Heritable Mutations Research Articles

The impact of tumor NGS testing on hereditary cancer risk assessment and population management in an integrated community health care system.

1517 Background: Next-generation sequencing (NGS) for tumor molecular profiling is used in Oncology to identify ‘actionable alterations’ for clinical trials or on/ off-label therapy. Tumor NGS can also reveal potentially heritable germline mutations. The frequency of such incidental germline mutations has been estimated to be 4-15%. The 2015 ASCO Statement supports communication of medically relevant incidental germline findings from somatic mutation profiling to patients (PTS). The impact of tumor NGS testing on hereditary cancer risk assessment programs in the context of a wider population management strategy is unknown. We sought to evaluate this within our Kaiser Permanente Northern California (KPNC) population with ready access to tumor NGS and an ongoing hereditary cancer risk assessment program. Methods: Kaiser Permanente Northern California (KPNC) is part of a large, integrated health care system. NGS at KPNC is performed in collaboration with STRATA Oncology, a precision oncology partnership. All NGS results are reviewed by a multidisciplinary KPNC Genomic Oncology Committee (GOC)which also includes genetic counselors and pathologists. We examined all NGS reports between November 2017 through December 2019 to determine the types of cancers tested, number with a possible germline mutation and number referred for genetic counseling and testing (GCT). Results: 4,825 PTS with advanced cancer underwent STRATA NGS testing. A total of 207 PTS (4.3%) were identified as potential germline mutation carriers, all 207 were recommended for GCT referral. Of these, 92 (45.0%) separately met 2020 NCCN Criteria for Genetic/Familial High-Risk Assessment (2020NG/FA), prior to tumor NGS; 115 (53.6%) did not and 3 (1.4%) had insufficient information. The cancers most frequently meeting NCCN criteria were pancreatic, breast and colon. Of the 92 PTS who met 2020NG/FA, 60 (65%) underwent GCT and 34 (57%) were confirmed to have a germline mutation. Of the 115 PTS that did not meet 2020NG/FA, 47 (41%) underwent GCT and 19 (40%) were confirmed to have a germline mutation. Overall germline mutations were confirmed in 16.5% of patients who did not meet 2020NG/FA and 37% who did. Conclusions: In our community-based integrated healthcare system, systematic review of next-generation sequencing results by an expert GOC led to more robust identification of germline mutation carriers and navigated them to appropriate GCT. Ongoing work will clarify data on cascade testing. We are currently developing automated workflows for GCT.

Heritable arrhythmias associated with abnormal function of cardiac potassium channels.

Cardiomyocytes express a surprisingly large number of potassium channel types. The primary physiological functions of the currents conducted by these channels are to maintain the resting membrane potential and mediate action potential repolarization under basal conditions and in response to changes in the concentrations of intracellular sodium, calcium, and ATP/ADP. Here, we review the diversity and functional roles of cardiac potassium channels under normal conditions and how heritable mutations in the genes encoding these channels can lead to distinct arrhythmias. We briefly review atrial fibrillation and J-wave syndromes. For long and short QT syndromes, we describe their genetic basis, clinical manifestation, risk stratification, traditional and novel therapeutic approaches, as well as insights into disease mechanisms provided by animal and cellular models.

Mutagenic Effect of Three Ion Beams on Rice and Identification of Heritable Mutations by Whole Genome Sequencing.

High-energy ion beams are known to be an effective and unique type of physical mutagen in plants. However, no study on the mutagenic effect of argon (Ar) ion beam radiation on rice has been reported. Genome-wide studies on induced mutations are important to comprehend their characteristics for establishing knowledge-based protocols for mutation induction and breeding, which are still very limited in rice. The present study aimed to investigate the mutagenic effect of three ion beams, i.e., Ar, carbon (C) and neon (Ne) on rice and identify and characterize heritable induced mutations by the whole genome sequencing of six M4 plants. Dose-dependent damage effects were observed on M1 plants, which were developed from ion beam irradiated dry seeds of two indica (LH15, T23) and two japonica (DS551, DS48) rice lines. High frequencies of chlorophyll-deficient seedlings and male-sterile plants were observed in all M2 populations (up to ~30% on M1 plant basis); plants from the seeds of different panicles of a common M1 plant appeared to have different mutations; the whole genome-sequencing demonstrated that there were 236–453 mutations in each of the six M4 plants, including single base substitutions (SBSs) and small insertion/deletions (InDels), with the number of SBSs ~ 4–8 times greater than that of InDels; SBS and InDel mutations were distributed across different genomic regions of all 12 chromosomes, however, only a small number of mutations (0–6) were present in exonic regions that might have an impact on gene function. In summary, the present study demonstrates that Ar, C and Ne ion beam radiation are all effective for mutation induction in rice and has revealed at the genome level the characteristics of the mutations induced by the three ion beams. The findings are of importance to the efficient use of ion beam radiation for the generation and utilization of mutants in rice.

