Coexistence of Retinitis Pigmentosa and Ataxia in Patients with PHARC, PCARP, and Oliver-McFarlane Syndromes.

Retinitis pigmentosa (RP) is an inherited retinal dystrophy caused by the loss of photoreceptors and retinal pigment epithelial atrophy, leading to severe visual impairment or blindness. RP can be classified as nonsyndromic or syndromic with complex clinical phenotypes. Three unrelated Polish probands affected with retinitis pigmentosa coexisting with cerebellar ataxia were recruited for this study. Clinical heterogeneity and delayed appearance of typical disease symptoms significantly prolonged the patients' diagnostic process. Therefore, many clinical and genetic tests have been performed in the past. Here, we provide detailed clinical and genetic analysis results of the patients. Whole-exome sequencing (WES) and targeted NGS analysis allow the identification of four novel and two previously reported variants in the following genes: ABHD12, FLVCR1, and PNPLA6. The use of next-generation sequencing (NGS) methods finally allowed for confirmation of the clinical diagnosis. Ultra-rare diseases such as PHARC, PCARP, and Oliver-McFarlane syndromes were diagnosed in patients, respectively. Our findings confirmed the importance of the application of next-generation sequencing methods, especially in ultra-rare genetic disorders with overlapping features.

DNAJC30 Gene Variants Are a Frequent Cause of a Rare Disease: Leber Hereditary Optic Neuropathy in Polish Patients.

Leber hereditary optic neuropathy (LHON) is a rare disorder causing a sudden painless loss of visual acuity in one or both eyes, affecting young males in their second to third decade of life. The molecular background of the LHON is up to 90%, genetically defined by a point mutation in mitochondrial DNA. Recently, an autosomal recessive form of LHON (LHONAR1, arLHON) has been discovered, caused by biallelic variants in the DNAJC30 gene. This study provides the results of the DNAJC30 gene analysis in a large group of 46 Polish patients diagnosed with LHON, together with the clinical characterization of the disease. The c.152A>G (p.Tyr51Cys) substitution in the DNAJC30 gene was detected in all the patients as homozygote or compound heterozygote. Moreover, we identified one novel variant, c.293A>G, p.(Tyr98Cys), as well as two ultra-rare DNAJC30 variants: c.293A>C, p.(Tyr98Ser), identified to date only in one individual affected with LHONAR1, and c.130_131delTC (p.Ser44ValfsTer8), previously described only in two patients with Leigh syndrome. The patients presented here represent the largest group of subjects with DNAJC30 gene mutations described to date. Based on our data, the autosomal recessive form of LHON caused by DNAJC30 gene mutations is more frequent than the mitochondrial form in Polish patients. The results of our study suggest that Sanger sequencing of the single-exon DNAJC30 gene should be a method of choice applied to identify a molecular background of clinically confirmed LHON in Polish patients. This approach will help to reduce the costs of molecular testing.

Molecular Re-Diagnosis with Whole-Exome Sequencing Increases the Diagnostic Yield in Patients with Non-Syndromic Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of disorders with progressive loss of photoreceptor and pigment epithelial function. Nineteen unrelated Polish probands clinically diagnosed with nonsyndromic RP were recruited to this study. We used whole-exome sequencing (WES) to identify potential pathogenic gene variants in molecularly undiagnosed RP patients, as a molecular re-diagnosis after having performed targeted NGS in the past. Targeted NGS allowed for identification of the molecular background in only 5 out of 19 patients. Fourteen patients who remained unsolved despite the targeted NGS were subjected to WES. WES revealed potentially causative variants in RP-related genes in another 12 patients. Together, NGS methods revealed the coexistence of causal variants affecting distinct RP genes in 17 out of 19 RP families, with a very high efficiency of 89%. With the improvement of NGS methods, including higher sequencing depth, broader target enrichment, and better bioinformatic analysis capabilities, the ratio of identified causal gene variants has significantly increased. Therefore, it is important to consider repeating high-throughput sequencing analysis in those patients in whom the previously performed NGS did not reveal any pathogenic variants. The study confirmed the efficiency and clinical utility of re-diagnosis with WES in molecularly undiagnosed RP patients.

