Frameshift Mutations In Genes Research Articles

Integrative genomic, proteomic and phenotypic studies of Leishmania donovani strains revealed genetic features associated with virulence and antimony-resistance

BackgroundLeishmaniasis is a neglected tropical disease affecting millions of people worldwide. Emerging drug resistance of Leishmania species poses threaten to the effective control and elimination programme of this neglected tropical disease.MethodsIn this work, we conducted drug-resistance testing, whole genome resequencing and proteome profiling for a recently reported clinical isolate with supposed drug resistance (HCZ), and two reference sensitive strains (DD8 and 9044) of Leishmania donovani, to explore molecular mechanisms underlying drug resistance in this parasite.ResultsWith reference to DD8 and 9044 strains, HCZ isolate showed higher-level virulence and clear resistance to antimonials in promastigote culture, infected macrophages and animal experiment. Pairwise genomic comparisons revealed genetic variations (86 copy number variations, 271 frameshift mutations in protein-coding genes and two site mutations in non-coding genes) in HCZ isolate that were absent from the reference sensitive strains. Proteomic analysis indicated different protein expression between HCZ isolate and reference strains, including 69 exclusively detected proteins and 82 consistently down-/upregulated molecules in the HCZ isolate. Integrative analysis showed linkage of 12 genomic variations (gene duplication, insertion and deletion) and their protein expression changes in HCZ isolate, which might be associated with pathogenic and antimony-resistant phenotype. Functional annotation analyses further indicated that molecules involved in nucleotide-binding, fatty acid metabolism, oxidation-reduction and transport might play a role in host-parasite interaction and drug-resistance.ConclusionsThis comprehensive integrative work provided novel insights into the genetic basis underlying virulence and resistance, suggesting new aspects to be investigated for a better intervention against L. donovani and associated diseases.

Experimental evolution reveals nitrate tolerance mechanisms in Desulfovibrio vulgaris.

Elevated nitrate in the environment inhibits sulfate reduction by important microorganisms of sulfate-reducing bacteria (SRB). Several SRB may respire nitrate to survive under elevated nitrate, but how SRB that lack nitrate reductase survive to elevated nitrate remains elusive. To understand nitrate adaptation mechanisms, we evolved 12 populations of a model SRB (i.e., Desulfovibrio vulgaris Hildenborough, DvH) under elevated NaNO3 for 1000 generations, analyzed growth and acquired mutations, and linked their genotypes with phenotypes. Nitrate-evolved (EN) populations significantly (p < 0.05) increased nitrate tolerance, and whole-genome resequencing identified 119 new mutations in 44 genes of 12 EN populations, among which six functional gene groups were discovered with high mutation frequencies at the population level. We observed a high frequency of nonsense or frameshift mutations in nitrosative stress response genes (NSR: DVU2543, DVU2547, and DVU2548), nitrogen regulatory protein C family genes (NRC: DVU2394-2396, DVU2402, and DVU2405), and nitrate cluster (DVU0246-0249 and DVU0251). Mutagenesis analysis confirmed that loss-of-functions of NRC and NSR increased nitrate tolerance. Also, functional gene groups involved in fatty acid synthesis, iron regulation, and two-component system (LytR/LytS) known to be responsive to multiple stresses, had a high frequency of missense mutations. Mutations in those gene groups could increase nitrate tolerance through regulating energy metabolism, barring entry of nitrate into cells, altering cell membrane characteristics, or conferring growth advantages at the stationary phase. This study advances our understanding of nitrate tolerance mechanisms and has important implications for linking genotypes with phenotypes in DvH.

Tight Junction-Related CLDN5 and CLDN6 Genes, and Gap Junction-Related GJB6 and GJB7 Genes Are Somatically Mutated in Gastric and Colorectal Cancers

