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1.  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
2.  Deletion of ameloblastin exon 6 is associated with amelogenesis imperfecta 
Human Molecular Genetics  2014;23(20):5317-5324.
Amelogenesis imperfecta (AI) describes a heterogeneous group of inherited dental enamel defects reflecting failure of normal amelogenesis. Ameloblastin (AMBN) is the second most abundant enamel matrix protein expressed during amelogenesis. The pivotal role of AMBN in amelogenesis has been confirmed experimentally using mouse models. However, no AMBN mutations have been associated with human AI. Using autozygosity mapping and exome sequencing, we identified genomic deletion of AMBN exon 6 in a second cousin consanguineous family with three of the six children having hypoplastic AI. The genomic deletion corresponds to an in-frame deletion of 79 amino acids, shortening the protein from 447 to 368 residues. Exfoliated primary teeth (unmatched to genotype) were available from family members. The most severely affected had thin, aprismatic enamel (similar to that reported in mice homozygous for Ambn lacking exons 5 and 6). Other teeth exhibited thicker but largely aprismatic enamel. One tooth had apparently normal enamel. It has been suggested that AMBN may function in bone development. No clinically obvious bone or other co-segregating health problems were identified in the family investigated. This study confirms for the first time that AMBN mutations cause non-syndromic human AI and that mouse models with disrupted Ambn function are valid.
doi:10.1093/hmg/ddu247
PMCID: PMC4168819  PMID: 24858907
3.  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
4.  Whole-exome sequencing, without prior linkage, identifies a mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta 
The conventional approach to identifying the defective gene in a family with an inherited disease is to find the disease locus through family studies. However, the rapid development and decreasing cost of next generation sequencing facilitates a more direct approach. Here, we report the identification of a frameshift mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta (AI). Whole-exome sequencing of three affected family members and subsequent filtering of shared variants, without prior genetic linkage, sufficed to identify the pathogenic variant. Simultaneous analysis of multiple family members confirms segregation, enhancing the power to filter the genetic variation found and leading to rapid identification of the pathogenic variant. LAMB3 encodes a subunit of Laminin-5, one of a family of basement membrane proteins with essential functions in cell growth, movement and adhesion. Homozygous LAMB3 mutations cause junctional epidermolysis bullosa (JEB) and enamel defects are seen in JEB cases. However, to our knowledge, this is the first report of dominant AI due to a LAMB3 mutation in the absence of JEB.
doi:10.1038/ejhg.2013.76
PMCID: PMC3865405  PMID: 23632796
amelogenesis; amelogenesis imperfecta; LAMB3; laminin-5
5.  A missense mutation in ITGB6 causes pitted hypomineralized amelogenesis imperfecta 
Human Molecular Genetics  2013;23(8):2189-2197.
We identified a family in which pitted hypomineralized amelogenesis imperfecta (AI) with premature enamel failure segregated in an autosomal recessive fashion. Whole-exome sequencing revealed a missense mutation (c.586C>A, p.P196T) in the I-domain of integrin-β6 (ITGB6), which is consistently predicted to be pathogenic by all available programmes and is the only variant that segregates with the disease phenotype. Furthermore, a recent study revealed that mice lacking a functional allele of Itgb6 display a hypomaturation AI phenotype. Phenotypic characterization of affected human teeth in this study showed areas of abnormal prismatic organization, areas of low mineral density and severe abnormal surface pitting in the tooth's coronal portion. We suggest that the pathogenesis of this form of AI may be due to ineffective ligand binding of ITGB6 resulting in either compromised cell–matrix interaction or compromised ITGB6 activation of transforming growth factor-β (TGF-β) impacting indirectly on ameloblast–ameloblast interactions and proteolytic processing of extracellular matrix proteins via MMP20. This study adds to the list of genes mutated in AI and further highlights the importance of cell–matrix interactions during enamel formation.
doi:10.1093/hmg/ddt616
PMCID: PMC3959822  PMID: 24319098
6.  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
7.  A new recessively inherited disorder composed of foveal hypoplasia, optic nerve decussation defects and anterior segment dysgenesis maps to chromosome 16q23.3-24.1 
Molecular Vision  2013;19:2165-2172.