Molecular background of Leber congenital amaurosis in a Polish cohort of patients-novel variants discovered by NGS.

Leber congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophies and the most frequent cause of congenital blindness in children. To date, 25 genes have been implicated in the pathogenesis of this rare disorder. Performing an accurate molecular diagnosis is crucial as gene therapy is becoming available. This study aimed to report the molecular basis of Leber congenital amaurosis, especially novel and rare variants in 27 Polish families with a clinical diagnosis of LCA fully confirmed by molecular analyses. Whole exome sequencing or targeted next-generation sequencing (NGS) of inherited retinal dystrophies-associated (IRD) genes was applied to identify potentially pathogenic variants. Bidirectional Sanger sequencing and quantitative PCR (qPCR) were carried out for validation and segregation analysis of the variants identified within the families. We identified 28 potentially pathogenic variants, including 11 novel, in 8 LCA genes: CEP290, CRB1, GUCY2D, NMNAT1, RPGRIP1, CRX, LRAT1, and LCA5. This study expands the mutational spectrum of the LCA genes. Moreover, these results, together with the conclusions from our previous studies, allow us to point to the most frequently mutated genes and variants in the Polish cohort of LCA patients.

The coincidence of two ultra-rare hereditary eye diseases: gyrate atrophy and Kjer optic atrophy - a surprising diagnosis based on next-generation sequencing.

Genetically determined ophthalmic diseases form a numerous and heterogenic group of disorders. Making the accurate clinical diagnosis of genetic eye disease is often a challenge for an ophthalmologist. In many cases, only genetic testing enables the establishment of the proper clinical diagnosis. Here we describe two ultra-rare diseases: gyrate atrophy of the choroid and retina (GACR) and Kjer-type optic atrophy coexisting in a 39-year-old Polish patient with severe visual impairment including a significant reduction of visual acuity and night blindness. Atrophic pigmented changes with large pigment deposits and chorioretinal atrophy with the retina's disturbed structure (with atrophic scarring changes and the epiretinal membrane) of both eyes were observed. Electroretinography (ERG) revealed extinguished responses. A Next-Generation Sequencing (NGS) panel comprising 275 retinal genes revealed a presence of potentially pathogenic variants in two genes: a homozygous variant c.1058G>A (p.Gly353Asp) in the OAT gene and a heterozygous variant c.1886C>G (p.Ser629Ter) in the OPA1 gene. The diagnosis established based on NGS is surprising because initially, several different diagnoses have been made, including high degenerative myopia, choroideremia, Leber congenital amaurosis, and severe, atypical retinitis pigmentosa. This report provides the unquestioned diagnostic value of the combination of chorioretinal imaging and the NGS technique. To our knowledge, this is the first and the only description of the coincidence of gyrate atrophy and Kjer-type optic atrophy.

Novel gene variants in Polish patients with Leber congenital amaurosis (LCA).