Low-dose X-ray imaging may increase the risk of neurodegenerative diseases

The hypothesis presented here explores the possibility that X-ray imaging commonly used in dental practices may be a shared risk factor for sporadic dementias and motor-neuron diseases. As the evidence will suggest, the brain is ill-equipped to manage the intrusion of low-dose ionizing radiation (IR) beyond that which is naturally occurring. When the brain’s antioxidant defenses are overwhelmed by IR, it produces an abundance of reactive oxygen species (ROS) that can lead to oxidative stress, mitochondrial dysfunction, loss of synaptic plasticity, altered neuronal structure and microvascular impairment that have been identified as early signs of neurodegeneration in Alzheimer’s disease, Parkinson’s, amyotrophic lateral sclerosis, vascular dementia and other diseases that progressively damage the brain and central nervous system. Although genes play a role in all outcomes, the focus here will be on the non-genetic processes at work. Common assumptions regarding the risks of low-dose IR will be addressed, such as: 1) comparing rapid, repeated bursts of man-made IR sent exclusively into the head to equivalent amounts of head-to-toe background IR over longer periods of time; 2) whether epidemiological studies that dismiss concerns regarding low-dose IR due to lack of evidence it causes cancer, heritable mutations or shortened life spans also apply to neurodegeneration; and 3) why even radiation-resistant neurons can be severely impacted by IR exposure, due to IR-induced injury to the processes they need to function. Also considered will be the difficulty of distinguishing the effects of dental X-ray exposure from similarly low amounts of background IR and where to find the evidence that they may, in fact, be responsible for neurodegeneration. Finally, the long-standing belief that whatever risks are inherent in dental radiography must be undertaken for the sake of oral health is challenged on two counts: 1) while dentists continue to drape their patients in lead-lined aprons, the most effective IR safety precautions are often ignored; and 2) there is an alternative dental imaging technology that does not use IR. While the thrust of this article will be on dental radiation and will touch on how age, gender, X-ray equipment and protocols may increase risk, chiropractic radiographs also will be considered because they focus exclusively on the central nervous system. If X-ray imaging is found to be associated with neurodegeneration, the risk-versus-benefit must be reevaluated, every means of reducing exposure implemented and imaging protocols revised.

Epidemiology of Bladder Cancer.

Based on the latest GLOBOCAN data, bladder cancer accounts for 3% of global cancer diagnoses and is especially prevalent in the developed world. In the United States, bladder cancer is the sixth most incident neoplasm. A total of 90% of bladder cancer diagnoses are made in those 55 years of age and older, and the disease is four times more common in men than women. While the average 5-year survival in the US is 77%, the 5-year survival for those with metastatic disease is a measly 5%. The strongest risk factor for bladder cancer is tobacco smoking, which accounts for 50–65% of all cases. Occupational or environmental toxins likewise greatly contribute to disease burden (accounting for an estimated 20% of all cases), though the precise proportion can be obscured by the fact bladder cancer develops decades after exposure, even if the exposure only lasted several years. Schistosomiasis infection is the common cause of bladder cancer in regions of Africa and the Middle East and is considered the second most onerous tropical pathogen after malaria. With 81% of cases attributable to known risk factors (and only 7% to heritable mutations), bladder cancer is a prime candidate for prevention strategies. Smoking cessation, workplace safety practices, weight loss, exercise and schistosomiasis prevention (via water disinfection and mass drug administration) have all been shown to significantly decrease the risk of bladder cancer, which poses a growing burden around the world.

Cas9-Mediated Gene-Editing in the Malaria Mosquito Anopheles stephensi by ReMOT Control.

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.

Health Disparities in Germline Genetic Testing for Cancer Susceptibility

Despite advances in testing for heritable cancer susceptibility gene mutations, health disparities persist. We review racial/ethnic and socioeconomic factors that affect access to genetic counseling and testing, which may influence the validity of results among diverse populations and the clinical utility of genetic testing. Differential access to genetic testing among racial/ethnic minorities including Hispanics and blacks compared with whites drives disparities in the clinical validity of results: specifically, variants of unknown significance (VUS) are notably more prevalent in minorities. Deficiencies in results interpretation by patients and providers are common with VUS, which may engender disparities in clinical utility. There is evidence of racial/ethnic disparities in genetic testing access and the clinical validity of results, which are threats to the clinical utility of testing for all populations. Concerted efforts are needed to decrease disparities in germline cancer genetic testing and results’ interpretation and management.