Purpose
We have previously described two families with unique phenotypes involving foveal hypoplasia. The first family (F1) presented with foveal hypoplasia and anterior segment dysgenesis, and the second family (F2) presented with foveal hypoplasia and chiasmal misrouting in the absence of albinism. A genome-wide linkage search in family F1 identified a 6.5 Mb locus for this disorder on chromosome 16q23.2–24.1. The aim of this study was to determine if both families have the same disorder and to see if family F2 is also linked to the 16q locus.
Methods
Family members underwent routine clinical examination. Linkage was determined by genotyping microsatellite makers and calculating logarithm of the odds (LOD) scores. Locus refinement was undertaken with single nucleotide polymorphism (SNP) microarray analysis.
Results
The identification of chiasmal misrouting in family F1 and anterior segment abnormalities in family F2 suggested that the families have the same clinical phenotype. This was confirmed when linkage analysis showed that family F2 also mapped to the 16q locus. The single nucleotide polymorphism microarray analysis excluded a shared founder haplotype between the families and refined the locus to 3.1 Mb.
Conclusions
We report a new recessively inherited syndrome consisting of foveal hypoplasia, optic nerve decussation defects and anterior segment dysgenesis, which we have abbreviated to FHONDA syndrome. The gene mutated in this disorder lies within a 3.1 Mb interval containing 33 genes on chromosome 16q23.3–24.1 (chr16:83639061 - 86716445, hg19).
PMCID: PMC3816992  PMID: 24194637
8.  Rapid Visualisation of Microarray Copy Number Data for the Detection of Structural Variations Linked to a Disease Phenotype 
PLoS ONE  2012;7(8):e43466.
Whilst the majority of inherited diseases have been found to be caused by single base substitutions, small insertions or deletions (<1Kb), a significant proportion of genetic variability is due to copy number variation (CNV). The possible role of CNV in monogenic and complex diseases has recently attracted considerable interest. However, until the development of whole genome, oligonucleotide micro-arrays, designed specifically to detect the presence of copy number variation, it was not easy to screen an individual for the presence of unknown deletions or duplications with sizes below the level of sensitivity of optical microscopy (3–5 Mb). Now that currently available oligonucleotide micro-arrays have in excess of a million probes, the problem of copy number analysis has moved from one of data production to that of data analysis. We have developed CNViewer, to identify copy number variation that co-segregates with a disease phenotype in small nuclear families, from genome-wide oligonucleotide micro-array data. This freely available program should constitute a useful addition to the diagnostic armamentarium of clinical geneticists.
doi:10.1371/journal.pone.0043466
PMCID: PMC3422275  PMID: 22912880
9.  Genetic Heterogeneity for Recessively Inherited Congenital Cataract Microcornea with Corneal Opacity 
The authors describe a new ocular phenotype, congenital cataract microcornea with corneal opacity, which is recessively inherited. This phenotype is genetically heterogeneous in the Pakistani population.
Purpose.
To investigate whether three consanguineous families from the Punjab province of Pakistan, with affected members with recessively inherited congenital cataract microcornea with corneal opacity, are genetically homogeneous.
Methods.
An ophthalmic examination was performed on each family member to establish the diagnosis. The two largest families were analyzed by homozygosity mapping using SNP arrays. Linkage was confirmed using polymorphic microsatellite markers, and logarithm of odds (LOD) scores were calculated. Candidate genes were prioritized using the ENDEAVOUR program.
Results.
