Bilineal inheritance of PKD1 abnormalities mimicking autosomal recessive polycystic disease

Background

Dominant polycystic kidney disease is common and usually presents clinically in adulthood. Recessive polycystic kidney disease is much less common and frequently presents antenatally or in the neonatal period with severe renal involvement. These are usually thought of as clinically distinct entities but diagnostic confusion is not infrequent.

Case-diagnosis/treatment

We describe an infant with antenatally diagnosed massive renal enlargement and oligohydramnios with no resolvable cysts on ultrasound scanning. He underwent bilateral nephrectomy because of respiratory compromise and poor renal function but died subsequently of overwhelming sepsis. Genetic analysis revealed that he had bilineal inheritance of abnormalities of PKD1 and no demonstrable abnormalities of PKD2 or PKHD1.

Conclusions

Biallelic inheritance of abnormalities of PKD1 may cause extremely severe disease resembling autosomal dominant polycystic kidney disease (ADPKD) which can result in diagnostic confusion. Accurate diagnosis is essential for genetic counseling.     

Type of PKD1 Mutation Influences Renal Outcome in ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) is heterogeneous with regard to genic and allelic heterogeneity, as well as phenotypic variability. The genotype-phenotype relationship in ADPKD is not completely understood. Here, we studied 741 patients with ADPKD from 519 pedigrees in the Genkyst cohort and confirmed that renal survival associated with PKD2 mutations was approximately 20 years longer than that associated with PKD1 mutations. The median age at onset of ESRD was 58 years for PKD1 carriers and 79 years for PKD2 carriers. Regarding the allelic effect on phenotype, in contrast to previous studies, we found that the type of PKD1 mutation, but not its position, correlated strongly with renal survival. The median age at onset of ESRD was 55 years for carriers of a truncating mutation and 67 years for carriers of a nontruncating mutation. This observation allows the integration of genic and allelic effects into a single scheme, which may have prognostic value.Autosomal dominant polycystic kidney disease (ADPKD) is the most common kidney disorder with a Mendelian inheritance pattern, with a prevalence ranging from 1/400 to 1/1000 worldwide.¹ ADPKD shows both locus and allelic heterogeneity. Two causative genes—PKD1, located at 16p13.3,² and PKD2, located at 4q21³—have been identified, and the ADPKD mutation database (http://pkdb.mayo.edu/) describes >1000 pathogenic mutations (929 in PKD1 and 167 in PKD2 as of June 5, 2012), not including our most recent data.⁴ADPKD also shows high phenotypic variability, as exemplified by the wide variation in the age at onset of ESRD,⁵ which is defined as the requirement of dialysis or transplantation. Genotype-phenotype correlation studies underscore two major issues. First, on average, ESRD occurs 20 years earlier in patients with PKD1 than those with PKD2,^6,7 indicating a genic influence on the ADPKD phenotype. Second, the position of the PKD1 mutation is associated with the age at ESRD onset,⁸ suggesting an allelic influence on ADPKD phenotype. However, these observations were made >10 years ago, when mutational analysis of the PKD1 and PKD2 genes (particularly of the nonunique portion of the PKD1 gene^2,9) was substantially less comprehensive and sophisticated than it is currently,^4,10 the methods for predicting the potential pathogenicity of missense mutations were in their infancy, and the studied patient cohorts were relatively small. To confirm (or refute) these earlier observations, we performed a genotype and phenotype correlation study using the Genkyst cohort, which comprises patients with ADPKD recruited from all private and public nephrology centers in the Brittany region, namely, the western part of France.     

A potentially crucial role of the <Emphasis Type="Italic">PKD1</Emphasis> C-terminal tail in renal prognosis

Background

Autosomal dominant polycystic disease (ADPKD) often results in renal failure. Recently, allelic influences of PKD1 mutation types on renal survival were extensively investigated. Here, we analyzed integrated influences of PKD1 mutation types and positions on renal survival.

Methods

We included 338 (82 pedigrees) and 72 (12 pedigrees) patients with PKD1 and PKD2 mutations, respectively, identified through comprehensive gene analysis of 101 probands with ADPKD. Genetic testing was performed using next-generation sequencing, long-range PCR, and multiplex ligation-dependent probe amplification. Pathogenic mutations were identified by a software package-integrated seven databases and provided access to five cloud-based computing systems.

Results

Mean renal survivals of carriers with PKD1 non-truncating-type mutations at positions upstream of G-protein-coupled receptor proteolytic site (GPS-upstream domain), transmembrane domain, or cytoplasmic C-terminal tail (CTT) domain were 70.2, 67.0, and 50.1 years, respectively (P < 0.0001); renal survival was shorter for mutation positions closer to CTT domain, suggesting its crucial role in renal prognosis. Furthermore, in truncating-type mutations, strong inactivation is anticipated on nucleotides downstream from the mutation site, implying CTT domain inactivation irrespective of mutation site. Shorter mean renal survival was found for PKD1 truncating-type than non-truncating-type mutation carriers (P = 0.0348); mean renal survival was not different between PKD1 3′- and 5′-region truncating-type mutation carriers (P = 0.4375), but was shorter in PKD1 3′-region than in 5′-region non-truncating-type mutation carriers (P = 0.0014). Variable strength of CTT domain inactivation might account for these results.

