Risk assessment and molecular mechanism of Sclerotium rolfsii resistance to boscalid.

Boscalid, a widely used SDHI fungicide, has been employed in plant disease control for over two decades. However, there is currently no available information regarding its antifungal activity against Sclerotium rolfsii and the potential risk of resistance development in this pathogen. In this study, we evaluated the sensitivity of 100 S. rolfsii strains collected from five different regions in China during 2018-2019 to boscalid using mycelial growth inhibition method and assessed the risk of resistance development. The EC50 values for boscalid ranged from 0.2994 µg/mL to 1.0766 µg/mL against the tested strains, with an average EC50 value of 0.7052 ± 0.1473 µg/mL. Notably, a single peak sensitivity baseline was curved, indicating the absence of any detected resistant strains. Furtherly, 10 randomly selected strains of S. rolfsii were subjected to chemical taming to evaluate its resistance risk to boscalid, resulting in the successful generation of six stable and inheritable resistant mutants. These mutants exhibited significantly reduced mycelial growth, sclerotia production, and virulence compared to their respective parental strains. Cross-resistance tests revealed a correlation between boscalid and flutolanil, benzovindiflupyr, pydiflumetofen, fluindapyr, and thifluzamide; however, no cross-resistance was observed between boscalid and azoxystrobin. Thus, we conclude that the development risk of resistance in S. rolfsii to boscalid is low. Boscalid can be used as an alternative fungicide for controlling peanut sclerotium blight when combined with other fungicides that have different mechanisms of action. Finally, the target genes SDHB, SDHC, and SDHD in S. rolfsii were initially identified, cloned and sequenced to elucidate the mechanism of S. rolfsii resistance to boscalid. Two mutation genotypes were found in the mutants: SDHD-D111H and SDHD-H121Y. The mutants carrying SDHD-H121Y exhibited moderate resistance, while the mutants with SDHD-D111H showed low resistance. These findings contribute to our comprehensive understanding of molecular mechanisms underlying plant pathogens resistance to SDHI fungicides.

Resistance risk assessment for benzovindiflupyr in Sclerotium rolfsii and transmission of resistance genes among population.

BACKGROUND: Sclerotium rolfsii is a destructive soil-borne fungal pathogen which is distributed worldwide. In previous study, the succinate dehydrogenase inhibitor (SDHI) fungicide benzovindiflupyr has been identified for its great antifungal activity against Sclerotium rolfsii. This study is aimed to investigate the resistance risk and mechanism of benzovindiflupyr in Sclerotium rolfsii. RESULTS: Eight stable benzovindiflupyr-resistant isolates were generated by fungicide adaptation. Although the obtained eight resistant isolates have a stronger pathogenicity than the parental sensitive isolate, they have a fitness penalty in the mycelial growth and sclerotia formation compared to the parental isolate. A positive cross-resistance existed in the resistant isolates between benzovindiflupyr and thifluzamide, carboxin, boscalid and isopyrazam. Three-point mutations, including SdhBN180D, SdhCQ68E and SdhDH103Y, were identified in the benzovindiflupyr-resistant isolates. However, molecular docking analysis indicated that only SdhDH103Y could influence the sensitivity of Sclerotium rolfsii to benzovindiflupyr. After mycelial co-incubation of resistant isolates and the sensitive isolate, resistance genes may be transmitted to the sensitive isolate. The in vivo efficacy of benzovindiflupyr and thifluzamide against benzovindiflupyr-resistant isolates was a little lower than that against the sensitive isolate but with no significant difference. CONCLUSION: The results suggested a low to medium resistance risk of Sclerotium rolfsii to benzovindiflupyr. However, once resistance occurs, it is possible to spread in the population of Sclerotium rolfsii. This study is helpful to understanding the risk and mechanism of resistance to benzovindiflupyr in multinucleate pathogens such as Sclerotium rolfsii. © 2024 Society of Chemical Industry.

Resistance to pydiflumetofen in Botrytis cinerea: risk assessment and detection of point mutations in sdh genes that confer resistance.

