In-frame Mutations Research Articles

An insertion mutation of ERBB2 enhances breast cancer cell growth and confers resistance to lapatinib through AKT signaling pathway.

ABSTRACTIn clinical practice, some breast cancer (BC) patients carry a rare ERBB2 in-frame insertion (p. Pro780_Tyr781insGlySerPro) and are resistant to anti-ERBB2 therapy. To explore the potential procarcinogenic role of this ERBB2 mutation, we conducted the present study using BC cells overexpressing wild-type (WT) ERBB2 or P780-Y781 ERBB2 [mutated (MT)]. MDA-MB-231 and MCF-7 cells were transfected with the following plasmids using a lentivirus system: negative control (ERBB2-NC), WT ERBB2 overexpression (ERBB2-WT), and P780-Y781 ERBB2 overexpression (ERBB2-MT). P780-Y781 ERBB2 conferred significant resistance to lapatinib, as assessed by cell viability and colony counts. Analysis of the cell cycle showed that the P780-Y781 ERBB2 group showed an elevated proportion of cells in S, G2, and M phases compared with WT ERBB2 when exposed to lapatinib. Following lapatinib treatment, phosphorylated AKT (p-AKT) was strongly upregulated in the P780-Y781 ERBB2 group. Among ERBB2+ patients, the P780-Y781 ERBB2 group showed increased levels of p-AKT. Furthermore, the AKT inhibitor perifosine effectively suppressed lapatinib resistance, as indicated by the lapatinib inhibition curve and results of the colony formation assay, and decreased AKT phosphorylation. Altogether, we discovered a procarcinogenic mutation of ERBB2 that enhances BC cell growth through AKT signaling and causes resistance to lapatinib. Patients with this in-frame insertion mutation of ERBB2 should be recommended other therapeutic strategies apart from ERBB2 tyrosine kinase inhibitors, in particular lapatinib.

Generation and characterisation of a COV434 cell clone carrying a monoallelic FecBB mutation introduced by CRISPR/Cas9

The FecBB mutation, which increases the ovulation quota, was identified in a hyperprolific sheep breed, and is associated with a single point mutation (c.A746G; p.Q249R) in bone morphogenetic protein receptor-type 1B (BMPR1B). However, the mechanism of action of the FecBB mutation remains unclear. Here, we describe the application of clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) to introduce the FecBB mutation into the human granulosa cell line COV434 via homology-directed repair (HDR), using single-stranded oligodeoxynucleotides (ssODNs) as template. Upon screening single cell-derived clones, we found one clone containing the FecBB mutation on one allele and knockout of BMPR1B on the other allele, and another clone harbouring an in-frame deletion on one allele and knockout of BMPR1B on the other. These two clones were subjected to functional analysis. We found that the FecBB mutation and knockout of BMPR1B could upregulate the expression of cumulus expansion-related genes, potentially mediated by enhanced activation of the Sma- and Mad-related 1/5 (SMAD1/5) pathway. The FecBB mutation, an in-frame mutation, and knockout of BMPR1B could impair the synthesis of oestradiol in COV434 cells, possibly through inhibition of the SMAD2/3 pathway. Our study provides a useful cell model for further investigation into the mechanism of action of the FecBB mutation.

Prevalence of ESR1 Mutation in Chinese ER-Positive Breast Cancer.

BackgroundESR1 mutation and its possible relation to endocrine therapy resistance in ER-positive breast cancers have been studied with respect to genetic sequencing data from Western patients but rarely from Chinese patients. This study aimed to investigate the prevalence of ESR1 mutation in Chinese primary and metastatic ER-positive breast cancer.MethodsTumor samples from 297 primary breast cancer (PBC) patients and blood samples from 43 metastatic breast cancer (MBC) patients were obtained to perform whole exon sequencing of the ESR1 gene through next-generation sequencing (NGS). Clinicopathological features of MBC patients were listed and grouped to explore potential factors in ESR1 mutations.ResultsA total of 15 ESR1 variations, including 11 point mutations, 1 in-frame deletion mutation, 1 synonymous mutation, and 2 amplifications were identified in 13 patients. The ESR1 mutation rate was 1% (3/297) in PBC patients and 18.6% (8/43) in MBC patients. All ESR1 point mutations occurred in the estrogen receptor ligand-binding domain. Six (54.5%) of the 11 point mutations were hotspot mutations. Among all MBC patients, the ESR1 mutation rate in those who had a treatment history using aromatase inhibitors (AI) was significantly higher than those who did not (25.8% versus 0%, P=0.015). Moreover, the ESR1 mutation rate in those who received AI treatment over a period of 12 months was significantly higher than in those whose treatment lasted less than 12 months [36.3% versus 0%, P<0.001].ConclusionESR1 mutations were more frequently observed in the circulating cell-free DNA of MBC patients than in PBC patients among the Chinese cohort, and higher among those pretreated with AI, suggesting that such mutations may undergo selection during AI treatment.