Conclusions

Aforementioned findings indicate that CTT domain’s crucial role in renal prognosis needs further investigation by larger studies (ClinicalTrials.gov; NCT02322385).

     

Reduced Ciliary Polycystin-2 in Induced Pluripotent Stem Cells from Polycystic Kidney Disease Patients with PKD1 Mutations

Heterozygous mutations in PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, cause autosomal dominant PKD (ADPKD), whereas mutations in PKHD1, which encodes fibrocystin/polyductin (FPC), cause autosomal recessive PKD (ARPKD). However, the relationship between these proteins and the pathogenesis of PKD remains unclear. To model PKD in human cells, we established induced pluripotent stem (iPS) cell lines from fibroblasts of three ADPKD and two ARPKD patients. Genetic sequencing revealed unique heterozygous mutations in PKD1 of the parental ADPKD fibroblasts but no pathogenic mutations in PKD2. Undifferentiated PKD iPS cells, control iPS cells, and embryonic stem cells elaborated primary cilia and expressed PC1, PC2, and FPC at similar levels, and PKD and control iPS cells exhibited comparable rates of proliferation, apoptosis, and ciliogenesis. However, ADPKD iPS cells as well as somatic epithelial cells and hepatoblasts/biliary precursors differentiated from these cells expressed lower levels of PC2 at the cilium. Additional sequencing confirmed the retention of PKD1 heterozygous mutations in iPS cell lines from two patients but identified possible loss of heterozygosity in iPS cell lines from one patient. Furthermore, ectopic expression of wild-type PC1 in ADPKD iPS-derived hepatoblasts rescued ciliary PC2 protein expression levels, and overexpression of PC1 but not a carboxy-terminal truncation mutant increased ciliary PC2 expression levels in mouse kidney cells. Taken together, these results suggest that PC1 regulates ciliary PC2 protein expression levels and support the use of PKD iPS cells for investigating disease pathophysiology.Polycystic kidney disease (PKD) is associated with defects of primary cilia and replacement of the normal kidney parenchyma with tubular epithelial cysts and fibrosis, leading to progressive deterioration of kidney function. PKD is among the world’s most common life-threatening genetic diseases, affecting approximately 1 in 600 people, and it is a significant contributor to CKD. Autosomal dominant PKD (ADPKD) causes end stage kidney disease by the age of 60 years in approximately 50% of adults with the disease, whereas autosomal recessive PKD (ARPKD) is a more rare form that typically presents earlier in life and causes significant childhood mortality. PKD may be considered a developmental disorder, with renal cysts becoming detectable in utero even in ADPKD.¹ In addition to kidney cysts, hepatic involvement is common, with liver cysts developing in many ADPKD patients and congenital hepatic fibrosis being a hallmark of ARPKD.^1,2ADPKD is inherited as heterozygous mutations in PKD1 or PKD2, whereas ARPKD is caused by biallelic mutations in PKHD1 (polycystic kidney and hepatic disease 1). These three genes encode transmembrane proteins, known as polycystin-1 (PC1), polycystin-2 (PC2), and fibrocystin/polyductin (FPC), respectively. PC1, PC2, and FPC form a receptor channel complex in membrane compartments including the primary cilium,^3,4 a sensory organelle on the apical cell surface, and loss of this localization pattern has been observed in cystic renal epithelia from humans.^5,6 Mutations in more than 50 gene products associated with the cilium cause a spectrum of related diseases known as the ciliopathies, most of which feature cystic kidneys.⁷ Ciliary trafficking signals have recently been identified at the carboxyl terminus of PC1 and the amino terminus of PC2, but the extent to which PC1 is involved in PC2 trafficking is not yet clear.^8–11 The abnormal phenotype in ADPKD has been attributed to loss of epithelial cell heterozygosity as a result of an additional somatic mutation or environmental insult (the two-hit hypothesis), although there is also genetic evidence for a haploinsufficiency model.^12–15There is a need for human disease-specific laboratory models for PKD to better understand disease and develop therapies, because animal models may not fully genocopy or phenocopy the human disease.^16,17 Primary cells taken from nephrectomized ADPKD kidneys have been linked to various epithelial cell phenotypes, but because these cells are derived from kidneys with advanced disease, it remains unclear whether these characteristics represent primary defects central to PKD etiology or secondary consequences of injury or dedifferentiation.^6,18–21 A powerful new technology, induced pluripotent stem (iPS) cells are adult somatic cells which have been reprogrammed into an embryonic pluripotent state.^22,23 The result is a next generation cell culture model that can differentiate into diverse cell types and complex tissues for the purposes of regenerative therapies or investigating disease. As for other hereditary diseases, iPS cells from patients with PKD can be examined for disease-specific abnormalities to better understand the pathophysiology of clinical mutations and screen for potential therapeutics.^7,24 PKD iPS cells derived from unaffected cell types, such as fibroblasts, might be expected to have fewer secondary phenotypes compared with cyst-lining epithelial cells, and they could be used to investigate PKD during development, when PKD disease genes are most highly expressed.^1,16,21,25 Their intrinsic pluripotency, ability to self-renew indefinitely, and immunocompatibility also make PKD iPS cells an attractive potential source for renal replacement tissue. As a first step in this direction, generation of iPS cells from one ADPKD patient was recently reported, although no disease phenotypes were described.²⁶ In our study, we generate iPS cell lines from ADPKD, ARPKD, and healthy control patients and evaluate their ability to ciliate, proliferate, and express PKD disease genes to establish a system in vitro for investigating human PKD. We identify reduced levels of PC2 at the primary cilium in undifferentiated iPS cells, differentiated somatic epithelial cells, and hepatoblasts as a consistent phenotype in three ADPKD patients with PKD1 mutations but not in ARPKD patients. Furthermore, we have found using ADPKD iPS-derived hepatoblasts and cultured kidney cells that wild-type but not mutant PC1 promotes PC2 localization to cilia.     

