Hotspot Mutations Research Articles

The chloroplast genome of Cephalanthera nanchuanica (Orchidaceae): comparative and phylogenetic analysis with other Neottieae species.

Cephalanthera nanchuanica is a terrestrial orchid species and has been red listed as a second-grade protected plant due to its limited distributions in China. Initially classified within a monotypicgenus Tangtsinia, this species was later reassigned to Cephalanthera based on morphological and molecular data. However, previous phylogenetic analyses of Cephalanthera using several segment sequences exhibited alow discriminatory power in delineating its relationships. In this study, we characterized and comparatively analyzed the complete chloroplast (cp) genome of C. nanchuanica with those of six previously reported Cephalanthera species. Our findings revealed that the cp genome of C. nanchuanica had the typical quadripartite structure, with a size of 161,365bp and a GC content of 37.27%. A total of 113 unique genes were annotated, among which nearly half of protein-encoding genes (RSCU > 1) showed a preference in codon usage. No structural rearrangements were observed among the cp genomes of Cephalanthera species, except for C. humilis, which displayed structural alterations due to gene loss, relocation, and inverted repeat (IR) expansion/contraction. The cp genomes of Cephalanthera species were highly conserved, with only a small number of SSRs detected, most of which preferred A/T bases. Comparative analysis of cp genomes indicated that IR and coding regions were less divergent than single copy and non-coding regions and eight mutational hotspots were identified. Phylogenetic analysis suggested that the tribe Neottieae was a monophyletic group, divided into five clades. Palmorchis was the earliest-diverging lineage, followed by Cephalanthera. Diplandrorchis was deeply nested within Neottia, forming a clade. Aphyllorchis and Limodorum formed another clade, sister to Epipactis. Within the Cephalanthera clade, C. nanchuanica was sister to C. falcata with astrong support. This study demonstrated that the cp genome characters of C. nanchuanica are highly similar to those of other Cephalanthera species, except for the mycoheterotrophic species C. humilis. Although the cp genomes of Cephalanthera species (excluding C. humilis) exhibited conservation in genome structure and sequence, SSR repeats and mutational hotspots were identified, which could potentially serve as as molecular markers for distinguishing Cephalanthera species. The phylogenetic analysis based on the protein-coding genes provided high-resolution support for the infrageneric classification. Therefore, cp genome data will be instrumental in resolving the phylogeny of the genus Cephalanthera.

Localised multiple spiradenomas are caused by somatic mosaicism in ALPK1 hotspot mutation.

Spiradenocarcinomas, a rare skin adnexal tumour, are linked to the ALPK1 hotspot mutation. This study reveals that spiradenocarcinoma, accompanied by multiple localized spiradenomas, is caused by somatic mosaicism in the ALPK1 mutation hotspot.

Sensitive and reliable detection of KIT p.D816V mutation in decalcified archival bone marrow trephines.

The majority of mastocytosis cases are characterized by an activating mutation in the KIT gene in codon 816. The detection of this alteration is of importance for proper diagnostic workup. Therefore, reliable and sensitive methods for the detection of KIT Codon 816 hotspot mutations in various types of patient samples are required. Since mutated cancer genes are often overexpressed, we evaluated the feasibility and sensitivity of KIT p.D816V detection by analysing mRNA/cDNA instead of genomic DNA. From 80 bone marrow trephines harboring a KIT p.D816 mutation, seven were only mutated by mRNA/cDNA pyrosequencing and 11 only by digital PCR analysis of genomic DNA. These results clearly demonstrate that detection of clinically relevant mutations in mRNA extracted from routinely processed decalcified archival bone marrow trephines is not only possible in a reliable fashion but under many circumstances advantageous. This enables the direct correlation of genomic data with high-quality morphological evaluation.

Abstract B009: Individualized-tumor-informed panel approach enables ultra-sensitive ctDNA-based minimal residue disease monitoring for high-risk pediatric cancers

