SDHB Research Articles

Mutations seen among patients with pheochromocytoma and paraganglioma at a referral center from India.

Determining the mutational status of susceptibility genes including RET, VHL, SDHx (SDHB, SDHC, SDHD) among patients with pheochromocytoma/paraganglioma (PCC/PGL) is gaining importance. These genes have not been systematically characterized among patients with PCC/PGL from India. The aim of the work was to screen the most frequently mutated genes among patients with PCC/PGL to determine the frequency and spectrum of mutations seen in this region. Fifty patients with PCC/PGL treated at our tertiary care hospital between January 2010 and June 2012 were screened for mutations in susceptibility genes using an algorithmic approach. Thirty-two percent (16/50) of patients were found to be positive for mutations including mutations among RET (n=4), VHL (n=6), SDHB (n=3), and SDHD (n=3) genes. None of these patients were positive for SDHC mutations. A significant association was found between young patients with bilateral tumors and VHL mutations (p=0.002). Two of the 3 patients with extra-adrenal SDHB associated tumors, had unique mutations, viz., c.436delT (exon 5) and c.788_857del (exon 8), one of which was malignant. High frequency of mutations seen among patients in this study emphasizes the need to consider mutational analysis among Indian patients with PCC/PGL.

Analysis of pheochromocytomas/paragangliomas from Eastern Slovakia

This multi centre observational cohort study gives a view about the occurrence, clinical and laboratory presentation, localization, histological type and genetic background of pheochromocytoma (PHEO) and paraganglioma (PGL) in Eastern Slovakia. It included 28 patients (18 women + 10 men), of which 23 were diagnosed to have PHEO (82,1%) and 7 patients (25%) suffered from PGL with retroperitoneal, inguinal/pelvic and mediastinal distribution. Arterial hypertension was the major symptom present in 86 % with slight dominance of paroxysmal form (58%). In 3 cases (10,7%), the diagnosis was gained after differentiation of adrenal incidentaloma in asymptomatic patients. Five patients (17,8%) were classified to have malignant form of the disease. 9 patients (32,1%) were confirmed to have hereditary form - five of them (17,8%) with familiar medullar thyroid cancer (FMTC) and mutations in RET gene classified as multiple endocrine neoplasia 2A and 4 patients (14,3%) with germline mutations of SDHB gene, respectively. There was found a relatively high occurrence of other co-morbidities: thyroid disease in 20 patients (71,4%), impairment of glucose metabolism in 11 patients (39,3%) and apart from FMTC, 4 patients (14,3%) suffered also from other malignancy. Together with a bigger size of the primary tumor (6,6 cm), higher concentrations of metanephrines and prevalence of extra-adrenal tumors, malignant and hereditary forms, we suppose genetic and environmental factors of Eastern Slovakia may play a role in the etiopathogenesis of the tumors.

Universal Genetic Screening Uncovers a Novel Presentation of an SDHAF2 Mutation

Hereditary pheochromocytoma/paraganglioma (PC/PGL) accounts for up to 60% of previously considered sporadic tumors. Guidelines suggest that phenotype should guide genetic testing. Next-generation sequencing technology can simultaneously sequence 9 of the 18 known susceptibility genes in a timely, cost-efficient manner. Our aim was to confirm that universal screening is superior to targeted testing in patients with histologically confirmed PC and PGL. In two tertiary referral hospitals in Ireland, NGS was carried out on all histologically confirmed cases of PC/PGL diagnosed between 2004 and 2013. The following susceptibility genes were sequenced: VHL, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and MAX. A multiplex ligation-dependent probe amplification analysis was performed in VHL, SDHB, SDHC, SDHD, and SDHAF2 genes to detect deletions and duplications. A total of 31 patients were tested, 31% (n = 10) of whom were found to have a genetic mutation. Of those patients with a positive genotype, phenotype predicted genotype in only 50% (n = 5). Significant genetic mutations that would have been missed in our cohort by phenotypic evaluation alone include a mutation in TMEM127, two mutations in SDHAF2, and two mutations in RET. Target testing would have identified three of the latter mutations based on age criteria. However, 20% of patients (n = 2) would not have satisfied any criteria for targeted testing including one patient with a novel presentation of an SDHAF2 mutation. This study supports the value of universal genetic screening for all patients with PC/PGL.

