Frameshift Research Articles

Clinical features and genetic analysis of 15 Chinese children with dent disease

Objective The clinical characteristics, genetic mutation spectrum, treatment strategies and prognoses of 15 children with Dent disease were retrospectively analyzed to improve pediatricians’ awareness of and attention to this disease. Methods We analyzed the clinical and laboratory data of 15 Chinese children with Dent disease who were diagnosed and treated at our hospital between January 2017 and May 2023 and evaluated the expression of the CLCN5 and OCRL1 genes. Results All 15 patients were male and complained of proteinuria, and the incidence of low-molecular-weight proteinuria (LMWP) was 100.0% in both Dent disease 1 (DD1) and Dent disease 2 (DD2) patients. The incidence of hypercalciuria was 58.3% (7/12) and 66.7% (2/3) in DD1 and DD2 patients, respectively. Nephrocalcinosis and nephrolithiasis were found in 16.7% (2/12) and 8.3% (1/12) of DD1 patients, respectively. Renal biopsy revealed focal segmental glomerulosclerosis (FSGS) in 1 patient, minimal change lesion in 5 patients, and small focal acute tubular injury in 1 patient. A total of 11 mutations in the CLCN5 gene were detected, including 3 missense mutations (25.0%, c.1756C > T, c.1166T > G, and c.1618G > A), 5 frameshift mutations (41.7%, c.407delT, c.1702_c.1703insC, c.137delC, c.665_666delGGinsC, and c.2200delG), and 3 nonsense mutations (25.0%, c.776G > A, c.1609C > T, and c.1152G > A). There was no significant difference in age or clinical phenotype among patients with different mutation types (p > 0.05). All three mutations in the OCRL1 gene were missense mutations (c.1477C > T, c.952C > T, and c.198A > G). Conclusion Pediatric Dent disease is often misdiagnosed. Protein electrophoresis and genetic testing can help to provide an early and correct diagnosis.

Patient-derived organoid elucidates the identical clonal origin of bilateral breast cancer with diverse molecular subtypes.

Bilateral breast cancer (BBC), an infrequent breast cancer subtype, has primarily been studied in terms of incidence, prognosis, and through comparative analysis of synchronous (SBBC) and metachronous (MBBC) manifestations. The advent and application of organoid technology hold profound implications for tumor research and clinical management. This study represents the pioneering use of organoid models in BBC research. We established organoid lines from two surgical tumor specimens of a BBC patient, with one line undergoing detailed pathological and genomic analysis. The BBC organoid from the right breast demonstrated a marker expression profile of ER (-), PR (-), HER-2 (0), and Ki67 index 10%, indicating that it may derived from the TNBC tissue. Whole Exome Sequencing (WES) displayed consistent set of Top10 cancer driver genes affected by missense mutations, frameshift mutation, or splice site mutations in three tumor tissues and the organoid samples. The organoids' single nucleotide polymorphisms (SNPs) were more closely aligned with the TNBC tissue than other tumor tissues. Evolutionary analysis suggested that different tumor regions might evolve from a common ancestral layer. In this case, the development of BBC organoids indicated that simultaneous lesions with diverse molecular profiles shared a high degree of consistency in key tumor-driving mutations. These findings suggest the feasibility of generating BBC organoids representing various molecular types, accurately replicating significant markers and driver mutations of the originating tumor. Consequently, organoids serve as a valuable in vitro model for exploring treatment strategies and elucidating the underlying mechanisms of BBC.

Deciphering the function of the fifth class of Gα proteins: regulation of ionic homeostasis as unifying hypothesis