Rapamycin Ameliorates PKD Resulting from Conditional Inactivation of Pkd1

Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD.Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the gradual replacement of normal renal parenchyma by cysts, which culminates in renal failure in approximately 50% of patients.¹ No effective drug treatment is available to slow the progression of ADPKD, which is primarily (85%) caused by mutations in the PKD1 gene encoding polycystin-1 (PC1).² Our previous results suggested that PC1 may regulate the kinase mammalian target of rapamycin (mTOR) via its interaction with tuberin.³ In addition, we have demonstrated that mTOR activity is low in the normal human kidney but strongly upregulated in renal cyst-lining epithelial cells in ADPKD.³ Finally, rapamycin treatment of four nonorthologous rodent PKD models resulted in inhibition of renal cyst growth, regression of kidney size, and preservation of renal function,^3–9 which led to the proposal that mTOR inhibitors, some of which are already in clinical use as immunosuppressants, may be effective in patients with ADPKD.^10–13 Indeed, four clinical trials have been initiated to test the efficacy of mTOR inhibitors in ADPKD.^12–14 Given the immunosuppressive and other adverse effects of mTOR inhibitors, it will be important to establish a compelling rationale for their use in patients with ADPKD.Previous studies used rodent PKD models with mutations in genes that encode proteins (polaris, bicaudal-C, samcystin, and folliculin) with poorly understood function and no known functional link to PC1.^3–9 We hypothesized that the normal function of these and other proteins involved in renal cystic diseases eventually converge on the mTOR pathway,¹³ but it has remained uncertain whether mTOR inhibition would be effective in human ADPKD.To overcome this limitation, we used a mouse model in which the orthologous Pkd1 gene is conditionally inactivated (Pkd1^cond/cond) by Cre-mediated recombination.^15,16 Initially, a Pkd1^cond/cond:MMTV^cre mouse line that resulted in infrequent renal cysts as a result of low renal Cre expression was generated¹⁶. We now report the development of a mouse line, Pkd1^cond/cond:Nestin^cre, in which the nestin promoter drives Cre expression.¹⁷ This results in a mosaic renal expression pattern, mimicking the situation in human ADPKD whereby random somatic, second-hit mutations affect the PKD1 locus,¹ and development of PKD with key features equivalent to the human disease. We report that rapamycin is highly effective in inhibiting all tested aspects of the disease phenotype, resulting in preservation of renal function.     

Double inhibition of cAMP and mTOR signalling may potentiate the reduction of cell growth in ADPKD cells

Background

ADPKD is a renal pathology caused by mutations of PKD1 and PKD2 genes, which encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC1 plays an important role regulating several signal transducers, including cAMP and mTOR, which are involved in abnormal cell proliferation of ADPKD cells leading to the development and expansion of kidney cysts that are a typical hallmark of this disease. Therefore, the inhibition of both pathways could potentiate the reduction of cell proliferation enhancing benefits for ADPKD patients.

Methods

The inhibition of cAMP- and mTOR-related signalling was performed by Cl-IB-MECA, an agonist of A3 receptors, and rapamycin, respectively. Protein kinase activity was evaluated by immunoblot and cell growth was analyzed by direct cell counting.

Results

The activation of A₃AR by the specific agonist Cl-IB-MECA causes a marked reduction of CREB, mTOR, and ERK phosphorylation in kidney tissues of Pkd1 ^flox/?: Ksp-Cre polycystic mice and reduces cell growth in ADPKD cell lines, but not affects the kidney weight. The combined sequential treatment with rapamycin and Cl-IB-MECA in ADPKD cells potentiates the reduction of cell proliferation compared with the individual compound by the inhibition of CREB, mTOR, and ERK kinase activity. Conversely, the simultaneous application of these drugs counteracts their effect on cell growth, because the inhibition of ERK kinase activity is lost.

