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2.  Phenotypic spectrum and prevalence of INPP5E mutations in Joubert Syndrome and related disorders 
European Journal of Human Genetics  2013;21(10):1074-1078.
Joubert syndrome and related disorders (JSRD) are clinically and genetically heterogeneous ciliopathies sharing a peculiar midbrain–hindbrain malformation known as the ‘molar tooth sign'. To date, 19 causative genes have been identified, all coding for proteins of the primary cilium. There is clinical and genetic overlap with other ciliopathies, in particular with Meckel syndrome (MKS), that is allelic to JSRD at nine distinct loci. We previously identified the INPP5E gene as causative of JSRD in seven families linked to the JBTS1 locus, yet the phenotypic spectrum and prevalence of INPP5E mutations in JSRD and MKS remain largely unknown. To address this issue, we performed INPP5E mutation analysis in 483 probands, including 408 JSRD patients representative of all clinical subgroups and 75 MKS fetuses. We identified 12 different mutations in 17 probands from 11 JSRD families, with an overall 2.7% mutation frequency among JSRD. The most common clinical presentation among mutated families (7/11, 64%) was Joubert syndrome with ocular involvement (either progressive retinopathy and/or colobomas), while the remaining cases had pure JS. Kidney, liver and skeletal involvement were not observed. None of the MKS fetuses carried INPP5E mutations, indicating that the two ciliopathies are not allelic at this locus.
doi:10.1038/ejhg.2012.305
PMCID: PMC3778343  PMID: 23386033
INPP5E; Joubert syndrome and related disorders; Meckel syndrome; ciliopathies
3.  Mutations in TJP2 cause progressive cholestatic liver disease 
Nature genetics  2014;46(4):326-328.
The elucidation of genetic causes of cholestasis has proved to be important in understanding the physiology and pathophysiology of the liver. Protein-truncating mutations in the tight junction protein 2 gene (TJP2) are shown to cause failure of protein localisation, with disruption of tight-junction structure leading to severe cholestatic liver disease. This contrasts with the embryonic-lethal knockout mouse, highlighting differences in redundancy in junctional complexes between organs and species.
doi:10.1038/ng.2918
PMCID: PMC4061468  PMID: 24614073
4.  ATMIN is a transcriptional regulator of both lung morphogenesis and ciliogenesis 
Development (Cambridge, England)  2014;141(20):3966-3977.
Initially identified in DNA damage repair, ATM-interactor (ATMIN) further functions as a transcriptional regulator of lung morphogenesis. Here we analyse three mouse mutants, Atmingpg6/gpg6, AtminH210Q/H210Q and Dynll1GT/GT, revealing how ATMIN and its transcriptional target dynein light chain LC8-type 1 (DYNLL1) are required for normal lung morphogenesis and ciliogenesis. Expression screening of ciliogenic genes confirmed Dynll1 to be controlled by ATMIN and further revealed moderately altered expression of known intraflagellar transport (IFT) protein-encoding loci in Atmin mutant embryos. Significantly, Dynll1GT/GT embryonic cilia exhibited shortening and bulging, highly similar to the characterised retrograde IFT phenotype of Dync2h1. Depletion of ATMIN or DYNLL1 in cultured cells recapitulated the in vivo ciliogenesis phenotypes and expression of DYNLL1 or the related DYNLL2 rescued the effects of loss of ATMIN, demonstrating that ATMIN primarily promotes ciliogenesis by regulating Dynll1 expression. Furthermore, DYNLL1 as well as DYNLL2 localised to cilia in puncta, consistent with IFT particles, and physically interacted with WDR34, a mammalian homologue of the Chlamydomonas cytoplasmic dynein 2 intermediate chain that also localised to the cilium. This study extends the established Atmin-Dynll1 relationship into a developmental and a ciliary context, uncovering a novel series of interactions between DYNLL1, WDR34 and ATMIN. This identifies potential novel components of cytoplasmic dynein 2 and furthermore provides fresh insights into the molecular pathogenesis of human skeletal ciliopathies.
doi:10.1242/dev.107755
PMCID: PMC4197704  PMID: 25294941
Asciz; Atmin; Ciliogenesis; Ciliopathy; Dynll1; Mouse
5.  The C2 Domains of Otoferlin, Dysferlin, and Myoferlin Alter the Packing of Lipid Bilayers 
Biochemistry  2013;52(33):10.1021/bi400432f.