Tight junction and gap junction are major cell junctions that play important roles in cellular communication and structural integrity. Alterations of these junctions are known to be involved in cancer pathogenesis. Claudins and connexins are major tight and gap junction proteins, but genetic alterations of these genes have not been reported in gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). Claudin genes CLDN5 and CKDN6, and connexin genes GJB6 and GJB7 have mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with MSI. We analyzed 79 GCs and 145 CRCs, and found CLDN5 frameshift mutations in 3 (3%) CRCs and 1 (2.9%) GC, CLDN6 frameshift mutations in 6 (6%) CRCs, GJB6 frameshift mutations in 5 (5%) CRCs and GJB7 frameshift mutation in one CRC (1%) with high MSI (MSI-H). We also analyzed intratumoral heterogeneity (ITH) of the frameshift mutations in 16 CRCs and found that CLDN6 and GJB6 frameshift mutations showed regional ITH in 2 (12.5%) and 2 (12.5%) cases, respectively. Our results show that CLDN5, CLDN6, GJB6 and GJB7 genes harbor not only frameshift mutations but also mutational ITH, which together may be features of GC and CRC with MSI-H. Based on the roles of cellular junctions in cancers, frameshift mutations of tight junction and gap junction genes might contribute to tumorigenesis by altering their functions in GC and CRC.

Increased frequency of FBN1 frameshift and nonsense mutations in Marfan syndrome patients with aortic dissection

BackgroundMarfan syndrome (MFS) is an inherited connective tissue disease that mainly involves Fibrillin‐1 (FBN1) mutations and aortic manifestations. In this study, we investigated the correlations between the FBN1 genotype–phenotype and aortic events (aortic dissection and aortic aneurysm) in patients with Marfan syndrome.MethodsGenotype and phenotype information was evaluated in 180 patients with MFS. DNA sequencing was performed on each patient. According to the clinical manifestation, these patients were split into two groups: the aortic dissection group and the aortic aneurysm group. Aortic wall tissue was obtained from Marfan patients who underwent surgery and was used for staining.ResultsA total of 180 patients with FBN1 mutations were grouped into four categories: 90 with missense mutations, 32 with splicing mutations, 29 with frameshift mutations, and 29 with nonsense mutations. There was a significantly higher frequency of frameshift and nonsense mutations observed in aortic dissection than in aortic aneurysm (25.58% vs. 4.35%, p = .005; 25.58% vs. 8.70%, p = .033, respectively;), while missense mutations showed a higher frequency in aortic aneurysm than in aortic dissection (69.57% vs. 32.56%, respectively; p < .001) and a higher rate of lens dislocation (34.78% vs. 13.95%, respectively; p = .008). Pathological staining showed that elastic fibers were sparser in patients with a frameshift and nonsense mutations, and the smooth muscle cells were sparser and more disorganized than those observed in patients with missense mutations.ConclusionThis study showed that FBN1 gene frameshift and nonsense mutations are more common in patients with aortic dissection and may have meaningful guidance for the treatment of Marfan syndrome patients.

High Prevalence of Antibiotic Resistance in Iranian Helicobacter pylori Isolates: Importance of Functional and Mutational Analysis of Resistance Genes and Virulence Genotyping.

The high prevalence of antibiotic resistance in Helicobacter pylori has become a great challenge in Iran. The genetic mutations that contribute to the resistance have yet to be precisely identified. This study aimed to investigate the prevalence of antibiotic resistance and virulence markers in Iranian H. pylori isolates and to analyze if there is any association between resistance and genotype. Antibiotic susceptibility patterns of 68 H. pylori isolates were investigated against metronidazole, clarithromycin, amoxicillin, rifampicin, ciprofloxacin, levofloxacin, and tetracycline by the agar dilution method. The frxA, rdxA, gyrA, gyrB, and 23S rRNA genes of the isolates were sequenced. The virulence genotypes were also determined using PCR. Metronidazole resistance was present in 82.4% of the isolates, followed by clarithromycin (33.8%), ciprofloxacin (33.8%), rifampicin (32.4%), amoxicillin (30.9%), levofloxacin (27.9%), and tetracycline (4.4%). Overall, 75% of the isolates were resistant to at least two antibiotics tested and considered as a multidrug resistance (MDR) phenotype. Most of the metronidazole-resistant isolates carried frameshift mutations in both frxA and rdxA genes, and premature termination occurred in positions Q5Stop and Q50Stop, respectively. Amino acid substitutions M191I, G208E, and V199A were predominantly found in gyrA gene of fluoroquinolone-resistant isolates. A2143G and C2195T mutations of 23S rRNA were found in four clarithromycin-resistant isolates. Interestingly, significant associations were found between resistance to metronidazole (MNZ) and cagA-, sabA-, and dupA-positive genotypes, with p = 0.0002, p = 0.0001, and p = 0.0001, respectively. Furthermore, a significant association was found between oipA “on” status and resistance to amoxicillin (AMX) (p = 0.02). The prevalence of H. pylori antibiotic resistance is high in our region, particularly that of metronidazole, clarithromycin, ciprofloxacin, and MDR. Simultaneous screening of virulence and resistance genotypes can help clinicians to choose the appropriate therapeutic regime against H. pylori infection.

