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.

Functional Study of SNCA p.V15A Variant: Further Linking α-Synuclein and Glucocerebrosidase.

BACKGROUND: SNCA p.V15A was reported in five families. In vitro models showed increased aggregation and seeding activity, mitochondrial damage, and apoptosis. Mutant flies had reduced flying ability and survival. OBJECTIVES: To clinically and functionally evaluate SNCA p.V15A in a large Italian family with Parkinson's disease (PD). METHODS: Genetic diagnosis was reached through next-generation sequencing. Pathogenicity was assessed by molecular dynamics simulation and biochemical studies on peripheral blood mononuclear cells (PBMCs). RESULTS: Five siblings carried SNCA p.V15A; three developed bradykinetic-rigid PD in their 50s with rapid motor progression and variable cognitive impairment. A fourth sibling had isolated mood disturbance, whereas the fifth was still unaffected at age 47. The mutant protein showed decreased stability and an unstable folded structure. Proband's PBMCs showed elevated total and phosphorylated α-synuclein (α-syn) levels and significantly reduced glucocerebrosidase activity. CONCLUSION: This study demonstrates accumulation of α-synV15A in PBMCs and strengthens the link between α-syn pathophysiology and glucocerebrosidase dysfunction. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Prodromal Cognitive Changes as a Prognostic Indicator of Forthcoming Huntington's Disease Severity: A Retrospective Longitudinal Study.

BACKGROUND: Cognitive changes in Huntington's disease (HD) precede motor manifestations. ENROLL-HD platform includes four cognitive measures of information processing speed (IPS). Our group is eager to seek clinical markers in the life stage that is as close as possible to the age of onset (ie, the so called prodromal HD phase) because this is the best time for therapeutic interventions. OBJECTIVES: Our study aimed to test whether cognitive scores in prodromal ENROLL-HD mutation carriers show the potential to predict the severity of motor and behavioral changes once HD became fully manifested. METHODS: From the global ENROLL-HD cohort of 21,343 participants, we first selected a premanifest Cohort#1 (ie, subjects with Total Motor Score (TMS) <10 and Diagnostic Confidence Level (DCL) <4, N = 1.222). From this cohort, we then focused on a prodromal Cohort#2 of subjects who were ascertained to phenoconvert into manifest HD at follow-up visits (ie, subjects from 6 ≤ TMS≤9 and DCL <4 to TMS≥10 and DCL = 4, n = 206). RESULTS: The main results of our study showed that low IPS before phenoconversion in Cohort#2 predicted the severity of motor and behavioral manifestations. By combining the four IPS cognitive measures (eg, the Categorical Verbal Fluency Test; Stroop Color Naming Test; Stroop Word Reading; Symbol Digit Modalities Test), we generated a Composite Cognition Score (CCS). The lower the CCS score the higher the TMS and the apathy scores in the same longitudinally followed-up patients after phenoconversion. CONCLUSIONS: CCS might represent a clinical instrument to predict the prognosis of mutation carriers who are close to manifesting HD.

Observation of a single protein by ultrafast X-ray diffraction.

The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.

The Notch1 signaling pathway directly modulates the human RANKL-induced osteoclastogenesis.

Notch signaling is an evolutionary conserved pathway with a key role in tissue homeostasis, differentiation and proliferation. It was reported that Notch1 receptor negatively regulates mouse osteoclast development and formation by inhibiting the expression of macrophage colony-stimulating factor in mesenchymal cells. Nonetheless, the involvement of Notch1 pathway in the generation of human osteoclasts is still controversial. Here, we report that the constitutive activation of Notch1 signaling induced a differentiation block in human mononuclear CD14+ cells directly isolated from peripheral blood mononuclear cells (PBMCs) upon in vitro stimulation to osteoclasts. Additionally, using a combined approach of single-cell RNA sequencing (scRNA-Seq) simultaneously with a panel of 31 oligo-conjugated antibodies against cell surface markers (AbSeq assay) as well as unsupervised learning methods, we detected four different cell stages of human RANKL-induced osteoclastogenesis after 5 days in which Notch1 signaling enforces the cell expansion of specific subsets. These cell populations were characterized by distinct gene expression and immunophenotypic profiles and active Notch1, JAK/STAT and WNT signaling pathways. Furthermore, cell-cell communication analyses revealed extrinsic modulators of osteoclast progenitors including the IL7/IL7R and WNT5a/RYK axes. Interestingly, we also report that Interleukin-7 receptor (IL7R) was a downstream effector of Notch1 pathway and that Notch1 and IL7R interplay promoted cell expansion of human RANKL-induced osteoclast progenitors. Taken together, these findings underline a novel cell pattern of human osteoclastogenesis, outlining the key role of Notch1 and IL-7R signaling pathways.