Trimeric G proteins transduce signals from a superfamily of receptors and each G protein controls a wide range of cellular and systemic functions. Their highly conserved alpha subunits fall in five classes, four of which have been well investigated (Gs, Gi, G12, Gq). In contrast, the function of the fifth class, Gv is completely unknown, despite its broad occurrence and evolutionary ancient origin (older than metazoans). Here we show a dynamic presence of Gv mRNA in several organs during early development of zebrafish, including the hatching gland, the pronephros and several cartilage anlagen, employing in situ hybridisation. Next, we generated a Gv frameshift mutation in zebrafish and observed distinct phenotypes such as reduced oviposition, premature hatching and craniofacial abnormalities in bone and cartilage of larval zebrafish. These phenotypes could suggest a disturbance in ionic homeostasis as a common denominator. Indeed, we find reduced levels of calcium, magnesium and potassium in the larvae and changes in expression levels of the sodium potassium pump atp1a1a.5 and the sodium/calcium exchanger ncx1b in larvae and in the adult kidney, a major osmoregulatory organ. Additionally, expression of sodium chloride cotransporter slc12a3 and the anion exchanger slc26a4 is altered in complementary ways in adult kidney. It appears that Gv may modulate ionic homeostasis in zebrafish during development and in adults. Our results constitute the first insight into the function of the fifth class of G alpha proteins.

Evolution towards extremely high β-lactam resistance in Mycobacterium abscessus outbreak strains.

Treatment of Mycobacterium abscessus pulmonary disease requires multiple antibiotics including intravenous β-lactams (e.g., imipenem, meropenem). M. abscessus produces a β-lactamase (BlaMab) that inactivates β-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance in M. abscessus and poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collected M. abscessus subsp. massiliense isolates from the index case of a CF center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC >512 μg/ml). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the β-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in β-lactamase activity and increased bla Mab mRNA level. Concurrent increase in transcription of preceding ppiA gene indicated upregulation of the entire operon in the resistant strains. Deletion of the porin mspA coincided with the first increase in MIC (from 8 to 32 μg/ml). A frameshift mutation in msp2 responsible for the rough colony morphology, and a SNP in ATP-dependent helicase hrpA co-occurred with the second increase in MIC (from 32 to 256 μg/ml). Increased BlaMab expression and enzymatic activity may have been due to altered regulation of the ppiA-bla Mab operon by the mutated HrpA alone, or in combination with other genes described above. This work supports using carbapenem/β-lactamase inhibitor combinations for treating M. abscessus, particularly imipenem resistant strains.

Venezuelan migration in South America: coordinating regional responses to a transboundary reception crisis

ABSTRACT Migration is a policy field that is prone to transboundary reception crises due to the weakness of inter-state cooperation and global governance institutions’ incomplete architecture. Research suggests that the regional level is a more realistic policy arena where cooperation to coordinate responses may emerge. Our paper aims to explore and analyse the conditions under which coordinated responses to transboundary reception crises are present in (or absent from) regional migration governance. Below we develop an analytical model based on crisis management scholarship to examine coordination capacities at the regional level. Empirically, we comparatively assess the ability of old regional institutions (i.e. MERCOSUR, the South American Conference of Migration) and new ones (i.e. the Quito Process) to orchestrate responses to receive Venezuelan migrants across the region. Our findings reveal that coordination capacities remain scarce as regional institutions navigate from non-crisis to crisis times. This is largely because there was a shift in policy framing regarding immigrant reception, to self-interested national, regional and extra-regional actors promoting new regional mechanisms, and to the increasing complexity of regional migration governance as (new) competing actors emerge. Our conceptual and empirical contributions provide insights into how governance processes in South America have changed, particularly during crises.

Frameshift mutation of LAMP2:c.667delT in a 17-year-old male with hypertrophic cardiomyopathy and dyslexia: a novel pathogenic variant for Danon disease.

Frameshift mutation of LAMP2:c.667delT in a 17-year-old male with hypertrophic cardiomyopathy and dyslexia: a novel pathogenic variant for Danon disease.

Creation and characterization of novel rat model for recessive dystrophic epidermolysis bullosa: Frameshift mutation of the Col7a1 gene leads to severe blistered phenotype.

Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.

Ribosome decision graphs for the representation of eukaryotic RNA translation complexity.