Autosomal recessive congenital cataract-microcornea with corneal opacity mapped to chromosome 10cen for family MEP57 and to either chromosomes 2ptel or 20p for family MEP60. For MEP57, the refined interval was 36.8 Mb flanked by D10S1208 (35.3 Mb) and D10S676 (72.1 Mb). For MEP60, the interval containing the mutation was either 6.7 Mb from the telomere of chromosome 2 to marker D2S281 or 3.8 Mb flanked by D20S906 (1.5 Mb) and D20S835 (5.3 Mb). Maximum multipoint LOD scores of 3.09, 1.94, and 3.09 were calculated at D10S567, D2S281, and D20S473 for families MEP57 and MEP60. Linkage to these loci was excluded for family MEP68. SLC4A11 was excluded as a candidate gene for the observed phenotype in MEP60.
Conclusions.
The authors have identified two new loci, one on chromosome 10cen and the other on 2ptel or 20p, that are associated with recessively inherited congenital cataract-microcornea with corneal opacity. This phenotype is genetically heterogeneous in the Pakistani population. Further genetic studies of this kind, combined with a detailed phenotypic description, will contribute to more precise classification criteria for anterior segment disease.
doi:10.1167/iovs.10-6776
PMCID: PMC3175982  PMID: 21474777
10.  Next generation sequencing identifies mutations in Atonal homolog 7 (ATOH7) in families with global eye developmental defects 
Human Molecular Genetics  2011;21(4):776-783.
The atonal homolog 7 (ATOH7) gene encodes a transcription factor involved in determining the fate of retinal progenitor cells and is particularly required for optic nerve and ganglion cell development. Using a combination of autozygosity mapping and next generation sequencing, we have identified homozygous mutations in this gene, p.E49V and p.P18RfsX69, in two consanguineous families diagnosed with multiple ocular developmental defects, including severe vitreoretinal dysplasia, optic nerve hypoplasia, persistent fetal vasculature, microphthalmia, congenital cataracts, microcornea, corneal opacity and nystagmus. Most of these clinical features overlap with defects in the Norrin/β-catenin signalling pathway that is characterized by dysgenesis of the retinal and hyaloid vasculature. Our findings document Mendelian mutations within ATOH7 and imply a role for this molecule in the development of structures at the front as well as the back of the eye. This work also provides further insights into the function of ATOH7, especially its importance in retinal vascular development and hyaloid regression.
doi:10.1093/hmg/ddr509
PMCID: PMC3263993  PMID: 22068589
11.  The Influence of Lamellar Orientation on Corneal Material Behavior: Biomechanical and Structural Changes in an Avian Corneal Disorder 
Pathological alteration to the material behavior of the avian cornea under inflation is described and correlated with changes in stromal collagen microstructure, yielding new insight into the influence of collagen fibril anisotropy on the cornea's biomechanical response to changes in IOP.
Purpose.
Retinopathy, globe enlarged (RGE) is an inherited genetic disease of chickens with a corneal phenotype characterized by loss of tissue curvature and changes in peripheral collagen fibril alignment. This study aimed to characterize the material behavior of normal and RGE chicken corneas under inflation and compare this with new spatial- and depth-resolved microstructural information to investigate how stromal fibril architecture determines corneal behavior under intraocular pressure (IOP).
Methods.
Six RGE chicken corneas and six age-matched normal controls were tested using trephinate inflation and their stress-strain behavior determined as a function of posterior pressure. Second harmonic generation mulitphoton microscopy was used to compare the in-plane appearance and degree of through-plane interlacing of collagen lamellae between normal and mutant corneas.
Results.
RGE corneas displayed a 30–130% increase in material stiffness [Etangent(RGE) = 0.94 ± 0.18 MPa to 3.09 ± 0.66 MPa; Etangent(normals) = 0.72 ± 0.13 MPa to 1.34 ± 0.35 MPa] (P ≤ 0.05). The normal in-plane disposition of anterior collagen in the peripheral cornea was altered in RGE but through-plane lamellar interlacing was unaffected.
Conclusions.
