Genetic Characterization of 191 Probands with Inherited Retinal Dystrophy by Targeted NGS Analysis.

Inherited retinal diseases (IRDs) represent a frequent cause of blindness in children and adults. As a consequence of the phenotype and genotype heterogeneity of the disease, it is difficult to have a specific diagnosis without molecular testing. To date, over 340 genes and loci have been associated with IRDs. We present the molecular finding of 191 individuals with IRD, analyzed by targeted next-generation sequencing (NGS). For 67 of them, we performed a family segregation study, considering a total of 126 relatives. A total of 359 variants were identified, 44 of which were novel. Genetic diagnostic yield was 41%. However, after stratifying the patients according to their clinical suspicion, diagnostic yield was higher for well-characterized diseases such as Stargardt disease (STGD), at 65%, and for congenital stationary night blindness 2 (CSNB2), at 64%. Diagnostic yield was higher in the patient group where family segregation analysis was possible (68%) and it was higher in younger (55%) than in older patients (33%). The results of this analysis demonstrated that targeted NGS is an effective method for establishing a molecular genetic diagnosis of IRDs. Furthermore, this study underlines the importance of segregation studies to understand the role of genetic variants with unknow pathogenic role.

Next-generation sequencing to genetically diagnose a diverse range of inherited eye disorders in 15 consanguineous families from Pakistan.

Inherited retinal dystrophies (IRDs) are characterized by photoreceptor dysfunction or degeneration. Clinical and phenotypic overlap between IRDs makes the genetic diagnosis very challenging and comprehensive genomic approaches for accurate diagnosis are frequently required. While there are previous studies on IRDs in Pakistan, causative genes and variants are still unknown for a significant portion of patients. Therefore, there is a need to expand the knowledge of the genetic spectrum of IRDs in Pakistan. Here, we recruited 52 affected and 53 normal individuals from 15 consanguineous Pakistani families presenting non-syndromic and syndromic forms of IRDs. We employed single molecule Molecular Inversion Probes (smMIPs) based panel sequencing and whole genome sequencing to identify the probable disease-causing variants in these families. Using this approach, we obtained a 93% genetic solve rate and identified 16 (likely) causative variants in 14 families, of which seven novel variants were identified in ATOH7, COL18A1, MERTK, NDP, PROM1, PRPF8 and USH2A while nine recurrent variants were identified in CNGA3, CNGB1, HGSNAT, NMNAT1, SIX6 and TULP1. The novel MERTK variant and one recurrent TULP1 variant explained the intra-familial locus heterogeneity in one of the screened families while two recurrent CNGA3 variants explained compound heterozygosity in another family. The identification of variants in known disease-associated genes emphasizes the utilization of time and cost-effective screening approaches for rapid diagnosis. The timely genetic diagnosis will not only identify any associated systemic issues in case of syndromic IRDs, but will also aid in the acceleration of personalized medicine for patients affected with IRDs.

Dark continuous noise from mutant G90D-rhodopsin predominantly underlies congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is an inherited retinal disease that causes a profound loss of rod sensitivity without severe retinal degeneration. One well-studied rhodopsin point mutant, G90D-Rho, is thought to cause CSNB because of its constitutive activity in darkness causing rod desensitization. However, the nature of this constitutive activity and its precise molecular source have not been resolved for almost 30 y. In this study, we made a knock-in (KI) mouse line with a very low expression of G90D-Rho (equal in amount to ~0.1% of normal rhodopsin, WT-Rho, in WT rods), with the remaining WT-Rho replaced by REY-Rho, a mutant with a very low efficiency of activating transducin due to a charge reversal of the highly conserved ERY motif to REY. We observed two kinds of constitutive noise: one being spontaneous isomerization (R*) of G90D-Rho at a molecular rate (R* s-1) 175-fold higher than WT-Rho and the other being G90D-Rho-generated dark continuous noise comprising low-amplitude unitary events occurring at a very high molecular rate equivalent in effect to ~40,000-fold of R* s-1 from WT-Rho. Neither noise type originated from G90D-Opsin because exogenous 11-cis-retinal had no effect. Extrapolating the above observations at low (0.1%) expression of G90D-Rho to normal disease exhibited by a KI mouse model with RhoG90D/WTand RhoG90D/G90D genotypes predicts the disease condition very well quantitatively. Overall, the continuous noise from G90D-Rho therefore predominates, constituting the major equivalent background light causing rod desensitization in CSNB.

