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Moumita Chaki,
Rannar Airik,
Amiya K Ghosh,
Rachel H Giles,
Rui Chen,
Gisela G Slaats,
Hui Wang,
Toby W Hurd,
Weibin Zhou,
Andrew Cluckey, [......],
Elizabeth Garner,
Katja Vanselow,
Jens S Andersen,
Joseph Shlomai,
Gudrun Nurnberg,
Peter Nurnberg,
Shawn Levy,
Agata Smogorzewska,
Edgar A Otto,
Friedhelm Hildebrandt
[show abstract]
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ABSTRACT: Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.
Cell 08/2012; 150(3):533-48. · 32.40 Impact Factor
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Weibin Zhou,
Edgar A Otto,
Andrew Cluckey,
Rannar Airik,
Toby W Hurd,
Moumita Chaki,
Katrina Diaz,
Francis P Lach,
Geoffrey R Bennett,
Heon Yung Gee, [......],
Tobias B Huber,
Andreas Friedl,
Gisela G Slaats,
Jaap A Joles,
Roel Goldschmeding,
Joseph Washburn,
Rachel H Giles,
Shawn Levy,
Agata Smogorzewska,
Friedhelm Hildebrandt
[show abstract]
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ABSTRACT: Chronic kidney disease (CKD) represents a major health burden. Its central feature of renal fibrosis is not well understood. By exome sequencing, we identified mutations in FAN1 as a cause of karyomegalic interstitial nephritis (KIN), a disorder that serves as a model for renal fibrosis. Renal histology in KIN is indistinguishable from that of nephronophthisis, except for the presence of karyomegaly. The FAN1 protein has nuclease activity and acts in DNA interstrand cross-link (ICL) repair within the Fanconi anemia DNA damage response (DDR) pathway. We show that cells from individuals with FAN1 mutations have sensitivity to the ICL-inducing agent mitomycin C but do not exhibit chromosome breakage or cell cycle arrest after diepoxybutane treatment, unlike cells from individuals with Fanconi anemia. We complemented ICL sensitivity with wild-type FAN1 but not with cDNA having mutations found in individuals with KIN. Depletion of fan1 in zebrafish caused increased DDR, apoptosis and kidney cysts. Our findings implicate susceptibility to environmental genotoxins and inadequate DNA repair as novel mechanisms contributing to renal fibrosis and CKD.
Nature Genetics 07/2012; 44(8):910-5. · 35.53 Impact Factor
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Bugsu Ovunc,
Edgar A Otto,
Virginia Vega-Warner,
Pawaree Saisawat,
Shazia Ashraf, Gokul Ramaswami,
Hanan M Fathy,
Dominik Schoeb,
Gil Chernin,
Robert H Lyons,
Engin Yilmaz,
Friedhelm Hildebrandt
[show abstract]
[hide abstract]
ABSTRACT: In two siblings of consanguineous parents with intermittent nephrotic-range proteinuria, we identified a homozygous deleterious frameshift mutation in the gene CUBN, which encodes cubulin, using exome capture and massively parallel re-sequencing. The mutation segregated with affected members of this family and was absent from 92 healthy individuals, thereby identifying a recessive mutation in CUBN as the single-gene cause of proteinuria in this sibship. Cubulin mutations cause a hereditary form of megaloblastic anemia secondary to vitamin B(12) deficiency, and proteinuria occurs in 50% of cases since cubilin is coreceptor for both the intestinal vitamin B(12)-intrinsic factor complex and the tubular reabsorption of protein in the proximal tubule. In summary, we report successful use of exome capture and massively parallel re-sequencing to identify a rare, single-gene cause of nephropathy.
Journal of the American Society of Nephrology 09/2011; 22(10):1815-20. · 9.66 Impact Factor
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[show abstract]
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ABSTRACT: Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, is the most frequent genetic cause for end-stage renal failure in the first three decades of life. Mutations in 13 genes (NPHP1-NPHP11, AHI1, and CC2D2A) cause NPHP with ubiquitous expression of the corresponding proteins consistent with the multiorgan involvement of NPHP-related diseases. The genotype-phenotype correlation in these ciliopathies can be explained by gene locus heterogeneity, allelism, and the impact of modifier genes. In some NPHP-related ciliopathies, the nature of the recessive mutations determines disease severity. In order to define the genotype-phenotype correlation more clearly, we evaluated a worldwide cohort of 440 patients from 365 families with NPHP-related ciliopathies, in whom both disease-causing alleles were identified. The phenotypes were ranked in the order of severity from degenerative to degenerative/dysplastic to dysplastic. A genotype of two null alleles caused a range of phenotypes, with an increasing order of severity of NPHP1, NPHP3, NPHP4, NPHP5, NPHP2, NPHP10, NPHP6, to AHI1. Only NPHP6 showed allelic influences on the phenotypes; the presence of two null mutations caused dysplastic phenotypes, whereas at least one missense allele rescued it to a milder degenerative phenotype. We also found nine novel mutations in the NPHP genes. Thus, our studies have important implications for genetic counseling and planning of renal replacement therapy.