This article demonstrates changes in corneal material behavior in RGE that are qualitatively consistent with microstructural collagen alterations identified both herein and previously. This study indicates that, in general, changes in stromal fibril orientation may significantly affect corneal material behavior and thereby its response to IOP.
doi:10.1167/iovs.10-5962
PMCID: PMC3101698  PMID: 21051696
12.  Three Gene-Targeted Mouse Models of RNA Splicing Factor RP Show Late-Onset RPE and Retinal Degeneration 
To investigate the pathogenesis of the RNA splicing factor forms of RP, the authors generated and characterized the retinal phenotypes of Prpf3-T494M, Prpf8-H2309P knockin mice, and evaluated the retinal ultrastructure of Prpf31-knockout mice. All three mouse models demonstrate degenerative changes in the RPE with age, suggesting that the RPE may be the primary cell type affected in the RNA splicing factor forms of RP.
Purpose.
Mutations in genes that produce proteins involved in mRNA splicing, including pre-mRNA processing factors 3, 8, and 31 (PRPF3, 8, and 31), RP9, and SNRNP200 are common causes of the late-onset inherited blinding disorder retinitis pigmentosa (RP). It is not known how mutations in these ubiquitously expressed genes lead to retina-specific disease. To investigate the pathogenesis of the RNA splicing factor forms of RP, the authors generated and characterized the retinal phenotypes of Prpf3-T494M, Prpf8-H2309P knockin mice. The retinal ultrastructure of Prpf31-knockout mice was also investigated.
Methods.
The knockin mice have single codon alterations in their endogenous Prpf3 and Prpf8 genes that mimic the most common disease causing mutations in human PRPF3 and PRPF8. The Prpf31-knockout mice mimic the null alleles that result from the majority of mutations identified in PRPF31 patients. The retinal phenotypes of the gene targeted mice were evaluated by electroretinography (ERG), light, and electron microscopy.
Results.
The RPE cells of heterozygous Prpf3+/T494M and Prpf8+/H2309P knockin mice exhibited loss of the basal infoldings and vacuolization, with accumulation of amorphous deposits between the RPE and Bruch[b]'s membrane at age two years. These changes were more severe in the homozygous mice, and were associated with decreased rod function in the Prpf3-T494M mice. Similar degenerative changes in the RPE were detected in Prpf31± mice at one year of age.
Conclusions.
The finding of similar degenerative changes in RPE cells of all three mouse models suggests that the RPE may be the primary cell type affected in the RNA splicing factor forms of RP. The relatively late-onset phenotype observed in these mice is consistent with the typical adult onset of disease in patients with RP.
doi:10.1167/iovs.10-5194
PMCID: PMC3053274  PMID: 20811066
13.  Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes 
Nature genetics  2010;42(7):619-625.
Joubert syndrome (JBTS), related disorders (JSRD) and Meckel syndrome (MKS) are ciliopathies. We now report that MKS2 and JBTS2 loci are allelic and due to mutations in TMEM216, encoding an uncharacterized tetraspan transmembrane protein. JBTS2 patients displayed frequent nephronophthisis and polydactytly, and two cases conformed to the Oro-Facio-Digital type VI phenotype, whereas skeletal dysplasia was common in MKS fetuses. A single p.R73L mutation was identified in all patients of Ashkenazi Jewish descent (n=10). TMEM216 localized to the base of primary cilia, and loss of TMEM216 in patient fibroblasts or following siRNA knockdown caused defective ciliogenesis and centrosomal docking, with concomitant hyperactivation of RhoA and Dishevelled. TMEM216 complexed with Meckelin, encoded by a gene also mutated in JSRD and MKS. Abrogation of tmem216 expression in zebrafish led to gastrulation defects that overlap with other ciliary morphants. The data implicate a new family of proteins in the ciliopathies, and further support allelism between ciliopathy disorders.
