Structural and Dynamic Analyses of Pathogenic Variants in PIK3R1 Reveal a Shared Mechanism Associated among Cancer, Undergrowth, and Overgrowth Syndromes.

The PI3K enzymes modify phospholipids to regulate cell growth and differentiation. Somatic variants in PI3K are recurrent in cancer and drive a proliferative phenotype. Somatic mosaicism of PIK3R1 and PIK3CA are associated with vascular anomalies and overgrowth syndromes. Germline PIK3R1 variants are associated with varying phenotypes, including immunodeficiency or facial dysmorphism with growth delay, lipoatrophy, and insulin resistance associated with SHORT syndrome. There has been limited study of the molecular mechanism to unify our understanding of how variants in PIK3R1 drive both undergrowth and overgrowth phenotypes. Thus, we compiled genomic variants from cancer and rare vascular anomalies and sought to interpret their effects using an unbiased physics-based simulation approach for the protein complex. We applied molecular dynamics simulations to mechanistically understand how genetic variants affect PIK3R1 and its interactions with PIK3CA. Notably, iSH2 genetic variants associated with undergrowth destabilize molecular interactions with the PIK3CA receptor binding domain in simulations, which is expected to decrease activity. On the other hand, overgrowth and cancer variants lead to loss of inhibitory interactions in simulations, which is expected to increase activity. We find that all disease variants display dysfunctions on either structural characteristics or intermolecular interaction energy. Thus, this comprehensive characterization of novel mosaic somatic variants associated with two opposing phenotypes has mechanistic importance and biomedical relevance and may aid in future therapeutic developments.

SeATAC: a tool for exploring the chromatin landscape and the role of pioneer factors.

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) reveals chromatin accessibility across the genome. Currently, no method specifically detects differential chromatin accessibility. Here, SeATAC uses a conditional variational autoencoder model to learn the latent representation of ATAC-seq V-plots and outperforms MACS2 and NucleoATAC on six separate tasks. Applying SeATAC to several pioneer factor-induced differentiation or reprogramming ATAC-seq datasets suggests that induction of these factors not only relaxes the closed chromatin but also decreases chromatin accessibility of 20% to 30% of their target sites. SeATAC is a novel tool to accurately reveal genomic regions with differential chromatin accessibility from ATAC-seq data.

FOXK1 regulates Wnt signalling to promote cardiogenesis.

AIMS: Congenital heart disease (CHD) is the most common genetic birth defect, which has considerable morbidity and mortality. We focused on deciphering key regulators that govern cardiac progenitors and cardiogenesis. FOXK1 is a forkhead/winged helix transcription factor known to regulate cell cycle kinetics and is restricted to mesodermal progenitors, somites, and heart. In the present study, we define an essential role for FOXK1 during cardiovascular development. METHODS AND RESULTS: We used the mouse embryoid body system to differentiate control and Foxk1 KO embryonic stem cells into mesodermal, cardiac progenitor cells and mature cardiac cells. Using flow cytometry, immunohistochemistry, cardiac beating, transcriptional and chromatin immunoprecipitation quantitative polymerase chain reaction assays, bulk RNA sequencing (RNAseq) and assay for transposase-accessible chromatin using sequencing (ATACseq) analyses, FOXK1 was observed to be an important regulator of cardiogenesis. Flow cytometry analyses revealed perturbed cardiogenesis in Foxk1 KO embryoid bodies (EBs). Bulk RNAseq analysis at two developmental stages showed a significant reduction of the cardiac molecular program in Foxk1 KO EBs compared to the control EBs. ATACseq analysis during EB differentiation demonstrated that the chromatin landscape nearby known important regulators of cardiogenesis was significantly relaxed in control EBs compared to Foxk1 KO EBs. Furthermore, we demonstrated that in the absence of FOXK1, cardiac differentiation was markedly impaired by assaying for cardiac Troponin T expression and cardiac contractility. We demonstrate that FOXK1 is an important regulator of cardiogenesis by repressing the Wnt/ß-catenin signalling pathway and thereby promoting differentiation. CONCLUSION: These results identify FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development. Mechanistically, FOXK1 represses Wnt signalling to promote the development of cardiac progenitor cells.

Etv2 regulates enhancer chromatin status to initiate Shh expression in the limb bud.

