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1.  Novel insights into the molecular pathogenesis of CYP4V2-associated Bietti's retinal dystrophy 
Bietti's crystalline dystrophy (BCD) is a rare, autosomal recessive retinal degenerative disease associated with mutations in CYP4V2. In this study, we describe the genetic and clinical findings in 19 unrelated BCD patients recruited from five international retinal dystrophy clinics. Patients underwent ophthalmic examinations and were screened for CYP4V2 mutations by Sanger sequencing and quantitative polymerase chain reaction (qPCR) copy number variation screening. Eight CYP4V2 mutations were found in 10/19 patients, including three patients in whom only monoallelic mutations were detected. Four novel mutations were identified: c.604G>A; p.(Glu202Lys), c.242C>G; p.(Thr81Arg), c.604+4A>G; p.(?), and c.1249dup; p.(Thr417Asnfs*2). In addition, we identified a heterozygous paternally inherited genomic deletion of at least 3.8 Mb, encompassing the complete CYP4V2 gene and several other genes, which is novel. Clinically, patients demonstrated phenotypic variability, predominantly showing choroidal sclerosis, attenuated vessels, and crystalline deposits of varying degrees of severity. To our knowledge, our study reports the first heterozygous CYP4V2 deletion and hence a novel mutational mechanism underlying BCD. Our results emphasize the importance of copy number screening in BCD. Finally, the identification of CYP4V2-negative patients with indistinguishable phenotypes from CYP4V2-positive patients might suggest the presence of mutations outside the coding regions of CYP4V2, or locus heterogeneity, which is unreported so far.
doi:10.1002/mgg3.109
PMCID: PMC4299712  PMID: 25629076
Bietti; crystalline dystrophy; CYP4V2; retinal dystrophy
2.  Comprehensive molecular diagnosis of 179 Leber congenital amaurosis and juvenile retinitis pigmentosa patients by targeted next generation sequencing 
Journal of medical genetics  2013;50(10):674-688.
Background
Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are inherited retinal diseases that cause early onset severe visual impairment. An accurate molecular diagnosis can refine the clinical diagnosis and allow gene specific treatments.
Methods
We developed a capture panel that enriches the exonic DNA of 163 known retinal disease genes. Using this panel, we performed targeted next generation sequencing (NGS) for a large cohort of 179 unrelated and prescreened patients with the clinical diagnosis of LCA or juvenile RP. Systematic NGS data analysis, Sanger sequencing validation, and segregation analysis were utilised to identify the pathogenic mutations. Patients were revisited to examine the potential phenotypic ambiguity at the time of initial diagnosis.
Results
Pathogenic mutations for 72 patients (40%) were identified, including 45 novel mutations. Of these 72 patients, 58 carried mutations in known LCA or juvenile RP genes and exhibited corresponding phenotypes, while 14 carried mutations in retinal disease genes that were not consistent with their initial clinical diagnosis. We revisited patients in the latter case and found that homozygous mutations in PRPH2 can cause LCA/juvenile RP. Guided by the molecular diagnosis, we reclassified the clinical diagnosis in two patients.
Conclusions
We have identified a novel gene and a large number of novel mutations that are associated with LCA/juvenile RP. Our results highlight the importance of molecular diagnosis as an integral part of clinical diagnosis.
doi:10.1136/jmedgenet-2013-101558
PMCID: PMC3932025  PMID: 23847139
3.  Next-Generation Sequencing–Based Molecular Diagnosis of a Chinese Patient Cohort With Autosomal Recessive Retinitis Pigmentosa 
Purpose.
Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease; therefore, an accurate molecular diagnosis is essential for appropriate disease treatment and family planning. The prevalence of RP in China had been reported at 1 in 3800, resulting in an estimated total of 340,000 Chinese RP patients. However, genetic studies of Chinese RP patients have been very limited. To date, no comprehensive molecular diagnosis has been done for Chinese RP patients. With the emergence of next-generation sequencing (NGS), comprehensive molecular diagnosis of RP is now within reach. The purpose of this study was to perform the first NGS-based comprehensive molecular diagnosis for Chinese RP patients.
Methods.
Thirty-one well-characterized autosomal recessive RP (arRP) families were recruited. For each family, the DNA sample from one affected member was sequenced using our custom capture panel, which includes 163 retinal disease genes. Variants were called, filtered, and annotated by our in-house automatic pipeline.
Results.