Conclusion

The double treatment with rapamycin and Cl-IB-MECA may have synergistic effects on the inhibition of cell proliferation in ADPKD cells suggesting that combined therapies could improve renal function in ADPKD patients.

     

Cost-effective PKHD1 genetic testing for autosomal recessive polycystic kidney disease

Background

Genetic diagnosis of autosomal recessive polycystic kidney disease (ARPKD) is challenging due to the length and allelic heterogeneity of the PKHD1 gene. Mutations appear to be clustered at specific exons, depending on the geographic origin of the patient. We aimed to identify the PKHD1 exons most likely mutated in Spanish ARPKD patients.

Methods

Mutation analysis was performed in 50 ARPKD probands and nine ARPKD-suspicious patients by sequencing PKHD1 exons arranged by their reported mutation frequency. Haplotypes containing the most frequent mutations were analyzed. Other PKD genes (HNF1B, PKD1, PKD2) were sequenced in PKHD1-negative cases.

Results

Thirty-six different mutations (concentrated in 24 PKHD1 exons) were detected, giving a mutation detection rate of 86 %. The screening of five exons (58, 32, 34, 36, 37) yielded a 54 % chance of detecting one mutation; the screening of nine additional exons (3, 9, 39, 61, 5, 22, 26, 41, 57) increased the chance to 76 %. The c.9689delA mutation was present in 17 (34 %) patients, all of whom shared the same haplotype. Two HNF1B mutations and one PKD1 variant were detected in negative cases.

Conclusions

Establishing a PKHD1 exon mutation profile in a specific population and starting the analysis with the most likely mutated exons might significantly enhance the efficacy of genetic testing in ARPKD. Analysis of other PKD genes might be considered, especially in suspicious cases.     

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.     

Scattered Deletion of PKD1 in Kidneys Causes a Cystic Snowball Effect and Recapitulates Polycystic Kidney Disease

In total, 1 in 1000 individuals carries a germline mutation in the PKD1 or PKD2 gene, which leads to autosomal dominant polycystic kidney disease (ADPKD). Cysts can form early in life and progressively increase in number and size during adulthood. Extensive research has led to the presumption that somatic inactivation of the remaining allele initiates the formation of cysts, and the progression is further accelerated by renal injury. However, this hypothesis is primarily on the basis of animal studies, in which the gene is inactivated simultaneously in large percentages of kidney cells. To mimic human ADPKD in mice more precisely, we reduced the percentage of Pkd1-deficient kidney cells to 8%. Notably, no pathologic changes occurred for 6 months after Pkd1 deletion, and additional renal injury increased the likelihood of cyst formation but never triggered rapid PKD. In mildly affected mice, cysts were not randomly distributed throughout the kidney but formed in clusters, which could be explained by increased PKD-related signaling in not only cystic epithelial cells but also, healthy-appearing tubules near cysts. In the majority of mice, these changes preceded a rapid and massive onset of severe PKD that was remarkably similar to human ADPKD. Our data suggest that initial cysts are the principal trigger for a snowball effect driving the formation of new cysts, leading to the progression of severe PKD. In addition, this approach is a suitable model for mimicking human ADPKD and can be used for preclinical testing.     

