Non-canonical Variants Research Articles

Synthesis of Non-canonical Tryptophan Variants via Rh-catalyzed C-H Functionalization of Anilines.

Tryptophan and its non-canonical variants play critical roles in pharmaceutical molecules and enzymes. Facile access to this privileged class of amino acids from readily available building blocks remains a long-standing challenge. Here, we report a regioselective synthesis of non-canonical tryptophans bearing C4-C7 substituents via Rh-catalyzed annulation between structurally diverse tert-butyloxycarbonyl (Boc)-protected anilines and alkynyl chlorides readily prepared from amino acid building blocks. This transformation harnesses Boc-directed C-H metalation and demetalation to afford a wide range of C2-unsubstituted indole products in a redox-neutral fashion. This umpolung approach compared to the classic Larock indole synthesis offers a novel mechanism for heteroarene annulation and will be useful for the synthesis of natural products and drug molecules containing non-canonical tryptophan residues in a highly regioselective manner.

Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa.

Porphyromonas gingivalis , a major oral pathobiont, evades canonical host pathogen clearance in human primary gingival epithelial cells (GECs) by initiating a non-canonical variant of autophagy consisting of Microtubule-associated protein 1A/1B-light chain 3 (LC3)-rich autophagosomes, which then act as replicative niches. Simultaneously, P. gingivalis inhibits apoptosis and oxidative-stress, including extracellular-ATP (eATP)-mediated reactive-oxygen-species (ROS) production via phosphorylating Heat Shock Protein 27 (HSp27) with the bacterial nucleoside-diphosphate-kinase (Ndk). Here, we have mechanistically identified that P. gingivalis -mediated induction of HSp27 is crucial for the recruitment of the LC3 isoform, LC3C, to drive the formation of live P. gingivalis -containing Beclin1-ATG14-rich autophagosomes that are redox sensitive and non-degrading. HSp27 depletions of both infected GECs and gingiva-mimicking organotypic-culture systems resulted in the collapse of P. gingivalis -mediated autophagosomes, and abolished P. gingivalis -induced LC3C-specific autophagic-flux in a HSp27-dependent manner. Concurrently, HSp27 depletion accompanied by eATP treatment abrogated protracted Beclin 1-ATG14 partnering and decreased live intracellular P. gingivalis levels. These events were only partially restored via treatments with the antioxidant N-acetyl cysteine (NAC), which rescued the cellular redox environment independent of HSp27. Moreover, the temporal phosphorylation of HSp27 by the bacterial Ndk results in HSp27 tightly partnering with LC3C, hindering LC3C canonical cleavage, extending Beclin 1-ATG14 association, and halting canonical maturation. These findings pinpoint how HSp27 pleiotropically serves as a major platform-molecule, redox regulator, and stepwise modulator of LC3C during P. gingivalis -mediated non-canonical autophagy. Thus, our findings can determine specific molecular strategies for interfering with the host-adapted P. gingivalis ' successful mucosal colonization and oral dysbiosis.

