Martin Gliem

Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.

Peptide-mediated desmoglein 3 crosslinking prevents pemphigus vulgaris autoantibody-induced skin blistering

In pemphigus vulgaris, a life-threatening autoimmune skin disease, epidermal blisters are caused by autoantibodies primarily targeting desmosomal cadherins desmoglein 3 (DSG3) and DSG1, leading to loss of keratinocyte cohesion. Due to limited insights into disease pathogenesis, current therapy relies primarily on nonspecific long-term immunosuppression. Both direct inhibition of DSG transinteraction and altered intracellular signaling by p38 MAPK likely contribute to the loss of cell adhesion. Here, we applied a tandem peptide (TP) consisting of 2 connected peptide sequences targeting the DSG adhesive interface that was capable of blocking autoantibody-mediated direct interference of DSG3 transinteraction, as revealed by atomic force microscopy and optical trapping. Importantly, TP abrogated autoantibody-mediated skin blistering in mice and was effective when applied topically. Mechanistically, TP inhibited both autoantibody-induced p38 MAPK activation and its association with DSG3, abrogated p38 MAPK-induced keratin filament retraction, and promoted desmosomal DSG3 oligomerization. These data indicate that p38 MAPK links autoantibody-mediated inhibition of DSG3 binding to skin blistering. By limiting loss of DSG3 transinteraction, p38 MAPK activation, and keratin filament retraction, which are hallmarks of pemphigus pathogenesis, TP may serve as a promising treatment option.

An update on the ocular phenotype in patients with pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is an inherited multi-system disorder characterized by ectopic mineralization and fragmentation of elastic fibers in the skin, the elastic laminae of blood vessels and Bruch’s membrane in the eye. Biallelic mutations in the ATP-binding cassette (ABC) transporter gene ABCC6 on chromosome 16 are responsible for the disease. The pathophysiology is incompletely understood. However, there is consent that a metabolic alteration leads to dysfunction in extracellular calcium homeostasis and subsequent calcification of connective tissues rich in elastic fibers. This review summarizes and aims at explaining the variety of phenotypic ocular findings in patients with PXE. Specialized imaging techniques including white light fundus photography, blue light autofluorescence, near-infrared confocal reflectance imaging, high resolution optical coherence tomography, fluorescein and indocyanine green (ICG) angiography have revealed characteristic lesions at the ocular fundus of PXE patients. These include the classic signs of angioid streaks, peau d’orange, comet lesions, and choroidal neovascularizations (CNVs), but also the more recently recognized features such as chorioretinal atrophy, subretinal fluid independent from CNV, pattern dystrophy-like changes, debris accumulation under the retinal pigment epithelium, reticular drusen and a decreased fluorescence on late phase ICG angiography.

Actin reorganization contributes to loss of cell adhesion in pemphigus vulgaris

In the human autoimmune blistering skin disease pemphigus vulgaris autoantibodies (PV-IgG), which are mainly directed against keratinocyte cell adhesion molecules desmoglein (Dsg) 3 and Dsg1, cause keratinocyte cell dissociation (acantholysis). Recent studies reported that loss of keratinocyte cell adhesion was accompanied by profound alterations of the actin cytoskeleton. Nevertheless, the relevance of actin reorganization in this process is unclear at present. In this study, we provide evidence for an important role of actin reorganization in pemphigus pathogenesis. In parallel to loss of cell adhesion and fragmentation of Dsg3 staining along cell borders, PV-IgG treatment resulted in striking changes in actin cytoskeleton organization. Moreover, in experiments using fluorescence recovery after photobleaching (FRAP), PV-IgG were detected to interfere with actin dynamics. Therefore, we investigated whether pharmacological manipulation of actin polymerization modulates pathogenic effects of PV-IgG. Pharmacological stabilization of actin filaments via jasplakinolide significantly blocked cell dissociation and Dsg3 fragmentation, whereas cytochalasin D-induced actin depolymerization strongly enhanced pathogenic effects of PV-IgG. To substantiate these findings, we studied whether the protective effects of Rho GTPases, which are potent regulators of the actin cytoskeleton and were shown to be involved in pemphigus pathogenesis, were dependent on modulation of actin dynamics. Cytotoxic necrotizing factor-1 (CNF-1)-mediated activation of Rho-GTPases enhanced the cortical junction-associated actin belt and blunted PV-IgG-induced cell dissociation. However, when actin polymerization was blocked under these conditions via addition of latrunculin B, the protective effects of CNF-1 were abrogated. Taken together, these experiments indicate that reorganization of cortical actin filaments is a critical step in PV-IgG-induced keratinocyte dissociation.