Genetic Variation of DKK3 May Modify Renal Disease Severity in ADPKD

Significant variation in the course of autosomal dominant polycystic kidney disease ( ADPKD) within families suggests the presence of effect modifiers. Recent studies of the variation within families harboring PKD1 mutations indicate that genetic background may account for 32 to 42% of the variance in estimated GFR (eGFR) before ESRD and 43 to 78% of the variance in age at ESRD onset, but the genetic modifiers are unknown. Here, we conducted a high-throughput single-nucleotide polymorphism (SNP) genotyping association study of 173 biological candidate genes in 794 white patients from 227 families with PKD1. We analyzed two primary outcomes: (1) eGFR and (2) time to ESRD (renal survival). For both outcomes, we used multidimensional scaling to correct for population structure and generalized estimating equations to account for the relatedness among individuals within the same family. We found suggestive associations between each of 12 SNPs and at least one of the renal outcomes. We genotyped these SNPs in a second set of 472 white patients from 229 families with PKD1 and performed a joint analysis on both cohorts. Three SNPs continued to show suggestive/significant association with eGFR at the Dickkopf 3 (DKK3) gene locus; no SNPs significantly associated with renal survival. DKK3 antagonizes Wnt/β-catenin signaling, which may modulate renal cyst growth. Pending replication, our study suggests that genetic variation of DKK3 may modify severity of ADPKD resulting from PKD1 mutations.Autosomal dominant polycystic kidney disease ( ADPKD) is the most common monogenic kidney disease worldwide, affecting one in 500 to 1000 births.^1,2 It is characterized by focal development of renal cysts in an age-dependent manner. Typically, only a few renal cysts are clinically detectable during the first three decades of life; however, by the fifth decade, tens of thousands of renal cysts of different sizes can be found in most patients.³ Progressive cyst expansion with age leads to massive enlargement and distortion of the normal architecture of both kidneys and, ultimately, ESRD in most patients. ADPKD is also associated with an increased risk for cardiac valvular defects, colonic diverticulosis, hernias, and intracranial arterial aneurysms. Overall, ADPKD accounts for approximately 5% of ESRD in North America.²Mutations of PKD1 and PKD2 respectively account for approximately 85% and approximately 15% of linkage-characterized European families. Polycystin-1 (PC-1) and PC-2, the proteins encoded by PKD1 and PKD2, respectively, function as a macromolecular complex and regulate multiple signaling pathways to maintain the normal tubular structure and function.¹ Monoclonal expansion of individual epithelial cells that have undergone a somatic “second hit” mutation, resulting in biallelic inactivation of either PKD1 or PKD2, seems to provide a major mechanism for focal cyst initiation,⁴ possibly through the loss of polycystin-mediated mechanosensory function in the primary cilium.⁵ In addition, a large prospective, observational study indicated that renal cysts in ADPKD expand exponentially with increasing age, and patients with large polycystic kidneys are at higher risk for developing kidney failure⁶; however, the key factors that modulate renal disease progression in ADPKD remain incompletely understood.Renal disease severity in ADPKD is highly variable, with the age of onset of ESRD ranging from childhood to old age.^7–11 A strong genetic locus effect has been noted in ADPKD. Adjusted for age and gender, patients with PKD1 have larger kidneys and earlier onset at ESRD than patients with PKD2 (mean age at ESRD 53.4 versus 72.7 years, respectively).^8,9 By contrast, a weak allelic effect (based on the 5′ versus 3′ location of the germline mutations) on renal disease severity may be present for PKD1¹⁰ but not PKD2.¹¹ Marked intrafamilial variability in renal disease is well documented in ADPKD and suggests a strong modifier effect.^10–15 In an extreme example, large polycystic kidneys were present in utero in one of a pair of dizygotic twins affected with the same germline PKD1 mutation, whereas the kidneys of the co-twin remained normal at 5 years of age.¹² Several studies have quantified the role of genetic background in the phenotypic expression of ADPKD. In a comparison of monozygotic twins and siblings, greater variance in the age of onset of ESRD in the siblings supported a role for genetic modifiers.¹³ Two other studies of intrafamilial disease variability in PKD1 have estimated that genetic factors may account for 32 to 42% of the variance of creatinine clearance before ESRD and 43 to 78% of the variance in age at ESRD.^14,15 The magnitude of the modifier gene effect from these studies suggests that mapping such factors is feasible. Here, we report the results of an association study of modifier genes for PKD1 renal disease severity.     

Patterns of Pancreatic Resection Differ Between Patients with Familial and Sporadic Pancreatic Cancer

Background

Although the increased risk of developing pancreatic cancer (PC) in families with a strong history of the disease is well known, characteristics and outcomes of patients with familial PC is not described well.

Aims

This study aims to evaluate outcomes following resection in patients with familial PC.

Methods

We studied 208 patients who underwent resection of PC from 2000 to 2007 and had prospectively completed family history questionnaires for the Biospecimen Resource for Pancreas Research at our institution. We compared clinical characteristics and outcomes of familial and sporadic PC patients.

Results

Familial (N?=?15) and sporadic PC patients (N?=?193) did not have significantly different demographics, pre-operative CA19-9, pre-operative weight loss, R0 status, or T-staging (all p??? 0.05). Familial PC patients had lower pre-operative total serum bilirubin concentrations (p?=?0.03) and lesions outside of the pancreatic head more frequently (p?=?0.02) than sporadic PC patients. There was no difference in survival at 2?years between familial and sporadic PC patients (p?=?0.52).

Conclusions

Familial PC patients appear to develop tumors outside of the pancreatic head more frequently than sporadic PC patients. This difference in tumor distribution may be due to a broader area of cancer susceptibility within the pancreas for familial PC patients.     