Twelve arRP families were successfully molecular diagnosed, achieving a diagnostic rate of approximately 40%. Interestingly, approximately 63% of the pathogenic mutations we identified are novel, which is higher than that observed in a similar study on European descent (45%). Moreover, the clinical diagnoses of two families were refined based on the pathogenic mutations identified in the patients.
Conclusions.
We conclude that comprehensive molecular diagnosis can be vital for an accurate clinical diagnosis of RP. Applying this tool on patients from different ethnic groups is essential for enhancing our knowledge of the global spectrum of RP disease-causing mutations.
Next-generation sequencing–based comprehensive molecular diagnosis of 31 families with autosomal recessive retinitis pigmentosa (arRP) was performed. This is the first such study of a Chinese arRP patient cohort, revealing a total of 10 novel putatively pathogenic mutations.
doi:10.1167/iovs.13-11672
PMCID: PMC3684217  PMID: 23661369
retinitis pigmentosa; next-generation sequencing; molecular diagnosis; Chinese population
4.  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
5.  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
6.  A (H1N1) pdm09 HA D222 variants associated with severity and mortality in patients during a second wave in Mexico 
Virology Journal  2013;10:41.
Background
Pandemic type A (H1N1) influenza arose in early 2009, probably in Mexico and the United States, and reappeared in North America in September for seven more months. An amino acid substitution in the hemagglutinin (HA), D222G, has been reported in a significant proportion of patients with a severe and fatal outcome. We studied the prevalence of HA222 substitutions in patients in Mexico during the second wave and its association with clinical outcome and pathogenicity in a mouse model.
Methods
The nucleotide sequences of hemagglutinin (HA) from viruses collected from 77 patients were determined including 50 severe and fatal cases and 27 ambulatory cases. Deep sequencing was done on 5 samples from severe or fatal cases in order to determine the quasispecies proportion. Weight loss and mortality due to infection with cultured influenza viruses were analyzed in a mouse model.
Results
Viruses from 14 out of 50 hospitalized patients (28%) had a non aspartic acid residue at the HA 222 position (nD222), while all 27 ambulatory patients had D222 (p = 0.0014). G222 was detected as sole species or in coexistence with N222 and D222 in 12 patients with severe disease including 8 who died. N222 in coexistence with D222 was detected in 1 patient who died and co-occurrence of A222 and V222, together with D222, was detected in another patient who died. The patients with a nD222 residue had higher mortality (71.4%), compared to the group with only D222 (22.2%, p = 0.0008). Four of the 14 viruses from hospitalized patients were cultured and intranasally infected into mice. Two viruses with G222 were lethal while a third virus, with G222, caused only mild illness in mice similar to the fourth virus that contained D222.
Conclusions
We confirm the elevated incidence of HA222 (H1N1)pdm09 variants in severe disease and mortality. Both clinical and mouse infection data support the idea that nD222 mutations contribute to increased severity of disease but additional determinants in disease outcome may be present.
doi:10.1186/1743-422X-10-41
PMCID: PMC3583722  PMID: 23369604
7.  Union Makes Strength: A Worldwide Collaborative Genetic and Clinical Study to Provide a Comprehensive Survey of RD3 Mutations and Delineate the Associated Phenotype 
PLoS ONE  2013;8(1):e51622.
Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration (RD), and the most common cause of incurable blindness diagnosed in children. It is occasionally the presenting symptom of multisystemic ciliopathies which diagnosis will require a specific care of patients. Nineteen LCA genes are currently identified and three of them account for both non-syndromic and syndromic forms of the disease. RD3 (LCA12) was implicated as a LCA gene based on the identification of homozygous truncating mutations in two LCA families despite the screening of large cohorts of patients. Here we provide a comprehensive survey of RD3 mutations and of their clinical expression through the screening of a cohort of 852 patients originating worldwide affected with LCA or early-onset and severe RD. We identified three RD3 mutations in seven unrelated consanguineous LCA families - i.e., a 2 bp deletion and two nonsense mutations – predicted to cause complete loss of function. Five families originating from the Southern Shores of the Mediterranean segregated a similar mutation (c.112C>T, p.R38*) suggesting that this change may have resulted from an ancient founder effect. Considering the low frequency of RD3 carriers, the recurrence risk for LCA in non-consanguineous unions is negligible for both heterozygote and homozygote RD3 individuals. The LCA12 phenotype in our patients is highly similar to those of patients with mutant photoreceptor-specific guanylate cyclase (GUCY2D/LCA1). This observation is consistent with the report of the role of RD3 in trafficking of GUCYs and gives further support to a common mechanism of photoreceptor degeneration in LCA12 and LCA1, i.e., inability to increase cytoplasmic cGMP concentration in outer segments and thus to recover the dark-state. Similar to LCA1, LCA12 patients have no extraocular symptoms despite complete inactivation of both RD3 alleles, supporting the view that extraocular investigations in LCA infants with RD3 mutations should be avoided.