Treatment of choroidal neovascularization due to angioid streaks: a comprehensive review.

Purpose: To evaluate different therapies for choroidal neovascularization (CNV) due to angioid streaks (AS). Methods: Studies were identified by a systematic literature search and were included in the analysis based on predefined criteria. Primary outcome measure was change in best-corrected visual acuity (BCVA). Results: Fifty-four relevant studies were identified and included mostly uncontrolled case series. No randomized controlled trials were available. Treatment with vascular endothelial growth factor inhibitors improved or stabilized BCVA in all case series. Photodynamic therapy slowed down disease progression with stabilization or decrease of BCVA. Individual BCVA and follow-up data for each treated eye were reported in >160 cases for both treatments, vascular endothelial growth factor inhibitors and photodynamic therapy. In a pooled analysis of those studies, the difference of mean change in BCVA between both treatment groups was estimated as approximately 6 lines (0.59 logMAR [95% confidence interval, 0.38-0.8; P < 0.0001]). A better baseline BCVA was associated with a better BCVA outcome (P < 0.0001). Laser photocoagulation yielded comparable results as photodynamic therapy but application was mostly restricted to extrafoveal lesions, was complicated by frequent recurrences, and led to more retinal damage with subsequent absolute scotomas. Combination therapies seem to be not superior to monotherapy. Conclusion: Intravitreal vascular endothelial growth factor inhibitors are currently the most effective treatment of CNV due to angioid streaks.

Quantitative Fundus Autofluorescence in Early and Intermediate Age-Related Macular Degeneration

IMPORTANCE Increased lipofuscin accumulation is assumed to be an important factor in the pathogenesis of age-related macular degeneration (AMD), although direct evidence for this hypothesis is missing. OBJECTIVE To quantitatively investigate lipofuscin-associated fundus autofluorescence (AF) in patients with early and intermediate AMD. DESIGN, SETTING, AND PARTICIPANTS A prospective, single-center, case-control study was conducted from August 1, 2014, to October 31, 2015, at a university referral center. Participants included 40 patients aged 65 years or younger and 108 individuals without eye disease serving as controls. All participants underwent quantitative fundus AF (qAF) imaging with a modified scanning laser ophthalmoscope equipped with an internal fluorescent reference. Mean qAF values of an 8-segment circular ring centered on the fovea (qAF8) were measured and compared between patients and controls. For subgroup analysis, drusen were categorized as soft drusen, cuticular drusen, and/or reticular pseudodrusen (RPD). MAIN OUTCOMES AND MEASURES The qAF8 levels. RESULTS In the 40 patients with AMD, mean (SD) age was 54.8 (5.6) years, and 32 (80%) were women. None of the investigated patients had qAF8 values above the 95% prediction interval (PI) of the 108 controls. In the soft drusen (28 [70%]) and cuticular drusen (8 [20%]) groups, qAF8 levels within the 95% PI were noted in 22 patients (79%; 95% CI, 60% to 90%) and 7 patients (88%; 95% CI, 51% to 99%) respectively. The qAF8 values in the RPD group (4 [10%]) were below the 95% PI in 3 patients (75%; 95% CI, 29% to 97%). Compared with the controls, statistical analysis revealed lower qAF8 values in the overall AMD cohort after adjusting for age (difference, -19.9% [95% CI, -25.6% to -12.7%], P < .001) as well as in all subgroups (soft drusen, -17.1% [95% CI, -24.1% to -9.5%], P < .001; cuticular drusen, -19.6% [95% CI, -30.3% to -7.2%], P = .003; and RPD, -34.5% [95% CI, -47.1% to -21.3%]; P < .001). CONCLUSIONS AND RELEVANCE The qAF8 measurements in this sample showed no increased lipofuscin-related fundus AF in patients with early and intermediate AMD. Lower qAF levels in certain subgroups may point to subnormal lipofuscin levels in the retinal pigment epithelium or, alternatively, limitations to detection of true retinal pigment epithelial lipofuscin content. The results of this study might expand the understanding of the pathogenesis of AMD and may have an effect on upcoming treatment trials that aim to modify lipofuscin accumulation.

