High-impact Mutations Research Articles

Recurrent high-impact mutations at cognate structural positions in class A G protein-coupled receptors expressed in tumors

G protein-coupled receptors (GPCRs) are the largest family of human proteins. They have a common structure and, signaling through a much smaller set of G proteins, arrestins, and effectors, activate downstream pathways that often modulate hallmark mechanisms of cancer. Because there are many more GPCRs than effectors, mutations in different receptors could perturb signaling similarly so as to favor a tumor. We hypothesized that somatic mutations in tumor samples may not be enriched within a single gene but rather that cognate mutations with similar effects on GPCR function are distributed across many receptors. To test this possibility, we systematically aggregated somatic cancer mutations across class A GPCRs and found a nonrandom distribution of positions with variant amino acid residues. Individual cancer types were enriched for highly impactful, recurrent mutations at selected cognate positions of known functional motifs. We also discovered that no single receptor drives this pattern, but rather multiple receptors contain amino acid substitutions at a few cognate positions. Phenotypic characterization suggests these mutations induce perturbation of G protein activation and/or β-arrestin recruitment. These data suggest that recurrent impactful oncogenic mutations perturb different GPCRs to subvert signaling and promote tumor growth or survival. The possibility that multiple different GPCRs could moonlight as drivers or enablers of a given cancer through mutations located at cognate positions across GPCR paralogs opens a window into cancer mechanisms and potential approaches to therapeutics.

EPCO-19. SYSTEMS BIOLOGY APPROACH ON MGMT-METHYLATED, IDH WILD-TYPE SHORT-TERM SURVIVORS REVEALS MUTATIONS IN THE BRCA1-MEDIATED DNA REPAIR SIGNALING PATHWAY

Abstract Numerous multi-omic studies have revealed the vast molecular complexity of glioblastoma; however, these studies have failed to identify actionable drivers for definitive therapy. Unlike conventional relational data analysis methods used to examine such data, systems biology approaches using graph databases have emerged as powerful tools with flexibility and scalability needed to reveal specific vulnerabilities. Here we demonstrate the utility of a patient-centric graph database in identifying novel factors associated with poor outcome (survival < 8 months) in patients with MGMT promoter methylated glioblastoma. Using a cohort of 112 patients from the TCGA database, we integrated high-impact mutations with MGMT methylation status. Network analysis revealed three subnetworks, consisting of (a) methylated patients, the M-network, (b) methylated and unmethylated patients, the MU-network, and (c) unmethylated patients, the U-network. In addition, querying the genes in the M-network revealed three key molecules in the DNA repair pathway, namely, FANC-A, FANC-E, and MYUTH, mutated in five patients (q-value < 2.3E-02). Moreover, we observed that BRCA1 mediates other critical signaling molecules in the MU-network. Interestingly, in intermediate (8-24 months) or long-term ( >24 months) survivors networks, this pathway is not implicated. In light of recent studies implicating BRCA1 modulating temozolomide resistance in GBM sphere-forming cells and BRCA1’s protein expression predicting survival in patients, this result suggests that mutations in BRCA1-mediated DNA repair pathways hinder response to chemotherapy despite methylated MGMT, reducing the survival in patients. Besides this novel result explaining low survival in MGMT methylated patients through a synthesis of epigenetic and genetic data, our framework provides a novel and compelling paradigm for data integration at various scales from molecular events in a single patient’s tumor to integrating the molecular profile (or genotype) with phenotypic characteristics at the population level. We will present additional insights derived from this analysis at the SNO annual meeting.

TP53 Aberrations and Outcomes in MBL and Untreated CLL

TP53 Aberrations and Outcomes in MBL and Untreated CLL

Comparison of Two Aspergillus oryzae Genomes From Different Clades Reveals Independent Evolution of Alpha-Amylase Duplication, Variation in Secondary Metabolism Genes, and Differences in Primary Metabolism.

Microbes (bacteria, yeasts, molds), in addition to plants and animals, were domesticated for their roles in food preservation, nutrition and flavor. Aspergillus oryzae is a domesticated filamentous fungal species traditionally used during fermentation of Asian foods and beverage, such as sake, soy sauce, and miso. To date, little is known about the extent of genome and phenotypic variation of A. oryzae isolates from different clades. Here, we used long-read Oxford Nanopore and short-read Illumina sequencing to produce a highly accurate and contiguous genome assemble of A. oryzae 14160, an industrial strain from China. To understand the relationship of this isolate, we performed phylogenetic analysis with 90 A. oryzae isolates and 1 isolate of the A. oryzae progenitor, Aspergillus flavus. This analysis showed that A. oryzae 14160 is a member of clade A, in comparison to the RIB 40 type strain, which is a member of clade F. To explore genome variation between isolates from distinct A. oryzae clades, we compared the A. oryzae 14160 genome with the complete RIB 40 genome. Our results provide evidence of independent evolution of the alpha-amylase gene duplication, which is one of the major adaptive mutations resulting from domestication. Synteny analysis revealed that both genomes have three copies of the alpha-amylase gene, but only one copy on chromosome 2 was conserved. While the RIB 40 genome had additional copies of the alpha-amylase gene on chromosomes III, and V, 14160 had a second copy on chromosome II and an third copy on chromosome VI. Additionally, we identified hundreds of lineage specific genes, and putative high impact mutations in genes involved in secondary metabolism, including several of the core biosynthetic genes. Finally, to examine the functional effects of genome variation between strains, we measured amylase activity, proteolytic activity, and growth rate on several different substrates. RIB 40 produced significantly higher levels of amylase compared to 14160 when grown on rice and starch. Accordingly, RIB 40 grew faster on rice, while 14160 grew faster on soy. Taken together, our analyses reveal substantial genome and phenotypic variation within A. oryzae.

