Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by the progressive development of kidney cysts, often resulting in end-stage renal disease (ESRD). This disorder is genetically ...heterogeneous with ∼7% of families genetically unresolved. We performed whole-exome sequencing (WES) in two multiplex ADPKD-like pedigrees, and we analyzed a further 591 genetically unresolved, phenotypically similar families by targeted next-generation sequencing of 65 candidate genes. WES identified a DNAJB11 missense variant (p.Pro54Arg) in two family members presenting with non-enlarged polycystic kidneys and a frameshifting change (c.166_167insTT) in a second family with small renal and liver cysts. DNAJB11 is a co-factor of BiP, a key chaperone in the endoplasmic reticulum controlling folding, trafficking, and degradation of secreted and membrane proteins. Five additional multigenerational families carrying DNAJB11 mutations were identified by the targeted analysis. The clinical phenotype was consistent in the 23 affected members, with non-enlarged cystic kidneys that often evolved to kidney atrophy; 7 subjects reached ESRD from 59 to 89 years. The lack of kidney enlargement, histologically evident interstitial fibrosis in non-cystic parenchyma, and recurring episodes of gout (one family) suggested partial phenotypic overlap with autosomal-dominant tubulointerstitial diseases (ADTKD). Characterization of DNAJB11-null cells and kidney samples from affected individuals revealed a pathogenesis associated with maturation and trafficking defects involving the ADPKD protein, PC1, and ADTKD proteins, such as UMOD. DNAJB11-associated disease is a phenotypic hybrid of ADPKD and ADTKD, characterized by normal-sized cystic kidneys and progressive interstitial fibrosis resulting in late-onset ESRD.
Autosomal-dominant polycystic kidney disease (ADPKD) is a common, progressive, adult-onset disease that is an important cause of end-stage renal disease (ESRD), which requires transplantation or ...dialysis. Mutations in PKD1 or PKD2 (∼85% and ∼15% of resolved cases, respectively) are the known causes of ADPKD. Extrarenal manifestations include an increased level of intracranial aneurysms and polycystic liver disease (PLD), which can be severe and associated with significant morbidity. Autosomal-dominant PLD (ADPLD) with no or very few renal cysts is a separate disorder caused by PRKCSH, SEC63, or LRP5 mutations. After screening, 7%–10% of ADPKD-affected and ∼50% of ADPLD-affected families were genetically unresolved (GUR), suggesting further genetic heterogeneity of both disorders. Whole-exome sequencing of six GUR ADPKD-affected families identified one with a missense mutation in GANAB, encoding glucosidase II subunit α (GIIα). Because PRKCSH encodes GIIβ, GANAB is a strong ADPKD and ADPLD candidate gene. Sanger screening of 321 additional GUR families identified eight further likely mutations (six truncating), and a total of 20 affected individuals were identified in seven ADPKD- and two ADPLD-affected families. The phenotype was mild PKD and variable, including severe, PLD. Analysis of GANAB-null cells showed an absolute requirement of GIIα for maturation and surface and ciliary localization of the ADPKD proteins (PC1 and PC2), and reduced mature PC1 was seen in GANAB+/− cells. PC1 surface localization in GANAB−/− cells was rescued by wild-type, but not mutant, GIIα. Overall, we show that GANAB mutations cause ADPKD and ADPLD and that the cystogenesis is most likely driven by defects in PC1 maturation.
Repeat expansions are responsible for over 40 monogenic disorders, and undoubtedly more pathogenic repeat expansions remain to be discovered. Existing methods for detecting repeat expansions in ...short-read sequencing data require predefined repeat catalogs. Recent discoveries emphasize the need for methods that do not require pre-specified candidate repeats. To address this need, we introduce ExpansionHunter Denovo, an efficient catalog-free method for genome-wide repeat expansion detection. Analysis of real and simulated data shows that our method can identify large expansions of 41 out of 44 pathogenic repeats, including nine recently reported non-reference repeat expansions not discoverable via existing methods.
Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited nephropathy responsible for 4%-10% of end-stage renal disease cases. Mutations in the genes encoding polycystin-1 (PC1, ...PKD1) or polycystin-2 (PC2, PKD2) cause ADPKD, and PKD1 mutations are associated with more severe renal disease. PC1 has been shown to form a complex with PC2, and the severity of PKD1-mediated disease is associated with the level of the mature PC1 glycoform. Here, we demonstrated that PC1 and PC2 first interact in the ER before PC1 cleavage at the GPS/GAIN site and determined that PC2 acts as an essential chaperone for PC1 maturation and surface localization. The chaperone function of PC2 was dependent on the presence of the distal coiled-coil domain and was disrupted by pathogenic missense mutations. In Pkd2-/- mice, complete loss of PC2 prevented PC1 maturation. In Pkd2 heterozygotes, the 50% PC2 reduction resulted in a nonequimolar reduction (20%-25%) of the mature PC1 glycoform. Interbreeding between various Pkd1 and Pkd2 models revealed that animals with reduced levels of functional PC1 and PC2 in the kidney exhibited severe, rapidly progressive disease, illustrating the importance of complexing of these proteins for function. Our results indicate that PC2 regulates PC1 maturation; therefore, mature PC1 levels are a determinant of disease severity in PKD2 as well as PKD1.
