BACKGROUND: Pathogenic variants in BRCA genes play a crucial role in the pathogenesis of ovarian cancer. Intronic variants of uncertain significance (VUS) may contribute to pathogenicity by affecting splicing. Currently, the significance of many intronic variants in BRCA has not been clarified, impacting patient treatment strategies and the management of familial cases.
METHODS: A retrospective study was conducted to analyze BRCA intronic VUS in a cohort of 707 unrelated ovarian cancer patients at a single institution from 2018 to 2023. Three splicing predictors were employed to analyze detected intronic VUS. Variants predicted to have splicing alterations were selected for further validation through minigene assays. Patient and familial investigations were conducted to comprehend cancer incidence within pedigrees and the application of poly (ADP-ribose) polymerase inhibitors (PARPi) by the patients. In accordance with the guidelines of the American College of Medical Genetics and Genomics (ACMG), the intronic VUS were reclassified based on minigene assay results and clinical evidence.
RESULTS: Approximately 9.8% (69/707) of patients were identified as carriers of 67 different VUS in BRCA1/2, with four intronic variants accounting for 6% (4/67) of all VUS. Splicing predictors indicated potential splicing alterations in splicing for BRCA1 c.4358-2A>G and BRCA2 c.475+5G>C variants. Minigene assays utilizing the pSPL3 exon trapping vector revealed that these variants induced changes in splicing sites and frameshift, resulting in premature termination of translation (p. Ala1453Glyfs and p. Pro143Glyfs). According to ACMG guidelines, BRCA1 c.4358-2A>G and BRCA2 c.475+5G>C were reclassified as pathogenic variants. Pedigree investigations were conducted on patients with BRCA1 c.4358-2A>G variant, and the detailed utilization of PARPi provided valuable insights into research on PARPi resistance.
CONCLUSIONS: Two intronic VUS were reclassified as pathogenic variants. A precise classification of variants is crucial for the effective treatment and management of both patients and healthy carriers.

Frontotemporal dementia (FTD) has genetic heterogeneity, and the endosomal ESCRTIII-complex subunit CHMP2B variant is a rare cause of FTD. The mutations in CHMP2B were first identified in a large Danish pedigree with autosomal dominant FTD, and have also been found in several individuals from Belgium, France, the United States, and Türkiye. In the Chinese population, cases of CHMP2B variant-associated FTD have never been reported.
The spectrum of clinical symptoms and the genetic analysis of the presented patient were identified and investigated. Besides this case, we assessed previously reported cases with CHMP2B gene mutations.
This study presents a Chinese patient harboring a novel heterozygous A-to-T variant (NM_014043:c.532-2A>T) in CHMP2B with a phenotype compatible with FTD. Although previous reports suggested cases of CHMP2B variant-associated FTD initially presented with personality changes and stereotypical movements at the age of 50, this case was characterized by psychosis involving delusion of persecution, auditory hallucination, and suspiciousness at the earlier onset age of 44. Minigene splicing assay revealed that the splice-site variant could result in the retention of intron 5.
This is the first case of CHMP2B variant-associated FTD reported in the Chinese population. The novel c.532-2A>T variant in the acceptor splice site of exon 6 retaining intron 5 was predicted to cause truncated protein and protein conformation changes. This discovery may expand the genetic and phenotypic spectrum of CHMP2B variant-associated FTD.

The diffusion-weighted SPLICE (split acquisition of fast spin-echo signals) sequence employs split-echo rapid acquisition with relaxation enhancement (RARE) readout to provide images almost free of geometric distortions. However, due to the varying T 2 $$ {}_2 $$ -weighting during k-space traversal, SPLICE suffers from blurring. This work extends a method for controlling the spatial point spread function (PSF) while optimizing the signal-to-noise ratio (SNR) achieved by adjusting the flip angles in the refocusing pulse train of SPLICE.
An algorithm based on extended phase graph (EPG) simulations optimizes the flip angles by maximizing SNR for a flexibly chosen predefined target PSF that describes the desired k-space density weighting and spatial resolution. An optimized flip angle scheme and a corresponding post-processing correction filter which together achieve the target PSF was tested by healthy subject brain imaging using a clinical 1.5 T scanner.
Brain images showed a clear and consistent improvement over those obtained with a standard constant flip angle scheme. SNR was increased and apparent diffusion coefficient estimates were more accurate. For a modified Hann k-space weighting example, considerable benefits resulted from acquisition weighting by flip angle control.
The presented flexible method for optimizing SPLICE flip angle schemes offers improved MR image quality of geometrically accurate diffusion-weighted images that makes the sequence a strong candidate for radiotherapy planning or stereotactic surgery.

Alternative splicing provides a broad strategy to amplify the genome. Yet how alternative splicing influences neurodevelopment or indeed which variants are translated at developmental choice points remains poorly explored. Here we focused on a gene important for neurodevelopment, the Lim homeodomain transcription factor, Lhx9. Lhx9 has two noncanonical splice variants, Lhx9a and Lhx9b which compared with the canonical variant Lhx9c have a truncated homeodomain and an alternative C-terminal sequence, suggesting that, if translated, these variants could differently impact on cellular function.
We created a unique antibody tool designed to selectively detect noncanonical Lhx9 variants (Lhx9ab) and used this to examine the protein expression dynamics in embryos. Lhx9ab variants were translated and dynamically expressed similarly between mouse and chicken at key developmental choice points in the spinal cord, limbs and urogenital ridge. Within the spinal cord, enrichment of Lhx9c vs Lhx9ab expression was observed during key migration and axonal projection choice points.
These data support the notion that the expression dynamics between canonical and noncanonical Lhx9 variants could play an important role in spinal neuron maturation. More broadly, determining the temporal dynamics of alternative protein variants is a key entry point to understand how splicing influences developmental processes.

