Fabry disease (FD) is an X-linked lysosomal storage disorder caused by pathogenic variants in the
GLA
gene. Although genetic testing is the gold standard for FD diagnosis, the identification of numerous splicing variants of unknown pathogenicity poses a significant diagnostic challenge. This uncertainty limits the clinical utility of sequencing in guiding patient management and therapeutic intervention.
A combination of in silico prediction tools and in vitro minigene splicing assays was utilized to explore the pathogenicity and underlying molecular mechanism of novel deep non-coding variants identified via long-read sequencing in clinically suspected but genetically unsolved patients.
Functional assays revealed that the large deep-intronic insertion c.640-613ins1715 fundamentally disrupts local splicing pattern and introduces new non-canonical splicing sites. It induces a complex array of aberrant events, including partial exon skipping, intron retention, and pseudoexon inclusion, generating five distinct abnormal transcripts alongside residual normal transcript. These aberrant transcripts introduce frameshifts predicted to yield truncated, non-functional proteins. Consequently, this variant was definitively classified as Pathogenic. Conversely, neither the two deep-intronic single nucleotide variants (c.640-363C > T and c.639+761 G > A) nor their cis configuration exhibited aberrant splicing in our minigene system. However, due to the proband’s later-onset FD phenotype, ambiguous population frequencies (gnomAD), and the potential for tissue-specific splicing missed by in vitro models, both variants remain classified as VUS.
This study expands the mutational spectrum of the
GLA
gene and underscores the complexity of deep-intronic variants. Our findings highlight the critical role of robust functional splicing assays in interpreting variant pathogenicity, establishing accurate genotype-phenotype correlations, and ultimately facilitating precision medicine for FD.
Jingwen Zhou, Aiping Mao, Jie Feng et al.· Orphanet Journal of Rare Dis...· 0 citations
The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) research consortium conducted a comprehensive study to characterize spliceogenic variants in BRCA1 exon 18. The absence of systematic RNA-based assessment for these variants has led to inconsistent interpretation, limiting accurate classification and management of individuals and their families. The splicing profile of 166 variants was assessed using minigene assays; 32 were additionally analyzed in blood-derived RNA from 51 individuals and 18 in mouse embryonic stem cell (mESC)-based assays to evaluate homology-directed repair (HDR) capacity. mRNA assessment by RT-PCR in blood samples and minigene assays showed a significant positive correlation, with splicing analysis in mESCs displaying highly concordant results. The mESC-based HDR assay showed that the in-frame exon 18 skipping (Δ18) transcript encodes a non-functional protein lacking rescue activity. Linear regression analysis using mESC splicing and functional data indicated that ≥59% of full-length (FL) levels and <34% of Δ18 were associated with benign HDR activity. These thresholds differ from those recommended by the ClinGen ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) specifications for applying BP7_strong(RNA): >30% functional transcripts or <70% non-functional transcripts. Incorporation of RNA splicing evidence into variant interpretation increased pathogenic (28.6%-31.7%) and benign (3.7%-24.4%) classifications while reducing likely pathogenic (19.5%-17.7%), uncertain (18.9%-8.5%), and likely benign (29.3%-17.7%) categories. Experimental mRNA profiling impacted the interpretation of 34% of variants and resolved uncertainty in approximately 10% of cases. Exon 18 skipping was less tolerated, indicating that the degree of splice perturbation required to impair BRCA1 function may depend on the nature of the resulting non-functional transcript.
Joanna Domènech-Vivó, Hélène Tubeuf, Romy L. S. Mesman et al.· American Journal of Human Ge...· 0 citations
A male infant with a severe FINCA-like phenotype is reported, including early-onset hemolytic anemia, pulmonary involvement, neurodevelopmental impairment, growth failure, recurrent infections, and fatal progression at 8.5 months.
A. Rozhkova, Anton Esibov, A. Borkovskaia et al.· International Journal of Mol...· 0 citations
BACKGROUND
Next-generation sequencing of cancer predisposition genes is routinely used in hereditary cancer diagnostics. However, a substantial fraction of detected variants remains clinically unresolved. Using a customised 77-gene panel, we analysed 2142 individuals and identified 384 pathogenic or likely pathogenic variants across 54 genes, corresponding to a diagnostic yield of approximately 18%. Despite this, 17% of cases carried variants of uncertain significance, many of which were suspected to affect pre-mRNA splicing and are particularly challenging to interpret due to the limited reliability of in silico predictions and lack of experimental evidence.