Liddle syndrome due to a novel mutation in the \u03b3 subunit of the epithelial sodium channel (ENaC) in family from Russia: a case report

BackgroundLiddle syndrome is a monogenic disease with autosomal dominant inheritance. Basic characteristics of this disease are hypertension, reduced concentration of aldosterone and renin activity, as well as increased excretion of potassium leading to low level of potassium in serum and metabolic alkalosis. The cause of Liddle syndrome is missense or frameshift mutations in SCNN1A, SCNN1B, or SCNN1G genes that encode epithelial sodium channel subunits.Case presentationWe describe a family with Liddle syndrome from Russia. 15-year-old proband has arterial hypertension, hypokalemia, hyporeninemia, metabolic alkalosis, but aldosterone level is within the normal range. At 12 years of age, arterial hypertension was noticed for the first time. We identified novel frameshift mutation c.1769delG (p.Gly590Alafs) in SCNN1G, which encodes the γ subunit of ENaC in vertebrates. The father and younger sister also harbor this heterozygous deletion. Treatment with amiloride of proband and his sister did not normalize the blood pressure, but normalized level of plasma renin activity.ConclusionsOur results expand the mutational spectrum of Liddle syndrome and provide further proof that the conserved PY motif is crucial to control of ENaC activity. Genetic analysis has implications for the management of hypertension, specific treatment with amiloride and counselling in families with Liddle syndrome.

SsODN-Mediated In-Frame Deletion with CRISPR/Cas9 Restores FVIII Function in Hemophilia A-Patient-Derived iPSCs and ECs

Given that the cDNA of F8 is too large to be packaged into adeno-associated virus (AAV) capsids, gene transfer of some versions of B-domain-deleted F8 (BDD-F8) for hemophilia A (HA) treatment has been attempted with promising results. Here, we describe an efficient gene correction via single-stranded-oligodeoxynucleotide (ssODN)-mediated in-frame deletion within the B domain of F8 with CRISPR/Cas9 in HA-patient-derived induced pluripotent stem cells (HA-iPSCs). The expression and activity of FVIII was restored in corrected HA-iPSC-derived induced endothelial progenitor cells (C-iEPCs) in vitro and in vivo. The bleeding phenotype was rescued in HA mice after C-iEPC infusion. Our results demonstrate an efficient approach for in situ gene correction via introduction of a tiny deletion using ssODN and CRISPR/Cas9 to reframe the F8 transcript and restore FVIII function in HA-iPSC-derived EPCs with potential clinical impact in HA gene therapy. For the first time, we demonstrated in vitro and in vivo the FVIII function that is encoded by the endogenous F8 gene with a partially deleted B domain. This work also suggests an applicable strategy for genetic correction of other gene frameshift mutations.

SAT-567 Mutational Profile of Hurthle Cell Carcinomas: Recurrent Mutations in Wnt/β-catenin Pathway Genes