Ferlins are large multi-C2 domain membrane proteins involved in membrane fusion and fission events. In this study we investigate the effects binding of the C2 domains of otoferlin, dysferlin and myoferlin have upon the structure of lipid bilayers. Fluorescence measurements indicate that multi-C2 domain constructs of myoferlin, dysferlin and otoferlin change the lipid packing of both small unilamellar vesicles and giant plasma membrane vesicles. The activities of these proteins were enhanced in the presence of calcium, and required negatively charged lipids like phosphatidylserine or phosphatidylglycerol for activity. Experiments on individual domains uncovered functional differences between the C2A domain of otoferlin as compared to dysferlin and myoferlin, and truncation studies suggest that the effects of each subsequent C2 domain on lipid ordering appear additive. Finally, we demonstrate that the activities of these proteins on membranes are insensitive to high salt concentrations, suggesting a non-electrostatic component to the interaction between ferlin C2 domains and lipid bilayers. Together, the data indicate that dysferlin, otoferlin, and myoferlin do not merely passively adsorb to membranes, but actively sculpt lipid bilayers, which would result in highly curved or distorted membrane regions that could facilitate membrane fusion, fission, or recruitment of other membrane trafficking proteins.
doi:10.1021/bi400432f
PMCID: PMC3826957  PMID: 23859474
otoferlin; dysferlin; myoferlin; laurdan; C2 domain
6.  Mutation Screening of Retinal Dystrophy Patients by Targeted Capture from Tagged Pooled DNAs and Next Generation Sequencing 
PLoS ONE  2014;9(8):e104281.
Purpose
Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies.
Methods
Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing.
Results
Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D).
Conclusions
Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics.
doi:10.1371/journal.pone.0104281
PMCID: PMC4136783  PMID: 25133751
7.  The role of primary cilia in the development and disease of the retina 
Organogenesis  2013;10(1):69-85.
The normal development and function of photoreceptors is essential for eye health and visual acuity in vertebrates. Mutations in genes encoding proteins involved in photoreceptor development and function are associated with a suite of inherited retinal dystrophies, often as part of complex multi-organ syndromic conditions. In this review, we focus on the role of the photoreceptor outer segment, a highly modified and specialized primary cilium, in retinal health and disease. We discuss the many defects in the structure and function of the photoreceptor primary cilium that can cause a class of inherited conditions known as ciliopathies, often characterized by retinal dystrophy and degeneration, and highlight the recent insights into disease mechanisms.
doi:10.4161/org.26710
PMCID: PMC4049897  PMID: 24162842
primary cilia; ciliopathy; inherted retinal conditions; photoreceptor development; retina; intraflagellar transport
8.  Variable expressivity of ciliopathy neurological phenotypes that encompass Meckel–Gruber syndrome and Joubert syndrome is caused by complex de-regulated ciliogenesis, Shh and Wnt signalling defects 
Human Molecular Genetics  2013;22(7):1358-1372.
The ciliopathies are a group of heterogeneous diseases with considerable variations in phenotype for allelic conditions such as Meckel–Gruber syndrome (MKS) and Joubert syndrome (JBTS) even at the inter-individual level within families. In humans, mutations in TMEM67 (also known as MKS3) cause both MKS and JBTS, with TMEM67 encoding the orphan receptor meckelin (TMEM67) that localizes to the ciliary transition zone. We now describe the Tmem67tm1(Dgen/H) knockout mouse model that recapitulates the brain phenotypic variability of these human ciliopathies, with categorization of Tmem67 mutant animals into two phenotypic groups. An MKS-like incipient congenic group (F6 to F10) manifested very variable neurological features (including exencephaly, and frontal/occipital encephalocele) that were associated with the loss of primary cilia, diminished Shh signalling and dorsalization of the caudal neural tube. The ‘MKS-like’ group also had high de-regulated canonical Wnt/β-catenin signalling associated with hyper-activated Dishevelled-1 (Dvl-1) localized to the basal body. Conversely, a second fully congenic group (F > 10) had less variable features pathognomonic for JBTS (including cerebellar hypoplasia), and retention of abnormal bulbous cilia associated with mild neural tube ventralization. The ‘JBTS-like’ group had de-regulated low levels of canonical Wnt signalling associated with the loss of Dvl-1 localization to the basal body. Our results suggest that modifier alleles partially determine the variation between MKS and JBTS, implicating the interaction between Dvl-1 and meckelin, or other components of the ciliary transition zone. The Tmem67tm1(Dgen/H) line is unique in modelling the variable expressivity of phenotypes in these two ciliopathies.