The application of ribosome profiling has revealed an unexpected abundance of translation in addition to that responsible for the synthesis of previously annotated protein-coding regions. Multiple short sequences have been found to be translated within single RNA molecules, within both annotated protein-coding and noncoding regions. The biological significance of this translation is a matter of intensive investigation. However, current schematic or annotation-based representations of mRNA translation generally do not account for the apparent multitude of translated regions within the same molecules. They also do not take into account the stochasticity of the process that allows alternative translations of the same RNA molecules by different ribosomes. There is a need for formal representations of mRNA complexity that would enable the analysis of quantitative information on translation and more accurate models for predicting the phenotypic effects of genetic variants affecting translation. To address this, we developed a conceptually novel abstraction that we term ribosome decision graphs (RDGs). RDGs represent translation as multiple ribosome paths through untranslated and translated mRNA segments. We termed the latter "translons." Nondeterministic events, such as initiation, reinitiation, selenocysteine insertion, or ribosomal frameshifting, are then represented as branching points. This representation allows for an adequate representation of eukaryotic translation complexity and focuses on locations critical for translation regulation. We show how RDGs can be used for depicting translated regions and for analyzing genetic variation and quantitative genome-wide data on translation for characterization of regulatory modulators of translation.

A Rare Consanguineous Case of Alazami Syndrome in a Jordanian Family: Clinical Presentation, Genetic Analysis, and Therapeutic Approaches - A Case Report

Objective: Alazami syndrome (AS) is an infrequent genetic disorder inherited in an autosomal recessive pattern, characterized by the presence of multiple congenital abnormalities. This study explores a case of a 4-year-old girl with AS, examining symptoms, genetic factors, and treatment efficacy. Case report: A 4-year-old girl, born to consanguineous Jordanian parents, displayed dysmorphic features including low birth weight, microcephaly, hyperthyroidism, short stature, blue sclera, triangular-shaped face, deep-set eyes, narrow palpebral fissures, and a prominent forehead. Examination revealed height (92 cm) and weight (7.7 kg) below the 5th and 3rd percentiles respectively. Blood tests and renal ultrasound were normal. Whole exome sequencing (WES) identified a homozygous eight-base pair deletion within exon 5 of the LARP7 gene on chromosome 4q25, confirming the diagnosis of AS, an autosomal recessive disorder. This variant induces frameshift mutations leading to premature stop codons, suggesting a probable mechanism of illness via loss of function. Treatment involving growth monitoring and therapy led to significant improvements in height, weight, and communication skills within three months. Conclusion: We describe a rare autosomal recessive AS case due to consanguinity, with a frameshift mutation in the LARP7 gene found via WES. Our AS treatment program effectively alleviates symptoms and enhances developmental progress.

Tying the knot: Unraveling the intricacies of the coronavirus frameshift pseudoknot.

Understanding and targeting functional RNA structures towards treatment of coronavirus infection can help us to prepare for novel variants of SARS-CoV-2 (the virus causing COVID-19), and any other coronaviruses that could emerge via human-to-human transmission or potential zoonotic (inter-species) events. Leveraging the fact that all coronaviruses use a mechanism known as -1 programmed ribosomal frameshifting (-1 PRF) to replicate, we apply algorithms to predict the most energetically favourable secondary structures (each nucleotide involved in at most one pairing) that may be involved in regulating the -1 PRF event in coronaviruses, especially SARS-CoV-2. We compute previously unknown most stable structure predictions for the frameshift site of coronaviruses via hierarchical folding, a biologically motivated framework where initial non-crossing structure folds first, followed by subsequent, possibly crossing (pseudoknotted), structures. Using mutual information from 181 coronavirus sequences, in conjunction with the algorithm KnotAli, we compute secondary structure predictions for the frameshift site of different coronaviruses. We then utilize the Shapify algorithm to obtain most stable SARS-CoV-2 secondary structure predictions guided by frameshift sequence-specific and genome-wide experimental data. We build on our previous secondary structure investigation of the singular SARS-CoV-2 68 nt frameshift element sequence, by using Shapify to obtain predictions for 132 extended sequences and including covariation information. Previous investigations have not applied hierarchical folding to extended length SARS-CoV-2 frameshift sequences. By doing so, we simulate the effects of ribosome interaction with the frameshift site, providing insight to biological function. We contribute in-depth discussion to contextualize secondary structure dual-graph motifs for SARS-CoV-2, highlighting the energetic stability of the previously identified 3_8 motif alongside the known dominant 3_3 and 3_6 (native-type) -1 PRF structures. Using a combination of thermodynamic methods and sequence covariation, our novel predictions suggest function of the attenuator hairpin via previously unknown pseudoknotted base pairing. While certain initial RNA folding is consistent, other pseudoknotted base pairs form which indicate potential conformational switching between the two structures.