Rare germline mutations identified by targeted next-generation sequencing of susceptibility genes in pheochromocytoma and paraganglioma.

Pheochromocytomas and paragangliomas have a highly diverse genetic background, with a third of the cases carrying a germline mutation in 1 of 14 identified genes. This study aimed to evaluate next-generation sequencing for more efficient genetic testing of pheochromocytoma and paraganglioma and to establish germline and somatic mutation frequencies for all known susceptibility genes. A targeted next-generation sequencing approach on an Illumina MiSeq instrument was used for a mutation analysis in 86 unselected pheochromocytoma and paraganglioma tumor samples. The study included the genes EGLN1, EPAS1, KIF1Bβ, MAX, MEN1, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL. RESULTS were verified in tumor and constitutional DNA with Sanger sequencing. In all cases with clinical syndromes or known germline mutations, a mutation was detected in the expected gene. Among 68 nonfamilial tumors, 32 mutations were identified in 28 of the samples (41%), including germline mutations in EGLN1, KIF1Bβ, SDHA, SDHB, and TMEM127 and somatic mutations in EPAS1, KIF1Bβ, MAX, NF1, RET, and VHL, including one double monoallelic EPAS1 mutation. Targeted next-generation sequencing proved to be fast and cost effective for the genetic analysis of pheochromocytoma and paraganglioma. More than half of the tumors harbored mutations in the investigated genes. Notably, 7% of the apparently sporadic cases carried germline mutations, highlighting the importance of comprehensive genetic testing. KIF1Bβ, which previously has not been investigated in a large cohort, appears to be an equally important tumor suppressor as MAX and TMEM127 and could be considered for genetic testing of these patients.

Fitness and Competitive Ability of Botrytis cinerea Field Isolates with Dual Resistance to SDHI and QoI Fungicides, Associated with Several sdhB and the cytb G143A Mutations

Respiration inhibitors such as the succinate dehydrogenase inhibitors (SDHIs) and the quinone outside inhibitors (QoIs) are fungicide classes with increasing relevance in gray mold control. However, recent studies have shown that dual resistance to both fungicide classes is a common trait in Botrytis cinerea populations from several hosts throughout the world. Resistance of B. cinerea to SDHIs is associated with several mutations in the sdhB, sdhC, and sdhD genes, while resistance to QoIs, in most cases, is associated with the G143A mutation in the cytb gene. The objective of the current study was to investigate the fitness and the competitive ability of B. cinerea field strains possessing one of the H272Y/R/L, N230I, or P225F sdhB substitutions and the G143A mutation of cytb. Fitness parameters measured were (i) mycelial growth and conidia germination in vitro, (ii) aggressiveness and sporulation capacity in vivo, (iii) sclerotia production in vitro and sclerotia viability under different storage conditions, and (iv) sensitivity to oxidative stress imposed by diquat treatments. The competitive ability of the resistant isolates was measured in the absence and presence of the SDHI fungicides boscalid and fluopyram selection pressure. The measurements of individual fitness components showed that the H272R/G143A isolates had the lower differences compared with the sensitive isolates. In contrast, the groups of H272Y/L/G143A, N230I/G143A, and P225F/G143A isolates showed reduced fitness values compared with the sensitive isolates. Isolates possessing only the cytb G143A substitution did not show any fitness cost. The competition experiments showed that, in the absence of fungicide selection pressure, after four disease cycles on apple fruit, the sensitive isolates dominated in the population in all the mixtures tested. In contrast, when the competition experiment was conducted under the selection pressure of boscalid, a gradual decrease in the frequency of sensitive isolates was observed, whereas the frequency of H272L and P225F isolates was increased. When the competition experiment was conducted in the presence of fluopyram, the sensitive isolates were eliminated even after the first disease cycle and the P225F mutants dominated in the population. Such results suggest that the sdhB mutations may have adverse effects on the mutants. The observed dominance of sensitive isolates in the competition experiments conducted in the absence of fungicides suggest that the application of SDHIs in alternation schemes may delay the selection or reduce the frequency of SDHI-resistant mutants.