Sonic hedgehog (Shh) is essential for limb development, and the mechanisms that govern the propagation and maintenance of its expression has been well studied; however, the mechanisms that govern the initiation of Shh expression are incomplete. Here we report that ETV2 initiates Shh expression by changing the chromatin status of the developmental limb enhancer, ZRS. Etv2 expression precedes Shh in limb buds, and Etv2 inactivation prevents the opening of limb chromatin, including the ZRS, resulting in an absence of Shh expression. Etv2 overexpression in limb buds causes nucleosomal displacement at the ZRS, ectopic Shh expression, and polydactyly. Areas of nucleosome displacement coincide with ETS binding site clusters. ETV2 also functions as a transcriptional activator of ZRS and is antagonized by ETV4/5 repressors. Known human polydactyl mutations introduce novel ETV2 binding sites in the ZRS, suggesting that ETV2 dosage regulates ZRS activation. These studies identify ETV2 as a pioneer transcription factor (TF) regulating the onset of Shh expression, having both a chromatin regulatory role and a transcriptional activation role.

Gain and loss of TASK3 channel function and its regulation by novel variation cause KCNK9 imprinting syndrome.

BACKGROUND: Genomics enables individualized diagnosis and treatment, but large challenges remain to functionally interpret rare variants. To date, only one causative variant has been described for KCNK9 imprinting syndrome (KIS). The genotypic and phenotypic spectrum of KIS has yet to be described and the precise mechanism of disease fully understood. METHODS: This study discovers mechanisms underlying KCNK9 imprinting syndrome (KIS) by describing 15 novel KCNK9 alterations from 47 KIS-affected individuals. We use clinical genetics and computer-assisted facial phenotyping to describe the phenotypic spectrum of KIS. We then interrogate the functional effects of the variants in the encoded TASK3 channel using sequence-based analysis, 3D molecular mechanic and dynamic protein modeling, and in vitro electrophysiological and functional methodologies. RESULTS: We describe the broader genetic and phenotypic variability for KIS in a cohort of individuals identifying an additional mutational hotspot at p.Arg131 and demonstrating the common features of this neurodevelopmental disorder to include motor and speech delay, intellectual disability, early feeding difficulties, muscular hypotonia, behavioral abnormalities, and dysmorphic features. The computational protein modeling and in vitro electrophysiological studies discover variability of the impact of KCNK9 variants on TASK3 channel function identifying variants causing gain and others causing loss of conductance. The most consistent functional impact of KCNK9 genetic variants, however, was altered channel regulation. CONCLUSIONS: This study extends our understanding of KIS mechanisms demonstrating its complex etiology including gain and loss of channel function and consistent loss of channel regulation. These data are rapidly applicable to diagnostic strategies, as KIS is not identifiable from clinical features alone and thus should be molecularly diagnosed. Furthermore, our data suggests unique therapeutic strategies may be needed to address the specific functional consequences of KCNK9 variation on channel function and regulation.

ETV2 functions as a pioneer factor to regulate and reprogram the endothelial lineage.

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and is thus relevant to the treatment of ischaemic diseases, injury-induced regeneration and solid tumour growth. Previously, we demonstrated that ETV2 is an essential transcription factor for the development of cardiac, endothelial and haematopoietic lineages. Here we report that ETV2 functions as a pioneer factor that relaxes closed chromatin and regulates endothelial development. By comparing engineered embryonic stem cell differentiation and reprogramming models with multi-omics techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and recruit BRG1. BRG1 recruitment remodelled chromatin around endothelial genes and helped to maintain an open configuration, resulting in increased H3K27ac deposition. Collectively, these results will serve as a platform for the development of therapeutic initiatives directed towards cardiovascular diseases and solid tumours.

Enhanced interpretation of 935 hotspot and non-hotspot RAS variants using evidence-based structural bioinformatics.

