Congenital glaucoma and CYP1B1: an old story revisited.

Primary congenital glaucoma is a trabecular meshwork dysgenesis with resultant increased intraocular pressure and ocular damage. CYP1B1 mutations remain the most common identifiable genetic cause. However, important questions about the penetrance of CYP1B1-related congenital glaucoma remain unanswered. Furthermore, mutations in other genes have been described although their exact contribution and potential genetic interaction, if any, with CYP1B1 mutations are not fully explored. In this study, we employed modern genomic approaches to re-examine CYP1B1-related congenital glaucoma. A cohort of 193 patients (136 families) diagnosed with congenital glaucoma. We identified biallelic CYP1B1 mutations in 80.8% (87.5 and 66.1% in familial and sporadic cases, respectively, p < 0.0086). The large family size of the study population allowed us to systematically examine penetrance of all identified alleles. With the exception of c.1103G>A (p.R368H), previously reported pathogenic mutations were highly penetrant (91.2%). We conclude from the very low penetrance and genetic epidemiological analyses that c.1103G>A (p.R368H) is unlikely to be a disease-causing recessive mutation in congenital glaucoma as previously reported. All cases that lacked biallelic CYP1B1 mutations underwent whole exome sequencing. No mutations in LTBP2, MYOC or TEK were encountered. On the other hand, mutations were identified in genes linked to other ophthalmic phenotypes, some inclusive of glaucoma, highlighting conditions that might phenotypically overlap with primary congenital glaucoma (SLC4A4, SLC4A11, CPAMD8, and KERA). We also encountered candidate causal variants in genes not previously linked to human diseases: BCO2, TULP2, and DGKQ. Our results both expand and refine the genetic spectrum of congenital glaucoma with important clinical implications.

Infantile Nephropathic Cystinosis: A Novel CTNS Mutation.

Cystinosis is a rare autosomal recessive metabolic disorder characterized by the accumulation of cystine in lysosomes, which results from defects in the carrier-mediated transport protein encoded by the CTNS gene. Infantile nephropathic cystinosis (INC) is one of the major complications of cystinosis. It is characterized by findings of Fanconi's syndrome within the first year of life. Here we report two patients with INC presenting with signs of Fanconi's syndrome and describe a novel CTNS mutation.

Novel phenotypes and loci identified through clinical genomics approaches to pediatric cataract.

Pediatric cataract is highly heterogeneous clinically and etiologically. While mostly isolated, cataract can be part of many multisystem disorders, further complicating the diagnostic process. In this study, we applied genomic tools in the form of a multi-gene panel as well as whole-exome sequencing on unselected cohort of pediatric cataract (166 patients from 74 families). Mutations in previously reported cataract genes were identified in 58% for a total of 43 mutations, including 15 that are novel. GEMIN4 was independently mutated in families with a syndrome of cataract, global developmental delay with or without renal involvement. We also highlight a recognizable syndrome that resembles galactosemia (a fulminant infantile liver disease with cataract) caused by biallelic mutations in CYP51A1. A founder mutation in RIC1 (KIAA1432) was identified in patients with cataract, brain atrophy, microcephaly with or without cleft lip and palate. For non-syndromic pediatric cataract, we map a novel locus in a multiplex consanguineous family on 4p15.32 where exome sequencing revealed a homozygous truncating mutation in TAPT1. We report two further candidates that are biallelically inactivated each in a single cataract family: TAF1A (cataract with global developmental delay) and WDR87 (non-syndromic cataract). In addition to positional mapping data, we use iSyTE developmental lens expression and gene-network analysis to corroborate the proposed link between the novel candidate genes and cataract. Our study expands the phenotypic, allelic and locus heterogeneity of pediatric cataract. The high diagnostic yield of clinical genomics supports the adoption of this approach in this patient group.

Characterizing the morbid genome of ciliopathies.

