Maternal uniparental isodisomy of chromosome 6 unmasks a novel variant in TULP1 in a patient with early onset retinal dystrophy

Purpose: Inherited retinal dystrophies are a clinically and genetically heterogeneous group of disorders. Molecular diagnosis has proven utility for affected individuals. In this study, we report an individual enrolled in the Australian Inherited Retinal Disease Registry and DNA Bank diagnosed with clinical features overlapping between Leber congenital amaurosis and retinitis pigmentosa. Methods: DNA from the proband was sequenced using a gene panel for inherited retinal disorders, and a single nucleotide polymorphism (SNP) array was conducted to detect the presence of deletions and uniparental disomy. Results: We identified a novel homozygous variant (c.524dupC, p.(Pro176ThrfsTer7)) in TULP1 resulting from maternal uniparental isodisomy of chromosome 6. The patient had clinical features consistent with biallelic pathogenic variants in TULP1, including congenital nystagmus, night blindness, non-recordable electroretinogram, mild myopia, and mild peripheral pigmentary changes in the fundus. Conclusions: This is the first report of uniparental disomy 6 and a homozygous variant in TULP1 associated with a rod-cone dystrophy. Molecular diagnosis of inherited retinal dystrophies is essential to inform the mode of transmission and clinical management, and to identify potential candidates for future gene-specific therapies.Emmanuelle Souzeau, Jennifer A. Thompson, Terri L. McLaren, John N. De Roach, Christopher P. Barnett, Tina M. Lamey, Jamie E. Crai

Characterising splicing defects of ABCA4 variants within exons 13–50 in patient-derived fibroblasts

The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T > C; 5603A > T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13–50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction

Determinants of disease penetrance in PRPF31-associated retinopathy

Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31, however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C>T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms

Pax genes in myogenesis: Alternate transcripts add complexity

Pax3 and Pax7 are powerful myogenic inducers and hence play a critical role in skeletal muscle development and regeneration. In this paper we discuss the role of Pax3 and Pax7 in dorsal patterning of the somite with subsequent determination of myogenic precursor cells for muscle formation within the developing embryo and in adult muscle. Recent evidence of the ability of stem cells to contribute to muscle regeneration in adult tissues, and the role of Pax7 in conversion of multipotent stem cells to the myogenic lineage are also discussed. Several tissue specific Pax7 transcripts that encode isoforms with different DNA binding characteristics and potentially distinct transactivation specificities are identified. The expression of a range of transcripts in the determination of different tissue lineages and distinct cell populations both in the embryo and in the adult indicates an extraordinary level of complexity. A detailed understanding of these molecules and their functions during embryogenesis and adult muscle formation is imperative for future stem cell therapies

Generation of two induced pluripotent stem cell lines from a patient with recessive inherited retinal disease caused by compound heterozygous mutations in SNRNP200

The human induced pluripotent stem cell (iPSC) lines LEIi015-A and LEIi015-B were derived from a patient with inherited retinal disease caused by compound heterozygous mutations in the SNRNP200 gene (c.[1792C>T];[3341T>C]). Dermal fibroblasts were transfected with episomal plasmids carrying transgenes encoding OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for P53. The clonal iPSC lines LEIi015-A and LEIi015-B expressed iPSC markers, were free from genomic alterations and demonstrated trilineage differentiation potential

Generation of two induced pluripotent stem cell lines from a patient with Stargardt disease caused by compound heterozygous mutations in the ABCA4 gene

Stargardt disease (STGD1) is the most common inherited retinal dystrophy and ABCA4 c.546-–10 T>C is the most commonly reported splice mutation. Here, we generated and characterized two induced pluripotent stem cell (iPSC) lines from a STGD1 patient with compound heterozygous mutations in ABCA4 (c.[5461-10 T > C;5603A > T];[4163 T > C;455G > A]). Episomal vectors containing OCT4, SOX2, KLF4, L-MYC, LIN28 and mp53DD were employed to conduct the reprogramming of patient-derived fibroblasts. Both lines had a normal karyotype, displayed iPSC morphology, expressed pluripotency markers and showed trilineage differentiation potential. These lines can provide a powerful platform for further investigating the pathophysiological consequences of mutations in ABCA4

Generation of an induced pluripotent stem cell line from a patient with Stargardt disease caused by biallelic c.[5461–10T>C;5603A>T];[6077T>C] mutations in the ABCA4 gene

Mutations in ABCA4 gene are causative for autosomal recessive Stargardt disease (STGD1), the most common inherited retinal dystrophy. Here, we report the generation of an induced pluripotent stem cell (iPSC) line from a STGD1 patient carrying biallelic c.[5461–10T>C;5603A>T];[6077T>C] mutations in the ABCA4 gene. Episomes carrying OCT4, SOX2, KLF4, L-MYC, LIN28 and mp53DD were employed for the reprogramming of patient-derived fibroblasts. This iPSC line expressed comparable pluripotency markers as in a commercially available human iPSC line, displayed normal karyotype and potential for trilineage differentiation, and were negative for both reprogramming episomes and mycoplasma test

Phenotype–genotype correlations in a pseudodominant Stargardt disease pedigree due to a novel ABCA4 deletion–insertion variant causing a splicing defect

