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1.  Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements 
Human genetics  2013;133(3):331-345.
Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy (CRD) patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.
doi:10.1007/s00439-013-1381-5
PMCID: PMC3945441  PMID: 24154662
retinitis pigmentosa; blindness; retinal genes; whole exome sequencing; next generation sequencing; retinal capture NGS
2.  Screening of a large cohort of Leber congenital amaurosis and retinitis pigmentosa patients identifies novel LCA5 mutations and new genotype-phenotype correlations 
Human mutation  2013;34(11):1537-1546.
To investigate the prevalence of sequence variants in LCA5 in patients with Leber congenital amaurosis (LCA), early onset rod-cone dystrophy (EORD) and autosomal recessive retinitis pigmentosa (RP), to delineate the ocular phenotypes, and to provide an overview of all published LCA5 variants in an online database._Patients underwent standard ophthalmic evaluations after providing informed consent. In selected patients, optical coherence tomography (OCT) and fundus autofluorescence imaging was possible. DNA samples from 797 unrelated patients with LCA and 211 with the various types of RP were screened by Sanger sequence analysis of all LCA5 exons and intron/exon junctions. Some LCA patients were pre-screened by APEX technology or selected based on homozygosity mapping. In silico analyses were performed to assess the pathogenicity of the variants. Segregation analysis was performed where possible. Published and novel LCA5 variants were collected, amended for their correct nomenclature, and listed in a Leiden Open Variation Database (LOVD). Sequence analysis identified 18 new probands with 19 different LCA5 variants. Seventeen of the 19 LCA5 variants were novel. Except for two missense variants and one splice site variant, all variants were protein-truncating mutations. Most patients expressed a severe phenotype, typical of LCA. However, some LCA subjects had better vision and intact inner segment/outer segment (IS/OS) junctions on OCT imaging. In two families with LCA5 variants, the phenotype was more compatible with EORD with affected individuals displaying preserved islands of RPE. One of these milder families harbored a homozygous splice site mutation, a second family was found to have a combination of a stop mutation and a missense mutation. This is the largest LCA5 study to date. We sequenced 1008 patients (797 with LCA, 211 with arRP) and identified 18 probands with LCA5 mutations. Mutations in LCA5 are a rare cause of childhood retinal dystrophy accounting for ~2% of disease in this cohort and the majority of LCA5 mutations are likely null. The LCA5 protein truncating mutations are predominantly associated with LCA. However, in two families with the milder EORD, the LCA5 gene analysis revealed a homozygous splice site mutation in one and a stop mutation in combination with a missense mutation in a second family, suggesting that this milder phenotype is due to residual function of lebercilin and expanding the currently known phenotypic spectrum to include the milder early onset RP. Some patients have remaining foveal cone structures (intact IS/OS junctions on OCT imaging) and remaining visual acuities, which may bode well for upcoming treatment trials.
doi:10.1002/humu.22398
PMCID: PMC4337959  PMID: 23946133
LCA; RP; retinal dystrophy; blindness; LCA5; lebercilin
3.  Safety and Proof-of-Concept Study of Oral QLT091001 in Retinitis Pigmentosa Due to Inherited Deficiencies of Retinal Pigment Epithelial 65 Protein (RPE65) or Lecithin:Retinol Acyltransferase (LRAT) 
PLoS ONE  2015;10(12):e0143846.