A case study of a liver transplant-treated patient with glycogen storage disease type Ia presenting with multiple inflammatory hepatic adenomas: an analysis of clinicopathologic and genetic data

BackgroundGlycogen storage disease (GSD) is a disease caused by excessive deposition of glycogen in tissues due to genetic disorders in glycogen metabolism. Glycogen storage disease type I (GSD-I) is also known as VonGeirk disease and glucose-6-phosphatase deficiency. This disease is inherited in an autosomal recessive manner, and both sexes can be affected. The main symptoms include hypoglycaemia, hepatomegaly, acidosis, hyperlipidaemia, hyperuricaemia, hyperlactataemia, coagulopathy and developmental delay.Case presentationHere, we present the case of a 13-year-old female patient with GSD Ia complicated with multiple inflammatory hepatic adenomas. She presented to the hospital with hepatomegaly, hypoglycaemia, and epistaxis. By clinical manifestations and imaging and laboratory examinations, we suspected that the patient suffered from GSD I. Finally, the diagnosis was confirmed by liver pathology and whole-exome sequencing (WES).WES revealed a synonymous mutation, c.648 G > T (p.L216 = , NM_000151.4), in exon 5 and a frameshift mutation, c.262delG (p.Val88Phefs*14, NM_000151.4), in exon 2 of the G6PC gene. According to the pedigree analysis results of first-generation sequencing, heterozygous mutations of c.648 G > T and c.262delG were obtained from the patient's father and mother.Liver pathology revealed that the solid nodules were hepatocellular hyperplastic lesions, and immunohistochemical (IHC) results revealed positive expression of CD34 (incomplete vascularization), liver fatty acid binding protein (L-FABP) and C-reactive protein (CRP) in nodule hepatocytes and negative expression of β-catenin and glutamine synthetase (GS). These findings suggest multiple inflammatory hepatocellular adenomas. PAS-stained peripheral hepatocytes that were mostly digested by PAS-D were strongly positive.This patient was finally diagnosed with GSD-Ia complicated with multiple inflammatory hepatic adenomas, briefly treated with nutritional therapy after diagnosis and then underwent living-donor liver allotransplantation.After 14 months of follow-up, the patient recovered well, liver function and blood glucose levels remained normal, and no complications occurred.ConclusionThe patient was diagnosed with GSD-Ia combined with multiple inflammatory hepatic adenomas and received liver transplant treatment. For childhood patients who present with hepatomegaly, growth retardation, and laboratory test abnormalities, including hypoglycaemia, hyperuricaemia, and hyperlipidaemia, a diagnosis of GSD should be considered. Gene sequencing and liver pathology play important roles in the diagnosis and typing of GSD.

Determinants of plasma levels of proglucagon and the metabolic impact of glucagon receptor signalling: a UK Biobank study