Background Deletion–insertion (delins) variants in the retina‐specific ATP‐binding cassette transporter gene, subfamily A, member 4 (ABCA4) accounts for <1% in Stargardt disease. The consequences of these delins variants on splicing cannot be predicted with certainty without supporting in vitro data. Methods Candidate ABCA4 variants were revealed by genetic and segregation analysis of a family with pseudodominant Stargardt disease using a commercial panel and Sanger sequencing. RNA extracted from patient‐derived fibroblasts was analyzed by RT‐PCR to evaluate splicing behavior of the ABCA4 variants. Results Affected members carrying the novel c.6031_6044delinsAGTATTTAACCAATATTT variant in exon 44 presented with contrasting phenotypes; from early‐onset cone‐rod dystrophy to late‐onset macular dystrophy. This variant resulted in a 56‐nucleotide deletion in the mutant allele by activation of a cryptic splice acceptor site which disrupts the reading frame and results in a premature termination codon (p.Ile2003LeufsTer41). If translated, the crucial functional domains near the C‐terminus would be truncated from the ABCA4 protein. Conclusion This work demonstrates the intrafamilial phenotypic variability in a pseudodominant Stargardt disease pedigree and the use of patient‐derived fibroblasts to evaluate the effect of a novel ABCA4 delins variant on splicing to complement in silico pathogenicity assessment

Association of Sex With Frequent and Mild ABCA4 Alleles in Stargardt Disease

Contains fulltext : 229362.pdf (Publisher’s version ) (Closed access)IMPORTANCE: The mechanisms behind the phenotypic variability and reduced penetrance in autosomal recessive Stargardt disease (STGD1), often a blinding disease, are poorly understood. Identification of the unknown disease modifiers can improve patient and family counseling and provide valuable information for disease management. OBJECTIVE: To assess the association of incompletely penetrant ABCA4 alleles with sex in STGD1. DESIGN, SETTING, AND PARTICIPANTS: Genetic data for this cross-sectional study were obtained from 2 multicenter genetic studies of 1162 patients with clinically suspected STGD1. Unrelated patients with genetically confirmed STGD1 were selected. The data were collected from June 2016 to June 2019, and post hoc analysis was performed between July 2019 and January 2020. MAIN OUTCOMES AND MEASURES: Penetrance of reported mild ABCA4 variants was calculated by comparing the allele frequencies in the general population (obtained from the Genome Aggregation Database) with the genotyping data in the patient population (obtained from the ABCA4 Leiden Open Variation Database). The sex ratio among patients with and patients without an ABCA4 allele with incomplete penetrance was assessed. RESULTS: A total of 550 patients were included in the study, among which the mean (SD) age was 45.7 (18.0) years and most patients were women (311 [57%]). Five of the 5 mild ABCA4 alleles, including c.5603A&gt;T and c.5882G&gt;A, were calculated to have incomplete penetrance. The women to men ratio in the subgroup carrying c.5603A&gt;T was 1.7 to 1; the proportion of women in this group was higher compared with the subgroup not carrying a mild allele (difference, 13%; 95% CI, 3%-23%; Pn=n.02). The women to men ratio in the c.5882G&gt;A subgroup was 2.1 to 1, and the women were overrepresented compared with the group carrying no mild allele (difference, 18%; 95% CI, 6%-30%; Pn=n.005). CONCLUSIONS AND RELEVANCE: This study found an imbalance in observed sex ratio among patients harboring a mild ABCA4 allele, which concerns approximately 25% of all patients with STGD1, suggesting that STGD1 should be considered a polygenic or multifactorial disease rather than a disease caused by ABCA4 gene mutations alone. The findings suggest that sex should be considered as a potential disease-modifying variable in both basic research and clinical trials on STGD1

Exploring microperimetry and autofluorescence endpoints for monitoring disease progression in PRPF31-associated retinopathy

Background Mutations in the splicing factor pre-messenger RNA processing factor 31 (PRPF31) gene cause autosomal dominant retinitis pigmentosa 11 (RP11) through a haplo-insufficiency mechanism. We describe the phenotype and progression of microperimetry and autofluorescence endpoints in an Indigenous Australian RP11 family. Patients and Methods Ophthalmic examination, optical coherence tomography, fundus autofluorescence and microperimetry were performed at baseline and every 6–12 months. Baseline and annual change in best-corrected visual acuity (BCVA), microperimetry mean sensitivity (MS) and number of scotoma loci, residual ellipsoid zone (EZ) span and hyperautofluorescent ring (HAR) area were reported. Next-generation and Sanger sequencing were performed in available members. Results 12 affected members from three generations were examined. Mean (SD, range) age at onset of symptoms was 11 (4.5, 4–19) years. MS declined steadily from the third decade and EZ span and HAR area declined rapidly during the second decade. Serial microperimetry showed negligible change in MS over 2–3 years. However, mean EZ span, near-infrared and short-wavelength HAR area reduction was 203 (6.4%) µm/year, 1.8 (8.7%) mm2/year and 1.1 (8.6%) mm2/year, respectively. Genetic testing was performed on 11 affected and 10 asymptomatic members and PRPF31 c.1205 C > A (p.Ser402Ter) mutation was detected in all affected and two asymptomatic members (non-penetrant carriers). Conclusions Our findings suggest that in the studied cohort, the optimal window for therapeutic intervention is the second decade of life and residual EZ span and HAR area can be considered as efficacy outcome measures. Further studies on larger samples with different PRPF31 mutations and longer follow-up duration are recommended