doi:10.1093/hmg/dds546
PMCID: PMC3596847  PMID: 23283079
9.  Mutation Analysis of 18 Nephronophthisis-associated Ciliopathy Disease Genes using a DNA Pooling and Next-Generation Sequencing Strategy 
Journal of medical genetics  2010;48(2):105-116.
Background
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 we devised a strategy of DNA pooling with consecutive massively parallel resequencing (MPR).
Methods
In 120 patients with severe NPHP-AC phenotypes we prepared 5 pools of genomic DNA with 24 patients each 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 a Illumina Genome-Analyzer and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease-based heteroduplex screening and confirmed by Sanger sequencing.
Results
For proof of principle we used DNA from patients with known mutations and demonstrated the detection of 22 out of 24 different alleles (92% sensitivity). MPR led to the molecular diagnosis in 30/120 patients (25%) and we identified 54 pathogenic mutations (27 novel) in 7 different NPHP-AC genes. Additionally, in 24 patients we only found single heterozygous variants of unknown significance.
Conclusions
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 our cohort indicates further extensive heterogeneity in NPHP-AC.
doi:10.1136/jmg.2010.082552
PMCID: PMC3913043  PMID: 21068128
Next-generation sequencing; Ciliopathy; Nephronophthisis
10.  Nephrocalcinosis (Enamel Renal Syndrome) Caused by Autosomal Recessive FAM20A Mutations 
Jaureguiberry, Graciana | De la Dure-Molla, Muriel | Parry, David | Quentric, Mickael | Himmerkus, Nina | Koike, Toshiyasu | Poulter, James | Klootwijk, Enriko | Robinette, Steven L. | Howie, Alexander J. | Patel, Vaksha | Figueres, Marie-Lucile | Stanescu, Horia C. | Issler, Naomi | Nicholson, Jeremy K. | Bockenhauer, Detlef | Laing, Christopher | Walsh, Stephen B. | McCredie, David A. | Povey, Sue | Asselin, Audrey | Picard, Arnaud | Coulomb, Aurore | Medlar, Alan J. | Bailleul-Forestier, Isabelle | Verloes, Alain | Le Caignec, Cedric | Roussey, Gwenaelle | Guiol, Julien | Isidor, Bertrand | Logan, Clare | Shore, Roger | Johnson, Colin | Inglehearn, Christopher | Al-Bahlani, Suhaila | Schmittbuhl, Matthieu | Clauss, François | Huckert, Mathilde | Laugel, Virginie | Ginglinger, Emmanuelle | Pajarola, Sandra | Spartà, Giuseppina | Bartholdi, Deborah | Rauch, Anita | Addor, Marie-Claude | Yamaguti, Paulo M. | Safatle, Heloisa P. | Acevedo, Ana Carolina | Martelli-Júnior, Hercílio | dos Santos Netos, Pedro E. | Coletta, Ricardo D. | Gruessel, Sandra | Sandmann, Carolin | Ruehmann, Denise | Langman, Craig B. | Scheinman, Steven J. | Ozdemir-Ozenen, Didem | Hart, Thomas C. | Hart, P. Suzanne | Neugebauer, Ute | Schlatter, Eberhard | Houillier, Pascal | Gahl, William A. | Vikkula, Miikka | Bloch-Zupan, Agnès | Bleich, Markus | Kitagawa, Hiroshi | Unwin, Robert J. | Mighell, Alan | Berdal, Ariane | Kleta, Robert
Nephron. Physiology  2013;122(0):1-6.