Paroxysmal Nocturnal Haemoglobinuria (PNH) Arising from Non-Canonical Mutations

Introduction Acquired somatic mutations in the X-linked PIGA gene are the most commonly described pathological mutations found in the serious haematopoietic disorder paroxysmal nocturnal haemoglobinuria (PNH), though incidental cases of mutations in PIGT, PIGB and PIGV have been reported. Mutations in the family of 20 PIG (phosphatidylinositol glycan) genes can disrupt biosynthesis of glycosylphosphatidylinositol (GPI) anchors, critical for tethering many important proteins, including the vital complement regulators CD55 and CD59, to the cell membrane. Their absence in PNH leads to complement dysregulation and complications including potentially life-threatening haemolysis and thrombosis, which may be mitigated by lifelong anti-complement therapy. From the cohort of 273 PNH patients currently on complement inhibitor therapy at the Leeds National PNH Centre we have identified 37 patients whose diagnostic flow cytometry report or clinical course differs significantly from expectation, particularly with evidence of inflammatory presentations, which may be indicative of an alternative, non- PIGA, aetiology. Methods Patients consented to the Leeds PNH Research Tissue Bank with unusual clinical and laboratory presentations were identified, genomic DNA was extracted from peripheral blood and initially screened by targeted high sensitivity NGS of the PIGA gene. From the first ten patient samples sequenced, three lacked any evidence of PIGA mutations and so whole exome sequencing (WES) was carried out. Results Patient 1:An 80 year old man, diagnosed with MDS with rearrangement of chromosomes 3 and 7 resulting in a dicentric chromosome and loss of chromosome 20q, was diagnosed with PNH following an increase in transfusion requirement. In addition to classical symptoms of PNH he was diagnosed with seronegative rheumatoid arthritis. Flow cytometry showed an unusual PNH phenotype with a large population of partially GPI-anchor deficient cells only (Table 1). Symptoms improved on eculizumab but he remained transfusion dependent and the disease later transformed to acute myeloid leukaemia. WES: exonic frameshift insertion in PIGT (figure1), pathogenic variant rs760395465. Patient 2:A 34 year old woman presented with a 13 year history of intermittent episodes of generalized urticaria associated with nausea, vomiting, general malaise and myalgia, increasing in intensity and latterly being associated with haemoglobinuria, lethargy and jaundice leading to a PNH diagnosis. Episodes were triggered by pregnancy or infections, but she remained asymptomatic between attacks. Flow cytometry revealed a PNH clone with a majority of cells being partially GPI deficient. She responds well to C5 inhibition with no further symptoms, including urticaria. WES: exonic frameshift insertion in PIGT (figure 1), pathogenic variant rs760395465. Patient 3: A 14 year old girl presented with intermittent episodes of urticaria and severe lethargy and was diagnosed with symptomatic significant transfusion-dependent PNH, with completely GPI-deficient cells. Diagnosis followed an episode of massive haemolysis resulting in transient acute renal failure requiring haemodialysis. She commenced anticoagulation initially until eculizumab was approved 2 years later; despite occasional episodes of breakthrough haemolysis she displays no further urticaria. She has also had symptoms of diffuse arthralgia. WES: stopgain mutation in PIGV (figure1), pathogenic variant rs145160045. Conclusions These cases add to the emerging picture of non- PIGA associated PNH cases and suggest that the presence of joint problems and episodic urticaria may be sufficient indication to prompt screening for alternative causative mutations. Similar to the previously reported PIGB mutated case, the patients with PIGT mutations only had partially deficient cells. It is important to fully characterise these emerging non-canonical variants in order to improve our understanding of the pathophysiology of this disorder and to provide more individualised care.

T Cell/Natural Killer (NK) Cell Transcriptional Profiles Are Associated with Response to Gilteritinib in FLT3-Mutated Acute Myeloid Leukemia