Family History of Renal Disease Severity Predicts the Mutated Gene in ADPKD

Mutations of PKD1 and PKD2 account for 85 and 15% of cases of autosomal dominant polycystic kidney disease (ADPKD), respectively. Clinically, PKD1 is more severe than PKD2, with a median age at ESRD of 53.4 versus 72.7 yr. In this study, we explored whether a family history of renal disease severity predicts the mutated gene in ADPKD. We examined the renal function (estimated GFR and age at ESRD) of 484 affected members from 90 families who had ADPKD and whose underlying genotype was known. We found that the presence of at least one affected family member who developed ESRD at age ≤55 was highly predictive of a PKD1 mutation (positive predictive value 100%; sensitivity 72%). In contrast, the presence of at least one affected family member who continued to have sufficient renal function or developed ESRD at age >70 was highly predictive of a PKD2 mutation (positive predictive value 100%; sensitivity 74%). These data suggest that close attention to the family history of renal disease severity in ADPKD may provide a simple means of predicting the mutated gene, which has prognostic implications.Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disorder, with a prevalence of one in 500 to 1000 in the general population. It is the third most common single cause of ESRD in the United States, accounting for 5% of people with ESRD.^1–5 ADPKD is genetically heterogeneous, with two disease genes (PKD1 on chromosome 16 and PKD2 on chromosome 4) accounting for most of the cases. Mutations of PKD1 and PKD2 are thought to account for 85 and 15% of cases, respectively, in linkage-characterized European populations.^6,7 Although the clinical manifestations overlap completely between two gene types, there is a strong locus effect on renal disease severity. Patients with PKD1 have significantly more severe renal disease than patients with PKD2, with larger kidneys and earlier onset at ESRD (median age 53.4 versus 72.7 yr, respectively).^8,9 By contrast, a weak allelic effect (based on the 5′ versus 3′ location of the germline mutations) on renal disease severity may exist for type 1¹⁰ but not type 2 ADPKD.¹¹ In addition, significant intrafamilial renal disease variability is evident, which is thought to be due to genetic and environmental modifiers.^11,12PKD1 is a large, complex gene containing 46 exons spanning 50 kb, with 33 of these exons at the 5′ end being duplicated elsewhere on chromosome 16. PKD2 is a single-copy gene, consisting of 15 exons spanning a 68-kb genomic region. There is marked allelic heterogeneity for both gene types, with 314 truncating mutations having been described in PKD1 and 91 truncating mutations in PKD2.^1,2 PKD1 encodes polycystin 1 (PC1), a large receptor-like protein, and PKD2 encodes polycystin 2 (PC2), a nonselective cation channel that transports calcium. Both PC1 and PC2 physically interact to form a complex that regulates intracellular levels of calcium and are located in the primary cilia of renal tubular cells. Recent studies suggested that the polycystin complex in the primary cilia of renal tubular cells serves as a mechanosensor for urine flow and that dysfunction of this mechanosensor may lead to cellular proliferation and cystogenesis.^1,2Recent advances in our understanding of the molecular pathobiology of ADPKD have led to the discovery of a number of drugs (e.g., tolvaptan, somatostatin, mammalian target of rapamycin inhibitors) that may target cyst growth and delay renal disease progression.^1,2 Several of these promising drugs are being or will be tested in clinical trials, and disease-modifying treatment may become a reality in the not-too-distant future.¹ In this context, the knowledge of ADPKD gene type may allow for the optimization of the design of such clinical trials, and identification of those affected individuals who are most likely to benefit from these novel therapies should they become available; however, the gene type is seldom known for most families in the clinical setting. Although molecular genetic testing, either by linkage or direct mutation analysis, can elucidate the gene type, such testing has its limitations.¹³ Linkage studies require DNA samples from several affected family members and are of limited utility in small families or de novo cases. Mutation-based screening for ADPKD is expensive and yields a definitive pathogenic mutation in only 42 to 63% of cases because the large and complex structure of PKD1 results in many unclassified missense variants whose pathogenicity often cannot be predicted with complete certainty.^14,15In this study, we explored whether renal disease severity can be used as clinical predictors of underlying gene type in families with ADPKD. To predict PKD1, we explored various cutoffs of early age at ESRD as indicative of severe renal disease. To predict PKD2, we explored various cutoffs of late age with renal sufficiency or at ESRD as indicative of milder renal disease. We then evaluated the performance characteristics of these cutoffs to define the optimal criteria for clinical prediction of ADPKD gene type.     

Role of Urinary Neutrophil Gelatinase–Associated Lipocalin for Predicting the Severity of Renal Functions in Patients With Autosomal-Dominant Polycystic Kidney Disease

Background

Autosomal-dominant polycystic kidney disease (ADPKD) has a feature of disruption of tubular integrity with increased cellular proliferation and apoptosis. There are several known tubular membrane proteins in the pathogenesis of ADPKD, and one of these proteins is the neutrophil gelatinase-associated lipocalin (NGAL). NGAL is a protein expressed on renal tubular cells of which production is markedly increased in response to harmful stimuli such as ischemia or toxicity.

Mutations in NOTCH2 in patients with Hajdu–Cheney syndrome

Summary

The Hajdu–Cheney syndrome is a very rare disease that affects several organ system, leading to severe osteoporosis and other abnormalities. We describe clinical and genetic findings of nine patients with this disease.

Introduction

The Hajdu–Cheney syndrome (HCS) is a rare autosomal dominant disorder characterized by severe osteoporosis, acroosteolysis of the distal phalanges, renal cysts, and other abnormalities. Recently, heterozygous mutations in NOTCH2 were identified as the cause of HCS.

Methods

Nine patients with typical presentations of HCS took part in this study: five affected patients from two small families and four sporadic cases. Peripheral blood DNA was obtained and exome sequencing performed in one affected individual per family and in all four sporadic cases. Sanger sequencing confirmed mutations in all patients.

Results

One of the identified mutations was introduced in a plasmid encoding NOTCH2. Wild-type and mutant NOTCH2 were transiently expressed in HEK293 cells to assess intracellular localization after ligand activation. Deleterious heterozygous mutations in the last NOTCH2 exon were identified in all patients; five of the six mutations were novel.