BACKGROUND: Ciliopathies are clinically diverse disorders of the primary cilium. Remarkable progress has been made in understanding the molecular basis of these genetically heterogeneous conditions; however, our knowledge of their morbid genome, pleiotropy, and variable expressivity remains incomplete. RESULTS: We applied genomic approaches on a large patient cohort of 371 affected individuals from 265 families, with phenotypes that span the entire ciliopathy spectrum. Likely causal mutations in previously described ciliopathy genes were identified in 85% (225/265) of the families, adding 32 novel alleles. Consistent with a fully penetrant model for these genes, we found no significant difference in their "mutation load" beyond the causal variants between our ciliopathy cohort and a control non-ciliopathy cohort. Genomic analysis of our cohort further identified mutations in a novel morbid gene TXNDC15, encoding a thiol isomerase, based on independent loss of function mutations in individuals with a consistent ciliopathy phenotype (Meckel-Gruber syndrome) and a functional effect of its deficiency on ciliary signaling. Our study also highlighted seven novel candidate genes (TRAPPC3, EXOC3L2, FAM98C, C17orf61, LRRCC1, NEK4, and CELSR2) some of which have established links to ciliogenesis. Finally, we show that the morbid genome of ciliopathies encompasses many founder mutations, the combined carrier frequency of which accounts for a high disease burden in the study population. CONCLUSIONS: Our study increases our understanding of the morbid genome of ciliopathies. We also provide the strongest evidence, to date, in support of the classical Mendelian inheritance of Bardet-Biedl syndrome and other ciliopathies.

Congenital hereditary endothelial dystrophy, not glaucoma, in a child with iris colobomas.

Corneal haze and elevated measured intraocular pressure in a young child with iris abnormalities are suggestive for glaucoma, but primary corneal disease is another possibility. We highlight the case of a 10-year-old boy with these clinical signs who was initially treated for glaucoma but in fact had congenital hereditary endothelial dystrophy, iris colobomas, and no glaucoma.

Exome-based case-control association study using extreme phenotype design reveals novel candidates with protective effect in diabetic retinopathy.

UNLABELLED: Diabetic retinopathy (DR) is a common clinical expression of diabetes mellitus-induced vasculopathy and is a major cause of vision loss. Significant gaps remain in our understanding of the molecular pathoetiology of DR, and it is hoped that human genetic approaches can reveal novel targets especially since DR is a heritable trait. Previous studies have focused on genetic risk factors of DR but their results have been mixed. In this study, we hypothesized that the use of the extreme phenotype design will increase the power of a genomewide search for "protective" genetic variants. We enrolled a small yet atypical cohort of 43 diabetics who did not develop DR a decade or more after diagnosis (cases), and 64 diabetics with DR (controls), all of similar ethnic background (Saudi). Whole-exome sequencing of the entire cohort was followed by statistical analysis employing combined multivariate and collapsing methods at the gene level, to identify genes that are enriched for rare variants in cases vs. CONTROLS: Three genes (NME3, LOC728699, and FASTK) reached gene-based genome-wide significance at the 10(-08) threshold (p value = 1.55 × 10(-10), 6.23 × 10(-10), 3.21 × 10(-08), respectively). Our results reveal novel candidate genes whose increased rare variant burden appears to protect against DR, thus highlighting them as attractive candidate targets, if replicated by future studies, for the treatment and prevention of DR. Extreme phenotype design when implemented in sequencing-based genome-wide case-control studies has the potential to reveal novel candidates with a smaller cohort size compared to standard study designs.

Expanding the clinical, allelic, and locus heterogeneity of retinal dystrophies.