Hürthle cell carcinomas (HCC) genomic characterization is evolving. Recent studies identified recurrent alterations in a few genes, chromosomal losses and mitochondrial DNA mutations. Gene expression studies demonstrated distinct profiles of PI3K-AKT-mTOR and Wnt/β-catenin pathways in widely invasive HCC. Potential driver mutations or mutations responsible for mitochondrion-rich phenotype are not completely defined. This study assessed HCC molecular profile using next-generation sequencing (NGS) of selected cancer-related genes and correlated with clinical characteristics and histological phenotypes. We evaluated 46 HCC and 8 adenomas (HCA) through a 97-gene NGS panel targeting MAPK, PI3K-AKT-mTOR, Wnt/β-catenin, Notch pathways, and other genes, such as TERT promoter and TP53. Variant callers Lofreq, Mutect and Lancet, identified somatic variants in matched carcinomas/normal thyroid, which were reviewed in Integrative Genomics Viewer. We searched genetic alterations in COSMIC70 database and predicted effects using in silico tools. Clinical and histological data were analyzed and compared to mutational profile. There were 46% of widely invasive HCC, 4.7% of which had lymph nodes and 9.5% distant metastases at diagnosis. On follow-up, 13% of patients had persistent/recurrent disease and 3% cancer-related deaths. Genetic alterations were identified in 54% of HCC, 221 single-nucleotide variants (SNV) and 6 insertions/deletions. Wnt/β-catenin pathway was most frequently involved (97 mutations in 41% HCC); FAT1 was the most recurrently mutated gene (31 mutations in 28% HCC), followed by APC (17 mutations in 15% HCC). Mutations in MAPK and mTOR pathways occurred in 30% and 22% of HCC, respectively. NRAS mutations were present in 3 HCC, so as 2 HCA. HRAS and KRAS mutations were found in one HCC each. There were 21 mutations described in COSMIC70, mostly in genes from Wnt and MAPK pathways: AKT2, APC, FGFR3, FLT3, GSK3B, HNF1A, IRS4, JAK2, KDR, KIT, KRAS, LRP6, MTOR, NOTCH2, NRAS and PIK3CG. Among remaining alterations, there were frameshift mutations in AGAP2, AXIN2 and FBXW7 genes, stop codons in ALK, CSNK1D, FAT1, NOTCH2 and TIMM 44, and 33 SNV predicted as deleterious. No BRAF or TP53 mutation was found, and there were 2 mutations in TERT promoter, different from C288T and C250T. We found no statistically significant association of clinical/histological features with mutational profile. In conclusion, HCC exhibited a wide range of mutations, mostly in Wnt/β-catenin pathway. FAT1, a tumor suppressor gene that controls mitochondrial activity to restrict cell growth, was the most frequently mutated gene, limited to HCC. Mutated FAT1 could activate Wnt/β-catenin pathway and provide growth advantage to mitochondrion-rich cells. We suppose FAT1 mutations are potential drivers and Wnt/β-catenin pathway plays a critical role in this distinct thyroid malignancy.

A novel elastin gene frameshift mutation in a Russian family with cutis laxa: a case report

BackgroundCutis laxa (CL) is a rare connective tissue disorder characterized by loose, redundant, inelastic and wrinkled skin. Patients develop a prematurely aged appearance. Inheritance can be autosomal dominant or autosomal recessive. The X-linked form is now classified in the group of copper transport diseases. Autosomal dominant CL is characterized by wrinkled, redundant and sagging, inelastic skin and in some cases is associated with internal organ involvement.Case presentationWe report a familial case of autosomal dominant CL, which includes a 33-year-old woman and her 11-year-old son with dry, thin and wrinkled skin that appeared prematurely aged. No serious involvement of internal organs was found. In both patients, we identified novel heterozygous mutation c.2323delG (p.Ala775fs) in exon 34 of elastin transcript NM_001278939.1. Similar frameshift mutations in the last exons of elastin gene were previously reported in patients with autosomal dominant CL.ConclusionsOur results show a novel frameshift mutation that was found in patients with cutis laxa. Exome sequencing is effective and useful technology for properly diagnosis of diseases with similar phenotype to ensure proper treatment is provided.

USA300 Staphylococcus aureus persists on multiple body sites following an infection

BackgroundUSA300 methicillin-resistant Staphylococcus aureus (MRSA) is a community- and hospital-acquired pathogen that frequently causes infections but also can survive on the human body asymptomatically as a part of the normal microbiota. We devised a comparative genomic strategy to track colonizing USA300 at different body sites after an initial infection. We sampled ST8 S. aureus from subjects at the site of a first known MRSA infection. Within 60 days of this infection and again 12 months later, each subject was tested for asymptomatic colonization in the nose, throat and perirectal region. 93 S. aureus strains underwent whole genome shotgun sequencing.ResultsAmong 28 subjects at the initial sampling time, we isolated S. aureus from the nose, throat and perirectal sites from 15, 11 and 15 of them, respectively. Twelve months later we isolated S. aureus from 9 subjects, with 6, 3 and 3 strains from the nose, throat and perirectal area, respectively. Genome sequencing revealed that 23 patients (ages 0–66 years) carried USA300 intra-subject lineages (ISLs), defined as having an index infection isolate and closely related colonizing strains. Pairwise distance between strains in different ISLs was 48 to 162 single nucleotide polymorphisms (SNPs) across the core regions of the chromosome, whereas within the same ISL it was 0 to 26 SNPs. Strains in ISLs from the same subject differed in plasmid and prophage content, and contained deletions that removed the mecA-containing SCCmec and ACME regions. Five strains contained frameshift mutations in agr toxin-regulating genes. Persistence of an ISL was not associated with clinical or demographic subject characteristics. We inferred that colonization with the ISL occurred about 18 weeks before the first assessment of asymptomatic colonization.ConclusionsClonal lineages of USA300 may continue to colonize people at one or more anatomic sites up to a year after an initial infection and experience loss of the SCCmec, loss and gain of other mobile genetic elements, and mutations in the agr operon.