Abstract 2191: Clear cell renal cell carcinoma molecular differences between Hispanic Americans and European Americans

Abstract Background: Hispanic Americans (HAs) in Arizona have a heavy burden of renal cell carcinoma (RCC) presenting at a younger age at diagnosis with higher incidence and mortality rates than European Americans (EAs). However, HAs are underrepresented in RCC research, and tumor molecular characteristics in this population are unknown. We report preliminary findings from a study of molecular differences between HAs and EAs exploring the molecular basis of early-onset clear cell RCC (ccRCC). Methods: Clinical records of patients who underwent surgical treatment for RCC between 2011 and 2020 at the University of Arizona Department of Urology was reviewed. A total of 539 patients were identified, and paraffin embedded surgical specimens of a subset of patients with ccRCC were selected for this study. Three exons of VHL gene, the most commonly altered gene in ccRCC, were screened for somatic mutations in a total of 117 patients. Thirty-three patients were included for TempO-Seq analysis to correlate molecular subtype with demographic and clinicopathologic characteristics. Results: Among 117 patients, 55 somatic mutations in coding regions of VHL were found in 42 patients (35.9%). Nine patients had more than 2 somatic mutations. There were 37 substitutions, 14 deletions, and 4 insertions. There was one patient with a mutation of an intron variant that was a splice acceptor. Mutations in the coding regions were more common in EAs (40.9%) than HAs (31%), but the difference was not statistically significant (P=0.32). For moderate or high impact mutations, EAs had mutations at a significantly higher frequency than HAs (39.4% vs. 20.5%, P=0.04). Patients with high impact mutations tend to be diagnosed before the age of 50 (37.5%) compared to patients without high impact mutations (23.9%). We were able to assign 32 out of 33 patients into molecular subtypes (ccA and ccB). Molecular subtype could not be assigned to one HA patient with high grade and advanced stage ccRCC. Compared to patients with ccB subtype, patients who had ccA subtype were younger (mean age of 52.2 vs. 61.4) and tend to be obese (55.6% vs. 28.6% body mass index ≥30). Molecular subtype, ccA, was more common in HAs than EAs (64.3% vs. 41.2%), but this difference was not statistically significant. Conclusion: Somatic mutations within the VHL gene often altered in early during ccRCC pathogenesis were less common in HAs, but ccA associated with early age of diagnosis and obesity was more common in HAs. Impact: HAs and EAs have different molecular basis of early-onset ccRCC which may impact clinical managements. Citation Format: Yuliang Chen, Karleen M. Meiklejohn, Nathan Ellis, Erika R. Bracamonte, Benjamin R. Lee, Ken Batai. Clear cell renal cell carcinoma molecular differences between Hispanic Americans and European Americans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2191.

Abstract 884: Tumor mutational load derived from recurrently mutated genes in chronic lymphocytic leukemia (CLL) predicts time-to-first treatment in high-count monoclonal B-cell lymphocytosis (HC MBL)