Structural modeling, mutation analysis, and in vitro expression of usherin, a major protein in inherited retinal degeneration and hearing loss

Usherin is the most common causative protein associated with autosomal recessive retinitis pigmentosa (RP) and Usher syndrome (USH), which are characterized by retinal degeneration alone and in combination with hearing loss, respectively. Usherin is essential for photoreceptor survival and hair cell bundle integrity. However, the molecular mechanism underlying usherin function in normal and disease conditions is unclear. In this study, we investigated structural models of usherin domains and localization of usherin pathogenic small in-frame mutations, mainly homozygous missense mutations. We found that usherin fibronectin III (FN3) domains and most laminin-related domains have a β-sandwich structure. Some FN3 domains are predicted to interact with each other and with laminin-related domains. The usherin protein may bend at some FN3 linker regions. RP- and USH-associated small in-frame mutations are differentially located in usherin domains. Most of them are located at the periphery of β-sandwiches, with some at the interface between interacting domains. The usherin laminin epidermal growth factor repeats adopt a rod-shaped structure, which is maintained by disulfide bonds. Most missense mutations and deletion of exon 13 in this region disrupt the disulfide bonds and may affect local protein folding. Despite low expression of the recombinant entire protein and protein fragments in mammalian cell culture, usherin FN3 fragments are more robustly expressed and secreted than its laminin-related fragments. Our findings provide new insights into the usherin structure and the disease mechanisms caused by pathogenic small in-frame mutations, which will help inform future experimental research on diagnosis, disease mechanisms, and therapeutic approaches.

Mutational Type and Configuration of an Individual Gene May Differentially Impact the Clinical and Phenotypic Features

Myelodysplastic syndromes (MDS) arise in older adults through the stepwise acquisition of multiple somatic mutations. The genetic heterogeneity that results includes mutations of diverse genes and their combinations, clonal hierarchy, genetic configuration (e.g., bi-allelic or compound heterozygous, hemizygous lesions), specific positions within a gene including canonical hotspots vs. other positions, and types of mutation (truncations vs. missense), all of which could differentially affect pathogenesis. Given the binary status (e.g. mutated vs. wild-type) used in many clinical analyses, the true impact of specific types of mutations may be obscured and their specific roles underestimated. Deep targeted NGS was carried out for a panel of the 36 most frequently mutated genes in 1,809 MDS patients (low-risk MDS (n=839) vs. high-risk MDS (n=607), MDS/MPN (n=212), and sAML n=151). Copy number alterations (CNA) were also evaluated by combining karyotyping, microarray, and digital copy number analysis. With a mean coverage of 862x, after removing SNPs and errors, 3,971 somatic mutations were identified, the most common (&gt;10% of cases) being TET2, SF3B1, ASXL1, del(5q), SRSF2, complex karyotype, and del(7q). For the purpose of this proof of concept analysis we focused on illustrative genes (TP53, RUNX1, TET2, and EZH2) affected by 2 recurrent hits. Bi-allelic TET2 or TP53 mutations were found in 15% (271/1,809) and 4% (72/1,809) of patients, respectively. TET2 and RUNX1 were most likely biallelic, whereas TP53 and EZH2 were most often affected by mutations and somatic deletion. Comparing the distribution of canonical vs. other types of mutations in genes, DNMT3A mutations affected the canonical site (R882) in 17% (35/203) of patients, were truncating in 39% (79/203) and missense in 44% (89/203) have also been found; deletions affecting the DNMT3A locus are rare. Within U2AF1, U2AF1Q157 are more frequent than U2AF1S34 (54% vs. 35%). Next, we checked correlation between these different types of mutations of one gene. 78 significant combinations were found. For instance, U2AF1Q157 mutations more commonly accompanied ASXL1 mutations and del(7q) and less frequently DNMT3A and BCOR mutations, trisomy8 and del(20) when compared to U2AF1S34 mutations [ASXL1 mutations 53% (42/80) in U2AF1Q157 vs. 16% (8/49) in U2AF1S34, P &lt; .0001]. TET2 Bi-allelic mutations were more commonly associated with ZRSR2 and SRSF2 mutations, and less frequently del(5q) when compared to TET2 mono-allelic mutations [SRSF2 mutations 29% (80/276) in TET2-bi vs. 15% (34/227) in TET2-mono, P = .003]. In addition, patients with SRSF2 missense mutations were more likely to have RUNX1 bi-allelic mutations than those with SRSF2 in-frame mutations. We evaluated the impact of different types of mutations and combinations of them on disease phenotypes and survival. We then evaluated the impact of different types of mutations and their combinations on clinical phenotypes including dichotomous morphological (MDS vs. MDS/MPN) features, progressive (low- vs. high risk) subtypes. EZH2 bi-allelic alterations were more commonly associated with myleoproliferative features` compared to EZH2 mono-allelic alteration (q=.016). TET2 bi-allelic alterations and truncating mutations were found more frequently in higher-risk subtypes than TET2 mono-allelic and missense mutations (q&lt;.001). In survival analyses, patients with DNMT3AR882 mutations had a poorer prognosis than those with truncating and the other missense mutations [P = .033, HR 1.86 (1.05-3.3)]. Next, using the PyClone bioanalytic pipeline, we recapitulated for each patient the clonal hierarchy and defined "dominant" vs. "secondary" mutations. DNMT3AR882 mutations were likely to be dominant/founder lesions compared to truncating or the other missense mutations: 77% (27/35) for R882 vs. 51% (40/79) for truncating vs. 45% (47/98) for the other missense, p=.0046. Specific dominant and secondary mutational pairs also differentially affected survival compared to the reverse configuration (q&lt;.1) including EZH2 and RUNX1 or BCOR and U2AF1 or RUNX1 and BCOR. In conclusion, we report a comprehensive analysis of various types and configurations of lesions of individual commonly affected genes. Our results indicate that establishment of clinical or phenotypic correlations requires consideration of the type, rank and configuration of somatic mutations. Disclosures Mukherjee: McGraw Hill Hematology Oncology Board Review: Other: Editor; Bristol-Myers Squibb: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy. Nazha:Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Tolero, Karyopharma: Honoraria; Abbvie: Consultancy; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Asahi Genomics: Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Qiagen Corporation: Patents &amp; Royalties; RegCell Corporation: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Kan Research Laboratory, Inc.: Consultancy. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

