Exploring non-coding genetic variability in ACE2: Functional annotation and in vitro validation of regulatory variants.

The surge in human whole-genome sequencing data has facilitated the study of non-coding region variations, yet understanding their biological significance remains a challenge. We used a computational workflow to assess the regulatory potential of non-coding variants, with a particular focus on the Angiotensin Converting Enzyme 2 (ACE2) gene. This gene is crucial in physiological processes and serves as the entry point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing coronavirus disease 19 (COVID-19). In our analysis, using data from the gnomAD population database and functional annotation, we identified 17 significant Single Nucleotide Variants (SNVs) in ACE2, particularly in its enhancers, promoters, and 3' untranslated regions (UTRs). We found preliminary evidence supporting the regulatory impact of some of these variants on ACE2 expression. Our detailed examination of two SNVs, rs147718775 and rs140394675, in the ACE2 promoter revealed that these co-occurring SNVs, when mutated, significantly enhance promoter activity, suggesting a possible increase in specific ACE2 isoform expression. This method proves effective in identifying and interpreting impactful non-coding variants, aiding in further studies and enhancing understanding of molecular bases of monogenic and complex traits.

APOGEE 2: multi-layer machine-learning model for the interpretable prediction of mitochondrial missense variants.

Mitochondrial dysfunction has pleiotropic effects and is frequently caused by mitochondrial DNA mutations. However, factors such as significant variability in clinical manifestations make interpreting the pathogenicity of variants in the mitochondrial genome challenging. Here, we present APOGEE 2, a mitochondrially-centered ensemble method designed to improve the accuracy of pathogenicity predictions for interpreting missense mitochondrial variants. Built on the joint consensus recommendations by the American College of Medical Genetics and Genomics/Association for Molecular Pathology, APOGEE 2 features an improved machine learning method and a curated training set for enhanced performance metrics. It offers region-wise assessments of genome fragility and mechanistic analyses of specific amino acids that cause perceptible long-range effects on protein structure. With clinical and research use in mind, APOGEE 2 scores and pathogenicity probabilities are precompiled and available in MitImpact. APOGEE 2's ability to address challenges in interpreting mitochondrial missense variants makes it an essential tool in the field of mitochondrial genetics.

Additional lesions identified by genomic microarrays are associated with an inferior outcome in low-risk chronic lymphocytic leukaemia patients.

We explored the relevance of genomic microarrays (GM) in the refinement of prognosis in newly diagnosed low-risk chronic lymphocytic leukaemia (CLL) patients as defined by isolated del(13q) or no lesions by a standard 4 probe fluorescence in situ hybridization (FISH) analysis. Compared to FISH, additional lesions were detected by GM in 27 of the 119 patients (22.7%). The concordance rate between FISH and GM was 87.4%. Discordant results between cytogenetic banding analysis (CBA) and GM were observed in 45/119 cases (37.8%) and were mainly due to the intrinsic characteristics of each technique. The presence of additional lesions by GM was associated with age > 65 years (p = 0.047), advanced Binet stage (p = 0.001), CLL-IPI score (p < 0.001), a complex karyotype (p = 0.004) and a worse time-to-first treatment in multivariate analysis (p = 0.009). Additional lesions by GM were also significantly associated with a worse time-to-first treatment in the subset of patients with wild-type TP53 and mutated IGHV (p = 0.025). In CLL patients with low-risk features, the presence of additional lesions identified by GM helps to identify a subset of patients with a worse outcome that could be proposed for a risk-adapted follow-up and for early treatment including targeted agents within clinical trials.

Potassium Channel KCNH1 Activating Variants Cause Altered Functional and Morphological Ciliogenesis.