BACKGROUND: Leber congenital amaurosis (LCA) is a rare retinal disease that is the most frequent cause of congenital blindness in children and the most severe form of inherited retinal dystrophies. To date, 25 genes have been implicated in the pathogenesis of LCA. As gene therapy is becoming available, the identification of potential treatment candidates is crucial. The aim of the study was to report the molecular basis of Leber congenital amaurosis in 22 Polish families. METHODS: Single Nucleotide Polymorphism-microarray for LCA genes or Next Generation Sequencing diagnostic panel for LCA genes (or both tests) were performed to identify potentially pathogenic variants. Bidirectional Sanger sequencing was carried out for validation and segregation analysis of the variants identified within the families. RESULTS: The molecular background was established in 22 families. From a total of 24 identified variants, 23 were predicted to affect protein-coding or splicing, including 10 novel variants. The variants were identified in 7 genes: CEP290, GUCY2D, RPE65, NMNAT1, CRB1, RPGRIP1, and CRX. More than one-third of the patients, with clinical LCA diagnosis confirmed by the results of molecular analysis, appeared to be affected with a severe form of the disease: LCA10 caused by the CEP290 gene variants. Intronic mutation c.2991+1655A>G in the CEP290 gene was the most frequent variant identified in the studied group. CONCLUSIONS: This study provides the first molecular genetic characteristics of patients with Leber congenital amaurosis from the previously unexplored Polish population. Our study expands the mutational spectrum as we report 10 novel variants identified in LCA genes. The fact that the most frequent causes of the disease in the studied group of Polish patients are mutations in one out of three genes that are currently the targets for gene therapy (CEP290, GUCY2D, and RPE65) strongly emphasizes the importance of the molecular background analyses of LCA in Polish patients.

Novel CHM mutations in Polish patients with choroideremia - an orphan disease with close perspective of treatment.

BACKGROUND: Choroideremia (CHM) is a rare X-linked recessive retinal dystrophy characterized by progressive chorioretinal degeneration in the males affected. The symptoms include night blindness in childhood, progressive peripheral vision loss and total blindness in the late stages. The disease is caused by mutations in the CHM gene encoding Rab Escort Protein 1 (REP-1). The aim of the study was to identify the molecular basis of choroideremia in five families of Polish origin. METHODS: Six male patients from five unrelated families of Polish ethnicity, who were clinically diagnosed with choroideremia, were examined in this study. An ophthalmologic examination performed in all the probands included: best-corrected visual acuity, slit-lamp examination, funduscopy, fluorescein angiography and perimetry. The entire coding region encompassing 15 exons and the flanking intronic sequences of the CHM gene were amplified with PCR and directly sequenced in all the patients. RESULTS: Five variants in the CHM gene were identified in the five families examined. Two of the variants were new: c.1175dupT and c.83C > G, while three had been previously reported. CONCLUSIONS: This study provides the first molecular genetic characteristics of patients with choroideremia from the previously unexplored Polish population.

Integrating clinical and genetic approaches in the diagnosis of 46,XY disorders of sex development.

46,XY differences and/or disorders of sex development (DSD) are clinically and genetically heterogeneous conditions. Although complete androgen insensitivity syndrome has a strong genotype-phenotype correlation, the other types of 46,XY DSD are less well defined, and thus, the precise diagnosis is challenging. This study focused on comparing the relationship between clinical assessment and genetic findings in a cohort of well-phenotyped patients with 46,XY DSD. The study was an analysis of clinical investigations followed by genetic testing performed on 35 patients presenting to a single center. The clinical assessment included external masculinization score (EMS), endocrine profiling and radiological evaluation. Array-comparative genomic hybridization (array-CGH) and sequencing of DSD-related genes were performed. Using an integrated approach, reaching the definitive diagnosis was possible in 12 children. The correlation between clinical and genetic findings was higher in patients with a more severe phenotype (median EMS 2.5 vs 6; P = 0.04). However, in 13 children, at least one variant of uncertain significance was identified, and most times this variant did not correspond to the original clinical diagnosis. In three patients, the genetic studies guided further clinical assessment which resulted in a reclassification of initial clinical diagnosis. Furthermore, we identified eight patients harboring variants in more than one DSD genes, which was not seen in controls (2.5%; P = 0.0003). In summary, taking into account potential challenges in reaching the definitive diagnosis in 46,XY DSD, only integrated approach seems to be the best routine practice.

Novel variants identified with next-generation sequencing in Polish patients with cone-rod dystrophy.