doi:10.1038/ng.594
PMCID: PMC2894012  PMID: 20512146
14.  Mutations in the Beta Propeller WDR72 Cause Autosomal-Recessive Hypomaturation Amelogenesis Imperfecta 
Healthy dental enamel is the hardest and most highly mineralized human tissue. Though acellular, nonvital, and without capacity for turnover or repair, it can nevertheless last a lifetime. Amelogenesis imperfecta (AI) is a collective term for failure of normal enamel development, covering diverse clinical phenotypes that typically show Mendelian inheritance patterns. One subset, known as hypomaturation AI, is characterised by near-normal volumes of organic enamel matrix but with weak, creamy-brown opaque enamel that fails prematurely after tooth eruption. Mutations in genes critical to enamel matrix formation have been documented, but current understanding of other key events in enamel biomineralization is limited. We investigated autosomal-recessive hypomaturation AI in a consanguineous Pakistani family. A whole-genome SNP autozygosity screen identified a locus on chromosome 15q21.3. Sequencing candidate genes revealed a point mutation in the poorly characterized WDR72 gene. Screening of WDR72 in a panel of nine additional hypomaturation AI families revealed the same mutation in a second, apparently unrelated, Pakistani family and two further nonsense mutations in Omani families. Immunohistochemistry confirmed intracellular localization in maturation-stage ameloblasts. WDR72 function is unknown, but as a putative β propeller is expected to be a scaffold for protein-protein interactions. The nearest homolog, WDR7, is involved in vesicle mobilization and Ca2+-dependent exocytosis at synapses. Vesicle trafficking is important in maturation-stage ameloblasts with respect to secretion into immature enamel and removal of cleaved enamel matrix proteins via endocytosis. This raises the intriguing possibility that WDR72 is critical to ameloblast vesicle turnover during enamel maturation.
doi:10.1016/j.ajhg.2009.09.014
PMCID: PMC2775821  PMID: 19853237
15.  Homozygous FOXE3 mutations cause non-syndromic, bilateral, total sclerocornea, aphakia, microphthalmia and optic disc coloboma 
Molecular Vision  2010;16:1162-1168.
Purpose
To investigate the genetic basis of recessively-inherited congenital, non syndromic, bilateral, total sclerocornea in two consanguineous pedigrees, one from the Punjab province of Pakistan and the other from the Tlaxcala province of Mexico.
Methods
Ophthalmic examinations were conducted on each family member to confirm their diagnosis and magnetic resonance imaging (MRI) or ultrasonography of the eyes was performed on some family members. Genomic DNA was analyzed by homozygosity mapping using the Affymetrix 6.0 SNP array and linkage was confirmed with polymorphic microsatellite markers. Candidate genes were sequenced.
Results
A diagnosis of autosomal recessive sclerocornea was established for 7 members of the Pakistani and 8 members of the Mexican pedigrees. In the Pakistani family we established linkage to a region on chromosome 1p that contained Forkhead Box E3 (FOXE3), a strong candidate gene since FOXE3 mutations had previously been associated with various anterior segment abnormalities. Sequencing FOXE3 identified the previously reported nonsense mutation, c.720C>A, p.C240X, in the Pakistani pedigree and a novel missense mutation which disrupts an evolutionarily conserved residue in the forkhead domain, c.292T>C, p.Y98H, in the Mexican pedigree. Individuals with heterozygous mutations had no ocular abnormalities. MRI or ultrasonography confirmed that the patients with sclerocornea were also aphakic, had microphthalmia and some had optic disc coloboma.
Conclusions
This is the fourth report detailing homozygous FOXE3 mutations causing anterior segment abnormalities in human patients. Previous papers have emphasized aphakia and microphthalmia as the primary phenotype, but we find that the initial diagnosis – and perhaps the only one possible in a rural setting – is one of non-syndromic, bilateral, total sclerocornea. Dominantly inherited anterior segment defects have also been noted in association with heterozygous FOXE3 mutations. However the absence of any abnormalities in the FOXE3 heterozygotes described suggests that genetic background and environmental factors plays a role in the penetrance of the mutant allele.
PMCID: PMC2901196  PMID: 20664696
16.  Analysis of candidate genes for macular telangiectasia type 2 
Molecular Vision  2010;16:2718-2726.
Purpose
To find the gene(s) responsible for macular telangiectasia type 2 (MacTel) by a candidate-gene screening approach.