Analysis of Heterozygous BRCA1 5382ins Founder Mutation in a Cohort of Egyptian Breast Cancer Female Patients Using Pyrosequencing Technique.

Background:Up to half of the heritable mutations in breast cancer (BC) are attributed to BRCA1 and BRCA2 genes. The mutation prevalence is variable based on ethnicity and may be influenced by founder mutations. The aim of this pilot study is to determine for the first time, the prevalence of BRCA1 5382insC founder mutation in a cohort of Egyptian familial breast cancer patients (FBC). Methods:Female patients were selected to have familial type of breast cancer. Twenty healthy females were included as a control group. Peripheral blood samples were withdrawn from all studied females and were analyzed for BRCA1 5382insC founder mutation detection using pyrosequencing technique. Results:Eighty Egyptian FBC females were eligible to be enrolled in the study with a mean age of 48.31 ± 10.97years.We found a BRCA1 5382insC mutation carrier frequency of 5% of total studied FBC patients (4 out of 80 patients) with 95% confidence interval (1.61-12.99). There was a high statistical significant difference between carriers and non-carriers concerning the number of affected family members by BC, (p=0.001). Conclusion: BRCA1 5382insC founder mutation is not uncommon among Egyptian FBC females. The carrier frequency is comparable to that reported worldwide; however it is lower than those from previous Egyptian studies using different molecular techniques. The strong association between the mutation and the number of affected family members suggest wider screening of the mutation among high risk families using the reliable pyrosequencing technique.

Mutation Induction, Detection and Breeding to Resist Viral and Fungal Diseases and Thermal Extremism for Vegetables

To artificially induce hereditary changes in plants, either physical or chemical agents are used. Ionizing radiation is a widely used physical agent to treat the seeds and other crops plant material to create heritable mutations.

Sperm Go to (Transcription) Extremes

Xia and colleagues show that heritable mutations are kept in check in the male germline partly by "transcriptional scanning," wherein the majority of genes are transcribed and therefore subject to transcription-coupled repair. They provide a new model for understanding the mechanisms of genome surveillance and evolution.

Ethyl Methanesulfonate as Inductor of Somaclonal Variants in Different Crops

Ethyl methanesulfonate is a chemical mutagen, which is currently being used in plant breeding, to increase genetic variability in genes of agronomic interest, of species useful in agriculture. It primarily causes single base point mutations by inducing guanine alkylation, resulting in GC to AT transitions. Its effect is different between clones of a genotype and between genotypes of the same species. This review presents the results obtained in recent research, where its effect on plant tissues, callus, and cells in suspension has been evaluated. Changes in the phenotypic expression of somaclonal variants were reported, involving morphology, production of secondary metabolites, changes in metabolic routes of resistance, tolerance to stress, increased seed yield, among others. In addition, this review compiles the doses and guidelines to consider before using this mutagen, which can serve as a guide for future trials in deciding the response variables, the type of plant explants and the selection of the study model. Mutant lines have allowed plant breeders to have a collection of plants with different characteristics, in places where the cultivar does not have its center of origin. It is important to note that it is still necessary to continue evaluating the heritability of mutations and their behaviour in the environment where they will be established, in order to obtain new varieties of plants that can be cultivated with uniformity in their genetic response.

Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production.

SummaryMicroRNAs (miRNAs) are 20‐24 nucleotides (nt) small RNAs functioning in eukaryotes. The length and sequence of miRNAs are not only related to the biogenesis of miRNAs but are also important for downstream physiological processes like ta‐siRNA production. To investigate these roles, it is informative to create small mutations within mature miRNA sequences. We used both TALENs (transcription activator‐like effector nucleases) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) to introduce heritable base pair mutations in mature miRNA sequences. For rice, TALEN constructs were built targeting five different mature miRNA sequences and yielding heritable mutations. Among the resulting mutants, mir390 mutant showed a severe defect in the shoot apical meristem (SAM), a shootless phenotype, which could be rescued by the wild‐type MIR390. Small RNA sequencing showed the two base pair deletion in mir390 substantially interfered with miR390 biogenesis. In Arabidopsis, CRISPR/Cas9‐mediated editing of the miR160* strand confirmed that the asymmetric structure of miRNA is not a necessary determinant for secondary siRNA production. CRISPR/Cas9 with double‐guide RNAs successfully generated mir160a null mutants with fragment deletions, at a higher efficiency than a single‐guide RNA. The difference between the phenotypic severity of miR160a mutants in Col‐0 versus Ler backgrounds highlights a diverged role for miR160a in different ecotypes. Overall, we demonstrated that TALENs and CRISPR/Cas9 are both effective in modifying miRNA precursor structure, disrupting miRNA processing and generating miRNA null mutant plants.