Aims/hypothesesGlucagon and glucagon-like peptide-1 (GLP-1) are derived from the same precursor; proglucagon, and dual agonists of their receptors are currently being explored for the treatment of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). Elevated levels of endogenous glucagon (hyperglucagonaemia) have been linked with hyperglycaemia in individuals with type 2 diabetes but are also observed in individuals with obesity and MASLD. GLP-1 levels have been reported to be largely unaffected or even reduced in similar conditions. We investigated potential determinants of plasma proglucagon and associations of glucagon receptor signalling with metabolic diseases based on data from the UK Biobank.MethodsWe used exome sequencing data from the UK Biobank for ~410,000 white participants to identify glucagon receptor variants and grouped them based on their known or predicted signalling. Data on plasma levels of proglucagon estimated using Olink technology were available for a subset of the cohort (~40,000). We determined associations of glucagon receptor variants and proglucagon with BMI, type 2 diabetes and liver fat (quantified by liver MRI) and performed survival analyses to investigate if elevated proglucagon predicts type 2 diabetes development.ResultsObesity, MASLD and type 2 diabetes were associated with elevated plasma levels of proglucagon independently of each other. Baseline proglucagon levels were associated with the risk of type 2 diabetes development over a 14 year follow-up period (HR 1.13; 95% CI 1.09, 1.17; n=1562; p=1.3×10−12). This association was of the same magnitude across strata of BMI. Carriers of glucagon receptor variants with reduced cAMP signalling had elevated levels of proglucagon (β 0.847; 95% CI 0.04, 1.66; n=17; p=0.04), and carriers of variants with a predicted frameshift mutation had higher levels of liver fat compared with the wild-type reference group (β 0.504; 95% CI 0.03, 0.98; n=11; p=0.04).Conclusions/interpretationOur findings support the suggestion that glucagon receptor signalling is involved in MASLD, that plasma levels of proglucagon are linked to the risk of type 2 diabetes development, and that proglucagon levels are influenced by genetic variation in the glucagon receptor, obesity, type 2 diabetes and MASLD. Determining the molecular signalling pathways downstream of glucagon receptor activation may guide the development of biased GLP-1/glucagon co-agonist with improved metabolic benefits.Data availabilityAll coding is available through https://github.com/nicwin98/UK-Biobank-GCGGraphical

A Novel Frameshift Mutation of HBB Causing Dominant β-Thalassemia in a Chinese Individual

We reported a rare β-thalassemia patient, a 41-year-old Chinese male with small cell hypopigmentation anemia, jaundice and splenomegaly as the main clinical symptoms. By using Next-Generation Sequencing (NGS), we identified a novel de novo HBB mutation(c.358_365dup, p.Phe123Alafs*39) which resulted in an abnormally prolonged β-globin chain comprising 159 amino acid residues. The secondary and three-dimensional structures of the β-globin predicted that the novel prolonged β-globin chain has a considerable risk of instability in the hemoglobin, and leads to clinical phenotype. This study contributes to the enrichment of the genetic pathogenic mutation database for thalassemia and underscores the significance of NGS in the screening of mutations for thalassemia families.

Streamlined Generation of CRISPR/Cas9-Mediated Single-Cell Knockout Clones in Murine Cell Lines.

Clonal cell lines harboring loss-of-function mutations in genes of interest are crucial for studying the cellular functions of the encoded proteins. Recent advances in genome engineering have converged on the CRISPR/Cas9 technology to quickly and reliably generate frameshift mutations in the target genes across various cell lines and species. Although high on-target cleavage efficiencies can be obtained reproducibly, screening and identifying clones with loss-of-function alleles remains a major bottleneck. Here, we describe a single sgRNA strategy to generate CRISPR/Cas9-mediated frameshift mutations in target genes of mammalian cell lines that can be easily and cost-effectively identified. Given the proliferation of workhorse cell lines such as N2a cells and the resulting clonal expansion of the cell type, our protocol can facilitate the isolation of knockout clonal cell lines and their genetic validation within a period of down to 6-8 weeks.

Abstract PO1-23-09: A bedside-to-bench translational analysis demonstrates that NF1 alterations promote CDK4/6 inhibitor (CDK4/6i) resistance in hormone receptor-positive (HR+) metastatic breast cancer (MBC)

