scholarly journals A global functional analysis of missense mutations reveals two major hotspots in the PALB2 tumor suppressor

2019 ◽  
Vol 47 (20) ◽  
pp. 10662-10677 ◽  
Author(s):  
Amélie Rodrigue ◽  
Guillaume Margaillan ◽  
Thiago Torres Gomes ◽  
Yan Coulombe ◽  
Gemma Montalban ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Tumor Suppressor ◽  
Homologous Recombination ◽  
Parp Inhibitor ◽  
Familial Pancreatic Cancer ◽  
Missense Mutations ◽  
Breast Cancer Patients ◽  
Missense Variants ◽  
Functional Assays ◽  
The Impact

Abstract While biallelic mutations in the PALB2 tumor suppressor cause Fanconi anemia subtype FA-N, monoallelic mutations predispose to breast and familial pancreatic cancer. Although hundreds of missense variants in PALB2 have been identified in patients to date, only a few have clear functional and clinical relevance. Herein, we investigate the effects of 44 PALB2 variants of uncertain significance found in breast cancer patients and provide detailed analysis by systematic functional assays. Our comprehensive functional analysis reveals two hotspots for potentially deleterious variations within PALB2, one at each terminus. PALB2 N-terminus variants p.P8L [c.23C>T], p.Y28C [c.83A>G], and p.R37H [c.110G>A] compromised PALB2-mediated homologous recombination. At the C-terminus, PALB2 variants p.L947F [c.2841G>T], p.L947S [c.2840T>C], and most strikingly p.T1030I [c.3089C>T] and p.W1140G [c.3418T>C], stood out with pronounced PARP inhibitor sensitivity and cytoplasmic accumulation in addition to marked defects in recruitment to DNA damage sites, interaction with BRCA2 and homologous recombination. Altogether, our findings show that a combination of functional assays is necessary to assess the impact of germline missense variants on PALB2 function, in order to guide proper classification of their deleteriousness.

scholarly journals In Planta Mutagenesis Determines the Functional Regions of the Wheat Puroindoline Proteins

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 853-860 ◽  
Author(s):  
Leila Feiz ◽  
Brian S. Beecher ◽  
John M. Martin ◽  
Michael J. Giroux
Keyword(s):  
Functional Analysis ◽  
Allelic Variation ◽  
Screening Method ◽  
Point Mutations ◽  
Missense Mutations ◽  
Grain Hardness ◽  
In Planta ◽  
Rich Region ◽  
Critical Function ◽  
The Impact

In planta analysis of protein function in a crop plant could lead to improvements in understanding protein structure/function relationships as well as selective agronomic or end product quality improvements. The requirements for successful in planta analysis are a high mutation rate, an efficient screening method, and a trait with high heritability. Two ideal targets for functional analysis are the Puroindoline a and Puroindoline b (Pina and Pinb, respectively) genes, which together compose the wheat (Triticum aestivum L.) Ha locus that controls grain texture and many wheat end-use properties. Puroindolines (PINs) together impart soft texture, and mutations in either PIN result in hard seed texture. Studies of the PINs' mode of action are limited by low allelic variation. To create new Pin alleles and identify critical function-determining regions, Pin point mutations were created in planta via EMS treatment of a soft wheat. Grain hardness of 46 unique PIN missense alleles was then measured using segregating F2:F3 populations. The impact of individual missense alleles upon PIN function, as measured by grain hardness, ranged from neutral (74%) to intermediate to function abolishing. The percentage of function-abolishing mutations among mutations occurring in both PINA and PINB was higher for PINB, indicating that PINB is more critical to overall Ha function. This is contrary to expectations in that PINB is not as well conserved as PINA. All function-abolishing mutations resulted from structure-disrupting mutations or from missense mutations occurring near the Tryptophan-rich region. This study demonstrates the feasibility of in planta functional analysis of wheat proteins and that the Tryptophan-rich region is the most important region of both PINA and PINB.

scholarly journals SMAD6 variants in craniosynostosis: genotype and phenotype evaluation

2020 ◽  
Vol 22 (9) ◽  
pp. 1498-1506 ◽  
Author(s):  
Eduardo Calpena ◽  
◽  
Araceli Cuellar ◽  
Krithi Bala ◽  
Sigrid M. A. Swagemakers ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Inhibitory Activity ◽  
Loss Of Function ◽  
Missense Variants ◽  
Common Polymorphism ◽  
Metopic Synostosis ◽  
Fold Enrichment ◽  
Unaffected Parent ◽  
The Impact