S.R. & A.C. equally contributed Background: Despite significative improvements in the management of FLT3-mutated AML and its prognosis since FLT3 inhibitors ( FLT3i) adoption, a significant proportion of patients results refractory or ultimately relapses after this targeted treatment. Gilteritinib is an oral and selective FLT3i with single-agent activity as salvage therapy in Relapsed/Refractory patients (R/R). The investigation of resistance mechanism to Gilteritinib is crucial to guide next therapeutic choices and improve drug effectiveness. Thus far, correlative studies have elucidated mainly intrinsic and mutational resistance mechanisms. However, less is known regarding potential extrinsic mechanisms and about the role of the immune system in influencing this FLT3i activity. Herein, we aimed at investigating intrinsic as well as novel extrinsic (immunological) mechanisms of resistance to Gilteritinib in routine clinical practice. Methods: A cohort of FLT3-mut AML patients treated with Gilteritinib at Hematology Institute of Bologna was selected. DNA/RNA were extracted from bone marrow (BM) samples pre-therapy and at first response assessment and screened by a custom NGS panel covering the full-coding sequence of 28 genes and a NanoString gene-expression analysis using the PanCancer IO 360 Panel. Data were collected in accordance with GCP and Helsinki declaration. NanoString data were acquired and analyzed using the nCounter FLEX Analysis System and the nSolver advanced analysis module (version 2.0.115). DESeq2 multi-factor design was employed to account for potential confounders. A public sc-RNA-seq dataset of newly diagnosed AMLs (Dufva et al. Cancer Cell 2020) was used to map DEGs between Responders and Non-Responders to Gilteritinib onto reference AML BM samples. Single-cell gene expression scores were computed using the UCell package in R. Results: Overall, 20 FLT3-mutated R/R AML patients receiving Gilteritinib as single-agent therapy were enrolled in this study. The median age at Gilteritinib therapy was 62 (range 33 - 77) years and 13/20 (65%) patients were male. Seventeen out of 20 (85 %) patients harbored a FLT3-ITD mutation, 2/20 (10 %) a TKD mutation and 1 (5 %) patient both mutations. Eleven out of 20 (55 %) patients had a concomitant NPM1 mutation. Patients were dichotomized into Responders (R) (n=9) and Non-Responders (NR) (n = 11) according to BM response. Intrinsic mechanisms of resistance were evaluated by NGS analyses on 13 patients (R=7 and NR=6). Firstly, FLT3 full-coding sequencing did not detect any clearly pathogenetic Non-Canonical (NC) mutation: M737I, M837I, P890L and P534L were four detected NC variants with a possible role in FLT3i sensitivity, however their low VAF (1-2 %) suggests a “passenger” role in this cohort. Secondly, evaluating variations in VAF of non- FLT3 variants detected pre- and post-therapy we observed a new variant onset in 4/6 (66.7 %) Gilteritinib NR patients, affecting genes involved in RAS pathway in all cases (1 NRAS, 2 KRAS and 1 PTPN11). Significant differences in immune transcriptional profiles were observed when comparing R and NR patients (R = 9 and NR = 11), including higher expression of genes reflecting T/NK function ( CD8A, GZMK, KLRK1, GNLY) and, more specifically, T-cell activity ( PRF1, EOMES, ZAP70, TNFSF8) in responders. Conversely, oncogenes involved in MAPK/Akt ( PROM1, ANGPT1, ANGPT2) or cell-cycle regulators ( CDKN2A, CCNA1) were overexpressed in NR patients (Fig. 1a). We next selected the top 30 DEGs between R and NR and we averaged gene expression across all BM samples. Gene set enrichment analysis (GSEA) revealed that approximately 25% of the significantly enriched pathways when using the top 30 DEGs as input were related to T-cell function / TCR signaling. By projecting the top 30 DEGs genes onto a sc-RNA-seq map of the AML tumor microenvironment, we found that they were largely expressed by CD8 + T EM, CD8 + T CM and NK cells (Fig. 1b). Summary/Conclusion: This study confirmed evolutionary trajectories often involving RAS pathway mutations as an intrinsic mechanism of resistance to Gilteritinib therapy. Of note, differences in immune transcriptomic profiles, involving mainly CD8+ T-Cells and NK-Cells, were associated with response, pointing to a previously unappreciated role of the immune system in mediating response to Gilteritinib.

High-Resolution Proximity-Guided Cytogenomics Improves Patient Stratification and Uncovers New Variants in Acute Myeloid Leukemia

Cytogenetic methods are a cornerstone of the modern diagnostic workflow for acute myeloid leukemia and related disorders. The goal of these methods is to identify structural genomic aberrations that drive the cancer or otherwise act as an indicator of patient risk. Despite their success, any one cytogenetic test is limited either in resolution, ability to provide unbiased survey of the genome, or ability to detect balanced aberrations. To address these limitations, we have applied proximity ligation sequencing (PLS) to characterize structural variants (SV) in AML genomes. PLS captures ultra-long-range sequence information without high-molecular-weight DNA and requires a modest (5-7x) sequence coverage of the genome. Applying this method to AML diagnostic specimens, we find that PLS identifies cytogenetically defined translocations (reciprocal and non-reciprocal) and inversions with specificity and sensitivity >0.95. The high resolution of PLS (<10kb) detected non-canonical variants missed by standard methods as well as structural variants below limits of detection by karyotyping, identifying variants in over half the patients previously determined to have a normal karyotype (Table 1). This included previously described variants of clinical significance as well as a recurrent inversion not previously described in AML. These observations highlight the effectiveness of PLS to provide high-resolution insights into known clinically relevant variants and discover variants missed by traditional methodologies.

Individualized tumor-informed circulating tumor DNA analysis for postoperative monitoring of non-small cell lung cancer.