Conclusion

Consistent with previous reports, all mutations are predicted to result in a loss of the proline/glutamic acid/serine/threonine sequence, which harbors signals for degradation, therefore suggesting activating mutations. One of the six mutations furthermore predicted disruption of the second nuclear localization signal of NOTCH2, but the mutant revealed normal nuclear localization after transfection, which is consistent with the proposed gain-of-function mechanism as the cause of this autosomal dominant disease. Our findings confirm that heterozygous NOTCH2 mutations are the cause of HCS and expand the mutational spectrum of this disorder.     

Polycystic Kidney Disease and Cancer after Renal Transplantation

Autosomal dominant polycystic kidney disease (ADPKD), the most common form of polycystic kidney disease (PKD), is a disorder with characteristics of neoplasia. However, it is not known whether renal transplant recipients with PKD have an increased risk of cancer. Data from the Scientific Registry of Transplant Recipients, which contains information on all solid organ transplant recipients in the United States, were linked to 15 population-based cancer registries in the United States. For PKD recipients, we compared overall cancer risk with that in the general population. We also compared cancer incidence in PKD versus non-PKD renal transplant recipients using Poisson regression, and we determined incidence rate ratios (IRRs) adjusted for age, sex, race/ethnicity, dialysis duration, and time since transplantation. The study included 10,166 kidney recipients with PKD and 107,339 without PKD. Cancer incidence in PKD recipients was 1233.6 per 100,000 person-years, 48% higher than expected in the general population (standardized incidence ratio, 1.48; 95% confidence interval [95% CI], 1.37 to 1.60), whereas cancer incidence in non-PKD recipients was 1119.1 per 100,000 person-years. The unadjusted incidence was higher in PKD than in non-PKD recipients (IRR, 1.10; 95% CI, 1.01 to 1.20). However, PKD recipients were older (median age at transplantation, 51 years versus 45 years for non-PKD recipients), and after multivariable adjustment, cancer incidence was lower in PKD recipients than in others (IRR, 0.84; 95% CI, 0.77 to 0.91). The reason for the lower cancer risk in PKD recipients is not known but may relate to biologic characteristics of ADPKD or to cancer risk behaviors associated with ADPKD.Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of inherited cystic renal disease and the fourth most common cause of ESRD in the United States.^1–3 There are currently>16,000 individuals with polycystic kidney disease (PKD, of which ADPKD is by far the most common type) living with a renal transplant in the United States.⁴ADPKD is a result of mutations in one of two genes: PKD1 and PKD2.^1,5,6 These genes are widely expressed in many tissues, consistent with the multiorgan pathology characterizing ADPKD. A key factor in cyst formation and enlargement in ADPKD is the abnormal proliferation of cyst epithelial cells in a cell-autonomous manner.^7,8 This cyst formation is associated with cellular dedifferentiation and is considered a neoplastic process driven by upregulated proto-oncogenes.^9–13 While published case reports document the occurrence of renal cell carcinomas (RCCs) in ADPKD-affected kidneys,^14,15 these tumors may be partly due to acquired renal cystic disease resulting from long-term dialysis.¹⁶ Because there do not appear to be widespread published reports of other cancers in patients with ADPKD, protective mechanisms might exist in ADPKD to prevent malignant transformation. Indeed, many oncogenes that promote cell proliferation also act as potent growth suppressors (e.g., Ras¹⁷) or inducers of apoptosis (e.g., Myc^18,19). Thus, there is uncertainty whether ADPKD mutations are associated with increased rates of kidney cancer or cancer in general.We therefore designed a study to compare cancer risk in kidney transplant recipients with PKD versus kidney recipients with other causes of ESRD. Organ transplant recipients are at increased risk of cancer, largely because of immunosuppressive therapy.²⁰ An increased risk of cancer in patients with PKD might be detectable in this high-risk cancer population. Alternatively, if there is no increased risk of cancer in ADPKD, the findings would suggest the need for further study to determine whether protective cellular mechanisms may be at work.     

Characterization of PKD Protein-Positive Exosome-Like Vesicles

Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1^del2/del2 mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease, affecting between 1:400 to 1:1000 individuals.^1,2 There are two genetic loci, PKD1 and PKD2, producing the proteins polycystin-1 (PC1)^3–5 and polycystin-2 (PC2),⁶ respectively. Autosomal recessive polycystic kidney disease (ARPKD), the most common cause of hereditary childhood PKD, is caused by mutations to PKHD1, which encodes fibrocystin/polyductin (FCP).^7,8 These three PKD proteins have been localized to primary cilia,^9,10 where the PC1/PC2 complex acts as a flow sensor on the cilium.¹¹ The role of FCP is less clear, but it complexes with PC2.^12,13Another site of PC1 expression is in urinary exosomes, small vesicles (50 to 100 nm in diameter) present in normal urine, that have been analyzed as a source of biomarkers for various renal diseases.^14,15 Urinary exosomes are thought to be end products of the multivesicular body (MVB)-sorting pathway in which membrane proteins are uniquely packaged into intraluminal vesicles (ILVs) within the MVB, some of which are secreted as exosomes when MVBs fuse with the apical plasma membrane.MVBs and exosomes have been shown to have a role in left/right (L/R) axis determination in the embryonic node. These MVBs, termed nodal vesicular parcels (NVPs), are released from the floor of the node and swept by nodal flow to the left side, where they interact with the “picket-fence” immotile cilia.¹⁶ Symmetry breaking is dependent on a PC2 Ca²⁺-dependent flux.^17,18Transmission electron microscopy studies of dilated bile ducts found in ARPKD mouse model Pkhd1^del2/del2 showed PC1+ exosome-like vesicles surrounding cholangiocyte primary cilia, whereas only occasional single ELVs were found attached to WT cilia.¹⁹ The observations of abundant PC1 in ELVs and of abnormal ELV accumulation in FCP-deficient mice led us to examine whether these may have a functional role in the urinary and biliary systems, analogous to the NVP in the node.     