Methods
Candidate genes were selected based on the following criteria: those known to cause or be associated with diseases with phenotypes similar to MacTel, genes with known function in the retinal vasculature or macular pigment transport, genes that emerged from expression microarray data from mouse models designed to mimic MacTel phenotype characteristics, and genes expressed in the retina that are also related to diabetes or hypertension, which have increased prevalence in MacTel patients. Probands from eight families with at least two affected individuals were screened by direct sequencing of 27 candidate genes. Identified nonsynonymous variants were analyzed to determine whether they co-segregate with the disease in families. Allele frequencies were determined by TaqMan analysis of the large MacTel and control cohorts.
Results
We identified 23 nonsynonymous variants in 27 candidate genes in at least one proband. Of these, eight were known single nucleotide polymorphisms (SNPs) with allele frequencies of >0.05; these variants were excluded from further analyses. Three previously unidentified missense variants, three missense variants with reported disease association, and five rare variants were analyzed for segregation and/or allele frequencies. No variant fulfilled the criteria of being causal for MacTel. A missense mutation, p.Pro33Ser in frizzled homolog (Drosophila) 4 (FZD4), previously suggested as a disease-causing variant in familial exudative vitreoretinopathy, was determined to be a rare benign polymorphism.
Conclusions
We have ruled out the exons and flanking intronic regions in 27 candidate genes as harboring causal mutations for MacTel.
PMCID: PMC3002960  PMID: 21179236
17.  Proteomic profiling of the retinal dysplasia and degeneration chick retina 
Molecular Vision  2010;16:7-17.
Purpose
In our previous paper we undertook proteomic analysis of the normal developing chick retina to identify proteins that were differentially expressed during retinal development. In the present paper we use the same proteomic approach to analyze the development and onset of degeneration in the retinal dysplasia and degeneration (rdd) chick. The pathology displayed by the rdd chick resembles that observed in some of the more severe forms of human retinitis pigmentosa.
Methods
Two-dimensional gel electrophoresis (pH 4–7), gel image analysis, and mass spectrometry were used to profile the developing and degenerating retina of the rdd and wild-type (wt) chick retina.
Results
Several proteins were identified by mass spectrometry that displayed differential expression between normal and rdd retina between embryonic day 12 (E12) and post-hatch day 1 (P1). Secernin 1 displayed the most significant variation in expression between rdd and wt retina; this may be due to differential phosphorylation in the rdd retina. Secernin 1 has dipeptidase activity and has been demonstrated to play a role in exocytosis; it has been shown to be overexpressed in certain types of cancer and has also been suggested as a potential neurotoxicologically relevant target. Its role in the retina and in particular its differential expression in the degenerate rdd retina remains unknown and will require further investigation. Other proteins that were differentially expressed in the rdd retina included valosin-containing protein, β-synuclein, stathmin 1, nucleoside diphosphate kinase, histidine triad nucleotide-binding protein, and 40S ribosomal protein S12. These proteins are reported to be involved in several cellular processes, including the ubiquitin proteasome pathway, neuroprotection, metastatic suppression, transcriptional and translational regulation, and regulation of microtubule dynamics.
Conclusions
This proteomic study is the first such investigation of the rdd retina and represents a unique data set that has revealed several proteins that are differentially expressed during retinal degeneration in the rdd chick. Secernin 1 showed the most significant differences in expression during this degeneration period. Further investigation of the proteins identified may provide insight into the complex events underlying retinal degeneration in this animal model.
