Identification of Novel Pathogenic Variants in Familial Adenomatous Polyposis Through Whole Genome Sequencing

An Integrated Whole-Genome Sequencing and In-House Targeted Testing Strategy Enhances Comprehensive Mutation Detection in Neurofibromatosis Type 1.

ABSTRACT Neurofibromatosis type 1 (NF1) is a common autosomal dominant neurocutaneous syndrome that affects 1 in 2,500-3,000 births. The phenotypic hallmarks include Lisch nodules, optic nerve gliomas, café-au-lait spots, axillary freckling, and benign neurofibromas. NF1 patients carry an 8-13% lifetime risk of malignant peripheral nerve sheath tumors, the leading cause of NF1 mortality. Current genetic testing detects NF1 variants in 95-97% of cases. In this prospective cohort of 103 cases, we used targeted NF1 sequencing by next-generation sequencing, multiplex ligation-dependent probe amplification (MLPA), and whole-genome sequencing to identify causative variants. Pathogenic or likely pathogenic variants were identified by in-house targeted testing in 93% of cases; six cases had large deletions detected by MLPA, and two had variants identified by whole-genome sequencing. This is the largest genetically confirmed Indian NF1 cohort reported, expanding the known mutational spectrum and demonstrating the utility of whole-genome sequencing for unresolved cases (~2%), as well as highlighting the clinical and genetic heterogeneity of NF1.

Genome sequencing reveals novel pathogenic deep-intronic PCDH15 variants, amenable to antisense oligonucleotide-based splice correction

Panel-based next-generation sequencing with copy number variant detection pipeline could increase the detection rate of hereditary colorectal cancer syndrome compared with Sanger sequencing

Panel-based NGS with CNV analysis was associated with a higher detection rate of clinically relevant variants than phenotype-driven Sanger sequencing in this single-institution cohort, and support the clinical utility of comprehensive germline testing for patients suspected of having hereditary colorectal cancer syndromes.

Expanding the Genomic Spectrum of NHLRC2-Associated FINCA Disease: Integrated Bioinformatic Characterization of a Novel Deep Intronic Variant Predicted to Activate a Pseudoexon

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.

Mapping intronic mutational hotspots by in silico mutagenesis enables single antisense oligonucleotide correction of multiple variants

Deep intronic variants remain an understudied class of pathogenic variation, primarily due to their absence from standard exome and gene panel datasets and the complexity of non-coding genome interpretation. We hypothesized that pathogenic deep intronic variants are not randomly distributed but instead cluster within intronic "hotspots" inherently prone to pseudoexon activation, and that mapping such regions could improve molecular diagnosis. Importantly, such intronic hotspots nominate targets for antisense oligonucleotide (ASO) therapy. Using X-linked Alport syndrome as a proof-of-concept model and based on our previous work, we screened unsolved patients across multiple European diagnostic centers for variants within a defined region of COL4A5 intron 6. We identified eight independent variants in more than 35 affected individuals from ten unrelated families, which led to two pseudoexon inclusion events, both using the same strong cryptic splice donor site. In all, RNA sequencing and/or minigene assays confirmed aberrant splicing, even when prediction tools were discordant or fell below clinical thresholds. A single ASO targeting the shared donor site restored normal COL4A5 mRNA and 5(IV) collagen protein expression in patient-derived cells regardless of the causative variant. Extending this analysis gene-wide using the AlphaGenome sequence-to-function model, we confirmed intron 6 as one of the most critical COL4A5 splicing hotspot and identified additional potential hotspots harboring novel predicted spliceogenic variants. This gene-agnostic framework establishes a systematic strategy for identifying intronic mutational hotspots and matching patients to scalable, mutation-agnostic ASO-based precision therapies.

Analysis of APC promoter 1B deletions in Russian families with familial adenomatous polyposis

Objective Familial adenomatous polyposis (FAP) is a severe autosomal dominant hereditary cancer syndrome. Patients develop hundreds of adenomatous polyps throughout the colon with the risk of colorectal cancer, if untreated, approaching 100%. FAP is caused by pathogenic germline variants in the APC gene. Deletions in the APC 1B promoter cause FAP in a small subgroup of patients. Previous studies suggested that the APC promoter deletions in unrelated FAP patients from the US and Italy are identical and may thus have spread from a single founder. The aim of this study was to investigate whether a similar founder effect can be detected in the Russian population. Patients and methods We performed whole-genome sequencing on five unrelated patients (three males and two females) with extensive (over 100) colon polyps, family history of FAP, and germline APC 1B promoter deletions previously detected by the multiplex ligation-dependent probe amplification (MLPA) and detected precise deletion boundaries. Results The patients carried deletions in the APC 1B promoter ranging from ~3 to ~122 kbp. We found no association between the deletion size and either the age of the onset or severity of the disease. All deletions were unique and no identical deletion boundaries were observed. However, in four patients, the right deletion breakpoints fell into a 1 kbp region downstream of the 1B promoter. The right breakpoints of several deletions detected in FAP patients from other countries also fell into this narrow region. Conclusion The APC 1B promoter deletions analyzed in this study had arisen independently and there is thus no evidence of a founder effect. Therefore, at least for the cohort of FAP patients with APC 1B promoter deletions studied here, WGS did not provide an added diagnostic benefit to MLPA aside from precisely determining the deletion breakpoints.