Abstract While the use of ctDNA-based minimal residual disease (MRD) monitoring is gaining popularity in adult cancers, this approach is not yet widely adopted in pediatric cancers (PCs). We developed a robust and sensitive assay to monitor ctDNA in PCs, which overcomes PC-specific challenges including low tumor mutational burden, paucity of hotspot mutations and low blood volume. To address these challenges, our innovations include personalized panel design that targets primary tumor mutations from each patient, a fixed panel targeting hotspot mutations, error suppression from duplex deep sequencing and customized pipeline for tumor burden estimation. We retrospectively analyzed 661 blood or cerebrospinal fluid (CSF) samples from 168 patients. We achieved an average detection limit of 0.005% ctDNA purity leading to MRD detected ranging from 0.001% to 100% with brain tumor consistently showing a low MRD. At disease diagnosis, progression and relapse, our assay identified ctDNA in over 93% of extra- cranial solid tumors, 90% of leukemia, 89% of sarcoma, and 90% of neuroblastoma. For brain tumors, we detected MRD in ∼90% of CSF samples, and in ∼60% of blood samples, a major advance over previous approaches. Importantly, we detected MRD in ∼ 20% of cases with a clinically complete response, predicting relapse up to 6 months before clinical confirmation. Overall, these results demonstrate that our monitoring assay for PCs is an important addition to standard of care for precision-guided treatment. Citation Format: Rob Salomon, Wenhan Chen, Charles Shale, Mojgan Toumari, Aileen Lowe, Wenyan Li, Jingwei Chen, Sajad Razavi-Bazaz, Paulette Barahona, Louise Cui, Chelsea Mayoh, Sam El-Kamand, Vanessa Tyrrell, Marie Wong-Erasmus, Loretta MS Lau, Noemi Fuentes-Bolanos, Edwin Cuppen, Dong-Anh Khoung-Quang, Jordan Staunton, Marion K Mateos, Toby Trahair, Michelle Haber, David S Ziegler, Paul Ekert, Peter Priestley, Mark Cowley. Individualized-tumor-informed panel approach enables ultra-sensitive ctDNA-based minimal residue disease monitoring for high-risk pediatric cancers [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B009.

Abstract A075: Circulating T-cell receptor repertoire analysis improves cancer early detection

Abstract Introduction: Cancer screening by liquid biopsy promises to detect cancer early when it can be cured. However, current ctDNA-based approaches detect only a minority of early-stage cancers. For liquid biopsy to realize its full potential for cancer early detection, sensitivity for earliest stage disease must be improved. Methods: We set out to improve sensitivity of liquid biopsy by exploiting tumor recognition by T cells through sequencing of the circulating T-cell receptor repertoire. Using gDNA extracted from blood buffy coats, we studied a cohort of 471 lung cancer patients (85% stage I) and 600 subjects without cancer enriched for older individuals with a history of smoking. We performed TCR beta chain sequencing to yield a median of 101,899 TCR clonotypes per sample (median 81,602 cancer / 112,966 controls) and built a TCR sequence similarity graph to cluster clonotypes into TCR repertoire functional units (RFUs). The TCR frequencies of RFUs were tested for association with cancer status and significantly associated RFUs were combined using an SVM model into a cancer score. Cancer RFU levels were also correlated to imputed subject HLA types for 32 HLA Class I and 38 Class II alleles. The model was evaluated by 10-fold cross-validation and compared to a ctDNA panel of 237 mutation hotspots in 154 lung cancer driver genes and to 17 lung cancer related protein biomarkers in 86 subjects. Results: We identified 372 cancer-associated TCR RFUs at FDR≤0.1, including 198 enriched in cancer samples (fold-change range 1.03 to 3.13) and 174 enriched in controls (fold- change 0.96 to 0.30). Levels of 265/372 (71%) RFUs were correlated to presence of an HLA allele at FDR ≤ 0.1. Of the total 2,770 significant HLA-RFU associations, 479 (17%) involved an HLA Class I allele, while 2,291 (83%) involved an HLA Class II allele, highlighting a potential role for CD4 helper or regulatory T cell antigen recognition in the cancer RFUs found. The RFU cancer score achieved a cross-validated test ROC AUC of 0.71 for cancer status prediction (0.71 Stage I / 0.70 Stage II-IV) with 49% of Stage I cancers detected at a specificity of 80%. Importantly, the cancer score was not dependent on sample source site, technical or biological variation in TCR repertoire depth, or lung cancer histology subtype. The TCR RFU score also distinguished lung cancer patients from control subjects with other conditions such as benign lung nodules and COPD. We observed an up to ∼20%-point gain in sensitivity for stage I cancer when TCR RFUs were added to ctDNA and proteins in a potential multi-analyte cancer screening test. By contrast, TCR analysis did not provide additional sensitivity for stage II-IV disease. Conclusion: We demonstrate detection of a significant fraction of early lung cancer cases from blood by analyzing the circulating TCR repertoire and show that this signal is complementary to established analytes such as proteins and ctDNA. A similar study for the detection of early colorectal cancer and advanced adenomas is under way. Citation Format: Yilong Li, Michelle Nahas, Dennis Stephens, Kate Froburg, Emma Hintz, Devin Champagne, Amaneet Lochab, Markus Brown, Jasper Braun, María Antonia Fortuño, María-del-Mar Ocón, Andrea Pasquier, Inés María Luque, Christopher W. Seder, Jeffrey A. Borgia, Luis Miguel Seijo, Luis M Montuenga, Roman Yelensky. Circulating T-cell receptor repertoire analysis improves cancer early detection [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A075.