We report a personalized tumor-informed technology, Patient-specific pROgnostic and Potential tHErapeutic marker Tracking (PROPHET) using deep sequencing of 50 patient-specific variants to detect molecular residual disease (MRD) with a limit of detection of 0.004%. PROPHET and state-of-the-art fixed-panel assays were applied to 760 plasma samples from 181 prospectively enrolled early stage non-small cell lung cancer patients. PROPHET shows higher sensitivity of 45% at baseline with circulating tumor DNA (ctDNA). It outperforms fixed-panel assays in prognostic analysis and demonstrates a median lead-time of 299days to radiologically confirmed recurrence. Personalized non-canonical variants account for 98.2% with prognostic effects similar to canonical variants. The proposed tumor-node-metastasis-blood (TNMB) classification surpasses TNM staging for prognostic prediction at the decision point of adjuvant treatment. PROPHET shows potential to evaluate the effect of adjuvant therapy and serve as an arbiter of the equivocal radiological diagnosis. These findings highlight the potential advantages of personalized cancer techniques in MRD detection.

Proteogenomics in Cancer: Then and Now.

For years, the paths of sequencing technologies and mass spectrometry have occurred in isolation, with each developing its own unique culture and expertise. These two technologies are crucial for inspecting complementary aspects of the molecular phenotype across the central dogma. Integrative multiomics strives to bridge the analysis gap among different fields to complete more comprehensive mechanisms of life events and diseases. Proteogenomics is one integrated multiomics field. Here in this review, we mainly summarize and discuss three aspects: workflow of proteogenomics, proteogenomics applications in cancer research, and the SWOT (Strengths, Weaknesses, Opportunities, Threats) analysis of proteogenomics in cancer research. In conclusion, proteogenomics has a promising future as it clarifies the functional consequences of many unannotated genomic abnormalities or noncanonical variants and identifies driver genes and novel therapeutic targets across cancers, which would substantially accelerate the development of precision oncology.

Splicing Analysis of MYO5B Noncanonical Variants in Patients with Low Gamma-Glutamyltransferase Cholestasis

Biallelic MYO5B variants have been associated with familial intrahepatic cholestasis (FIC) with low serum gamma-glutamyltransferase (GGT). Intronic or synonymous variants outside of canonical splice sites (hereinafter referred to as noncanonical variants) with uncertain significance were identified in MYO5B posing a challenge in clinical interpretation. This study is aimed at assessing the effects of these variants on premessenger RNA (pre-mRNA) splicing to improve recognition of pathogenic spliceogenic variants in MYO5B and better characterize the MYO5B genetic variation spectrum. Disease-associated MYO5B noncanonical variants were collected from the literature or newly identified low GGT cholestasis patients. In silico splicing predictions were performed to prioritize potential pathogenic variants. Minigene splicing assays were performed to determine their splicing patterns, with confirmation by blood RNA analysis in one case. Eleven (five novel) noncanonical variants with uncertain significance were identified. Minigene splicing assays revealed that three variants (c.2090+3A>T, c.2414+5G>T, and c.613-11G>A) caused complete aberrations, five variants (c.2349A>G/p.(=), c.4221G>A/p.(=), c.1322+5G>A, c.1669-35A>C, and c.3045+3A>T) caused predominant aberrations, and three variants (c.4852+11A>G, c.455+8T>C, and c.2415-6C>G) had no effect on pre-mRNA splicing. Patient-derived RNA analysis showed consistent results. Based on our results, eight variants were reclassified as likely pathogenic and three as likely benign. Combining the clinical features and the above analysis, the diagnosis of MYO5B-associated FIC could be made in three new patients. In conclusion, we characterized the splicing patterns of MYO5B noncanonical variants and suggest that RNA analysis should be routinely included in clinical diagnostics to provide essential evidence for the interpretation of variants.

A real-world study of ALK fusion detection, treatment patterns, and survival outcomes of patients with advanced non–small-cell lung cancer (aNSCLC) in Brazil.

