Noncanonical ß-catenin interactions promote leukemia-initiating activity in early T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a T-cell malignancy characterized by cell subsets and enriched with leukemia-initiating cells (LICs). ß-Catenin modulates LIC activity in T-ALL. However, its role in maintaining established leukemia stem cells remains largely unknown. To identify functionally relevant protein interactions of ß-catenin in T-ALL, we performed coimmunoprecipitation followed by liquid chromatography-mass spectrometry. Here, we report that a noncanonical functional interaction of ß-catenin with the Forkhead box O3 (FOXO3) transcription factor positively regulates LIC-related genes, including the cyclin-dependent kinase 4, which is a crucial modulator of cell cycle and tumor maintenance. We also confirm the relevance of these findings using stably integrated fluorescent reporters of ß-catenin and FOXO3 activity in patient-derived xenografts, which identify minor subpopulations with enriched LIC activity. In addition, gene expression data at the single-cell level of leukemic cells of primary patients at the time of diagnosis and minimal residual disease (MRD) up to 30 days after the standard treatments reveal that the expression of ß-catenin- and FOXO3-dependent genes is present in the CD82+CD117+ cell fraction, which is substantially enriched with LICs in MRD as well as in early T-cell precursor ALL. These findings highlight key functional roles for ß-catenin and FOXO3 and suggest novel therapeutic strategies to eradicate aggressive cell subsets in T-ALL.

COVID-19 Specific Immune Markers Revealed by Single Cell Phenotypic Profiling.

COVID-19 is a viral infection, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and characterized by a complex inflammatory process and clinical immunophenotypes. Nowadays, several alterations of immune response within the respiratory tracts as well as at the level of the peripheral blood have been well documented. Nonetheless, their effects on COVID-19-related cell heterogeneity and disease progression are less defined. Here, we performed a single-cell RNA sequencing of about 400 transcripts relevant to immune cell function including surface markers, in mononuclear cells (PBMCs) from the peripheral blood of 50 subjects, infected with SARS-CoV-2 at the diagnosis and 27 healthy blood donors as control. We found that patients with COVID-19 exhibited an increase in COVID-specific surface markers in different subsets of immune cell composition. Interestingly, the expression of cell receptors, such as IFNGR1 and CXCR4, was reduced in response to the viral infection and associated with the inhibition of the related signaling pathways and immune functions. These results highlight novel immunoreceptors, selectively expressed in COVID-19 patients, which affect the immune functionality and are correlated with clinical outcomes.

Associations between Allelic Variants of the Human IgH 3' Regulatory Region 1 and the Immune Response to BNT162b2 mRNA Vaccine.

The escalation of Coronavirus disease 2019 (COVID-19) has required the development of safe and effective vaccines against the severe acute respiratory syndrome coronavirus 2-associated (SARS-CoV-2), which is the causative agent of the disease. Here, we determined the levels of antibodies, antigen-specific B cells, against a recombinant GFP-tagged SARS-CoV-2 spike (S) protein and total T and NK cell subsets in subjects up to 20 days after the injection of the BNT162b2 (Pfizer-BioNTech) vaccine using a combined approach of serological and flow cytometry analyses. In former COVID-19 patients and highly responsive individuals, a significant increase of antibody production was detected, simultaneous with an expansion of antigen-specific B cell response and the total number of NK-T cells. Additionally, through a genetic screening of a specific polymorphic region internal to the 3' regulatory region 1 (3'RR1) of human immunoglobulin constant-gene (IgH) locus, we identified different single-nucleotide polymorphic (SNP) variants associated with either highly or lowly responsive subjects. Taken together, these results suggest that favorable genetic backgrounds and immune profiles support the progression of an effective response to BNT162b2 vaccination.

Cerebrospinal fluid levels of L-glutamate signal central inflammatory neurodegeneration in multiple sclerosis.

Excessive extracellular concentrations of L-glutamate (L-Glu) can be neurotoxic and contribute to neurodegenerative processes in multiple sclerosis (MS). The association between cerebrospinal fluid (CSF) L-Glu levels, clinical features, and inflammatory biomarkers in patients with MS remains unclear. In 179 MS patients (relapsing remitting, RR, N = 157; secondary progressive/primary progressive, SP/PP, N = 22), CSF levels of L-Glu at diagnosis were determined and compared with those obtained in a group of 40 patients with non-inflammatory/non-degenerative disorders. Disability at the time of diagnosis, and after 1 year follow-up, was assessed using the Expanded Disability Status Scale (EDSS). CSF concentrations of lactate and of a large set of pro-inflammatory and anti-inflammatory molecules were explored. CSF levels of L-Glu were slightly reduced in MS patients compared to controls. In RR-MS patients, L-Glu levels correlated with EDSS after 1 year follow-up. Moreover, in MS patients, significant correlations were found between L-Glu and both CSF levels of lactate and the inflammatory molecules interleukin (IL)-2, IL-6, and IL-1 receptor antagonist. Altered expression of L-Glu is associated with disability progression, oxidative stress, and inflammation. These findings identify CSF L-Glu as a candidate neurochemical marker of inflammatory neurodegeneration in MS.