E-cadherin and Src associate with extradesmosomal Dsg3 and modulate desmosome assembly and adhesion

Desmosomes provide strong intercellular cohesion essential for the integrity of cells and tissues exposed to continuous mechanical stress. For desmosome assembly, constitutively synthesized desmosomal cadherins translocate to the cell–cell border, cluster and mature in the presence of Ca2+ to stable cell contacts. As adherens junctions precede the formation of desmosomes, we investigated in this study the relationship between the classical cadherin E-cadherin and the desmosomal cadherin Desmoglein 3 (Dsg3), the latter of which is indispensable for cell–cell adhesion in keratinocytes. By using autoantibodies from patients with the blistering skin disease pemphigus vulgaris (PV), we showed in loss of function studies that E-cadherin compensates for effects of desmosomal disassembly. Overexpression of E-cadherin reduced the loss of cell cohesion induced by PV autoantibodies and attenuated activation of p38 MAPK. Silencing of E-cadherin abolished the localization of Dsg3 at the membrane and resulted in a shift of Dsg3 from the cytoskeletal to the non-cytoskeletal protein pool which conforms to the notion that E-cadherin regulates desmosome assembly. Mechanistically, we identified a complex consisting of extradesmosomal Dsg3, E-cadherin, β-catenin and Src and that the stability of this complex is regulated by Src. Moreover, Dsg3 and E-cadherin are phosphorylated on tyrosine residues in a Src-dependent manner and Src activity is required for recruiting Dsg3 to the cytoskeletal pool as well as for desmosome maturation towards a Ca2+-insensitive state. Our data provide new insights into the role of E-cadherin and the contribution of Src signaling for formation and maintenance of desmosomal junctions.

Reticular Pseudodrusen in Sorsby Fundus Dystrophy

PURPOSE To investigate the association of reticular pseudodrusen (RPD) with Sorsby fundus dystrophy (SFD). DESIGN Prospective, monocenter, cross-sectional case series. SUBJECTS Sixteen patients of 4 unrelated families with SFD caused by mutations in TIMP3. METHODS All subjects underwent multimodal imaging including near-infrared (NIR) reflectance and fundus autofluorescence with a confocal scanning laser ophthalmoscope and spectral-domain optical coherence tomography (SD OCT). MAIN OUTCOME MEASURES Prevalence, topographic distribution, and phenotype of RPD. RESULTS Mean age of the investigated patients was 56.8 years (range, 23-78 years). Reticular pseudodrusen were identified frequently in SFD patients in the sixth decade of life (5 of 7 [71%]) and were absent in younger (n = 3) or older (n = 6) patients. They were most abundant in the superior quadrant and spared the foveal region. Reticular pseudodrusen appeared as yellowish round to oval (dot subtype; n = 5) or confluent, wriggled (ribbon subtype; n = 3) lesions, sometimes forming irregular networks. Reticular pseudodrusen were hyporeflective on NIR reflectance and hypofluorescent on fundus autofluorescence imaging. They appeared as subretinal deposits on SD OCT imaging. Other lesions, such as peripheral pseudodrusen and soft drusen, were present less frequently. CONCLUSIONS Reticular pseudodrusen are a frequent finding in patients with SFD. Although SFD patients with RPD are younger, distribution and phenotype of RPD are similar to those observed in patients with age-related macular degeneration. The association of RPD with SFD implicates a role of Bruchs membrane, the Bruchs membrane-retinal pigment epithelium interface, or both in the pathogenesis of RPD.