Genetic analysis for a haemophilia B family with multi nucleotides deletion mutation of F9 gene

Objective To conduct genetic diagnosis and prenatal diagnosis for a haemophilia B family with multi-nucleotides deletion mutation of F9 gene. Methods This is a genetic analysis. Whole exon mutation of the F9 gene was analyzed by PCR and Sanger sequencing for seven patients with the family of hemophilia B who consulted doctors in Henan Province People′s Hospital in April 2013. Suspected mutation was verified among non-hemophilia B members of the family and 100 healthy controls to rule out genetic polymorphism of the F9 gene. The above-mentioned detection results of hemophilia B gene, the pathogenic mutation of F9 gene in the family was clarified, and prenatal diagnosis was conducted for the female carriers in the family.It is recommended that the fetal gene detection should be conducted in amniotic fluid in the mid-term pregnancy of the female carriers of hemophilia, and then they can be informed of the non-hemophilia B fetus by the results of the gene detection. Results PCR and sequencing analysis has identified a deletion mutation of F9 gene c. 185_188delGAGA[p.Glu62Asnfs*41]in seven hemophilia B patients. This mutation induced F9 gene frame shift mutation which led to early termination of F9 gene translation because there was a termination codon TAA at the 41th codon after the mutation site. The same mutation was not found among the non-hemophilia B members of the family and the 100 healthy controls. There were eight female carriers and nine female non-carriers in the family. Upon prenatal diagnosis, the Y chromosome sex-determining gene (SRY) in amniotic fluid was positive and no deletion mutation was observed in the F9 gene c. 185_188. Conclusion The pathogenic mutation of F9 gene in the family was identified, which was helpful for prenatal diagnosis in female carriers.(Chin J Lab Med, 2018, 41: 675-679) Key words: Hemophilia B; Gene deletion; Prenatal diagnosis; Genetic testing

Reduced Abd-B Hox function during kidney development results in lineage infidelity

Hox genes can function as key drivers of segment identity, with Hox mutations in Drosophila often resulting in dramatic homeotic transformations. In addition, however, they can serve other essential functions. In mammals, the study of Hox gene roles in development is complicated by the presence of four Hox clusters with a total of 39 genes showing extensive functional overlap. In this study, in order to better understand shared core Hox functions, we examined kidney development in mice with frameshift mutations of multiple Abd-B type Hox genes. The resulting phenotypes included dramatically reduced branching morphogenesis of the ureteric bud, premature depletion of nephron progenitors and abnormal development of the stromal compartment. Most unexpected, however, we also observed a cellular level lineage infidelity in nephron segments. Scattered cells within the proximal tubules, for example, expressed genes normally expressed only in collecting ducts. Multiple combinations of inappropriate nephron segment specific marker expression were found. In some cases, cells within a tubule showed incorrect identity, while in other cases cells showed ambiguous character, with simultaneous expression of genes associated with more than one nephron segment. These results give evidence that Hox genes have an overlapping core function at the cellular level in driving and/or maintaining correct differentiation decisions.

Genomic Sequencing of Japanese Plum (Prunus salicina Lindl.) Mutants Provides a New Model for Rosaceae Fruit Ripening Studies.