Molecular dynamics analysis of superoxide dismutase 1 mutations suggests decoupling between mechanisms underlying ALS onset and progression.

Mutations in the superoxide dismutase 1 (SOD1) gene are the second most common known cause of ALS. SOD1 variants express high phenotypic variability and over 200 have been reported in people with ALS. It was previously proposed that variants can be broadly classified in two groups, 'wild-type like' (WTL) and 'metal binding region' (MBR) variants, based on their structural location and biophysical properties. MBR variants, but not WTL variants, were associated with a reduction of SOD1 enzymatic activity. In this study we used molecular dynamics and large clinical datasets to characterise the differences in the structural and dynamic behaviour of WTL and MBR variants with respect to the wild-type SOD1, and how such differences influence the ALS clinical phenotype. Our study identified marked structural differences, some of which are observed in both variant groups, while others are group specific. Moreover, collecting clinical data of approximately 500 SOD1 ALS patients carrying variants, we showed that the survival time of patients carrying an MBR variant is generally longer (∼6 years median difference, p < 0.001) with respect to patients with a WTL variant. In conclusion, our study highlighted key differences in the dynamic behaviour between WTL and MBR SOD1 variants, and between variants and wild-type SOD1 at an atomic and molecular level, that could be further investigated to explain the associated phenotypic variability. Our results support the hypothesis of a decoupling between mechanisms of onset and progression of SOD1 ALS, and an involvement of loss-of-function of SOD1 with the disease progression.

Integrative CUT&Tag-RNA-Seq analysis of histone variant macroH2A1-dependent orchestration of human induced pluripotent stem cell reprogramming.

Aim: Human induced pluripotent stem cells (iPSCs) are inefficiently derived from somatic cells by overexpression of defined transcription factors. Overexpression of H2A histone variant macroH2A1.1, but not macroH2A1.2, leads to increased iPSC reprogramming by unclear mechanisms. Materials & methods: Cleavage under targets and tagmentation (CUT&Tag) allows robust epigenomic profiling of a low cell number. We performed an integrative CUT&Tag-RNA-Seq analysis of macroH2A1-dependent orchestration of iPSCs reprogramming using human endothelial cells. Results: We demonstrate wider genome occupancy, predicted transcription factors binding, and gene expression regulated by macroH2A1.1 during reprogramming, compared to macroH2A1.2. MacroH2A1.1, previously associated with neurodegenerative pathologies, specifically activated ectoderm/neural processes. Conclusion: CUT&Tag and RNA-Seq data integration is a powerful tool to investigate the epigenetic mechanisms occurring during cell reprogramming.

Identification of a Novel FOXP1 Variant in a Patient with Hypotonia, Intellectual Disability, and Severe Speech Impairment.