Abstract B027: Combining total cell-free DNA (cfDNA) and circulating tumor (ctDNA) to enhance the clinical sensitivity of ddPCR assays to detect minimal residual disease (MRD) in stage IIIB/C/IV melanoma patients on adjuvant immunotherapy in CheckMate 238

Abstract Background: In patients (pts) with resected, stage III/IV melanoma receiving adjuvant immunotherapy, pre-treatment (pre-Tx) and on-treatment (on-Tx) ctDNA measurements are associated with relapse free survival; however, clinical adoption has been hindered by suboptimal assay sensitivity. Methods: We used analytically validated, mutation-specific ddPCR assays to measure ctDNA and cfDNA in pre- and on-Tx plasma samples from pts with resected stage IIIB/C/IV melanoma enrolled in CheckMate 238 (NCT02388906) receiving either adjuvant Nivolumab (NIVO) or Ipilimumab (IPI). Assay choice was personalized based on detection of one of the 7 melanoma hot-spot mutations in pt tumors: BRAF V600E/K, NRAS Q61R/L/K, or TERT promotor C228T or C250T. We evaluated associations between ctDNA detection, or high cfDNA based on cutpoint analysis (>6,000 copies/ml of plasma) and recurrence-free survival (RFS), pre-Tx tumor biomarkers including tumor cell PD-L1 expression, IFNg gene signature, CD8 infiltration, and tumor mutational burden. Survival analysis was performed using univariable and multivariable Kaplan-Meier and Cox regression models. Results: Assay mutations were identified in 87% of resected tumors. In the pre-Tx samples, ctDNA alone was detected in 94/753 (12%) pts. The detection rate increased to 20% when assay positivity was defined as either detectable ctDNA or high cfDNA. The sensitivity of ctDNA detection to predict recurrence ≤6 months(m) from pre-Tx samples increased from 25% (ctDNA alone) to 33% when high cfDNA was added to detectable ctDNA; specificity decreased to 84% from 91% (ctDNA alone). The patient subgroup defined by the combined metric of ctDNA+ or high cfDNA in pre- Tx samples was significantly enriched with higher stage of disease, elevated LDH, tumor cell PD-L1 expression < 5%, and tumor IFNg-RNA signature < median. Pts with detectable pre-Tx ctDNA or high cfDNA was associated with shorter RFS in both NIVO (median RFS 20.5 m [95%CI, 2.88, 59.15]) and IPI (median RFS 7.75 m [95%CI, 2.76, 24.08]) arms compared to those with undetectable ctDNA and low cfDNA (NIVO (median RFS 47.95 m [95%CI, 11.09, 59.89]) and IPI (median RFS 24.15 m [95%CI, 5.78, 58.38])). In a multivariable model that included sex, age, treatment, and IFNg gene signature, ctDNA+ or cfDNA high was an independent predictor of shorter RFS [HR 1.624 (95%CI, 1.275, 2.068), p<0.001]. When combining pre-Tx and on-Tx measurements at week 3 (w3) and w7, the assay performance generally increased compared to pre-Tx alone. For example, pre-Tx assay positivity had a 38% positive predictive value (PPV) and 81% negative predictive value (NPV) to predict recurrence at <6 m. In pts who were undetectable at pre-Tx but became positive at w3 and remained positive at w7, PPV increased to 62% and NPV increased to 90%. Conclusions: Incorporating quantitative measurements of cfDNA with ctDNA detection in the evaluation of pre-Tx and on- Tx plasma samples improves the clinical performance of mutation-specific ddPCR assays to detect MRD in patients with resected stage III and stage IV melanoma. Citation Format: Mahrukh M Syeda, Jennifer M Wiggins, Josephine Alegun, Saim Ali, Soutrik Mandal, Tracy (Hao) Tang, Keyur Desai, Sonia Dolfi, Daniel J Tenney, Paolo A Ascierto, James Larkin, Michele Del Vecchio, Jeffrey S Weber, David Polsky. Combining total cell-free DNA (cfDNA) and circulating tumor (ctDNA) to enhance the clinical sensitivity of ddPCR assays to detect minimal residual disease (MRD) in stage IIIB/C/IV melanoma patients on adjuvant immunotherapy in CheckMate 238 [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B027.

