomal dominant (ADPKD) and autosomal recessive (ARPKD)--are highly variable in penetrance. This is in terms of severity of renal disease, which can range from neonatal death to adequate function into old age, characteristics of the liver disease, and other extrarenal manifestations in ADPKD. Influences of the germline mutation are at the genic and allelic levels, but intrafamilial variability indicates that genetic background and environmental factors are also key. In ADPKD, the gene involved, PKD1 or PKD2, is a major factor, with ESRD occurring 20 yr later in PKD2. Mutation position may also be significant, especially in terms of the likelihood of vascular events, with 5' mutations most detrimental. Variance component analysis in ADPKD populations indicates that genetic modifiers are important, but few such factors (beyond co-inheritance of a TSC2 mutation) have been identified. Hormonal influences, especially associated with more severe liver disease in female individuals, indicate a role for nongenetic factors. In ARPKD, the combination of mutations is critical to the phenotypic outcome. Patients with two truncating mutations have a lethal phenotype, whereas the presence of at least one missense change can be compatible with life, indicating that many missense changes are hypomorphic alleles that generate partially functional protein. Clues from animal models and other forms of PKD highlight potential modifiers. The information that is now available on both genes is of considerable prognostic value with the prospects from the ongoing genetic revolution that additional risk factors will be revealed.     

PKD2 mutations in a Czech population with autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is genetically heterogeneous and caused by mutations in at least three different loci. Based on linkage analysis, mutations in the PKD2 gene are responsible for approximately 15% of the cases. PKD2-linked ADPKD is supposed to be a milder form of the disease, its mean age of end-stage renal failure (ESRF) approximately 20 years later than PKD1. METHODS: We screened all coding sequences of the PKD2 gene in 115 Czech patients. From dialysis centres in the Czech Republic and from the Department of Nephrology of the General Hospital in Prague, we selected 52 patients (29 males, 23 females), who reached ESRF after the age of 63, and 10 patients (three males, seven females) who were not on renal replacement therapy at that age. The age of 63 was used as the cut-off because it is between the recently published ages of onset of ESRF for PKD1 and PKD2. From PKD families we also selected 53 patients (26 males, 27 females) who could be linked to either the PKD1 or PKD2 genes by linkage analysis. An affected member from each family was analysed by heteroduplex analysis (HA) for all 15 coding regions. Samples exhibiting shifted bands on gels were sequenced. RESULTS: We detected 22 mutations (six new mutations)-14 mutations in 62 patients (23%) with mild clinical manifestations, eight in 53 families (15%) with possible linkage to both PKD genes. As the detection rate of HA is approximately 70-80%, we estimate the prevalence of PKD2 cases in the Czech ADPKD population to be 18-20%. We identified nonsense mutations in eight patients (36.5%), frameshifting mutations in 12 patients (54.5%) and missense mutations in two patients (9%). CONCLUSION: In this study in the Czech population we identified 22 mutations (six of which were new mutations). The prevalence of PKD2 cases was 18-20% and the mean age of ESRF was 68.3 years. An at-least weak hot spot in exon 1 of the PKD2 gene was found.     

Putative mutation of PKD1 gene responsible for autosomal dominant polycystic kidney disease in a Chinese family

Autosomal dominant polycystic kidney disease (ADPKD) is a common and severe renal disease. Mutations of PKD1 and PKD2 genes are responsible for approximately 85% and 15% of ADPKD cases, respectively. In the present study, PKD1 and PKD2 genes were analyzed in a large Chinese family with ADPKD using denaturing high‐performance liquid chromatography and DNA sequencing. A novel mutation, c.3623‐3624insGTGT in exon 15 of the PKD1 gene, was identified in all nine affected family members, but not in any unaffected consanguineous relatives or 100 unrelated controls. These findings suggest that the unique 4 bp insertion, c.3623‐3624insGTGT, in the PKD1 gene might be the pathogenic mutation responsible for the disease in this family.     