Purpose: The aim of this study was to identify the molecular genetic basis of cone-rod dystrophy in 18 unrelated families of Polish origin. Cone-rod dystrophy is one of the inherited retinal dystrophies, which constitute a highly heterogeneous group of disorders characterized by progressive dysfunction of photoreceptors and retinal pigment epithelium (RPE) cells. Methods: The study group was composed of four groups of patients representing different Mendelian inheritance of the disease: autosomal dominant (AD), autosomal recessive (AR), X-linked recessive (XL), and autosomal recessive or X-linked recessive (AR/XL). The combined molecular strategy included Sanger sequencing of the RPGR-ORF15 gene (three families with XL and three families with the AR/XL mode of inheritance), mutation-specific microarray analysis of the ABCA4 gene (five families with the AR mode of inheritance and two families with the AR/XL mode of inheritance), targeted next-generation sequencing (NGS) of inherited retinal disease-associated (IRD) genes (seven families with the AD mode of inheritance and five families with the AR mode of inheritance), and whole exome sequencing, performed in select families who had been mutation-negative in the analysis with the targeted NGS panel (one family with the AD mode of inheritance, one family with the AR mode of inheritance, and two families with the AR/XL mode of inheritance). Results: Based on this combined strategy, we managed to identify potentially causative variants in seven out of 18 families with CRD. Five of these variants are novel: c.3142_3143dupAA, p.(Glu1049Argfs*41) in the RPGR-ORF15 gene, two variants: c.1612delT, p.(Trp538Glyfs*15) and c.2389dupG, p.(Ile798Hisfs*20) in the PROM1 gene in one family, c.592A>C, p.(Ser198Arg) in the PRPH2 gene and the variant c.1691A>G, p.(Asp564Gly) in the ATF6 gene that we have already reported to be pathogenic. NGS on the IRD panel allowed the molecular basis of CRD to be identified in four out of 14 families with a total detection rate of 38%. WES allowed identification of the molecular genetic basis of CRD in one family. Conclusions: This is the first report on the spectrum of disease genes and pathogenic variants causing CRD in the Polish population. The study presents five novel variants identified in four genes and therefore, broadens the spectrum of probable pathogenic variants associated with CRD.

Autosomal recessive cone-rod dystrophy can be caused by mutations in the ATF6 gene.

Inherited retinal dystrophies (IRDs) are clinically and genetically highly heterogeneous, making clinical diagnosis difficult. The advances in high-throughput sequencing (ie, panel, exome and genome sequencing) have proven highly effective on defining the molecular basis of these disorders by identifying the underlying variants in the respective gene. Here we report two siblings affected by an IRD phenotype and a novel homozygous c.1691A>G (p.(Asp564Gly)) ATF6 (activating transcription factor 6A) missense substitution identified by whole exome sequencing analysis. The pathogenicity of the variant was confirmed by functional analyses done on patients' fibroblasts and on recombinant p.(Asp564Gly) protein. The ATF6Asp564Gly/Asp564Gly variant shows impaired production of the ATF6 cleaved transcriptional activator domain in response to endoplasmic reticulum stress. Detailed phenotypic examination revealed extinguished cone responses but also decreased rod responses together with the ability to discriminate some colours suggestive rather for cone-rod dystrophy than achromatopsia.

Co-occurrence of Jalili syndrome and muscular overgrowth.

Jalili syndrome is a rare disorder inherited in an autosomal recessive pattern manifesting as a combination of cone-rod dystrophy including progressive loss of visual acuity, color blindness, photophobia, and amelogenesis imperfecta with hypoplastic, immature, or hypocalcified dental enamel. It is caused by mutations in CNNM4, which encodes the ancient conserved domain protein 4. Here we report three brothers with Jalili syndrome and muscle overgrowth of the legs. Myopathic changes were found in needle electromyography. Mutational analysis showed in all three brothers a novel likely pathogenic homozygous missense substitution in exon 1 (c.1076T>C, p.(Leu359Pro)) of CNNM4. Both parents were carriers for the variant. In order to exclude other causative variants that could modify the patients' phenotype we performed exome sequencing and MLPA analysis of the DMD gene in Patient 1. These analyses did not identify any additional variants. Our results expand the mutational spectrum associated with Jalili syndrome and suggest that mild myopathy with muscle overgrowth of the legs could be a newly identified manifestation of the disorder.