Kidney International 08/2011; 80(11):1239-45. · 6.61 Impact Factor
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Francesc R Garcia-Gonzalo,
Kevin C Corbit,
María Salomé Sirerol-Piquer, Gokul Ramaswami,
Edgar A Otto,
Thomas R Noriega,
Allen D Seol,
Jon F Robinson,
Christopher L Bennett,
Dragana J Josifova,
José Manuel García-Verdugo,
Nicholas Katsanis,
Friedhelm Hildebrandt,
Jeremy F Reiter
[show abstract]
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ABSTRACT: Mutations affecting ciliary components cause ciliopathies. As described here, we investigated Tectonic1 (Tctn1), a regulator of mouse Hedgehog signaling, and found that it is essential for ciliogenesis in some, but not all, tissues. Cell types that do not require Tctn1 for ciliogenesis require it to localize select membrane-associated proteins to the cilium, including Arl13b, AC3, Smoothened and Pkd2. Tctn1 forms a complex with multiple ciliopathy proteins associated with Meckel and Joubert syndromes, including Mks1, Tmem216, Tmem67, Cep290, B9d1, Tctn2 and Cc2d2a. Components of this complex co-localize at the transition zone, a region between the basal body and ciliary axoneme. Like Tctn1, loss of Tctn2, Tmem67 or Cc2d2a causes tissue-specific defects in ciliogenesis and ciliary membrane composition. Consistent with a shared function for complex components, we identified a mutation in TCTN1 that causes Joubert syndrome. Thus, a transition zone complex of Meckel and Joubert syndrome proteins regulates ciliary assembly and trafficking, suggesting that transition zone dysfunction is the cause of these ciliopathies.
Nature Genetics 07/2011; 43(8):776-84. · 35.53 Impact Factor
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Erica E Davis,
Qi Zhang,
Qin Liu,
Bill H Diplas,
Lisa M Davey,
Jane Hartley,
Corinne Stoetzel,
Katarzyna Szymanska, Gokul Ramaswami,
Clare V Logan, [......],
Joseph G Gleeson,
Eamonn R Maher,
Tania Attié-Bitach,
Hélène Dollfus,
Colin A Johnson,
Eric D Green,
Richard A Gibbs,
Friedhelm Hildebrandt,
Eric A Pierce,
Nicholas Katsanis
[show abstract]
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ABSTRACT: Ciliary dysfunction leads to a broad range of overlapping phenotypes, collectively termed ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifier alleles to clinically distinct disorders. Here we show that mutations in TTC21B, which encodes the retrograde intraflagellar transport protein IFT139, cause both isolated nephronophthisis and syndromic Jeune asphyxiating thoracic dystrophy. Moreover, although resequencing of TTC21B in a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations showed a significant enrichment of pathogenic alleles in cases (P < 0.003), suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy cases. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies and interact in trans with other disease-causing genes and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of inherited disorders.
Nature Genetics 01/2011; 43(3):189-96. · 35.53 Impact Factor
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Edgar A Otto, Gokul Ramaswami,
Sabine Janssen,
Moumita Chaki,
Susan J Allen,
Weibin Zhou,
Rannar Airik,
Toby W Hurd,
Amiya K Ghosh,
Matthias T Wolf,
Bernd Hoppe,
Thomas J Neuhaus,
Detlef Bockenhauer,
David V Milford,
Neveen A Soliman,
Corinne Antignac,
Sophie Saunier,
Colin A Johnson,
Friedhelm Hildebrandt
[show abstract]
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ABSTRACT: Nephronophthisis associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative mutations in NPHP-AC have been described for 18 different genes, rendering mutation analysis tedious and expensive. To overcome the broad genetic locus heterogeneity, a strategy of DNA pooling with consecutive massively parallel resequencing (MPR) was devised.