Abstract Background: CDK4/6i are part of the standard management of HR+ MBC. The MAPK pathway has been implicated in mediating resistance to CDK4/6 blockade (Wander 2020). A variety of molecular alterations dysregulate MAPK signaling, including NF1 mutations (NF1-MUT) (Wallace 2012), though NF1 has not been implicated in CDK4/6i resistance. To assess this, we analyzed retrospective clinical data from multiple institutions, real-world data (RWD) from the GuardantINFORM database, and preclinical models of HR+ breast tumors. The primary objective of this study was to characterize clinical outcomes of CDK4/6i in patients (pts) with NF1-MUT MBC, and to validate those findings in laboratory models. Methods: Pts with pathogenic (p)NF1-MUT were identified via sequencing of circulating tumor DNA (ctDNA) or tumor tissue. Pathogenic was defined as nonsense, insertion/deletion, frameshift, or oncogenic missense mutations. Progression free survival (PFS) on CDK4/6i was analyzed, and intrinsic resistance was defined as progression < 6 months on 1st line (1L) CDK4/6i or < 3 mo on 2nd line and beyond (2L+). Acquired resistance was defined as PFS >6 mo (1L) or >3 mo (2L+) with a NF1 alteration detected post-progression. RWD from the GuardantINFORM dataset had Guardant360 (G360) ctDNA testing linked with claims data on >37,000 pts with advanced or MBC tested between June 2014 and March 2023. Pts on CDK4/6i within 90 days pre-G360 were stratified as NF1-MUT (n = 28) vs. NF1-non-mutant (NF1-nonMUT) (n = 1133) and analyzed for real-world time-to-next-treatment (rwTTNT) and real-world overall survival (rwOS). Propensity score weighting for age, sex, line of therapy, and year of G360 were used to create a balanced control group. To determine whether NF1 loss-of-function alterations were causal of CDK4/6i resistance, we used CRISPR/Cas9 to delete NF1 in MCF7 breast cancer cells. MCF7 wild-type (WT) and two NF1-knockout (KO) clones were treated with palbociclib ± estrogen deprivation, followed by cell viability assays. MAPK and PI3K/AKT pathway activation were measured by immunoblot analysis. Results: Across 4 institutions, 40 pts with HR+, HER2- MBC expressing pNF1-MUT and prior CDK4/6i were included. The mean age at MBC diagnosis was 56 years, and the majority of pts received no prior treatment in the metastatic setting before CDK4/6i (range 0 - 5 prior lines). Intrinsic or acquired resistance to CDK4/6i was seen in 29/40 (73%) tumors harboring pNF1-MUT. We analyzed pts with baseline pNF1-MUT who received 1L CDK4/6i plus endocrine therapy (n = 13) and found that the median PFS was 7.7 months (range 2 - 18 mo). We next examined GuardantINFORM RWD and discovered a significant difference in rwTTNT and rwOS on CDK4/6i for NF1-MUT (n = 28) vs. weighted NF1-nonMUT (n = 28) tumors. Mean age (62 years) and level of pretreatment (majority 2L+) were similar between the groups. Compared to NF1-nonMUT, NF1-MUT pts had a shorter median rwTTNT on CDK4/6i regimens (4.2 vs. 12.4 mo, weighted log-rank p< 0.0001) and worse rwOS (15.8 vs 45.2 mo, weighted log-rank p=0.016). In MCF7 cells, NF1-KO exhibited a palbociclib IC50 11- to 13-fold higher than NF1-WT controls. In estrogen-deprived media, treatment with 250 nM palbociclib for 6 days reduced MCF7 NF1-WT cell viability 75% but only 5-20% in NF1-KO cells. As expected, phosphorylation of ERK and AKT were strongly induced in NF1-KO cells relative to NF1-WT. Conclusions: Using multicenter clinical and real-world evidence, we demonstrate that NF1-MUT is associated with inferior duration and worse outcomes on CDK4/6i in MBC. Pts with baseline pNF1-MUT on 1L CDK4/6i and endocrine therapy had a median PFS below typical responses expected in clinical practice. A link between NF1 loss and CDK4/6i resistance was supported by in vitro experiments in HR+ breast cancer cells. NF1 deletion was accompanied by activation of ERK and AKT, therefore, future directions include investigating alternative or combination treatment strategies targeting the MAPK and/or PI3K/AKT pathways in these tumors. *MRL and RCP contributed equally. ABH and SAW are co-senior authors. Citation Format: Maxwell Lloyd, M Rosario Chica-Parrado, Todd Knepper, Chang-Ching Lin, Emily Podany, Christine Walko, Fabiana Napolitano, Caroline Weipert, Jiemin Liao, Arielle Medford, Lianne Ryan, Jennifer Keenan, Nicole Zhang, Shiyuan Liu, Gerburg Wulf, Katherine Clifton, Cynthia Ma, Hyo Han, Leif Ellisen, Aditya Bardia, Carlos Arteaga, Ariella Hanker, Seth Wander. A bedside-to-bench translational analysis demonstrates that NF1 alterations promote CDK4/6 inhibitor (CDK4/6i) resistance in hormone receptor-positive (HR+) metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-23-09.