TMOD-22. LOSS OF PTEN AND TRP53 IN OLIGODENDROCYTE PROGENITORS LEADS TO GLIOMA FORMATION

Abstract Tumor suppressors PTEN and TP53 are the most commonly mutated anti-oncogenes in human gliomas after TERT mutation. 38% and 17% of total 4023 samples of gliomas in TCGA show TP53 and PTEN mutations respectively. TP53 signaling pathway plays an important role in the suppression of tumor progression PTEN negatively regulates PI3 triphosphate levels and suppresses AKT/PKB pathways. In-frame and missense mutations in PTEN constitutively activates the proliferative pathway and give a survival advantage. Mice with conditional mutation of PTEN and TP53 in neural stem cells directed by hGFAP-Cre develop high-grade gliomas. Our recent study indicates that primitive oligodendrocyte-progenitor intermediates (Pri-OPC) are abundant and hyperproliferative cells and can be progressively reprogrammed towards a stem-like state susceptible to malignant transformation. We hypothesize that mutations in PTEN and TP53 in pri-OPC lineage cells can lead to glioma formation. OPCs distribute across the whole adult brain and exhibit a much higher number than NSCs that are restricted to very small niche areas. Olig1 and Olig2 mark pri-OPC and oligodendrocyte lineage cells. In this study, we inactivated the PTEN/TP53 gene in mouse pri-OPCs using Olig1-Cre or Olig2tva-Cre. The incidence of glioma formation was 100%. Both the tumor formed in these models display multifocal, high-grade, diffuse and infiltrating gliomas. Though tumors are derived from similar cell sources, they vary in the expression and extent of oncogenic pathways. Our transcriptomics profiling at different stages of tumorigenesis reveals that progressive activation of oncogenic signaling pathways establishes a tumorigenic state to promote malignant transformation, suggesting a dynamic process of tumorigenesis caused by the cooperative action of the PTEN and TP53 loss in the pri-OPC lineage.

Infrared biomarkers of impaired cystic fibrosis transmembrane regulator protein biogenesis.

