Verheij and colleagues were the first to describe two patients harboring de novo overlapping 8q24.3 microdeletions.1 The phenotype was characterized by coloboma, cardiac and renal defect, limb abno...

Our study aimed to characterize miR-221 and 483-3p dysregulation and to correlate their expression with clinical outcomes in patients submitted in first instance to surgical therapy (naïve patients) for esophageal adenocarcinoma (EAC). MicroRNA (miRNAs) are small, noncoding RNAs that play important roles in several biological processes by fine-tuning gene expression1. In a cohort of well-characterized EAC, we analyzed the expression of miRNAs. RNA was extracted from formalin-embedded (FFPE) surgical specimens of normal gastric tissues and EACs, classified according to BIM/GIM classification (BIM=Barrett’s intestinal metaplasia, GIM=Gastric intestinal metaplasia): 43 BIM-/GIM-, 32 BIM+/GIM-, 2 BIM+/GIM+, 9 BIM-/GIM+). Expression of 754 human miRNAs was profiled in 8 cases with TaqMan MicroRNA Array card A1.1/B3.0. miR-221 and 483-3p validation was performed with single TaqMan probes on all 85 FFPE samples. miR-221 and miR-483-3p were also analyzed in a cohort of 39 fresh-frozen biopsies (29 EAC and 10 normal esophageal tissues). In 3 EAC cell-lines (OE-19, OE-33, FLO-1)2 we tested the expression of miR-221 and miR-483-3p3 and of two major targets (PTEN and SMAD4, respectively). Correlations between miRNAs and clinical outcomes were calculated using Kruskal-Wallis, Mann-Whitney, t-Student’s and Kaplan-Meier tests. miR-221 and 483-3p resulted upregulated from the MicroRNA Array card analysis. This increase was confirmed in two independent cohorts (FFPE-cohort: miR-483-3p p=0.0065; fresh-frozen-cohort: miR-483-3p p<0.0001, miR-221 p=0.0062). Statistical analysis showed an upregulation of both miRNAs, in particular miR-483-3p (p<0.0001), in BIM-/GIM-, compared to BIM+/GIM-. miR-221 and 483-3p up- regulation correlated with reduced cancer-specific survival in FFPE samples (miR-483-3p p=0.053, miR-221 p=0.001). We demonstrated a significantly increase for miR-483-3p (p=0.01) with a trend toward reduction of SMAD4 expression in FLO-1 compared to other cell-lines. We identified an up-regulation of miR-221 and miR-483-3p, which might contribute to cancer progression. In the most aggressive cell line, FLO-1, we observed an increased expression of miR-483-3p, with SMAD4 reduction. Further studies are warranted to elucidate in depth the role of miR-221 and miR- 483-3p in EAC.

Background: Exome (ES) and genome sequencing (GS) are powerful tools for diagnosing neurodevelopmental disorders (NDDs), yet sequencing coverage failures can leave clinically relevant variants undetected. Analyzing the distribution of coverage gaps across sequencing approaches and batches is therefore informative for diagnostic accuracy. Methods: We analyzed sequencing data from 43 NDD patients across four ES runs, including 14 individuals sequenced by both ES (Twist Human-Core-Exome-v1.3) and GS. Low-coverage regions (LCRs) were defined as target intervals with mean depth <20 x, and z-scores < −1.96 were used to identify batch-specific systematic LCRs. LCRs were clinically annotated using OMIM and SysNDD databases. Results: LCR patterns were highly consistent within each ES batch but were characterized by extreme variability between batches. Higher global mean coverage increased intra-batch consistency, but batches sequenced at a commonly accepted yield in clinical sequencing (>100 x mean coverage) showed thousands of batch-specific LCRs. LCR patterns substantially diverged between ES and GS, displaying preferential impact on different genes. Although a restricted group of genes accumulates LCRs disproportionately, most LCRs are broadly dispersed throughout the genome. LCRs were not systematically associated with features such as GC content and genomic location (e.g., exon 1). Interestingly, LCRs affected OMIM/SysNDD genes and occasionally overlapped ClinVar pathogenic variants, indicating potential impact on diagnostic sensitivity. Conclusion: The global distribution of coverage gaps appears strongly influenced by batch-specific effects, making the occurrence of LCRs partly unpredictable even within clinically relevant gene sets. These findings support systematic assessment of LCRs as a component of quality evaluation in diagnostic sequencing workflows.

Background: Founder mutations in MYBPC3 may contribute substantially to the genetic burden of hypertrophic cardiomyopathy (HCM) and provide important insights into genotype–phenotype correlations and population-specific disease mechanisms. In this study, we investigated two recurrent canonical splice-site variants, MYBPC3 c.1458-1G>A and c.3331-1G>A, identified in patients with HCM from the Emilia-Romagna region of Northern Italy. Methods: Ninety-one unrelated patients with HCM carrying either MYBPC3c.1458-1G>A or c.3331-1G>A were analyzed. Haplotype reconstruction was performed to assess a possible founder effect and estimate the approximate age of the shared ancestral allele. Functional characterization was carried out by RNA sequencing of myocardial tissue to evaluate the impact of the variants on splicing. Clinical and phenotypic data were compared with those of carriers of other truncating MYBPC3 variants or pathogenic variants in other sarcomeric genes. Results: Both variants, classified as pathogenic according to ACMG criteria, shared a conserved variant-specific core haplotype. Founder age was estimated at approximately 10.5 generations (~262 years), consistent with a regional founder effect. RNA sequencing demonstrated aberrant splicing for both variants, resulting in premature termination codons and presumably subsequent nonsense-mediated mRNA decay. Clinically, carriers showed delayed disease onset, with a mean onset in the fifth decade of life, and a comparatively milder phenotype, including a lower incidence of sudden cardiac death, than patients carrying other truncating MYBPC3 variants or pathogenic variants in other sarcomeric genes. Both variants exhibited partial penetrance (approximately 63–67%) and age-dependent variable expressivity. Conclusions:MYBPC3 c.1458-1G>A and c.3331-1G>A represent novel founder alleles associated with HCM in Northern Italy. Identification of these locally prevalent variants improves molecular diagnosis, family screening, and supports the development of variant-targeted therapeutic approaches.