Restoring vision in inherited retinal degenerations remains an unmet medical need. In mice exhibiting a genetically engineered block of the visual cycle, vision was recently successfully restored by oral administration of 9-cis-retinyl acetate (QLT091001). Safety and visual outcomes of a once-daily oral dose of 40 mg/m2/day QLT091001 for 7 consecutive days was investigated in an international, multi-center, open-label, proof-of-concept study in 18 patients with RPE65- or LRAT-related retinitis pigmentosa. Eight of 18 patients (44%) showed a ≥20% increase and 4 of 18 (22%) showed a ≥40% increase in functional retinal area determined from Goldmann visual fields; 12 (67%) and 5 (28%) of 18 patients showed a ≥5 and ≥10 ETDRS letter score increase of visual acuity, respectively, in one or both eyes at two or more visits within 2 months of treatment. In two patients who underwent fMRI, a significant positive response was measured to stimuli of medium contrast, moving, pattern targets in both left and right hemispheres of the occipital cortex. There were no serious adverse events. Treatment-related adverse events were transient and the most common included headache, photophobia, nausea, vomiting, and minor biochemical abnormalities. Measuring the outer segment length of the photoreceptor layer with high-definition optical coherence tomography was highly predictive of treatment responses with responders having a significantly larger baseline outer segment thickness (11.7 ± 4.8 μm, mean ± 95% CI) than non-responders (3.5 ± 1.2 μm). This structure-function relationship suggests that treatment with QLT091001 is more likely to be efficacious if there is sufficient photoreceptor integrity.
Trial Registration
ClinicalTrials.gov NCT01014052
doi:10.1371/journal.pone.0143846
PMCID: PMC4687523  PMID: 26656277
4.  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
5.  Mutations in the unfolded protein response regulator ATF6 cause the cone dysfunction disorder achromatopsia 
Nature genetics  2015;47(7):757-765.
Achromatopsia (ACHM) is an autosomal recessive disorder characterized by color blindness, photophobia, nystagmus and severely reduced visual acuity. Using homozygosity mapping and whole-exome and candidate gene sequencing, we identified ten families carrying six homozygous and two compound-heterozygous mutations in the ATF6 gene (encoding activating transcription factor 6A), a key regulator of the unfolded protein response (UPR) and cellular endoplasmic reticulum (ER) homeostasis. Patients had evidence of foveal hypoplasia and disruption of the cone photoreceptor layer. The ACHM-associated ATF6 mutations attenuate ATF6 transcriptional activity in response to ER stress. Atf6−/− mice have normal retinal morphology and function at a young age but develop rod and cone dysfunction with increasing age. This new ACHM-related gene suggests a crucial and unexpected role for ATF6A in human foveal development and cone function and adds to the list of genes that, despite ubiquitous expression, when mutated can result in an isolated retinal photoreceptor phenotype.
doi:10.1038/ng.3319
PMCID: PMC4610820  PMID: 26029869
6.  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
7.  A Dominant Mutation in Hexokinase 1 (HK1) Causes Retinitis Pigmentosa 
Purpose.
To identify the cause of retinitis pigmentosa (RP) in UTAD003, a large, six-generation Louisiana family with autosomal dominant retinitis pigmentosa (adRP).
Methods.
A series of strategies, including candidate gene screening, linkage exclusion, genome-wide linkage mapping, and whole-exome next-generation sequencing, was used to identify a mutation in a novel disease gene on chromosome 10q22.1. Probands from an additional 404 retinal degeneration families were subsequently screened for mutations in this gene.
Results.
Exome sequencing in UTAD003 led to identification of a single, novel coding variant (c.2539G>A, p.Glu847Lys) in hexokinase 1 (HK1) present in all affected individuals and absent from normal controls. One affected family member carries two copies of the mutation and has an unusually severe form of disease, consistent with homozygosity for this mutation. Screening of additional adRP probands identified four other families (American, Canadian, and Sicilian) with the same mutation and a similar range of phenotypes. The families share a rare 450-kilobase haplotype containing the mutation, suggesting a founder mutation among otherwise unrelated families.
Conclusions.
We identified an HK1 mutation in five adRP families. Hexokinase 1 catalyzes phosphorylation of glucose to glucose-6-phosphate. HK1 is expressed in retina, with two abundant isoforms expressed at similar levels. The Glu847Lys mutation is located at a highly conserved position in the protein, outside the catalytic domains. We hypothesize that the effect of this mutation is limited to the retina, as no systemic abnormalities in glycolysis were detected. Prevalence of the HK1 mutation in our cohort of RP families is 1%.