The mid-infrared (IR) spectra of human cystic fibrosis (CF) cells acquired by Fourier transform infrared microspectroscopy were compared with those of non-CF cells. Within the 1700 to 1480 cm-1 spectral domain of amides, unsupervised explorative principal component analysis identified a few variables reflecting quantitative and qualitative vibrations arising from protein secondary structures and amino acid side chains. Their pattern reflected α-helix to β-sheet transitions in bronchial epithelial cells and in immortalized peripheral blood mononuclear cells from patients with R1162X missense or in-frame F508del mutations in the cystic fibrosis transmembrane regulator gene (Cftr). Similar transitions have been described in IR spectra of cells, tissues and body fluids of patients affected with some neurodegenerative diseases characterized by the accumulation of misfolded protein aggregates. The variables pattern was able to distinguish CF cells from non-CF cells and was modified by molecular compounds used to rescue the unbalanced folding process of mutated cystic fibrosis transmembrane regulator (CFTR) anion channel. To our knowledge, this is the first experimental evidence of spectroscopic biomarkers of the impaired biogenesis of CFTR by IR microanalysis in the spectra of human CF bronchial epithelial and lymphoblastoid cells.

Universal stress proteins contribute Edwardsiella piscicida adversity resistance and pathogenicity and promote blocking host immune response

Universal stress proteins (Usps) exist ubiquitously in bacteria and other organisms. Usps play an important role in adaptation of bacteria to a variety of environmental stresses. There is increasing evidence that Usps facilitate pathogens to adapt host environment and are involved in pathogenicity. Edwardsiella piscicida (formerly included in E. tarda) is a severe fish pathogen and infects various important economic fish including tilapia (Oreochromis niloticus). In E. piscicida, a number of systems and factors that are involved in stress resistance and pathogenesis were identified. However, the function of Usps in E. piscicida is totally unknown. In this study, we examined the expressions of 13 usp genes in E. piscicida and found that most of these usp genes were up-regulated expression under high temperature, oxidative stress, acid stress, and host serum stress. Particularly, among these usp genes, usp13, exhibited dramatically high expression level upon several stress conditions. To investigate the biological role of usp13, a markerless usp13 in-frame mutant strain, TX01Δusp13, was constructed. Compared to the wild type TX01, TX01Δusp13 exhibited markedly compromised tolerance to high temperature, hydrogen peroxide, and low pH. Deletion of usp13 significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis showed that the inactivation of usp13 significantly impaired the ability of E. piscicida to invade into host cell and infect host tissue. Introduction of a trans-expressed usp13 gene restored the lost virulence of TX01Δusp13. In support of these results, host immune response induced by TX01 and TX01Δusp13 was examined, and the results showed reactive oxygen species (ROS) levels in TX01Δusp13-infected macrophages were significantly higher than those in TX01-infected cells. The expression level of several cytokines (IL-6, IL-8, IL-10, TNF-α, and CC2) in TX01Δusp13-infected fish was significantly higher than that in TX01-infected fish. These results suggested that the deletion of usp13 attenuated the ability of bacteria to overcome the host immune response to pathogen infection. Taken together, our study indicated Usp13 of E. piscicida was not only important participant in adversity resistance, but also was essential for E. piscicida pathogenicity and contributed to block host immune response to pathogen infection.

HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida

Edwardsiella piscicida is a severe fish pathogen. Haem utilization systems play an important role in bacterial adversity adaptation and pathogenicity. In this study, a speculative haem utilization protein, HutZEp, was characterized in E. piscicida. hutZEp is encoded with two other genes, hutW and hutX, in an operon that is similar to the haem utilization operon hutWXZ identified in V. cholerae. However, protein activity analysis showed that HutZEp is probably not related to hemin utilization. To explore the biological role of HutZEp, a markerless hutZEp in-frame mutant strain, TX01ΔhutZ, was constructed. Deletion of hutZEp did not significantly affect bacterial growth in normal medium, in iron-deficient conditions, or in the presence of haem but significantly retarded bacterial biofilm growth. The expression of known genes related to biofilm growth was not affected by hutZEp deletion, which indicated that HutZEp was probably a novel factor promoting biofilm formation in E. piscicida. Compared to the wild-type TX01, TX01ΔhutZ exhibited markedly compromised tolerance to acid stress and host serum stress. Pathogenicity analysis showed that inactivation of hutZEp significantly impaired the ability of E. piscicida to invade and reproduce in host cells and to infect host tissue. In contrast to TX01, TX01ΔhutZ was defective in blocking host macrophage activation. The expression of hutZEp was directly regulated by the ferric uptake regulator Fur. This study is the first functional characterization of HutZ in a fish pathogen, and these findings suggested that HutZEp is essential for E. piscicida biofilm formation and contributes to host infection.

Clinical Characteristics and Outcomes of Non-small Cell Lung Cancer Patients with HER2 Alterations in Korea.