Abstract Clinical practice guidelines recommend that individuals with HC MBL are evaluated annually for evidence of progression as it has been shown that they progress to develop CLL requiring therapy at a rate of 1-5%/year. Next-generation sequencing identified 59 genes recurrently mutated in CLL, and we previously showed that the tumor mutational load (TML), i.e., the total number of these genes mutated, is associated with a shorter time to first treatment (TTT) among 112 HC MBLs independent of the CLL international prognostic index (CLL-IPI), an established prognostic score. Herein, we continue to support our finding in additional HC MBLs and report the pooled results across the two cohorts. From the Mayo Clinic CLL Resource, we sequenced a total 167 HC MBL (112 from the initial round and 55 from additional sequencing). We extracted DNA from CD5+/CD19+ B-cells and sequenced the coding regions of 59 driver genes. Somatic mutations were called using MuTect2 in tumor-only mode. Germline variants were eliminated based on minor allele frequencies >0.01% and by those identified in public databases, unless present in known CLL mutation hotspots. After filtering, we computed the TML based on the number of genes with high impact (nonsense, frameshift and splicing) and hotspot mutations. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI), adjusted for sex and CLL-IPI. Stratified analyses were conducted by low/intermediate risk and high/very high CLL-IPI. Of the 167 HC MBL, low risk CLL-IPI comprised 60%, intermediate risk 28%, high risk 12%; and none were very-high risk. The most commonly mutated genes were SF3B1 (11%), NOTCH1 (9%), TP53 (7%), BIRC3 (7%), and MYD88 (6%); and 52 (31%) individuals had mutations in at least one gene. The median follow-up was 4.4 years, and 26 of the 167 individuals progressed to CLL requiring therapy. HC MBL with ≥2 mutated genes had a 28% 5-year risk of needing therapy compared to 6% among those with no mutated genes. Similar to our initial finding (HR=1.53, CI:1.12-2.07; P=0.007), we found a shorter TTT in the additional HC MBLs (HR=4.63, CI:1.57-13.7, P=0.006), and in the pooled cohort (HR=1.72; CI:1.29-2.28, P=0.0002). When we stratified by CLL-IPI risk, TML remained associated with TTT among individuals with low/intermediate CLL-IPI risk in the additional HC MBLs (HR=3.52, CI:1.11-11.2, P=0.03) and in the pooled cohort (HR=2.25, CI:1.34-3.77, P=0.002). A non-significant 2.8 fold association was observed with high risk CLL-IPI in the pooled cohort. In conclusion, the TML along with the CLL-IPI provide clinicians with a more accurate prognostic information regarding disease progression requiring therapy, especially for those MBLs with low to intermediate risk based on CLL-IPI. If validated, these findings may modify current practice guidelines for individuals with HC MBL. Citation Format: Geffen Kleinstern, Cecilia Bonolo de Campos, Nicholas J. Boddicker, Daniel R. O'Brien, Shulan Tian, Chantel Barney, Kari G. Rabe, Cristine Allmer, Laura Bruins, Aaron D. Norman, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, Huihuang Yan, James R. Cerhan, Neil E. Kay, Celine Vachon, Esteban Braggio, Susan L. Slager. Tumor mutational load derived from recurrently mutated genes in chronic lymphocytic leukemia (CLL) predicts time-to-first treatment in high-count monoclonal B-cell lymphocytosis (HC MBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 884.

Prediction of Clinically Relevant Mutations in (MDS): A Report on Behalf of the MDS Clinical Research Consortium (CRC)

Abstract Introduction: Somatic mutations are common in MDS and mutation profiling is becoming increasingly standard as part of the initial diagnostic evaluation. While clinical reports often indicate the predicted prognostic significance of specific mutations identified, they do not predict the degree of clinical impact or how these predictions actually correlate with clinical outcomes. We investigated the utility of the Ensembl Variant Effector Predictor (VEP) tool in correlating specific mutations with outcomes in MDS. Methods: MDS patients from the MDS CRC with available molecular genetic data by next-generation sequencing were included in the analysis. Mutations present at diagnosis were obtained, converted to HGVS notation and then categorized using the VEP tool into mutations with predicted high, moderate, or low impact based on sequence variants and structural variants including copy number changes or insertion or deletions. (McLaren , Genome Biol. 2016). We also utilized SIFT, which predicts amino acid substitution affect on protein function based on degree of conservation of residues. Neither tool accounts for VAF. The mutation categories were correlated with survival in a univariate log-rank test. Complete remission (CR) status was defined per IWG 2006. Cox proportional hazards model of time from diagnosis to death or loss of follow up with mutation category, age, IPSS classification was applied. Results: Clinical characteristics of the 963 included patients are summarized in Table 1. We categorized mutations of the following 6 prognostic genes, identified within 6 months of initial MDS diagnosis, using Ensembl VEP: ASXL1, DNMT3A, EZH2, RUNX1, TET2, and TP53. The distribution and predicted impact of the mutations are summarized in Table 2. None of the mutations were predicted to have a low impact. In our cohort, onlymutations in DNMT3A and TP53 were correlated with worse overall survival (OS). (HR 1.67, p The mutational profile of the six genes was characterized in 270 patients tested within 6 months of starting treatment with azacitidine or decitabine. The distribution and predicted impact of the mutations are summarized in Table 2. Similar to mutations characterized at diagnosis, worse OS was associated with high impact DNMT3A mutations (HR 3.39, p=0.002, CI 1.57-7.34), but not with mutations predicted to have a moderate impact (HR 1.16, p=0.63, CI=0.63-2.17). Among patients with TP53 mutations treated with hypomethylating agents, adverse OS were associated with moderate impact mutations (HR 2.23, p= Conclusions: While testing for myeloid-associated mutations is becoming standard practice as part of the diagnostic evaluation of MDS patients, interpretation of the sequencing results is complex and may be subject to technical variations that are obscure to clinicians. Presence of an abnormal mutation by itself is not enough to predict prognosis; predicted impact along with clinical characteristics need to be incorporated to establish their relative impact on clinical outcomes. Download : Download high-res image (213KB) Download : Download full-size image Disclosures Lee: Clinipace: Consultancy; Baxalta: Consultancy; Alexion Pharmaceuticals: Consultancy; Amgen: Consultancy. Komrokji: Celgene: Honoraria; Novartis: Honoraria, Speakers Bureau. Steensma: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Onconova: Consultancy; Janssen: Consultancy, Research Funding; Incyte: Equity Ownership; H3 Biosciences: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy. Ritchie: Pfizer: Consultancy, Other: Research funding to my institution; NS Pharma: Other: Research funding to my institution; Bristol-Myers Squibb: Other: Research funding to my institution; Astellas Pharma: Other: Research funding to my institution; Novartis: Consultancy, Other: Research funding to my institution, and travel, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Celgene: Consultancy, Other: Travel, Speakers Bureau. Hassane: Cellectis: Research Funding. Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees. Roboz: AbbVie, Agios, Amgen, Amphivena, Array Biopharma Inc., Astex, AstraZeneca, Celator, Celgene, Clovis Oncology, CTI BioPharma, Genoptix, Immune Pharmaceuticals, Janssen Pharmaceuticals, Juno, MedImmune, MEI Pharma, Novartis, Onconova, Pfizer, Roche Pharmace: Consultancy; Cellectis: Research Funding.