Mutations in a novel gene, hexokinase 1 (HK1), account for 1% of cases of autosomal dominant retinitis pigmentosa.
doi:10.1167/iovs.14-15419
PMCID: PMC4224580  PMID: 25190649
retinitis pigmentosa; hexokinase; inherited retinal dystrophy
8.  CYP1B1 mutations in patients with primary congenital glaucoma from Saudi Arabia 
BMC Medical Genetics  2014;15:109.
Background
CYP1B1 is the most commonly mutated gene in primary congenital glaucoma (PCG). This study was undertaken to identify mutations in CYP1B1 in the Western region of Saudi Arabia.
Methods
Blood of patients who had typical findings of PCG, were screened by direct sequencing of all coding exons and splice junctions of the CYP1B1 gene.
Results
34 patients were studied; 18 patients belonged to 8 families, and 16 patients were non-familial, isolated PCG. Consanguinity was found in 27/34 (79.4%) of cases. All patients were diagnosed to have bilateral PCG at birth except one child, who had glaucoma in the right eye. More males (61.8%) were affected than females (38.2%). 79.4% (27/34) of patients were solved with pathogenic mutations and 20.6% (7/34) remained unsolved. Of the solved ones, 22.2% (6/27) of patients carry a pathogenic allele on one allele while the other allele remained yet to be determined. Direct sequencing of exon 2 revealed two pathogenic variants (p.Gly61Glu, p.Glu229Lys). P.Gly61Glu substitution was found both homozygously in 63% (17/27) of cases, and heterozygously in one patient. P.Glu229Lys variant was found heterozygous in 3.7% (1/27) of cases. One pathogenic variant (p.Arg469Trp) was found in exon 3, and is present homozygously in 14.8% (4/27) of cases while four patients have this variant heterozygously. All mutations were reported previously in the Saudi population, except p.Glu229Lys. Severe cases were associated with p.Gly61Glu, and p.Arg469Trp in 50% and 30% of ten patients respectively.
Conclusions
This study confirms that CYP1B1 mutations are the most frequent cause of PCG in the Saudi population, with p.Gly61Glu being the major disease-associated mutation. P.Glu229Lys is a newly discovered mutation in our PCG patients. Patient lacking mutation in CYP1B1 gene seems likely, to have another genetic loci involved in the pathogenesis of the disease, and need further study. Genetic studies of recessive diseases such as PCG is important in consanguineous populations, since it will increase awareness and allows genetic counseling to be offered to patients and their relatives. This will not only reduce the disease to be inherited to future generations, but will also reduce the disease burden in the community.
doi:10.1186/s12881-014-0109-2
PMCID: PMC4258803  PMID: 25261878
Congenital; Glaucoma; Gene; Mutation; CYP1B1
9.  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
10.  Visual Acuity Changes in Patients with Leber Congenital Amaurosis (LCA) and Mutations in the CEP290 Gene 
JAMA ophthalmology  2013;131(2):178-182.
Objective
To evaluate changes in visual acuity (VA) over time in patients with Leber congenital amaurosis (LCA) and mutations in the CEP290 gene.
Patients
Forty-three patients with LCA and CEP290 mutations participated.
Methods
VA was determined at the initial and most recent visits.
Main Outcome Measures
The best-corrected VA at the initial and most recent visits, and the correlation between age and VA.
Results
At the initial visit, 14 patients had measurable chart VA in the better-seeing eye, 25 patients had non-measurable chart VA, and VA was not assessed in 4 young patients. At the most recent visit, 15 patients had measurable chart VA and 28 had non-measurable chart VA. The average duration between the two visits was 10.4 years (range: 2 to 47 years). For patients with measurable chart VA, the median logMAR value at the initial visit, 0.75 (range: 0.10 to 2.30), and most recent visit, 0.70 (range: 0.10 to 2.00), did not differ significantly (p > 0.05). There was no significant relationship between VA and age.
