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The biallelic pathogenic repeat (AAGGG)400–2000 intronic expansion in the RFC1 gene has been recently described as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and as a major cause of late‐onset ataxia. Since then, many heterozygous carriers have been identified, with an estimated allele frequency of 0.7% to 4% in the healthy population. Here, we describe in two affected CANVAS sisters the presence of the nonsense c.724C > T p.(Arg242*) variant in compound heterozygosity with the pathogenic repeat expansion in the RFC1 gene. Further RNA analysis demonstrated a reduced expression of the p.Arg242* allele in patients confirming an efficient nonsense‐mediated mRNA decay. We also highlight the importance of considering the sequencing of the RFC1 gene for the diagnosis, especially in patients with CANVAS diagnosis carriers of the AAGGG repeat expansion. [ABSTRACT FROM AUTHOR]

PAX6 haploinsufficiency causes aniridia, a congenital eye disorder that involves the iris, and foveal hypoplasia. Comprehensive screening of the PAX6 locus, including the non-coding regions, by next-generation sequencing revealed four deep-intronic variants with potential effects on pre-RNA splicing. Nevertheless, without a functional analysis, their pathogenicity could not be established. We aimed to decipher their impact on the canonical PAX6 splicing using in vitro minigene splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6 constructs were generated, and minigene assays were carried out. An aberrant splicing pattern was observed for two variants in intron 6, c.357+136G>A and c.357+334G>A. In both cases, several exonization events, such as pseudoexon inclusions and partial intronic retention, were observed due to the creation or activation of new/cryptic non-canonical splicing sites, including a shared intronic donor site. In contrast, two variants identified in intron 11, c.1032+170A>T and c.1033-275A>C, seemed not to affect splicing processes. We confirmed the high complexity of alternative splicing of PAX6 exon 6, which also involves unreported cryptic intronic sites. Our study highlights the importance of integrating functional studies into diagnostic algorithms to decipher the potential implication of non-coding variants, usually classified as variants of unknown significance, thus allowing variant reclassification to achieve a conclusive genetic diagnosis. [ABSTRACT FROM AUTHOR]

Disruption of the autism susceptibility candidate 2 (AUTS2) gene through genomic rearrangements, copy number variations (CNVs), and intragenic deletions and mutations, has been recurrently involved in syndromic forms of developmental delay and intellectual disability, known as AUTS2 syndrome. The AUTS2 gene plays an important role in regulation of neuronal migration, and when altered, associates with a variable phenotype from severely to mildly affected patients. The more severe phenotypes significantly correlate with the presence of defects affecting the C‐terminus part of the gene. This article reports a new patient with a syndromic neurodevelopmental disorder, who presents a deletion of 30 nucleotides in the exon 9 of the AUTS2 gene. Importantly, this deletion includes the transcription start site for the AUTS2 short transcript isoform, which has an important role in brain development. Gene expression analysis of AUTS2 full‐length and short isoforms revealed that the deletion found in this patient causes a remarkable reduction in the expression level, not only of the short isoform, but also of the full AUTS2 transcripts. This report adds more evidence for the role of mutated AUTS2 short transcripts in the development of a severe phenotype in the AUTS2 syndrome. [ABSTRACT FROM AUTHOR]