Achromatopsia mutations target sequential steps of ATF6 activation.

Achromatopsia is an autosomal recessive disorder characterized by cone photoreceptor dysfunction. We recently identified activating transcription factor 6 (ATF6) as a genetic cause of achromatopsia. ATF6 is a key regulator of the unfolded protein response. In response to endoplasmic reticulum (ER) stress, ATF6 migrates from the ER to Golgi to undergo regulated intramembrane proteolysis to release a cytosolic domain containing a basic leucine zipper (bZIP) transcriptional activator. The cleaved ATF6 fragment migrates to the nucleus to transcriptionally up-regulate protein-folding enzymes and chaperones. ATF6 mutations in patients with achromatopsia include missense, nonsense, splice site, and single-nucleotide deletion or duplication changes found across the entire gene. Here, we comprehensively tested the function of achromatopsia-associated ATF6 mutations and found that they group into three distinct molecular pathomechanisms: class 1 ATF6 mutants show impaired ER-to-Golgi trafficking and diminished regulated intramembrane proteolysis and transcriptional activity; class 2 ATF6 mutants bear the entire ATF6 cytosolic domain with fully intact transcriptional activity and constitutive induction of downstream target genes, even in the absence of ER stress; and class 3 ATF6 mutants have complete loss of transcriptional activity because of absent or defective bZIP domains. Primary fibroblasts from patients with class 1 or class 3 ATF6 mutations show increased cell death in response to ER stress. Our findings reveal that human ATF6 mutations interrupt distinct sequential steps of the ATF6 activation mechanism. We suggest that increased susceptibility to ER stress-induced damage during retinal development underlies the pathology of achromatopsia in patients with ATF6 mutations.

Differential diagnosis of Norrie disease and X-linked familial exudative vitreoretinopathy (XL-FEVR) based on clinical and molecular evaluation.

Background: Molecular analysis of the NDP gene to confirm and precise the clinical diagnosis in two patients with X-linked familial exudative vitreoretinopathy (XL-FEVR). Material and methods: We report two patients from unrelated families with NDP gene mutations: a 14-month-old boy (p1) who was found to have severe exudative vitreoretinopathy and a 4-year-old boy with exudative vitreoretinopathy (p2). An extensive clinical examination of the probands, including slit-lamp examination, B-mode ultrasonography and magnetic resonance imaging was conducted, along with genetic analysis of NDP gene. Results: Clinical findings in patient 1 included no light perception, total retinal detachment and hyperplastic primary vitreous in both eyes. The genetic analysis of the NDP gene enabled to identify the novel frameshift mutation c.222_c223insCG in p1 leading to the premature stop codon and production of aberrant norrin protein. In P2, clinical presentation included high myopia with astigmatism, unilateral fibrous bands and retinal detachment. Genetic testing revealed known point mutation c.362G>A leading to amino-acid alteration and improper protein. Conclusions: Mutation screening of NDP gene identified two different mutations in this region, one of which has not been previously reported.

The first case of Oguchi disease, type 2 in a Polish patient with confirmed GRK1 gene mutation.

UNLABELLED: Oguchi disease type 2 is a rare autosomal recessive form of congenital stationary night blindness. A typical feature of this disorder is a golden-brown discoloration of the fundus called Mizuo-Nakamura phenomenon, which disappears after prolonged dark adaptation and reappears shortly after the onset of light. MATERIAL AND METHODS: A 13-year-old boy exhibiting the clinical features of congenital stationary night blindness, was examined. Ophthalmic examination including slit-lamp biomicroscopy, perimetry and funduscopy was performed. Additionally, the full-field electroretinography and molecular testing for congenital stationary night blindness using the Single Nucleotide Polymorphism microarray technique were performed. RESULTS: The ophthalmic examination showed normal visual acuity, normal anterior segment of both eyes and full visual fields. The eye fundus examination showed a typical golden-brownish discoloration of the peripheral retina (disappearing after long dark adaptation) with no pigment deposits. Full-field electroretinography showed reduced amplitudes of both waves under scotopic conditions, while under photopic conditions both shape and parameters of the record were within the normal limits. The Single Nucleotide Polymorphism microarray revealed a homozygous deletion: c.1607161 OdelCGGA in GRK1 gene. This frameshift mutation introduces a stop codon (p.Asp537Valfs*542) and results in deletion of terminal 22 amino acid residues of retinal kinase protein. CONCLUSIONS: This is the first molecular evidence for GRK1 gene mutation in a Polish patient with Oguchi disease type 2. The identification of the c.1607_1610delCGGA mutation in a patient with Oguchi disease confirms the pathogenicity of this variant.