PURPOSE: Retinal dystrophies (RD) are heterogeneous hereditary disorders of the retina that are usually progressive in nature. The aim of this study was to clinically and molecularly characterize a large cohort of RD patients. METHODS: We have developed a next-generation sequencing assay that allows known RD genes to be sequenced simultaneously. We also performed mapping studies and exome sequencing on familial and on syndromic RD patients who tested negative on the panel. RESULTS: Our panel identified the likely causal mutation in >60% of the 292 RD families tested. Mapping studies on all 162 familial RD patients who tested negative on the panel identified two novel disease loci on Chr2:25,550,180-28,794,007 and Chr16:59,225,000-72,511,000. Whole-exome sequencing revealed the likely candidate as AGBL5 and CDH16, respectively. We also performed exome sequencing on negative syndromic RD cases and identified a novel homozygous truncating mutation in GNS in a family with the novel combination of mucopolysaccharidosis and RD. Moreover, we identified a homozygous truncating mutation in DNAJC17 in a family with an apparently novel syndrome of retinitis pigmentosa and hypogammaglobulinemia. CONCLUSION: Our study expands the clinical and allelic spectrum of known RD genes, and reveals AGBL5, CDH16, and DNAJC17 as novel disease candidates.Genet Med 18 6, 554-562.

Phenotypes of Recessive Pediatric Cataract in a Cohort of Children with Identified Homozygous Gene Mutations (An American Ophthalmological Society Thesis).

PURPOSE: To assess for phenotype-genotype correlations in families with recessive pediatric cataract and identified gene mutations. METHODS: Retrospective review (2004 through 2013) of 26 Saudi Arabian apparently nonsyndromic pediatric cataract families referred to one of the authors (A.O.K.) and for which recessive gene mutations were identified. RESULTS: Fifteen different homozygous recessive gene mutations were identified in the 26 consanguineous families; two genes and five families are novel to this study. Ten families had a founder CRYBB1 deletion (all with bilateral central pulverulent cataract), two had the same missense mutation in CRYAB (both with bilateral juvenile cataract with marked variable expressivity), and two had different mutations in FYCO1 (both with bilateral posterior capsular abnormality). The remaining 12 families each had mutations in 12 different genes (CRYAA, CRYBA1, AKR1E2, AGK, BFSP2, CYP27A1, CYP51A1, EPHA2, GCNT2, LONP1, RNLS, WDR87) with unique phenotypes noted for CYP27A1 (bilateral juvenile fleck with anterior and/or posterior capsular cataract and later cerebrotendinous xanthomatosis), EPHA2 (bilateral anterior persistent fetal vasculature), and BFSP2 (bilateral flecklike with cloudy cortex). Potential carrier signs were documented for several families. CONCLUSIONS: In this recessive pediatric cataract case series most identified genes are noncrystallin. Recessive pediatric cataract phenotypes are generally nonspecific, but some notable phenotypes are distinct and associated with specific gene mutations. Marked variable expressivity can occur from a recessive missense CRYAB mutation. Genetic analysis of apparently isolated pediatric cataract can sometimes uncover mutations in a syndromic gene. Some gene mutations seem to be associated with apparent heterozygous carrier signs.

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes.

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

Accelerating novel candidate gene discovery in neurogenetic disorders via whole-exome sequencing of prescreened multiplex consanguineous families.

Our knowledge of disease genes in neurological disorders is incomplete. With the aim of closing this gap, we performed whole-exome sequencing on 143 multiplex consanguineous families in whom known disease genes had been excluded by autozygosity mapping and candidate gene analysis. This prescreening step led to the identification of 69 recessive genes not previously associated with disease, of which 33 are here described (SPDL1, TUBA3E, INO80, NID1, TSEN15, DMBX1, CLHC1, C12orf4, WDR93, ST7, MATN4, SEC24D, PCDHB4, PTPN23, TAF6, TBCK, FAM177A1, KIAA1109, MTSS1L, XIRP1, KCTD3, CHAF1B, ARV1, ISCA2, PTRH2, GEMIN4, MYOCD, PDPR, DPH1, NUP107, TMEM92, EPB41L4A, and FAM120AOS). We also encountered instances in which the phenotype departed significantly from the established clinical presentation of a known disease gene. Overall, a likely causal mutation was identified in >73% of our cases. This study contributes to the global effort toward a full compendium of disease genes affecting brain function.

Recessive mutations in LEPREL1 underlie a recognizable lens subluxation phenotype.