Conclusions
Patients with LCA and CEP290 mutations had a wide spectrum of VA that was not related to age or length of follow-up. Severe VA loss was observed in most, but not all, patients in the first decade. These data will help clinicians provide counseling on VA changes in patients with CEP290 mutations and could be of value for future treatment trials.
doi:10.1001/2013.jamaophthalmol.354
PMCID: PMC3688627  PMID: 23411883
11.  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
12.  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
13.  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
14.  Retinal Phenotypes in Patients Homozygous for the G1961E Mutation in the ABCA4 Gene 
Purpose.
We evaluated the pathogenicity of the G1961E mutation in the ABCA4 gene, and present the range of retinal phenotypes associated with this mutation in homozygosity in a patient cohort with ABCA4-associated phenotypes.
Methods.
Patients were enrolled from the ABCA4 disease database at Columbia University or by inquiry from collaborating physicians. Only patients homozygous for the G1961E mutation were enrolled. The entire ABCA4 gene open reading frame, including all exons and flanking intronic sequences, was sequenced in all patients. Phenotype data were obtained from clinical history and examination, fundus photography, infrared imaging, fundus autofluorescence, fluorescein angiography, and spectral domain-optical coherence tomography. Additional functional data were obtained using the full-field electroretinogram, and static or kinetic perimetry.
Results.
We evaluated 12 patients homozygous for the G1961E mutation. All patients had evidence of retinal pathology consistent with the range of phenotypes observed in ABCA4 disease. The latest age of onset was recorded at 64 years, in a patient diagnosed initially with age-related macular degeneration (AMD). Of 6 patients in whom severe structural (with/without functional) fundus changes were detected, 5 had additional, heterozygous or homozygous, variants detected in the ABCA4 gene.
Conclusions.
Homozygous G1961E mutation in ABCA4 results in a range of retinal pathology. The phenotype usually is at the milder end of the disease spectrum, with severe phenotypes linked to the presence of additional ABCA4 variants. Our report also highlights that milder, late-onset Stargardt disease may be confused with AMD.
There is still debate as to the pathogenicity of homozygous G1961E mutation in the ABCA4 gene. We present 12 patients, homozygous for G1961E mutation, with retinal disease. In 6 cases, additional mutations were detected in ABCA4 and tended to yield more severe disease phenotypes.
doi:10.1167/iovs.11-9166
PMCID: PMC3394687  PMID: 22661473
16.  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
17.  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
18.  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
19.  Disruption of intraflagellar protein transport in photoreceptor cilia causes Leber congenital amaurosis in humans and mice 
The Journal of Clinical Investigation  2011;121(6):2169-2180.
The mutations that cause Leber congenital amaurosis (LCA) lead to photoreceptor cell death at an early age, causing childhood blindness. To unravel the molecular basis of LCA, we analyzed how mutations in LCA5 affect the connectivity of the encoded protein lebercilin at the interactome level. In photoreceptors, lebercilin is uniquely localized at the cilium that bridges the inner and outer segments. Using a generally applicable affinity proteomics approach, we showed that lebercilin specifically interacted with the intraflagellar transport (IFT) machinery in HEK293T cells. This interaction disappeared when 2 human LCA-associated lebercilin mutations were introduced, implicating a specific disruption of IFT-dependent protein transport, an evolutionarily conserved basic mechanism found in all cilia. Lca5 inactivation in mice led to partial displacement of opsins and light-induced translocation of arrestin from photoreceptor outer segments. This was consistent with a defect in IFT at the connecting cilium, leading to failure of proper outer segment formation and subsequent photoreceptor degeneration. These data suggest that lebercilin functions as an integral element of selective protein transport through photoreceptor cilia and provide a molecular demonstration that disrupted IFT can lead to LCA.
doi:10.1172/JCI45627
PMCID: PMC3104757  PMID: 21606596
20.  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
21.  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
23.  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
24.  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
25.  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

Results 1-25 (28)