Missense but mis-spliced: germline TP53 variant c.671A > C (p.E224A) and the path from uncertainty to pathogenicity.

A Lynch syndrome patient with multiple cancers who was found to have a novel pathogenic variant generating aberrant splicing of MLH1.

Lynch syndrome is an autosomal dominant cancer predisposition syndrome caused by inactivating germline variants in DNA mismatch repair genes. Here, we describe a woman who developed colorectal, gastric, and endometrial cancers and was found to have a rare missense variant, MLH1:c.545G > T/p.Arg182Met. The variant, which was located at the last nucleotide of MLH1exon 6, was initially reported by a clinical testing laboratory as being a variant of uncertain significance. In silico analyses predicted that it would cause splicing aberration, and reverse-transcription polymerase chain reaction analysis of total RNA from the patient's peripheral blood cells identified an aberrant MLH1 transcript that skipped the entire exon 6 and caused generation of a premature stop codon (p.Glu153Phefs*8). In accordance with the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines, we reclassified the variant as likely pathogenic. Further additional analysis enabled carrier diagnosis in the patient's relatives. Genetics summary Disorder: Lynch syndrome Ethnicity of the patient: Japanese Gene: MLH1 (19 exons) GenBank accession number: NM_000249.4 Chromosomal assignment: 3p21.3 Type of DNA variant: a germline splice aberration variant Mutation: c.545G > T in exon 6 of MLH1 Methods of mutation detection: PCR-sequencing, RT-PCR.

Characterisation of a novel p53 mutation (p53N263Tfs*7) associated with li-fraumeni syndrome

Li-Fraumeni Syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in the TP53 tumour suppressor gene, which encodes the multifunctional transcription factor p53. p53 is the most commonly mutated protein in human cancer, with the majority occurring within the DNA-binding domain, often disrupting transcriptional activity and resulting in a loss-of-function. Here, we characterise the novel p53 N263Tfs*7 truncated mutant, identified as a germline mutation from a patient with LFS who developed breast cancer. Functional assays revealed a partial loss-of-function across key cellular processes, including proliferation, cell death, cell motility, and transcriptional transactivation. This mutant lacked a dominant-negative effect, distinguishing it from common DNA-binding domain missense mutations. Our findings demonstrate that oncogenesis in LFS can be driven by partial impairment of functional p53, rather than dominant-negative or gain-of-function mutations alone. This underscores the clinical significance of recognising subtle TP53 variants for the refined molecular classification and clinical prediction of cancer risk in TP53 mutation carriers.

Splice-altering TP53 missense mutations as drivers of dysfunction and targets for RNA-based therapy.

TP53 missense mutations introduce single amino acid substitutions in the p53 protein, which can lead to diverse functional consequences. However, their post-transcriptional impact, particularly on RNA splicing, remains underexplored. Herein, we analyzed cancer genome databases and identified 34 TP53 missense and synonymous mutations capable of generating de novo-splice sites. Using minigene assays, we confirmed the splicing-altering potential of several mutations, including c.178 C > A, c.182 A > G, c.318 C > G, c.356 C > G, c.362 C > A, c.551 A > G, and c.922 C > G. To assess the physiological relevance of these splicing changes, we developed mutant TP53 knock-in cell models using bacterial artificial chromosome DNA-mediated homologous recombination. These missense mutations can function as frameshift or hypomorphic mutations due to aberrant splicing, significantly compromising TP53 mRNA integrity. Meanwhile, severe impairment of constitutive splicing suggests that it correlates with an increased likelihood of nonsense-mediated mRNA decay for mutation-driven alternative transcripts carrying a frameshifted premature stop codon. By designing antisense morpholino oligomers targeting de-novo splice site, we were able to restore csV1 expression and enhance p53 function. This reclassification of select p53 mutations from simple missense to splicing-disruptive mutations reveals their underlying loss-of-function mechanisms and highlights their therapeutic reversibility. Our findings present a novel framework for RNA-based therapeutic strategies aimed at correcting splicing defects in TP53-mutant cancers.

A Novel 5-bp c.433-1_436delGAGTA (p.Ser145Lysfs*9) Frameshift Deletion in C22orf31 Associated with Global Developmental Delay and Microcephaly in a Saudi Consanguineous Family

Intellectual disability (ID) affects approximately 1-3% of the global population, with a higher prevalence reported in consanguineous populations due to autosomal recessive variants. The C22orf31 gene is one of the important candidate genes that is expressed in the brain and is associated with global developmental delays and ID. We aimed to identify the genetic basis of ID, seizure, and microcephaly in a Saudi consanguineous family. Whole exome sequencing was performed on the affected individual from a consanguineous Saudi family, followed by Sanger validation and bioinformatics prediction. Our results showed a novel homozygous 5-base pair (bp) deletion NM_015370.1 (c.433-1_436delGAGTA; p.Ser145Lysfs*9) in the C22orf31 gene. The gene is important and the identified mutation in the C22orf31 gene may disrupt the canonical splice acceptor site and cause a frameshift, leading to a premature termination codon (PTC) that is predicted to trigger nonsense-mediated decay and complete protein loss. The patient manifested developmental delays and seizures along with microcephaly. In conclusion, we report a novel 5-bp deletion in the C22orf31 gene in a Saudi patient. Functional studies and identification of additional families are needed to confirm the role of C22orf31 in disease pathogenesis. This novel finding expands the mutational and clinical spectrum of C22orf31 mutation-related neurodevelopmental disorders in Saudi Arabia.

Whole-Genome Sequencing Identifies Potentially Causative Variants in SOX30, AKAP4, and RNF220 in Non-Obstructive Azoospermia

The co-occurrence of variants in AKAP4 and RNF220 may suggest an oligogenic etiology of NOA and contribute to the phenotypic variability associated with AKAP4 variants.

A novel pathogenic synonymous DHCR7 variant unveiled by aberrant splicing in Smith-Lemli-Opitz syndrome