Abstract 4127919: Disease-associated variants in DSP -encoded desmoplakin are common, yet low penetrant alleles associated with development of myocarditis and cardiomyopathy at a population level

Intro: DSP- encoded desmoplakin is implicated in a distinct form of arrhythmic cardiomyopathy (ACM), often involving the LV and characterized by myocardial inflammation. Conventional ACM diagnostic criteria has poor sensitivity for DSP -associated ACM, thus highlighting the need to identify the full spectrum of phenotypic risk associated with disease-associated DSP variants. Objective: To analyze the genotypic and phenotypic spectrum of DSP -mediated disease at a large population level. Methods: UK Biobank participants with exome sequencing (ES) were included. Variants were filtered by gene evidence category, yielding 3 groups: predicted deleterious (pDel); subset with predicted loss of function (LOF); and ultra-filtered subset by ClinVar 2* pathogenic/likely pathogenic (P/LP). Phenotypic penetrance was analyzed with DSP -neg individuals as control. Variant location analysis assessed LOFs by susceptibility to nonsense mediated decay region and missenses by amino acid sequence to identify mutational hotspots where P/LPs localize. Results: Out of 200,580 with ES, 1407 DSP carriers had pDel, 168 with LOF, and 44 with ClinVar 2* P/LP. DSP carriers had higher burden of myocarditis, cardiomyopathy (CM), and heart failure. A progressive enrichment in myocarditis and CM was observed by more stringent variant filtering in DSP carriers compared to control. A higher proportion of DSP carriers had myocarditis – 0.28% (4) of the pDels, 1.8% (3) of LOFs, and 4.5% (2) compared to control 0.07% (p<0.05). A parallel trend noted the proportion of DSP carriers with CM growing from 1.4% (19) to 5.4% (9) and 6.8% (3) in the pDel, LOF, and ClinVar 2* P/LPs respectively compared to 0.6% in the control group (p<0.01). Location analysis of missense variants revealed that P/LPs localized in higher proportions (74%) to key domains compared to non-P/LPs (50%; p<0.001), highlighting prognostic value of predicting pathogenicity of missense DSP variants by mutation hotspots. Conclusions: While DSP variants are relatively common in the population, variant presence is associated with low penetrance. Enhancing clinical penetrance with increasingly stringent filtering of DSP variants emphasizes the importance of variant evaluation in clinical risk assessment. This first-in-kind population study of DSP provides insights into novel risk prediction factors, allowing us to predict pathogenicity by type and location of mutation and using variant filtering strategy to better assess phenotypic penetrance.

Assembly, Annotation, and Comparative Analysis of Mitochondrial Genomes in Trichoderma

Trichoderma is a widely studied ascomycete fungal genus, including more than 400 species. However, genetic information on Trichoderma is limited, with most species reporting only DNA barcodes. Mitochondria possess their own distinct DNA that plays a pivotal role in molecular function and evolution. Here, we report 42 novel mitochondrial genomes (mitogenomes) combined with 18 published mitogenomes of Trichoderma. These circular mitogenomes exhibit sizes of 26,276–94,608 bp, typically comprising 15 core protein-coding genes (PCGs), 2 rRNAs, and 16–30 tRNAs; however, the number of endonucleases and hypothetical proteins encoded in the introns of PCGs increases with genome size enlargement. According to the result of phylogenetic analysis of the whole mitogenome, these strains diverged into six distinct evolutionary branches, supported by the phylogeny based on 2830 single-copy nuclear genes. Comparative analysis revealed that dynamic Trichoderma mitogenomes exhibited variations in genome size, gene number, GC content, tRNA copy, and intron across different branches. We identified three mutation hotspots near the regions encoding nad3, cox2, and nad5 that caused major changes in the mitogenomes. Evolutionary analysis revealed that atp9, cob, nad4L, nad5, and rps3 have been influenced by positive selection during evolution. This study provides a valuable resource for exploring the important roles of the genetic and evolutionary dynamics of Trichoderma mitogenome in the adaptive evolution of biocontrol fungi.

Mutant PP2A Induces IGFBP2 Secretion to Promote Development of High-Grade Uterine Cancer.