Background/Aims
Calcium homeostasis requires regulated cellular and interstitial systems interacting to modulate the activity and movement of this ion. Disruption of these systems in the kidney results in nephrocalcinosis and nephrolithiasis, important medical problems whose pathogenesis is incompletely understood.
Methods
We investigated 25 patients from 16 families with unexplained nephrocalcinosis and characteristic dental defects (amelogenesis imperfecta, gingival hyperplasia, impaired tooth eruption). To identify the causative gene, we performed genome-wide linkage analysis, exome capture, next-generation sequencing, and Sanger sequencing.
Results
All patients had biallelic FAM20A mutations segregating with the disease; 20 different mutations were identified.
Conclusions
This autosomal recessive disorder, also known as enamel renal syndrome, of FAM20A causes nephrocalcinosis and amelogenesis imperfecta. We speculate that all individuals with biallelic FAM20A mutations will eventually show nephrocalcinosis.
doi:10.1159/000349989
PMCID: PMC3782194  PMID: 23434854
Nephrolithiasis; Urolithiasis; Amelogenesis imperfecta; FAM20B; FAM20C
11.  Evolutionarily Assembled cis-Regulatory Module at a Human Ciliopathy Locus 
Science (New York, N.Y.)  2012;335(6071):966-969.
Neighboring genes are often coordinately expressed within cis-regulatory modules, but evidence that nonparalogous genes share functions in mammals is lacking. Here, we report that mutation of either TMEM138 or TMEM216 causes a phenotypically indistinguishable human ciliopathy, Joubert syndrome. Despite a lack of sequence homology, the genes are aligned in a head-to-tail configuration and joined by chromosomal rearrangement at the amphibian-to-reptile evolutionary transition. Expression of the two genes is mediated by a conserved regulatory element in the noncoding intergenic region. Coordinated expression is important for their interdependent cellular role in vesicular transport to primary cilia. Hence, during vertebrate evolution of genes involved in ciliogenesis, nonparalogous genes were arranged to a functional gene cluster with shared regulatory elements.
doi:10.1126/science.1213506
PMCID: PMC3671610  PMID: 22282472
12.  Mutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration 
Nature genetics  2012;44(9):1035-1039.
Leber congenital amaurosis (LCA) is a blinding retinal disease that presents within the first year after birth. Using exome sequencing, we identified mutations in the nicotinamide adenine dinucleotide (NAD) synthase gene NMNAT1 encoding nicotinamide mononucleotide adenylyltransferase 1 in eight families with LCA, including the family in which LCA was originally linked to the LCA9 locus. Notably, all individuals with NMNAT1 mutations also have macular colobomas, which are severe degenerative entities of the central retina (fovea) devoid of tissue and photoreceptors. Functional assays of the proteins encoded by the mutant alleles identified in our study showed that the mutations reduce the enzymatic activity of NMNAT1 in NAD biosynthesis and affect protein folding. Of note, recent characterization of the slow Wallerian degeneration (Wlds) mouse model, in which prolonged axonal survival after injury is observed, identified NMNAT1 as a neuroprotective protein when ectopically expressed. Our findings identify a new disease mechanism underlying LCA and provide the first link between endogenous NMNAT1 dysfunction and a human nervous system disorder.
doi:10.1038/ng.2356
PMCID: PMC3657614  PMID: 22842230
13.  A meckelin–filamin A interaction mediates ciliogenesis 
Human Molecular Genetics  2011;21(6):1272-1286.