doi:10.1371/journal.pone.0051622
PMCID: PMC3538699  PMID: 23308101
9.  Combining Cep290 and Mkks ciliopathy alleles in mice rescues sensory defects and restores ciliogenesis  
The Journal of Clinical Investigation  2012;122(4):1233-1245.
Cilia are highly specialized microtubule-based organelles that have pivotal roles in numerous biological processes, including transducing sensory signals. Defects in cilia biogenesis and transport cause pleiotropic human ciliopathies. Mutations in over 30 different genes can lead to cilia defects, and complex interactions exist among ciliopathy-associated proteins. Mutations of the centrosomal protein 290 kDa (CEP290) lead to distinct clinical manifestations, including Leber congenital amaurosis (LCA), a hereditary cause of blindness due to photoreceptor degeneration. Mice homozygous for a mutant Cep290 allele (Cep290rd16 mice) exhibit LCA-like early-onset retinal degeneration that is caused by an in-frame deletion in the CEP290 protein. Here, we show that the domain deleted in the protein encoded by the Cep290rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy Bardet-Biedl syndrome disrupted this interaction. In zebrafish embryos, combined subminimal knockdown of mkks and cep290 produced sensory defects in the eye and inner ear. Intriguingly, combinations of Cep290rd16 and Mkksko alleles in mice led to improved ciliogenesis and sensory functions compared with those of either mutant alone. We propose that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of the sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a possible novel approach for treating human ciliopathies.
doi:10.1172/JCI60981
PMCID: PMC3314468  PMID: 22446187
10.  TOPORS, implicated in retinal degeneration, is a cilia-centrosomal protein 
Human Molecular Genetics  2010;20(5):975-987.
We recently reported that mutations in the widely expressed nuclear protein TOPORS (topoisomerase I-binding arginine/serine rich) are associated with autosomal dominant retinal degeneration. However, the precise localization and a functional role of TOPORS in the retina remain unknown. Here, we demonstrate that TOPORS is a novel component of the photoreceptor sensory cilium, which is a modified primary cilium involved with polarized trafficking of proteins. In photoreceptors, TOPORS localizes primarily to the basal bodies of connecting cilium and in the centrosomes of cultured cells. Morpholino-mediated silencing of topors in zebrafish embryos demonstrates in another species a comparable retinal problem as seen in humans, resulting in defective retinal development and failure to form outer segments. These defects can be rescued by mRNA encoding human TOPORS. Taken together, our data suggest that TOPORS may play a key role in regulating primary cilia-dependent photoreceptor development and function. Additionally, it is well known that mutations in other ciliary proteins cause retinal degeneration, which may explain why mutations in TOPORS result in the same phenotype.
doi:10.1093/hmg/ddq543
PMCID: PMC3033188  PMID: 21159800
11.  Ocular and extra-ocular features of patients with Leber congenital amaurosis and mutations in CEP290 
Molecular Vision  2012;18:412-425.
Purpose
This study investigated the centrosomal protein, 290-KD (CEP290) associated genotype and ocular and extra-ocular phenotype in 18 patients with Leber congenital amaurosis (LCA).
Methods
Eighteen patients with LCA from 14 families with mutations in the CEP290 gene were identified with sequencing or with heteroduplex analysis. Ophthalmic examinations were performed on all patients. Scans of the central nervous system were reassessed in three patients and obtained in two. Renal function was evaluated in all patients. Ultrasonography of the kidneys was performed in six patients.