Heritable Collagen Mutations Causing Varicose Veins in a Large Three-Generation Family

Introduction - Primary varicose veins (PVV) constitute an important both health and social problem. In developed countries their prevalence vary from 25 to 50% in women and from 7% to 40% in men and may lead to serious complications. To date, no specific cause for the development of varicose veins has been identified, even though a various environmental and genetic risk factors have been suggested to contribute to their formation. Epidemiological research highlighted a trend of familial clustering and associated increased risk of disease development.

Methods for the generation of heritable germline mutations in the disease vector Culex quinquefasciatus using clustered regularly interspaced short palindrome repeats‐associated protein 9

Culex quinquefasciatus is a vector of many diseases that adversely impact human and animal health; however, compared to other mosquito vectors limited genome engineering technologies have been characterized for this vector. Clustered regularly interspaced short palindrome repeats-associated protein 9 (CRISPR-Cas9) based technologies are a powerful tool for genome engineering and functional genetics and consequently have transformed genetic studies in many organisms. Our objective was to improve upon the limited technologies available for genome editing in C. quinquefasciatus to create a reproducible and straightforward method for CRISPR-Cas9-targeted mutagenesis in this vector. Here we describe methods to achieve high embryo survival and mutagenesis rates and we provide details on the injection supplies and procedures, embryo handling and guide RNA (gRNA) target designs. Through these efforts, we achieved embryo survival rates and germline mutagenesis rates that greatly exceed previously reported rates in this vector. This work is also the first to characterize the white gene marker in this species, which is a valuable phenotypic marker for future transgenesis or mutagenesis of this vector. Overall, these tools provide the framework for future functional genetic studies in this important disease vector and may support the development of future gene drive and genetic technologies that can be used to control this vector.

Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish.

Inconsistent activity limits the use of CRISPR-Cas9 in zebrafish. We show supernumerary guanine nucleotides at the 5' ends of single guide RNAs (sgRNAs) account for diminished CRISPR-Cas9 activity in zebrafish embryos. Genomic sequences can be targeted consistently with extremely high efficiency using Cas9 ribonucleoproteins (RNPs) containing either a sgRNA molecule or a synthetic crRNA:tracrRNA duplex that perfectly matches the protospacer target site. Following injection of zebrafish eggs with such RNPs, virtually every copy of a targeted locus harbors an induced indel mutation. Loss of gene function is often complete, as F0 embryos closely resemble true null mutants without detectable non-specific effects. Mosaicism is sufficiently low in F0 embryos that cell non-autonomous gene functions can be probed effectively and redundant activities of genes can be uncovered when two genes are targeted simultaneously. Finally, heritable deletion mutations of at least 50 kbp can be readily induced using pairs of duplex guide RNPs targeted to a single chromosome.

Steroid Receptor Coactivator-1 Expression in Pheochromocytoma: Clinicopathologic Correlation and Potential Diagnostic Pitfall.

Pheochromocytoma is a relatively uncommon tumor, and the histomorphologic and biochemical features that may portend malignant behavior have poor overall consensus across various proposed classification systems. Steroid receptor coactivator-1 (SRC-1) is a nuclear protein that mediates transcriptional activity. Current diagnostic applications of SRC-1 are limited, and include distinguishing adrenocortical carcinoma (ACC) from renal cell carcinoma, and other mimickers. SRC-1 expression in pheochromocytoma has not been previously studied. Pheochromocytoma cases were retrieved from our Urological Pathology database and expert consultation files of the senior author, from 2015 to 2019. Clinicopathological data were obtained. SRC-1 expression was scored systematically. Thirty-eight cases were included, with a female predominance, and a mean age of 52 years (range, 16 to 75 y). Seven patients had heritable mutations including RET (n=3), VHL (2), SDHB (1), and ATM and PDGFRA (1). Two patients developed clinical metastasis, who individually had ATM and PDGFRA mutations, and SDHB p.V140F mutation. All heritable tumors were positive for SRC-1, including diffuse/strong staining and intensity in the VHL cases, and diffuse staining with variable intensity in RET cases. Diffuse positivity was seen in most of our heritable cases, providing evidence for a putative link between RET and downstream SRC-1 signaling. An inverse relationship was observed between SRC-1 expression and Pheochromocytoma of the Adrenal Gland Scaled Score/tumor size, suggesting that SRC-1 phenotype may become muted in pheochromocytomas that have malignant potential. SRC-1 expression in aggressive pheochromocytomas, may also be a potential diagnostic pitfall in view of the fact that these tumors may be misinterpreted as ACC in the primary or metastatic setting.