MKS3, encoding the transmembrane receptor meckelin, is mutated in Meckel–Gruber syndrome (MKS), an autosomal-recessive ciliopathy. Meckelin localizes to the primary cilium, basal body and elsewhere within the cell. Here, we found that the cytoplasmic domain of meckelin directly interacts with the actin-binding protein filamin A, potentially at the apical cell surface associated with the basal body. Mutations in FLNA, the gene for filamin A, cause periventricular heterotopias. We identified a single consanguineous patient with an MKS-like ciliopathy that presented with both MKS and cerebellar heterotopia, caused by an unusual in-frame deletion mutation in the meckelin C-terminus at the region of interaction with filamin A. We modelled this mutation and found it to abrogate the meckelin–filamin A interaction. Furthermore, we found that loss of filamin A by siRNA knockdown, in patient cells, and in tissues from FlnaDilp2 null mouse embryos results in cellular phenotypes identical to those caused by meckelin loss, namely basal body positioning and ciliogenesis defects. In addition, morpholino knockdown of flna in zebrafish embryos significantly increases the frequency of dysmorphology and severity of ciliopathy developmental defects caused by mks3 knockdown. Our results suggest that meckelin forms a functional complex with filamin A that is disrupted in MKS and causes defects in neuronal migration and Wnt signalling. Furthermore, filamin A has a crucial role in the normal processes of ciliogenesis and basal body positioning. Concurrent with these processes, the meckelin–filamin A signalling axis may be a key regulator in maintaining correct, normal levels of Wnt signalling.
doi:10.1093/hmg/ddr557
PMCID: PMC3284117  PMID: 22121117
14.  Stereotactic body radiation therapy with concurrent full-dose gemcitabine for locally advanced pancreatic cancer: a pilot trial demonstrating safety 
Background
Concurrent chemoradiation is a standard option for locally advanced pancreatic cancer (LAPC). Concurrent conventional radiation with full-dose gemcitabine has significant toxicity. Stereotactic body radiation therapy (SBRT) may provide the opportunity to administer radiation in a shorter time frame with similar efficacy and reduced toxicity. This Pilot study assessed the safety of concurrent full-dose gemcitabine with SBRT for LAPC.
Methods
Patients received gemcitabine, 1000 mg/m2 for 6 cycles. During week 4 of cycle 1, patients received SBRT (25 Gy delivered in five consecutive daily fractions of 5 Gy prescribed to the 75-83% isodose line). Acute and late toxicities were assessed using NIH CTCAE v3. Tumor response was assessed by RECIST. Patients underwent an esophagogastroduodenoscopy at baseline, 2, and 6 months to assess the duodenal mucosa. Quality of life (QoL) data was collected before and after treatment using the QLQ-C30 and QLQ-PAN26 questionnaires.
Results
Between September 2009 and February 2011, 11 patients enrolled with one withdrawal during radiation therapy. Patients had grade 1 to 2 gastrointestinal toxicity from the start of SBRT to 2 weeks after treatment. There were no grade 3 or greater radiation-related toxicities or delays for cycle 2 of gemcitabine. On endoscopy, there were no grade 2 or higher mucosal toxicities. Two patients had a partial response. The median progression free and overall survival were 6.8 and 12.2 months, respectively. Global QoL did not change between baseline and immediately after radiation treatment.
Conclusions
SBRT with concurrent full dose gemcitabine is safe when administered to patients with LAPC. There is no delay in administration of radiation or chemotherapy, and radiation is completed with minimal toxicity.