Cerebral arterial fenestrations: a common phenomenon in unexplained subarachnoid haemorrhage

Background

Fenestrations of intracranial arteries are variants resulting from incomplete fusion of vessels during development with unknown clinical significance. They are best visualised with 3D rotational angiography (3DRA).

Objective

In a prospective consecutive series of patients with suspected aneurysms, 3DRA was performed to identify not only the potential bleeding source but also to assess the frequency and location of any fenestrations of intracranial arteries.

Methods

In 287 consecutive patients with possible intracranial aneurysms (accidental discovery or previous history of SAH), 3DRAs were prospectively performed, and the location of subarachnoid haemorrhage was assessed by CT.

Results

Of 174 patients presenting with SAH, 153 had saccular aneurysms, and in 21 cases (12.1 %), no source of bleeding was found. In 20 of these 21 patients with "unexplained SAH" (95.2 %) an arterial fenestration was detected in the neighbourhood of the clot. The incidence of fenestration in the 153 aneurysmal SAH patients was 22.9 %, and it was 23.3 % in 266 patients with intracranial aneurysms (113 accidental and 153 ruptured).

Conclusions

Arterial fenestration was detected in 22.9 % of ruptured cerebral aneurysms, in contrast with 95.2 % in patients with unexplained SAH, the difference being statisctically significant (p?<?0.01). Fenestration is a developmental defect, a structural wall weakness possibly making the vessel prone to rupture. Its incidence of nearly 100 % may suggest a connection with idiopathic SAH. The presented data indicate that arterial fenestrations are generally overlooked, and they can be considered as one of the candidates for the source of idiopathic SAH.     

Familial renal glucosuria: a clinicogenetic study of 23 additional cases

Background

Familial renal glucosuria (FRG) is an inherited renal tubular disorder characterized by persistent isolated glucosuria in the absence of hyperglycemia that is caused by mutations in the sodium-glucose cotransporter SGLT2 coding gene, SLC5A2.

Objective

We conducted molecular and phenotype analyses of a cohort of 23 unrelated Korean children with FRG.

Methods

Mutational analysis of the SLC5A2 gene was conducted in this multicenter study organized by the Korean Society of Pediatric Nephrology.

Results

A total of 21 different SLC5A2 mutations were detected, including 19 novel mutations. All patients had at least one mutated allele; ten patients had homozygous or compound heterozygous mutations and 13 patients had a single heterozygous mutation. Most mutations were private. Patients with two mutations were diagnosed earlier with larger amounts of urinary glucose excretion than patients with single mutations. Pedigree analysis data were consistent with the inheritance of a codominant trait with incomplete penetrance.

Conclusions

These findings extend the allelic heterogeneity in FRG and confirm previous observations of inheritance and genotype–phenotype correlation in patients with this disease.     

Stent salvage using the Enterprise stent for procedure-related complication during coil embolization of ruptured intracranial aneurysms

Background

Despite accumulated experience and improved understanding of the tools, endovascular treatment of intracranial aneurysms continues to have risks linked to the technique itself, and induces procedure-related complications. The purpose of this study was to report our series of stent salvage using the Enterprise stent for procedure-related complication during coil embolization in patients with ruptured intracranial aneurysms.

Methods

Parent artery thrombosis, parent artery dissection, and coil protrusion were considered to be the procedure-related complications. There were 18 consecutive cases (3 unruptured and 15 ruptured aneurysms) with procedure-related complications rescued by the Enterprise stent from December 2008 to December 2011. Follow-up angiography was performed in 14 of the 15 patients with ruptured aneurysms between 6 and 30 months (mean 14.6 months) after the procedure.

Results

The procedure-related complications were parent artery dissection (n?=?1), parent artery thrombosis (n?=?4), and coil protrusion (n?=?10). There was no complication related to delivering or deploying of the Enterprise stent. Initial radiographic results showed 8 cases of complete occlusion and 7 cases of neck remnant. There was no change in the angiographic results during the follow-up periods.

Conclusions

Facing with procedure-related complications during coil embolization of ruptured intracranial aneurysms, the closed-cell designed Enterprise stent might be a useful option for the salvage technique by restoring blood flow and minimizing thromboembolic events.