Alpha-synuclein stepwise aggregation reveals features of an early onset mutation in Parkinson\u2019s disease

Amyloid formation is a process involving interconverting protein species and results in toxic oligomers and fibrils. Aggregated alpha-synuclein (αS) participates in neurodegenerative maladies, but a closer understanding of the early αS polymerization stages and polymorphism of heritable αS variants is sparse still. Here, we distinguished αS oligomer and protofibril interconversions in Thioflavin T polymerization reactions. The results support a hypothesis reconciling the nucleation-polymerization and nucleation-conversion-polymerization models to explain the dissimilar behaviors of wild-type and the A53T mutant. Cryo-electron microscopy with a direct detector shows the polymorphic nature of αS fibrils formed by heritable A30P, E46K, and A53T point mutations. By showing that A53T rapidly nucleates competent species, continuously elongates fibrils in the presence of increasing amounts of seeds, and overcomes wild-type surface requirements for growth, our findings place A53T with features that may explain the early onset of familial Parkinson’s disease cases bearing this mutation.

34.1 GENETIC MUTATIONS AND ENVIRONMENTAL FACTORS THAT PROMOTE ADVERSE NEURODEVELOPMENTAL OUTCOMES IN PRECLINICAL MODELS

Developmental exposure to environmental neurotoxicants, such as polychlorinated biphenyls (PCBs), is implicated in the pathogenesis of neurodevelopmental disorders (NDDs). Mechanistic studies have shown that PCBs alter calcium-dependent signaling pathways linked to activity-dependent dendritic growth. Mutations in calcium signaling molecules are associated with increased NDD risk, so we are using mouse models to test the hypothesis that heritable mutations that alter the fidelity of calcium signals influence neurodevelopmental outcomes after developmental PCB exposure.

Morphological, cytological and genetic analyzes of the 'sango' mutant with the defects in basidiocarp development in edible mushroom Pleurotus pulmonarius.

A spontaneous, morphological variation 'sango' was observed in the progeny of a Pleurotus pulmonarius (Fr.) Quél. wild-type basidiocarp (also known as fruiting body) collected from the field. This variant developed wart- and coral-like structures instead of normal basidiocarps. Microscopic analysis showed that the sango phenotype had defects in the differentiation of the pileus and hymenium. Basidiocarp phenotypic data analysis in the progenies revealed that the sango trait is a heritable mutation character controlled by a single recessive gene. This mutation locus was mapped on linkage group III of a previously constructed genetic linkage map by amplified fragment length polymorphism (AFLP) technique in P. pulmonarius. Four AFLP markers identified by bulked segregant analysis showed linkage to the sango mutation locus, with the genetic distance ranging from 0 to 2.1 cM. Of these markers, one marker was co-segregated with the sango mutation locus. This knowledge will be a useful foundation for practical breeding as well as for elucidating molecular mechanisms in basidiocarp development of main edible mushrooms.

Protozoan persister-like cells and drug treatment failure.

Antimicrobial treatment failure threatens our ability to control infections. In addition to antimicrobial resistance, treatment failures are increasingly understood to derive from cells that survive drug treatment without selection of genetically heritable mutations. Parasitic protozoa, such as Plasmodium species that cause malaria, Toxoplasma gondii and kinetoplastid protozoa, including Trypanosoma cruzi and Leishmania spp., cause millions of deaths globally. These organisms can evolve drug resistance and they also exhibit phenotypic diversity, including the formation of quiescent or dormant forms that contribute to the establishment of long-term infections that are refractory to drug treatment, which we refer to as 'persister-like cells'. In this Review, we discuss protozoan persister-like cells that have been linked to persistent infections and discuss their impact on therapeutic outcomes following drug treatment.