Large-scale genomic analysis reveals the genetic cost of chicken domestication

BackgroundSpecies domestication is generally characterized by the exploitation of high-impact mutations through processes that involve complex shifting demographics of domesticated species. These include not only inbreeding and artificial selection that may lead to the emergence of evolutionary bottlenecks, but also post-divergence gene flow and introgression. Although domestication potentially affects the occurrence of both desired and undesired mutations, the way wild relatives of domesticated species evolve and how expensive the genetic cost underlying domestication is remain poorly understood. Here, we investigated the demographic history and genetic load of chicken domestication.ResultsWe analyzed a dataset comprising over 800 whole genomes from both indigenous chickens and wild jungle fowls. We show that despite having a higher genetic diversity than their wild counterparts (average π, 0.00326 vs. 0.00316), the red jungle fowls, the present-day domestic chickens experienced a dramatic population size decline during their early domestication. Our analyses suggest that the concomitant bottleneck induced 2.95% more deleterious mutations across chicken genomes compared with red jungle fowls, supporting the “cost of domestication” hypothesis. Particularly, we find that 62.4% of deleterious SNPs in domestic chickens are maintained in heterozygous states and masked as recessive alleles, challenging the power of modern breeding programs to effectively eliminate these genetic loads. Finally, we suggest that positive selection decreases the incidence but increases the frequency of deleterious SNPs in domestic chicken genomes.ConclusionThis study reveals a new landscape of demographic history and genomic changes associated with chicken domestication and provides insight into the evolutionary genomic profiles of domesticated animals managed under modern human selection.

Whole-exome sequencing analysis to identify novel potential pathogenetic mutations in fetuses with abnormal brain structure.

BackgroundGenetic mutations in genes related to the production, migration, or differentiation of cortical neurons can result in malformations of cortical development (MCDs). However, a large number of MCD-related pathogenetic mutations remain unknown. This study aimed to investigate the genetic cause of MCDs and to identify the new MCD-associated mutations through whole‐exome sequencing (WES) in fetuses with abnormal brain structure.MethodsCord venous blood samples were collected from 11 fetuses with MCDs. Whole-genome DNA was extracted from the blood, and WES was performed. Single nucleotide substitutions, insertions, and deletions were detected by bioinformatics analysis. Genetic mutations in genes associated with MCD were identified.ResultsA total of 1035 genes with high-impact genetic variants in at least 1 fetus were identified. The results of gene ontology enrichment analysis were consistent with those of previous studies and also indicated new potential MCD-related pathogenetic genetic mutations. Genes with high-impact mutations in multiple fetuses, such as CTDSP2 and C-terminal binding protein 2 (CTBP2), were more likely to be the genes affecting normal brain development.ConclusionsThis study has characterized variations in fetuses with MCDs and identified potential genetic mutations causing MCDs. Our findings extend the mutation spectrum of MCDs and provide a promising source for the identification of MCD-related pathogenetic mutations.

High impact mutation FN1 affects cartilage integrity via aberrant binding to collagen type II