BACKGROUND: Gray mold caused by Botrytis cinerea Pers. is one of the most significant airborne diseases. It can infest a wide range of crops, causing significant losses in yield and quality worldwide. Pydiflumetofen, a new generation succinate dehydrogenase inhibitor (SDHI), is currently being registered in China to control gray mold in a variety of crops. The baseline sensitivity, resistance risk, and resistance mechanism of Botrytis cinerea to pydiflumetofen were assessed in this study. RESULTS: A total of 138 strains of B. cinerea from 10 different regions were tested for their sensitivity to pydiflumetofen, and the mean EC50 value was 0.0056 µg mL-1 . Eight mutants were obtained by fungicide adaption from five sensitive parental isolates, and the resistance factor (RF) ranged from 51 to 135. The mutants exhibited strong adaptive traits in conidial production, conidial germination, and pathogenicity. Positive cross-resistance was only observed between other SDHIs (i.e. boscalid, fluopyram, and isopyrazam). Two different types of pydiflumetofen-resistant mutants were identified: point mutation P225L in sdhB and double mutation G85A and I93V in sdhC. The in vivo control efficacy of pydiflumetofen on the resistant mutants carrying P225L in sdhB as well as G85A and I93V in sdhC was significantly decreased to 52.62% and 32.27%, respectively. CONCLUSION: The fitness was significantly higher for all pydiflumetofen-resistant mutants than the corresponding parental. Two types of point mutations, sdhB-P225L and sdhC-G85A and I93V, might confer resistance to pydiflumetofen in B. cinerea. A precautionary resistance management strategy should be implemented. © 2021 Society of Chemical Industry.

Sensitivity assessment and SDHC-I86F mutation frequency of Phakopsora pachyrhizi populations to benzovindiflupyr and fluxapyroxad fungicides from 2015 to 2019 in Brazil.

BACKGROUND: Fungicides of the succinate dehydrogenase inhibitors (SDHIs) group have been used in soybean to control Asian soybean rust (ASR) caused by Phakopsora pachyrhizi. Fungal populations with less sensitivity to SDHI fungicides have been reported since 2015. RESULTS: In this study, fungal sensitivity to benzovindiflupyr (BZV) and fluxapyroxad (FXD) was assessed using a total of 770 P. pachyrhizi populations sampled over four soybean growing seasons. Cross-resistance, intrinsic activity, and frequency of SDHC-I86F mutation were also analyzed. The average effective concentration to inhibit 50% (EC50 ) and SDHC-I86F frequency increased over the 2015/2016, 2016/2017, 2017/2018 and 2018/2019 soybean-seasons. Fourteen P. pachyrhizi populations had the EC50 value above 10 mg L-1 for both carboxamides. No difference was found in intrinsic active to BZV and FXD fungicides for sensitive P. pachyrhizi populations. For P. pachyrhizi classified as less sensitive BZV showed the highest fungitoxicity effect. High frequency of the C-I86F mutation was observed in samples collected in volunteer soybean plants. The maximum frequency of SDHC-I86F mutation in the population was 50% and resulting in ASR populations with low sensitivity to SDHIs. A low correlation between bioassay and SDHC-I86F mutation was observed possible due to the dikaryotic nature of rust fungi or other mutations in the other succinate dehydrogenase subunits. CONCLUSION: The present work provides an overview of a large sampling size of P. pachyrhizi populations and their performance over the four crop seasons. The high frequency of SDHC-I86F mutation and low sensitivity to SDHIs are widely distributed in the main soybean growing regions in Brazil and present in volunteer plants in the soybean-free period. Further detailed studies are needed to identify novel point mutations affecting the effectiveness of SDHIs. © 2021 Society of Chemical Industry.

Field Assessment of Six Point-Mutations in SDH Subunit Genes Conferring Varying Resistance Levels to SDHIs in Clarireedia spp.

