Long-read sequencing uncovers novel pathogenic duplications in the PRPH2 gene in patients with macular dystrophy

Purpose: Detailed genetic characterization of three individuals from two families with macular dystrophy caused by exon 2 duplication of the PRPH2 gene.Methods: Two of the affected members (P1, P2) from one family were treated in the Helsinki University Hospital, Finland and one (P3) at Loma Linda University Eye Institute, CA, USA. The patients underwent comprehensive ophthalmic examination, and their genomic DNA was subjected to macular (P2) and retinal dystrophy (P3) gene panels or targeted genetic testing (P1). Additionally, Oxford nanopore adaptive sequencing was used for characterizing and mapping the breakpoints of the identified partial duplication of the PRPH2 gene.Results: Based on the ophthalmic examination, the mother (P1) of the Finnish family was diagnosed with butterfly‐shaped pattern dystrophy (BPD) and the daughter (P2) with vitelliform macular dystrophy. P3 (male) from the USA was diagnosed with BPD in the right eye and central areolar choroidal dystrophy in the left eye. Exome‐based genetic testing indicated that all patients are heterozygous for the same 482 bp duplication c.(581+1_582‐1)_(828+1_829‐1)dup between introns 1 and 2 of the PRPH2 gene, duplicating exon 2 (NM_000322.5). In‐depth analysis of the patients DNA samples by long‐read adaptive nanopore sequencing revealed the duplication to be ∼4kb in size. For all patients the 3’ breakpoint (BP) is located directly upstream from an intronic alu‐element (AluYM1). In the Finnish family the 5’BP resides in a novel alu‐element (AluYa5) lacking from the hg38 reference genome. The P3 lacks the novel‐alu element found in the Finnish family and instead the 5’BP resides in a known alu‐element (AluSq), located some 300bp upstream.Conclusions: We report here that the macular dystrophy in the two families is caused by duplication involving exon 2 of the PRPH2 gene. The alu‐elements at the breakpoints indicate non‐allelic homologous recombination as a possible mechanism for the duplication event.

Purpose: Detailed genetic characterization of three individuals from two families with macular dystrophy caused by exon 2 duplication of the PRPH2 gene.Methods: Two of the affected members (P1, P2) from one family were treated in the Helsinki University Hospital, Finland and one (P3) at Loma Linda University Eye Institute, CA, USA. The patients underwent comprehensive ophthalmic examination, and their genomic DNA was subjected to macular (P2) and retinal dystrophy (P3) gene panels or targeted genetic testing (P1). Additionally, Oxford nanopore adaptive sequencing was used for characterizing and mapping the breakpoints of the identified partial duplication of the PRPH2 gene.Results: Based on the ophthalmic examination, the mother (P1) of the Finnish family was diagnosed with butterfly‐shaped pattern dystrophy (BPD) and the daughter (P2) with vitelliform macular dystrophy. P3 (male) from the USA was diagnosed with BPD in the right eye and central areolar choroidal dystrophy in the left eye. Exome‐based genetic testing indicated that all patients are heterozygous for the same 482 bp duplication c.(581+1_582‐1)_(828+1_829‐1)dup between introns 1 and 2 of the PRPH2 gene, duplicating exon 2 (NM_000322.5). In‐depth analysis of the patients DNA samples by long‐read adaptive nanopore sequencing revealed the duplication to be ∼4kb in size. For all patients the 3’ breakpoint (BP) is located directly upstream from an intronic alu‐element (AluYM1). In the Finnish family the 5’BP resides in a novel alu‐element (AluYa5) lacking from the hg38 reference genome. The P3 lacks the novel‐alu element found in the Finnish family and instead the 5’BP resides in a known alu‐element (AluSq), located some 300bp upstream.Conclusions: We report here that the macular dystrophy in the two families is caused by duplication involving exon 2 of the PRPH2 gene. The alu‐elements at the breakpoints indicate non‐allelic homologous recombination as a possible mechanism for the duplication event.