Intergenic sequences harboring potential enhancer elements contribute to Axenfeld-Rieger syndrome by regulating PITX2.

Recent studies have uncovered that noncoding sequence variants may relate to Axenfeld-Rieger syndrome (ARS), a rare developmental anomaly with genetic heterogeneity. However, how these genomic regions are functionally and structurally associated with ARS is still unclear. In this study, we performed genome-wide linkage analysis and whole-genome sequencing in a Chinese family with ARS and identified a heterozygous deletion of about 570 kb (termed LOH-1) in the intergenic sequence between paired-like homeodomain transcription factor 2 (PITX2) and family with sequence similarity 241 member A. Knockout of LOH-1 homologous sequences caused ARS phenotypes in mice. RNA-Seq and real-time quantitative PCR revealed a significant reduction in Pitx2 gene expression in LOH-1-/- mice, while forkhead box C1 expression remained unchanged. ChIP-Seq and bioinformatics analysis identified a potential enhancer region (LOH-E1) within LOH-1. Deletion of LOH-E1 led to a substantial downregulation of the PITX2 gene. Mechanistically, we found a sequence (hg38 chr4:111,399,594-111,399,691) that is on LOH-E1 could regulate PITX2 by binding to RAD21, a critical component of the cohesin complex. Knockdown of RAD21 resulted in reduced PITX2 expression. Collectively, our findings indicate that a potential enhancer sequence that is within LOH-1 may regulate PITX2 expression remotely through cohesin-mediated loop domains, leading to ARS when absent.

In Vivo Assessment of Retinal Phenotypes in Axenfeld-Rieger Syndrome.

Purpose: Axenfeld-Rieger syndrome (ARS) is characterized by ocular anomalies including posterior embryotoxon, iridocorneal adhesions, corectopia/iris hypoplasia, and developmental glaucoma. Although anterior segment defects and glaucoma contribute to decreased visual acuity, the role of potential posterior segment abnormalities has not been explored. We used high-resolution retinal imaging to test the hypothesis that individuals with ARS have posterior segment pathology. Methods: Three individuals with FOXC1-ARS and 10 with PITX2-ARS completed slit-lamp and fundus photography, optical coherence tomography (OCT), OCT angiography, and adaptive optics scanning light ophthalmoscopy (AOSLO). Quantitative metrics were compared to previously published values for individuals with normal vision. Results: All individuals demonstrated typical anterior segment phenotypes. Average ganglion cell and inner plexiform layer thickness was lower in PITX2-ARS, consistent with the glaucoma history in this group. A novel phenotype of foveal hypoplasia was noted in 40% of individuals with PITX2-ARS (but none with FOXC1-ARS). Moreover, the depth and volume of the foveal pit were significantly lower in PITX2-ARS compared to normal controls, even excluding individuals with foveal hypoplasia. Analysis of known foveal hypoplasia genes failed to identify an alternative explanation. Foveal cone density was decreased in one individual with foveal hypoplasia and normal in six without foveal hypoplasia. Two individuals (one from each group) demonstrated non-foveal retinal irregularities with regions of photoreceptor anomalies on OCT and AOSLO. Conclusions: These findings implicate PITX2 in the development of the posterior segment, particularly the fovea, in humans. The identified posterior segment phenotypes may contribute to visual acuity deficits in individuals with PITX2-ARS.