Results
Eight patients (from five families) carried the c.2991+1655A>G mutation homozygously. Nine solitary patients carried this variant combined with a nonsense, frameshift, or splice site mutation on the second allele. One new nonsense mutation was identified: c.1078C>T. Fourteen patients (from 12 families) had been completely blind from birth or had light perception. The best-recorded visual acuity was 20/200. Peripheral fundus changes appeared to be progressive with a relatively preserved posterior pole. Novel ophthalmic features for the CEP290 phenotype were Coats-like exudative vasculopathy in two patients, a small chorioretinal coloboma in one patient, and well defined, small, atrophic spots at the level of the retinal pigment epithelium causing a dot-like appearance in five patients. Some CEP290 patients exhibited systemic abnormalities. We found abnormal proprioception in two patients and mild mental retardation in one. One patient was infertile due to immobile spermatozoa. No renal abnormalities were detected.
Conclusions
CEP290-associated LCA has a severe, progressive, and clinically identifiable phenotype. Distinct extra-ocular findings were noted, which may be attributed to ciliary dysfunction.
PMCID: PMC3283211  PMID: 22355252
12.  Homozygosity Mapping in Patients with Cone–Rod Dystrophy: Novel Mutations and Clinical Characterizations 
Homozygosity mapping in CRD patients led to the identification of novel mutations in ABCA4, CERKL, and PROM1, showing that this method may be a powerful tool for identifying genetic defects, even in nonconsanguineous patients. Clinical examinations showed a variety of retinal phenotypes.
Purpose.
To determine the genetic defect and to describe the clinical characteristics in a cohort of mainly nonconsanguineous cone–rod dystrophy (CRD) patients.
Methods.
One hundred thirty-nine patients with diagnosed CRD were recruited. Ninety of them were screened for known mutations in ABCA4, and those carrying one or two mutations were excluded from further research. Genome-wide homozygosity mapping was performed in the remaining 108. Known genes associated with autosomal recessive retinal dystrophies located within a homozygous region were screened for mutations. Patients in whom a mutation was detected underwent further ophthalmic examination.
Results.
Homozygous sequence variants were identified in eight CRD families, six of which were nonconsanguineous. The variants were detected in the following six genes: ABCA4, CABP4, CERKL, EYS, KCNV2, and PROM1. Patients carrying mutations in ABCA4, CERKL, and PROM1 had typical CRD symptoms, but a variety of retinal appearances on funduscopy, optical coherence tomography, and autofluorescence imaging.
Conclusions.
Homozygosity mapping led to the identification of new mutations in consanguineous and nonconsanguineous patients with retinal dystrophy. Detailed clinical characterization revealed a variety of retinal appearances, ranging from nearly normal to extensive retinal remodeling, retinal thinning, and debris accumulation. Although CRD was initially diagnosed in all patients, the molecular findings led to a reappraisal of the diagnosis in patients carrying mutations in EYS, CABP4, and KCNV2.
doi:10.1167/iovs.10-5797
PMCID: PMC3061516  PMID: 20554613
13.  Candidate exome capture identifies mutation of SDCCAG8 as the cause of a retinal-renal ciliopathy 
Nature genetics  2010;42(10):840-850.
Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders featuring dysplasia or degeneration preferentially in kidney, retina, and cerebellum. Here we combine homozygosity mapping with candidate gene analysis by performing “ciliopathy candidate exome capture” followed by massively-parallel sequencing. We detect 12 different truncating mutations of SDCCAG8 in 10 NPHP-RC families. We demonstrate that SDCCAG8 is localized at both centrioles and directly interacts with NPHP-RC-associated OFD1. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in 3D renal cell cultures. This work identifies SDCCAG8 loss of function as a novel cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.
doi:10.1038/ng.662
PMCID: PMC2947620  PMID: 20835237
15.  Differential Macular Morphology in Patients with RPE65-, CEP290-, GUCY2D-, and AIPL1-Related Leber Congenital Amaurosis 
This study evaluated possible correlations between the microanatomic macular structures, as measured by spectral-domain optical coherence tomography in Leber congenital amaurosis patients with mutations in RPE65, CEP290, GUCY2D, or AIPL1. Phenotypic variations of macular microstructures were observed among LCA patients with different genotypes.
Purpose.
To evaluate genotypic and macular morphologic correlations in patients with RPE65-, CEP290-, GUCY2D-, or AIPL1-related Leber congenital amaurosis (LCA) using spectral-domain optical coherence tomography (SD-OCT).
Methods.
SD-OCT macular scans were performed in 21 patients, including 10 with RPE65, 7 with CEP290, 3 with GUCY2D, and 1 with AIPL1 mutations. An image processing software was used to manually draw segmentation lines by three observers. Lamellar structure was evaluated based on the number of retinal layers on segmented images. Total retinal thickness was measured at the central macular and perifoveal areas by using an automated algorithm.