e18655 Background: Clinical trials have firmly established that ALK tyrosine kinase inhibitors (TKIs) are safe and effective in aNSCLC, with substantial anti-tumor activity of multiple agents across different treatment lines. Real-world ALK fusion diagnosis and targeted therapy effectiveness have not been described in Brazilian private healthcare setting, with ALK TKIs approved since 2016. Methods: This study retrospectively explored the patterns of use and outcome after initial exposure to ALK TKIs in patients with ALK positive aNSCLC in a real-world clinical cohort from Oncoclínicas Group, Brazil's largest network of community oncology practices, between 2016 and 2022. For data collection we used a platform where structured EHR variables are integrated with unstructured elements from physician notes using technology-based abstraction and expert curation. We also describe molecular epidemiology and ALK fusion variant prevalence for a subset of patients tested with an in-house RNA sequencing assay (GS180, ArcherDx) at Oncoclínicas Precision Medicine Lab from 2020 to 2022. Results: Out of 100 ALK-positive aNSCLC patients treated with ALK TKIs, median age was 60 years and 62% were female. Overall, median number of treatment lines was 2, with 29% receiving 3 or more regimens. In first line, 64% of the patients received an ALK TKI (58% crizotinib, 33% alectinib, and 9% brigatinib, ceritinib or lorlatinib). The remaining 36% were treated with chemo +/- immunotherapy first, and 28% switched to an ALK TKI right after driver diagnosis. When comparing first line treatment practice in 2020-2022 versus 2016-2019 we detected major reduction in crizotinib as first ALK TKI (83% versus 35%). Overall, 14% received 2 or more sequential ALK TKIs. With a median follow-up of 12 months, median Overall Survival (mOS) from start of first ALK TKI was 28 months (CI95% 18-NR). In first line, exposure to alectinib was associated with numerically higher mOS (36 months) when compared to crizotinib (18 months, HR = 0.58, CI95% 0.2-1.6). From 592 cases of non-squamous NSCLC cases with in-house RNA sequencing, ALK fusions were detected in 5.2%. EML4-ALK fusion was found in 84%, with most common variants being E5:A20 (non-canonical) in 41% and E13:E20 (v1) in 38%. We also found novel fusions partners HIP1, KLC1, LTK and STRN in 16%. Conclusions: The largest clinico-genomics cohort in Latin America reveals that the impact of ALK TKIs in ALK fusion aNSCLC may not be as profound in the real world as in clinical trials. Fast access to molecular diagnosis and exposure to ALK TKI in first line are being incentivized. EML4-ALK variant prevalence in Brazilian patients with aNSCLC is different from published cohorts, and the effect that non-canonical variants may have on ALK TKI activity must be studied further.

Abstract 1039: Individualized tumor-informed circulating tumor DNA (ctDNA) analysis for postoperative monitoring of non-small cell lung cancer (NSCLC) - the MEDAL study

Abstract Background: Through parallel testing and comparison of personalized and fixed panel minimal residual disease (MRD) assays, to establish the best technique and application strategy of dynamic MRD detection for prognosis prediction and disease assessment among non-small cell lung cancer (NSCLC) patients. Method: We analyzed 760 plasma samples from prospectively enrolled 181 patients with NSCLC recruited to the MEDAL study (NCT03634826), with disease stage I (63%), II (19%) and III (18%). 80% were adenocarcinomas. Plasma samples were collected at baseline (n=157), landmark 3-day and 1-month (n=334), and longitudinal points (n=248) were analyzed. Additional plasma was collected after relapse for 14 patients (n=21). Median follow-up was 1092 days, and 48 patients progressed. We employed a novel personalized tumor-informed technology named PROPHET using deep sequencing of 50 patient-specific variants. The PROPHET was developed to detect MRD with a limit of detection (LoD) of 0.004% and sample-level specificity of greater than 99% in the analytical validation. Detection and quantification of MRD through tumor-informed (TI) and tumor-agnostic (TA) fixed panel assays in the same samples were conducted for a head-to-head comparison. Results: ctDNA was detected by PROPHET prior to treatment in 45% of samples (83%, 75% and 23% for disease stage III, II and I), and showed a higher positive rate than the TI and TA assays (22% and 19%). PROPHET identified 30 more ctDNA positive patients with a median ctDNA fraction of 0.01% at baseline. From the landmark single test, the sensitivity was 45%; integrating longitudinal time points increased the sensitivity to 85%. Landmark PROPHET status was the only risk factor other than clinical features to predict the clinical relapse (p<0.001, multivariate Cox model). MRD positive patients defined by non-canonical variants (n=8) had similar disease-free survival (DFS) as MRD positive patients defined by canonical variants (n=9). Landmark PROPHET-based MRD status combined with clinical TNM stage outperformed TNM stage for prediction of prognosis (p<0.001). Longitudinal MRD achieved negative predictive value (NPV) of 99% with an interval of 150 days, and demonstrated 299 days of longer lead-time than other state-of-the-art fixed-panel assays. Among 16 patients with equivocal radiological diagnosis, all the MRD positive patients relapsed (n=6). Among relapsed patients received next-line treatments, 7 ctDNA negative patients survived or died other disease, 67% (2/3) ctDNA positive patients died from cancer. Sensitivity for bone and brain metastasis was 100% (11/11) and 50% (2/4), respectively. Conclusion: The sensitive tumor-informed personalized MRD approach could provide advantages in prognosis prediction at landmark and disease assessment during surveillance for NSCLC patients. Citation Format: Kezhong Chen, Chenyang Wang, Haifeng Shen, Xi Li, Yichen Jin, Shuailai Wu, Fujun Qiu, Qiang Lu, Di Peng, Shuai Fang, Bing Li, Juan Lv, Jinlei Song, Yang Wang, Shannon Chuai, Zhihong Zhang. Individualized tumor-informed circulating tumor DNA (ctDNA) analysis for postoperative monitoring of non-small cell lung cancer (NSCLC) - the MEDAL study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1039.