Abstract PO2-13-02: Detection of SPEN mutations in advanced breast cancer by circulating tumor cell-free DNA

Abstract Background: Alterations in the SPEN gene are rare in primary breast cancer (~3%, ref: TCGA). SPEN is a hormone-inducible transcriptional repressor with known functions in orchestrating X-inactivation in females. Additionally, SPEN has been implicated as a potential estrogen receptor co-repressor and molecular partner of NCOR2 as well as epigenetic modifiers such as KMT2D and HDACs via C-terminal interactions. SPEN loss-of-function is associated with tamoxifen resistance in preclinical models. However, the landscape of SPEN alterations in advanced breast cancer remains poorly described. The primary objective of this study was to evaluate the frequency and different types of SPEN alterations in metastatic breast cancer (MBC). Methods: A query was performed on all patients at an academic institution with available genomic testing from Guardant360, a next-generation sequencing cell-free DNA (cfDNA) assay, evaluating 500 genes in total. Patients with MBC were identified, and specific alterations in SPEN as well as co-alterations were extracted. Silent and germline mutations were excluded from analysis. Retrospective review was conducted to determine clinicopathologic characteristics such as age, hormone and HER2 receptor status, menopausal status, and therapy at time of the detected mutation. Results: Among 366 patients with MBC and available cfDNA, 8.5% (n=31) had an alteration detected in the SPEN gene. Median age of patients with SPEN mutations was 66 years (range 33-92). A majority of patients (90%) had HR+ disease (n=28/31), with HR-/HER2+ disease and TNBC additionally representing 3% (n=1/31) and 6% (n=2/31) of cases, respectively. SNVs were most frequent, comprising 89% (n=42/47) of all alterations. Indels were additionally identified in 11% (n=5/47), and no CNVs or fusions were noted. Nonsense and frameshift mutations represented 8/47 (17%) and 2/47 (4%) of all alterations respectively. No recurrent mutational hotspots were detected. The most common co-alterations in descending order were PIK3CA, TP53, ERBB2, ARID1A, MDM4, PPM1D, NF1, DNMT3A, ATM, and ESR1. Association between SPEN mutations and clinical outcomes will be presented at the meeting. Conclusions: SPEN mutations were detected mostly in patients with HR+/HER2- MBC and at a higher frequency than reported in primary disease via TCGA. SNVs were common, though it remains unclear whether these comprise driver versus passenger mutations. While no mutational hotspots were identified, several novel loss-of-function truncating mutations were identified, which warrant further validation of functional and clinical significance. SPEN alterations in metastatic breast cancer Alterations detected by cell-free DNA sequencing in patients with metastatic breast cancer and annotated by type of alteration and receptor subtype. Citation Format: Charles Dai, Haley Barnes, Arielle Medford, Annika Putur, Jennifer Keenan, Beverly Moy, Seth Wander, Ryan Corcoran, Aditya Bardia. Detection of SPEN mutations in advanced breast cancer by circulating tumor cell-free DNA [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-02.

Disruption of the c-terminal serine protease domain of Fam111a does not alter calcium homeostasis in mice.

FAM111A gene mutations cause Kenney-Caffey syndrome (KCS) and Osteocraniostenosis (OCS), conditions characterized by short stature, low serum ionized calcium (Ca2+), low parathyroid hormone (PTH), and bony abnormalities. The molecular mechanism mediating this phenotype is unknown. The c-terminal domain of FAM111A harbors all the known disease-causing variations and encodes a domain with high homology to serine proteases. However, whether this serine protease domain contributes to the maintenance of Ca2+ homeostasis is not known. We hypothesized the disruption of the serine protease domain of FAM111A would disrupt Ca2+ homeostasis. To test this hypothesis, we generated with CRISPR/Cas9, mice with a frameshift insertion (c.1450insA) or large deletion (c.1253-1464del) mutation in the Fam111a serine protease domain. Serum-ionized Ca2+ and PTH levels were not significantly different between wild type, heterozygous, or homozygous Fam111a mutant mice. Additionally, there were no significant differences in fecal or urine Ca2+ excretion, intestinal Ca2+ absorption or overall Ca2+ balance. Only female homozygous (c.1450insA), but not heterozygous mice displayed differences in bone microarchitecture and mineral density compared to wild-type animals. We conclude that frameshift mutations that disrupt the c-terminal serine protease domain do not induce a KCS or OCS phenotype in mice nor alter Ca2+ homeostasis.