To identify the economic impact of pediatric patients with clinical indications of genetic disease (GD) on the US health-care system.
Using the 2012 Kids' Inpatient Database, we identified pediatric ...inpatient discharges with International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes linked to genetic disease, including well-established genetic disorders, neurological diseases, birth defects, and other physiological or functional abnormalities with a genetic basis. Cohort characteristics and health-care utilization measures were analyzed. Discharges with a GD-associated primary diagnosis were used to estimate the minimum burden; discharges with GD-associated primary or secondary codes established the maximum burden.
Of 5.85 million weighted discharges, 2.6-14% included GD-associated ICD-9-CM codes. For these discharges, mean total costs were $16,000-77,000 higher (P < 0.0001) in neonates and $12,000-17,000 higher (P < 0.0001) in pediatric patients compared with background, corresponding to significantly higher total charges and lengths of stay. Aggregate total charges for suspected GD accounted for $14 to $57 billion (11-46%) of the "national bill" for pediatric patients in 2012.
Pediatric inpatients with diagnostic codes linked to genetic disease have a significant and disproportionate impact on resources and costs in the US health-care system.
Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of ESRD. Affected individuals inherit a defective copy of either PKD1 or PKD2, which encode polycystin-1 (PC1) or polycystin-2 ...(PC2), respectively. PC1 and PC2 are secreted on urinary exosome-like vesicles (ELVs) (100-nm diameter vesicles), in which PC1 is present in a cleaved form and may be complexed with PC2. Here, label-free quantitative proteomic studies of urine ELVs in an initial discovery cohort (13 individuals with PKD1 mutations and 18 normal controls) revealed that of 2008 ELV proteins, 9 (0.32%) were expressed at significantly different levels in samples from individuals with PKD1 mutations compared to controls (P<0.03). In samples from individuals with PKD1 mutations, levels of PC1 and PC2 were reduced to 54% (P<0.02) and 53% (P<0.001), respectively. Transmembrane protein 2 (TMEM2), a protein with homology to fibrocystin, was 2.1-fold higher in individuals with PKD1 mutations (P<0.03). The PC1/TMEM2 ratio correlated inversely with height-adjusted total kidney volume in the discovery cohort, and the ratio of PC1/TMEM2 or PC2/TMEM2 could be used to distinguish individuals with PKD1 mutations from controls in a confirmation cohort. In summary, results of this study suggest that a test measuring the urine exosomal PC1/TMEM2 or PC2/TMEM2 ratio may have utility in diagnosis and monitoring of polycystic kidney disease. Future studies will focus on increasing sample size and confirming these studies. The data were deposited in the ProteomeXchange (identifier PXD001075).
Autosomal-dominant polycystic kidney disease (ADPKD) is a common, progressive, adult-onset disease that is an important cause of end-stage renal disease (ESRD), which requires transplantation or ...dialysis. Mutations in PKD1 or PKD2 (~85% and ~15% of resolved cases, respectively) are the known causes of ADPKD. Extrarenal manifestations include an increased level of intracranial aneurysms and polycystic liver disease (PLD), which can be severe and associated with significant morbidity. Autosomal-dominant PLD (ADPLD) with no or very few renal cysts is a separate disorder caused by PRKCSH, SEC63, or LRP5 mutations. After screening, 7%-10% of ADPKD-affected and ~50% of ADPLD-affected families were genetically unresolved (GUR), suggesting further genetic heterogeneity of both disorders. Whole-exome sequencing of six GUR ADPKD-affected families identified one with a missense mutation in GANAB, encoding glucosidase II subunit a (GIIa). Because PRKCSH encodes GIIβ, GANAB is a strong ADPKD and ADPLD candidate gene. Sanger screening of 321 additional GUR families identified eight further likely mutations (six truncating), and a total of 20 affected individuals were identified in seven ADPKD- and two ADPLD-affected families. The phenotype was mild PKD and variable, including severe, PLD. Analysis of GANAB-null cells showed an absolute requirement of GIIa for maturation and surface and ciliary localization of the ADPKD proteins (PC1 and PC2), and reduced mature PC1 was seen in GANAB+/- cells. PC1 surface localization in GANAB-/- cells was rescued by wild-type, but not mutant, GIIa. Overall, we show that GANAB mutations cause ADPKD and ADPLD and that the cystogenesis is most likely driven by defects in PC1 maturation.
Cerebral palsy is a common neurodevelopmental disorder affecting movement and posture that often co-occurs with other neurodevelopmental disorders. Individual cases of cerebral palsy are often ...attributed to birth asphyxia; however, recent studies indicate that asphyxia accounts for less than 10% of cerebral palsy cases.
To determine the molecular diagnostic yield of exome sequencing (prevalence of pathogenic and likely pathogenic variants) in individuals with cerebral palsy.
