Hadas Newman

Non-syndromic retinitis pigmentosa due to mutations in the mucopolysaccharidosis type IIIC gene, heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT)

Retinitis pigmentosa (RP), the most common form of inherited retinal degeneration, is clinically and genetically heterogeneous and can appear as syndromic or non-syndromic. Mucopolysaccharidosis type IIIC (MPS IIIC) is a lethal disorder, caused by mutations in the heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) gene and characterized by progressive neurological deterioration, with retinal degeneration as a prominent feature. We identified HGSNAT mutations in six patients with non-syndromic RP. Whole exome sequencing (WES) in an Ashkenazi Jewish Israeli RP patient revealed a novel homozygous HGSNAT variant, c.370A>T, which leads to partial skipping of exon 3. Screening of 66 Ashkenazi RP index cases revealed an additional family with two siblings homozygous for c.370A>T. WES in three Dutch siblings with RP revealed a complex HGSNAT variant, c.[398G>C; 1843G>A] on one allele, and c.1843G>A on the other allele. HGSNAT activity levels in blood leukocytes of patients were reduced compared with healthy controls, but usually higher than those in MPS IIIC patients. All patients were diagnosed with non-syndromic RP and did not exhibit neurological deterioration, or any phenotypic features consistent with MPS IIIC. Furthermore, four of the patients were over 60 years old, exceeding by far the life expectancy of MPS IIIC patients. HGSNAT is highly expressed in the mouse retina, and we hypothesize that the retina requires higher HGSNAT activity to maintain proper function, compared with other tissues associated with MPS IIIC, such as the brain. This report broadens the spectrum of phenotypes associated with HGSNAT mutations and highlights the critical function of HGSNAT in the human retina.

The prevalence of retinal and optical coherence tomography findings in preeclamptic women.

Purpose: To evaluate retinal and optical coherence tomography findings and establish their prevalence in preeclamptic women. Methods: Twenty-seven preeclamptic women who underwent clinical examinations with both slit-lamp biomicroscopy and optical coherence tomography were prospectively studied. Results: Three of the 4 women (14.8%) identified by clinical examinations as having retinal findings related to preeclampsia had visual disturbances. Optical coherence tomography revealed retinal pathology in 2 women (7.4%) consisting of retinal edema, subretinal fluid, photoreceptors irregularities, and lesions at the retinal pigment epithelium level (Elschnig spots). The mean retinal nerve fiber layer thickness was significantly higher in women with pathologic funduscopic findings (P = 0.009) than in women with normal eye examinations. All women with ocular findings had severe preeclampsia and higher systolic blood pressure than women without ocular findings (P = 0.03). Conclusion: The prevalence of retinal involvement is high in severe preeclampsia and low in asymptomatic preeclampsia. Preeclamptic women diagnosed as having ocular involvement should be evaluated and monitored by optical coherence tomography, provided that their systemic condition allows it. Findings of retinal nerve fiber layer thickening in these women may indicate occult central nervous system involvement.

Involvement of CD24 in Angiogenesis in a Mouse Model of Oxygen-Induced Retinopathy

Purpose: To investigate a possible involvement of CD24 in vascular remodeling and angiogenesis in retinopathy of prematurity (ROP) in a mouse model of oxygen-induced retinopathy. Materials and methods: 17 CD24 knockout (KO) and 12 wild-type (WT) C57BL/6 mice were used. Group 1 mice were exposed to oxygen concentrations of 75 ± 2% from postnatal day (P) 7 to P12. Group 2 mice were raised in room air. At P17, all mice underwent fluorescein-conjugated-dextran perfusion and were sacrificed. The flat-mounted retinas were scored manually and digitally by a new computerized algorithm, according to blood vessel obliteration, tortuosity, vascular tufts and neovascularization formation. Results: Fifty four retinal whole mounts were available for analysis and scoring. Group 1 retinas had significantly higher values of vaso-obliteration, tufts, neovascularization, vessel tortuosity and higher mean retinopathy scores than Group 2 retinas (KO mice: 9.0 ± 0.27 vs. 0.74 ± 0.2, respectively, P < 0.0001; WT mice: 7.58 ± 0.40 vs. 1.17 ± 0.27, respectively, P < 0.0001). Manual scoring in Group 1 revealed higher values of neovascularization, tortuosity and mean retinopathy scores in KO mice vs. WT mice (9.0 ± 0.27 vs. 7.58 ± 0.40, respectively, P = 0.009). Digital scoring revealed a higher neovascularization score in KO mice as well (13.72 ± 0.82% vs. 8.06 ± 0.27%, P < 0.0001). All mice had similar vaso-obliteration areas. There were no significant differences between KO and WT mice in Group 2. Conclusions: Absence of CD24 may have a deleterious effect on angiogenesis occurring in the second stage of ROP development, though its role in vessel obliteration during the first stage of ROP is probably limited.