Dollar spot, caused by Clarireedia spp. (formerly Sclerotinia homoeocarpa F.T. Bennett), is the most economically important turfgrass disease causing considerable damage on golf courses. While cultural practices are available for reducing dollar spot infection, chemical fungicide use is often necessary for maintaining optimal turf quality. Since the release of boscalid in 2003, the succinate dehydrogenase inhibitor (SDHI) class has become an invaluable tool for managing dollar spot. However, resistance to this class has recently been reported in Clarireedia spp. and many other plant pathogenic fungi. After SDHI field failure on four golf courses and one university research plot, a total of six unique SDH mutations conferring differential in vitro sensitivities to SDHIs have been identified in Clarireedia spp. In 2018 and 2019, turf research plots were inoculated with sensitive, non-mutated isolates of Clarireedia spp., as well as resistant isolates harboring each unique identified mutation. Fungicide efficacy trials were conducted on inoculated plots to assess differential sensitivity to five SDHI active ingredients (boscalid, fluxapyroxad, isofetamid, fluopyram, and pydiflumetofen) across mutations under field conditions. Results indicate unique mutations are associated with distinct SDHI field efficacy profiles as shown in in-vitro sensitivity assays. Isolate populations with B subunit mutations (H267Y/R) were more sensitive to fluopyram, whereas isolate populations with C subunit mutations (C-G91R, C-G150R) showed resistance to all SDHIs tested. Mutation-associated differential sensitivity observed under field conditions indicates a need for a nation-wide survey and frequent monitoring of SDHI sensitivity of dollar spot populations on golf courses in the USA. Further, the information gained from this study will be useful in providing sustainable management recommendations for controlling site-specific resistant populations of Clarireedia spp.

Site-directed mutagenesis of the succinate dehydrogenase subunits B and D from Corynespora cassiicola reveals different fitness costs and sensitivities to succinate dehydrogenase inhibitors.

Carboxamide fungicides target succinate dehydrogenase (SDH). Recently published monitoring studies have shown that Corynespora cassiicola isolates are resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B and D subunits. We confirmed, by site-directed mutagenesis of the sdhB and sdhD genes, that each of the mutations identified in the field strains of C. cassiicola conferred resistance to boscalid and, in some cases, cross-resistance to other SDHIs (fluopyram, carboxin and penthiopyrad). Analyses of the enzyme activity and sdhB and sdhD gene expression show that modifications (SdhB_H278Y and SdhD_H105R) that result in a decline in SDH enzyme activity may be complemented by gene overexpression. The SdhB_H278Y, SdhB_I280V and SdhD_H105R mutants suffered large fitness penalties based on their biological properties, including conidia production and germination, mycelial growth, pathogenicity or survival abilities under environment stress. However, fitness cost was not found in the SdhB_H278R, SdhD_D95E and SdhD_G109V mutants. In the evaluation of resistance to boscalid in 2018 and 2019, the frequency of the SdhD_D95E and SdhD_G109V genotypes in the Liaoning and Shandong provinces changed dramatically compared with 2005-2017, from low resistance frequency (0.53% for D95E and 2.53% for G109V) to dominant resistance frequency (17.28% for D95E and 15.38% for G109V). Considering both the fitness and increased frequency of these genotypes, we may infer that the SdhD_D95E and SdhD_G109V mutants will be the dominant resistance mutants in field.

Resistance risk assessment for a novel succinate dehydrogenase inhibitor pydiflumetofen in Fusarium asiaticum.

BACKGROUND: Fusarium asiaticum is one of predominant pathogens of Fusarium head blight (FHB) in China. Pydiflumetofen (Pyd) is a novel succinate dehydrogenase inhibitor (SDHI) which has been commercialized in China for the controlling of wheat FHB since 2019. In the current study, a risk assessment of the pydiflumetofen-resistance selected in Fusarium asiaticum was investigated. RESULTS: One PydMR mutant [resistance factor (RF) < 80] and four PydHR mutants (RF > 3000) were generated by fungicide-taming from 1000 mycelial discs of the wild-type strain 2021. Nucleotide sequences alignment results of FaSdh from the wild-type strain and resistant mutants showed that all the mutations were categorized into three genotypes, i.e. FaSdhBH248Y from PydMR mutant, both FaSdhC1 A64V and FaSdhC1 R67K from PydHR mutants. All the resistant mutants possessed no fitness penalty based on the data of mycelial linear growth, conidiation and virulence. In addition, the FaSdhC1 A64V mutants showed positive cross-resistance between pydiflumetofen and boscalid or thifluzamide, but no cross-resistance between pydiflumetofen and Y13149 or Y12196, while the FaSdhC1 R67K mutants exhibited positive cross-resistance between pydiflumetofen and boscalid, thifluzamide or Y12196, and no cross-resistance between pydiflumetofen and Y13149. Furthermore, positive cross-resistance between the five tested SDHIs was detected in the FaSdhBH248Y mutants. CONCLUSION: The results suggest a moderate to high resistance risk of F. asiaticum to pydiflumetofen, and provide essential data for monitoring the emergence of resistance and resistance management strategies for pydiflumetofen, which will be useful for scientific application of this fungicide in China.