Purpose: By applying exome sequencing to an extended early-onset osteoarthritis (OA) family followed by linkage analysis, a high impact, likely causal mutation (C518F) was identified in the fibronectin (FN1) gene. Founded by the hypothesis that the underlying pathway in which this high impact mutation is causing OA may likely be extrapolated to common, age related, OA disease pathways we here studied the effects of the identified high impact mutation on neo-cartilage formation. Hereto the identified FN1 mutation was introduced in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome engineering technology while employing these cells to an established 3D in vitro chondrogenesis model. Methods: hiPSCs were electroporated with gRNA/Cas9 complex targeting FN1 and single-strand oligodeoxynucleotide template containing the specific FN1 mutation, after which the hiPSCs were plated at low seeding density to obtain clonal colonies. After 14 days, the multicellular colonies were picked and transferred into a 96-wells plate for clonal screening and expansion. Positive clones were identified by PCR followed by HincIII digestion and confirmed by Sanger sequencing. Mesodermal lineage differentiation towards induced chondroprogenitor cells (iCPCs) was performed by stepwise changes of the culture medium. After 14 days, iCPC aggregates were manually picked and chondrogenesis was initiated in 3D using chondrogenic differentiation medium. Pellets were refreshed twice per week and medium and pellets were collected following 5 weeks of chondrogenesis. Simultaneously, conditioned medium of the wildtype and mutant FN1 pellets was collected. After concentrating the medium, a collagen type II solid-phase binding assay was performed, to compare binding affinity of wildtype and mutant FN1 to collagen type II. Results: Sequencing confirmed that out of 274 hiPSC clones 14 (5.1%) were heterozygous and 3 (1.1%) were homozygous for the C518F FN1 mutation. To investigate the effect of the mutation on chondrogenesis, in vitro derived neo-cartilage constructs of wildtype, heterozygous, and homozygous FN1 mutant hiPSCs were generated. As can be seen in Figure 1, we observed a different morphology in the 3D neo-cartilage pellets formed by homo- and heterozygous FN1 mutated cells as compared to those of wildtype, being that the mutant chondrogenic pellets are more protrusive and frail. Alcian blue and collagen type II staining revealed that the frail matrix was not cartilage. On the other hand, neo-cartilage produced by the iCPCs showed comparable staining intensities between the mutant and wildtype groups. To study whether the C518F mutation, located in the gelatin-binding domain of fibronectin, affected binding to collagen type II, we next determined binding affinity between collagen type II and wildtype, heterozygous and homozygous mutated fibronectin. As shown in Figure 2, we observed a significant (P = 0.002) dose response reduction in binding as function of C518F mutated fibronectin. To study the effect of the mutation on integrity of neo-cartilage formed, we next explored chondrocyte signaling as result of the mutation by performing RT-qPCR. To adjust for differences in efficiency between neo-cartilage formation we used COL2A1 and COL1A1 as housekeeping genes of cartilage tissue. Notably, ADAMTS-5 and RUNX2 showed a dose response significant upregulation, while ACAN, and FN1 expression were downregulated with the C518F mutated as compared to wildtype neo-cartilage chondrocytes. Conclusions: By introducing a high impact OA mutation in human iPSCs while utilizing them in an established 3D in vitro chondrogenesis model we showed that the binding capacity between fibronectin and collagen type II is essential for proper cartilage formation. Moreover, as reflected by the aberrant chondrocyte signaling towards a hypertrophic state, such improper binding makes the cartilage prone to an OA state. Together our work merits further exploration of fibronectin as potential target for therapeutic interventions. We advocate that restoring or maintaining proper binding between fibronectin and collagen type 2 should be the focus of such a quest.

A disease-risk mutation in COL6A3 alters the biologic response of chondrocytes to hyper-physiologic mechanical loading

Purpose: We set out to determine the interacting effect of a high-impact osteoarthritis mutation in COL6A3 and hyper-physiologic mechanical stress on chondrocyte signaling. Hereto, we differentiated wild-type and COL6A3-mutated human induced pluripotent stem cells (hiPSCs) with an established 3D, in vitro chondrogenesis protocol. We hypothesized that the amino-acid change in the von Willebrand factor 8 of COL6A3 affects cartilage integrity by an aberrant mechanobiologic response of chondrocytes, particularly under hyper-physiologic mechanical stress.

What is the genetic architecture of major psychiatric disorders?

Taken as a whole, psychiatric disorders are considered as complex genetic disorders. There are clear genetic mutations and susceptibility factors to these disorders. However, these form the full spectrum of impact, frequency, and mutation type. With rare large scale chromosomal rearrangements and copy number mutations of high impact at one end, and common single nucleotide variations of minor impact at the other. This multitude of variation type also means that different epidemiological study designs are needed to test the genetic component of these disorders, from familial forms, to common population level studies. This process has been facilitated by advances in genomic analysis, that enable the measuring of genetic variation at a greater depth in a greater number of individuals and has led to a boom in genetic information. This has given us a greater understanding of the genetic aetiology of psychiatric disorders and how they are biologically related to each other. How this information can be translated to the clinics, can now be considered. Genetic testing in psychiatric disorders, is currently possible for certain disorders and mutation types, but is not universally advised. Much still remains to be understood about population level genetic risk factors before they could conceivably be utilised in the clinic. Whereas genetic testing of high impact mutations could be of use to the clinical programs, and are actively tested for in clinics across Europe.DisclosureNo significant relationships.