It has recently been described that the Japanese plum “Santa Rosa” bud sport series contains variations in ripening pattern: climacteric, suppressed-climacteric and non-climacteric types. This provides an interesting model to study the role of ethylene and other key mechanisms governing fruit ripening, softening and senescence. The aim of the current study was to investigate such differences at the genomic level, using this series of plum bud sports, with special reference to genes involved in ethylene biosynthesis, signal transduction, and sugar metabolism. Genomic DNA, isolated from leaf samples of six Japanese plum cultivars (“Santa Rosa”, “July Santa Rosa”, “Late Santa Rosa”, “Sweet Miriam”, “Roysum”, and “Casselman”), was used to construct paired-end standard Illumina libraries. Sequences were aligned to the Prunus persica genome, and genomic variations (SNPs, INDELS, and CNV's) were investigated. Results determined 12 potential candidate genes with significant copy number variation (CNV), being associated with ethylene perception and signal transduction components. Additionally, the Maximum Likelihood (ML) phylogenetic tree showed two sorbitol dehydrogenase genes grouping into a distinct clade, indicating that this natural group is well-defined and presents high sequence identity among its members. In contrast, the ethylene group, which includes ACO1, ACS1, ACS4, ACS5, CTR1, ERF1, ERF3, and ethylene-receptor genes, was widely distributed and clustered into 10 different groups. Thus, ACS, ERF, and sorbitol dehydrogenase proteins potentially share a common ancestor for different plant genomes, while the expansion rate may be related to ancestral expansion rather than species-specific events. Based on the distribution of the clades, we suggest that gene function diversification for the ripening pathway occurred prior to family extension. We herein report all the frameshift mutations in genes involved in sugar transport and ethylene biosynthesis detected as well as the gene CNV implicated in ripening differences.

CRISPR/Cas9-Based Genome Editing of Transcription Factor Genes in Marchantia polymorpha.

Plant transcription factors (TFs) belong to a wide variety of gene families. The systematic and rapid establishment of knockout lines of TF genes is critical for functional genetics. Genome engineering techniques for dissecting out the molecular function of TFs have been dramatically improved by CRISPR/Cas9-based genome editing technology. In the CRISPR/Cas9 system, Cas9 functions as a Cas9-gRNA ribonucleoprotein complex and uses its DNA endonuclease activity to induce the cleavage of the genome, which is targeted by gRNA. Double-strand breaks sometimes induce insertions and deletions at the target site, leading to frameshift mutations of TF genes. In this chapter, we describe a detailed protocol for the targeted mutagenesis of TFs using the CRISPR/Cas9 system, specifically in the case of Marchantia polymorpha, an emerging model plant for functional genomics. The CRISPR/Cas9 system is highly versatile for targeting genomic sequences because only an alteration of the gRNA sequence is needed to change target sequences. The labor and cost required to establish genome-edited lines are low enough that multiple mutants of TF genes can be generated in one laboratory. The CRISPR/Cas9-based genome editing technique consists of four steps: (1) gRNA design; (2) vector construction; (3) transformation; and (4) isolation of genome-edited lines. This manuscript focuses mainly on the strategy of gRNA design, the workflow for off-target searches, and the selection and identification of genome-edited lines by genotyping. Although we describe a protocol for M. polymorpha, the basic strategy of generating genome-edited lines of TF genes should be applicable widely to other plants.

Abstract IA4: The role of inflammation and DNA mismatch repair in colorectal cancer

Abstract Anti-inflammatory drugs, such as aspirin, have been identified as agents that can serve a primary preventative approach towards lessening the burden of colorectal cancer (CRC), as well as be effective secondary prevention for CRC recurrence. Genetic and epigenetic events that drive the pathogenesis of CRC influences a relationship with the immune system that modifies cancer behavior and patient prognosis, as well as the effectiveness of anti-inflammatory drugs and other agents for chemoprevention. Approximately 15% of sporadic CRCs manifest defective DNA mismatch repair (MMR) function due to epigenetic inactivation of the MMR gene MLH1, allowing cancer cells to accumulate hundreds of somatic frameshift mutations in genes that contain microsatellite sequences. Most of the coding microsatellite frameshift mutations are translated into novel truncated proteins, which act as neo-antigens to the patient's immune system, with the patient self-immunizing against the tumor. This defective MMR-driven inflammatory response slows the ability of the CRC to advance and metastasize, and patients present at an earlier stage and survive longer compared to CRC patients with competent MMR tumors. MMR-defective CRCs acquire PD-1 expression, making the tumor more susceptible to immune checkpoint blockade. However, these patients show muted response to adjuvant 5-fluorouracil (5-FU) therapy. There is evidence that aspirin is chemopreventive in Lynch syndrome, a familial form of MMR deficiency, but needs to be studied further. Additionally, approximately 50% of all CRCs manifest dysfunction of the MMR protein MSH3 as a consequence of inflammation and release of the pro-inflammatory cytokine IL-6 from intimately-located immune cells. The isolated MSH3 dysfunction allows cancer cells to accumulate non-mononucleotide microsatellite frameshift mutations that are likely not very immunogenic, as well as DNA double strand breaks as a consequence of loss of MSH3s contribution to homologous recombination repair. These CRC patients are more apt to be advanced stage and possess metastases, demonstrate poor survival, and more common among African American patients. Isolated loss of MSH3 function does not hinder a patient's response to 5-FU, and the effectiveness of immune checkpoint blockade and anti-inflammatory agents has not been tested to date. Specific chromosome breakage points such as at chromosome 9p24.2 are enriched in liver metastases from CRCs with MSH3 dysfunction and modify patient outcome by increasing survival over patients with MSH3 dysfunction alone. Overall, the MMR status of the CRC can inform approach to patient care, and the outcome difference depends on if the MMR defect drives an immune response, or if acquired inflammation causes disruption of MMR via MSH3 cellular mis-localization. Citation Format: John M. Carethers. The role of inflammation and DNA mismatch repair in colorectal cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA4.