High performance liquid chromatography determination of L-glutamate, L-glutamine and glycine content in brain, cerebrospinal fluid and blood serum of patients affected by Alzheimer's disease.

Altered glutamatergic neurotransmission is thought to play a crucial role in the progression of Alzheimer's disease (AD). Accordingly, the identification of peculiar biochemical patterns reflecting AD-related synaptopathy in blood and cerebrospinal fluid (CSF) could have relevant diagnostic and prognostic implications. In this study, we measured by High-Performance Liquid Chromatography the amount of glutamate, glutamine and glycine in post-mortem brain samples of AD patients, as well as in CSF and blood serum of drug-free subjects encompassing the whole AD clinical spectrum (pre-clinical AD, n = 18, mild cognitive impairment-AD, n = 29, dementia AD, n = 30). Interestingly, we found that glutamate and glycine levels, as well as total tau protein content, were significantly reduced in the superior frontal gyrus of patients with AD, compared with non-demented controls. No significant change was also found in glutamate, glutamine and glycine CSF concentrations between AD patients and neurological controls. Remarkably, serum glutamate levels were significantly higher in patients affected by early AD phases compared to controls, and were negatively correlated with CSF total tau levels. Conversely, serum glutamine concentration was significantly increased in AD patients, with a negative correlation with MMSE performances. Finally, we reported a significant correlation between serum L-glutamate concentrations and CDR score in female but not in male cohort of AD subjects. Overall, our results suggest that serum glutamate and glutamine levels in AD patients could vary across disease stages, potentially reflecting the progressive alteration of glutamatergic signaling during neurodegenerative processes.

Cerebrospinal fluid and serum d-serine concentrations are unaltered across the whole clinical spectrum of Alzheimer's disease.

The diagnosis of Alzheimer's disease (AD) relies on the presence of amyloidosis and tauopathy, as reflected in cerebrospinal fluid (CSF), independently from the clinical stage. Recently, CSF d-serine has been proposed as a possible new AD biomarker, reflecting dysfunctional activation of neuronal glutamatergic N-methyl-d-aspartate receptor (NMDAR). In this study, we measured blood serum and CSF concentration of two NMDAR modulators, such as d-serine and d-aspartate, in a cohort of drug-free subjects encompassing the whole AD clinical spectrum. In addition, we also analyzed d-serine levels in a cohort of post-mortem AD and control cortex samples. We reported unaltered serum and CSF concentrations of d-serine and d-aspartate in AD patients both during the AD progression and compared to non-demented controls. Accordingly, no correlation was detected between serum or CSF d-serine content and mini-mental state examination or Clinical Dementia Rating. Similarly, cortical d-serine levels were also unaltered in post-mortem samples of AD patients. Overall, our results failed to confirm previous findings indicating the CSF d-serine as a novel biomarker for AD.

Dysfunctional d-aspartate metabolism in BTBR mouse model of idiopathic autism.

BACKGROUND: Autism spectrum disorders (ASD) comprise a heterogeneous group of neurodevelopmental conditions characterized by impairment in social interaction, deviance in communication, and repetitive behaviors. Dysfunctional ionotropic NMDA and AMPA receptors, and metabotropic glutamate receptor 5 activity at excitatory synapses has been recently linked to multiple forms of ASD. Despite emerging evidence showing that d-aspartate and d-serine are important neuromodulators of glutamatergic transmission, no systematic investigation on the occurrence of these D-amino acids in preclinical ASD models has been carried out. METHODS: Through HPLC and qPCR analyses we investigated d-aspartate and d-serine metabolism in the brain and serum of four ASD mouse models. These include BTBR mice, an idiopathic model of ASD, and Cntnap2-/-, Shank3-/-, and 16p11.2+/- mice, three established genetic mouse lines recapitulating high confidence ASD-associated mutations. RESULTS: Biochemical and gene expression mapping in Cntnap2-/-, Shank3-/-, and 16p11.2+/- failed to find gross cerebral and serum alterations in d-aspartate and d-serine metabolism. Conversely, we found a striking and stereoselective increased d-aspartate content in the prefrontal cortex, hippocampus and serum of inbred BTBR mice. Consistent with biochemical assessments, in the same brain areas we also found a robust reduction in mRNA levels of d-aspartate oxidase, encoding the enzyme responsible for d-aspartate catabolism. CONCLUSIONS: Our results demonstrated the presence of disrupted d-aspartate metabolism in a widely used animal model of idiopathic ASD. GENERAL SIGNIFICANCE: Overall, this work calls for a deeper investigation of D-amino acids in the etiopathology of ASD and related developmental disorders.

Inversion variants in human and primate genomes.