A retrospective cohort study of patients with cerebral palsy that included a clinical laboratory referral cohort with data accrued between 2012 and 2018 and a health care-based cohort with data accrued between 2007 and 2017.
Exome sequencing with copy number variant detection.
The primary outcome was the molecular diagnostic yield of exome sequencing.
Among 1345 patients from the clinical laboratory referral cohort, the median age was 8.8 years (interquartile range, 4.4-14.7 years; range, 0.1-66 years) and 601 (45%) were female. Among 181 patients in the health care-based cohort, the median age was 41.9 years (interquartile range, 28.0-59.6 years; range, 4.8-89 years) and 96 (53%) were female. The molecular diagnostic yield of exome sequencing was 32.7% (95% CI, 30.2%-35.2%) in the clinical laboratory referral cohort and 10.5% (95% CI, 6.0%-15.0%) in the health care-based cohort. The molecular diagnostic yield ranged from 11.2% (95% CI, 6.4%-16.2%) for patients without intellectual disability, epilepsy, or autism spectrum disorder to 32.9% (95% CI, 25.7%-40.1%) for patients with all 3 comorbidities. Pathogenic and likely pathogenic variants were identified in 229 genes (29.5% of 1526 patients); 86 genes were mutated in 2 or more patients (20.1% of 1526 patients) and 10 genes with mutations were independently identified in both cohorts (2.9% of 1526 patients).
Among 2 cohorts of patients with cerebral palsy who underwent exome sequencing, the prevalence of pathogenic and likely pathogenic variants was 32.7% in a cohort that predominantly consisted of pediatric patients and 10.5% in a cohort that predominantly consisted of adult patients. Further research is needed to understand the clinical implications of these findings.
Dent disease 1 (DD1) is caused by mutations in the CLCN5 gene encoding a voltage-gated electrogenic nCl−/H+ exchanger ClC-5. Using ion-selective microelectrodes and Xenopus oocytes, here we studied ...Cl−/H+ coupling properties of WT ClC-5 and four DD1-associated variants (S244L, R345W, Q629*, and T657S), along with trafficking and localization of ClC-5. WT ClC-5 had a 2Cl−/H+ exchange ratio at a Vh of +40 mV with a Cl−out of 104 mm, but the transport direction did not reverse with a Cl−out of 5 mm, indicating that ClC-5-mediated exchange of two Cl− out for one H+ in is not permissible. We hypothesized that ClC-5 and H+-ATPase are functionally coupled during H+-ATPase–mediated endosomal acidification, crucial for ClC-5 activation by depolarizing endosomes. ClC-5 transport that provides three net negative charges appeared self-inhibitory because of ClC-5's voltage-gated properties, but shunt conductance facilitated further H+-ATPase–mediated endosomal acidification. Thus, an on-and-off “burst” of ClC-5 activity was crucial for preventing Cl− exit from endosomes. The subcellular distribution of the ClC-5:S244L variant was comparable with that of WT ClC-5, but the variant had a much slower Cl− and H+ transport and displayed an altered stoichiometry of 1.6:1. The ClC-5:R345W variant exhibited slightly higher Cl−/H+ transport than ClC-5:S244L, but co-localized with early endosomes, suggesting decreased ClC-5:R345W membrane trafficking is perhaps in a fully functional form. The truncated ClC-5:Q629* variant displayed the lowest Cl−/H+ exchange and was retained in the endoplasmic reticulum and cis-Golgi, but not in early endosomes, suggesting the nonsense mutation affects ClC-5 maturation and trafficking.
Aberrant activation of the Hedgehog (Hh) signaling pathway, through which the GLI family of transcription factors (TF) is stimulated, is commonly observed in cancer cells. One well-established ...mechanism of this increased activity is through the inactivation of Suppressor of Fused (SUFU), a negative regulator of the Hh pathway. Relief from negative regulation by SUFU facilitates GLI activity and induction of target gene expression. Here, we demonstrate a novel role for SUFU as a promoter of GLI activity in pancreatic ductal adenocarcinoma (PDAC). In non-ciliated PDAC cells unresponsive to Smoothened agonism, SUFU overexpression increases GLI transcriptional activity. Conversely, knockdown (KD) of SUFU reduces the activity of GLI in PDAC cells. Through array PCR analysis of GLI target genes, we identified B-cell lymphoma 2 (BCL2) among the top candidates down-regulated by SUFU KD. We demonstrate that SUFU KD results in reduced PDAC cell viability, and overexpression of BCL2 partially rescues the effect of reduced cell viability by SUFU KD. Further analysis using as a model GLI1, a major TF activator of the GLI family in PDAC cells, shows the interaction of SUFU and GLI1 in the nucleus through previously characterized domains. Chromatin immunoprecipitation (ChIP) assay shows the binding of both SUFU and GLI1 at the promoter of BCL2 in PDAC cells. Finally, we demonstrate that SUFU promotes GLI1 activity without affecting its protein stability. Through our findings, we propose a novel role of SUFU as a positive regulator of GLI1 in PDAC, adding a new mechanism of Hh/GLI signaling pathway regulation in cancer cells.
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