Uterine serous carcinoma (USC) and uterine carcinosarcoma (UCS) tumors are uniquely aggressive, suggesting that the primary tumor is intrinsically equipped to disseminate and metastasize. Previous work identified mutational hotspots within PPP2R1A, which encodes the Aα scaffolding subunit of protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine phosphatase. Two recurrent heterozygous PPP2R1A mutations, P179R and S256F, occur exclusively within high-grade subtypes of uterine cancer and can drive tumorigenesis and metastasis. Elucidation of the mechanisms by which PP2A-Aα mutants promote tumor development and progression could help identify therapeutic opportunities. Here, we showed that expression of these mutants in USC/UCS cell-lines enhanced tumor-initiating capacity, drove a hybrid epithelial-to-mesenchymal (EM) plasticity phenotype, and elevated secretion of the tumorigenic cytokine IGFBP2. Therapeutic targeting of the IGFBP2/IGF1R signaling axis using small molecules and genetic approaches resulted in marked tumor growth inhibition. Mechanistically, PP2A regulated IGFBP2 expression through the transcription factor, NF-κB, which harbors a B56 recognition motif. Collectively, these results identify a role for PP2A in regulating paracrine cancer cell signaling that can be targeted to block the initiation and metastasis of high-grade uterine cancer.

EPCO-09. DICER1-MUTANT PRIMARY INTRACRANIAL SARCOMA: ASSESSING THE ONCOGENIC ROLE OF MIRNA DYSREGULATION

Abstract Cross-cancer profiling has shown that mutations in human DICER1 are recurrent in diverse cancers including intracranial sarcoma. Due to the rarity of these tumors, treatment options for these patients remain poorly defined. DICER1 encodes an endoribonuclease that processes the hairpin precursor microRNAs (miRNAs) into functionally mature miRNAs. In particular, hotspot mutations in the RNase IIIb domain of DICER1 are predicted to confer an oncogenic role although the precise mechanism is unknown. In a case of a 28-year-old male who presented with multicentric right frontal dural and parenchymal enhancing masses and underwent partial resection, pathology showed a DICER1-mutant primary intracranial sarcoma with a hotspot E1813G mutation in the RNase IIIb domain. Additionally, due to a family history of an unknown fatal brain tumor in his mother, the patient had additional molecular testing which revealed a germline BRCA2 mutation. After surgery, the patient received intensity-modulated radiation therapy followed by 6 cycles of ICE (ifosfamide, carboplatin, etoposide) chemotherapy with significant radiographic response and clinical improvement. He remains progression-free on radiological surveillance for 13 months since diagnosis. The treatment was well tolerated with clinically manageable myelosuppression (including grade 3 thrombocytopenia). This is the first reported case of an adult DICER1-mutant primary intracranial sarcoma patient with a germline BRCA2 mutation although the interaction between these oncogenic mechanisms is unclear. Based on the role of DICER1 in miRNA processing and to test the hypothesize that DICER1 mutations drive primary intracranial sarcoma through dysregulation of miRNA function, we have established a multi-institutional collaboration that is collecting and profiling DICER 1 mutant primary intracranial sarcoma cases from across the United States preliminary results of which will be presented. Identification of a miRNA-driven oncogenic mechanism in these tumors may yield novel targeted approaches beyond intensive chemotherapy for these patients.

A protein vaccine of RBD integrated with immune evasion mutation shows broad protection against SARS-CoV-2.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge and evade immunity, resulting in breakthrough infections in vaccinated populations. There is an urgent need for the development of vaccines with broad protective effects. In this study, we selected hotspot mutations in the receptor-binding domain (RBD) that contribute to immune escape properties and integrated them into the original RBD protein to obtain a complex RBD protein (cRBD), and we found cRBDs have broad protective effects against SARS-CoV-2 variants. Three cRBDs were designed in our study. Compared with the BA.1 RBD protein, the cRBDs induced the production of higher levels of broader-spectrum neutralizing antibodies, suggesting stronger and broader protective efficacy. In viral challenge experiments, cRBDs were more effective than BA.1 RBD in attenuating lung pathologic injury. Among the three constructs, cRBD3 showed optimal broad-spectrum and protective effects and is a promising candidate for a broad-spectrum SARS-CoV-2 vaccine. In conclusion, immunization with cRBDs triggered immunity against a wide range of variants, including those that emerged after we had completed designing the cRBDs. This study preliminarily explores and validates the feasibility of incorporating hotspot mutations that contribute to immune evasion into the RBD to expand the activity spectrum of antigen-induced antibodies.

Concurrent POLE hotspot mutations and mismatch repair deficiency in endometrial cancer: Defining cancer drivers

Concurrent POLE hotspot mutations and mismatch repair deficiency in endometrial cancer: Defining cancer drivers

KRASG12D-Mutated Metastatic Colorectal Cancer: Clinical, Molecular, Immunologic, and Prognostic Features of a New Emerging Targeted Alteration.

