Summary

Summary

in ion homeostasis. To a certain extent, BEST1 mutations and the associated phenotypes

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comply with a genotype-phenotype correlation model.

Section 3.2 is a detailed clinical and molecular genetic analysis of 20 patients with Best vitelliform macular dystrophy from 15 different families, who all carried a mutation in the BEST1 gene. Eight different BEST1 mutations were found, including two novel mutations. A broad phenotypic variability was observed, even in association with a single BEST1 mutation. As much as 60% of the macular lesions could not be classified as a typical stage of Best vitelliform macular dystrophy. These findings complicated the establishment of distinct genotype-phenotype correlations. FAF and optical coherence tomography, especially when used in combination, proved to be very useful non-invasive imaging methods for the phenotyping and follow-up of Best vitelliform macular dystrophy patients. These imaging techniques are able to visualize abnormalities within vitelliform lesions that are not seen on ophthalmoscopy and fluorescein angiography. As such, FAF and optical coherence tomography also provide a valuable insight into the pathogenesis of Best vitelliform macular dystrophy.

Section 3.3 describes the clinical and genetic findings in multifocal vitelliform dystrophy. Multifocal vitelliform dystrophy is shown to be both clinically and genetically heterogeneous. Fifteen patients with multifocal vitelliform lesions were studied, as well as their affected family members. Nine of these 15 patients (60%) carried a mutation in the BEST1 gene. Seven different BEST1 mutations were identified, including 4 novel mutations. The electro-oculogram was abnormal in all patients with a BEST1 mutation. The age at onset of visual loss was highly variable, as was the number and size of the vitelliform lesions outside the macula. However, the appearance of the lesions outside the macula was quite similar to the central vitelliform lesion on ophthalmoscopy, FAF, and optical coherence tomography, despite the fact that they were smaller. The findings in this study indicate that a multifocal vitelliform response is associated with, but not exclusive to, mutations in the BEST1 gene. Multifocal vitelliform dystrophy in patients with a BEST1 mutation and an abnormal electro-oculogram can be considered a multifocal variant of Best vitelliform macular dystrophy.

In Chapter 4, section 4.1 serves as a review of the peripherin/RDS gene and the broad spectrum of retinal dystrophies caused by mutations in this gene. The peripherin/RDS protein is a structural protein that plays an important role in the morphogenesis of the photoreceptor outer segments. Mutations in the peripherin/RDS gene may first of all cause various autosomal dominant macular dystrophies. These include three phenotypes that have been classified as pattern dystrophies: butterfly-shaped pigment dystrophy, adultonset foveomacular vitelliform dystrophy, and multifocal pattern dystrophy simulating Stargardtdisease/fundusflavimaculatus.Otherperipherin/RDS-relatedmaculardystrophies are central areolar choroidal dystrophy and age-related macular degeneration (AMD)- like late-onset macular dystrophy. Apart from these macular dystrophies, peripherin/RDS mutations may also cause cone-rod dystrophy, which shares ophthalmoscopic and FAF features with central areolar choroidal dystrophy. In addition, mutations in peripherin/ RDS are among the most frequently identified mutations in autosomal dominant retinitis pigmentosa. Finally, a specific peripherin/RDS mutation causes digenic retinitis

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pigmentosa, when co-inherited with a mutation in the ROM1 gene. A single peripherin/ RDS mutation may cause an intriguingly broad range of phenotypes, even within a single family, which makes it difficult to recognize consistent genotype-phenotype correlations in peripherin/RDS-related retinal dystrophies.

Section 4.2 is the largest clinical and genetic study that has thus far been published on central areolar choroidal dystrophy (CACD), describing a group of 103 CACD patients. Follow-up data were available for 42% of the patients, with a follow-up period up to 35 years. This specific macular dystrophy was shown to be caused by autosomal dominant inheritance of a p.Arg142Trp peripherin/RDS mutation in 95% of the patients in our study. This high percentage of p.Arg142Trp mutations is most likely due to the fact that peripherin/RDS p.Arg142Trp is a relatively frequent founder mutation in the southeast region of the Netherlands. The remaining CACD patients, who were members of the same family, carried a p.Arg172Gln mutation in peripherin/RDS. Lesions corresponded to typical CACD stages in virtually all patients. Peripherin/RDS p.Arg142Trp-associated CACD was shown to be a central cone dystrophy phenotype. A remarkable variability in disease severity was observed, and non-penetrance was seen up to the age of 64, in up to 21% of mutation carriers. The overlapping age at onset and similar clinical features of CACD and atrophic age-related macular degeneration, together with the decreased penetrance of the p.Arg142Trp peripherin/RDS mutation, can make the differential diagnosis between these conditions challenging.