Autosomal dominant polycystic kidney disease with minimal clinical expression unlinked to the PKD1 locus

Autosomal dominant polycystic kidney disease (ADPKD) is causedby mutations at the PKD1 locus in most families. This locushas been assigned to the short arm of chromosome 16 by linkageanalysis. It has been estimated that approximately 5% of familieshave a disease that does not map to this locus and most of thesefamilies have clinical features indistinguishable from the diseasecaused by PKD1 mutations. We report a large three-generationCaucasian family from Northern Ireland with ADPKD in whom allaffected individuals (age range 22–68) were normotensiveand only the two eldest had mild renal impairment. Linkage wasexcluded between the disease and both the alpha-globin genecomplex and the microsatellite marker D16S283. This family confirmsthat phenotypic heterogeneity exists between unlinked familiesand that certain non-PKD1 mutations cause mild disease expression.Many such individuals may therefore remain undetected and theincidence of families with ADPKD who have non-PKDl mutationsmay be greater than previously estimated.     

Characterization of large rearrangements in autosomal dominant polycystic kidney disease and the PKD1/TSC2 contiguous gene syndrome

Large DNA rearrangements account for about 8% of disease mutations and are more common in duplicated genomic regions, where they are difficult to detect. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2. PKD1 is located in an intrachromosomally duplicated region. A tuberous sclerosis gene, TSC2, lies immediately adjacent to PKD1 and large deletions can result in the PKD1/TSC2 contiguous gene deletion syndrome. To rapidly identify large rearrangements, a multiplex ligation-dependent probe amplification assay was developed employing base-pair differences between PKD1 and the six pseudogenes to generate PKD1-specific probes. All changes in a set of 25 previously defined deletions in PKD1, PKD2 and PKD1/TSC2 were detected by this assay and we also found 14 new mutations at these loci. About 4% of the ADPKD patients in the CRISP study were found to have gross rearrangements, and these accounted for about a third of base-pair mutation negative families. Sensitivity of the assay showed that about 40% of PKD1/TSC contiguous gene deletion syndrome families contained mosaic cases. Characterization of a family found to be mosaic for a PKD1 deletion is discussed here to illustrate family risk and donor selection considerations. Our assay improves detection levels and the reliability of molecular testing of patients with ADPKD.     

A complete mutation screen of the ADPKD genes by DHPLC

BACKGROUND: Genetic analysis is a useful diagnostic tool in autosomal dominant polycystic kidney disease (ADPKD), especially when imaging results are equivocal. However, molecular diagnostics by direct mutation screening has proved difficult in this disorder due to genetic and allelic heterogeneity and complexity of the major locus, PKD1. METHODS: A protocol was developed to specifically amplify the exons of PKD1 and PKD2 from genomic DNA as 150 to 450 bp amplicons. These fragments were analyzed by the technique of denaturing high-performance liquid chromatography (DHPLC) using a Wave Fragment Analysis System (Transgenomics) to detect base-pair changes throughout both genes. DHPLC-detected changes were characterized by sequencing. RESULTS: Cost effective and sensitive mutation screening of the entire coding regions of PKD1 and PKD2 by DHPLC was optimized. All base-pair mutations to these genes that we previously characterized were detected as an altered DHPLC profile. To assess this method for routine diagnostic use, samples from a cohort of 45 genetically uncharacterized ADPKD patients were analyzed. Twenty-nine definite mutations were detected, 26 PKD1, 3 PKD2 and a further five possible missense mutations were characterized leading to a maximal detection rate of 76%. A high level of polymorphism of PKD1 also was detected, with 71 different changes defined. The reproducibility of the DHPLC profile enabled the recognition of many common polymorphisms without the necessity for re-sequencing. CONCLUSIONS: DHPLC has been demonstrated to be an efficient and effective means for gene-based molecular diagnosis of ADPKD. Differentiating missense mutations and polymorphisms remains a challenge, but family-based segregation analysis is helpful.