PurposeHuman epidermal growth factor receptor 2 (HER2) alterations are found in approximately 1%-3% of non-small cell lung cancers (NSCLCs). We evaluated the clinical features and outcomes of NSCLC harboring HER2 alteration detected by next-generation sequencing (NGS) in Korea.Materials and MethodsA total of 1,108 patients who were diagnosed with NSCLC between December 2015 and December 2017 were screened and analyzed by NGS. Medical records were reviewed retrospectively to analyze the clinical characteristics and outcomes from various treatments.ResultsHER2 alterations were identified in 36 NSCLC patients. Of the patients, 22 (61.1%) had an exon 20 in-frame insertion mutation, 15 (41.7%) had HER2 amplification, and one had both. The median patient age was 58 years, 55.6% were male, and 50.0% were never-smokers. Adenocarcinoma was predominant (88.9%). The most common metastatic site was bone (58.3%), and 66.7% of patients were stage IV at initial diagnosis. Six patients (16.7%) had a coexistent sensitizing epidermal growth factor receptor (EGFR) mutation, and two patients (5.6%) had anaplastic lymphoma kinase (ALK) rearrangement. With a median 14 months of follow-up, the median progression-free survival of first-line treatment was 6 months (95% confidence interval, 4.172 to 7.828), and median overall survival was not reached. The proportions of adenocarcinoma, never-smokers, and metastasis to the liver were higher in the exon 20 in-frame insertion mutation group, whereas coexistence of EGFR mutation was more frequently found in the HER2 amplification group.ConclusionHER2-altered NSCLC showed distinct clinical features. Moreover, different characteristics were identified between the HER2 in-frame insertion mutation group and the HER2 amplification group.

A deep intronic splice mutation of STAT3 underlies hyper IgE syndrome by negative dominance

Heterozygous in-frame mutations in coding regions of human STAT3 underlie the only known autosomal dominant form of hyper IgE syndrome (AD HIES). About 5% of familial cases remain unexplained. The mutant proteins are loss-of-function and dominant-negative when tested following overproduction in recipient cells. However, the production of mutant proteins has not been detected and quantified in the cells of heterozygous patients. We report a deep intronic heterozygous STAT3 mutation, c.1282-89C>T, in 7 relatives with AD HIES. This mutation creates a new exon in the STAT3 complementary DNA, which, when overexpressed, generates a mutant STAT3 protein (D427ins17) that is loss-of-function and dominant-negative in terms of tyrosine phosphorylation, DNA binding, and transcriptional activity. In immortalized B cells from these patients, the D427ins17 protein was 2 kDa larger and 4-fold less abundant than wild-type STAT3, on mass spectrometry. The patients' primary B and T lymphocytes responded poorly to STAT3-dependent cytokines. These findings are reminiscent of the impaired responses of leukocytes from other patients with AD HIES due to typical STAT3 coding mutations, providing further evidence for the dominance of the mutant intronic allele. These findings highlight the importance of sequencing STAT3 introns in patients with HIES without candidate variants in coding regions and essential splice sites. They also show that AD HIES-causing STAT3 mutant alleles can be dominant-negative even if the encoded protein is produced in significantly smaller amounts than wild-type STAT3.

Comparative genomic profiling of paired tissue and blood samples from advanced non-small cell lung cancer patients.

e20521 Background: While tissue-based next generation sequencing (NGS) ushered in a new era of precision medicine, profiling of circulating tumor DNA (ctDNA) has also emerged as a minimally invasive alternative to inform clinical decisions. In non-small cell lung cancer (NSCLC), data describing comparison between mutational profiles of paired tissue and blood samples remained scarce. Methods: Matched formalin-fixed paraffin-embedded tissue and peripheral blood samples (≤14 days apart) were collected from treatment naïve advanced NSCLC patients. Genomic profiling was performed using whole exome sequencing (WES) for tissues and a 150-gene panel for ctDNA, with a mean sequencing depth of 239× and 3417× respectively. Tumor- and blood-based tumor mutational burdens (tTMB and bTMB) were defined as the sum of missense, stop-loss, in-frame and frameshift mutations in protein coding regions and the number of somatic single nucleotide variations and indels in targeted coding regions respectively. Maximum somatic allele frequency (MSAF) was also determined for each case as a measure of ctDNA quantity. Results: Matched tissue and blood samples from 48 patients were included for sequencing. A total of 410 mutations were identified, where 84 (20.5%) were covered in both sample types. Sixty-three (15.4%) alterations were only detected in tissues while 263 (64.1%) were exclusively seen in ctDNA. The most frequently altered genes were SMARCA2 (62.5%), TP53 (54.2%), and AR (47.9%) in ctDNA and were TP53 (37.5%) and EGFR (22.9%) in tissues. Aberrations in all genes, except for FAT1 and KRAS, occurred at a higher frequency in ctDNA. The median tTMB and bTMB were 75 and 7, respectively. bTMB displayed a moderate linear relationship with tTMB as indicated by a Spearman correlation of 0.62 (P &lt; 0.01). Patients with bTMB ≥ 7 had significantly higher MSAF (Mann-Whitney P &lt; 0.001), suggesting a positive correlation between bTMB and ctDNA fraction in blood. Conclusions: Mutational profiles as well as TMB levels were in general consistent between blood and tissue samples, further corroborating a role for ctDNA testing as a complementary approach to tissue testing in advanced NSCLC.