Multimodal imaging and genetic characteristics of autosomal recessive bestrophinopathy.

PURPOSE: To report the ocular manifestations, multimodal imaging characteristics and genetic testing results of six patients with autosomal recessive bestrophinopathy (ARB). METHODS: This was an observational case series including 12 eyes of 6 patients who were diagnosed with ARB. All patients underwent a complete ophthalmic examination including refraction, slit-lamp biomicroscopy, dilated fundus examination, fundus autofluorescence, optical coherence tomography and electrooculography. BEST1 gene sequencing was also performed for all patients. RESULTS: The mean age was 22.8years and the male-female ratio was 0.50. All ARB patients had a hyperopic refractive error. A spectrum of fundus abnormalities, including multifocal yellowish subretinal deposits in the posterior pole, subfoveal accumulation of vitelliform material and cystoid macular edema, was observed. Fundus autofluorescence imaging demonstrated marked hyperautofluorescence corresponding to the yellowish subretinal deposits. Optical coherence tomography revealed serous retinal detachment, intraretinal cysts, brush border appearance caused by elongation of the outer segments of photoreceptors, and hyperreflective dome-shaped deposits at the level of the retinal pigment epithelium. Fundus fluorescein angiography showed hyperfluorescence with staining of the yellowish subretinal deposits. Electrooculography showed reduced Arden ratio in all patients. In addition, biallelic pathogenic variants in the BEST1 gene were detected in all patients. CONCLUSION: ARB is a rare autosomal recessive inherited retinal disorder with biallelic pathogenic variants in the BEST1 gene and may present with a wide range of ocular abnormalities that may not be easily diagnosed. Multimodal retinal imaging in conjunction with EOG is helpful to establish the correct diagnosis.

[Features of genetic mutations in children with high myopia combined with peripheral retinal degenerations]. / Osobennosti mutatsii v genakh u detei s vysokoi blizorukost'yu, sochetayushcheisya s perifericheskimi vitreokhorioretinal'nymi distrofiyami.

Degenerative changes in the peripheral regions of the ocular fundus allow a closer look at both the role of collagen genes and their mutations in children with high myopia. PURPOSE: The study investigates the features of genetic mutations in children with high myopia combined with peripheral retinal degenerations. MATERIAL AND METHODS: Study group was formed from the database of genetic studies of the Scientific and Clinical Center OOO Oftalmic, which consists of 4362 patients referred for medical genetic counseling and molecular genetic testing from 2016 to 2021. Selection criteria were: male and female patients, aged 5-18 years old, who had the following clinical signs: high myopia (>6.00 D) and the presence of peripheral retinal degenerations (PRD). The study considered both isolated cases of ophthalmic pathology, as well as its syndromic forms. The final selection included 40 children. All patients had consulted with a geneticist. Whole-exome sequencing (WES), next generation sequencing (NGS), and single gene sequencing were conducted by taking 5 mL of peripheral venous blood and extracting deoxyribonucleic acid (DNA). RESULTS: In patients with isolated cases of ophthalmic pathology (peripheral retinal degenerations and high myopia) with a confirmed genetic diagnosis, mutations in the COL2A1 gene were detected in 77.4% of cases, and in the COL11A1 gene - in 22.6% of cases. In Stickler syndrome with a confirmed genetic diagnosis, mutations in the COL2A1 gene were detected in 33.3% of cases. In Marshall syndrome, the mutation in the COL11A1 gene was detected in 11.1% of cases. In children with Ehlers-Danlos, Knobloch type 1, Cohen, Marfan, Wagner syndromes mutations in the genes COL5A1, COL18A1, VPS13B, FBN1, VCAN were detected in 55.6% of cases. In 33.3% of cases of Knobloch type 1, Cohen, Wagner syndromes the mutation is found in both copies of the gene (i.e., in both chromosomes), which leads to the development of peripheral retinal degenerations with high myopia. CONCLUSION: The results of the conducted molecular genetic testing expand our understanding of the mutation spectrum in the genes of children with both isolated cases of ophthalmic pathology, as well as syndromic pathology.