The primary cilium is a non-motile sensory organelle that extends from the surface of most vertebrate cells and transduces signals regulating proliferation, differentiation, and migration. Primary cilia dysfunctions have been observed in cancer and in a group of heterogeneous disorders called ciliopathies, characterized by renal and liver cysts, skeleton and limb abnormalities, retinal degeneration, intellectual disability, ataxia, and heart disease and, recently, in autism spectrum disorder, schizophrenia, and epilepsy. The potassium voltage-gated channel subfamily H member 1 (KCNH1) gene encodes a member of the EAG (ether-à-go-go) family, which controls potassium flux regulating resting membrane potential in both excitable and non-excitable cells and is involved in intracellular signaling, cell proliferation, and tumorigenesis. KCNH1 missense variants have been associated with syndromic neurodevelopmental disorders, including Zimmermann-Laband syndrome 1 (ZLS1, MIM #135500), Temple-Baraitser syndrome (TMBTS, MIM #611816), and, recently, with milder phenotypes as epilepsy. In this work, we provide evidence that KCNH1 localizes at the base of the cilium in pre-ciliary vesicles and ciliary pocket of human dermal fibroblasts and retinal pigment epithelial (hTERT RPE1) cells and that the pathogenic missense variants (L352V and R330Q; NP_002229.1) perturb cilia morphology, assembly/disassembly, and Sonic Hedgehog signaling, disclosing a multifaceted role of the protein. The study of KCNH1 localization, its functions related to primary cilia, and the alterations introduced by mutations in ciliogenesis, cell cycle coordination, cilium morphology, and cilia signaling pathways could help elucidate the molecular mechanisms underlying neurological phenotypes and neurodevelopmental disorders not considered as classical ciliopathies but for which a significant role of primary cilia is emerging.

MiRLog and dbmiR: Prioritization and functional annotation tools to study human microRNA sequence variants.

The recent identification of noncoding variants with pathogenic effects suggests that these variations could underlie a significant number of undiagnosed cases. Several computational methods have been developed to predict the functional impact of noncoding variants, but they exhibit only partial concordance and are not integrated with functional annotation resources, making the interpretation of these variants still challenging. MicroRNAs (miRNAs) are small noncoding RNA molecules that act as fine regulators of gene expression and play crucial functions in several biological processes, such as cell proliferation and differentiation. An increasing number of studies demonstrate a significant impact of miRNA single nucleotide variants (SNVs) both in Mendelian diseases and complex traits. To predict the functional effect of miRNA SNVs, we implemented a new meta-predictor, MiRLog, and we integrated it into a comprehensive database, dbmiR, which includes a precompiled list of all possible miRNA allelic SNVs, providing their biological annotations at nucleotide and miRNA levels. MiRLog and dbmiR were used to explore the genetic variability of miRNAs in 15,708 human genomes included in the gnomAD project, finding several ultra-rare SNVs with a potentially deleterious effect on miRNA biogenesis and function representing putative contributors to human phenotypes.

Correlating Neuroimaging and CNVs Data: 7 Years of Cytogenomic Microarray Analysis on Patients Affected by Neurodevelopmental Disorders.

The aim of this study was to evaluate the relationship between neurodevelopmental disorders, brain anomalies, and copy number variations (CNVs) and to estimate the diagnostic potential of cytogenomical microarray analysis (CMA) in individuals neuroradiologically characterized with intellectual developmental disorders (IDDs) isolated or associated with autism spectrum disorders (ASDs) and epilepsy (EPI), all of which were identified as a "synaptopathies." We selected patients who received CMA and brain magnetic resonance imaging (MRI) over a 7-year period. We divided them into four subgroups: IDD, IDD + ASD, IDD + EPI, and IDD + ASD + EPI. The diagnostic threshold of CMA was 16%. The lowest detection rate for both CMA and brain anomalies was found in IDD + ASD, while MRI was significantly higher in IDD and IDD + EPI subgroups. CMA detection rate was significantly higher in patients with brain anomalies, so CMA may be even more appropriate in patients with pathological MRI, increasing the diagnostic value of the test. Conversely, positive CMA in IDD patients should require an MRI assessment, which is more often associated with brain anomalies. Posterior fossa anomalies, both isolated and associated with other brain anomalies, showed a significantly higher rate of CMA positive results and of pathogenic CNVs. In the next-generation sequencing era, our study confirms once again the relevant diagnostic output of CMA in patients with IDD, either isolated or associated with other comorbidities. Since more than half of the patients presented brain anomalies in this study, we propose that neuroimaging should be performed in such cases, particularly in the presence of genomic imbalances.

Landscapes of cellular phenotypic diversity in breast cancer xenografts and their impact on drug response.