Homozygosity for a Recessive Loss-of-Function Mutation of the NRL Gene Is Associated With a Variant of Enhanced S-Cone Syndrome

Purpose To investigate the genetic basis for severe visual complaints by Bukharan Jewish patients with oculopharyngeal muscular dystrophy (OPMD). Methods Polymerase chain reaction amplification and direct sequencing were used to test for NRL, PABPN1, and NR2E3 mutations. Complete ophthalmic examination included best-corrected visual acuity, biomicroscopic examination, optical coherence tomography, and fundus autofluorescence. Detailed electroretinography (ERG) testing was conducted including expanded International Society for Clinical Electrophysiology of Vision protocol for light-adapted and dark-adapted conditions, measurements of S-cone function, and ON-OFF light-adapted ERG. Results The index patients were homozygotes for both a dominant mutation of the PABPN1 gene, (GCN)13, and a recessive mutation of the NRL gene, p.R31X, on chromosome 14q11.1, leading to early-onset OPMD accompanied by night blindness and reduced visual acuity. No mutations were found in the NR2E3 gene. Both patients were of Bukharan Jewish origin, but from unrelated families. Electroretinography responses of both patients were dominated by short-wavelength-sensitive mechanisms, with no detectable rod function, similar to the ERG responses of individuals with enhanced S-cone syndrome (ESCS) due to NR2E3 mutations. Heterozygotes for the PABPN1 and NRL mutations demonstrated normal fundi and ERG responses. Conclusions Homozygosity for the recessive NRL mutation described here appears to be associated with a distinct retinal phenotype, demonstrating ERG characteristics similar to those of ESCS patients. This report expands the spectrum of NRL recessive mutations, as well as the genetic spectrum of ESCS, and indicates a new syndrome of OPMD with an ESCS-like phenotype.

Nonsyndromic Retinitis Pigmentosa in the Ashkenazi Jewish Population: Genetic and Clinical Aspects

PURPOSE To analyze the genetic and clinical findings in retinitis pigmentosa (RP) patients of Ashkenazi Jewish (AJ) descent, aiming to identify genotype-phenotype correlations. DESIGN Cohort study. PARTICIPANTS Retinitis pigmentosa patients from 230 families of AJ origin. METHODS Sanger sequencing was performed to detect specific founder mutations known to be prevalent in the AJ population. Ophthalmologic analysis included a comprehensive clinical examination, visual acuity (VA), visual fields, electroretinography, color vision testing, and retinal imaging by OCT, pseudocolor, and autofluorescence fundus photography. MAIN OUTCOME MEASURES Inheritance pattern and causative mutation; retinal function as assessed by VA, visual fields, and electroretinography results; and retinal structural changes observed on clinical funduscopy as well as by pseudocolor, autofluorescence, and OCT imaging. RESULTS The causative mutation was identified in 37% of families. The most prevalent RP-causing mutations are the Alu insertion (c.1297_8ins353, p.K433Rins31*) in the male germ cell-associated kinase (MAK) gene (39% of families with a known genetic cause for RP) and c.124A>G, p.K42E in dehydrodolichol diphosphate synthase (DHDDS) (33%). Additionally, disease-causing mutations were identified in 11 other genes. Analysis of clinical parameters of patients with mutations in the 2 most common RP-causing genes revealed that MAK patients had better VA and visual fields at relatively older ages in comparison with DHDDS patients. Funduscopic findings of DHDDS patients matched those of MAK patients who were 20 to 30 years older. Patients with DHDDS mutations were referred for electrophysiologic evaluation at earlier ages, and their cone responses became nondetectable at a much younger age than MAK patients. CONCLUSIONS Our AJ cohort of RP patients is the largest reported to date and showed a substantial difference in the genetic causes of RP compared with cohorts of other populations, mainly a high rate of autosomal recessive inheritance and a unique composition of causative genes. The most common RP-causing genes in our cohort, MAK and DHDDS, were not described as major causative genes in other populations. The clinical data show that in general, patients with biallelic MAK mutations had a later age of onset and a milder retinal phenotype compared with patients with biallelic DHDDS mutations.

Prevalence of choroidal nevus and retinal pigment epithelial alterations in vitiligo patients

PurposeTo investigate ocular manifestations in patients with vitiligo by multimodal imaging, including optical coherence tomography (OCT), color fundus photography, and fundus autofluorescence (FAF).MethodsIn this prospective, observational clinical study, vitiligo patients underwent ophthalmologic and dermatologic clinical assessment and imaging by spectral-domain OCT, FAF, and color fundus imaging. Ocular echography was performed as indicated. Statistical analysis was performed using paired T test and Pearson correlation.ResultsA total of 61 eyes of 31 vitiligo patients were examined. Ocular findings consisted of choroidal nevi (n = 10, 32%), of which four (40%) were bilateral; two patients (6.5%) had a prominent choroidal pattern, two (6.5%) had hypopigmentary retinal pigment epithelium (RPE) lesions, and one (3.2%) had peripapillary atrophy of the RPE. Choroidal nevi were demonstrated only in eyes of patients with generalized vitiligo and were more common with upper body involvement (p = 0.02) and more prevalent in women (p = 0.02). Hypopigmentary lesions were detected in two patients and demonstrated on OCT as RPE atrophy and as photoreceptor/RPE changes.ConclusionsIn this case series, vitiligo patients had a higher rate of choroidal nevi than previously reported. The hypopigmentary vitiliginous fundus lesions were depicted on OCT as photoreceptor and RPE atrophy. These findings may suggest the advisability of regular ocular monitoring for vitiligo patients.