96 Validation of the candidate mutation responsible for embryonic lethality in Holstein haplotype 2 carriers

Holstein haplotype 2 (HH2) is embryonic lethal and carried by 1.21% of the US Holstein population. Using next-generation sequencing, we identified a high-impact frameshift mutation in intraflagellar protein 80 (IFT80) as the putative causal mutation. In bovine embryos, IFT80 expression begins at the 8-cell stage and decreases by the blastocyst stage. We hypothesised that the loss of function of IFT80 early in development causes the lethal phenotype. The aim of this study was to mimic the mutation observed invivo using a CRISPR-Cas9 approach to determine its effect on embryo development. Two guide RNAs (gRNAs) were designed to disrupt exon 11 (Ex11), one before and one after the known IFT80 mutation site, creating a 317-nucleotide (nt) cut to facilitate genotyping. Then, gRNAs annealed to a tracr-Cas9mRNA complex were delivered to 1-cell embryos by microinjection. Each replicate contained control embryos injected with only Cas9mRNA and treated embryos injected with gRNAs targeting IFT80. Embryos from each group were collected at the 8-cell stage for genotyping and gene expression analysis (n=47), or on Day 8 to validate genotypes of embryos left to develop (n=50). DNA sequences containing gRNA target sequences were amplified and visualised on an agarose gel. IFT80 expression was determined in biallelic embryos (n=13) using quantitative PCR and normalized to GAPDH. Primers were designed for the transcript regions before and after gRNAs target sequences, exons 9 and 12, respectively. Expression data were analysed using SAS software (v. 9.4; SAS Institute Inc.) using PROC GLM and LSMEANS to determine expression differences. Biallelic samples (n=9) were Sanger-sequenced (SS) and aligned with the reference sequence to determine exact cut sites. Protein amino acid (AA) sequences were predicted using SS data. Protein models were constructed using the I-Tasser platform, and then aligned and visualised using PyMol 2.4. Biallelic edits showed a significant decrease in exon 12 expression (P<0.05), and no difference in exon 9 compared with controls (P>0.05), indicating that the transcript was severely affected downstream of the edited sites. The reference protein model contained 777 AA, whereas the biallelic sample with the most accurate cut sites yielded a 385-AA protein, indicating that the mutation severely altered protein conformation and possible function. Embryos injected with CRISPR-Cas9 targeting Ex11 arrested at the 8-cell stage and failed to form blastocysts. Day 8 embryos were genotyped (n=24) and 58% were biallelic, 21% were monoallelic, and 21% appeared wild-type. Given the high rate of edits, the observed embryonic arrest is likely due to disruption of IFT80, and wild-type embryos may contain small edits not visible by gel. In conclusion, generation of CRISPR-Cas9 IFT80 knockouts demonstrated that the frameshift mutation in Ex11 results in a seemingly nonfunctional protein that is responsible for the embryonic lethality seen in HH2 carriers. Future research is needed to determine how IFT80 regulates embryonic development. This research was supported by USDA-NIFA National Needs Fellowship, USDA-NIFA AFRI Grant No. 2019-67015-28998.

A Retrospective Comparison of In Silico Pharmaceutical Recommendations with Tumor Board Recommendations in Pediatric Oncology

Background and Hypothesis: 
 The objective of this study was to analyze available whole genome sequencing from an adolescent male patient diagnosed with osteosarcoma (OS) in 2014. OS is a primary bone malignancy that most commonly affects the pediatric population. Precision medicine techniques provide new opportunities to improve treatment of OS patients. Pharmaceutical annotation tools such as PharmacoDB and DGIdb can help indicate chemotherapy agents that may benefit patients based on their molecular profiles. We hypothesize that these tools can indicate genome-specific chemotherapy agents for OS after genomic data has been aligned and analyzed. 
 
 Project Methods: 
 A PDX pipeline and retrospective study were performed that identified and compared pharmaceutical treatment options from software tools with the chemotherapy provided. Gene alignment and variant calling were used to process and analyze DNA sequencing data; germline and somatic mutations were also identified. Ensembl VEP was used for variant annotation. PharmacoDB and DGIdb were then applied to identify potentially beneficial medications. 
 
 Results: 
 Gene variant annotation indicated 54 potentially high impact mutations. Of these, DGIdb identified 15 drug-gene interactions. PharmacoDB identified no drugs that target any of the genes containing the 54 high impact mutations. For the entire mutated gene list, DGIdb identified 398 drug-gene interactions. After gene set enrichment, DGIdb identified medications targeting genes of pathways such as “O-glycan processing” and “Diseases of glycosylation”. Potentially harmful variants in the NPRL3 gene were identified. Because NPRL3 is a component of the Gator1 complex that serves as a negative regulator of mammalian target of rapamycin complex 1 (mTORC1), the identified variants in NPRL3 could have played a role in the patient’s OS. 
 
 Potential Impact: 
 This study will foster future collaborations to evaluate the pharmaceutical tool recommendations for this patient’s derived cell lines. These efforts will determine the efficacy of and identify improvements for computational treatment recommendation systems.

Genotype of CDC73 germline mutation determines risk of parathyroid cancer.