Abstract Purpose Enrichment of heterozygous missense and truncating SMAD6 variants was previously reported in nonsyndromic sagittal and metopic synostosis, and interaction of SMAD6 variants with a common polymorphism nearBMP2 (rs1884302) was proposed to contribute to inconsistent penetrance. We determined the occurrence of SMAD6 variants in all types of craniosynostosis, evaluated the impact of different missense variants on SMAD6 function, and tested independently whether rs1884302 genotype significantly modifies the phenotype. Methods We performed resequencing of SMAD6 in 795 unsolved patients with any type of craniosynostosis and genotyped rs1884302 in SMAD6-positive individuals and relatives. We examined the inhibitory activity and stability of SMAD6 missense variants. Results We found 18 (2.3%) different rare damaging SMAD6 variants, with the highest prevalence in metopic synostosis (5.8%) and an 18.3-fold enrichment of loss-of-function variants comparedwith gnomAD data (P < 10−7). Combined with eight additional variants, ≥20/26 were transmitted from an unaffected parent but rs1884302 genotype did not predict phenotype. Conclusion Pathogenic SMAD6 variants substantially increase the risk of both nonsyndromic and syndromic presentations of craniosynostosis, especially metopic synostosis. Functional analysis is important to evaluate missense variants. Genotyping of rs1884302 is not clinically useful. Mechanisms to explain the remarkable diversity of phenotypes associated with SMAD6 variants remain obscure.

Characterization of tumor mutation load (TML) in solid tumors.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 11517-11517 ◽  
Author(s):  
Mohamed E. Salem ◽  
Joanne Xiu ◽  
Heinz-Josef Lenz ◽  
Michael B. Atkins ◽  
Philip Agop Philip ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Gene Mutations ◽  
Predictive Biomarker ◽  
Suppressor Gene ◽  
Older Age ◽  
Patient Treatment ◽  
Missense Mutations ◽  
Driver Genes ◽  
Primary Nsclc ◽  
The Impact

11517 Background: Rapid advances in immunotherapy have created a need for biomarkers to improve patient treatment selection. TML is proposed as a potential predictive biomarker due to its association with tumor immunogenicity. Methods: TML was assessed in 8020 tumors from 14 different cancers using somatic nonsynonymous missense mutations sequenced with a 592-gene panel. High TML was set at ≥ 17 mutations per megabase (mt/MB) based on an established concordance ( > 99%) with MSI-High in colorectal cancer (CRC). Results: Mean TML was highest in melanoma (Mel; 21 mt/MB), NSCLC (11 mt/MB), and bladder cancer (BLC; 11 mt/MB), whereas prostate cancer (PC), pancreas adenocarcinoma (PA), and renal cell carcinoma (RCC) had the lowest levels (all 6 mt/MB). High TML was seen most frequently in Mel (36%), NSCLC (15%), BLC (15%), and anal cancer (SCCA; 9%); and least frequently in PA (1.6%) and RCC (0.5%). Primary NSCLC carried lower TML than its brain metastases (11 vs. 16 mt/MB, p < 0.001). Older age was associated with higher TML in Mel (p = 0.001), CRC (p = 0.009), breast cancer (BC; p = 0.01), and NSCLC (p = 0.02). Higher TML was seen in males than in females for Mel (p = 0.002) and NSCLC (p < 0.001). Presence of mutations in oncogenic driver genes such as EGFR, ALK, ROS1 RET fusions, cMET exon 14 skipping correlated with lower TML in NSCLC (6.9 vs. 12 mt/MB, p < 0.001), as did BRAF and NRAS mutations in Mel (17 vs. 26, p = 0.003). Conversely, mutations in tumor suppressor genes such as ARID1A (CRC, NSCLC, and BLC) and NF1 (BC, CRC, Mel, BLC, and NSCLC) were associated with higher TML (p < 0.05). MSI-high was correlated with high TML in CRC and gastric cancers (p < 0.05). Conclusions: TML varied significantly among different cancers. High TML was associated with older age, absence of oncogenic mutations and presence of tumor suppressor gene mutations. Future studies will assess the impact of TML on clinical outcome and establish its role in selecting patients for immunotherapy. [Table: see text]

scholarly journals Classifying Variants of Undetermined Significance in BRCA2 with Protein Likelihood Ratios