The landscape of RET alterations from 56,970 adult patients with cancer: Clinical implications.

3106 Background: Activating receptor-tyrosine kinase rearranged during transfection ( RET) mutations and fusions have been recognized as potent drivers of oncogenesis. Recent identification of highly potent and selective RET inhibitors holds great promise in the management of RET-dependent tumors. Here we present a comprehensive analysis of RET alterations in pan-cancer adult malignancies. Methods: We analyzed 59,347 samples from 56,970 patients available from AACR Project GENIE (Cancer Discov. 2017) database for the prevalence of RET fusions, mutations, and copy number alterations in diverse cancer types. Results: A total of 1414 RET alterations were detected, including 91 fusions (6.4%), 1166 missense mutations (82.5%), 136 truncating mutations (9.6%), and 21 in-frame mutations (1.5%). RET fusions were observed in 0.15% of tumor samples and were most commonly identified in non-small cell lung cancer, thyroid cancer, colorectal cancer, prostate cancer, and gastric cancer (62.6%, 18.6%, 5.5%, 4.4%, 3.3% of identified RET fusions, respectively). RET fusions were significantly co-altered with MAPK3/ERK1 (p=0.045), SETD2 (p=1.36E-07 ), and EIF4E (p=0.045), while there was a negative association between RET fusions and EGFR (p=0.009634) , TP53 (p=0.02267), and KRAS (p=2.53E-05) alterations. Most common RET gene upstream partners were KIF5B, CCDC6, and NCOA4 (42.9%, 24.2%, 7.7% of identified RET fusions, respectively). RET missense mutations were found in 2.0% of tumor samples; 136 (11.7%) of identified missense mutations, including 8 RET gatekeeper V804M/L mutations, were characterized as likely oncogenic, 12 (1.0%) as likely benign, and 1018 (87.3%) as variants of unknown significance using OncoKB database. RET amplifications occurred in 1.5% of tested samples. Conclusions: While RET fusions represent extremely rare events in multiple cancers, RET missense mutations occur in 2% of malignancies. Most RET missense variants are described as variants of unknown significance, limiting the impact of precision oncology for the majority of patients with RET alterations. Further functional characterization of RET variants is warranted. MAPK pathway co-alterations in patents with RET fusions may present a strategy for future therapeutic combinations.

CBP/p300 antagonises EGFR-Ras-Erk signalling and suppresses increased Ras-Erk signalling-induced tumour formation in mice.

CREB-binding protein (CBP) and p300 have oncogenic properties; both co-operate with pro-oncogenic transcription factors downstream of Ras-Erk signalling to support cell proliferation. By contrast, missense, truncating and in-frame mutations of CBP/p300 are found frequently in some human cancers, including cutaneous squamous cell carcinomas that originate from epidermal keratinocytes. Data support that dysfunction of CBP/p300 contributes to keratinocyte hyperproliferation and tumourigenesis; however, the mechanism by which dysfunction of CBP/p300 affects keratinocytes is unknown. Here, we used mice harbouring keratinocyte-specific genetic modifications to examine the role of CBP/p300 in the epidermis. While a single copy of either Crebbp or Ep300 was necessary and sufficient for maintaining epidermal development, reduced expression of CBP/p300 strengthened the Ras-Erk signalling-induced hyperplastic phenotype of epidermal keratinocytes. Reduced CBP/p300 expression increased ligand-induced EGFR activity while decreasing basal expression of Mig6, a negative regulator of EGFR. A reduction in CBP/p300, in combination with increased Ras-Erk signalling, also promoted epidermal tumour formation in mice. Thus, our findings support that CBP/p300 acts as a tumour suppressor in epidermal keratinocytes by counteracting EGFR-Ras-Erk signalling. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Apparent synonymous mutation F9 c.87A>G causes secretion failure by in-frame mutation with aberrant splicing