Section 4.3 is the first study that specifically analyzed multifocal pattern dystrophy simulating Stargardt disease/fundus flavimaculatus (MPD), an autosomal dominant pattern dystrophy of the retina. Mutations in the peripherin/RDS gene were found to be the major cause of this phenotype. We describe nine different peripherin/RDS mutations, including six novel mutations, that were found in 10 different MPD families. All patients with peripherin/RDS-related MPD showed a retinal dystrophy characterized by irregular yellow-white flecks in the posterior pole. These flecks were highly similar to those observed in the fundus flavimaculatus subtype of Stargardt disease, which is caused by autosomal recessive mutations in the ABCA4 gene. Clinical characteristics of MPD that may help to distinguish MPD from Stargardt disease are the autosomal dominant inheritance pattern, the relatively late age at onset of visual loss, and the absence of a “dark choroid” on fluorescein angiography. However, the decreased penetrance and markedlyvariableexpressivityofseveraloftheseperipherin/RDSmutationsmaycomplicate the differentiation between MPD and Stargardt disease. In these cases, analysis of the ABCA4 and peripherin/RDS genes is especially helpful.

Chapter 5 discusses the clinical and molecular genetic findings in phenotypes associated with variants in the complement factor H (CFH) gene, with an emphasis on the phenotypes associated with drusen, the hallmark lesions in age-related macular degeneration. Drusen are yellow-white deposits between the RPE and Bruch’s membrane.

The CFH protein is a multifunctional protein, that primarily plays a role in the inhibition 7 of excessive activation of the alternative pathway of the complement cascade. The

complement cascade is an essential part of innate immunity.

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Section 5.1 extensively reviews the spectrum of phenotypes associated with variants in the CFH gene. This phenotypic spectrum includes renal phenotypes, such as membranoproliferative glomerulonephritis and atypical hemolytic uremic syndrome, as well as ocular phenotypes, including basal laminar drusen and AMD. In addition, several overlapping clinical entities associated with CFH gene variants are discussed. An interesting common feature of age-related macular degeneration, basal laminar drusen, and membranoproliferative glomerulonephritis, is the presence of drusen, although these drusen appear with a different age at onset. This common feature of drusen may be explained by a partially similar pathogenetic background, involving an abnormally active alternative complement pathway.

The phenotypic consequences of CFH variants depend on their differential impact on the regulatory function of plasmaand surface-bound CFH. Therefore, distinct genotypephenotype correlations can be observed. In this thesis, we discuss these correlations, and we propose a genotype-phenotype correlation model for CFH-related diseases.

In section 5.2, the role of the CFH gene was evaluated in 30 patients from different families with early-onset basal laminar drusen. The phenotype of basal laminar drusen is characterized by an innumerable amount of small drusen in the macula, and often scattered throughout the entire fundus. These drusen correspond to a characteristic “stars-in-the-sky” picture on the fluorescein angiogram. We show that basal laminar drusen is a genetically heterogeneous phenotype, as we found four different CFH gene mutations in five basal laminar drusen families. Our findings strongly support a recessive disease model in this subgroup of patients with basal laminar drusen. In these families, individuals develop early-onset basal laminar drusen when they carry a CFH mutation on one allele and the CFH p.Tyr402His risk variant on the other allele. The presence of a CFH mutation in the absence of the p.Tyr402His risk variant may contribute to the development of age-related macular degeneration at a later age. Thus, basal laminar drusen and age-related macular degeneration appear to belong to a spectrum of diseases, characterized by drusen, that are associated with either monogenic or multifactorial inheritance of variants in the CFH gene.

Chapter 6 is a general discussion of the findings in this thesis. Similarities and differencesbetweensimilarphenotypesarediscussed,basedontheirclinical,genetic,and pathophysiologicalcharacteristics.Theproposedgenotype-phenotypecorrelationmodels are discussed. This general discussion also attempts to shed a light on the phenomena of phenotypic variability and non-penetrance that were regularly observed with the genes in this thesis, by discussing possible genetic and environmental modifying factors. Agerelated macular degeneration is the example par excellence of a multifactorial retinal disease associated with complement activation and drusen. Therefore, it is also discussed in the light of the important contributing genetic and environmental factors, and their pathophysiological consequences. Finally, future perspectives on gene therapy and other possible therapeutic approaches are discussed.

A profound knowledge on the clinical, genetic, and pathophysiologic characteristics of the hereditary retinal diseases described in this thesis is important. After all, such

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knowledge enables optimal patient information and genetic counseling. In addition, it may facilitate the application and evaluation of future therapeutic strategies in these diseases. A thorough insight in the genetic and phenotypic characteristics of hereditary retinal disease may determine which patients are most eligible for treatments such as anti-angiogenic and gene therapy.

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