Mutation of the CDC73 gene, which encodes parafibromin, has been linked with parathyroid cancer. However, no correlation between genotypes of germline CDC73 mutations and the risk of parathyroid cancer has been known. In this study, subjects with germline CDC73 mutations were identified from the participants of two clinical protocols at National Institutes of Health (Discovery Cohort) and from the literature (Validation Cohort). The relative risk of developing parathyroid cancer was analyzed as a function of CDC73 genotype, and the impact of representative mutations on structure of parafibromin was compared between genotype groups. A total of 419 subjects, 68 in Discovery Cohort and 351 in Validation Cohort, were included. In both cohorts, percentages of CDC73 germline mutations that predicted significant conformational disruption or loss of expression of parafibromin (referred as 'high-impact mutations') were significantly higher among the subjects with parathyroid cancers compared to all other subjects. The Kaplan-Meier analysis showed that high-impact mutations were associated with a 6.6-fold higher risk of parathyroid carcinoma compared to low-impact mutations, despite a similar risk of developing primary hyperparathyroidism between two groups. Disruption of the C-terminal domain (CTD) of parafibromin is directly involved in predisposition to parathyroid carcinoma, since only the mutations impacting this domain were associated with an increased risk of parathyroid carcinoma. Structural analysis revealed that a conserved surface structure in the CTD is universally disrupted by the mutations affecting this domain. In conclusion, high-impact germline CDC73 mutations were found to increase risk of parathyroid carcinoma by disrupting the CTD of parafibromin.

MYBPC3 Haplotype Linked to Hypertrophic Cardiomyopathy in Rhesus Macaques (Macaca mulatta).

In humans, abnormal thickening of the left ventricle of the heart clinically defines hypertrophic cardiomyopathy (HCM), a common inherited cardiovascular disorder that can precede a sudden cardiac death event. The wide range of clinical presentations in HCM obscures genetic variants that may influence an individual's susceptibility to sudden cardiac death. Although exon sequencing of major sarcomere genes can be used to detect high-impact causal mutations, this strategy is successful in only half of patient cases. The incidence of left ventricular hypertrophy (LVH) in a managed research colony of rhesus macaques provides an excellent comparative model in which to explore the genomic etiology of severe HCM and sudden cardiac death. Because no rhesus HCM-associated mutations have been reported, we used a next-generation genotyping assay that targets 7 sarcomeric rhesus genes within 63 genomic sites that are orthologous to human genomic regions known to harbor HCM disease variants. Amplicon sequencing was performed on 52 macaques with confirmed LVH and 42 unrelated, unaffected animals representing both the Indian and Chinese rhesus macaque subspecies. Bias-reduced logistic regression uncovered a risk haplotype in the rhesus MYBPC3 gene, which is frequently disrupted in both human and feline HCM; this haplotype implicates an intronic variant strongly associated with disease in either homozygous or carrier form. Our results highlight that leveraging evolutionary genomic data provides a unique, practical strategy for minimizing population bias in complex disease studies.

Genomic variation between PRSV resistant transgenic SunUp and its progenitor cultivar Sunset

BackgroundThe safety of genetically transformed plants remains a subject of scrutiny. Genomic variants in PRSV resistant transgenic papaya will provide evidence to rationally address such concerns.ResultsIn this study, a total of more than 74 million Illumina reads for progenitor ‘Sunset’ were mapped onto transgenic papaya ‘SunUp’ reference genome. 310,364 single nucleotide polymorphisms (SNPs) and 34,071 small Inserts/deletions (InDels) were detected between ‘Sunset’ and ‘SunUp’. Those variations have an uneven distribution across nine chromosomes in papaya. Only 0.27% of mutations were predicted to be high-impact mutations. ATP-related categories were highly enriched among these high-impact genes. The SNP mutation rate was about 8.4 × 10− 4 per site, comparable with the rate induced by spontaneous mutation over numerous generations. The transition-to-transversion ratio was 1.439 and the predominant mutations were C/G to T/A transitions. A total of 3430 nuclear plastid DNA (NUPT) and 2764 nuclear mitochondrial DNA (NUMT) junction sites have been found in ‘SunUp’, which is proportionally higher than the predicted total NUPT and NUMT junction sites in ‘Sunset’ (3346 and 2745, respectively). Among all nuclear organelle DNA (norgDNA) junction sites, 96% of junction sites were shared by ‘SunUp’ and ‘Sunset’. The average identity between ‘SunUp’ specific norgDNA and corresponding organelle genomes was higher than that of norgDNA shared by ‘SunUp’ and ‘Sunset’. Six ‘SunUp’ organelle-like borders of transgenic insertions were nearly identical to corresponding sequences in organelle genomes (98.18 ~ 100%). None of the paired-end spans of mapped ‘Sunset’ reads were elongated by any ‘SunUp’ transformation plasmid derived inserts. Significant amounts of DNA were transferred from organelles to the nuclear genome during bombardment, including the six flanking sequences of the three transgenic insertions.ConclusionsComparative whole-genome analyses between ‘SunUp’ and ‘Sunset’ provide a reliable estimate of genome-wide variations and evidence of organelle-to-nucleus transfer of DNA associated with biolistic transformation.

High-Throughput Genotyping of Resilient Tomato Landraces to Detect Candidate Genes Involved in the Response to High Temperatures.

