Background: Inherited retinal dystrophies (IRDs) are a genetically heterogeneous group of conditions, with approximately 40% of cases remaining unresolved after initial genetic testing. This study aimed to assess the impact of a personalised genomic approach integrating whole-exome sequencing (WES) reanalysis, whole-genome sequencing (WGS), customised gene panels and functional assays to improve diagnostic yield in unresolved cases. Subjects/Methods: We retrospectively reviewed a cohort of 597 individuals with IRDs, including 525 probands and 72 affected relatives. Among the 221 genetically unresolved cases, a subset of 101 was selected for stepwise re-evaluation. This included WES reanalysis with updated virtual panels, WGS in selected cases and targeted sequencing of complex regions. Variant interpretation was refined using updated classification criteria, segregation analysis and functional assays such as mRNA and minigene/midigene studies. Results: An initial diagnostic yield of 59.6% (313/525) was achieved through first-tier genetic testing. Re-evaluation of the 101 prioritised cases resulted in 42 new diagnoses in probands and resolution of 7 more familial cases, yielding 49 additional diagnoses among previously unresolved patients (48.5%). This increased the overall diagnostic rate for probands to 67.6% (355/525). Functional assays confirmed pathogenicity of variants in ABCA4 , ATF6 , REEP6, and TULP1 , while WGS enabled the detection of structural and deep intronic variants, further enhancing diagnostic accuracy. Conclusions: A patient-centred, stepwise genomic approach significantly improved the molecular diagnosis of IRDs. This strategy supports the clinical utility of periodic WES reanalysis and targeted use of customised panels, WGS and functional assays. The proposed workflow is scalable and applicable to routine clinical practice, contributing to precision medicine in IRDs.

Introduction:TULP1 exemplifies the remarkable clinical and genetic heterogeneity observed in inherited retinal dystrophies. Our research describes the clinical and molecular characteristics of a patient manifesting an atypical retinal dystrophy pattern, marked by the identification of both a previously unreported and a rarely encountered TULP1 variant.Methods: Whole-exome sequencing was performed to identify potential causative variants. The pathogenicity of the identified TULP1 variants was evaluated through in silico predictors and a minigene splice assay, specifically designed to assess the effect of the unreported TULP1 variant.Results: We identified two TULP1 gene variants in a patient exhibiting unusual and symmetrical alterations in both retinas, characterized by an increase in autofluorescence along the distribution of retinal vessels. These variants included a known rare missense variant, c.1376T>C, and a novel splice site variant, c.822G>T. For the latter variant (c.822G>T), we conducted a minigene splice assay that demonstrated the incorporation of a premature stop codon. This finding suggests a likely activation of the nonsense-mediated mRNA decay mechanism, ultimately resulting in the absence of protein production from this allele. Segregation analysis confirmed that these variants were in trans.Discussion: Our data support that individuals with biallelic TULP1 variants may present with a unique pattern of macular degeneration and periarteriolar vascular pigmentation. This study highlights the importance of further clinical and molecular characterization of TULP1 variants to elucidate genotype–phenotype correlations in the context of inherited retinal dystrophies.

Background: Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disease inherited in an autosomal dominant manner. Disease-causing variants in endoglin (ENG) and activin A receptor type II-like 1 (ACVRL1) genes are detected in more than 90% of the patients undergoing molecular testing. The identification of variants of unknown significance is often seen as a challenge in clinical practice that makes family screening and genetic counseling difficult. Here, we show that the implementation of cDNA analysis to assess the effect of splice site variants on mRNA splicing is a powerful tool. Methods: Gene panel sequencing of genes associated with HHT and other arteriovenous malformation-related syndromes was performed. To evaluate the effect of the splice site variants, cDNA analysis of ENG and ACVRL1 genes was carried out. Results: three novel splice site variants were identified in ENG (c.68-2A > T and c.1311+4_1311+8del) and ACVLR1 (c.526-6C > G) genes correspondingly in three individuals with HHT that met ≥ 3 Curaçao criteria. All three variants led to an aberrant splicing inducing exon skipping (ENG:c.68-2A > T and ACVRL1:c.526-6C > G) or intron retention (ENG:c.1311+4_1311+8del) allowing the confirmation of the predicted effect on splicing and the reclassification from unknown significance to pathogenic/likely pathogenic of two of them. Conclusions: RNA analysis should be performed to assess and/or confirm the impact of variants on splicing. The molecular diagnosis of HHT patients is crucial to allow family screening and accurate genetic counseling. A multidisciplinary approach including clinicians and geneticists is crucial when dealing with patients with rare diseases.

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder most commonly caused by pathogenic variants in ACVRL1 or ENG. Most pathogenic variants are coding missense, nonsense, splice-site variants, or small insertions/deletions, as well as exon-level deletions or duplications, and are typically detectable by next-generation sequencing (NGS). However, balanced structural rearrangements and deep intronic variants are not reliably detected by standard exome-based approaches and may contribute to genetically unresolved cases. We investigated four unrelated families with clinical features of HHT in whom prior whole exome sequencing (WES) was inconclusive. All affected individuals reported recurrent miscarriages, and further clinical review identified prior karyotype analyses. These cytogenetic findings prompted targeted reanalysis of WES datasets to assess potential disruption of HHT-associated genes. All four families carried an apparently balanced translocation, t(9;12)(p21.2;q13.13). Breakpoint mapping revealed disruption of a CT-rich region within intron 9 of ACVRL1, a known mutational hotspot. Reanalysis of WES data supported ACVRL1 disruption as the likely pathogenic mechanism. The translocation co-segregated with disease in all families and was absent in unaffected relatives. All carriers fulfilled clinical diagnostic criteria for HHT. A history of recurrent miscarriages emerged as a key clinical clue that prompted cytogenetic investigation and enabled molecular diagnosis. We identify a recurrent translocation, t(9;12)(p21.2;q13.13), that disrupts intron 9 of ACVRL1 and is undetectable by standard exome-based approaches, as a previously unrecognised cause of HHT in genetically unresolved patients. Our findings highlight and support the need to extend genomic testing beyond conventional exome sequencing in unresolved HHT cases.

Growing evidence suggests genetic basis in familial and other types of fibrotic interstitial lung disease (ILD). Genetics may improve diagnostic accuracy a

Background/Objectives: Biallelic pathogenic variants in the CEP290 gene are typically associated with severe, early-onset inherited retinal dystrophies (IRDs) in both syndromic and non-syndromic forms. This study explores the phenotypic variability of non-syndromic IRDs associated with CEP290 variants, focusing on two siblings with biallelic variants, one of whom exhibits a remarkably mild phenotype, thereby expanding the clinical spectrum. Methods: Whole-exome sequencing (WES) and mRNA analysis were performed to identify and characterize CEP290 variants in the siblings. Comprehensive ophthalmologic evaluations assessed retinal function and disease progression. Results: Two CEP290 variants, a frameshift (c.955del, p.(Ser319LeufsTer16)) and a missense (c.5777G>C, p.(Arg1926Pro)), were identified in trans in both siblings. Despite sharing the same genetic variants, the sister exhibited significantly preserved retinal function, while the brother presented with a more severe, progressive retinal dystrophy. Conclusions: This study broadens the phenotypic spectrum of non-syndromic CEP290-related IRDs, demonstrating variability in disease severity ranging from mild to severe. These findings highlight the importance of personalized monitoring and tailored management strategies based on individual clinical presentations of CEP290-related IRDs.