The FOXP subfamily includes four different transcription factors: FOXP1, FOXP2, FOXP3, and FOXP4, all with important roles in regulating gene expression from early development through adulthood. Haploinsufficiency of FOXP1, due to deleterious variants (point mutations, copy number variants) disrupting the gene, leads to an emerging disorder known as "FOXP1 syndrome", mainly characterized by intellectual disability, language impairment, dysmorphic features, and multiple congenital abnormalities with or without autistic features in some affected individuals (MIM 613670). Here we describe a 10-year-old female patient, born to unrelated parents, showing hypotonia, intellectual disability, and severe language delay. Targeted resequencing analysis allowed us to identify a heterozygous de novo FOXP1 variant c.1030C>T, p.(Gln344Ter) classified as likely pathogenetic according to the American College of Medical Genetics and Genomics guidelines. To the best of our knowledge, our patient is the first to date to report carrying this stop mutation, which is, for this reason, useful for broadening the molecular spectrum of FOXP1 clinically relevant variants. In addition, our results highlight the utility of next-generation sequencing in establishing an etiological basis for heterogeneous conditions such as neurodevelopmental disorders and providing additional insight into the phenotypic features of FOXP1-related syndrome.

GLUT-1 changes in paediatric Huntington disease brain cortex and fibroblasts: an observational case-control study.

BACKGROUND: Paediatric Huntington disease with highly expanded mutations (HE-PHD; >80 CAG repeats) presents atypically, compared to adult-onset Huntington disease (AOHD), with neurodevelopmental delay, epilepsy, abnormal brain glucose metabolism, early striatal damage, and reduced lifespan. Since genetic GLUT-1 deficiency syndrome shows a symptom spectrum similar to HE-PHD, we investigated the potential role of the two main glucose transporters, GLUT-1 and GLUT-3, in HE-PHD. METHODS: We compared GLUT-1 and GLUT-3 protein expression in HE-PHD, juvenile-onset (JOHD), and AOHD brains (n = 2; n = 3; n = 6) and periphery (n = 3; n = 2; n = 2) versus healthy adult controls (n = 6; n = 6). We also investigated mitochondrial complexes and hexokinase-II protein expression. FINDINGS: GLUT-1 and GLUT-3 expression were significantly lower in HE-PHD frontal cortex (p = 0.009, 95% [CI 13.4, 14.7]; p = 0.017, 95% [CI 14.2, 14.5]) versus controls. In fibroblasts, GLUT-1 and GLUT-3 expression were lower compared to controls (p < 0.0001, 95% [CI 0.91, 1.09]; p = 0.046, 95% [CI 0.93, 1.07]). In the frontal cortex, this occurred without evidence of extensive neuronal degeneration. Patients with HE-PHD had deregulated mitochondrial complex expression, particularly complexes II-III, levels of which were lower in frontal cortex versus controls (p = 0.027, 95% [CI 17.1, 17.6]; p = 0.002, 95% CI [16.6, 16.9]) and patients with AOHD (p = 0.052, 95% [CI 17.0, 17.6]; p = 0.002, 95% [CI 16.6, 16.7]). Hexokinase-II expression was also lower in HE-PHD frontal cortex and striatum versus controls (p = 0.010, 95% [CI 17.8, 18.2]; p = 0.045, 95% [CI 18.6, 18.7]) and in frontal cortex versus patients with AOHD (p = 0.013, 95% [CI 17.7, 18.1]). Expression JOHD levels were consistently different to those of HE-PHD but similar to those of AOHD. INTERPRETATION: Our data suggest a dysfunctional hypometabolic state occurring specifically in paediatric Huntington disease brains. FUNDING: '5 × 1000' Personal Income Tax donation to LIRH Foundation; Italian Ministry of HealthRC2301MH04 and RF-2016-02364123 to CSS.

Multiple-core-hole resonance spectroscopy with ultraintense X-ray pulses.

Understanding the interaction of intense, femtosecond X-ray pulses with heavy atoms is crucial for gaining insights into the structure and dynamics of matter. One key aspect of nonlinear light-matter interaction was, so far, not studied systematically at free-electron lasers-its dependence on the photon energy. Here, we use resonant ion spectroscopy to map out the transient electronic structures occurring during the complex charge-up pathways of xenon. Massively hollow atoms featuring up to six simultaneous core holes determine the spectra at specific photon energies and charge states. We also illustrate how different X-ray pulse parameters, which are usually intertwined, can be partially disentangled. The extraction of resonance spectra is facilitated by the possibility of working with a constant number of photons per X-ray pulse at all photon energies and the fact that the ion yields become independent of the peak fluence beyond a saturation point. Our study lays the groundwork for spectroscopic investigations of transient atomic species in exotic, multiple-core-hole states that have not been explored previously.