For many years, inversions have been proposed to be a direct driving force in speciation since they suppress recombination when heterozygous. Inversions are the most common large-scale differences among humans and great apes. Nevertheless, they represent large events easily distinguishable by classical cytogenetics, whose resolution, however, is limited. Here, we performed a genome-wide comparison between human, great ape, and macaque genomes using the net alignments for the most recent releases of genome assemblies. We identified a total of 156 putative inversions, between 103 kb and 91 Mb, corresponding to 136 human loci. Combining literature, sequence, and experimental analyses, we analyzed 109 of these loci and found 67 regions inverted in one or multiple primates, including 28 newly identified inversions. These events overlap with 81 human genes at their breakpoints, and seven correspond to sites of recurrent rearrangements associated with human disease. This work doubles the number of validated primate inversions larger than 100 kb, beyond what was previously documented. We identified 74 sites of errors, where the sequence has been assembled in the wrong orientation, in the reference genomes analyzed. Our data serve two purposes: First, we generated a map of evolutionary inversions in these genomes representing a resource for interrogating differences among these species at a functional level; second, we provide a list of misassembled regions in these primate genomes, involving over 300 Mb of DNA and 1978 human genes. Accurately annotating these regions in the genome references has immediate applications for evolutionary and biomedical studies on primates.

Discovery of large genomic inversions using long range information.

BACKGROUND: Although many algorithms are now available that aim to characterize different classes of structural variation, discovery of balanced rearrangements such as inversions remains an open problem. This is mainly due to the fact that breakpoints of such events typically lie within segmental duplications or common repeats, which reduces the mappability of short reads. The algorithms developed within the 1000 Genomes Project to identify inversions are limited to relatively short inversions, and there are currently no available algorithms to discover large inversions using high throughput sequencing technologies. RESULTS: Here we propose a novel algorithm, VALOR, to discover large inversions using new sequencing methods that provide long range information such as 10X Genomics linked-read sequencing, pooled clone sequencing, or other similar technologies that we commonly refer to as long range sequencing. We demonstrate the utility of VALOR using both pooled clone sequencing and 10X Genomics linked-read sequencing generated from the genome of an individual from the HapMap project (NA12878). We also provide a comprehensive comparison of VALOR against several state-of-the-art structural variation discovery algorithms that use whole genome shotgun sequencing data. CONCLUSIONS: In this paper, we show that VALOR is able to accurately discover all previously identified and experimentally validated large inversions in the same genome with a low false discovery rate. Using VALOR, we also predicted a novel inversion, which we validated using fluorescent in situ hybridization. VALOR is available at https://github.com/BilkentCompGen/VALOR.

Palindromic GOLGA8 core duplicons promote chromosome 15q13.3 microdeletion and evolutionary instability.

Recurrent deletions of chromosome 15q13.3 associate with intellectual disability, schizophrenia, autism and epilepsy. To gain insight into the instability of this region, we sequenced it in affected individuals, normal individuals and nonhuman primates. We discovered five structural configurations of the human chromosome 15q13.3 region ranging in size from 2 to 3 Mb. These configurations arose recently (∼0.5-0.9 million years ago) as a result of human-specific expansions of segmental duplications and two independent inversion events. All inversion breakpoints map near GOLGA8 core duplicons-a ∼14-kb primate-specific chromosome 15 repeat that became organized into larger palindromic structures. GOLGA8-flanked palindromes also demarcate the breakpoints of recurrent 15q13.3 microdeletions, the expansion of chromosome 15 segmental duplications in the human lineage and independent structural changes in apes. The significant clustering (P = 0.002) of breakpoints provides mechanistic evidence for the role of this core duplicon and its palindromic architecture in promoting the evolutionary and disease-related instability of chromosome 15.

An emerging phenotype of interstitial 15q25.2 microdeletions: clinical report and review.

Interstitial deletions of chromosome 15q25.2 are rare. To date, only nine patients with microdeletions within this chromosomal region have been described. Here, we report on a girl with severe speech and psychomotor delay, behavioral problems and mild dysmorphic features with a 1.6 Mb deletion in 15q25.2 region. In order to study the parental origin of the rearrangement, we analyzed selected SNPs in the deleted area in the patient and her parents, showing Mendelian incompatibilities suggesting a de novo deletion on the chromosome of maternal origin. By comparing the clinical and molecular features of our patient with five previously reported cases of an overlapping deletion, we suggest that 15q25.2 deletion is an emerging syndrome characterized by a distinct although variable spectrum of clinical manifestations, including mild dysmorphic features, neurodevelopmental delay, and a recognizable pattern of congenital malformation. Furthermore, our patient is the second one in which a behavioral phenotype characterized by hyperactivity, anxiety, and autistic features was reported, indicating that these features might be part of this new syndromic condition. Breakpoints of the deletion in the patient reported here are useful to better define the smallest region of overlap (SRO) among all the patients. Selected genes that are present in the hemizygous state and which might be important for the phenotype of these patients, are discussed in context of the clinical features. In conclusion, our patient increases the knowledge about the molecular and phenotypic consequences of interstitial 15q25.2 deletions, highlighting that deletions of this region may be responsible for a new microdeletion syndrome.