PMCID: PMC2805419  PMID: 20069063
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.  Loss of the Metalloprotease ADAM9 Leads to Cone-Rod Dystrophy in Humans and Retinal Degeneration in Mice 
Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction.
doi:10.1016/j.ajhg.2009.04.005
PMCID: PMC2681008  PMID: 19409519
20.  Mutations in the Beta Propeller WDR72 Cause Autosomal-Recessive Hypomaturation Amelogenesis Imperfecta 
Healthy dental enamel is the hardest and most highly mineralized human tissue. Though acellular, nonvital, and without capacity for turnover or repair, it can nevertheless last a lifetime. Amelogenesis imperfecta (AI) is a collective term for failure of normal enamel development, covering diverse clinical phenotypes that typically show Mendelian inheritance patterns. One subset, known as hypomaturation AI, is characterised by near-normal volumes of organic enamel matrix but with weak, creamy-brown opaque enamel that fails prematurely after tooth eruption. Mutations in genes critical to enamel matrix formation have been documented, but current understanding of other key events in enamel biomineralization is limited. We investigated autosomal-recessive hypomaturation AI in a consanguineous Pakistani family. A whole-genome SNP autozygosity screen identified a locus on chromosome 15q21.3. Sequencing candidate genes revealed a point mutation in the poorly characterized WDR72 gene. Screening of WDR72 in a panel of nine additional hypomaturation AI families revealed the same mutation in a second, apparently unrelated, Pakistani family and two further nonsense mutations in Omani families. Immunohistochemistry confirmed intracellular localization in maturation-stage ameloblasts. WDR72 function is unknown, but as a putative β propeller is expected to be a scaffold for protein-protein interactions. The nearest homolog, WDR7, is involved in vesicle mobilization and Ca2+-dependent exocytosis at synapses. Vesicle trafficking is important in maturation-stage ameloblasts with respect to secretion into immature enamel and removal of cleaved enamel matrix proteins via endocytosis. This raises the intriguing possibility that WDR72 is critical to ameloblast vesicle turnover during enamel maturation.
doi:10.1016/j.ajhg.2009.09.014
PMCID: PMC2775821  PMID: 19853237
21.  Mutations in CNNM4 Cause Jalili Syndrome, Consisting of Autosomal-Recessive Cone-Rod Dystrophy and Amelogenesis Imperfecta 
The combination of recessively inherited cone-rod dystrophy (CRD) and amelogenesis imperfecta (AI) was first reported by Jalili and Smith in 1988 in a family subsequently linked to a locus on chromosome 2q11, and it has since been reported in a second small family. We have identified five further ethnically diverse families cosegregating CRD and AI. Phenotypic characterization of teeth and visual function in the published and new families reveals a consistent syndrome in all seven families, and all link or are consistent with linkage to 2q11, confirming the existence of a genetically homogenous condition that we now propose to call Jalili syndrome. Using a positional-candidate approach, we have identified mutations in the CNNM4 gene, encoding a putative metal transporter, accounting for the condition in all seven families. Nine mutations are described in all, three missense, three terminations, two large deletions, and a single base insertion. We confirmed expression of Cnnm4 in the neural retina and in ameloblasts in the developing tooth, suggesting a hitherto unknown connection between tooth biomineralization and retinal function. The identification of CNNM4 as the causative gene for Jalili syndrome, characterized by syndromic CRD with AI, has the potential to provide new insights into the roles of metal transport in visual function and biomineralization.
doi:10.1016/j.ajhg.2009.01.009
PMCID: PMC2668026  PMID: 19200525
22.  Loss of the Metalloprotease ADAM9 Leads to Cone-Rod Dystrophy in Humans and Retinal Degeneration in Mice 
Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction.
doi:10.1016/j.ajhg.2009.04.005
PMCID: PMC2681008  PMID: 19409519
23.  Ultrastructural changes in the retinopathy, globe enlarged (rge) chick cornea☆ 
Journal of Structural Biology  2009;166(2):195-204.