Structural origin and rational development of bright red noncanonical variants of green fluorescent protein.

The incorporation of noncanonical amino acids (ncAAs) into fluorescent proteins is promising for red-shifting their fluorescence and benefiting tissue imaging with deep penetration and low phototoxicity. However, ncAA-based red fluorescent proteins (RFPs) have been rare. The 3-aminotyrosine modified superfolder green fluorescent protein (aY-sfGFP) represents a recent advance, yet the molecular mechanism for its red-shifted fluorescence remains elusive while its dim fluorescence hinders applications. Herein, we implement femtosecond stimulated Raman spectroscopy to obtain structural fingerprints in the electronic ground state and reveal that aY-sfGFP possesses a GFP-like instead of RFP-like chromophore. Red color of aY-sfGFP intrinsically arises from a unique "double-donor" chromophore structure that raises ground-state energy and enhances charge transfer, notably differing from the conventional conjugation mechanism. We further developed two aY-sfGFP mutants (E222H and T203H) with significantly improved (∼12-fold higher) brightness by rationally restraining the chromophore's nonradiative decay through electronic and steric effects, aided by solvatochromic and fluorogenic studies of the model chromophore in solution. This study thus provides functional mechanisms and generalizable insights into ncAA-RFPs with an efficient route for engineering redder and brighter fluorescent proteins.

Spanish HTT gene study reveals haplotype and allelic diversity with possible implications for germline expansion dynamics in Huntington disease.

We aimed to determine the genetic diversity and molecular characteristics of the Huntington disease (HD) gene (HTT) in Spain. We performed an extended haplotype and exon one deep sequencing analysis of the HTT gene in a nationwide cohort of population-based controls (n = 520) and families with symptomatic individuals referred for HD genetic testing. This group included 331 HD cases and 140 carriers of intermediate alleles. Clinical and family history data were obtained when available. Spanish normal alleles are enriched in C haplotypes (40.1%), whereas A1 (39.8%) and A2 (31.6%) prevail among intermediate and expanded alleles, respectively. Alleles ≥ 50 CAG repeats are primarily associated with haplotypes A2 (38.9%) and C (32%), which are also present in 50% and 21.4%, respectively, of HD families with large intergenerational expansions. Non-canonical variants of exon one sequence are less frequent, but much more diverse, in alleles of ≥27 CAG repeats. The deletion of CAACAG, one of the six rare variants not observed among smaller normal alleles, is associated with haplotype C and appears to correlate with larger intergenerational expansions and early onset of symptoms. Spanish HD haplotypes are characterized by a high genetic diversity, potentially admixed with other non-Caucasian populations, with a higher representation of A2 and C haplotypes than most European populations. Differences in haplotype distributions across the CAG length range support differential germline expansion dynamics, with A2 and C showing the largest intergenerational expansions. This haplotype-dependent germline instability may be driven by specific cis-elements, such as the CAACAG deletion.