Severe isolated exudative vitreoretinopathy caused by biallelic FZD4 variants.

Familial exudative vitreoretinopathy (FEVR) is linked to disruption of the Norrin/Frizzled-4 signaling pathway, which plays an important role in retinal angiogenesis. Severe or complete knock-down of proteins in the pathway also causes syndromic forms of the condition. Both heterozygous and biallelic pathogenic variants in the FZD4 gene, encoding the pathway's key protein frizzled-4, are known to cause FEVR. However, it is not clear what effect different FZD4 variants have, and whether extraocular features should be expected in those with biallelic pathogenic FZD4 variants. Biallelic FZD4 variants were found in a young boy with isolated, severe FEVR. His parents were heterozygous for one variant each and reported normal vision. In-vitro studies of the two variants, demonstrated that it was the combination of the two which led to severe inhibition of the Norrin/Frizzled-4 pathway. Our observations demonstrate that biallelic FZD4-variants are associated with a severe form of FEVR, which does not necessarily include extraocular features. In addition, variants causing severe FEVR in combination, may have no or minimal effect in heterozygous parents as non-penetrance is also a major feature in dominant FZD4-FEVR disease. This underscores the importance of genetic testing of individuals and families with FEVR.

Deciphering a crucial dimeric interface governing Norrin dimerization and the pathogenesis of familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a hereditary eye disease that could cause blindness. It has been established that Norrin forms dimers to activate ß-catenin signaling, yet the core interface for Norrin dimerization and the precise mechanism by which Norrin dimerization contributes to the pathogenesis of FEVR remain elusive. Here, we report an NDP variant, c.265T>C (p.Phe89Leu), that interrupted ß-catenin signaling by disrupting Norrin dimerization. Structural and functional analysis revealed that the Phe-89 of one Norrin monomer interacts with Pro-98, Ser-101, Arg-121, and Ile-123 of another, forming two core symmetrical dimerization interfaces that are pivotal for the formation of a "hand-by-arm" dimer. Intriguingly, we proved that one of the two core symmetrical interfaces is sufficient for dimerization and activation of ß-catenin signaling, with a substantial contribution from the Phe-89/Pro-98 interaction. Further functional analysis revealed that the disruption of both dimeric interfaces eliminates potential binding sites for LRP5, which could be partially restored by over-expression of TSPAN12. In conclusion, our findings unveil a core dimerization interface that regulates Norrin/LRP5 interaction, highlighting the essential role of Norrin dimerization on ß-catenin signaling and providing potential therapeutic avenues for the treatment of FEVR.

Genetic Characteristics and Clinical Manifestations of Foveal Hypoplasia in Familial Exudative Vitreoretinopathy.

PURPOSE: This study aimed to ascertain the occurrence of foveal hypoplasia (FH) in individuals diagnosed with familial exudative vitreoretinopathy (FEVR). DESIGN: Retrospective cohort study. METHODS: In this study, FEVR families and sporadic cases were diagnosed at the Eye and ENT Hospital, Fudan University, between 2017 and 2023. All patients attended routine ophthalmologic examinations and genetic screenings. The classification of FH was determined using optical coherence tomography (OCT) scans. The FH condition was classified into 2 subgroups: group A (FH being limited to the inner layers) and group B (FH affecting the outer layers). A total of 102 eyes from 58 patients were suitable for analysis. RESULTS: Forty-nine mutations in LRP5, FZD4, NDP, TSPAN12, KIF11, CTNNB1, and ZNF408 were examined and detected, with 26 of them being novel. Forty-seven eyes (46.1%) revealed FH. The majority (53.2%) were due to the typical grade 1 FH. Patients with mutations in LRP5 and KIF11 were found to exhibit a higher prevalence of FH (P = .0088). Group B displayed the lowest visual acuity compared with group A (P = .048) and the group without FH (P < .001). The retinal arteriolar angle in group B was significantly smaller than in group A (P = .001) and those without FH (P < .001). CONCLUSIONS: This study offers a new diagnostic approach and expands the spectrum of FEVR mutations. LRP5 and KIF11 were found to be more susceptible to causing FH in patients with FEVR. FEVR eyes with FH exhibited both greater visual impairment and reduced retinal arteriolar angles. The assessment of foveal status in patients with FEVR should be valued.