In the current study, we report computational scores for advancing genomic interpretation of disease-associated genomic variation in members of the RAS family of genes. For this purpose, we applied 31 sequence- and 3D structure-based computational scores, chosen by their breadth of biophysical properties. We parametrized our data by assembling a numerically homogenized experimentally-derived dataset, which when use in our calculations reveal that computational scores using 3D structure highly correlate with experimental measures (e.g., GAP-mediated hydrolysis RSpearman = 0.80 and RAF affinity Rspearman = 0.82), while sequence-based scores are discordant with this data. Performing all-against-all comparisons, we applied this parametrized modeling approach to the study of 935 RAS variants from 7 RAS genes, which led us to identify 4 groups of mutations according to distinct biochemical scores within each group. Each group was comprised of hotspot and non-hotspot KRAS variants, indicating that poorly characterized variants could functionally behave like pathogenic mutations. Combining computational scores using dimensionality reduction indicated that changes to local unfolding propensity associate with changes in enzyme activity by genomic variants. Hence, our systematic approach, combining methodologies from both clinical genomics and 3D structural bioinformatics, represents an expansion for interpreting genomic data, provides information of mechanistic value, and that is transferable to other proteins.

Computational modeling reveals key molecular properties and dynamic behavior of disruptor of telomeric silencing 1-like (DOT1L) and partnering complexes involved in leukemogenesis.

Disruptor of telomeric silencing 1-like (DOT1L) is the only non-SET domain histone lysine methyltransferase (KMT) and writer of H3K79 methylation on nucleosomes marked by H2B ubiquitination. DOT1L has elicited significant attention because of its interaction or fusion with members of the AF protein family in blood cell biology and leukemogenic transformation. Here, our goal was to extend previous structural information by performing a robust molecular dynamic study of DOT1L and its leukemogenic partners combined with mutational analysis. We show that statically and dynamically, D161, G163, E186, and F223 make frequent time-dependent interactions with SAM, while additional residues T139, K187, and N241 interact with SAM only under dynamics. Dynamics models reveal DOT1L, SAM, and H4 moving as one and show that more than twice the number of DOT1L residues interacts with these partners, relative to the static structure. Mutational analyses indicate that six of these residues are intolerant to substitution. We describe the dynamic behavior of DOT1L interacting with AF10 and AF9. Studies on the dynamics of a heterotrimeric complex of DOT1L1-AF10 illuminated describe coordinated motions that impact the relative position of the DOT1L HMT domain to the nucleosome. The molecular motions of the DOT1L-AF9 complex are less extensive and highly dynamic, resembling a swivel-like mechanics. Through molecular dynamics and mutational analysis, we extend the knowledge previous provided by static measurements. These results are important to consider when describing the biochemical properties of DOT1L, under normal and in disease conditions, as well as for the development of novel therapeutic agents.

De novo coding variants in the AGO1 gene cause a neurodevelopmental disorder with intellectual disability.

BACKGROUND: High-impact pathogenic variants in more than a thousand genes are involved in Mendelian forms of neurodevelopmental disorders (NDD). METHODS: This study describes the molecular and clinical characterisation of 28 probands with NDD harbouring heterozygous AGO1 coding variants, occurring de novo for all those whose transmission could have been verified (26/28). RESULTS: A total of 15 unique variants leading to amino acid changes or deletions were identified: 12 missense variants, two in-frame deletions of one codon, and one canonical splice variant leading to a deletion of two amino acid residues. Recurrently identified variants were present in several unrelated individuals: p.(Phe180del), p.(Leu190Pro), p.(Leu190Arg), p.(Gly199Ser), p.(Val254Ile) and p.(Glu376del). AGO1 encodes the Argonaute 1 protein, which functions in gene-silencing pathways mediated by small non-coding RNAs. Three-dimensional protein structure predictions suggest that these variants might alter the flexibility of the AGO1 linker domains, which likely would impair its function in mRNA processing. Affected individuals present with intellectual disability of varying severity, as well as speech and motor delay, autistic behaviour and additional behavioural manifestations. CONCLUSION: Our study establishes that de novo coding variants in AGO1 are involved in a novel monogenic form of NDD, highly similar to the recently reported AGO2-related NDD.

SASH3 variants cause a novel form of X-linked combined immunodeficiency with immune dysregulation.

Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3), also called SH3-containing lymphocyte protein (SLY1), is a putative adaptor protein that is postulated to play an important role in the organization of signaling complexes and propagation of signal transduction cascades in lymphocytes. The SASH3 gene is located on the X-chromosome. Here, we identified 3 novel SASH3 deleterious variants in 4 unrelated male patients with a history of combined immunodeficiency and immune dysregulation that manifested as recurrent sinopulmonary, cutaneous, and mucosal infections and refractory autoimmune cytopenias. Patients exhibited CD4+ T-cell lymphopenia, decreased T-cell proliferation, cell cycle progression, and increased T-cell apoptosis in response to mitogens. In vitro T-cell differentiation of CD34+ cells and molecular signatures of rearrangements at the T-cell receptor α (TRA) locus were indicative of impaired thymocyte survival. These patients also manifested neutropenia and B-cell and natural killer (NK)-cell lymphopenia. Lentivirus-mediated transfer of the SASH3 complementary DNA-corrected protein expression, in vitro proliferation, and signaling in SASH3-deficient Jurkat and patient-derived T cells. These findings define a new type of X-linked combined immunodeficiency in humans that recapitulates many of the abnormalities reported in mice with Sly1-/- and Sly1Δ/Δ mutations, highlighting an important role of SASH3 in human lymphocyte function and survival.

Molecular mechanics and dynamic simulations of well-known Kabuki syndrome-associated KDM6A variants reveal putative mechanisms of dysfunction.

BACKGROUND: Kabuki syndrome is a genetic disorder that affects several body systems and presents with variations in symptoms and severity. The syndrome is named for a common phenotype of faces resembling stage makeup used in a Japanese traditional theatrical art named kabuki. The most frequent cause of this syndrome is mutations in the H3K4 family of histone methyltransferases while a smaller percentage results from genetic alterations affecting the histone demethylase, KDM6A. Because of the rare presentation of the latter form of the disease, little is known about how missense changes in the KDM6A protein sequence impact protein function. RESULTS: In this study, we use molecular mechanic and molecular dynamic simulations to enhance the annotation and mechanistic interpretation of the potential impact of eleven KDM6A missense variants found in Kabuki syndrome patients. These variants (N910S, D980V, S1025G, C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W, and R1351Q) are predicted to be pathogenic, likely pathogenic or of uncertain significance by sequence-based analysis. Here, we demonstrate, for the first time, that although Kabuki syndrome missense variants are found outside the functionally critical regions, they could affect overall function by significantly disrupting global and local conformation (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), chemical environment (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), and/or molecular dynamics of the catalytic domain (all variants). In addition, our approaches predict that many mutations, in particular C1153R, could allosterically disrupt the key enzymatic interactions of KDM6A. CONCLUSIONS: Our study demonstrates that the KDM6A Kabuki syndrome variants may impair histone demethylase function through various mechanisms that include altered protein integrity, local environment, molecular interactions and protein dynamics. Molecular dynamics simulations of the wild type and the variants are critical to gain a better understanding of molecular dysfunction. This type of comprehensive structure- and MD-based analyses should help develop improved impact scoring systems to interpret the damaging effects of variants in this protein and other related proteins as well as provide detailed mechanistic insight that is not currently predictable from sequence alone.

Defining the genotypic and phenotypic spectrum of X-linked MSL3-related disorder.

PURPOSE: We sought to delineate the genotypic and phenotypic spectrum of female and male individuals with X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). METHODS: Twenty-five individuals (15 males, 10 females) with causative variants in MSL3 were ascertained through exome or genome sequencing at ten different sequencing centers. RESULTS: We identified multiple variant types in MSL3 (ten nonsense, six frameshift, four splice site, three missense, one in-frame-deletion, one multi-exon deletion), most proven to be de novo, and clustering in the terminal eight exons suggesting that truncating variants in the first five exons might be compensated by an alternative MSL3 transcript. Three-dimensional modeling of missense and splice variants indicated that these have a deleterious effect. The main clinical findings comprised developmental delay and intellectual disability ranging from mild to severe. Autism spectrum disorder, muscle tone abnormalities, and macrocephaly were common as well as hearing impairment and gastrointestinal problems. Hypoplasia of the cerebellar vermis emerged as a consistent magnetic resonance image (MRI) finding. Females and males were equally affected. Using facial analysis technology, a recognizable facial gestalt was determined. CONCLUSION: Our aggregated data illustrate the genotypic and phenotypic spectrum of X-linked, MSL3-related disorder (Basilicata-Akhtar syndrome). Our cohort improves the understanding of disease related morbidity and allows us to propose detailed surveillance guidelines for affected individuals.