A sociophonetic approach to the acquisition of Spanish rhotics in a bilingual community

This study explores Spanish-English bilingual children’s acquisition of Spanish rhotics. The children’s productions are compared to a group of Spanish-English bilingual adults who represent the Spanish spoken in the same community: Albuquerque, New Mexico. In their narrations of a wordless picture-book, both children and adults produce more non-canonical than canonical variants. Binary logistic regressions run on 817 rhotics produced by 21 children, ages 3-9-years-old, and 6 adults demonstrate that non-canonical variants, instead of canonical variants are more likely to occur as Spanish use increases and in word medial position. The results point towards the propagation of a working change in Spanish rhotics that sees both articulatory and sociolinguistic motivations. Implications for research on phonetic-phonological development are also discussed

The Specificity of Formation and Use of Patronyms in the Orthodox Parish of Rajsk in the Podlasie Region in the Late 19th Century

Widespread use of patronymic names among the Orthodox residents of Podlasie in the second half of the 19th and early 20th centuries is conditioned by its administrative affiliation to the Russian Empire and consequently, the prevalence of the Russian naming formula. This paper discusses patronymics (otchestva) attested in the Rajsk parish registers in the late 19th century. Researching the structure (including word-formation, phonetic, and graphic features) and etymology of these names, the author discovers that most of patronyms were derived from the Orthodox variants of proper names (e.g. Ilіin, Sofronіev, Filimonov; Aleksіeva, Stefanova), yet some refer to the non-canonical variants of baptismal names (e.g. Bonifatov, Tarasov; Demianova, Sebestіanova, Trokhimova), differing from the original variants by phonetic alternations. From derivation perspective, short forms of patronyms (so-called semi-patronymics, poluotchestva) e.g. Ignatіev, Matfeev, Fomov; Kondratova, Stakhіeva prevail over the full forms (Petrovich, Savvich; Epifanіevna, Ippolitovna). The presence of patronyms in the personal data allows to trace family traditions of naming children spanning three generations. Regardless of the fact that all analyzed records were made by one person, the names showcase a noticeable variability in terms of motivation, word-formation, orthography, and phonetics. It was not uncommon for the different patronymics to refer to one and the same bearer, highlighting the phenomenon of several co-existing name variants in the period in question.

Emerging Role of isomiRs in Cancer: State of the Art and Recent Advances.

The advent of Next Generation Sequencing technologies brought with it the discovery of several microRNA (miRNA) variants of heterogeneous lengths and/or sequences. Initially ascribed to sequencing errors/artifacts, these isoforms, named isomiRs, are now considered non-canonical variants that originate from physiological processes affecting the canonical miRNA biogenesis. To date, accurate IsomiRs abundance, biological activity, and functions are not completely understood; however, the study of isomiR biology is an area of great interest due to their high frequency in the human miRNome, their putative functions in cooperating with the canonical miRNAs, and potential for exhibiting novel functional roles. The discovery of isomiRs highlighted the complexity of the small RNA transcriptional landscape in several diseases, including cancer. In this field, the study of isomiRs could provide further insights into the miRNA biology and its implication in oncogenesis, possibly providing putative new cancer diagnostic, prognostic, and predictive biomarkers as well. In this review, a comprehensive overview of the state of research on isomiRs in different cancer types, including the most common tumors such as breast cancer, colorectal cancer, melanoma, and prostate cancer, as well as in the less frequent tumors, as for example brain tumors and hematological malignancies, will be summarized and discussed.

An Improved Top-Down Mass Spectrometry Characterization of Chlamydomonas reinhardtii Histones and Their Post-translational Modifications.

We present an updated analysis of the linker and core histone proteins and their proteoforms in the green microalga Chlamydomonas reinhardtii by top-down mass spectrometry (TDMS). The combination of high-resolution liquid chromatographic separation, robust fragmentation, high mass spectral resolution, the application of a custom search algorithm, and extensive manual analysis enabled the characterization of 86 proteoforms across all four core histones H2A, H2B, H3, and H4 and the linker histone H1. All canonical H2A paralogs, which vary in their C-termini, were identified, along with the previously unreported noncanonical variant H2A.Z that had high levels of acetylation and C-terminal truncations. Similarly, a majority of the canonical H2B paralogs were identified, along with a smaller noncanonical variant, H2B.v1, that was highly acetylated. Histone H4 exhibited a novel acetylation profile that differs significantly from that found in other organisms. A majority of H3 was monomethylated at K4 with low levels of co-occuring acetylation, while a small fraction of H3 was trimethylated at K4 with high levels of co-occuring acetylation.