Phenotyping and genotyping inherited retinal diseases: Molecular genetics, clinical and imaging features, and therapeutics of macular dystrophies, cone and cone-rod dystrophies, rod-cone dystrophies, Leber congenital amaurosis, and cone dysfunction syndromes.

Inherited retinal diseases (IRD) are a leading cause of blindness in the working age population and in children. The scope of this review is to familiarise clinicians and scientists with the current landscape of molecular genetics, clinical phenotype, retinal imaging and therapeutic prospects/completed trials in IRD. Herein we present in a comprehensive and concise manner: (i) macular dystrophies (Stargardt disease (ABCA4), X-linked retinoschisis (RS1), Best disease (BEST1), PRPH2-associated pattern dystrophy, Sorsby fundus dystrophy (TIMP3), and autosomal dominant drusen (EFEMP1)), (ii) cone and cone-rod dystrophies (GUCA1A, PRPH2, ABCA4, KCNV2 and RPGR), (iii) predominant rod or rod-cone dystrophies (retinitis pigmentosa, enhanced S-Cone syndrome (NR2E3), Bietti crystalline corneoretinal dystrophy (CYP4V2)), (iv) Leber congenital amaurosis/early-onset severe retinal dystrophy (GUCY2D, CEP290, CRB1, RDH12, RPE65, TULP1, AIPL1 and NMNAT1), (v) cone dysfunction syndromes (achromatopsia (CNGA3, CNGB3, PDE6C, PDE6H, GNAT2, ATF6), X-linked cone dysfunction with myopia and dichromacy (Bornholm Eye disease; OPN1LW/OPN1MW array), oligocone trichromacy, and blue-cone monochromatism (OPN1LW/OPN1MW array)). Whilst we use the aforementioned classical phenotypic groupings, a key feature of IRD is that it is characterised by tremendous heterogeneity and variable expressivity, with several of the above genes associated with a range of phenotypes.

Complex balanced intrachromosomal rearrangement involving PITX2 identified as a cause of Axenfeld-Rieger Syndrome.

Axenfeld-Rieger Syndrome (ARS) type 1 is a rare autosomal dominant condition characterized by anterior chamber anomalies, umbilical defects, dental hypoplasia, and craniofacial anomalies, with Meckel's diverticulum in some individuals. Here, we describe a clinically ascertained female of childbearing age with ARS for whom clinical targeted sequencing and deletion/duplication analysis followed by clinical exome and genome sequencing resulted in no pathogenic variants or variants of unknown significance in PITX2 or FOXC1. Advanced bioinformatic analysis of the genome data identified a complex, balanced rearrangement disrupting PITX2. This case is the first reported intrachromosomal rearrangement leading to ARS, illustrating that for patients with compelling clinical phenotypes but negative genomic testing, additional bioinformatic analysis are essential to identify subtle genomic abnormalities in target genes.

Canine and Feline Models of Inherited Retinal Diseases.

