Phenotype Variability in Czech Patients Carrying PAX6 Disease-Causing Variants

The aim of this study was to report PAX6 disease-causing variants in six Czech families, to describe the associated phenotypes, and to perform functional assessment of the splice site variants. Detailed ophthalmic examination was performed. The PAX6 coding region was directly sequenced in three probands. Two probands were analysed by exome sequencing and one by genome sequencing. The effect of two variants on pre-mRNA splicing was evaluated using an exon trapping assay. Six different heterozygous PAX6 variants were identified, with c.111_120del and c.1183+1G˃T being novel. Both c.1183+1G˃T and c.1032+1G>A were proved to cause aberrant splicing with exon skipping and subsequent frameshift. The phenotypic features were variable between and within families. One individual, aged 31 years, presented with mild unilateral ptosis accompanied by aniridia in the right eye, partial aniridia in the left eye, and bilateral congenital cataracts, without marked foveal hypoplasia. Bilateral microcornea, partial aniridia, congenital cataracts, and a large posterior segment coloboma were found in another proband, aged 32 years. One child, aged 8 years, had bilateral high myopia, optic nerve colobomas, anterior polar cataracts, but no iris defects. Another individual, aged 46 years, had bilateral congenital ptosis, iris hypoplasia, keratopathy with marked fibrovascular pannus, anterior polar cataract, and foveal hypoplasia combined with impaired glucose tolerance. However, his daughter, aged 11 years, showed classical features of aniridia. Our study extends the genetic spectrum of PAX6 disease-causing variants and confirms that the associated phenotypic features may be very broad and different to the 'classical' aniridia

IPSC-Derived Corneal Endothelial-like Cells Act as an Appropriate Model System to Assess the Impact of SLC4A11 Variants on Pre-mRNA Splicing

Purpose: To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing. Methods: Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts. Results: In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele. Conclusions: This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants

Multi-center real-world comparison of the fully automated Idylla (TM) microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer

Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla (TM) MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla (TM) testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla (TM) results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla (TM) MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had >= 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla (TM) MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla (TM) MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla (TM) MSI Assay and routine tests.Peer reviewe

Multi-center real-world comparison of the fully automated Idylla (TM) microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer

Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla (TM) MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla (TM) testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla (TM) results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla (TM) MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had >= 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla (TM) MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla (TM) MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla (TM) MSI Assay and routine tests.Peer reviewe

Multi-center real-world comparison of the fully automated Idylla (TM) microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer

Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla (TM) MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla (TM) testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla (TM) results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla (TM) MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had >= 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla (TM) MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla (TM) MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla (TM) MSI Assay and routine tests