Caveolin-1 frame-shift mutation induces multifunctional alterations in human fibroblasts that are exosome-mediated

Caveolins, Cav1-3, are oligomeric proteins critical for the formation of membrane caveolae that regulate cell physiology. A de novo frameshift mutation (Cav1F160X) in Cav1 (Cav1M) was described in a patient with a progeria-like phenotype, lipodystrophy, and pulmonary hypertension. We hypothesize that Cav1M presents as a dominant negative protein that perturbs cellular physiology, particularly nuclear morphology, mitochondrial function, plasma membrane structure, and exocytic behavior. Transmission electron microscopy (TEM) of patient-derived dermal fibroblasts (Cav1M) showed abnormal nuclear shape, increased rough endoplasmic reticulum, and an increased number of autophagosomes. Cav1M cells present an abnormal mitochondrial size and shape, accompanied by a reduced mitochondrial capacity for energy production detected by Seahorse real-time oximetry analysis. In addition, we observed in the patient’s fibroblasts an altered pattern of Cav1 expression and distribution, with less Cav1 in caveolae membrane fractions, together with a reduced membrane cholesterol concentration and number of caveolae. Electron paramagnetic spectroscopy shows an altered localization of proteins commonly found in caveolae fractions and increased membrane fluidity. Interestingly, Cav1M fibroblasts showed 4-fold increase in exosome secretion. Nanoparticle tracking and TEM analysis revealed a significant reduction in nanoparticle size in Cav1M-derived exosomes. Normal human dermal fibroblasts (HDF) treated to express Cav1M (Cav1M1-159) or exogenously exposed to exosomes isolated from Cav1M fibroblasts showed altered nuclear morphology, exosome secretion profile, proteomics, and miRNA analysis. Our results indicate that mutations in the C-terminus of Cav1 lead to significant cellular alterations at different levels that can lead to morpho-functional features, energetic imbalances, membrane ultrastructure abnormalities, and exosome secretion and signaling alterations. These findings suggest a novel role of Cav1 that links several aspects of cell physiology, from plasma and nuclear membrane dynamics to exocytic behavior, that have potential implications in cellular communication impacting physiology and pathophysiology. 5R01HL091071. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pre-problem families: predictive analytics and the future as the present

Predictive analytics is seen as a way of identifying the risk of future problems in families. Integral to such automated predictive analysis is a shift in time frames that redraws the relationship between families and the state, to potentially intervene on an anticipatory basis of ‘what hasn’t happened but might’. In the process, human subjects are reformulated as disembodied objects of data-driven futures. The article explains this process and fills a significant gap in knowledge about parents’ views of this development. We draw on group and individual discussions with parents across Great Britain to consider their understanding of predictive analytics and how comfortable they are with it. Parents’ concerns focused on inaccuracies in the data used for prediction, the unfair risk of false positives and false negatives, the deterministic implications of the past predicting the future, and the disturbing potential of being positioned in what was a pre-problem space. We conclude with policy implications.

Generation of human induced pluripotent stem cell lines derived from four Rett syndrome patients with MECP2 mutations

Rett syndrome is characterized by severe global developmental impairments with autistic features and loss of purposeful hand skills. Here we show that human induced pluripotent stem cell (hiPSC) lines derived from four Japanese female patients with Rett syndrome are generated from peripheral blood mononuclear cells using Sendai virus vectors. The generated hiPSC lines showed self-renewal and pluripotency and carried heterozygous frameshift, missense, or nonsense mutations in the MECP2 gene. Since the molecular pathogenesis caused by MECP2 dysfunction remains unclear, these cell resources are useful tools to establish disease models and develop new therapies for Rett syndrome.