Results.
All three patients with GUCY2D mutations (age range, 20–53 years) retained six retinal layers with visible photoreceptor inner/outer segment juncture (PSJ). However, the preservation of lamellar structures did not parallel better visual acuity. Patients with other mutations had poorly defined PSJ and disorganized retinal lamellar structures, where only one to three retinal layers could be observed. Patients with CEP290 mutations trended to have retention of the outer nuclear layer at the fovea and macular thickening, especially at younger ages. In patients with RPE65 (age range, 20–71 years) and AIPL1 mutations (age, 22 years), macular thickness was markedly decreased. Disorganization of retinal lamellar structures in the RPE65 group trended toward a worsening with increasing age.
Conclusions.
Variations of macular microstructures were observed among LCA patients with different genotypes. Disorganization of retinal lamellar structure was generally age related. Preservation of retinal microanatomic structures may not be associated with better visual acuity.
doi:10.1167/iovs.09-3734
PMCID: PMC2868490  PMID: 19959640
16.  AHI1 is required for outer segment development and is a modifier for retinal degeneration in nephronophthisis 
Nature genetics  2010;42(2):175-180.
Photoreceptor degeneration is a common feature of ciliopathies, owing to the importance of the highly specialized ciliary structure of these cells. Absence of AHI1, which encodes a cilium-localized protein, has been shown to cause a form of Joubert syndrome highly penetrant for retinal degeneration1,2. We show that Ahi1 knockout mice fail to form outer segments (OS), and show abnormal distribution of opsin throughout photoreceptors. Apoptotic cell death occurs rapidly between 2-4 weeks of age and is significantly delayed by reduced dosage of opsin. This phenotype also displays dosage-sensitive genetic interactions with Nphp1, another ciliopathy gene. Although not a primary cause of retinal blindness in humans, an allele of AHI1 modifies the relative risk of retinal degeneration greater than 7 fold within a nephronophthisis cohort. Our data support context-specific roles for AHI1 as a contributor to retinopathy and may explain a proportion of the variability of retinal phenotypes observed in nephronophthisis.
doi:10.1038/ng.519
PMCID: PMC2884967  PMID: 20081859
17.  PDZD7 is a modifier of retinal disease and a contributor to digenic Usher syndrome  
The Journal of Clinical Investigation  2010;120(6):1812-1823.
Usher syndrome is a genetically heterogeneous recessive disease characterized by hearing loss and retinitis pigmentosa (RP). It frequently presents with unexplained, often intrafamilial, variability of the visual phenotype. Although 9 genes have been linked with Usher syndrome, many patients do not have mutations in any of these genes, suggesting that there are still unidentified genes involved in the syndrome. Here, we have determined that mutations in PDZ domain–containing 7 (PDZD7), which encodes a homolog of proteins mutated in Usher syndrome subtype 1C (USH1C) and USH2D, contribute to Usher syndrome. Mutations in PDZD7 were identified only in patients with mutations in other known Usher genes. In a set of sisters, each with a homozygous mutation in USH2A, a frame-shift mutation in PDZD7 was present in the sister with more severe RP and earlier disease onset. Further, heterozygous PDZD7 mutations were present in patients with truncating mutations in USH2A, G protein–coupled receptor 98 (GPR98; also known as USH2C), and an unidentified locus. We validated the human genotypes using zebrafish, and our findings were consistent with digenic inheritance of PDZD7 and GPR98, and with PDZD7 as a retinal disease modifier in patients with USH2A. Pdzd7 knockdown produced an Usher-like phenotype in zebrafish, exacerbated retinal cell death in combination with ush2a or gpr98, and reduced Gpr98 localization in the region of the photoreceptor connecting cilium. Our data challenge the view of Usher syndrome as a traditional Mendelian disorder and support the reclassification of Usher syndrome as an oligogenic disease.
doi:10.1172/JCI39715
PMCID: PMC2877930  PMID: 20440071
18.  A common allele in RPGRIP1L is a modifier of retinal degeneration in ciliopathies 
Nature genetics  2009;41(6):739-745.