ADAR1 RNA editing enzyme regulates R-loop formation and genome stability at telomeres in cancer cells

ADAR1 is involved in adenosine-to-inosine RNA editing. The cytoplasmic ADAR1p150 edits 3’UTR double-stranded RNAs and thereby suppresses induction of interferons. Loss of this ADAR1p150 function underlies the embryonic lethality of Adar1 null mice, pathogenesis of the severe autoimmune disease Aicardi-Goutières syndrome, and the resistance developed in cancers to immune checkpoint blockade. In contrast, the biological functions of the nuclear-localized ADAR1p110 remain largely unknown. Here, we report that ADAR1p110 regulates R-loop formation and genome stability at telomeres in cancer cells carrying non-canonical variants of telomeric repeats. ADAR1p110 edits the A-C mismatches within RNA:DNA hybrids formed between canonical and non-canonical variant repeats. Editing of A-C mismatches to I:C matched pairs facilitates resolution of telomeric R-loops by RNase H2. This ADAR1p110-dependent control of telomeric R-loops is required for continued proliferation of telomerase-reactivated cancer cells, revealing the pro-oncogenic nature of ADAR1p110 and identifying ADAR1 as a promising therapeutic target of telomerase positive cancers.

Molecular heterogeneity of pyruvate kinase deficiency.

Red cell pyruvate kinase (PK) deficiency is the most common glycolytic defect associated with congenital non-spherocytic hemolytic anemia. The disease, transmitted as an autosomal recessive trait, is caused by mutations in the PKLR gene and is characterized by molecular and clinical heterogeneity; anemia ranges from mild or fully compensated hemolysis to life-threatening forms necessitating neonatal exchange transfusions and/or subsequent regular transfusion support; complications include gallstones, pulmonary hypertension, extramedullary hematopoiesis and iron overload. Since identification of the first pathogenic variants responsible for PK deficiency in 1991, more than 300 different variants have been reported, and the study of molecular mechanisms and the existence of genotype-phenotype correlations have been investigated in-depth. In recent years, during which progress in genetic analysis, next-generation sequencing technologies and personalized medicine have opened up important landscapes for diagnosis and study of molecular mechanisms of congenital hemolytic anemias, genotyping has become a prerequisite for accessing new treatments and for evaluating disease state and progression. This review examines the extensive molecular heterogeneity of PK deficiency, focusing on the diagnostic impact of genotypes and new acquisitions on pathogenic non-canonical variants. The recent progress and the weakness in understanding the genotype-phenotype correlation, and its practical usefulness in light of new therapeutic opportunities for PK deficiency are also discussed.

300 Assessment of Non-Canonical Splicing Variants in ABCC6 and Comparison with Bioinformatics Predictions

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by inactivating mutations in the ABCC6 gene predominantly expressed in hepatocytes. Different types of mutations have been identified in ABCC6 including canonical splice site mutations affecting pre-mRNA splicing. Non-canonical splicing variants near or within exon-intron boundary in ABCC6 are under-recognized due to the lack of liver biopsies from patients carrying such variants, and consequently, inability to determine their consequences on splicing by transcriptional analysis. The interpretation of such variants has primarily relied on the prediction by in silico algorithms; often such programs do not provide consensus predictions. Among seven non-canonical splicing variants identified in ABCC6 in patients with PXE, five variants were found to affect splicing in in vitro minigene splicing assays in transfected HepG2 cells. Four of the variants, c.1868-5T>G, c.2070+5G>A, c.3735G>A (p.E1245=), and c.3883-46A>G, caused skipping of the respective adjacent exon. One variant, c.3883-6G>A, created a new splicing donor site resulting in a 4-bp retention from the intronic region. The results provide experimental evidence that these non-canonical variants disrupt splicing, suggesting the presence of pathogenic mutations beyond the canonical splice sites, including silent variants in the coding region.

Symmetry and asymmetry in the Hebrew copula construction

The copula construction in Hebrew has received much attention in the linguistic literature. Nevertheless, one non-canonical variant has been largely neglected. In this variant the copula, flanked by two NPs, exhibits agreement with the post-copular NP, contrary to the canonical variant, where the agreement controller is the initial NP. This phenomenon challenges the notion of subject and its relation to agreement. The current corpus-based study investigates the word order and agreement patterns exhibited by the Hebrew copular constructions and shows that their distribution is largely motivated by information structure considerations. The proposed analysis accounts for the syntactic symmetry and semantic asymmetry between the two NPs.