doi:10.1186/1748-717X-8-44
PMCID: PMC3607991  PMID: 23452509
Pancreatic cancer; Gemcitabine; Radiation; Stereotactic body radiation therapy; SBRT; CyberKnife; Quality of life; QLQ-C30; QLQ-PAN26; Upper endoscopy
15.  Nephrocalcinosis (Enamel Renal Syndrome) Caused by Autosomal Recessive FAM20A Mutations 
Jaureguiberry, Graciana | De la Dure-Molla, Muriel | Parry, David | Quentric, Mickael | Himmerkus, Nina | Koike, Toshiyasu | Poulter, James | Klootwijk, Enriko | Robinette, Steven L. | Howie, Alexander J. | Patel, Vaksha | Figueres, Marie-Lucile | Stanescu, Horia C. | Issler, Naomi | Nicholson, Jeremy K. | Bockenhauer, Detlef | Laing, Christopher | Walsh, Stephen B. | McCredie, David A. | Povey, Sue | Asselin, Audrey | Picard, Arnaud | Coulomb, Aurore | Medlar, Alan J. | Bailleul-Forestier, Isabelle | Verloes, Alain | Le Caignec, Cedric | Roussey, Gwenaelle | Guiol, Julien | Isidor, Bertrand | Logan, Clare | Shore, Roger | Johnson, Colin | Inglehearn, Christopher | Al-Bahlani, Suhaila | Schmittbuhl, Matthieu | Clauss, François | Huckert, Mathilde | Laugel, Virginie | Ginglinger, Emmanuelle | Pajarola, Sandra | Spartà, Giuseppina | Bartholdi, Deborah | Rauch, Anita | Addor, Marie-Claude | Yamaguti, Paulo M. | Safatle, Heloisa P. | Acevedo, Ana Carolina | Martelli-Júnior, Hercílio | dos Santos Netos, Pedro E. | Coletta, Ricardo D. | Gruessel, Sandra | Sandmann, Carolin | Ruehmann, Denise | Langman, Craig B. | Scheinman, Steven J. | Ozdemir-Ozenen, Didem | Hart, Thomas C. | Hart, P. Suzanne | Neugebauer, Ute | Schlatter, Eberhard | Houillier, Pascal | Gahl, William A. | Vikkula, Miikka | Bloch-Zupan, Agnès | Bleich, Markus | Kitagawa, Hiroshi | Unwin, Robert J. | Mighell, Alan | Berdal, Ariane | Kleta, Robert
Nephron. Physiology  2013;122(1-2):1-6.
Background/Aims
Calcium homeostasis requires regulated cellular and interstitial systems interacting to modulate the activity and movement of this ion. Disruption of these systems in the kidney results in nephrocalcinosis and nephrolithiasis, important medical problems whose pathogenesis is incompletely understood.
Methods
We investigated 25 patients from 16 families with unexplained nephrocalcinosis and characteristic dental defects (amelogenesis imperfecta, gingival hyperplasia, impaired tooth eruption). To identify the causative gene, we performed genome-wide linkage analysis, exome capture, next-generation sequencing, and Sanger sequencing.
Results
All patients had bi-allelic FAM20A mutations segregating with the disease; 20 different mutations were identified.
Conclusions
This au-tosomal recessive disorder, also known as enamel renal syndrome, of FAM20A causes nephrocalcinosis and amelogenesis imperfecta. We speculate that all individuals with biallelic FAM20A mutations will eventually show nephrocalcinosis.
doi:10.1159/000349989
PMCID: PMC3782194  PMID: 23434854
Nephrolithiasis; Urolithiasis; Amelogenesis imperfecta; FAM20B; FAM20C

16.  Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling 
Chaki, Moumita | Airik, Rannar | Ghosh, Amiya K. | Giles, Rachel H. | Chen, Rui | Slaats, Gisela G. | Wang, Hui | Hurd, Toby W. | Zhou, Weibin | Cluckey, Andrew | Gee, Heon-Yung | Ramaswami, Gokul | Hong, Chen-Jei | Hamilton, Bruce A. | Červenka, Igor | Ganji, Ranjani Sri | Bryja, Vitezslav | Arts, Heleen H. | van Reeuwijk, Jeroen | Oud, Machteld M. | Letteboer, Stef J.F. | Roepman, Ronald | Husson, Hervé | Ibraghimov-Beskrovnaya, Oxana | Ysunaga, Takayuki | Walz, Gerd | Eley, Lorraine | Sayer, John A. | Schermer, Bernhard | Liebau, Max C. | Benzing, Thomas | Le Corre, Stephanie | Drummond, Iain | Joles, Jaap A. | Janssen, Sabine | Allen, Susan J. | Natarajan, Sivakumar | O Toole, John F. | Attanasio, Massimo | Saunier, Sophie | Antignac, Corinne | Koenekoop, Robert K. | Ren, Huanan | Lopez, Irma | Nayir, Ahmet | Stoetzel, Corinne | Dollfus, Helene | Massoudi, Rustin | Gleeson, Joseph G. | Andreoli, Sharon P. | Doherty, Dan G. | Lindstrad, Anna | Golzio, Christelle | Katsanis, Nicholas | Pape, Lars | Abboud, Emad B. | Al-Rajhi, Ali A. | Lewis, Richard A. | Lupski, James R. | Omran, Heymut | Lee, Eva | Wang, Shaohui | Sekiguchi, JoAnn M. | Saunders, Rudel | Johnson, Colin A. | Garner, Elizabeth | Vanselow, Katja | Andersen, Jens S. | Shlomai, Joseph | Nurnberg, Gudrun | Nurnberg, Peter | Levy, Shawn | Smogorzewska, Agata | Otto, Edgar A. | Hildebrandt, Friedhelm
Cell  2012;150(3):533-548.