Two for the price of one: Attacking the energetic-metabolic hub of mycobacteria to produce new chemotherapeutic agents.

Cellular bioenergetics is an area showing promise for the development of new antimicrobials, antimalarials and cancer therapy. Enzymes involved in central carbon metabolism and energy generation are essential mediators of bacterial physiology, persistence and pathogenicity, lending themselves natural interest for drug discovery. In particular, succinate and malate are two major focal points in both the central carbon metabolism and the respiratory chain of Mycobacterium tuberculosis. Both serve as direct links between the citric acid cycle and the respiratory chain due to the quinone-linked reactions of succinate dehydrogenase, fumarate reductase and malate:quinone oxidoreductase. Inhibitors against these enzymes therefore hold the promise of disrupting two distinct, but essential, cellular processes at the same time. In this review, we discuss the roles and unique adaptations of these enzymes and critically evaluate the role that future inhibitors of these complexes could play in the bioenergetics target space.

Resistance risk assessment for Fusarium graminearum to pydiflumetofen, a new succinate dehydrogenase inhibitor.

BACKGROUND: Pydiflumetofen is a new generation succinate dehydrogenase inhibitor currently undergoing the process of registration in China for the control of Fusarium head blight in wheat. A resistance risk assessment of Fusarium graminearum to pydiflumetofen was undertaken in this study. RESULTS: A total of 75 pydiflumetofen-resistant mutants were generated through spontaneous selection and displayed high resistance with an average resistance factor (RF) value of 78. Four mutants were generated through UV mutagenesis and displayed very high resistance with an RF value >1000. The sequence analysis results for Sdh genes and fitness studies revealed the existence of four types of mutations. In particular, 32 spontaneous selection mutants (SP mutants) had an arginine (R) to histidine (H) transition at position 86 in FGSdhC, resulting in seriously reduced fitness. Seven SP mutants had an R to cysteine (C) transition at position 86 in FGSdhC, resulting in reduced fitness. Thirty-six SP mutants had an alanine (A) to valine (V) transition at position 83 in FGSdhC and had no fitness penalties. The efficacy of pydiflumetofen towards a mutant carrying A83V in FGSdhC in vivo was significantly decreased at 42.7%. Four UV mutants had no mutations on all Sdh genes and no fitness penalties. Cross-resistance among boscalid, fluopyram and pydiflumetofen was observed. CONCLUSION: Sdhc mutations were found and other target site resistance may be present in laboratory PR mutants of F. graminearum. An overall moderate risk of resistance development in F. graminearum was recommended for pydiflumetofen. © 2019 Society of Chemical Industry.

Secreted Flavin Cofactors for Anaerobic Respiration of Fumarate and Urocanate by Shewanella oneidensis: Cost and Role.