Generation of Large Vortex-Free Superfluid Helium Nanodroplets.

Superfluid helium nanodroplets are an ideal environment for the formation of metastable, self-organized dopant nanostructures. However, the presence of vortices often hinders their formation. Here, we demonstrate the generation of vortex-free helium nanodroplets and explore the size range in which they can be produced. From x-ray diffraction images of xenon-doped droplets, we identify that single compact structures, assigned to vortex-free aggregation, prevail up to 10^{8} atoms per droplet. This finding builds the basis for exploring the assembly of far-from-equilibrium nanostructures at low temperatures.

The ubiquitin ligase TRIM32 promotes the autophagic response to Mycobacterium tuberculosis infection in macrophages.

Mycobacterium tuberculosis (Mtb) is known to evade host immune responses and persist in macrophages for long periods. A mechanism that the host uses to combat Mtb is xenophagy, a selective form of autophagy that targets intracellular pathogens for degradation. Ubiquitination of Mtb or Mtb-containing compartments is a key event to recruit the autophagy machinery and mediate the bacterial delivery to the lysosome. This event relies on the coordinated and complementary activity of different ubiquitin ligases, including PARKIN, SMURF1, and TRIM16. Because each of these factors is responsible for the ubiquitination of a subset of the Mtb population, it is likely that additional ubiquitin ligases are employed by macrophages to trigger a full xenophagic response during Mtb infection. In this study, we investigated the role TRIM proteins whose expression is modulated in response to Mtb or BCG infection of primary macrophages. These TRIMs were ectopically expressed in THP1 macrophage cell line to assess their impact on Mtb replication. This screening identified TRIM32 as a novel player involved in the intracellular response to Mtb infection, which promotes autophagy-mediated Mtb degradation. The role of TRIM32 in xenophagy was further confirmed by silencing TRIM32 expression in THP1 cells, which causes increased intracellular growth of Mtb associated to impaired Mtb ubiquitination, reduced recruitment of the autophagy proteins NDP52/CALCOCO2 and BECLIN 1/BECN1 to Mtb and autophagosome formation. Overall, these findings suggest that TRIM32 plays an important role in the host response to Mtb infection through the induction of autophagy, representing a promising target for host-directed tuberculosis therapies.

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.

Mucosal Microbiota from Colorectal Cancer, Adenoma and Normal Epithelium Reveals the Imprint of Fusobacterium nucleatum in Cancerogenesis.

An increasing amount of evidence suggests the emerging role of the gut microbiota in the development of colorectal cancer (CRC). This study aimed to elucidate the architecture of microbial communities within normal and neoplastic colonic mucosa. METHODS: Microbiota were analyzed by NGS and by an ensemble of metagenomics analysis tools in a total of 69 tissues from 9 patients with synchronous colorectal neoplasia and adenomas (27 specimens: 9 from normal tissues, 9 adenomas, and 9 tumours), 16 patients with only colonic adenomas (32 specimens: 16 from normal tissues and 16 adenomas), and from healthy subjects (10 specimens of normal mucosa). RESULTS: Weak differences were observed in alpha and beta metrics among the synchronous tissues from CRC and controls. Through pairwise differential abundance analyses of sample groups, an increasing trend of Rikenellaceae, Pseudomonas and Fusobacterium, and decreasing trends of Staphylococcus, Actinobacillus and Gemmiger were observed in CRC, while Staphylococcus and Bifidobacterium were decreased in patients with only adenomas. At RT-qPCR analysis, Fusobacterium nucleatum was significantly enriched in all the tissues of subjects with synchronous colorectal neoplasia. CONCLUSION: Our findings provide a comprehensive view of the human mucosa-associated gut microbiota, emphasizing global microbial diversity mostly in synchronous lesions and proving the constant presence of Fusobacterium nucleatum, with its ability to drive carcinogenesis.