IntroductionHemophilia B is an X-linked recessive bleeding disorder caused by coagulation factor IX (FIX) gene (F9) mutations. Several F9 synonymous mutations have been known to cause hemophilia B; however, the deleterious mechanisms underlying the development of hemophilia B have not been completely understood. To elucidate the molecular pathogenesis causing hemophilia B, we investigated the synonymous F9 mutation: c.87A>G, p.(Thr29=). Materials and methodsThe influence of F9 c.87A>G on mRNA splicing was analyzed by exon-trap assay and in silico prediction. We prepared FIX expression vectors using mutant F9 cDNA and transfected HepG2 cells to investigate intracellular transport and extracellular secretion of FIX. Intracellular kinetics of the expressed FIX was examined by treatment with the proteasome inhibitor MG132. ResultsExon-trap analysis revealed that F9 c.87A>G resulted in almost (99.1%) aberrant splicing (r.83_88del). In silico analysis predicted that F9 c.87A>G influenced the splicing pattern by generating an available aberrant 5′ splice site. The aberrant F9 mRNA (r.83_88del) was translated to a mutant FIX p.Cys28_Val30delinsPhe with an in-frame mutation at the signal peptide cleavage site. Simultaneously, a small amount (0.9%) of mutant F9 r.87A>G translating into WT FIX p.Thr29 = was also observed. The mutant FIX was abnormally retained in the endoplasmic reticulum (ER) and was not extracellularly secreted. It appeared to be intracellularly degraded via proteasome-dependent degradation machinery. ConclusionF9 c.87A>G was found to cause abnormal mRNA splicing, r.83_88del, and produce the mutant FIX p.Cys28_Val30delinsPhe. The mutant FIX is an abnormal protein with extracellular secretory defects and is intracellularly eliminated via proteasome-dependent ER-associated degradation.

True MEN1 or phenocopy? Evidence for geno-phenotypic correlations in MEN1 syndrome

PurposeMultiple endocrine neoplasia type 1 is a rare tumor syndrome caused by germline mutations of MEN1 gene. Phenotype varies widely, and no definitive correlation with the genotype has been observed. Mutation-negative patients with MEN1-associated tumors represent phenocopies. By comparing mutation-positive and mutation-negative patients, we aimed to identify phenotype features predictive for a positive genetic test and to evaluate the role of MEN1 mutations in phenotype modulation.MethodsMutation screeening of MEN1 gene by Sanger sequencing and assessment of clinical data of 189 consecutively enrolled probands and relatives were performed at our national and European Reference Center. Multiple ligation probe amplification analysis of MEN1 gene and Sanger sequencing of CDKN1B were carried out in clinically suspicious but MEN1-negative cases.ResultsTwenty-seven probands and twenty family members carried MEN1 mutations. Five mutations have not been described earlier. Pronouncedly high number of phenocopies (>70%) was observed. Clinical suspicion of MEN1 syndrome emerged at significantly earlier age in MEN1-positive compared to MEN1-negative probands. Gastroenteropancreatic neuroendocrine tumors developed significantly earlier and more frequently in carriers compared to non-carriers. Probands with high-impact (frameshift, nonsense, large deletions) mutations, predicted to affect menin function significantly, developed GEP-NETs more frequently compared to low-impact (inframe and missense) mutation carriers.ConclusionsMEN1 phenocopy is common and represents a significant confounder for the genetic testing. GEP-NET under 30 years best predicted a MEN1 mutation. The present study thus confirmed a previous proposal and suggested that GEP-NET under 30 years should be considered as a part of the indication criteria for MEN1 mutational analysis.

Thioredoxin H (TrxH) contributes to adversity adaptation and pathogenicity of Edwardsiella piscicida

Thioredoxins (Trxs) play an important role in defending against oxidative stress and keeping disulfide bonding correct to maintain protein function. Edwardsiella piscicida, a severe fish pathogen, has been shown to encode several thioredoxins including TrxA, TrxC, and TrxH, but their biological roles remain unknown. In this study, we characterized TrxH of E. piscicida (named TrxHEp) and examined its expression and function. TrxHEp is composed of 125 residues and possesses typical thioredoxin H motifs. Expression of trxHEp was upregulated under conditions of oxidative stress, iron starvation, low pH, and during infection of host cells. trxHEp expression was also regulated by ferric uptake regulator (Fur), an important global regulatory of E. piscicida. Compared to the wild type TX01, a markerless trxHEp in-frame mutant strain TX01∆trxH exhibited markedly compromised tolerance of the pathogen to hydrogen peroxide, acid stress, and iron deficiency. Deletion of trxHEp significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis shows that the inactivation of trxHEp significantly impaired the ability of E. piscicida to invade host cells, reproduce in macrophages, and infect host tissues. Introduction of a trans-expressed trxH gene restored the lost virulence of TX01∆trxH. There is likely to be a complex relationship of functional complementation or expression regulation between TrxH and another two thioredoxins, TrxA and TrxC, of E. piscicida. This is the first functional report of TrxH in fish pathogens, and the findings suggest that TrxHEp is essential for coping with adverse circumstances and contributes to host infection of E. piscicida.