The THOC2 gene encodes THO complex subunit 2, a subunit of the Transcription-Export (TREX) complex which binds specifically to splice messenger ribonucleic acid (mRNAs) to facilitate mRNA export. Mutations in the THOC2 gene have been described to lead to X-linked mental retardation syndrome type 12/35 (XLMR-12/35) (MIM#300957). Here, we describe for the first time a recurrent arthrogryposis multiplex congenita phenotype (AMC) in two male fetuses in a family. Exome sequencing identified a novel pathogenic variation chrX: 122761817_122761820delTGAC (genome assembly GRCh37 format) or c.2482-1_2484delGTCA (as per Genbank transcript ID NM_001081550) in theTHOC2 gene. This variant affects the consensus acceptor splice site between intron 22 and exon 23. This is the most severe phenotype described in THOC2 gene-related disease till date. This case report expands the clinical phenotype of THOC2 gene related defects.

To identify the pathogenic mechanism of the c.244G>T mutation in NR5A1 gene found in a Chinese patient with 46, XY disorders of sex development (DSD).
Genomic DNA was extracted from a Chinese 46, XY DSD patient. Targeted next-generation and Sanger sequencing were performed to investigate and validate the gene mutation causing 46, XY DSD, respectively. In silico tools were used to predict the pathogenicity of the variant. Dual luciferase reporter gene assay and minigene splicing reporter assay were used to identify the pathogenicity of the variant.
A novel heterozygous variant, c.244G>T (p.Ala82Ser), in NR5A1 gene was detected in the 46, XY DSD patient. Four of five silico tools predicting pathogenicity of missense variants indicated that the variant was pathogenic. However, in vitro functional study showed that p.Ala82Ser did not affect the transcriptional activity of NR5A1. In silico tools predicting the potential splicing loci revealed that c.244G>T led to aberrant splicing of NR5A1 RNA. Minigene splicing reporter assay confirmed that c.244G>T resulted in the deletion of exon2 or deletion of 19 nucleotides in 3\' end of exon2.
Mutation of c.244G>T in NR5A1 results in 46, XY DSD by inducing abnormal splicing of NR5A1 RNA instead of amino acid substitution of NR5A1.

Pathogenic variants in the X-chromosome Aristaless-related homeobox (ARX) gene contribute to intellectual disability, epilepsy, and associated comorbidities in affected males. Here, we report a novel splice variant in ARX in a family with three affected individuals. The proband had early onset developmental and epileptic encephalopathy, his brother and mother had severe and mild intellectual disability, respectively. Massively parallel sequencing identified a novel c.1449-1G>C in intron 4 of the ARX gene, predicted to abolish the splice acceptor site, retaining intron 4 and leading to a premature termination codon immediately after exon 4. As exon 5 is the last exon of the ARX gene, the premature termination codon at position p.L484* would be predicted to escape nonsense-mediated mRNA decay, potentially producing at least some C-terminally truncated protein. Analysis of cDNA from patient lymphoblastoid cells confirmed retention of intron 4 and loss of detectable expression of ARX mRNA across exon 4 to exon 5. We review published cases of variants that lead to altered or early termination of the ARX protein, but not complete loss of function, and are associated with phenotypes of intellectual disability and infantile onset developmental and epileptic encephalopathies, including Ohtahara and West syndromes. Taken together, this novel splice variant retaining intron 4 is likely to be the cause of the early onset developmental and epileptic encephalopathy in the proband.

Ataxia-telangiectasia (A-T) is a recessive disorder caused by biallelic pathogenic variants of ataxia-telangiectasia mutated (ATM). This disease is characterized by progressive ataxia, telangiectasia, immune deficiency, predisposition to malignancies, and radiosensitivity. However, hypomorphic variants may be discovered associated with very atypical phenotypes, raising the importance of evaluating their pathogenic effects. In this study, multiple functional analyses were performed on lymphoblastoid cell lines from 36 patients, comprising 49 ATM variants, 24 being of uncertain significance. Thirteen patients with atypical phenotype and presumably hypomorphic variants were of particular interest to test strength of functional analyses and to highlight discrepancies with typical patients. Western-blot combined with transcript analyses allowed the identification of one missing variant, confirmed suspected splice defects and revealed unsuspected minor transcripts. Subcellular localization analyses confirmed the low level and abnormal cytoplasmic localization of ATM for most A-T cell lines. Interestingly, atypical patients had lower kinase defect and less altered cell-cycle distribution after genotoxic stress than typical patients. In conclusion, this study demonstrated the pathogenic effects of the 49 variants, highlighted the strength of KAP1 phosphorylation test for pathogenicity assessment and allowed the establishment of the Ataxia-TeLangiectasia Atypical Score to predict atypical phenotype. Altogether, we propose strategies for ATM variant detection and classification.

GNE myopathy is a rare autosomal recessive myopathy caused by bi-allelic mutations in GNE. We report the case of a 36-year-old man who presented with typical clinical and pathological features of GNE myopathy including distal dominant muscle weakness from the age of 29 and numerous rimmed vacuoles on muscle biopsy. Targeted next-generation sequencing revealed a novel synonymous mutation, c.1500A>G (p.G500=), together with a common Japanese mutation c.620A>T (p.D207V). The cDNA analysis of the biopsied muscle revealed that this synonymous mutation creates a cryptic splice donor site that causes aberrant splicing. This report will expand our understanding of the genetic heterogeneity of GNE myopathy emphasizing the importance of interpreting synonymous variants in genetic testing.

There are several in silico programs that endeavor to predict the functional impact of an individual\'s sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping vector, such as pSPL3, an investigator can test whether a splice site sequence change leads to altered RNA splicing, through expression of reference and variant mini-genes in mammalian cells and analysis of the resultant RNA products.