Harmonic radiation contribution and X-ray transmission at the Small Quantum Systems instrument of European XFEL.

Transmission measurements of the soft X-ray beamline to the Small Quantum Systems (SQS) scientific instrument at the SASE3 undulator of European XFEL are presented. Measurements are reported for a wide range of photon energies (650 eV to 2400 eV), using X-ray gas monitors as well as a bolometric radiometer. The results are in good agreement with simulations for the beam transport and show a transmission of up to 80% over the whole photon energy range. The contribution of second- and third-harmonic radiation of the soft X-ray undulator is determined at selected photon energies by performing transmission measurements using a gas absorber to provide variable attenuation of the incoming photon flux. A comparison of the results with semi-analytic calculations for the generation of free-electron laser pulses in the SASE3 undulator reveals an influence of apertures along the beam transport on the exact harmonic content to be accounted for at the experiment. The second-harmonic content is measured to be in the range of 0.1% to 0.3%, while the third-harmonic contributed a few percent to the SASE3 emission. For experiments at the SQS instrument, these numbers can be reduced through specific selections of the mirror reflection angles.

The beam transport system for the Small Quantum Systems instrument at the European XFEL: optical layout and first commissioning results.

The Small Quantum Systems instrument is one of the six operating instruments of the European XFEL, dedicated to the atomic, molecular and cluster physics communities. The instrument started its user operation at the end of 2018 after a commissioning phase. The design and characterization of the beam transport system are described here. The X-ray optical components of the beamline are detailed, and the beamline performances, transmission and focusing capabilities are reported. It is shown that the X-ray beam can be effectively focused as predicted by ray-tracing simulations. The impact of non-ideal X-ray source conditions on the focusing performances is discussed.

Recurrent, founder and hypomorphic variants contribute to the genetic landscape of Joubert syndrome.

BACKGROUND: Joubert syndrome (JS) is a neurodevelopmental ciliopathy characterised by a distinctive mid-hindbrain malformation, the 'molar tooth sign'. Over 40 JS-associated genes are known, accounting for two-thirds of cases. METHODS: While most variants are novel or extremely rare, we report on 11 recurring variants in seven genes, including three known 'founder variants' in the Ashkenazi Jewish, Hutterite and Finnish populations. We evaluated variant frequencies in ~550 European patients with JS and compared them with controls (>15 000 Italian plus gnomAD), and with an independent cohort of ~600 JS probands from the USA. RESULTS: All variants were markedly enriched in the European JS cohort compared with controls. When comparing allele frequencies in the two JS cohorts, the Ashkenazim founder variant (TMEM216 c.218G>T) was significantly enriched in American compared with European patients with JS, while MKS1 c.1476T>G was about 10 times more frequent among European JS. Frequencies of other variants were comparable in the two cohorts. Genotyping of several markers identified four novel European founder haplotypes.Two recurrent variants (MKS1 c.1476T>G and KIAA0586 c.428delG), have been detected in homozygosity in unaffected individuals, suggesting they could act as hypomorphic variants. However, while fibroblasts from a MKS1 c.1476T>G healthy homozygote showed impaired ability to form primary cilia and mildly reduced ciliary length, ciliary parameters were normal in cells from a KIAA0586 c.428delG healthy homozygote. CONCLUSION: This study contributes to understand the complex genetic landscape of JS, explain its variable prevalence in distinct geographical areas and characterise two recurrent hypomorphic variants.

Further case of enlarged spinal nerve roots in KRAS-related Noonan syndrome.

Noonan syndrome (NS) belongs to RASopathies, a family of disorders caused by unregulated signaling through the RAS-MAPK pathway. Herein, we report on an individual with molecularly confirmed diagnosis of NS showing asymptomatic enlarged spinal nerve roots, which are distinctive features of neurofibromatosis type 1. To date, a total of 16 patients with neurogenic tumors resembling neurofibromas/schwannomas and a molecularly confirmed diagnosis of a non-NF1 RASopathy have been reported, adding this further feature shared among RASopathies.