Structural bioinformatics enhances mechanistic interpretation of genomic variation, demonstrated through the analyses of 935 distinct RAS family mutations.

MOTIVATION: Protein-coding genetic alterations are frequently observed in Clinical Genetics, but the high yield of variants of uncertain significance remains a limitation in decision making. RAS-family GTPases are cancer drivers, but only 54 variants, across all family members, fall within well-known hotspots. However, extensive sequencing has identified 881 non-hotspot variants for which significance remains to be investigated. RESULTS: Here, we evaluate 935 missense variants from seven RAS genes, observed in cancer, RASopathies and the healthy adult population. We characterized hotspot variants, previously studied experimentally, using 63 sequence- and 3D structure-based scores, chosen by their breadth of biophysical properties. Applying scores that display best correlation with experimental measures, we report new valuable mechanistic inferences for both hot-spot and non-hotspot variants. Moreover, we demonstrate that 3D scores have little-to-no correlation with those based on DNA sequence, which are commonly used in Clinical Genetics. Thus, combined, these new knowledge bear significant relevance. AVAILABILITY AND IMPLEMENTATION: All genomic and 3D scores, and markdown for generating figures, are provided in our supplemental data. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Novel destabilizing Dynactin variant (DCTN1 p.Tyr78His) in patient with Perry syndrome.

INTRODUCTION: Perry syndrome, also recognized as Perry disease, is a rare autosomal dominant disorder characterized by midlife-onset atypical parkinsonism, apathy or depression, respiratory failure and weight loss caused by a mutation in the Dynactin (DCTN1) gene. CASE DESCRIPTION: A fifty-six years-old adopted male presented with atypical parkinsonism with bradykinesia and postural instability, apathy, weight loss, and recurrent respiratory failure due to central hypoventilation requiring tracheostomy. METHODS AND RESULTS: Clinical workup revealed a novel DCTN1 p.Tyr78His variant. Using bioinformatic protein structure modeling, we compare our patient's variant to known DCTN1 mutations and predict protein stability of each variant at the CAP-Gly domain of p150Glued. All eight variants causing Perry syndrome, as well as Tyr78His, are located at site expected to interact with MAPRE1 tail and are predicted to be destabilizing. Variants causing atypical parkinsonism with incomplete Perry syndrome phenotype (K56R and K68E) are not significantly destabilizing in silico. CONCLUSION: We propose p.Tyr78His as the ninth pathogenic DCTN1 variant causing Perry syndrome. Bioinformatic protein modeling may provide additional window to understand and interpret DCTN1 variants, as we observed non-destabilizing variants to have different phenotype than destabilizing variants.

Interaction between Mas1 and AT1RA contributes to enhancement of skeletal muscle angiogenesis by angiotensin-(1-7) in Dahl salt-sensitive rats.

The heptapeptide angiotensin-(1-7) (Ang-(1-7)) is protective in the cardiovascular system through its induction of vasodilator production and angiogenesis. Despite acting antagonistically to the effects of elevated, pathophysiological levels of angiotensin II (AngII), recent evidence has identified convergent and beneficial effects of low levels of both Ang-(1-7) and AngII. Previous work identified the AngII receptor type I (AT1R) as a component of the protein complex formed when Ang-(1-7) binds its receptor, Mas1. Importantly, pharmacological blockade of AT1R did not alter the effects of Ang-(1-7). Here, we use a novel mutation of AT1RA in the Dahl salt-sensitive (SS) rat to test the hypothesis that interaction between Mas1 and AT1R contributes to proangiogenic Ang-(1-7) signaling. In a model of hind limb angiogenesis induced by electrical stimulation, we find that the restoration of skeletal muscle angiogenesis in SS rats by Ang-(1-7) infusion is impaired in AT1RA knockout rats. Enhancement of endothelial cell (EC) tube formation capacity by Ang-(1-7) is similarly blunted in AT1RA mutant ECs. Transcriptional changes elicited by Ang-(1-7) in SS rat ECs are altered in AT1RA mutant ECs, and tandem mass spectrometry-based proteomics demonstrate that the protein complex formed upon binding of Ang-(1-7) to Mas1 is altered in AT1RA mutant ECs. Together, these data support the hypothesis that interaction between AT1R and Mas1 contributes to proangiogenic Ang-(1-7) signaling.