Systematic Screening of BEST1 and PRPH2 in Juvenile and Adult Vitelliform Macular Dystrophies: A Rationale for Molecular Analysis

Frequency and Clinical Pattern of Vitelliform Macular Dystrophy Caused by Mutations of Interphotoreceptor Matrix IMPG1 and IMPG2 Genes

Vitelline macular dystrophy (also known as Best disease) is a progressive, chronic disease of the macula (central retina) at the back of the eye. Best disease is characterised by the development of a lesion in the retinal pigment epithelium and emerging as one of the leading causes of blindness in both juvenile and adult cases. This disease has an autosomal dominant pattern of inheritance with varied penetrance and expressivity. Diagnosis of this disease is delayed due to the fact that the individual may be asymptomatic for several years and the disease can be identified only after visual acuity reduces, during which the disease could have progressed to later stages. Mutations observed in the BEST1 and PRPH2 genes are involved in development of the disease. In this article, a review of the history, genetics and pathophysiology, diagnosis and treatment is outlined for further understanding of this disease. Key Concepts: Macular dystrophy is a rare, genetic condition of the eye, wherein the macula, which is the central part of the retina, is damaged due to successive buildup of eye pigments within the epithelial cells of the retina. There are two main types of this disease: Best disease, which is seen in juvenile cases, and adult‐onset macular dystrophy. Best disease is also known as early‐onset or juvenile vitelliform macular dystrophy. It is an autosomal dominant disorder characterised by the accumulation of specific eye pigment material in the subretinal space, which leads to the formation of a lesion on the macula. Adult‐onset vitelliform macular dystrophy is a disease of the retina wherein central vision loss occurs in the fourth or fifth decade of life. It is an autosomal dominant disorder. It is a dystrophy of the retinal pigment epithelium causing lesions on the macula. VMD2 gene, also known as the BEST1 gene, codes for a protein called bestrophin. This protein functions as a chloride ion channel in the eye and its dysfunction leads to buildup of pigments and eventual loss of central vision. PRPH2 gene codes for a protein called peripherin. This protein is essential for light sensing in the retina. Mutations in this gene lead to central vision loss.

Tuberous sclerosis complex (TSC) is a relatively common autosomal dominant disorder characterized by multiple dysplastic organ lesions and neuropsychiatric symptoms caused by loss-of-function mutation of either TSC1 or TSC2. The genetic diagnosis of inherited diseases, including TSC, in the clinical field is widespread using next-generation sequencing. The mutations in protein-coding exon tend to be verified because mutations directly cause abnormal protein. However, it is relatively difficult to verify mutations in the intron region because it is required to investigate whether the intron mutations affect the abnormal splicing of transcripts. In this study, we developed a target-capture full-length double-stranded cDNA sequencing method using Nanopore long-read sequencer (Nanopore long-read target sequencing). This method revealed the occurrence of intron mutation in the TSC2 gene and found that the intron mutation produces novel intron retention splicing transcripts that generate truncated proteins. The protein-coding transcripts were decreased due to the expression of the novel intron retention transcripts, which caused TSC in patients with the intron mutation. Our results indicate that Nanopore long-read target sequencing is useful for the detection of mutations and confers information on the full-length alternative splicing of transcripts for genetic diagnosis.

Objective To observe the autofluorescence (AF) manifestation in children with hereditary retinal diseases. Methods The clinical data of 22 children (aged from 5 to 14 years) with hereditary retinal diseases were retrospectively analyzed. There were 8 children (16 eyes) with Best vitelliform macular dystrophy, 3 children (6 eyes) with Stargardt macular dystrophy, 3 children (6 eyes) with macular cone dystrophy, 5 children (10 eyes) with primary retinitis pigmentosa, and 3 children (6 eyes) with X-linked juvenile retinoschisis. The routine clinical examinations included present history, family history, visual acuity, silt-lamp microscopy, indirect ophthalmoscopy, color fundus photography and fundus autofluorescence angiography (FAF). Some patients received fundus fluorescein angiography (FFA),electroretinogram (ERG), electrooculogram (EOG), and ocular coherence tomography (OCT). The characteristics of AF in all the children were analyzed, and were compared with the images of color fundus and/or FFA. Results Symmetry round macular fluorescent weak or absent area was found in all Stargardt disease and cone dystrophy. Weak AF area with surrounded circular increased AF was found in 2 children (4 eyes) with cone dystrophy and 1 child (2 eyes) with Stargardt macular dystrophy. A central round area with regular or irregular intense AF was observed in Best vitelliform macular dystrophy. RP children showed increased AF out of the macular region. Cellular or granular strong AF was found in the fovea of 3 children (5 eyes) with X-linked juvenile retinoschisis. Conclusion The children with hereditary retinal diseases had special AF changes. Key words: Retinal diseases/congenital; Eye diseases, hereditary; Fluorescence; Fluorescein angiography