Carrier frequency analysis of mutations causing autosomal-recessive-inherited retinal diseases in the Israeli population

Inherited retinal diseases (IRDs) are heterogeneous phenotypes caused by variants in a large number of genes. Disease prevalence and the frequency of carriers in the general population have been estimated in only a few studies, but are largely unknown. To this end, we developed two parallel methods to calculate carrier frequency for mutations causing autosomal-recessive (AR) IRDs in the Israeli population. We created an SQL database containing information on 178 genes from gnomAD (including genotyping of 5706 Ashkenazi Jewish (AJ) individuals) and our cohort of >2000 families with IRDs. Carrier frequency for IRD variants and genes was calculated based on allele frequency values and the Hardy–Weinberg (HW) equation. We identified 399 IRD-causing variants in 111 genes in Israeli patients and AJ controls. For the AJ subpopulation, gnomAD and HW-based regression analysis showed high correlation, therefore allowing one to use HW-based data as a reliable estimate of carrier frequency. Overall, carrier frequency per subpopulation ranges from 1/2.2 to 1/9.6 individuals, with the highest value obtained for the Arab-Muslim subpopulation in Jerusalem reaching an extremely high carrier rate of 44.7%. Carrier frequency per gene ranges from 1/31 to 1/11994 individuals. We estimate the total carrier frequency for AR-IRD mutations in the Israeli population as over 30%, a relatively high carrier frequency with marked variability among subpopulations. Therefore, these data are highly important for more reliable genetic counseling and genetic screening. Our method can be adapted to study other populations, either based on allele frequency data or cohort of patients.

Purification and characterization of human dehydrodolychil diphosphate synthase (DHDDS) overexpressed in E. coli

Protein asparagine (N)-linked glycosylation is a post-translational modification that occurs in the endoplasmic reticulum; it plays an important role in protein folding, oligomerization, quality control, sorting, and transport. Accordingly, disorders of glycosylation may affect practically every organ system. Dehydrodolichyl diphosphate synthase (DHDDS) is an eukaryotic cis prenyltransferase (cis-PT) that catalyzes chain elongation of farnesyl diphosphate via multiple condensations with isopentenyl diphosphate to form dehydrodolichyl diphosphate, a precursor for the glycosyl carrier dolichylpyrophophate involved in N-linked glycosylation. Mutations in DHDDS were shown to result in retinitis pigmentosa, ultimately leading to blindness, but the exact molecular mechanism by which the mutations affect DHDDS function remains elusive. In addition, bacterial cis-PT homologs are involved in bacterial wall synthesis and are therefore potential targets for new antibacterial agents. However, as eukaryotic cis-PT were not thoroughly characterized structurally and functionally, rational design of prokaryotic cis-PT specific drugs is currently impossible. Here, we present a simple protocol for purification of functionally active human DHDDS under non-denaturating conditions using a codon-optimized construct. The purified protein forms a stable homodimer, similar to its bacterial homologs, and shows time- and substrate-dependent activity. Purification of this protein requires the presence of a detergent for protein solubility. The protocol described here may be utilized for the overexpression of other eukaryotic cis-PT. Future structural and functional studies of the recombinant DHDDS may shed light on the mechanisms underlying DHDDS-related retinitis pigmentosa and lead to novel therapeutic approaches.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Optical Coherence Tomography: An Adjunctive Tool for Differentiating between Choroidal Melanoma and Metastasis

Purpose. To investigate the value of optical coherence tomography (OCT) for differentiation between choroidal melanoma and metastasis based on characteristics of the anterior choroidal surface and the chorioretinal interface. Methods. This retrospective observational case series included 29 patients with untreated choroidal melanomas and 21 patients with untreated choroidal metastases. Regularity and lobularity characteristics of the anterior choroidal surface were evaluated in a masked manner. Retinal and retinal pigment epithelium (RPE) findings were documented as well. Results. OCT demonstrated a regular and smooth anterior choroidal surface in 89.7% of the eyes with melanoma and in 47.6% of the eyes with metastasis (p = 0.002; sensitivity = 89.7%; specificity = 52.4%). The anterior choroidal contour was lobulated in 81.0% of the eyes with metastasis versus 17.2% of the eyes with melanoma (p < 0.001; sensitivity = 82.8%; specificity = 81.0%). RPE thickness and neuroretinal characteristics (e.g., retinal thickness, the presence of cysts, and the presence of subretinal fluid) were similar in both choroidal tumors. Conclusion. OCT may serve as a noninvasive adjunctive tool for the differential diagnosis of choroidal tumors. Choroidal melanomas usually demonstrate regular surfaces on OCT, while choroidal metastases usually have an irregular and lobulated surface.