In the cornea, the precise organisation of fibrillar collagen and associated proteoglycans comprising the stromal extracellular matrix plays a major role in governing tissue form and function. Recently, abnormal collagen alignment was noted in the misshapen corneas of mature chickens affected by the retinopathy, globe enlarged (rge) mutation. Here we further characterize corneal ultrastructural changes as the rge eye develops post-hatch. Wide-angle X-ray scattering disclosed alteration to dominant collagen lamellae directions in the rge chick cornea, compared to age-matched controls. These changes accompanied eye globe enlargement and corneal flattening in affected birds, manifesting as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Collagen intermolecular separation was unchanged in rge. However, small-angle X-ray scattering results suggest collagen fibril separation and diameter increase more rapidly towards the corneal periphery in rge at 3 months post-hatch compared to controls, although central collagen fibril diameter was unchanged. By transmission electron microscopy utilising cuprolinic blue stain, the morphology and distribution of stromal proteoglycans were unaltered in rge corneas otherwise demonstrating abnormal collagen fibril organisation. From a numerical simulation of tissue mechanics, progressive remodelling of stromal collagen in rge during globe enlargement post-hatch appears to be related to the corneal morphometric changes presented by the disease.
doi:10.1016/j.jsb.2009.01.009
PMCID: PMC2680986  PMID: 19258040
Corneal stroma; Chick; Collagen fibril; Biomechanics; Proteoglycan
24.  Mutations in CNNM4 Cause Jalili Syndrome, Consisting of Autosomal-Recessive Cone-Rod Dystrophy and Amelogenesis Imperfecta 
The combination of recessively inherited cone-rod dystrophy (CRD) and amelogenesis imperfecta (AI) was first reported by Jalili and Smith in 1988 in a family subsequently linked to a locus on chromosome 2q11, and it has since been reported in a second small family. We have identified five further ethnically diverse families cosegregating CRD and AI. Phenotypic characterization of teeth and visual function in the published and new families reveals a consistent syndrome in all seven families, and all link or are consistent with linkage to 2q11, confirming the existence of a genetically homogenous condition that we now propose to call Jalili syndrome. Using a positional-candidate approach, we have identified mutations in the CNNM4 gene, encoding a putative metal transporter, accounting for the condition in all seven families. Nine mutations are described in all, three missense, three terminations, two large deletions, and a single base insertion. We confirmed expression of Cnnm4 in the neural retina and in ameloblasts in the developing tooth, suggesting a hitherto unknown connection between tooth biomineralization and retinal function. The identification of CNNM4 as the causative gene for Jalili syndrome, characterized by syndromic CRD with AI, has the potential to provide new insights into the roles of metal transport in visual function and biomineralization.
doi:10.1016/j.ajhg.2009.01.009
PMCID: PMC2668026  PMID: 19200525
25.  Mutations in the RP1 gene causing autosomal dominant retinitis pigmentosa 
Human molecular genetics  1999;8(11):2121-2128.
Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration that affects ~1 in 3500 people worldwide. Recently we identified the gene responsible for the RP1 form of autosomal dominant retinitis pigmentosa (adRP) at 8q11–12 and found two different nonsense mutations in three families previously mapped to 8q. The RP1 gene is an unusually large protein, 2156 amino acids in length, but is comprised of four exons only. To determine the frequency and range of mutations in RP1 we screened probands from 56 large adRP families for mutations in the entire gene. After preliminary results indicated that mutations seem to cluster in a 442 nucleotide segment of exon 4, an additional 194 probands with adRP and 409 probands with other degenerative retinal diseases were tested for mutations in this region alone. We identified eight different disease-causing mutations in 17 of the 250 adRP probands tested. All of these mutations are either nonsense or frameshift mutations and lead to a severely truncated protein. Two of the eight different mutations, Arg677X and a 5 bp deletion of nucleotides 2280–2284, were reported previously, while the remaining six mutations are novel. We also identified two rare missense changes in two other families, one new polymorphic amino acid substitution, one silent substitution and a rare variant in the 5′-untranslated region that is not associated with disease. Based on this study, mutations in RP1 appear to cause at least 7% (17/250) of adRP. The 5 bp deletion of nucleotides 2280–2284 and the Arg677X nonsense mutation account for 59% (10/17) of these mutations. Further studies will determine whether missense changes in the RP1 gene are associated with disease, whether mutations in other regions of RP1 can cause forms of retinal disease other than adRP and whether the background variation in either the mutated or wild-type RP1 allele plays a role in the disease phenotype.
PMCID: PMC2585827  PMID: 10484783

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