To assess the occurrence of PRPH2 mutations in patients presenting macular dystrophies and to describe their phenotype-genotype correlation. A total of 32 sporadic cases and 13 individuals from 5 families were studied. The patients presented early onset drusen, suspected pattern dystrophy (including adult-onset foveomacular vitelliform dystrophy [AOFVD]), or any presumed macular dystrophy producing neovascularization or atrophic changes documented before patients reached 50 years of age. In case of atrophy, this could be confined to the macula, which was considered to be central areolar choroidal dystrophy (CACD), or extend to the midperiphery of the retina, which we called diffuse macular dystrophy (DMD). Clinical workup and analysis of PRPH2, EFEMP1, and TIMP3 genes were done. Four mutations of the PRPH2 gene were found in 3 sporadic cases and 3 families (n = 11). A p.R46X mutation, previously described in CACD, was found in 3 members of a family with AOFVD and in a sporadic case with DMD. A p.L45F mutation, described before in retinitis pigmentosa, was found in a sporadic case of AOFVD. A p.R195L mutation previously described in CACD was found in 2 members of a family with CACD. The latter was found in a family and a sporadic case (from the same village as the family) and all of them presented DMD. A new p.V2091 mutation was found in a patient with AOFVD. New phenotypes were found for known mutations. No phenotype variation was observed in the members of the 3 families. A new mutation in PRPH2 gene was found.

Germline Loss-of-Function Mutations in MDM4 Cause p53-Dependent Hematopoietic Cell Death in Patients with Variable Bone Marrow Failure Phenotypes

Screening for splice site mutation c.828+3A>T in the peripherin 2 (PRPH2) gene should be a high priority in families with highly variable retinal dystrophies. The correction of missplicing is a potential therapeutic target. To determine the prevalence, genetic origin, and molecular mechanism of a donor c.828+3A>T mutation in the PRPH2 (peripherin 2, retinal degeneration slow) gene in individuals with retinal dystrophies. Case-control study that took place at the University of Texas Health Science Center, the University of Iowa, and the Retina Foundation of the Southwest, from January 1, 1987, to August 1, 2014, including affected individuals from 200 families with a diagnosis of autosomal dominant retinitis pigmentosa, 35 families with unspecified macular dystrophies, and 116 families with pattern dystrophy. Participants were screened for the c.828+3A>T mutation by restriction-enzyme digest, single-strand conformational polymorphism screening, or bidirectional sequencing. Haplotypes of polymorphic markers flanking the PRPH2 locus and sequence variants within the gene were determined by denaturing gel electrophoresis or automated capillary-based cycle sequencing. The effect of the splice site mutation on the PRPH2 transcript was analyzed using NetGene2, a splice prediction program and by the reverse transcription polymerase chain reaction of illegitimate transcripts from peripheral white blood cells. Results of testing for splice site mutation, haplotypes, and alternate transcripts. The PRPH2 mutation was found in 97 individuals of 19 independently ascertained families with a clinical diagnosis of retinitis pigmentosa, macular dystrophy, and/or pattern dystrophy. All affected individuals also shared a rare haplotype of approximately 644 kilobase pairs containing the c.828+3A>T mutation, which extends from the short tandem repeat polymorphism D6S282 to c.1013G>A (rs434102, a single-nucleotide polymorphism) in exon 3 of PRPH2, suggesting this mutation is from a common ancestor and is a founder mutation. It has a prevalence of 2% in families diagnosed as having autosomal dominant retinitis pigmentosa and 10% in families with variable clinical diagnosis of pattern, macular, and retinal dystrophies. Individuals with the c.828+3A>T mutation expressed a PRPH2 transcript not found in control participants and that was consistent with abnormal splicing. The PRPH2 c.828+3A>T splice site mutation is a frequent cause of inherited retinal dystrophies and is owing to the founder effect. The likely cause of disease is the missplicing of the PRPH2 message that results in a truncated protein product. Identifying the genetic etiology assists in more accurate management and possible future therapeutic options.

16S rRNA long-read nanopore sequencing is feasible and reliable for endometrial microbiome analysis

Vitelliform macular dystrophy (VMD) is a group of macular dystrophy characterized by the subretinal accumulation of yellow yolk-like materials which predominantly affect the macula. Best vitelliform macular dystrophy is among the most common autosomal dominant (AD) retinal dystrophy, caused by mutations in the BEST1 gene. Since first identification of BEST1 gene in 1998, molecular biology and pathophysiology of BEST1 gene and vitelliform macular dystrophy were studied. Recent advances in genetic analysis have described over 200 different human BEST1 mutations to date, associated with a broad spectrum of ocular diseases, called bestrophinopathy. However, the genotype-phenotype correlation in VMD is largely unexplored. Genetic test is clinically important in the diagnosis of VMD because the clinical features of VMD are similar to those of exudative age-related macular degeneration (AMD), choroidal neovascularization (CNV), or central serous chorioretinopathy (CSC). Here, in addition to describing the clinical characteristics of VMD, this chapter focuses on the clinical genetics of BEST1 gene in VMD.