Fundus albipunctatus: review of the literature and report of a novel RDH5 gene mutation affecting the invariant tyrosine (p.Tyr175Phe).

Fundus albipunctatus (FA) is a rare, congenital form of night blindness with rod system impairment, characterised by the presence of numerous small, white-yellow retinal lesions. FA belongs to a heterogenous group of so-called flecked retina syndromes. This disorder shows autosomal recessive inheritance and is caused mostly by mutations in the RDH5 gene. This gene encodes the enzyme that is a part of the visual cycle, the 11-cis retinol dehydrogenase. This study is a brief review of the literature on FA and a report of the first molecular evidence for RDH5 gene mutation in a Polish patient with this rare disorder. We present a novel pathogenic RDH5 gene mutation in a 16-year-old female patient with symptoms of night blindness. The patient underwent ophthalmological examinations, including colour vision testing, fundus photography, automated visual field testing, full-field electroretinography (ERG) and spectral optical coherent tomography (SOCT). The patient showed typical FA ERG records, the visual field was constricted and fundus examination revealed numerous characteristic, small, white-yellowish retinal lesions. DNA sequencing of the RDH5 gene coding sequence (exons 2-5) enabled the detection of the homozygous missense substitution c.524A > T (p.Tyr175Phe) in exon 3. This is the first report of RDH5 gene mutation that affects the invariant tyrosine, one of the most conserved amino acid residues in short-chain alcohol dehydrogenases/reductases (SDRs), crucial for these enzymes' activity. The location of this substitution, together with its predicted influence on the protein function, indicate that the p.Tyr175Phe mutation is the cause of FA in our patient.

Five novel CNGB3 gene mutations in Polish patients with achromatopsia.

PURPOSE: To identify the genetic basis of achromatopsia (ACHM) in four patients from four unrelated Polish families. METHODS: In this study, we investigated probands with a clinical diagnosis of ACHM. Ophthalmologic examinations, including visual acuity testing, color vision testing, and full-field electroretinography (ERG), were performed in all patients (with the exception of patient p4, who had no ERG). Direct DNA sequencing encompassing the entire coding region of the CNGB3 gene, eight exons of the GNAT2 gene, and exons 5-7 of the CNGA3 gene was performed. Segregation analysis for the presence and independent inheritance of two mutant alleles was performed in the three families available for study. RESULTS: All patients showed typical achromatopsia signs and symptoms. Sequencing helped detect causative changes in the CNGB3 gene in all probands. Eight different mutations were detected in the CNGB3 gene, including five novel mutations: two splice site mutations (c.1579-1G>A and c.494-2A>T), one nonsense substitution (c.1194T>G), and two frame-shift mutations (c.393_394delGCinsTCCTGGTGA and c.1366delC). We also found three mutations: one splice site (c.1578+1G>A) and two frame-shift deletions that had been previously described (c.819_826del and c.1148delC). All respective parents were shown to be heterozygous carriers for the mutation detected in their children. CONCLUSIONS: The present study reports five novel mutations in the CNGB3 gene, and thus broadens the spectrum of probably pathogenic mutations associated with ACHM. Together with molecular data, we provide a brief clinical description of the affected individuals.