Long-term deep phenotyping in inherited ocular disease novel genotype-phenotype correlations

Inherited retinal dystrophies (IRDs) are a heterogenous group of hereditary disorders characterized by a predominantly progressive degeneration of the photoreceptors and/or retinal pigment epithelium (RPE) leading to often profound visual loss. With a prevalence of 1:3000 individuals, they are the leading cause of visual impairment and blindness in the Western European population, afflicting children and working-age adults, and thus not as rare as previously considered. To date, over 280 genes have been identified to cause both non-syndromic and syndromic forms of inherited retinal dystrophies (https://sph.uth.edu/retnet). Therefore, each genetic subtype may be exceedingly rare. Nevertheless, genetic heterogeneity, variable clinical expression of certain genes with high inter- and intrafamilial variability, the possibility of incomplete penetrance and the additional impact of modifier genes and environmental factors complicate matters. As such, one rapidly ends up with a melting pot of very different diseases and phenotypes. IRDs have long been considered as a group of disorders for which no treatments or cures were available. But the present is definitely a time of great promise. Sound preclinical data recently resulted in the first reimbursed gene augmentation therapy Luxturna and the development of many other ongoing clinical treatment trials. We are on the doorstep of a new era where patients have a possibility that they won’t experience the full course of their disease. DNA and RNA therapies, optogenetics, stem cell transplantation and neurotrophic factors are expected to undergo major developments. Despite the latter, insight into the phenotypic spectrum and the disease course remains scarce for several IRD subtypes. This thesis provides a detailed clinical description of several subtypes of inherited ocular disease. Adequate insight into the clinical characteristics, variability, and long-term evolution of these diseases is crucial in establishing an adequate diagnosis and in counselling patients on their prognosis.This knowledge will prove to be vital in the design of future therapeutic trials, providing a comparison between the natural evolution and course of disease after treatment with innovative therapies.We describe the longitudinal phenotypic findings of autosomal dominant late-onset retinal degeneration (L-ORD) related to a heterozygous mutation in the C1QTNF5 gene. L-ORD is extremely rare. It is estimated that 110 patients have been diagnosed worldwide. In the majority of patients, LORD is related to the c.489C>G p.(Ser169Arg) variant in C1QTNF5. Twenty-six of these 110 patients were the subject of this thesis and underwent an annual detailed clinical characterization with a mean follow-up period of 8 years (1 – 37 years). A novel variant, c.562C>A p.(Pro188Thr), was identified, confirming the genetic heterogeneity of L-ORD. The detection of this causal variant was required to unequivocally confirm the clinical diagnosis in the patients, to identify future treatment options, to inform patients about the risk disease in family members and to offer the option of presymptomatic testing, preconceptual advice and prenatal or pre-implantation diagnosis, and to exclude the involvement of other organ systems.Although one disease, L-ORD presented initially as a spectrum of three, rather completely different, phenotypes, creating a striking clinical variation between family members. We classified them into 3 categories. The presence of these 3 completely different presenting phenotypes of L-ORD could render making a correct clinical diagnosis potentially challenging. Depending on the presenting phenotype and the disease phase at presentation, L-ORD can easily be mistaken for another disease. Only after years of evolution, a mixture of different retinal features will progress to a common, late phenotype of generalized chorioretinal atrophy. Patients of course are keen to hear about prognosis. The progression rate differed depending on the phenotype at presentation.Other differences compared to historical knowledge on L-ORD comprised the cause of vision loss and the anterior segment findings. L-ORD patients are thought to suffer vision loss related to the development of macular subretinal neovascularization. In our cohort however, most patients developed macular atrophy. The presence of long, anteriorly inserted lens zonules (LAZ) is considered to be pathognomonic for L-ORD. LAZ are thought to cause iris transillumination and subsequent glaucoma. LAZ are absent in the large majority of our L-ORD patients. Iris transillumination however is present, in combination with poor pupillary dilatation, and can therefore be considered to arise independently of the presence of LAZ. Glaucoma is not a major issue in our cohort.A major finding in our study is that evolution of disease is rather linear at first, with progression of disease accelerating considerably beyond a certain clinical stage. Once BCVA reduces, a period of only 1 to 3 years precedes loss of both central and peripheral sight. As the next step, attempts will be made to develop therapeutic modalities. In IRDs with a dominant-negative effect, promising strategies include silencing of the mutant allele and selective removal of the mutant protein. Projecting an individual patient’s phenotype against the findings of this natural history