Naturally occurring inherited retinal diseases (IRDs) in cats and dogs provide a rich source of potential models for human IRDs. In many cases, the phenotypes between the species with mutations of the homologous genes are very similar. Both cats and dogs have a high-acuity retinal region, the area centralis, an equivalent to the human macula, with tightly packed photoreceptors and higher cone density. This and the similarity in globe size to that of humans means these large animal models provide information not obtainable from rodent models. The established cat and dog models include those for Leber congenital amaurosis, retinitis pigmentosa (including recessive, dominant, and X-linked forms), achromatopsia, Best disease, congenital stationary night blindness and other synaptic dysfunctions, RDH5-associated retinopathy, and Stargardt disease. Several of these models have proven to be important in the development of translational therapies such as gene-augmentation therapies. Advances have been made in editing the canine genome, which necessitated overcoming challenges presented by the specifics of canine reproduction. Feline genome editing presents fewer challenges. We can anticipate the generation of specific cat and dog IRD models by genome editing in the future.

Exome sequencing-aided precise diagnosis of four families with type I Stickler syndrome.

BACKGROUND: Stickler syndrome is a multisystemic disorder characterized by ophthalmological and non-ophthalmological abnormalities, frequently misdiagnosed due to high clinical heterogeneity. Stickler syndrome type I (STL1) is predominantly caused by mutations in the COL2A1 gene. METHODS: Exome sequencing and co-segregation analysis were utilized to scrutinize 35 families with high myopia, and pathogenic mutations were identified. Mutant COL2A1 was overexpressed in cells for mechanistic study. A retrospective genotype-phenotype correlation analysis was further conducted. RESULTS: Two novel pathogenic mutations (c.2895+1G>C and c.3505G>A (p.Val1169Ile)) and two reported mutations (c.1597C>T (p.Arg533*) and c.1693C>T (p.Arg565Cys)) in COL2A1 were identified causing STL1. These mutations are all in the G-X-Y triplet, and c.2895+1G>C contributed to aberrant RNA splicing. COL2A1 mutants tended to form large aggregates in the endoplasmic reticulum (ER) and elevated ER stress. Additionally, mutations c.550G>A (p.Ala184Thr) and c.2806G>A (p.Gly936Ser) in COL2A1 were found in high myopia families, but were likely benign, although c.2806G>A (p.Gly936Ser) is on G-X-Y triplet. Moreover, genotype-phenotype correlation analysis revealed that mutations in exon 2 mainly contribute to retinal detachment, whereas mutations in the collagen alpha-1 chain region of COL2A1 tend to cause non-ophthalmologic symptoms. CONCLUSION: This study broadens the COL2A1 gene mutation spectrum, provides evidence for ER stress caused by pathogenic COL2A1 mutations and highlights the importance of non-ophthalmological examination in clinical diagnosis of high myopia.

Use of vitreous phenotype as a key clinical marker to identify Ocular-only Stickler syndrome in a family with Marfan syndrome.

This clinical report describes a family with both Marfan and ocular-only Stickler syndromes. We report 2 cases of ocular-only Stickler syndrome and 2 cases of Marfan syndrome concurrent with ocular-only Stickler syndrome. Type 1 Stickler syndrome and Marfan syndrome share many clinical similarities, and it can be difficult to differentiate them solely based on clinical presentation. Vitreous phenotyping allows for the identification of vitreous anomalies pathognomonic of Stickler syndrome, which can guide future gene sequencing. Having the accurate diagnosis of Marfan or type 1 Stickler syndrome is important, as patients with type 1 Stickler syndrome have higher rates of retinal detachment and will benefit from prophylaxis.

HIGH MYOPIA IS COMMON IN PATIENTS WITH X-LINKED RETINOPATHIES: Myopic Maculopathy Analysis.