Determinants of hemoglobin variability in stable peritoneal dialysis patients

Significant within-patient hemoglobin (Hb) level variability is well recognized in particularly hemodialysis patients. Several factors such as hospitalizations, intercurrent diseases and IV iron therapy are found to be related to Hb variability (Hb-var). In this observational study, we aimed to identify predictors and outcome of Hb-var in peritoneal dialysis (PD) patients without hospitalization, intercurrent disease and IV iron therapy during the study period. All patients were in the maintenance phase of short-acting erythropoiesis-stimulating agents (ESAs) therapy. The target range of Hb was 11-12 g/dL according to KDOQI Guidelines in 2007. The desired range of Hb was 11-12.5 g/dL. Patients' demographic and laboratory data were collected at baseline. Atherosclerotic disease was assessed using carotid intima-media thickness (CIMT). We assessed Hb variability with various methods using SD Hbmean, SD Hbrange and the velocity of Hb change. Hb deflectpositive, Hb deflectnegative, Hb values and ESA dosing were recorded monthly for 6 months. This study included 50 prevalent PD patients (mean age 46.9 ± 13.7 years, 25 women). The mean velocity of Hb change was negatively correlated with age and positively correlated with frequent ESA dose changes. Higher albumin and residual renal function (RRF) were also positively correlated with Hb deflectpositive. Patients with CIMT ≥0.7 cm had lower SD Hb range compared to CIMT <0.7 cm. Cumulative survival was better in patients with Hb levels consistently ≥10 g/dL compared to patients who had Hb <10 g/dL for at least 1 month. However, Hb-var was not associated with mortality. In PD patients without hospitalization, intercurrent disease(s) or IV iron therapy, young age, higher albumin or RRF and lower CIMT were associated with greater oscillations in response to ESA therapy. Careful and appropriate ESA dose changes considering these parameters could minimize Hb variability in these patients.

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 sdhBH272Y 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.

Molecular characterisation and detection of resistance to succinate dehydrogenase inhibitor fungicides in Botryotinia fuckeliana (Botrytis cinerea)

Succinate dehydrogenase inhibitors (SDHIs), interfering with fungal respiration, are considered to be fungicides at medium to high risk of resistance. Boscalid was the first molecule belonging to the SDHIs that was introduced for the control of Botryotinia fuckeliana. A range of different target-site mutations leading to boscalid resistance have been found in field populations of the fungus. The different types of mutation confer different cross-resistance profiles towards novel SDHIs, such as the recently introduced fungicide fluopyram. This study combines the determination of cross-resistance profiles and the setting-up of methods for fast molecular detection of the mutations. By means of in vitro tests, a range of SdhB mutations were characterised for resistance levels towards boscalid and fluopyram. SdhB mutations conferring P225L and P225F substitutions conferred high resistance to boscalid and high or moderate resistance to fluopyram respectively. Mutants carrying the N230I replacement were moderately resistant to both SDHIs. Substitutions at position H272 responsible for a high level of resistance to boscalid conferred sensitivity (H272R), hypersensitivity (H272Y) or moderate resistance (H272V) to fluopyram. Allele-specific (AS) PCR was developed and used for genotyping 135 B. fuckeliana isolates. The assay confirmed the strict association between resistance profiles and allelic variants of the SdhB gene. Real-time AS-PCR proved to be sensitive and specific for quantitative detection of different SDHI-resistant genotypes. Fluopyram-resistant mutants are currently rarely detected in the field sprayed with boscalid, but this may change with intensive exposure of the fungal population to fluopyram. PCR assays/methods developed in the study provide tools for fast monitoring of field populations and observing possible changes in population composition following fluopyram introduction, useful for the setting-up of appropriate preventive measures.

High‐resolution melting analysis for rapid detection and characterization of Botrytis cinerea phenotypes resistant to fenhexamid and boscalid

A novel, high‐resolution melting (HRM) analysis was developed to detect single nucleotide polymorphisms (SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty‐six single‐spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine (F) with serine (S) or valine (V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdhB gene resulted in the replacement of histidine (H) with arginine (R) or tyrosine (Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high‐resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B. cinerea populations.