study of L-ORD would allow identification of the optimal window for intervention long before central macular function and vision are lost.Leber congenital amaurosis (LCA) and early-onset retinal dystrophy (EORD) are situated at the severe end of the phenotypic spectrum of rod-cone dystrophy, leading to blindness from birth or in the first few years of life. Phenotypically, RDH12-related LCA/EORD is characterized by macular atrophy with yellowish discolouration, and the typical retinitis pigmentosa characteristics of a pale optic disc, attenuation of the retinal vasculature, peripheral bone-spiculae-like intraretinal pigment migration and an extinguished full-field electroretinography (ffERG). Typical, additional characteristics linked to the RDH12 gene involve peripapillary sparing of retinal structure, patchy preservation of the peripheral retina, and preserved para-arteriolar retina.We identified a unique cohort of seven patients with biallelic RDH12 mutations with the novel finding of mild disease that was unrecognizable from the classic RDH12-phenotype. This mild disease did not involve the entire retina as is usual, but was instead limited to either the central macula or the posterior pole. As such, we expanded the phenotypic spectrum of RDH12-related disease. Two subgroups were identified based on the clinical presentation. In patients with an RDH12-maculopathy, retinal features were strictly confined within the vascular arcades and the ffERG was normal. The intermediate rodcone subtype was defined by a macular dystrophy with additional patchy involvement of the retina beyond the vascular arcades, but limited to the posterior pole, and recordable ffERG responses beyond puberty.Of note, the maculopathy evolved only very mildly during longitudinal follow-up. The ffERG results remained normal. Together, this hinted at long-term preservation of peripheral visual function and was important regarding prognosis. Moreover, patients had a variable degree of foveal sparing. The presence and amount of foveal sparing determined the onset of clinical symptoms.The central atrophy in patients with an intermediate phenotype demonstrated jagged borders extending beyond the vascular arcades. Again, imaging hinted at initially better foveal quality. The typical RDH12 feature of peripapillary sparing of retinal tissue is seen clearly in the younger patients. In contrast to the very mild progression of the maculopathy, patients with an intermediate phenotype did show evolution of disease.Extensive clinical phenotyping, combined with in-depth genotyping, will help establish the full spectrum of disease phenotypes related to each disease gene. Providing essential information regarding thesemilder phenotypes and their genotype–phenotype correlations facilitates correct interpretation of genetic findings in IRDs and will be of utmost importance as novel therapeutic options are being developed and ideal candidates for treatment within the ideal window of treatment to conserve functional vision need to be selected.CRB1 is another gene responsible for Leber congenital amaurosis or early-onset retinal dystrophy. We described the clinical spectrum and the natural disease course of CRB1-related retinal dystrophy in a large Belgian cohort of 40 patients.On the one hand, the phenotype was characterized by the classic retinitis pigmentosa features, such as optic disc pallor, attenuated vessels, and peripheral, bone-spiculae-like, intraretinal pigment migration. On the other hand, this study additionally confirmed the presence of characteristic CRB1 features such as fine, yellow-white, punctate spots, hyperopia, nanophthalmos, a shallow anterior chamber, peripheral, peri-arteriolar preservation of the RPE, optic disc drusen and a Coats-like exudative vasculopathy. OCT often demonstrated the presence of inner retinal thickening.Combining these characteristic phenotypic features with the classic retinitis pigmentosa features in a patient in the clinic, allows for targeted testing of the CRB1 gene.If you combine these characteristic phenotypic features with the classic retinitis pigmentosa features in a patient in the clinic, it allows for targeted testing of the CRB1 gene.Finally, CRB1-related retinal dystrophy also displays a maculopathy as a mild-end spectrum phenotype.Only for this maculopathy, a clear genotype-phenotype correlation could be established, as all patients harbour the p.(Ile167_Gly169del) mutation in CRB1.Leber hereditary optic neuropathy (LHON) is a blinding disorder characterized by degeneration of the retinal ganglion cells, leading to painless, acute or subacute, profound central vision loss. More than 90% of LHON in the Caucasian population can be explained by one of three common variants m.11778G>A (MT-ND4), m.14484T>C (MT-ND6) and m.3460G>A (MT-ND1). Whole mitochondrial DNA(mtDNA) genome sequencing did not reveal one of these primary variants in the mtDNA, but rather the homoplasmic m.14502T>C (MT-ND6) variant in the family we studied. In the Asian population, the m.14502T>C variant is mainly known as a secondary or modifying variant but has also been reported as a primary pathogenic variant, in the absence of any other mtDNA alterations. Protein modelling predictions performed in our study suggested a mild effect on protein structure. Family members were invited for a clinical workup to further clarify the causal relationship between the m.14502T>C variant and the presence of an optic