Shewanella oneidensis strain MR-1, a facultative anaerobe and model organism for dissimilatory metal reduction, uses a periplasmic flavocytochrome, FccA, both as a terminal fumarate reductase and as a periplasmic electron transfer hub for extracellular respiration of a variety of substrates. It is currently unclear how maturation of FccA and other periplasmic flavoproteins is achieved, specifically in the context of flavin cofactor loading, and the fitness cost of flavin secretion has not been quantified. We demonstrate that deletion of the inner membrane flavin adenine dinucleotide (FAD) exporter Bfe results in a 23% slower growth rate than that of the wild type during fumarate respiration and an 80 to 90% loss in fumarate reductase activity. Exogenous flavin supplementation does not restore FccA activity in a Δbfe mutant unless the gene encoding the periplasmic FAD hydrolase UshA is also deleted. We demonstrate that the small Bfe-independent pool of FccA is sufficient for anaerobic growth with fumarate. Strains lacking Bfe were unable to grow using urocanate as the sole electron acceptor, which relies on the periplasmic flavoprotein UrdA. We show that periplasmic flavoprotein maturation occurs in careful balance with periplasmic FAD hydrolysis, and that the current model for periplasmic flavin cofactor loading must account for a Bfe-independent mechanism for flavin transport. Finally, we determine that the metabolic burden of flavin secretion is not significant during growth with flavin-independent anaerobic electron acceptors. Our work helps frame the physiological motivations that drove evolution of flavin secretion by ShewanellaIMPORTANCEShewanella species are prevalent in marine and aquatic environments, throughout stratified water columns, in mineral-rich sediments, and in association with multicellular marine and aquatic organisms. The diversity of niches shewanellae can occupy are due largely to their respiratory versatility. Shewanella oneidensis is a model organism for dissimilatory metal reduction and can respire a diverse array of organic and inorganic compounds, including dissolved and solid metal oxides. The fumarate reductase FccA is a highly abundant multifunctional periplasmic protein that acts to bridge the periplasm and temporarily store electrons in a variety of respiratory nodes, including metal, nitrate, and dimethyl sulfoxide respiration. However, maturation of this central protein, particularly flavin cofactor acquisition, is poorly understood. Here, we quantify the fitness cost of flavin secretion and describe how free flavins are acquired by FccA and a homologous periplasmic flavoprotein, UrdA.

Simple, reliable, and time-efficient colorimetric method for the assessment of mitochondrial function and toxicity.

Mitochondria are organelles involved in the production of cellular energy, regulation of Ca2+ and redox signaling, and are critical for normal functioning of eukaryotic cells. The dysfunction of mitochondria has been implicated in a wide range of diseases, including metabolic and neurodegenerative disorders and different types of cancers. To better understand the role of mitochondria in healthy and disease states, the development of efficient and reliable tools for the assessment of mitochondrial function is particularly important. Janus green B (JG-B) is a supravital lipophilic cationic dye which, in its oxidized form, has a green-blue color. As JG-B is taken up and reduced by metabolically active mitochondria, the dye has been used for assessing the purity, integrity and metabolic activity of mitochondria with microscopy-based methods. Here we present a simple, time- and cost-efficient JG-B-based colorimetric assay for assessing mitochondrial function, activity and toxicity. The method is based upon reduction of JG-B by mitochondrial dehydrogenases to diethylsafranine, which is pink colored and has a maximum absorption at 550 nm. In this proof of principle study, using in vitro mitochondrial preparations isolated from rat brain, we provide evidence that monitoring JG-B conversion to diethylsafranine can be used as a reliable and robust indicator of mitochondrial activity and toxicity. Because of its simplicity and efficiency in terms of costs and time, this assay has a wide potential in analytical as well as therapeutic areas of biomedical research.

Assessment of liver antioxidant status and mitochondrial membrane composition of Plasmodium berghei-infected mice treated with selected antimalarials.

The present study was aimed at determining and comparing the effects of Artecxin (ART), P - Alaxin (P-ALA), Lonart (LON) and Chloroquine (CQ) on oxidative stress parameters and mitochondrial membrane composition in the course of malaria infection. Six groups of five mice each categorized as healthy control (non-parasitized non-treated group), parasitized-non-treated (PnT), parasitized-chloroquine-treated (positive control), parasitized-Artecxin, -Lonart and -P-Alaxin-treated groups were used for the study. Hepatic antioxidant status was assessed with levels of malondialdehyde (MDA) and reduced glutathione (GSH) as well as activity of superoxide dismutase (SOD) and catalase (CAT) in the post mitochondrial and mitochondrial fractions. Mitochondrial membrane integrity was also evaluated with activity of succinate dehydrogenase and levels of phospholipids, cholesterol and proteins in the liver mitochondria. Results revealed that treatment of parasitized mice with the antimalarial drugs significantly (p<0.05) decreased hepatic malondialdehyde (MDA) and mitochondrial membrane phospholipids compared to parasitized untreated group. On the other hand, significantly (p<0.05) elevated succinate dehydrogenase (SDH) activity, mitochondrial membrane cholesterol level, GSH concentration, catalase (CAT) and superoxide dismutase (SOD) activity in the post mitochondrial fraction were obtained. Thus, antimalarial drugs distort mitochondrial membrane integrity and electron transfer but reduce the malaria-induced oxidative stress on the host.