Detection of HRD gene mutations and copy number changes in cfDNA from prostate cancer patients.

e16546 Background: Recent genomic surveys of prostate cancer have identified somatic mutations in metastatic castration-resistant prostate cancer (mCRPC). In this study, we examined mCRPC patients for AR aberrations and mutations in the HRD (homologous recombination DNA-repair) pathway which may confer platinum or PARP inhibitor sensitivity. Methods: A novel targeted-hybrid-capture NGS assay capable of identifying deleterious mutations, copy number amplification and gene copy loss, was applied to circulating, cell-free DNA (cfDNA) extracted from plasma samples from 20 mCRPC patients. Samples were collected between 3/2010 – 10/2015 and stored at -20C. The gene panel used in the assay included AR and several genes in the HRD pathway — ATM, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK2, FANCA, HDAC2, NBN, PALB2, and RAD51. Sequencing libraries created with the cfDNA extracted from 2.8-4 mLs of plasma had an average unique read coverage depth of 2282 genome equivalents (range 445 – 5136, median 2181). Results: Somatic variations were observed in 17 of the 20 samples analyzed. Copy number variation (CNV) was observed in 11 samples. AR amplification, linked to resistance to abiraterone and enzalutamide, was observed in 30% (6) of samples. Canonical AR ligand binding domain mutations, such as T787A and L702H were also detected. ATM, BRCA1, BRCA2, HDAC2, and FANCA gene deletions were also detected, as well as frameshift, nonsense, and other deleterious mutations in HRD genes. Significant CNV in multiple genes was observed in at least 5 patient samples. Mutations were detected across the entire collection date range, speaking to the robustness of cfDNA. Conclusions: Non-invasive tumor mCRPC genotyping appears to be feasible. The ability to detect HRD gene alterations suggests cfDNA testing may be suitable for the detection of HRD pathway defects and overall genome instability.

Frameshift mutational target gene analysis identifies similarities and differences in constitutional mismatch repair-deficiency and Lynch syndrome.

Mismatch-repair deficient (MMR-D) malignancies include Lynch Syndrome (LS), which is secondary to germline mutations in one of the MMR genes, and the rare childhood-form of constitutional mismatch repair-deficiency (CMMR-D); caused by bi-allelic MMR gene mutations. A hallmark of LS-associated cancers is microsatellite instability (MSI), characterized by coding frameshift mutations (cFSM) in target genes. By contrast, tumors arising in CMMR-D patients are thought to display a somatic mutation pattern differing from LS. This study has the main goal to identify cFSM in MSI target genes relevant in CMMR-D and to compare the spectrum of common somatic mutations, including alterations in DNA polymerases POLE and D1 between LS and CMMR-D. CMMR-D-associated tumors harbored more somatic mutations compared to LS cases, especially in the TP53 gene and in POLE and POLD1, where novel mutations were additionally identified. Strikingly, MSI in classical mononucleotide markers BAT40 and CAT25 was frequent in CMMR-D cases. MSI-target gene analysis revealed mutations in CMMR-D-associated tumors, some of them known to be frequently hit in LS, such as RNaseT2, HT001, and TGFβR2. Our results imply a general role for these cFSM as potential new drivers of MMR-D tumorigenesis.