neuropathy. Three out of 4 siblings presented with clinical or subclinical manifestations of optic neuropathy. In the absence of other nuclear or mitochondrial DNA pathogenic variants, it is hypothesized that the m.14502T>C variant causes the LHON phenotype in this family.Considering that phenotypic expression depends on additional environmental factors, smoking and alcohol are thought to have triggered the clinical manifestation of disease in affected family members of our study. Lifestyle adaptations such as smoking and alcohol cessation have been strongly advised in preventing visual impairment.This thesis shows that long-term deep phenotyping, sometimes over many years, provides insight in different disease characteristics, functional data, variability, and mild-end-of-the-spectrum disease and will be adamant in the development of future therapeutic strategies.Public defense: 2022-09-06Inherited retinal dystrophies (IRDs) are a heterogenous group of hereditary disorders characterized by a predominantly progressive degeneration of the photoreceptors and/or retinal pigment epithelium (RPE) leading to often profound visual loss. With a prevalence of 1:3000 individuals, they are the leading cause of visual impairment and blindness in the Western European population, afflicting children and working-age adults, and thus not as rare as previously considered. To date, over 280 genes have been identified to cause both non-syndromic and syndromic forms of inherited retinal dystrophies (https://sph.uth.edu/retnet). Therefore, each genetic subtype may be exceedingly rare. Nevertheless, genetic heterogeneity, variable clinical expression of certain genes with high inter- and intrafamilial variability, the possibility of incomplete penetrance and the additional impact of modifier genes and environmental factors complicate matters. As such, one rapidly ends up with a melting pot of very different diseases and phenotypes. IRDs have long been considered as a group of disorders for which no treatments or cures were available. But the present is definitely a time of great promise. Sound preclinical data recently resulted in the first reimbursed gene augmentation therapy Luxturna and the development of many other ongoing clinical treatment trials. We are on the doorstep of a new era where patients have a possibility that they won’t experience the full course of their disease. DNA and RNA therapies, optogenetics, stem cell transplantation and neurotrophic factors are expected to undergo major developments. Despite the latter, insight into the phenotypic spectrum and the disease course remains scarce for several IRD subtypes. This thesis provides a detailed clinical description of several subtypes of inherited ocular disease. Adequate insight into the clinical characteristics, variability, and long-term evolution of these diseases is crucial in establishing an adequate diagnosis and in counselling patients on their prognosis.This knowledge will prove to be vital in the design of future therapeutic trials, providing a comparison between the natural evolution and course of disease after treatment with innovative therapies.We describe the longitudinal phenotypic findings of autosomal dominant late-onset retinal degeneration (L-ORD) related to a heterozygous mutation in the C1QTNF5 gene. L-ORD is extremely rare. It is estimated that 110 patients have been diagnosed worldwide. In the majority of patients, LORD is related to the c.489C>G p.(Ser169Arg) variant in C1QTNF5. Twenty-six of these 110 patients were the subject of this thesis and underwent an annual detailed clinical characterization with a mean follow-up period of 8 years (1 – 37 years). A novel variant, c.562C>A p.(Pro188Thr), was identified, confirming the genetic heterogeneity of L-ORD. The detection of this causal variant was required to unequivocally confirm the clinical diagnosis in the patients, to identify future treatment options, to inform patients about the risk disease in family members and to offer the option of presymptomatic testing, preconceptual advice and prenatal or pre-implantation diagnosis, and to exclude the involvement of other organ systems.Although one disease, L-ORD presented initially as a spectrum of three, rather completely different, phenotypes, creating a striking clinical variation between family members. We classified them into 3 categories. The presence of these 3 completely different presenting phenotypes of L-ORD could render making a correct clinical diagnosis potentially challenging. Depending on the presenting phenotype and the disease phase at presentation, L-ORD can easily be mistaken for another disease. Only after years of evolution, a mixture of different retinal features will progress to a common, late phenotype of generalized chorioretinal atrophy. Patients of course are keen to hear about prognosis. The progression rate differed depending on the phenotype at presentation.Other differences compared to historical knowledge on L-ORD comprised the cause of vision loss and the anterior segment findings. L-ORD patients are thought to suffer vision loss related to the development of macular subretinal neovascularization. In our cohort however, most patients developed macular atrophy. The presence of long, anteriorly inserted lens zonules (LAZ) is considered to be pathognomonic for L-ORD. LAZ are thought to cause iris transillumination and subsequent glaucoma. LAZ are absent in the large majority of our