Zimmermann, Michael T.

ABSTRACT The cohesin complex, composed of SMC1, SMC3, RAD21, and STAG1/STAG2, is essential for chromosome cohesion, DNA repair, and transcriptional regulation. Pathogenic variants in cohesin components cause cohesinopathies. The classical characteristics of cohesinopathies include developmental delay (DD), intellectual disability (ID), feeding difficulties, hypotonia, short stature, hearing loss, and dysmorphic features. Here, we present a 5‐year‐old boy with classical cohesinopathy features, including DD/ID and feeding difficulties, along with non‐classical features such as hypoglycemia, recurrent fever, and immunodeficiency. Trio exome sequencing identified a novel de novo missense variant of uncertain significance (NM_005862.3:c.643G>A(p.Val215Ile)) in the STAG1 gene. The variant localizes to the RAD21 interaction interface, and molecular dynamics (MD) simulations revealed conformational changes comparable to other STAG1 variants reported as likely pathogenic in patients, supporting a deleterious effect which may disrupt the STAG1‐RAD21 interaction interface. This case expands the phenotypic and molecular spectrum of STAG1‐related cohesinopathy and advances our understanding of the disease mechanism.

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) arises predominantly from activating KRAS mutations, yet individual genetic variants differ markedly in signaling output and clinical impact. G12D, the most prevalent variant, strongly drives oncogenic programs, whereas G12R signals less efficiently through the AKT and ERK pathways and is associated with longer patient survival than G12D-driven PDAC. Methods: To elucidate how these differences influence early cellular transformation, we expressed a panel of KRAS mutants in non-cancerous pancreatic ductal epithelial cells as a model of early PDAC initiation and profiled transcriptional and phospho-proteomic responses. We next examined whether epigenetic differences translate into mutation-specific changes in nuclear organization using quantitative imaging of G12D- and G12R-expressing nuclei at 24 and 48 h. Results: Each variant established a unique regulatory program enriched for chromatin remodelers, histone modifiers, and nuclear structural factors, indicating that variant-specific KRAS signaling rapidly develops divergent epigenetic states. Integrated transcriptomic and phospho-proteomic analyses identified G12D and G12R as the most divergent variants. G12D induced pronounced nuclear remodeling, including increased nuclear size, irregular morphology, and reorganization of the nucleolus and spliceosome, consistent with extensive chromatin and transcriptional reprogramming. In contrast, G12R elicited a weaker response, with minimal or delayed structural changes. Conclusions: Together, these findings demonstrate that KRAS mutational context in pancreatic ductal epithelial cells shapes early transcriptional reprogramming that actively remodels nuclear architecture and nuclear sub-compartments. This work establishes nuclear structural remodeling as a structural state of KRAS-driven epigenetic dysregulation during PDAC initiation.

Background and Aims: Metabolic dysfunction–associated steatotic liver disease affects about a third of adults worldwide and is projected soon to be the leading cause of liver cirrhosis. It occurs when fat accumulates in hepatocytes and can progress to metabolic dysfunction–associated steatohepatitis, liver cirrhosis, and HCC. Metabolic dysfunction–associated steatotic liver disease pathogenesis is believed to involve a combination of genetic and environmental risk factors. Single nucleotide polymorphisms have been implicated, but non-syndromic monogenic causes are lacking. Approach and Results: We identified a novel genetic variant in a familial case of metabolic dysfunction–associated steatohepatitis and performed deep variant functional analysis, including protein modeling, dynamics, and cell-based assays to assess molecular mechanisms of dysfunction and altered cellular signaling. We analyzed exome sequencing data of 3904 individuals with steatotic liver disease (SLD) to identify additional cases and establish the link between specific gene variants and SLD diagnosis. We discovered and functionally validated the NM_000245.4:c.3505A>T; p.(Ile1169Phe) variant in the MET (mesenchymal-epithelial transition) kinase domain as a monogenic cause of SLD. Subsequently, we detected additional ultra-rare, previously uninterpreted, and likely deleterious variants in MET from screening sequencing data. Among individuals with confirmed SLD based on electronic record review, 1.1% (45/3904) had rare predicted deleterious MET variants. Eight of 45 (17.7%) individuals had predicted deleterious variants in the MET kinase domain confirmed to be functionally like the familial case variant. Conclusions: We report the first germline nonmalignant rare MET -driven disease, a monogenic form of SLD.

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.