Despite rapid advances in disease gene identification, the predictive power of the genotype remains limited, in part due to poorly understood effects of second-site modifiers. Here we demonstrate that a polymorphic coding variant of RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein-1 like), a ciliary gene mutated in Meckel-Gruber (MKS) and Joubert (JBTS) syndromes, is associated with the development of retinal degeneration in patients with ciliopathies caused by mutations in other genes. As part of our resequencing efforts of the ciliary proteome, we identified several putative loss of function RPGRIP1L mutations, including one common variant, A229T. Multiple genetic lines of evidence showed this allele to be associated with photoreceptor loss in ciliopathies. Moreover, we show that RPGRIP1L interacts biochemically with RPGR, loss of which causes retinal degeneration, and that the 229T-encoded protein significantly compromises this interaction. Our data represent an example of modification of a discrete phenotype of syndromic disease and highlight the importance of a multifaceted approach for the discovery of modifier alleles of intermediate frequency and effect.
doi:10.1038/ng.366
PMCID: PMC2783476  PMID: 19430481
19.  An USH2A founder mutation is the major cause of Usher syndrome type 2 in Canadians of French origin and confirms common roots of Quebecois and Acadians 
Congenital hearing loss affects approximately one child in 1000. About 10% of the deaf population have Usher syndrome (USH). In USH, hearing loss is complicated by retinal degeneration with onset in the first (USH1) or second (USH2) decade. In most populations, diagnostic testing is hampered by a multitude of mutations in nine genes. We have recently shown that in French Canadians from Quebec, USH1 largely results from a single USH1C founder mutation, c.216G>A (‘Acadian allele'). The genetic basis of USH2 in Canadians of French descent, however, has remained elusive. Here, we have investigated nine USH2 families from Quebec and New Brunswick (the former Acadia) by haplotype analyses of the USH2A locus and sequencing of the three known USH2 genes. Seven USH2A mutations were identified in eight patients. One of them, c.4338_4339delCT, accounts for 10 out of 18 disease alleles (55.6%). This mutation has previously been reported in an Acadian USH2 family, and it was found in homozygous state in the three Acadians of our sample. As in the case of c.216G>A (USH1C), a common haplotype is associated with c.4338_4339delCT. With a limited number of molecular tests, it will now be possible in these populations to estimate whether children with congenital hearing impairment of different degrees will develop retinal disease – with important clinical and therapeutic implications. USH2 is the second example that reveals a significant genetic overlap between Quebecois and Acadians: in contrast to current understanding, other genetic disorders present in both populations are likely based on common founder mutations as well.
doi:10.1038/ejhg.2008.143
PMCID: PMC2985947  PMID: 18665195
Usher syndrome type 2; USH2A; founder population; Quebec; Acadia
20.  Deafblindness in French Canadians from Quebec: a predominant founder mutation in the USH1C gene provides the first genetic link with the Acadian population 
Genome Biology  2007;8(4):R47.
Genetic characterisation of 15 French Canadian patients from different regions of the province of Quebec who were clinically diagnosed as USH1 reveals that carriers of the c.216G>A-allele haplotype belong to the early founders of both the Acadian and the Quebec population.
Background
Usher syndrome type 1 (USH1) is the leading cause of deafblindness. In most populations, many private mutations are distributed across the five known USH1 genes. We investigated patients from the French Canadian population of Quebec (approximately 6 million people) that descends from about 8,500 French settlers who colonized the St Lawrence River valley between 1608 and 1759. We hypothesized that founder mutations in USH1 genes exist in this population.
Results
We have genetically characterized 15 patients from different regions of Quebec who were clinically diagnosed as USH1. Of these cases, 60% carried mutations of the USH1C gene, a genetic subtype that is rare outside the Acadian population. We have discovered a founder effect of the c.216G>A mutation, which has previously been designated the 'Acadian allele' because it accounts for virtually all Acadian USH1 cases. It represents 40% of disease alleles in Quebec, and a carrier of c.216G>A was identified in the general population. Mutations in other genes, except CDH23, are very rare.
Conclusion
Based on our findings, approximately 0.5% of congenitally deaf children in Quebec are at risk of developing retinal degeneration due to homozygosity for c.216G>A. Although the Acadians and French Canadians from Quebec are descended from French ancestors, they have always been considered genetically distinct. The genetic conditions common in Quebec are generally not found in Acadians, or they are due to different mutations. Our results, however, show that carriers of the c.216G>A allele haplotype belonged to the early founders of both the Acadian and the Quebec population.
doi:10.1186/gb-2007-8-4-r47
PMCID: PMC1895989  PMID: 17407589

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