ZeoR: A Candidate for Assays Testing Enzymatic Activities and Off-Target Effects of Gene-Editing Enzymes.

The target lengths of gene-editing enzymes vary from approximately 20 to 80 nucleotides. The ideal reporter genes should be able to tolerate the insertion of target sequences to differentiate the phenotypic changes between in-frame and frame shift mutations for evaluating the enzymatic activities and off-target effects of gene-editing enzymes such as zinc finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats-associated system. This study demonstrated that the zeocin resistance gene (ZeoR) tolerated the insertion of long targets for a variety of tests using a strategy of inserting tandem repeats of NNT. Four genes such as CCR5, AR, HPV 16E7 early gene, and EGFR applications of ZeoR in quantitatively analyzing the off-targets of CRISPR were successful. The data indicated that evaluating the enzymatic activities and off-target effects with assays employing ZeoR had great potential in facilitating the application of gene editing to human gene therapy.

FLNC mutations in patients with cardiomyopathies: Prevalence and genotype-phenotype correlations

Mutations in FLNC encoding filamin C have been firstly reported to cause dominant myofibrillar and distal myopathy. More recently, dominant pathogenic variants in FLNC have also been linked to the development of isolated cardiac phenotypes. Only few publications on cohorts with FLNC pathogenic variants are published and the pathophysiology of FLNC-related cardiomyopathy is poorly understood. The aim of this retrospective study is to establish the prevalence of mutation in the FLNC gene in the different subtypes of cardiomyopathies and search for genotype-phenotype associations. DNAs from a cohort of 1150 unrelated index-patients with an isolated cardiomyopathy (700 hypertrophic, 300 dilated, 50 restrictive cardiomyopathies, and 100 left ventricle non-compactions) have been sequenced on a custom panel of 52 cardiomyopathy disease-causing genes. A FLNC pathogenic mutation was identified in 28 patients corresponding to a prevalence ranging from 1 to 8% depending on the cardiomyopathy subtypes. Truncating mutations were always identified in patients with dilated cardiomyopathy, while mis-sense or in-frame mutations were found in other phenotypes. In the cohort, nine patients (32%) were implanted with an automatic defibrillator. In 7 families (25%), history of sudden cardiac death (SCD) before 50 years was reported. A personal or family history of SCD was significantly higher in patients with truncating variants than in patients carrying missense variants ( P = 0.01). Acute heart failure was observed in 7 patients (25%). Four patients died of cardiac cause including 3 from SCD and 1 from heart failure. This work highlights the role of FLNCin studied cardiomyopathy subtypes. A correlation between the type of the mutation and the cardiomyopathy subtype was observed as well as with SCD risk. These new data should be taken into consideration for patient's management and primary prevention of sudden cardiac death.

A novel in-frame mutation in CLN3 leads to Juvenile neuronal ceroid lipofuscinosis in a large Pakistani family

Aim: Neuronal ceroid lipofuscinosis (NCLs) are the most common neurodegenerative disorders, with global incidence of 1 in 100,000 live births. NCLs affect central nervous system, primarily cerebellar and cerebral cortices. Juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease, is the most common form of NCLs. JNCL is primarily caused by pathogenic mutations in CLN3 gene, which encodes a transporter transmembrane protein of uncertain function. The 1.02 kb deletion is the most common mutation in CLN3 that results in frame shift and a premature termination leading to nonfunctional protein. Here, we invetigated a large consanguineous family consisting of four affected individuals with clincal symptoms suggestive of Juvenile neuronal ceroid lipofuscinosis.Materials and methods: We conducted clinial and radilogical investigation of the family and performed NGS based Gene Panel sequencing comprising of five hundred and forty five candidate genes to characterize it at genetic level.Results: We identified a novel homozygous c.181_183delGAC mutation in the CLN3 gene seggregating witht the disorder in the family. The mutation induces in-frame deletion, deleting one amino acid (p.Asp61del) in CLN3 protein. The deleted amino acid aspartic acid plays an important role as general acid in enzymes active centers as well as in maintaining the ionic character of proteins.Conclusion: Our finding adds to genetic variability of Juvenile neuronal ceroid lipofuscinosis associated with CLN3 gene and a predicted CLN3 protein interacting domain site.