KRASG12D mutation (mut) occurs in about 10%-12% of metastatic colorectal cancer (mCRC). Recently, novel KRASG12D inhibitors have been developed and are currently under investigation in phase I/II clinical trials in solid tumors including mCRC. We aimed at performing a comprehensive characterization of clinical, molecular, immunologic, and prognostic features of KRASG12D-mutated mCRC to inform the design and the interpretation of future trials. We performed a pooled analysis of phase III TRIBE and TRIBE2 studies comparing 5-fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI)/bevacizumab (bev) to doublets (5-fluorouracil, leucovorin, and oxaliplatin or 5-fluorouracil, leucovorin, and irinotecan)/bev. One hundred and thirty-six (16%) of 854 patients with available KRASG12D mutational status were KRASG12D mutated. KRASG12D-mutated patients had more frequently right-sided primary tumor and were less likely to present liver-only metastases with respect to other RAS mutated and all-wild-type (wt) patients. Compared with the BRAFV600E-mutated group, KRASG12D-mutated patients had more frequently left-sided primary tumor, resected primary tumor at the time of diagnosis, and Eastern Cooperative Oncology Group performance status 0. KRASG12D-mutated patients had better prognosis than BRAFV600E-mutated and worse prognosis than all wt patients. No prognostic difference was evident between KRASG12D mut and other RAS mut patients overall or according to other specific KRAS or NRAS hotspot mutations. No interaction effect was observed between KRASG12D mut and the benefit provided by FOLFOXIRI/bev compared with doublets/bev. PIK3CA mut were reported more frequently among KRASG12D-mutated tumors compared with both other RAS mut and all wt. A detail estimation of KRASG12D mut mCRC patients' characteristics and expected outcomes may be useful when planning future studies in this subgroup. The high prevalence of PI3K/PTEN/Akt pathway activating alterations may affect the efficacy of targeted strategies.

Somatic GNAS mutations in acromegaly: prevalence, clinical features and gender differences

Background: Somatic GNAS mutations are acknowledged as a significant etiological factor for acromegaly. However, the relationship between GNAS mutation status, clinical characteristics and gender has not been adequately investigated. This study aims to address these gaps by examining GNAS mutations and delineating the detailed clinical profile of affected patients within a Chinese acromegaly cohort. Methods: Our study encompassed 97 individuals newly diagnosed with acromegaly, who underwent surgical treatment between May 2015 and January 2022. We obtained DNA from frozen pituitary adenomas to screen for GNAS hotspot mutations and assessed the associated clinical characteristics. Results: In our cohort, 44.3% (43/97) of patients exhibited somatic GNAS mutations. Patients with mutations were predominantly male (58.1% vs. 33.3%, p=0.015), experienced longer diagnosis delays [72.0 (48.0, 120.0) vs 36.0 (21.0, 75.0) months, p=0.002], had smaller maximum tumor diameters (1.75±0.83 vs. 2.23±0.89 cm, p=0.008), and demonstrated higher rates of GH secretion per unit tumor volume [18.93 (9.67, 30.12) vs 10.91 (2.80, 20.40) ng/mL/cm3, p=0.005]. Regarding gender-specific differences, GNAS mutations in male patients were linked to significantly higher baseline GH levels [24.40 (14.40, 36.30) vs. 10.55 (5.25, 16.95) ng/mL, p=0.002], while female patients with mutations had notably smaller tumor sizes (1.55±0.55 cm vs. 2.32±0.85 cm, p<0.001). Conclusion: GNAS mutations are prevalent among Chinese acromegaly patients, correlating with reduced pituitary tumor sizes and enhanced GH secretion functions. Our findings underscore the influence of gender on the clinical manifestations of GNAS mutations. Accordingly, we recommend that future clinical and foundational researches on acromegaly give heightened consideration to gender-specific differences.

A chip-based universal strategy to realize multiplex PCR by using wax films for sealing and controllable release of primers

Simultaneous detection of multiple nucleic acid targets from a single sample is a common requirement in molecular diagnosis and basic research. Dividing a bulky polymerase chain reaction (PCR) into many isolated small reaction units through microfluidic technology is commonly used to realize this goal. However, previous microfluidic platforms for multiplex PCR suffer from complex structures and strict operation requirements. In this study, a chip-based universal strategy is constructed to realize multiplex PCR by using wax films for sealing and controllable release of prespotted primers. This microfluidic chip contains twenty-four reaction chambers connected in series by a single channel, and the bottom of each reaction chamber is covered by a wax film generated by solvent volatilization. These wax films can prevent the prespotted primers from being flushed away by the reaction mixture during the injection process. After thermal blocking of the connecting channel to isolate each reaction chamber, the chip is placed on a flat thermal cycler. The wax films melt during the denaturing step of PCR so that the primers are released and mixed with the reaction mixture, a process seamlessly compatible with PCR thermal cycling. After fluid flow simulation and carefully examining its basic performance, this chip was applied to genotype seven deafness-associated hotspot mutations using a competitive allele-specific PCR assay. The genotyping results of clinical samples using this chip were totally concordant with those obtained by Sanger sequencing, demonstrating the practical utility of this universal strategy for multiplex PCR detection.