A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish.

ABSTRACTClustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.

Abstract A006: Frameshift peptide neoantigens as vaccine targets in microsatellite-unstable cancers

Abstract Background: Deficient DNA mismatch repair (MMR) boosts the accumulation of frameshift mutations in genes encompassing coding microsatellites (cMS). This results in translation of proteins with mutation-induced frameshift peptide (FSP) neoantigens rendering MMR-deficient microsatellite-unstable (MSI) cancers highly immunogenic. Patients with MSI cancers and healthy individuals affected by Lynch syndrome, an inherited prediposition for MSI cancers, develop specific immune responses against these neoantigens. MSI cancers are unique in tumor immunology, because they express a defined set of long neoantigens that result from functionally relevant driver mutations and therefore are shared by the majority of MSI cancers. Consequently, MSI cancers in Lynch syndrome are an ideal model to evaluate the concept of cancer vaccines, which, with the increasing knowledge about mutational antigens in a wide variety of cancer types, can potentially be applied to many human cancer types. We here report the results of a clinical phase I/IIa trial as the first step to translate this concept into the clinical application. Methods: The vaccination protocol comprised 3 cycles of 4 subcutaneous applications of FSP antigens (frameshift variants of the coding microsatellite-containing genes AIM2, HT001, TAF1B) mixed with Montanide ISA-51 VG over a 6 month period. Inclusion criteria were history of MSI-H colorectal cancer (UICC stage III or IV) and completion of standard chemotherapy. Phase I of the trial evaluated safety and toxicity as the primary endpoint (6 patients), phase IIa addressed the induction of cellular and humoral immune responses (16 patients). Results: Significant induction of FSP-specific immune responses against one or more FSP antigens was observed in all patients vaccinated per protocol. No vaccination-induced systematic severe adverse events occurred. Few patients had stage IV disease and were evaluable according to RECIST. One heavily pretreated patient with bulky metastases showed a stable disease and stable CEA levels over 7 months under the study treatment. Conclusions: Vaccination with FSPs is well tolerated and leads to the induction of humoral and cellular immune responses. FSP vaccination represents a promising novel approach for treatment of MSI cancer patients and for tumor prevention in Lynch syndrome, allowing the evaluation of the concept of preventive cancer vaccines in an ideal model scenario of a defined high-risk patient population. Note: This abstract was not presented at the conference. Citation Format: Magnus von Knebel Doeberitz, Matthias Kloor, Miriam Reuschenbach, Claudia Pauligk, Mohammad-Reza Rafiyan, Salah-Eddin Al Batran, Julia Karbach, Mirjam Tariverdian, Elke Jaeger. Frameshift peptide neoantigens as vaccine targets in microsatellite-unstable cancers [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A006.

Clinical characteristics and the identification of novel mutations of COL1A1 and COL1A2 in 61Chinese patients with osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is an inherited connective tissue disorder characterized by brittle bone fractures. The aim of the present study was to investigate the pathogenic gene mutation spectrum and clinical manifestations of mutations in collagen type I, alpha 1 (COL1A1) and collagen typeI, alpha2 (COL1A2) genes in Chinese patients with OI. A total of 61unrelated Chinese OI patients with COL1A1 and COL1A2 mutations were recruited. All the exons and the exon-intron boundaries of the COL1A1 and COL1A2 genes were amplified and directly sequenced and lumbar spine bone mineral density was measured by dual‑energy X‑ray absorptiometry. The mutations of the 61probands included 33missense mutations, 8nonsense mutations, 7splicing variants and 13frameshift mutations in COL1A1 and COL1A2 genes. A total of 25novel mutations were identified, including 18 in COL1A1 and 7in COL1A2. The mutations p.Gly257Arg, p.Gly767Ser and p.Gly821Ser in COL1A1 and p.Gly337Ser in COL1A2 may be located at a mutation hotspot for human OI due to the high repetition rate in OI patients. Family history was positive for OI in 33probands (54%). All probands had suffered fractures and the most common fracture site was the femur. A total of 49probands presented with blue sclerae (80.3%), 20probands suffered from dentinogenesis imperfecta (32.8%) and 1patient had hearing loss (1.6%). These findings may improve understanding of the pathogenic gene mutation spectrum and the clinical manifestations of mutations of COL1A1 and COL1A2 genes in Chinese patients with OI.