Herein, we report the clinical cases of two affected first-degree relatives from a family with highly variable macular dystrophy, expanding the known phenotype spectrum with mutations in the thyroid hormone receptor beta gene (THRB). Multimodal retinal imaging included wide-field fundus photography, fundus autofluorescence (FAF), spectral domain optical coherence tomography (SD-OCT) imaging, performed alongside functional testing (visual fields, electroretinogram (ERG)), metabolic blood analyses, and genetic testing of both cases. A 67-year-old female patient presenting with reading difficulties and visual impairment since childhood was referred for evaluation and counseling for potential treatment options. Extensive ophthalmologic examination, including multimodal retinal imaging and functional testing, revealed an occult macular dystrophy. Her 39-year-old son reported similar visual symptoms in combination with mild photophobia. In multimodal retinal imaging, he also showed a macular dystrophy but with a vitelliform phenotype. Genetic testing identified the heterozygous pathogenic variant c.283+1G>A in the thyroid hormone receptor beta gene (THRB) in both patients. This report shows a high intrafamilial variability of macular dystrophy caused by a heterozygous THRB mutation, which has only recently been recognized as a cause of macular dystrophy. Here, we describe a novel clinical presentation characterized by a vitelliform lesion, expanding the phenotypic spectrum of THRB-associated macular dystrophy.

ABSTRACTBackground/Objectives: To reveal the underlying genetic defect in a complex family affected with different clinical features of inherited retinal dystrophy, we carried out whole exome sequencing followed by confirmatory molecular tests.Materials and Methods: Complete ophthalmic examinations were performed for available affected family members. Whole exome sequencing, bioinformatics analysis, Sanger sequencing confirmation, and segregation analysis were done to identify the causative mutation.Results: Clinical findings suggested fundus flavimaculatus as an early clinical feature progressing to an extensive chorioretinal atrophy involving the macula and mid-periphery of the fundus in one parent and central areolar chorioretinal dystrophy (CACD) as the most probable clinical diagnosis in another parent. Macular pattern dystrophy for one of their daughters and a Leber congenital amaurosis (LCA) like phenotype for the daughter with an early onset retinal dystrophy (EORD) phenotype was suggested. We found a known pathogenic nonsense variation in the PRPH2 gene (NM_000322: p.Gln239Ter). The parents with end stage fundus flavimaculatus and CACD diagnosis and their daughter with macular pattern dystrophy were heterozygous for the identified variant. The daughter affected with EORD/LCA like retinal dystrophy was homozygous for the same variation.Conclusions: In this family, the same pathogenic variant in PRPH2 gene showed a wide range of clinical features of extensive chorioretinal macular atrophy with flecks as fundus falvimaculatus to CACD and macular pattern dystrophy in the heterozygous inheritance pattern and early onset/LCA like retinal dystrophy in the patient who was homozygous for the causative variant.

BackgroundOccult Macular Dystrophy (OMD), primarily caused by retinitis pigmentosa 1-like 1 (RP1L1) variants, is a complex retinal disease characterised by progressive vision loss and a normal fundus appearance. This study aims to investigate the diverse phenotypic expressions and genotypic correlations of OMD in Chinese patients, including a rare case of Vitelliform Macular Dystrophy (VMD) associated with RP1L1.MethodsWe analysed seven OMD patients and one VMD patient, all with heterozygous pathogenic RP1L1 variants. Clinical assessments included Best Corrected Visual Acuity (BCVA), visual field testing, Spectral Domain Optical Coherence Tomography (SD-OCT), multifocal Electroretinograms (mfERGs), and microperimetry. Next-generation sequencing was utilised for genetic analysis.ResultsThe OMD patients displayed a range of phenotypic variability. Most (5 out of 7) had the RP1L1 variant c.133 C > T; p.R45W, associated with central vision loss and specific patterns in SD-OCT and mfERG. Two patients exhibited different RP1L1 variants (c.3599G > T; p.G1200V and c.2880G > C; p.W960C), presenting milder phenotypes. SD-OCT revealed photoreceptor layer changes, with most patients showing decreased mfERG responses in the central rings. Interestingly, a unique case of VMD linked to the RP1L1 variant was observed, distinct from traditional OMD presentations.ConclusionsThis study highlights the phenotypic diversity within OMD and the broader spectrum of RP1L1-associated macular dystrophies, including a novel association with VMD. The findings emphasise the complexity of RP1L1 variants in determining clinical manifestations, underscoring the need for comprehensive genetic and clinical evaluations in macular dystrophies.