Risk assessment of maternally inherited SDHD paraganglioma and phaeochromocytoma.

BACKGROUND: Germline mutations in the SDHD tumour suppressor gene (11q23.1) predispose to phaeochromocytomas and paragangliomas (PPGL) mainly on a paternal transmission. However, PPGL have been recently reported in three carriers of a maternally inherited SDHD mutation. OBJECTIVE: To assess the risk of PPGL occurrence on maternal transmission of SDHD mutation. METHODS: Pedigrees of 80 SDHD-related families have been reviewed. 35 asymptomatic subjects carrying a maternally transmitted SDHD mutation were identified. 20 of them accepted to benefit from a PPGL imaging screening. RESULTS: A unique histologically proven biochemically negative phaeochromocytoma has been diagnosed in a 35-year-old woman. Molecular investigations carried out on tumour tissue revealed that the loss of heterozygosity encompassed the paternally derived q arm and the maternally derived p arm of chromosome 11. CONCLUSIONS: This study demonstrates that the risk of developing PPGL for a subject carrying a germline SDHD mutation on the maternal allele remains a rare scenario but does exist. Our data suggest an adjustment of current genetic counselling and clinical care recommendations for at-risk subjects. A targeted familial genetic test should be proposed from the age of 18 years to every subject having a mother carrying a germline SDHD mutation and a first medical workup, including imaging, should be recommended to SDHD-positive mutation carriers.

Fitness measurement reveals contrasting costs in homologous recombinant mutants of Botrytis cinerea resistant to succinate dehydrogenase inhibitors.

Botrytis cinerea is a pathogenic ascomycete fungus that causes gray mold on many crops. Chemical control remains the principal method for curbing this disease. However, fungicide efficacy may be compromised by the selection of resistant strains. Assessments of the fitness of resistant strains is important, to evaluate the risk of their establishment in populations. Strains resistant to boscalid, the first succinate dehydrogenase inhibitor (SDHI) registered for the treatment of gray mold on grapevine in France, have recently been detected in the field. Most of these strains harbor mutations of the sdhB gene, encoding subunit B of SDH. In this study, we used sdhB recombinant mutants to investigate the impact of mutations conferring SDHI resistance on the fitness of B. cinerea. We have shown that sdhB mutations (except for the sdhB(H272Y) mutation) affect SDH activity and respiration rate. Our results suggest that different sdhB mutations have different effects on fitness. In particular, mutants displaying an inhibition of SDH activity do not suffer the same effects on fitness. We discuss the results in the context of mutant frequencies in field populations and the possible occurrence of compensatory mechanisms that modulate fitness losses.

Usefulness of Succinate dehydrogenase B (SDHB) immunohistochemistry in guiding mutational screening among patients with pheochromocytoma-paraganglioma syndromes.

Genetic testing of pheochromocytomas (PCC) and paragangliomas (PGL), although expensive, is gradually becoming a part of the routine laboratory investigation for patients with PCC-PGL syndrome. Recently, Succinate dehydrogenase B (SDHB) immunochemistry has been shown to be an excellent indicator of germline mutations in the SDH genes and could help significantly reduce cost. This study assesses the utility of SDHB immunohistochemical analysis when used to guide genetic analysis, with emphasis on cost benefits it could provide in a resource-limited setting. Forty-four cases of PCC/PGL characterized by genetic analysis were included to determine their SDHB expression pattern by immunohistochemistry. SDHB antibody expression was negative among three cases each, with SDHB and SDHD mutations. Immunohistochemistry results were positive for all three cases of RET, a single case of neurofibromatosis and for two cases with Von Hippel-Lindau (VHL) mutations while the remaining two cases with VHL mutations showed a diffuse 'cytoplasmic blush'. Thirty of the remaining 31 samples demonstrated positive staining and were negative for mutations, while a lone sample that was negative for staining and mutation was not included in the final analysis as the internal control for the sample was not adequately stained. Cost analysis in our settings showed that triaging with SDHB immunohistochemistry could potentially reduce costs by USD 320-500 per patient. SDHB immunohistochemistry, when used as a guide to genetic testing, can significantly reduce the effort, time and costs of testing among patients with PCC-PGL, a huge benefit in resource limited settings.