Abstract Histone H3 lysine 9 methylation (H3K9me), which is written by the Euchromatic Histone Lysine Methyltransferases EHMT1 and EHMT2 and read by the heterochromatin protein 1 (HP1) chromobox (CBX) protein family, is dysregulated in many types of cancers. Approaches to inhibit regulators of this pathway are currently being evaluated for therapeutic purposes. Thus, knowledge of the complexes supporting the function of these writers and readers during the process of cell proliferation is critical for our understanding of their role in carcinogenesis. Here, we immunopurified each of these proteins and used mass spectrometry to define their associated non-histone proteins, individually and at two different phases of the cell cycle, namely G1/S and G2/M. Our findings identify novel binding proteins for these writers and readers, as well as corroborate known interactors, to show the formation of distinct protein complex networks in a cell cycle phase-specific manner. Furthermore, there is an organizational switch between cell cycle phases for interactions among specific writer–reader pairs. Through a multi-tiered bioinformatics-based approach, we reveal that many interacting proteins exhibit histone mimicry, based on an H3K9-like linear motif. Gene ontology analyses, pathway enrichment, and network reconstruction inferred that these comprehensive EHMT and CBX-associated interacting protein networks participate in various functions, including transcription, DNA repair, splicing, and membrane disassembly. Combined, our data reveals novel complexes that provide insight into key functions of cell cycle-associated epigenomic processes that are highly relevant for better understanding these chromatin-modifying proteins during cell cycle and carcinogenesis.

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.

Abstract Motivation Genomic data are prevalent, leading to frequent encounters with uninterpreted variants or mutations with unknown mechanisms of effect. Researchers must manually aggregate data from multiple sources and across related proteins, mentally translating effects between the genome and proteome, to attempt to understand mechanisms. Materials and methods P2T2 presents diverse data and annotation types in a unified protein-centric view, facilitating the interpretation of coding variants and hypothesis generation. Information from primary sequence, domain, motif, and structural levels are presented and also organized into the first Paralog Annotation Analysis across the human proteome. Results Our tool assists research efforts to interpret genomic variation by aggregating diverse, relevant, and proteome-wide information into a unified interactive web-based interface. Additionally, we provide a REST API enabling automated data queries, or repurposing data for other studies. Conclusion The unified protein-centric interface presented in P2T2 will help researchers interpret novel variants identified through next-generation sequencing. Code and server link available at github.com/GenomicInterpretation/p2t2.

431 Background: Patients (pts) with localized PC do not routinely undergo comprehensive genomic profiling (CGP) unless they develop recurrent or metastatic disease. KRAS is the most frequently mutated gene in PC, however, the impact of different KRAS mutations in localized PC has not been well characterized. We interrogated our genomic database to analyze the KRAS status in PC pts who presented with localized disease at diagnosis (Dx). Methods: We identified PC pts at our institution who underwent CGP utilizing the Foundation One CDx assay and had localized disease at initial Dx; these pts were categorized into resectable/borderline resectable PC (LPC) and locally advanced PC (LAPC). All pts with LPC and LAPC underwent neoadjuvant chemotherapy and chemoradiation prior to possible surgery (all intended therapy - AIT). Tissue from metastatic sites was used for CGP in pts who developed recurrent/metastatic disease before or after completion of AIT. The primary tumor was used for CGP in pts who completed AIT without subsequent relapse or in the absence of adequate metastatic tissue. Effect of each gene on response and survival outcomes was estimated using proportional odds and Cox regression analysis, respectively, adjusting for stage. Results: 75 pts were identified, median age at Dx was 65 years, 59% were male, 65% had a primary tumor in the pancreatic head. 38 (86%) pts with LPC completed AIT compared to 21 (68%) pts with LAPC (p<0.001). KRAS mutation was detected in 95% (71/75) of pts– 94% (67/71) in codon 12 and 6% (4/71) in codon 61. The various KRAS mutations and their association with completion of AIT is summarized in the table. The likelihood of completing AIT did not differ based on KRAS wildtype (WT) vs mutated status (p =1.00), the mutated codon (codon 12 vs. codon 61; p =1.00) or the individual KRAS point mutations (p = 0.7); however, all patients with G12A (N= 1), G12C (N=1), G12L (N=1) and G12R (N=11) mutations completed AIT. KRAS status (mutated vs. WT) and the individual KRAS mutations were not associated with overall survival (OS) after adjusting for stage (p= 0.13 and p = 0.26 respectively). Median OS for patients with LPC and LAPC, was 39 months (mos) and 29 mos respectively. Conclusions: KRAS status and individual KRAS mutations did not have an impact on completing AIT or mOS; however, these findings need to be interpreted with caution due to the inherent biases involved in such analyses. The clinical significance and functional relevance of KRAS G12A, G12C, G12L and G12R mutations, though relatively rare, needs further characterization as well as mechanistic elucidation. [Table: see text]