Congenital aniridia is a complex disease, characterized mainly by iris and foveal hypoplasia, but patients show great clinical variability with overlapping of different anterior and posterior segment anomalies. More than 90% of patients carry heterozygous variants in the PAX6 gene, a highly conserved transcriptional regulator that plays a key role in normal ocular development. Loss‐of‐function variants causing premature stop codons are the most frequent within the broad mutational spectrum of PAX6, with more than 750 different mutations identified to date. 11p13 microdeletions altering PAX6 or its downstream regulatory region have been found in ~25% of patients. Iris abnormalities are also present in various aniridia‐mimicking conditions, such as FOXC1 and PITX2‐related anterior segment dysgenesis, among other minor genes. Therefore, this is now recognized that genetic and clinical diagnosis of patients with suspected aniridia is challenging. Genetic testing has proven to be crucial for differential diagnosis. Here, we update best practices to improve genetic testing and clinical management of aniridia using more cost‐effective next‐generation sequencing analysis. Recent advances in genomic sequencing contributed to increasing diagnostic yield in congenital aniridia and allied conditions, from 70 to 95% as we demonstrated in a cohort of Spanish patients, and also in a "naïve" cohort of Hungarian patients without previous genetic testing. NGS allowed us to identify pathogenic PAX6 variants in patients with an atypical or incomplete presentation of aniridia, such as isolated foveal or optic nerve hypoplasia. In addition, genomic approaches contributed to elucidating new molecular mechanisms underlying the etiopathogenesis of congenital aniridia involving non‐coding PAX6 variants and revealed an unexpected role of mosaic variants in the intrafamilial variable expressivity of aniridia. Emerging long‐read sequencing techniques may provide insight into hidden sources of variation at PAX6 locus in some patients with unexplained genetic causes of aniridia. Nanopore‐based long‐read whole genome sequencing unveiled cryptic balanced chromosomal structural variants that directly disrupt PAX6 or its regulatory elements. In conclusion, NGS‐based genetic testing is crucial to ensure a correct and timely clinical diagnosis, and provide better prognosis and management of patients with aniridia or aniridia‐like, besides the further benefits to families by allowing prenatal and preimplantation genetic testing. Genetic findings would also be critical in defining eligible patients for future clinical trials. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) is widely used to treat non-melanoma skin cancer. However, some patients affected with squamous cell carcinoma (SCC) do not respond adequately to PDT with methyl-δ-aminolevulinic acid (MAL-PDT) and the tumors acquire an infiltrative phenotype and became histologically more aggressive, less differentiated, and more fibroblastic. To search for potential factors implicated in SCC resistance to PDT, we have used the SCC-13 cell line (parental) and resistant SCC-13 cells obtained by repeated MAL-PDT treatments (5th and 10th PDT-resistant generations). Xenografts assays in immunodeficient mice showed that the tumors generated by resistant cells were bigger than those induced by parental cells. Comparative genomic hybridization array (aCGH) showed that the three cell types presented amplicons in 3p12.1 CADM2, 7p11.2 EFGR, and 11q13.3 CCND1 genes. The 5th and 10th PDT-resistant cells showed an amplicon in 5q11.2 MAP3K1, which was not present in parental cells. The changes detected by aCGH on CCND1, EFGR, and MAP3K1 were confirmed in extracts of SCC-13 cells by reverse-transcriptase PCR and by western blot, and by immunohistochemistry in human biopsies from persistent tumors after MAL-PDT. Our data suggest that genomic imbalances related to CCND1, EFGR, and particularly MAP3K1 seem to be involved in the development of the resistance of SCC to PDT. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) constitutes a cancer treatment modality based on the administration of a photosensitizer, which accumulates in tumor cells. The subsequent irradiation of the tumoral area triggers the formation of reactive oxygen species responsible for cancer cell death. One of the compounds approved in clinical practice is methyl-aminolevulinate (MAL), a protoporphyrin IX (PpIX) precursor intermediate of heme synthesis. We have identified the mitotic catastrophe (MC) process after MAL-PDT in HeLa human carcinoma cells. The fluorescence microscopy revealed that PpIX was located mainly at plasma membrane and lysosomes of HeLa cells, although some fluorescence was also detected at endoplasmic reticulum and Golgi apparatus. Cell blockage at metaphase-anaphase transition was observed 24 h after PDT by phase contrast microscopy and flow cytometry. Mitotic apparatus components evaluation by immunofluorescence and Western blot indicated: multipolar spindles and disorganized chromosomes in the equatorial plate accompanied with dispersion of centromeres and alterations in aurora kinase proteins. The mitotic blockage induced by MAL-PDT resembled that induced by two compounds used in chemotherapy, taxol and nocodazole, both targeting microtubules. The alterations in tumoral cells provided evidence of MC induced by MAL-PDT, resolving mainly by apoptosis, directly or through the formation of multinucleate cells.