Usefulness of negative and weak-diffuse pattern of SDHB immunostaining in assessment of SDH mutations in paragangliomas and pheochromocytomas.

This is a confirmatory study about usefulness of SDHB and SDHA immunostaining in assessment of SDH mutations in paragangliomas and pheochromocytomas. Paraganglioma/pheochromocytoma syndrome (PGL/PCC syndrome) consists of different entities, associated with germline mutations in five different genes: SDHD, SDHAF2, SDHC, SDHA and SDHB. It has been suggested that negative immunostaining of SDHB can be taken as an indicator of the presence of a mutation in one of the five SDH genes. We have performed SDHB and SDHA immunohistochemical staining in a series of paragangliomas and pheochromocytomas from 64 patients. The patients had been previously checked for mutations in SDHD, SDHC and SDHB, but also for mutation in RET and VHL. All 14 patients with SDH mutations (9 with SDHB and 5 with SDHD mutations) exhibited negative or weak-diffuse SDHB staining pattern in tumour tissue, whereas cells of the 23 RET mutated and 8 VHL mutated tumours showed a positive SDHB immunostaining. Sixteen of the patients that did not exhibit a mutation in any gene showed positive SDHB immunostaining in tumour tissue, while only three of the patients without mutation exhibited negative staining. All patients exhibited positive pattern of SDHA immunostaining. The results confirm the value of SDHB immunohistochemical status in assessment of germline mutations in PGL/PCC syndrome.

A comprehensive next generation sequencing-based genetic testing strategy to improve diagnosis of inherited pheochromocytoma and paraganglioma.

CONTEXT: Pheochromocytomas and paragangliomas are notable for a high frequency of inherited cases, many of which present as apparently sporadic tumors. OBJECTIVE: The objective of this study was to establish a comprehensive next generation sequencing (NGS)-based strategy for the diagnosis of patients with pheochromocytoma and paraganglioma by testing simultaneously for mutations in MAX, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL. DESIGN: After the methodology for the assay was designed and established, it was validated on DNA samples with known genotype and then patients were studied prospectively. SETTING: The study was performed in a diagnostic genetics laboratory. PATIENTS: DNA samples from 205 individuals affected with adrenal or extraadrenal pheochromocytoma/head and neck paraganglioma (PPGL/HNPGL) were analyzed. A proof-of-principle study was performed using 85 samples known to contain a variant in 1 or more of the genes to be tested, followed by prospective analysis of an additional 120 samples. MAIN OUTCOME MEASURES: We assessed the ability to use an NGS-based method to perform comprehensive analysis of genes implicated in inherited PPGL/HNPGL. RESULTS: The proof-of-principle study showed that the NGS assay and analysis gave a sensitivity of 98.7%. A pathogenic mutation was identified in 16.6% of the prospective analysis cohort of 120 patients. CONCLUSIONS: A comprehensive NGS-based strategy for the analysis of genes associated with predisposition to PPGL and HNPGL was established, validated, and introduced into diagnostic service. The new assay provides simultaneous analysis of 9 genes and allows more rapid and cost-effective mutation detection than the previously used conventional Sanger sequencing-based methodology.

Evaluation of SDHB, SDHD and VHL gene susceptibility testing in the assessment of individuals with non-syndromic phaeochromocytoma, paraganglioma and head and neck paraganglioma.