4050 Background: Somatic mutations in KRAS, HRAS, NRAS (extended RAS) and BRAF have prognostic and predictive impact in pts with mCRC. We analyzed the prognostic impact of specific somatic mutations in extended RAS and BRAF. Methods: We retrospectively reviewed the electronic medical records of pts with mCRC at our institution who underwent comprehensive genomic profiling (CGP) utilizing the Foundation One assay. DNA was extracted from clinical specimens and CGP was performed on hybrid-capture, adaptor ligation-based libraries for up to 315 genes plus 47 introns from 19 genes frequently rearranged in cancer. BRAF mutations were classified as class I, II and III according to accepted nomenclature. Fisher’s exact test and Kaplan Meier estimates were used for statistical analyses. This project was approved by the Medical College of Wisconsin Institutional Review Board. Results: 273 pts were identified - median age at diagnosis was 57, 48% were male. Somatic mutations in extended RAS were found in 138 (50%) pts, majority being mutations in KRAS (46%). Among pts with KRAS mutations, codon 12, 13, 61 and 146 mutations accounted for 73%, 11%, 4% and 6% respectively while KRAS G12C mutations accounted for 9%. BRAF mutations were detected in 22 (8%) pts - BRAF V600E and non–V600E mutations accounting for 4.4% and 3.6% respectively. Among pts with BRAF mutations, 17 (77%) were kinase domain mutations, 16 of which could be further classified as class I (12/16), II (1/16) and III (3/16). Median overall survival (mOS) for the entire cohort was 26.4 months (mo). KRAS mutated pts had a mOS of 25.8 mo; pts with KRAS G12C mutation had a mOS of 23 mo compared to 27.1 mo for pts with other KRAS mutations (p < 0.001).Pts with BRAF mutation had a mOS of 26.2 mo; pts with BRAF V600E mutation had a mOS of 14.1 mo compared to 30.6 mo for pts with BRAF non-V600E mutations (p = 0.1). Conclusions: The poor prognosis of pts with KRAS G12C and BRAF V600E mutations compared to pts with other KRAS and BRAF mutations merit further biologic characterization with functional assays. Individualized therapeutic strategies must be conceptualized for mCRC pts with specific RAS/BRAF mutations, considering their widely disparate prognosis and putative downstream signaling mechanisms. Dynamic molecular simulation to understand conformational changes in proteins associated with specific mutations will be pivotal to optimizing precision therapeutic strategies.

ABSTRACT Inactivating mutations of the ENPP1 gene are associated with generalized arterial calcification of infancy (GACI) and less often autosomal-recessive hypophosphatemic rickets type 2 (ARHR2). We aimed to investigate the spectrum of phenotypes in a family with monoallelic and biallelic mutations of ENPP1 after identification through whole exome sequencing of a 54-year-old female with biallelic mutation of ENPP1, c.323G &gt; T; p.Cys108Phe and c.1441C &gt; T; p.Arg481Trp. Including the proband, 2 subjects had biallelic mutations, 5 had monoallelic mutations, and 2 had no mutation of ENPP1. The maternal mutation, a known pathogenic variant associated with GACI, was found in 3 subjects with monoallelic mutations, while the paternal mutation, which was not previously reported, was present in 2 subjects with monoallelic mutations. Both subjects with biallelic mutations had bowing of bilateral femurs, periarticular mineral deposition, normocalcemic primary hyperparathyroidism with multigland parathyroidectomy, increased carotid intima-media thickness, and enthesopathy was also noted in one subject. Intact FGF23 was elevated in both subjects with biallelic mutations, while C-terminal FGF23 was only elevated in one and PPi was reduced in one. Subjects with monoallelic mutations did not have periarticular calcifications or bone deformities. To conclude, patients with biallelic GACI causing mutations can survive well into adulthood, and despite the same biallelic ENPP1 pathogenic variants, clinical and biochemical manifestations can significantly differ, and include enthesopathy and primary hyperparathyroidism, which have not been previously described. Although carriers of monoallelic ENPP1 variants appear unaffected by classic disease manifestations, there may be subtle biochemical and clinical findings that warrant further investigation. © 2019 American Society for Bone and Mineral Research.