METHODS
To address this diagnostic gap, we developed a streamlined minigene-based workflow for rapid functional evaluation of splicing variants and applied it retrospectively. The approach relies on synthetic DNA and recombination-based cloning, eliminating the need for patient-derived RNA and enabling efficient construct generation within a clinically compatible timeframe. Computational prioritisation using AlphaGenome was integrated to support variant selection, while experimental assays provided direct evidence of splicing outcomes.
RESULTS
Application of this strategy allowed the reclassification of previously unresolved variants and clarified cases with discordant computational evidence. Importantly, the workflow is designed for implementation in routine diagnostic settings, with a turnaround time aligned with clinical reporting requirements.
CONCLUSION
This approach provides a robust and scalable framework for functional interpretation of splicing variants, improving diagnostic resolution and supporting more informed clinical decision-making in hereditary cancer genetics.
Noemi Calandra, Elisabetta Mereu, P. Ogliara et al.· Journal of Medical Genetics· 0 citations
X-linked hypophosphatemia (XLH) is a rare, genetically determined disorder of phosphate metabolism, most commonly caused by mutations in the PHEX gene. These mutations lead to overexpression of the phosphaturic hormone FGF23, resulting in renal phosphate wasting and impaired bone mineralization. In up to 16% of clinically diagnosed cases, no causative variant can be identified using standard sequencing approaches. We report on a female patient with a clearly defined clinical XLH phenotype, in whom no causative mutation had been detected over several years despite extensive genetic testing. The aim was to identify a previously undetected genetic cause using extended DNA and RNA methods. After unremarkable short-read whole exome sequencing (WES), short-read whole genome sequencing (WGS) was performed. For confirmation of splice effect, RNA was extracted from peripheral blood, amplified via RT-PCR, and analyzed using Nanopore long-read sequencing. A novel deep intronic variant in the PHEX gene (c.2070 + 601C>T) was identified and confirmed as de novo. The variant caused two aberrant transcripts with pseudoexon inclusions, each leading to a premature stop codon. This aberrant splicing supports the pathogenicity of the variant in the context of a loss-of-function mechanism. Following molecular diagnosis, the patient was successfully initiated on Burosumab therapy, resulting in clinical improvement. This case highlights the diagnostic value of comprehensive genomic analysis and subsequent RNA sequencing for identifying and analyzing deep intronic variants in genetically unexplained cases of XLH. The findings expand the known PHEX mutation spectrum and emphasize the importance of re-evaluating patients with a strong clinical diagnosis but previously negative genetic results. In the future, such technologies may play a crucial role in improving diagnostics for rare monogenic diseases.
Susanne Spranger, Helene Faust, Patricia Duffek et al.· Frontiers in Endocrinology· 0 citations
Familial adenomatous polyposis (FAP) is an autosomal dominant colorectal cancer predisposition syndrome most commonly caused by pathogenic variants in the APC gene. Although targeted gene panel sequencing is routinely used, some FAP cases remain genetically unresolved due to limitations in detecting structural and deep intronic variants. We investigated two unrelated kindreds with clinically evident but genetically unexplained FAP using targeted panel sequencing and whole exome sequencing. Pedigree‐based whole genome sequencing (WGS) was performed to detect cryptic variants, with in silico splice prediction and transcript‐level validation using RT‐PCR, Sanger sequencing, and RT‐qPCR of blood‐derived RNA. WGS uncovered two novel pathogenic APC variants missed by prior testing. In Kindred 1, a heterozygous large deletion encompassing exon 10 was detected and confirmed to cause exon skipping, resulting in a frameshift and premature termination codon. In Kindred 2, a deep intronic complex deletion‐insertion variant was identified between exons 11 and 12, predicted to create novel splice sites. Transcript analysis demonstrated aberrant pseudoexon activation with a 2‐base‐pair deletion, leading to a frameshift and premature termination. Both variants segregated with disease within the respective families and were classified as pathogenic according to ACMG guidelines based on loss‐of‐function effects and functional RNA evidence. Our findings demonstrate that cryptic pathogenic APC variants beyond the detection limits of routine genetic testing can be resolved through WGS combined with transcript‐level validation. This study highlights the importance of transcript‐aware variant interpretation and supports the integration of genome‐wide sequencing and RNA‐based analyses into the diagnostic evaluation of genetically unexplained FAP.
Sung-Ryung Choi, Min Jung Kim, Ja-Lok Ku et al.· Human Mutation· 0 citations