The heterogeneity of breast cancer plays a major role in drug response and resistance and has been extensively characterized at the genomic level. Here, a single-cell breast cancer mass cytometry (BCMC) panel is optimized to identify cell phenotypes and their oncogenic signalling states in a biobank of patient-derived tumour xenograft (PDTX) models representing the diversity of human breast cancer. The BCMC panel identifies 13 cellular phenotypes (11 human and 2 murine), associated with both breast cancer subtypes and specific genomic features. Pre-treatment cellular phenotypic composition is a determinant of response to anticancer therapies. Single-cell profiling also reveals drug-induced cellular phenotypic dynamics, unravelling previously unnoticed intra-tumour response diversity. The comprehensive view of the landscapes of cellular phenotypic heterogeneity in PDTXs uncovered by the BCMC panel, which is mirrored in primary human tumours, has profound implications for understanding and predicting therapy response and resistance.

GDF5 mutation case report and a systematic review of molecular and clinical spectrum: Expanding current knowledge on genotype-phenotype correlations.

INTRODUCTION: Brachydactyly is a bone development abnormality presenting with variable phenotypes and different transmission patterns. Mutations in GDF5 (Growth and Differentiation Factor 5, MIM *601146) account for a significant amount of cases. Here, we report on a three-generation family, where the proband and the grandfather have an isolated brachydactyly with features of both type A1 (MIM #112500) and type C (MIM #113100), while the mother shows only subtle hand phenotype signs. MATERIALS AND METHODS: Whole Exome Sequencing (WES) was performed on the two affected individuals. An in-depth analysis of GDF5 genotype-phenotype correlations was performed through literature reviewing and retrieving information from several databases to elucidate GDF5-related molecular pathogenic mechanisms. RESULTS: WES analysis disclosed a pathogenic variant in GDF5 (NM_000557.5:c.157dup; NP_000548.2:p.Leu53Profs*41; rs778834209), segregating with the phenotype. The frameshift variant was previously associated with Brachydactyly type C (MIM #113100), in heterozygosity, and with the severe Grebe type chondrodysplasia (MIM #200700), in homozygosity. In-depth analysis of literature and databases allowed to retrieve GDF5 mutations and correlations to phenotypes. We disclosed the association of 49 GDF5 pathogenic mutations with eight phenotypes, with both autosomal dominant and recessive transmission patterns. Clinical presentations ranged from severe defects of limb morphogenesis to mild redundant ossification. We suggest that such clinical gradient can be linked to a continuum of GDF5-activity variation, with loss of GDF5 activity underlying bone development defects, and gain of function causing disorders with excessive bone formation. CONCLUSIONS: Our analysis of GDF5 pathogenicity mechanisms furtherly supports that mutation and zygosity backgrounds resulting in the same level of GDF5 activity may lead to similar phenotypes. This information can aid in interpreting the potential pathogenic effect of new variants and in supporting an appropriate genetic counseling.

Heterozygous nonsense ARX mutation in a family highlights the complexity of clinical and molecular diagnosis in case of chromosomal and single gene disorder co-inheritance.

BACKGROUND: Corpus callosum agenesis (ACC) is one of the most frequent Central Nervous System (CNS) malformations. However, genetics underlying isolated forms is still poorly recognized. Here, we report on two female familial cases with partial ACC. The proband shows isolated partial ACC and a mild neurodevelopmental phenotype. A fetus from a previous interrupted pregnancy exhibited a complex phenotype including partial ACC and the occurrence of a de novo 17q12 microduplication, which was interpreted as probably disease-causing. METHODS: A trio-based clinical exome sequencing (CES) was performed. RESULTS: Clinical exome sequencing data analysis led to identifying a heterozygous nonsense variant (NM_139058.3:c.922G>T; NP_620689.1:p.Glu308Ter) in the aristaless related homeobox gene (ARX) in the proband, with a putative de novo occurrence, producing a hypothetical protein lacking two essential domains. Sanger analysis confirmed the wild-type status of both parents in different tissues, and disclosed the occurrence of the nonsense variant in the fetus of the interrupted pregnancy, suggesting a formerly unrecognized contribution of the ARX mutation to the fetus' phenotype and gonadal or gonadosomatic mosaicism in one of the parents. CONCLUSION: This study describes the phenotype associated with a heterozygous loss of function variant in ARX. Moreover, it highlights the importance of investigating both chromosomal and genetic contributions in cases of complex syndromic phenotypes involving CNS.

TLR4 T399I Polymorphism and Endometriosis in a Cohort of Italian Women.