2008 ◽  
Vol 6 ◽  
pp. CIN.S618 ◽  
Author(s):  
Rachel Karchin ◽  
Mukesh Agarwal ◽  
Andrej Sali ◽  
Fergus Couch ◽  
Mary S. Beattie
Keyword(s):  
Genetic Testing ◽  
Protein Structure ◽  
Tumor Suppressor ◽  
Likelihood Ratio ◽  
Cancer Susceptibility ◽  
Suppressor Gene ◽  
Medical Genetics ◽  
Missense Mutations ◽  
Likelihood Ratios ◽  
The Impact

Background Missense (amino-acid changing) variants found in cancer predisposition genes often create difficulties when clinically interpreting genetic testing results. Although bioinformatics has developed approaches to predicting the impact of these variants, many of these approaches have not been readily applicable in the clinical setting. Bioinformatics approaches for predicting the impact of these variants have not yet found their footing in clinical practice because 1) interpreting the medical relevance of predictive scores is difficult; 2) the relationship between bioinformatics “predictors” (sequence conservation, protein structure) and cancer susceptibility is not understood. Methodology/Principal Findings We present a computational method that produces a probabilistic likelihood ratio predictive of whether a missense variant impairs protein function. We apply the method to a tumor suppressor gene, BRCA2, whose loss of function is important to cancer susceptibility. Protein likelihood ratios are computed for 229 unclassified variants found in individuals from high-risk breast/ovarian cancer families. We map the variants onto a protein structure model, and suggest that a cluster of predicted deleterious variants in the BRCA2 OB1 domain may destabilize BRCA2 and a protein binding partner, the small acidic protein DSS1. We compare our predictions with variant “re-classifications” provided by Myriad Genetics, a biotechnology company that holds the patent on BRCA2 genetic testing in the U.S., and with classifications made by an established medical genetics model [ 1 ]. Our approach uses bioinformatics data that is independent of these genetics-based classifications and yet shows significant agreement with them. Preliminary results indicate that our method is less likely to make false positive errors than other bioinformatics methods, which were designed to predict the impact of missense mutations in general. Conclusions/Significance Missense mutations are the most common disease-producing genetic variants. We present a fast, scalable bioinformatics method that integrates information about protein sequence, conservation, and structure in a likelihood ratio that can be integrated with medical genetics likelihood ratios. The protein likelihood ratio, together with medical genetics likelihood ratios, can be used by clinicians and counselors to communicate the relevance of a VUS to the individual who has that VUS. The approach described here is generalizable to regions of any tumor suppressor gene that have been structurally determined by X-ray crystallography or for which a protein homology model can be built.

Association of ATM mutations in metastatic prostate cancer with differential genomic alteration profiles from homologous recombination deficient and proficient tumors.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 5063-5063
Author(s):  
Charles J. Ryan ◽  
Julie Elaine McGrath ◽  
Joanne Xiu ◽  
Justin Hwang ◽  
Chadi Nabhan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Homologous Recombination ◽  
Parp Inhibitor ◽  
Therapy Resistance ◽  
Genomic Alteration ◽  
Clinical Activity ◽  
Missense Mutations ◽  
Therapeutic Trials ◽  
Whole Transcriptome Sequencing