PURPOSE: To uncover the homozygous recessive gene mutation underlying familial lens subluxation and/or juvenile lens opacities in four sisters from a consanguineous family. METHODS: Prospective family study (clinical phenotyping; homozygosity-analysis-guided candidate gene testing). RESULTS: The proband was a 14-year-old girl with long-standing poor vision, bilateral temporal lens subluxation, lens opacities, and axial high myopia. There were no syndromic findings, and fibrillin-1 sequencing was normal. Three sisters, also non-syndromic, had undergone bilateral juvenile lens surgery (two for juvenile cataract, 1 for lens subluxation) within the first two decades of life. Both sisters who had cataract surgery developed bilateral post-operative retinal detachments and one had documented lens instability during cataract surgery. Genetic analysis revealed the phenotype to segregate with a novel homozygous recessive mutation in LEPREL1 (c.292delC; p.Gly100Alafs*104). Recessive mutations in this gene were recently highlighted as a cause for axial myopia and early-onset cataract in two families for whom some affected members also had ectopia lentis and/or post-operative retinal detachments. CONCLUSIONS: Recessive LEPREL1 mutations should be recognized as part of the differential diagnosis of lens subluxation. The associated phenotype is non-syndromic and distinguishable from other causes of ectopia lentis in the context of its additional features: juvenile lens opacities, axial myopia, and a predisposition to retinal tears/detachment following intraocular surgery.

Lens subluxation and retinal dysfunction in a girl with homozygous VSX2 mutation.

OBJECTIVE: To describe a unique lens subluxation phenotype in a child from a consanguineous family and to determine its genetic basis. METHODS: Ophthalmologic examination (including ocular biometry and electroretinography [ERG] for the proband) and autozygosity-analysis-guided exome sequencing for the family; confirmatory candidate gene sequencing in the family and ethnically matched controls. RESULTS: An otherwise healthy 3-year-old Saudi Arabian girl with poor vision since birth had smooth irides, lens subluxation, cone-rod dysfunction, and high myopia - features resembling Knobloch syndrome but differing in regard to direction of lens subluxation (superior rather than temporal) and the pattern of chorioretinal atrophy (without vitreous condensations or distinct macular atrophy). Autozygome-guided exome sequencing revealed the girl to harbor a homozygous exon 5 mutation in the ocular transcription factor gene visual homeobox 2 (VSX2) [c.773delA; p.Lys258SerfsX44] that was heterozygous in the unaffected brother and parents and absent in 100 healthy ethnically matched controls and on-line databases. Previously reported VSX2 mutations have affected the DNA-binding domains and only been associated with microphthalmia. Unlike previously reported mutations, the current VSX2 mutation is downstream to the protein's DNA binding domains. CONCLUSIONS: The phenotype of this girl is unique and suggests a normal regulatory role for VSX2 in iris, zonule, and cone-rod development. For a consanguineous family with suspected recessive ocular disease but without a clear candidate gene, autozygome-guided exome analysis is a powerful technique, even when only a single patient is affected.

IFT27, encoding a small GTPase component of IFT particles, is mutated in a consanguineous family with Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy with multisystem involvement. So far, 18 BBS genes have been identified and the majority of them are essential for the function of BBSome, a protein complex involved in transporting membrane proteins into and from cilia. Yet defects in the identified genes cannot account for all the BBS cases. The genetic heterogeneity of this disease poses significant challenge to the identification of additional BBS genes. In this study, we coupled human genetics with functional validation in zebrafish and identified IFT27 as a novel BBS gene (BBS19). This is the first time an intraflagellar transport (IFT) gene is implicated in the pathogenesis of BBS, highlighting the genetic complexity of this disease.

Childhood cone-rod dystrophy with macular cystic degeneration from recessive CRB1 mutation.