PURPOSE: High myopia can occur as a single or syndromic condition. The aim of this study was to evaluate the refractive error and myopic maculopathy in patients with X-linked retinopathies. METHODS: Whole exome sequencing, Sanger sequencing, and comprehensive ocular examinations were performed in patients with X-linked retinopathies. RESULTS: A total of 17 patients were recruited, including six with CACNA1F, seven with RPGR, three with NYX, and one with OPN1MW mutations. The diagnoses were congenital stationary night blindness (6), cone-rod dystrophy (4), retinitis pigmentosa (4), achromatopsia (1), Leber congenital amaurosis (1), and myopia (1). Myopia was present in 88.2% patients, and 64.7% patients had high myopia. Gene analysis showed that high myopia was present in 80% patients with CACNA1F, 100% patients with NYX, and 57.1% patients with RPGR mutations. In the ATN classification, 64.7% of the patients were A1T0N0 and 35.3% were A0T0N0. The refractive errors progressed over time, even in patients with congenital stationary night blindness. Two females with heterozygous de novo RPGR mutations presented with retinitis pigmentosa or cone rod dystrophy combined with high myopia. CONCLUSION: High myopia is common in patients with X-linked retinopathies, and myopic maculopathy was only mild atrophy without traction and neovascularization.

[The role of endothelin-1 in the pathogenesis of familial exudative vitreoretinopathy]. / Rol' endotelina-1 v patogeneze semeinoi ekssudativnoi vitreoretinopatii.

Familial exudative vitreoretinopathy (FEVR) is a rare hereditary disease characterized by pathological retinal vascularization with a progressive and variable course. The mechanisms of disease progression remain unclear. One substance that plays an important role in the pathogenesis of retinal vascular diseases is endothelin (ET). It was found that tissue hypoxia enhances the expression of the gene encoding ET-1, and ET-1 can be locally produced in the eye. PURPOSE: The study evaluates the possible role of endothelin-1 in the pathogenesis of FEVR. MATERIAL AND METHODS: The study included 85 patients with FEVR aged from 1 months to 17 years who were examined in Helmholtz National Medical Research Center of Eye Diseases. The concentration of ET-1 was evaluated in 19 patients with FEVR in the blood serum (n=17), lacrimal fluid (n=18) and 16 patients from the control group. RESULTS: The median of ET-1 in the lacrimal fluid in patients with FEVR was 13.74 pg/mL, respectively, which exceeded the same indicator of the control group 4.66 pg/mL by 2.5 times (p<0.001). The median of ET-1 in the blood serum exceeded the control group by 2.4 times (21.61 pg/mL and 9.21 pg/mL, respectively, p<0.001). CONCLUSIONS: An increase in the concentration of ET-1 in the lacrimal fluid and blood serum of patients with FEVR in comparison with the control group indicates its involvement in the pathogenesis of the disease.

Genetic detection of two novel LRP5 pathogenic variants in patients with familial exudative vitreoretinopathy.

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a genetic eye disorder that leads to abnormal development of retinal blood vessels, resulting in vision impairment. This study aims to identify pathogenic variants by targeted exome sequencing in 9 independent pedigrees with FEVR and characterize the novel pathogenic variants by molecular dynamics simulation. METHODS: Clinical data were collected from 9 families with FEVR. The causative genes were screened by targeted next-generation sequencing (TGS) and verified by Sanger sequencing. In silico analyses (SIFT, Polyphen2, Revel, MutationTaster, and GERP + +) were carried out to evaluate the pathogenicity of the variants. Molecular dynamics was simulated to predict protein conformation and flexibility transformation alterations on pathogenesis. Furthermore, molecular docking techniques were employed to explore the interactions and binding properties between LRP5 and DKK1 proteins relevant to the disease. RESULTS: A 44% overall detection rate was achieved with four variants including c.4289delC: p.Pro1431Argfs*8, c.2073G > T: p.Trp691Cys, c.1801G > A: p.Gly601Arg in LRP5 and c.633 T > A: p.Tyr211* in TSPAN12 in 4 unrelated probands. Based on in silico analysis and ACMG standard, two of them, c.4289delC: p.Pro1431Argfs*8 and c.2073G > T: p.Trp691Cys of LRP5 were identified as novel pathogenic variants. Based on computational predictions using molecular dynamics simulations and molecular docking, there are indications that these two variants might lead to alterations in the secondary structure and spatial conformation of the protein, potentially impacting its rigidity and flexibility. Furthermore, these pathogenic variants are speculated to potentially influence hydrogen bonding interactions and could result in an increased binding affinity with the DKK1 protein. CONCLUSIONS: Two novel genetic variants of the LRP5 gene were identified, expanding the range of mutations associated with FEVR. Through molecular dynamics simulations and molecular docking, the potential impact of these variants on protein structure and their interactions with the DKK1 protein has been explored. These findings provide further support for the involvement of these variants in the pathogenesis of the disease.