OBJECTIVES: Research studies have reported that about a third of individuals with phaeochromocytoma/paraganglioma (PPGL) have an inherited predisposition, although the frequency of specific mutations can vary between populations. We evaluated VHL, SDHB and SDHD mutation testing in cohorts of patients with non-syndromic PPGL and head and neck paraganglioma (HNPGL). DESIGN: Prospective, observational evaluation of NHS practice. PATIENTS: Individuals with PPGL/HNPGL referred to a supraregional genetics testing service over a 10-year period. MEASUREMENTS: Clinical (age, tumour site, malignancy, etc.), mutation frequencies and characteristics. RESULTS: A total of 501 probands with PPGL (n = 413) or HNPGL (n = 88) were studied. Thirty-one percent of patients with PPGL presented had a pathogenic mutation in SDHB, SDHD or VHL. Mutation detection rates were highest in those with a positive family history (62%), malignancy (53%), multiple tumours (33%) or PGL (44%). Twenty-eight percent of individuals with a single sporadic phaeochromocytoma had a mutation. Overall, 63% of patients with HNPGL had a mutation (92% of those with a family history, 89% of those with multicentric tumours and 34% of those with a single sporadic HNPGL). Penetrance was calculated in 121 SDHB mutation-positive probands and 187 of their mutation-positive relatives. Most relatives were asymptomatic and lifetime penetrance in non-proband SDHB mutation carriers was <50%. CONCLUSIONS: Practice-based evaluations of genetic testing in PPGL reveal high mutation detection rates. Although clinical criteria can be used to prioritize mutation testing, mutations were detected in 'low risk groups' indicating a need for comprehensive and inexpensive genetic testing strategies for PPGL and HNPGL.

Somatic mutation analysis of the SDHB, SDHC, SDHD, and RET genes in the clinical assessment of sporadic and hereditary pheochromocytoma.

Systemic analysis of somatic mutations of other susceptibility genes in syndromic tumors as well as apparently sporadic tumors in well-characterized specimens is lacking. Its clinical relevance has not been studied. Our objective was to determine the frequency of second allele inactivation in syndromic tumors and determine the frequency and potential clinical impact of somatic mutations and loss of heterozygosity (LOH) of the known susceptibility genes in syndromic and sporadic tumors. Nine tumor specimens from clinically characterized VHL mutation, five from SDHB mutation, four from SDHD mutation, two from RET mutation carriers, and eight from apparently sporadic cases were analyzed. Tumor DNA mutation screening of the SDHx, VHL, and RET genes and LOH analyses of the SDHx and VHL genes were performed. The Yates-corrected chi-squared test was used for comparison of the clinical data and the molecular-genetic results. Second allele inactivation in tumors was identified in 83% of VHL, 80% of SDHB, and 50% of SDHD specimen. High prevalence of VHL (6/6, p=0.024) and SDHB (7/7, p=0.018) somatic mutations has been identified in the sporadic group compared to all others. In the group of the VHL tumors the SDHB somatic events were significantly lower (2/6; p=0.045). In 18/19 (95%) of cases, we were able to demonstrate the presence of at least two concomitant affected susceptibility genes. We conclude that LOH is the most prevalent second allele-inactivating event. SDHB and VHL somatic mutation might play a role in the sporadic forms of tumor development. There is no clinical impact of mutation screening or LOH analysis of tumor specimens.

Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat.

Chemical control of Septoria leaf blotch, caused by Mycosphaerella graminicola, is essential to ensure wheat yield and food security in most European countries. Mycosphaerella graminicola has developed resistance to several classes of fungicide and, with the efficacy of azoles gradually declining over time, new modes of action and/or improvements in host varietal resistance are urgently needed to ensure future sustainable disease control. Several new-generation carboxamide fungicides with broad-spectrum activity have recently been introduced into the cereal market. Carboxamides inhibit succinate dehydrogenase (Sdh) of the mitochondrial respiratory chain (complex II) but, because of their single-site specificity, these fungicides may be prone to resistance development. The objective of this study was to assess the risk of resistance development to different Sdh inhibitor (SDHI) fungicides in M. graminicola. UV mutagenesis was conducted to obtain a library of carboxin-resistant mutants. A range of SDHI resistance-conferring mutations was found in Sdh subunits B, C and D. Pathogenicity studies with a range of Sdh variants did not detect any fitness costs associated with these mutations. Most of the amino acid residues identified (e.g. B-S221P/T, B-H267F/L/N/Y, B-I269V and D-D129E/G/T) are directly involved in forming the cavity in which SDHI fungicides bind. Docking studies of SDHI fungicides in structural models of wild-type and mutated Sdh complexes also indicated which residues were important for the binding of different SDHI fungicides and showed a different binding for fluopyram. The predictive power of the model was also shown. Further diagnostic development, enabling the detection of resistant alleles at low frequencies, and cross-resistance studies will aid the implementation of anti-resistance strategies to prolong the cost-effectiveness and lifetime of SDHI fungicides.