SUMMARY
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, hitherto not implicated in ciliopathies. 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. We identify TTBK2, CCDC92, NPHP3 and DVL3 as novel CEP164 interaction partners. Our findings link degenerative diseases of kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.
doi:10.1016/j.cell.2012.06.028
PMCID: PMC3433835  PMID: 22863007
17.  A year of progress 
doi:10.1308/003588413X13511609956976
PMCID: PMC3964627
18.  Planar Cell Polarity Acts Through Septins to Control Collective Cell Movement and Ciliogenesis 
Science (New York, N.Y.)  2010;329(5997):1337-1340.
The planar cell polarity (PCP) signaling pathway governs collective cell movements duringvertebrate embryogenesis, and certain PCP proteins are also implicated in the assembly ofcilia. The septins are cytoskeletal proteins controlling behaviors such as cell division and migration. Here, we identified control of septin localization by the PCP protein Fritz as a crucial control point for both collective cell movement and ciliogenesis in Xenopus embryos. We also linked mutations in human Fritz to Bardet-Biedl and Meckel-Gruber syndromes, a notable link given that other genes mutated in these syndromes also influence collective cell movement and ciliogenesis. These findings shed light on the mechanisms by which fundamental cellular machinery, such as the cytoskeleton, is regulated during embryonic development and human disease.
doi:10.1126/science.1191184
PMCID: PMC3509789  PMID: 20671153
19.  Founder mutations and genotype-phenotype correlations in Meckel-Gruber syndrome and associated ciliopathies 
Cilia  2012;1:18.
Background
Meckel-Gruber syndrome (MKS) is an autosomal recessive lethal condition that is a ciliopathy. MKS has marked phenotypic variability and genetic heterogeneity, with mutations in nine genes identified as causative to date.
Methods
Families diagnosed with Meckel-Gruber syndrome were recruited for research studies following informed consent. DNA samples were analyzed by microsatellite genotyping and direct Sanger sequencing.
Results
We now report the genetic analyses of 87 individuals from 49 consanguineous and 19 non-consanguineous families in an unselected cohort with reported MKS, or an associated severe ciliopathy in a kindred. Linkage and/or direct sequencing were prioritized for seven MKS genes (MKS1, TMEM216, TMEM67/MKS3, RPGRIP1L, CC2D2A, CEP290 and TMEM237) selected on the basis of reported frequency of mutations or ease of analysis. We have identified biallelic mutations in 39 individuals, of which 13 mutations are novel and previously unreported. We also confirm general genotype-phenotype correlations.
Conclusions
TMEM67 was the most frequently mutated gene in this cohort, and we confirm two founder splice-site mutations (c.1546 + 1 G > A and c.870-2A > G) in families of Pakistani ethnic origin. In these families, we have also identified two separate founder mutations for RPGRIP1L (c. 1945 C > T p.R649X) and CC2D2A (c. 3540delA p.R1180SfsX6). Two missense mutations in TMEM67 (c. 755 T > C p.M252T, and c. 1392 C > T p.R441C) are also probable founder mutations. These findings will contribute to improved genetic diagnosis and carrier testing for affected families, and imply the existence of further genetic heterogeneity in this syndrome.
doi:10.1186/2046-2530-1-18
PMCID: PMC3579735  PMID: 23351400
Meckel-Gruber syndrome; Genotype-phenotype; Founder mutation
20.  CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium 
Nature Genetics  2012;44(2):193-199.