5063 Background: ATM mutations, one of a family of DNA repair defects prevalent in prostate cancer, have been included in a list of actionable mutations for PARP inhibitor (PARPi) therapeutic trials. Despite preclinical evidence, PARPi have shown minimal clinical activity in ATM mutant prostate cancer (ATMmPCa). The present analysis explores co-occurring genomic alterations that may drive outcomes of metastatic PCa (mPCa) patients with tumors harboring ATM mutations and provide clues for understanding therapy resistance and potential targets. Methods: This study included molecular profiling analysis of 1375 cases of mPCa. Tumors were analyzed using next-generation sequencing (NGS), whole transcriptome sequencing (WTS), and immunohistochemistry (IHC) (Caris Life Sciences, Phoenix, AZ). dMMR/MSI-H status was determined by IHC, NGS, and fragment analysis and tumor mutational burden (TMB) was calculated based on somatic nonsynonymous missense mutations. We performed differential gene expression analysis of HR-associated transcripts such as ATR, PARP1-3, RAD50, RAD51A/B/C/D and RAD54. Significance was determined using the ꭓ2 test and Benjamini-Hochberg method. Results: Fifty-nine (4.2%) cases harbored pathogenic ATM mutations, 84 (6.2%) harbored BRCA2 mutations. 1018 tumors (74%) were deemed homologous recombination proficient (HRP) and 155 tumors (11.3%) were HR Deficient (HRD); harboring one or more mutation in HR-related genes excluding ATM and BRCA2. The mutation rate of TP53 was significantly lower in ATMmPCa (12.0%) compared to BRCA2mPCa (35%), HRD (35%) and HRP (46.6%) tumors. ATMmPCa showed higher rates of SMAD2 (3.7%/1%) and FLCN (5.2%/0.3%) alterations compared to HRP cases. PARP1 and RAD51D gene expression was reduced in ATMmPCa compared to HRP (p < 0.05) and BRCA2mPCa ( p < 0.05) tumors, respectively. No differences in gene expression levels were detected for ATR, PARP2, PARP3, RAD50, and RAD54. Chromosomal segments demonstrating differential CNA in ATMmPCa vs HRP, HRD, or BRCA2mPCa included FGF19, FGF4, PTPN11, ALDH2, DAXX, BCL7A, CCND1, BMPR1A and MEF2B (Q-value < 0.05 determined by ꭓ2). The most common CNA in ATMmPCa was CCND1, present in approximately 13% (7/55) of cases. Compared to BRCA2mPCa and HRD cases, ATMmPCa cases are less likely to display markers of immunotherapy response such as dMMR/MSI-H or TMB ≥10 mutations/MB. Conclusions: ATMmPCa demonstrated several differences in co-occurring alterations compared to BRCA2mPCa, HRD and HRP mPCa. ATMmPCa tumors were less likely to harbor alterations in TP53 compared to BRCA2, HRD or HRP tumors. CNA in ATMmPCa occurred in 9 genes across distinct mPCa molecular subtypes and were enriched for those associated with the 11q13 amplicon harboring Cyclin D1. The FGF and PTPN11 related pathways are potentially targetable pathways in ATMmPC and may merit further study.

scholarly journals Functional analysis of novel A20 variants in patients with atypical inflammatory diseases

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Saori Kadowaki ◽  
Kunio Hashimoto ◽  
Toyoki Nishimura ◽  
Kenichi Kashimada ◽  
Tomonori Kadowaki ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Gene Activity ◽  
Hek293 Cells ◽  
Missense Mutations ◽  
Reporter Gene Activity ◽  
Missense Variants ◽  
Functional Assays ◽  
Iκb Kinase ◽  
Gene Assay

Abstract Background A20 haploinsufficiency (HA20) is an early-onset autoinflammatory disease caused by mutations in the TNFAIP3 gene, which encodes the protein A20. Numerous truncating mutations in the TNFAIP3 gene have been reported in HA20 patients, whereas fewer missense variants have had their pathogenicity confirmed. Here, we evaluated the pathogenic significance of three previously unreported missense variants of the TNFAIP3 gene in suspected cases of HA20. Methods We obtained the clinical features and immunological data of three patients with missense variants (Glu192Lys, Ile310Thr, and Gln709Arg) of unknown significance of TNFAIP3. We then performed in vitro functional assays including analysis of nuclear factor (NF)-κB reporter gene activity, detection of A20 expression and phosphorylation of A20 by IκB kinase β (IKKβ), and K63-deubiquitination assay using TNFAIP3-deficient HEK293 cells. Three known pathogenic missense mutations reported previously were also investigated. Results The inhibitory effect on NF-κB reporter gene activity was significantly disrupted by A20 Glu192Lys and the three known mutations. The variants Ile310Thr and Gln709Arg did not show a difference from the wild type in any of the assays performed in this study. Conclusions Among the three variants in the TNFAIP3 gene, Glu192Lys was interpreted as being likely pathogenic, but Ile310Thr and Gln709Arg as being not pathogenic (uncertain significance and likely benign, respectively), based on the American College of Medical Genetics and Genomics standards and guidelines. Our study highlights the necessity of performing in vitro functional assays, notably, NF-κB reporter gene assay, to evaluate the pathogenicity of TNFAIP3 missense variants for the accurate diagnosis of HA20.