In 120 patients with severe NPHP-AC phenotypes, five pools of genomic DNA with 24 patients each were prepared which were used as templates in order to PCR amplify all 376 exons of 18 NPHP-AC genes (NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, TMEM67, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, TTC21B, MKS1, and XPNPEP3). PCR products were then subjected to MPR on an Illumina Genome-Analyser and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease based heteroduplex screening and confirmed by Sanger sequencing.
For proof of principle, DNA from patients with known mutations was used and detection of 22 out of 24 different alleles (92% sensitivity) was demonstrated. MPR led to the molecular diagnosis in 30/120 patients (25%) and 54 pathogenic mutations (27 novel) were identified in seven different NPHP-AC genes. Additionally, in 24 patients only single heterozygous variants of unknown significance were found.
The combined approach of DNA pooling followed by MPR strongly facilitates mutation analysis in broadly heterogeneous single gene disorders. The lack of mutations in 75% of patients in this cohort indicates further extensive heterogeneity in NPHP-AC.
Journal of Medical Genetics 11/2010; 48(2):105-16. · 6.36 Impact Factor
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Sabine Janssen, Gokul Ramaswami,
Erica E Davis,
Toby Hurd,
Rannar Airik,
Jennifer M Kasanuki,
Lauren Van Der Kraak,
Susan J Allen,
Philip L Beales,
Nicholas Katsanis,
Edgar A Otto,
Friedhelm Hildebrandt
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ABSTRACT: Bardet-Biedl syndrome (BBS) is a rare, primarily autosomal-recessive ciliopathy. The phenotype of this pleiotropic disease includes retinitis pigmentosa, postaxial polydactyly, truncal obesity, learning disabilities, hypogonadism and renal anomalies, among others. To date, mutations in 15 genes (BBS1-BBS14, SDCCAG8) have been described to cause BBS. The broad genetic locus heterogeneity renders mutation screening time-consuming and expensive. We applied a strategy of DNA pooling and subsequent massively parallel resequencing (MPR) to screen individuals affected with BBS from 105 families for mutations in 12 known BBS genes. DNA was pooled in 5 pools of 21 individuals each. All 132 coding exons of BBS1-BBS12 were amplified by conventional PCR. Subsequent MPR was performed on an Illumina Genome Analyzer II™ platform. Following mutation identification, the mutation carrier was assigned by CEL I endonuclease heteroduplex screening and confirmed by Sanger sequencing. In 29 out of 105 individuals (28%), both mutated alleles were identified in 10 different BBS genes. A total of 35 different disease-causing mutations were confirmed, of which 18 mutations were novel. In 12 additional families, a total of 12 different single heterozygous changes of uncertain pathogenicity were found. Thus, DNA pooling combined with MPR offers a valuable strategy for mutation analysis of large patient cohorts, especially in genetically heterogeneous diseases such as BBS.
Human Genetics 10/2010; 129(1):79-90. · 5.07 Impact Factor
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Edgar A Otto,
Toby W Hurd,
Rannar Airik,
Moumita Chaki,
Weibin Zhou,
Corinne Stoetzel,
Suresh B Patil,
Shawn Levy,
Amiya K Ghosh,
Carlos A Murga-Zamalloa, [......],
Gudrun Nürnberg,
Peter Nürnberg,
Eric A Pierce,
Peter K Jackson,
Corinne Antignac,
Sophie Saunier,
Ronald Roepman,
Helene Dollfus,
Hemant Khanna,
Friedhelm Hildebrandt
[show abstract]
[hide abstract]
ABSTRACT: Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders that feature dysplasia or degeneration occurring preferentially in the kidney, retina and cerebellum. Here we combined homozygosity mapping with candidate gene analysis by performing 'ciliopathy candidate exome capture' followed by massively parallel sequencing. We identified 12 different truncating mutations of SDCCAG8 (serologically defined colon cancer antigen 8, also known as CCCAP) in 10 families affected by NPHP-RC. We show that SDCCAG8 is localized at both centrioles and interacts directly with OFD1 (oral-facial-digital syndrome 1), which is associated with NPHP-RC. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in three-dimensional renal cell cultures. This work identifies loss of SDCCAG8 function as a cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.
Nature Genetics 10/2010; 42(10):840-50. · 35.53 Impact Factor