Comprehensive Genetic Analysis Unraveled the Missing Heritability and a Founder Variant of BEST1 in a Chinese Cohort With Autosomal Recessive Bestrophinopathy.

Purpose: To describe the genetic landscape of BEST1 for a large Chinese cohort with autosomal recessive bestrophinopathy (ARB), identify the missing heritability, and report a common Chinese founder variant. Methods: We recruited 65 patients from 63 families with a clinical diagnosis of ARB. All patients underwent ophthalmic examinations and comprehensive genetic analyses, including Sanger DNA sequencing of BEST1 and whole genome sequencing (WGS). The effects of deep intronic variants (DIVs) on splicing were assessed using in vitro splicing assays in HEK293T cells and patient-derived peripheral blood mononuclear cells. Haplotype mapping was performed for 17 unrelated patients harboring variant c.867+97G>A. Results: We identified 54 distinct disease-causing variants of BEST1 in 63 pedigrees, 62 probands with biallelic variants, and one family with monoallelic variants. Sanger DNA sequencing of BEST1 initially detected 51 variants in 61 pedigrees, including 19 probands with one heterozygous variant. Subsequent WGS, combined with supplementary Sanger sequencing, revealed three missing DIVs (c.1101-491A>G, c.867+97G>A, and c.867+97G>T) in 20 families. The novel DIV c.1101-491A>G caused an abnormal splicing resulting in a 204-nt pseudoexon (PE) insertion, whereas c.867+97G>A/T relatively strengthened an alternative donor site, resulting in a 203-nt intron retention (IR). The PE and IR generated a premature termination codon downstream. Haplotype analysis identified c.867+97G>A as a common founder variant with an allele frequency of 16%. Conclusions: Our results expand the pathogenic variant spectrum of BEST1, and DIVs can explain almost all of the missing heritability. The c.867+97G>A DIV is a common founder variant for Chinese patients with ARB.

LONG-TERM CLINICAL OUTCOMES AND GENOTYPE-PHENOTYPE CORRELATION IN FAMILIAL EXUDATIVE VITREORETINOPATHY IN A TERTIARY REFERRAL CENTER.

BACKGROUND/PURPOSE: To evaluate clinical outcomes and assess genotype-phenotype correlations in patients with familial exudative vitreoretinopathy (FEVR). METHODS: Clinical charts of 40 patients with FEVR were reviewed. FEVR was staged per Pendergast and Trese, and retinal dragging and folds further classified per Yaguchi et al. We performed whole-exome sequencing and compared clinical characteristics between genetic-positive and genetic-negative groups. RESULTS: The mean duration of follow-up was 5.4 years (range: 0.33, 15) for genetic-positive and 6.9 (range: 1, 20) for genetic-negative patients. The mean age at diagnosis was 5.6 years (0.25, 27) for genetic-positive and 6.0 (0, 32) for genetic-negative patients. Genetic-positive patients reported 100% full-term births and genetic-negative patients reported 45% full-term births ( P = 0.0012). There were more patients with retinal folds with all major vessels affected (Yaguchi's Group 4) in genetic-positive compared with genetic-negative patients (21.4% vs. 2.6%, P = 0.045). TSPAN12 was the most common (57.1%) genetic mutation in our population of which 50% exhibited asymmetric presentation. CONCLUSION: Patients who test positive for a typical FEVR gene mutation reported more term births and had more severe disease by Yaguchi's classification. TSPAN12 was the most common genetic mutation in our population and had highly asymmetrical disease.