BACKGROUND: Endometriosis is a widespread multifactorial disease in which environmental, genetic, and epigenetic factors contribute to the phenotype. Single Nucleotide Polymorphisms (SNPs) in genes implicated in pivotal molecular mechanisms have been investigated as susceptible risk factors in distinct populations. Among these, Toll-like receptor 4 (TLR4) represents a good candidate due to its role in the immune/inflammatory response and endometriosis pathogenesis. METHODS: The TRL4 gene T399I SNP (C/T transition, rs4986791) was investigated in 236 Italian endometriosis patients and 150 controls by using the PCR-RFLP method. One-tailed Fisher's exact test was used to compare differences between categorical variables. T399I genotype distribution was evaluated for Hardy-Weinberg equilibrium in both groups using the Chi-squared test for given probabilities. RESULTS: Fisher's exact test comparing C and T allele frequencies showed a difference in the frequency of T alleles between patients and controls (OR = 1.96, 95% confidence interval 0.91-4.23; p-value = 0.0552). Genotype frequencies did not show any significant difference between patients and controls. The homozygous TT genotype was observed in 2% of endometriosis women and not in controls. CONCLUSIONS: Our results show that the TLR4 rs4986791 T variant may be considered a genetic risk factor for endometriosis in Italian women. More extensive studies in other populations are needed to confirm this result.

Prenatal whole exome sequencing detects a new homozygous fukutin (FKTN) mutation in a fetus with an ultrasound suspicion of familial Dandy-Walker malformation.

BACKGROUND: Posterior fossa malformations are among the most diagnosed central nervous system (CNS) anomalies detected by ultrasound (US) in prenatal age. We identified the pathogenic gene mutation in a male fetus of 17 weeks of gestation with US suspicion of familial Dandy-Walker spectrum malformation, using Next Generation Sequencing approach in prenatal diagnosis. METHODS: Whole exome sequencing (WES) approach has been performed on fetal genomic DNA. After reads preprocessing, mapping, variant calling, and annotation, a filtering strategy based on allelic frequency, recessive inheritance, and phenotypic ontologies has been applied. A fetal magnetic resonance imaging (MRI) at 18 weeks of gestation has been performed. An in silico analysis of a potential causative missense variant in the fukutin protein has been carried out through a structural modeling approach. RESULTS: We identified a new homozygous missense mutation in fukutin gene (FKTN, NM_006731.2: c.898G>A; NP_006722.2: p.Gly300Arg). Fetal MRI supported molecular findings. Structural modeling analyses indicated a potential pathogenetic mechanism of the variant, through a reduced activation of the sugar moieties, which in turn impairs transfer to dystroglycan and thus its glycosylation. These findings pointed to a redefinition of the US suspicion of recurrence of Dandy-Walker malformation (DWM) to a muscular dystrophy-dystroglycanopathy type A4. CONCLUSIONS: The present case confirmed WES as a reliable tool for the prenatal identification of the molecular bases of early-detected CNS malformations.

Whole exome sequencing in an Italian family with isolated maxillary canine agenesis and canine eruption anomalies.

OBJECTIVE: The aim of this study was the clinical and molecular characterization of a family segregating a trait consisting of a phenotype specifically involving the maxillary canines, including agenesis, impaction and ectopic eruption, characterized by incomplete penetrance and variable expressivity. DESIGN: Clinical standardized assessment of 14 family members and a whole-exome sequencing (WES) of three affected subjects were performed. WES data analyses (sequence alignment, variant calling, annotation and prioritization) were carried out using an in-house implemented pipeline. Variant filtering retained coding and splice-site high quality private and rare variants. Variant prioritization was performed taking into account both the disruptive impact and the biological relevance of individual variants and genes. Sanger sequencing was performed to validate the variants of interest and to carry out segregation analysis. RESULTS: Prioritization of variants "by function" allowed the identification of multiple variants contributing to the trait, including two concomitant heterozygous variants in EDARADD (c.308C>T, p.Ser103Phe) and COL5A1 (c.1588G>A, p.Gly530Ser), specifically associated with a more severe phenotype (i.e. canine agenesis). Differently, heterozygous variants in genes encoding proteins with a role in the WNT pathway were shared by subjects showing a phenotype of impacted/ectopic erupted canines. CONCLUSIONS: This study characterized the genetic contribution underlying a complex trait consisting of isolated canine anomalies in a medium-sized family, highlighting the role of WNT and EDA cell signaling pathways in tooth development.