PURPOSE: To describe three siblings with childhood cone-rod dystrophy and macular cystic degeneration in a family with apparently variable phenotypes of CRB1-related recessive retinal dystrophy. METHODS: Ophthalmologic examination (including electroretinography (ERG), ocular coherence tomography (OCT), and intravenous fluorescein angiography when possible) and homozygosity analysis guided candidate gene testing. RESULTS: When the proband was evaluated at 7 years old for progressive visual loss, fundus exam was unremarkable (including no macular thickening clinically or by OCT) but ERG revealed cone-rod dysfunction with an electronegative waveform. Four years later repeat examination was significant for bilateral macular cystic degeneration and immediate family members were evaluated. Both the older sister (15 years old) and the younger brother (7 years old) had cone-rod dystrophy with macular cystic degeneration. Both the father (45 years old) and mother (35 years old) had had early adult-onset nyctalopia with later eventual loss of central vision; examination revealed dystrophic retinas with mostly peripheral clumped and/or nummular pigment and macular atrophy. ERG for both the older sister and younger brother confirmed cone-rod dysfunction (without an electronegative waveform) and was non-recordable for both the parents. Homozygosity analysis guided candidate gene analysis and confirmatory Sanger sequencing for the family uncovered a homozygous CRB1 mutation (c.80G > T [p.Cys27Phe]) in affected family members. CONCLUSIONS: The phenotypic spectrum of recessive CRB1 mutation includes childhood cone-rod dystrophy with macular cystic degeneration and the associated ERG can be electronegative.

Corneal enlargement without optic disk cupping in children with recessive CYP1B1 mutations.

Corneal enlargement during the first 3 years of life can be a sign of early childhood glaucoma and optic nerve head cupping is a useful confirmatory finding. We report 3 children with corneal enlargement without optic nerve head cupping who had recessive CYP1B1 mutations, the most common identifiable cause of primary congenital glaucoma. One child later developed unilateral Haab striae, still in the absence of optic disk cupping. These cases illustrate that CYP1B1-related corneal changes can occur in young children without visible optic nerve head damage.

The syndrome of microcornea, myopic chorioretinal atrophy, and telecanthus (MMCAT) is caused by mutations in ADAMTS18.

One of us recently described an apparently novel ocular syndrome characterized by microcornea, myopic chorioretinal atrophy, and telecanthus (MMCAT) in a number of Saudi families. Consistent with the presumed pseudodominant inheritance in one of the original families, we show that MMCAT maps to a single autozygous locus on chr16q23.1 in which exome sequencing revealed a homozygous missense change in ADAMTS18. Direct sequencing of this gene in four additional probands with the same phenotype revealed three additional homozygous changes in ADAMTS18 including two nonsense mutations. Reassuringly, the autozygomes of all probands overlap on the same chr16q23.1 locus, further supporting the positional mapping of MMCAT to ADAMTS18. ADAMTS18 encodes a member of a family of metalloproteinases that are known for their role in extracellular matrix remodeling, and previous work has shown a strong expression of Adamts18 in the developing eye. Our data suggest that ADAMTS18 plays an essential role in early eye development and that mutations therein cause a distinct eye phenotype that is mainly characterized by microcornea and myopia.

Mutations in LRPAP1 are associated with severe myopia in humans.

Myopia is an extremely common eye disorder but the pathogenesis of its isolated form, which accounts for the overwhelming majority of cases, remains poorly understood. There is strong evidence for genetic predisposition to myopia, but determining myopia genetic risk factors has been difficult to achieve. We have identified Mendelian forms of myopia in four consanguineous families and implemented exome/autozygome analysis to identify homozygous truncating variants in LRPAP1 and CTSH as the likely causal mutations. LRPAP1 encodes a chaperone of LRP1, which is known to influence TGF-ß activity. Interestingly, we observed marked deficiency of LRP1 and upregulation of TGF-ß in cells from affected individuals, the latter being consistent with available data on the role of TGF-ß in the remodeling of the sclera in myopia and the high frequency of myopia in individuals with Marfan syndrome who characteristically have upregulation of TGF-ß signaling. CTSH, on the other hand, encodes a protease and we show that deficiency of the murine ortholog results in markedly abnormal globes consistent with the observed human phenotype. Our data highlight a role for LRPAP1 and CTSH in myopia genetics and demonstrate the power of Mendelian forms in illuminating new molecular mechanisms that may be relevant to common phenotypes.