Tubulin glutamylation is a post-translational modification (PTM) occurring predominantly on ciliary axonemal tubulin and has been suggested to be important for ciliary function 1,2. However, its relationship to disorders of the primary cilium, termed ‘ciliopathies’, has not been explored. Here, in Joubert syndrome (JBTS) 3, we identify the JBTS15 locus and the responsible gene as CEP41, encoding a centrosomal protein of 41 KDa 4. We show that CEP41 is localized to the basal body/primary cilium, and regulates the ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme 5. Depletion of CEP41 causes ciliopathy-related phenotypes in zebrafish and mouse, and induces cilia axonemal glutamylation defects. Our data identify loss of CEP41 as a cause of JBTS ciliopathy and highlight involvement of tubulin PTM in pathogenesis of the ciliopathy spectrum.
doi:10.1038/ng.1078
PMCID: PMC3267856  PMID: 22246503
21.  The transition zone: an essential functional compartment of cilia 
Cilia  2012;1:10.
Recent studies of the primary cilium have begun to provide further insights into ciliary ultrastructure, with an emerging picture of complex compartmentalization and molecular components that combine in functional modules. Many proteins that are mutated in ciliopathies are localized to the transition zone, a compartment of the proximal region of the cilium. The loss of these components can disrupt ciliary functions such as the control of protein entry and exit from the cilium, the possible trafficking of essential ciliary components, and the regulation of signaling cascades and control of the cell cycle. The discovery of functional modules within the primary cilium may help in understanding the variable phenotypes and pleiotropy in ciliopathies.
doi:10.1186/2046-2530-1-10
PMCID: PMC3555838  PMID: 23352055
Cilia; Transition zone; IFT; Ciliopathies
22.  Cross-cultural development of an EORTC questionnaire to assess health-related quality of life in patients with testicular cancer: the EORTC QLQ-TC26 
Quality of Life Research  2012;22(2):369-378.
Objective
Testicular cancer (TC) is the most common cancer in young men, and its incidence is increasing. The low mortality rate makes quality of life (QOL) an important issue in this patient group. This study aimed to develop a supplementary module of the EORTC QLQ-C30 questionnaire to assess TC-specific aspects of QOL.
Methods
Questionnaire development was conducted according to guidelines from the EORTC Quality of Life Group. Phase I comprised generation of QOL issues relevant to TC patients through a literature search and interviews with patients and experts. Phase II included operationalization and assessment of item relevance. In phase III, items were pre-tested in a cross-cultural sample to assess issues such as understandability and intrusiveness of items.
Results
In phase I and II, an initial list of 69 QOL issues possibly relevant to TC patients was refined through patient and expert interviews. The remaining 37 issues were operationalized into items and assessed for relevance and priority in an expert sample (n = 28) and a patient sample (n = 62) from Austria, Canada and the Netherlands. After revision of the item list, 26 items were considered eligible for pre-testing in phase III, in which 156 patients from Australia, Austria, Italy and Spain participated. All items passed criteria for pre-testing, thus forming the new EORTC QLQ-TC26.
Conclusion
The newly developed EORTC QLQ-TC26 is now available in several languages to assess QOL in TC patients receiving treatment and in TC survivors. Phase IV of questionnaire development will comprise international field testing, including extensive analysis of psychometric characteristics of the EORTC QLQ-TC26.
doi:10.1007/s11136-012-0147-1
PMCID: PMC3576569  PMID: 22407356
Testicular cancer; Quality of life; Questionnaire; eortc qlq-tc26
23.  Mechanism and function of synaptotagmin-mediated membrane apposition 
SUMMARY
Synaptotagmin-I (syt) is a Ca2+ sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of syt was its ability to aggregate membranes in response to Ca2+. However, the mechanism and function of syt-mediated membrane aggregation are poorly understood. Here, we demonstrate that syt-mediated vesicle aggregation is driven by trans interactions between syt molecules bound to different membranes. We observed a strong correlation between the ability of Ca2+-syt to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes - using non-syt proteins - also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that syt drives the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion competent heterodimers, in an aggregation-independent manner. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of Ca2+-syt-regulated, SNARE-catalyzed fusion reactions.
doi:10.1038/nsmb.2075
PMCID: PMC3130839  PMID: 21642967
24.  e for editing 
doi:10.1308/003588411X12851639108277
PMCID: PMC3293258

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