L-ORD patients. Iris transillumination however is present, in combination with poor pupillary dilatation, and can therefore be considered to arise independently of the presence of LAZ. Glaucoma is not a major issue in our cohort.A major finding in our study is that evolution of disease is rather linear at first, with progression of disease accelerating considerably beyond a certain clinical stage. Once BCVA reduces, a period of only 1 to 3 years precedes loss of both central and peripheral sight. As the next step, attempts will be made to develop therapeutic modalities. In IRDs with a dominant-negative effect, promising strategies include silencing of the mutant allele and selective removal of the mutant protein. Projecting an individual patient’s phenotype against the findings of this natural history study of L-ORD would allow identification of the optimal window for intervention long before central macular function and vision are lost.Leber congenital amaurosis (LCA) and early-onset retinal dystrophy (EORD) are situated at the severe end of the phenotypic spectrum of rod-cone dystrophy, leading to blindness from birth or in the first few years of life. Phenotypically, RDH12-related LCA/EORD is characterized by macular atrophy with yellowish discolouration, and the typical retinitis pigmentosa characteristics of a pale optic disc, attenuation of the retinal vasculature, peripheral bone-spiculae-like intraretinal pigment migration and an extinguished full-field electroretinography (ffERG). Typical, additional characteristics linked to the RDH12 gene involve peripapillary sparing of retinal structure, patchy preservation of the peripheral retina, and preserved para-arteriolar retina.We identified a unique cohort of seven patients with biallelic RDH12 mutations with the novel finding of mild disease that was unrecognizable from the classic RDH12-phenotype. This mild disease did not involve the entire retina as is usual, but was instead limited to either the central macula or the posterior pole. As such, we expanded the phenotypic spectrum of RDH12-related disease. Two subgroups were identified based on the clinical presentation. In patients with an RDH12-maculopathy, retinal features were strictly confined within the vascular arcades and the ffERG was normal. The intermediate rodcone subtype was defined by a macular dystrophy with additional patchy involvement of the retina beyond the vascular arcades, but limited to the posterior pole, and recordable ffERG responses beyond puberty.Of note, the maculopathy evolved only very mildly during longitudinal follow-up. The ffERG results remained normal. Together, this hinted at long-term preservation of peripheral visual function and was important regarding prognosis. Moreover, patients had a variable degree of foveal sparing. The presence and amount of foveal sparing determined the onset of clinical symptoms.The central atrophy in patients with an intermediate phenotype demonstrated jagged borders extending beyond the vascular arcades. Again, imaging hinted at initially better foveal quality. The typical RDH12 feature of peripapillary sparing of retinal tissue is seen clearly in the younger patients. In contrast to the very mild progression of the maculopathy, patients with an intermediate phenotype did show evolution of disease.Extensive clinical phenotyping, combined with in-depth genotyping, will help establish the full spectrum of disease phenotypes related to each disease gene. Providing essential information regarding thesemilder phenotypes and their genotype–phenotype correlations facilitates correct interpretation of genetic findings in IRDs and will be of utmost importance as novel therapeutic options are being developed and ideal candidates for treatment within the ideal window of treatment to conserve functional vision need to be selected.CRB1 is another gene responsible for Leber congenital amaurosis or early-onset retinal dystrophy. We described the clinical spectrum and the natural disease course of CRB1-related retinal dystrophy in a large Belgian cohort of 40 patients.On the one hand, the phenotype was characterized by the classic retinitis pigmentosa features, such as optic disc pallor, attenuated vessels, and peripheral, bone-spiculae-like, intraretinal pigment migration. On the other hand, this study additionally confirmed the presence of characteristic CRB1 features such as fine, yellow-white, punctate spots, hyperopia, nanophthalmos, a shallow anterior chamber, peripheral, peri-arteriolar preservation of the RPE, optic disc drusen and a Coats-like exudative vasculopathy. OCT often demonstrated the presence of inner retinal thickening.Combining these characteristic phenotypic features with the classic retinitis pigmentosa features in a patient in the clinic, allows for targeted testing of the CRB1 gene.If you combine these characteristic phenotypic features with the classic retinitis pigmentosa features in a patient in the clinic, it allows for targeted testing of the CRB1 gene.Finally, CRB1-related retinal dystrophy also displays a maculopathy as a mild-end spectrum phenotype.Only for this maculopathy, a clear genotype-phenotype correlation could be established, as all patients harbour the p.(Ile167_Gly169del) mutation in CRB1.Leber hereditary optic neuropathy (LHON) is a blinding disorder characterized by degeneration of the retinal ganglion cells, leading to painless, acute or subacute, profound central vision loss. More