Comparative plastomes sheds light on phylogeny of Weigela.

Weigela Thunb. is a genus in the family Caprifoliaceae. All species in this genus have high ornamental and medicinal value. However, the genetic divergence between species and the phylogeny within Weigela is still unclear. Therefore, we sequenced and analyzed four plastomes from four different Weigela species to reveal the genetic divergence among species of this genus, and the phylogeny within Weigela. The four plastomes from Weigela ranged from 156,909 bp to 157,739 bp in size, and presented a typical circular quadripartite structure. Each complete plastome contained a pair of inverted repeat regions (23,592~24,957 bp), a larger single-copy (LSC) region (89,922~90,229 bp), and a small single-copy (SSC) region (17,668~20,429 bp). We identified three types of repeats, corresponding to 268 forward repeats, 128 palindromic repeats, and 867 tandem repeats, for a total of 1,263 long repeats. A total of 352 SSRs were identified from the four plastomes, and most of them were concentrated in the LSC region and the noncoding regions. Mononucleotide repeat units were the most frequently detected types of repeats, of which A/T repeat units were the most abundant. Three mutational hotspots (trnH-psbA, trnR-ndhF, and trnN-ndhF) were identified as candidate barcodes for Weigela species. Weigela belongs to Diervilloideae located at an early diverging position in the Caprifoliaceae. Within Weigela, W. japonica and W. floribunda were sister with W. subsessilis and W. florida. This study revealed the plastome structure and variation of four well-known Weigela species, and found three candidate barcodes for further study of four well-known Weigela species. In addition, the phylogenetic location of Weigela within the Caprifoliaceae was identified.

Somatic DICER1-Mutant Benign Thyroid Nodules in Adults: A Group of Follicular Nodular Disease With Continuous Growth.

Germline DICER1 mutations cause familial multinodular goiter (MNG). However, the prevalence of somatic DICER1 mutations in non-MNG benign thyroid nodules and their characteristics remain unknown. Adult-onset thyroid nodules with a pathological diagnosis were genotyped via targeted sequencing. DICER1-mutant nodules were assessed clinically and pathologically. Organoids were established to investigate follicular formation and growth. Transcriptomic analysis was conducted to evaluate transcriptional features, which were validated by immunofluorescence. Among 931 adult-onset thyroid nodules, we identified 13 benign thyroid nodules with DICER1 hotspot mutations. The majority harbored a somatic DICER1 hotspot mutation with a somatic DICER1 truncating variant. Clinically, 38.5% of the DICER1-mutant nodules exhibited substantial growth. DICER1-mutant nodules with durations longer than 2 years were substantially enlarged (P=.0448). Pathologically, all DICER1-mutant nodules were defined as thyroid follicular nodular disease (TFND). The TFND nodules with DICER1 mutations grew faster than those with wild-type DICER1. Organoid culture of a DICER1-mutant nodule revealed increased active follicular formation. Compared with the normal thyroid tissues, the DICER1-mutant nodules had comparable thyroid differentiation scores, significantly higher ERK scores (P=.0141) and lower epithelial‒mesenchymal transition scores (P=.0001). Moreover, the expression of genes related to follicular polarity, such as CDH16, SLC5A5, TSHR and TPO, was downregulated in the DICER1-mutant nodules. Somatic DICER1 two-hit mutations represent a notable percentage in adult TFND patients, and DICER1-mutant benign thyroid nodules were characterized by continuous growth.

Detection of PIK3CA hotspot mutations in canine mammary tumors using droplet digital PCR: tissue validation and liquid biopsy feasibility

Domestic dogs (Canis lupus familiaris) serve as valuable translational models for human cancer research due to their biological similarities. Canine mammary tumors (CMTs), frequently diagnosed in female dogs, share various characteristics with human breast cancers. This study investigates the PIK3CA (H1047R) mutation in CMTs using droplet digital PCR (ddPCR) and explores the potential of liquid biopsy for non-invasive detection. We analyzed 80 formalin-fixed, paraffin-embedded (FFPE) CMT tissue samples and compared ddPCR results with next-generation sequencing (NGS) data, achieving high concordance. Plasma and serum samples were also assessed for mutation concordance with tissue results. Our findings indicate a higher frequency of the PIK3CA (H1047R) mutations in benign and grade I malignant CMTs compared to more aggressive malignancies. The ddPCR assay demonstrated high sensitivity and specificity, with plasma testing showing 78.6% sensitivity and 87.5% specificity, and serum testing showing 66.7% sensitivity and 90.0% specificity. These results highlight the viability of liquid biopsy as a minimally invasive method for monitoring PIK3CA mutations in canine patients. The study suggests that liquid biopsy techniques hold significant promise for improving the early detection and monitoring of canine cancers, warranting further research to refine these methods and explore their applications in canine cancer diagnostics and treatment.