Sorsby Fundus Dystrophy: Novel Mutations, Novel Phenotypic Characteristics, and Treatment Outcomes

PURPOSE To report novel TIMP3 mutations, and to characterize the ocular phenotype of Sorsby fundus dystrophy (SFD), including a novel early sign for the disease and to report the effect of anti-VEGF therapy. METHODS Twenty-one probands of three unrelated families with SFD were investigated using wide-field imaging, confocal laser scanning ophthalmoscopy with autofluorescence imaging, optical coherence tomography (OCT), indocyanine green-angiography (ICG-A), and molecular diagnostic for causative mutations. RESULTS Molecular genetic analysis revealed two novel (p.Tyr174Cys, p.Tyr177Cys) and one previously described (p.Tyr182Cys) missense mutations in TIMP3. In families with p.Tyr177Cys and p.Tyr182Cys, metamorphopsia and/or decrease in visual acuity were the initial symptoms occurring at approximately the sixth decade of life. The p.Tyr174Cys mutation carriers had first symptoms at approximately the third decade with dark adaptation problems and visual field defects. The ocular phenotype included drusen-like deposits, rapidly progressive geographic atrophy, choroidal neovascularization (CNV), and polypoidal choroidal neovascularization (PCV). Late disease manifestations were uniform with widespread chorioretinal atrophy, fibrosis, and choroidal thinning. Three asymptomatic young carriers of a TIMP3 mutation with otherwise normal findings on funduscopy and retinal imaging showed a characteristically reduced fluorescence on late-phase ICG-A images. This phenotypic sign was more pronounced and widespread in later disease stages. Patients with CNV or PCV showed a favorable response to therapy with intravitreally injected bevacizumab. CONCLUSIONS This study expands the spectrum of mutations in the TIMP3 gene and associated phenotypic findings. Imaging using late-phase ICG-A may be useful for early identification of individuals at risk for developing SFD. Intravitreal anti-VEGF therapy if initiated timely is effective in SFD patients with CNV.

Monoallelic ABCA4 Mutations Appear Insufficient to Cause Retinopathy: A Quantitative Autofluorescence Study

PURPOSE To investigate the effect of ABCA4 mutation status on lipofuscin-related quantitative autofluorescence (qAF) in humans and on bisretinoid accumulation in mice. METHODS Genotyped parents (n = 26; age 37-64 years) of patients with biallelic ABCA4-related retinopathy underwent in-depth retinal phenotyping including qAF imaging as a surrogate measure for RPE lipofuscin accumulation. In addition, bisretinoids as the main components of autofluorescent lipofuscin at the ocular fundus were quantified in Abca4-/-, Abca4+/-, and wild-type mice. RESULTS Index patients showed a retinal phenotype characteristic for ABCA4-related retinopathy, including increased qAF levels. In contrast, qAF measures in carriers of only one ABCA4 mutation were not different from age-matched controls in this sample, and there was no difference between truncating and missense mutations. Also, none of these carriers presented an abnormal phenotype on conventional imaging. One parent with ABCA4-related retinopathy and increased qAF carried an additional ABCA4 mutation, explaining the phenotype under a recessive disease model (pseudodominance). Biochemical analysis in the mouse model revealed direct downstream products (A2PE-H2, at-RALdimer-PE) of the ABCA4 substrate N-Ret-PE to be similar in wild-type and Abca4+/- mice. Both bisretinoids were 12- to 18-fold increased in Abca4-/- mice. Levels of A2E and A2PE in Abca4+/- mice were in between those measured in wild-type and Abca4-/- mice. CONCLUSIONS This study indicates that carriers of monoallelic ABCA4 mutations are phenotypically normal. However, biochemical analysis in the Abca4-deficient mouse model suggests detectable effects of one mutation in ABCA4 on the molecular level. The findings may have implications for therapeutic approaches such as gene replacement therapy.