Impact analysis of missense variants on heat shock protein offarmed carp rohu, Labeo rohita (Hamilton, 1822)

2016 ◽  
Author(s):  
Prajna Pradhan ◽  
Kiran Dashrath Rasal ◽  
Dibyashree Swain ◽  
Pranati Swain ◽  
Jitendra Kumar Sundaray ◽  
...  
Keyword(s):  
Heat Shock ◽  
Impact Analysis ◽  
Labeo Rohita ◽  
Strong Association ◽  
Interaction Mechanism ◽  
Missense Mutations ◽  
Missense Variants ◽  
Shock Proteins ◽  
The Impact

The progress in the computational field is advantageous for investigating the impact of missense variants/mutation on the protein structure-function. The impact analyses of variants on the genes/proteins using in vivo laboratory methods are laborious and time consuming. Thus, the present study was performed for investigating the impact of missense mutations on the Heat-shock proteins (HSP70) of farmed carp, rohu, Labeo rohita. We have used several sequence-based computational tools/algorithms such as SIFT, PANTHER, PROVEAN and I-Mutant2.0. We have depicted that all mutations (p.G6V, p.A56I, and p.A159T) in the HSP70 were deleterious. The 3D model of HSP70 of rohu was generated using Modeller9.14 and subsequently validated using SAVEs server. The Ramachandran plot shown that shifting of residues in the mutant structure towards the disallowed region due to mutation as compared to native counterpart. The ERAT score and PROSA score, also given clues of deteriorating quality of mutant protein HSP70. Moreover, STRING9.1 shown that HSP70 protein interacting with several proteins and strong association was observed with two proteins, hsp90 and dnajb (Hsp40 homolog). The RMSD was obtained 0.04Å between native and mutant structure. The present study will helpful for understanding the impact of missense mutations on the HSP70 of rohu using in vivo methods. This study enriched for further exploring disturbances of protein-protein interaction mechanism as well as associated molecular pathways.

Functional analysis of patient-derived PALB2 missense variants of uncertain significance.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 1531-1531
Author(s):  
Shijie Wu ◽  
Jiaojiao Zhou ◽  
Yiding Chen
Keyword(s):  
Breast Cancer ◽  
Functional Analysis ◽  
In Silico ◽  
Cancer Genetic Counseling ◽  
Wild Type ◽  
Variants Of Uncertain Significance ◽  
Missense Variants ◽  
Pathogenic Variants ◽  
Uncertain Significance ◽  
The Impact

1531 Background: Inherited PALB2 pathogenic variants are associated with an increased lifetime risk for breast cancer development. However, the interpretation of numerous PALB2 missense variants of uncertain significance (VUS) identified in germline genetic testing remains a challenge. Here, we assessed the impact of breast cancer patient-derived VUS on PALB2 function and identified pathogenic PALB2 missense variants that may increase cancer risk. Methods: A total of seven potentially pathogenic PALB2 VUS identified in 2,279 breast cancer patients were selected for functional analysis. All these selected VUS were assessed by SIFT, Align-GVGD, and PolyPhen2 in silico and were predicted to be deleterious by at least two in silico algorithms. The p.L35P [c.104T > C] variant was also included, for which pathogenicity has been recently confirmed. The effects of the VUS on the homologous recombination (HR) activity of PALB2 were tested by U2OS/DR-GFP reporting system. Functional characterization was further validated by protein co-immunoprecipitation and RAD51 recruitment assay. Results: PALB2 variants p.L24F [c.72G > C] and p.L35P [c.104T > C] showed the most significant disruption to the HR activity of PALB2 relative to the wild-type condition, retaining only 52.2% ( p = 0.0013) and 8.5% ( p < 0.0001) of HR activity respectively. Moderate but statistically significant HR deficiency was observed for four other variants (p.P405A [c.1213C > G], p.T1012I [c.3035C > T], p.E1018D [c.3054G > C], and p.T1099M [c.3296C > T]). We found no statistical differences for the p.K628N [c.1884G > T] and p.R663C [c.1987C > T] in the HR activity compared to wild-type PALB2. The p.L24F and p.L35P variants compromised the BRCA1-PALB2 interaction and reduced RAD51 foci formation in response to DNA damage. Conclusions: We have identified a novel patient-derived pathogenic PALB2 missense variant, p.L24F [c.72G > C], that compromises PALB2-mediated HR activity. We suggest the integration of the identified pathogenic variants into breast cancer genetic counseling and individualized treatment regimens for better clinical outcomes.

scholarly journals The impact of PTEN tumor suppressor gene on acquiring resistance to tamoxifen treatment in breast cancer patients

2012 ◽  
Vol 13 (12) ◽  
pp. 1165-1174 ◽  
Author(s):  
Nikola Tanic ◽  
Zorka Milovanovic ◽  
Nasta Tanic ◽  
Radan Dzodic ◽  
Zorica Juranic ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Suppressor ◽  
Cancer Patients ◽  
Tumor Suppressor Gene ◽  
Suppressor Gene ◽  
Tamoxifen Treatment ◽  
Breast Cancer Patients ◽  
The Impact
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