Revealing SARS-CoV-2 Mpro mutation cold and hot spots: Dynamic residue network analysis meets machine learning

Deciphering the effect of evolutionary mutations of viruses and predicting future mutations is crucial for designing long-lasting and effective drugs. While understanding the impact of current mutations on protein drug targets is feasible, predicting future mutations due to natural evolution of viruses and environmental pressures remains challenging. Here, we leveraged existing mutation data during the evolution of the SARS-CoV-2 protein drug target main protease (Mpro) to test the predictive power of dynamic residue network (DRN) analysis in identifying mutation cold and hot spots. We conducted molecular dynamics simulations on the Mpro of SARS-CoV-2 (Wuhan strain) and calculated eight DRN metrics (averaged BC, CC, DC, EC, ECC, KC, L, PR), each of which identifies a unique network feature within the protein. The sets of residues with the highest and lowest values for each metric, comprising potential cold and hot spots, were compared to published biochemical analyses and per residue mutation frequencies observed across five SARS-CoV-2 lineages, encompassing a total of 191,878 sequences. Individual DRN metrics displayed only modest power to predict the mutation frequency of individual residues. However, integrating the eight DRN metrics with additional structural and sequence-derived metrics allowed us to develop machine learning models which significantly improved the prediction of residue mutation frequency. While further refinements should enhance accuracy, we demonstrated a robust method to understand pathogen evolution. This approach can also guide the development of long-lasting drugs by targeting functional residues located in and near active site, and allosteric sites, that are less prone to mutations.

Comparative and phylogenetic analysis of the chloroplast genomes of four commonly used medicinal cultivars of Chrysanthemums morifolium

‘Boju’ and ‘Huaiju’ are cultivars of the Chrysanthemum (Chrysanthemum morifolium Ramat.) in the family Asteraceae, valued for their medicinal, tea, and ornamental properties, and valued by individuals. However, the yield and quality of medicinal chrysanthemums are limited by the characteristics of the germplasm resources, including the identification at the varieties and cultivation levels. Currently, research characterizing the chloroplast genomes of medicinal Chrysanthemum flowers is relatively limited. This study conducted chloroplast whole-genome sequencing on two cultivars of Chrysanthemum, ‘Boju’ and ‘Huaiju’, and compared them with the previously published chloroplast genomes of ‘Hangbaiju’ and ‘Gongju’. The study analyzed the chloroplast genome structures of these four medicinal Chrysanthemums, identifying mutation hotspots and clarifying their phylogenetic relationships. The chloroplast genome sizes of four medicinal Chrysanthemum cultivation products ranged from 151,057 to 151,109 bp, with GC content ranging from 37.45% to 37.76%. A total of 134 genes were identified, including 89 protein-coding genes, 37 ribosomal RNA genes, and 8 transfer RNA genes. Comparative genomic analysis revealed 159 large repeat sequences, 276 simple sequence repeats, 1 gene, and 8 intergenic regions identified as highly variable regions. Nucleotide diversity (Pi) values were high (≥ 0.004) for the petN-psbM, trnR-UCU-trnT-GGU, trnT-GGU-psbD, ndhC-trnV-UCA, ycf1, ndhI-ndhG, trnL-UGA-rpl32, rpl32-ndhF, and ndhF-ycf1 fragments, aiding in variety identification. Phylogenetic analysis revealed consistent results between maximum likelihood and Bayesian inference trees, showing that the four medicinal Chrysanthemum cultivars, along with their wild counterparts and related species, evolved as a monophyletic group, forming a sister clade to Artemisia and Ajania. Among the six Chrysanthemum species, the wild Chrysanthemum diverged first (Posterior probability = 1, bootstrap = 1,000), followed by Ajania, while C. indicum and C. morifolium clustered together (Bootstrap = 100), indicating their closest genetic relationship. The chloroplast whole-genome data and characteristic information provided in this study can be used for variety identification, genetic conservation, and phylogenetic analysis within the family Asteraceae.