The selection of tolerant varieties is a powerful strategy to ensure highly stable yield under elevated temperatures. In this paper, we report the phenotypic and genotypic characterization of 10 tomato landraces to identify the best performing under high temperatures. The phenotyping of five yield-related traits allowed us to select one genotype that exhibits highly stable yield performances in different environmental conditions. Moreover, a Genotyping-by-Sequencing approach allowed us to explore the genetic variability of the tested genotypes. The high and stable yielding landrace E42 was the most polymorphic one, with ~49% and ~47% private SNPs and InDels, respectively. The effect of 26,113 mutations on proteins’ structure was investigated and it was discovered that 37 had a high impact on the structure of 34 proteins of which some are putatively involved in responses to high temperatures. Additionally, 129 polymorphic sequences aligned against tomato wild species genomes revealed the presence in the genotype E42 of several introgressed regions deriving from S. pimpinellifolium. The position on the tomato map of genes affected by moderate and high impact mutations was also compared with that of known markers/QTLs (Quantitative Trait Loci) associated with reproductive and yield-related traits. The candidate genes/QTLs regulating heat tolerance in the selected landrace E42 could be further investigated to better understand the genetic mechanisms controlling traits for high and stable yield trait under high temperatures.

Analysis of gene variants in the GASH/Sal model of epilepsy

Epilepsy is a complex neurological disorder characterized by sudden and recurrent seizures, which are caused by various factors, including genetic abnormalities. Several animal models of epilepsy mimic the different symptoms of this disorder. In particular, the genetic audiogenic seizure hamster from Salamanca (GASH/Sal) animals exhibit sound-induced seizures similar to the generalized tonic seizures observed in epileptic patients. However, the genetic alterations underlying the audiogenic seizure susceptibility of the GASH/Sal model remain unknown. In addition, gene variations in the GASH/Sal might have a close resemblance with those described in humans with epilepsy, which is a prerequisite for any new preclinical studies that target genetic abnormalities. Here, we performed whole exome sequencing (WES) in GASH/Sal animals and their corresponding controls to identify and characterize the mutational landscape of the GASH/Sal strain. After filtering the results, moderate- and high-impact variants were validated by Sanger sequencing, assessing the possible impact of the mutations by “in silico” reconstruction of the encoded proteins and analyzing their corresponding biological pathways. Lastly, we quantified gene expression levels by RT-qPCR. In the GASH/Sal model, WES showed the presence of 342 variations, in which 21 were classified as high-impact mutations. After a full bioinformatics analysis to highlight the high quality and reliable variants, the presence of 3 high-impact and 15 moderate-impact variants were identified. Gene expression analysis of the high-impact variants of Asb14 (ankyrin repeat and SOCS Box Containing 14), Msh3 (MutS Homolog 3) and Arhgef38 (Rho Guanine Nucleotide Exchange Factor 38) genes showed a higher expression in the GASH/Sal than in control hamsters. In silico analysis of the functional consequences indicated that those mutations in the three encoded proteins would have severe functional alterations. By functional analysis of the variants, we detected 44 significantly enriched pathways, including the glutamatergic synapse pathway. The data show three high-impact mutations with a major impact on the function of the proteins encoded by these genes, although no mutation in these three genes has been associated with some type of epilepsy until now. Furthermore, GASH/Sal animals also showed gene variants associated with different types of epilepsy that has been extensively documented, as well as mutations in other genes that encode proteins with functions related to neuronal excitability, which could be implied in the phenotype of the GASH/Sal. Our findings provide valuable genetic and biological pathway data associated to the genetic burden of the audiogenic seizure susceptibility and reinforce the need to validate the role of each key mutation in the phenotype of the GASH/Sal model.

Genome sequencing of ion-beam-induced mutants facilitates detection of candidate genes responsible for phenotypes of mutants in rice

Ion beams are physical mutagens used for plant and microbe breeding that cause mutations via a mechanism distinct from those of chemical mutagens or gamma rays. We utilized whole-exome sequencing of rice DNA in order to understand the properties of ion beam-induced mutations in a genome-wide manner. DNA libraries were constructed from selected carbon-ion-beam-induced rice mutants by capturing with a custom probes covering 66.3 M bases of nearly all exons and miRNAs predicted in the genome. A total of 56 mutations, including 24 single nucleotide variations, 23 deletions, and 5 insertions, were detected in five mutant rice lines (two dwarf and three early-heading-date mutants). The mutations were distributed among all 12 chromosomes, and the average mutation frequency in the M1 generation was estimated to be 2.7 × 10−7 per base. Many single base insertions and deletions were associated with homopolymeric repeats, whereas larger deletions up to seven base pairs were observed at polynucleotide repeats in the DNA sequences of the mutation sites. Of the 56 mutations, six were classified as high-impact mutations that caused a frame shift or loss of exons. A gene that was functionally related to the phenotype of the mutant was disrupted by a high-impact mutation in four of the five lines tested, suggesting that whole-exome sequencing of ion-beam-irradiated mutants could facilitate the detection of candidate genes responsible for the mutant phenotypes.