Phenotypic Variability and Risk of Malignancy inSDHC-Linked Paragangliomas: Lessons From Three Unrelated Cases With an Identical Germline Mutation (p.Arg133*)

Mutations in the four subunits of succinate dehydrogenase (SDH) are the cause for the hereditary paraganglioma (PGL) syndrome types 1-4 and are associated with multiple and recurrent pheochromocytomas and PGLs. SDHC mutations most frequently result in benign, nonfunctional head-and neck PGLs (HNPGLs). The malignant potential of SDHC mutations remains unclear to date. We report a patient with malignant PGL carrying a SDHC mutation and compare her case with two others of the same genotype but presenting with classic benign HNPGLs. Loss of heterozygosity (LOH) was demonstrated in the malignant PGL tissue. In three unrelated patients referred for routine genetic testing, SDHB, SDHC, and SDHD genes were sequenced, and gross deletions were excluded by multiplex ligation-dependent probe amplification (MLPA). LOH was determined by pyrosequencing-based allele quantification and SDHB immunohistochemistry. In a patient with a nonfunctioning thoracic PGL metastatic to the bone, the lungs, and mediastinal lymph nodes, we detected the SDHC mutation c.397C>T predicting a truncated protein due to a premature stop codon (p.Arg133*). We demonstrated LOH and loss of SDHB protein expression in the malignant tumor tissue. The two other patients also carried c.397C>T, p.Arg133*; they differed from each other with respect to their tumor characteristics, but both showed benign HNPGLs. We describe the first case of a malignant PGL with distant metastases caused by a SDHC germline mutation. The present case shows that SDHC germline mutations can have highly variable phenotypes and may cause malignant PGL, although malignancy is probably rare.

The Adrenal Medulla and Extra-adrenal Paraganglia: Then and Now

The past 25 years have witnessed revolutionary changes in the care of patients with pheochromocytomas and extra-adrenal paragangliomas. Germline mutations of at least 13 genes are now associated with tumor development, a greater degree of hereditary susceptibility than for any other human neoplasm. Somatic mutations, either of the same genes or of several additional ones with closely related functions, are also increasingly recognized. Clinicians are now aware of the genetic implications of a pheochromocytoma or paraganglioma. All patients are therefore offered genetic testing and receive lifelong surveillance. Almost all of the mutated genes have well-described correlations with clinical and biochemical phenotypes. Tumors arising in patients with mutations of the SDHB gene have at least a 30 % chance of metastasizing and typically produce norepinephrine and/or dopamine. Assay of plasma-free metanephrines serves as a highly sensitive and specific biochemical screen for the presence of catecholamine-producing tumors, and the dopamine metabolite methoxytyramine serves as a useful marker for detecting minimally functional tumors or their metastases. New functional imaging techniques provide highly sensitive tumor localization. In addition to differential diagnosis, pathologists play new roles in helping to identify hereditary disease and guiding the sequence of genetic testing.

Site‐directed mutagenesis of the P225, N230 and H272 residues of succinate dehydrogenase subunit B from Botrytis cinerea highlights different roles in enzyme activity and inhibitor binding

Carboxamide fungicides target succinate dehydrogenase (SDH). Recent field monitoring studies have identified Botrytis cinerea isolates resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B subunit. We confirmed, by site-directed mutagenesis of the sdhB gene, that each of the mutations identified in field strains conferred resistance to boscalid in B.cinerea, and in some cases cross-resistance to other SDHIs (fluopyram, carboxin). Enzyme inhibition studies showed that the studied modifications (SdhB_P225T/L/F, N230I, H272Y/R/L) affected the inhibition of SDH activity by SDHIs, directly contributing to resistance. Our results confirm the importance of H272, P225 and N230 for carboxamide binding. Modifications of P225 and N230 conferred resistance to the four carboxamides tested (boscalid, fluopyram, carboxin, bixafen). Modifications of H272 had differential effects on the susceptibility of SDH to SDHIs. SdhB(H272L) , affected susceptibility to all SDHIs, SdhB(H272R) conferred resistance to all SDHIs tested except fluopyram, and SdhB(H272Y) conferred fluopyram hypersensitivity. Affinity-binding studies with radiolabelled fluopyram revealed strong correlations among the affinity of SDHIs for SDH, SDH inhibition and in vivo growth inhibition in the wild type. The sdhB(H272Y) mutation did not affect SDH and respiration activities, whereas all the other mutations affected respiration by decreasing SDH activity.

GSTT1 Copy Number Gain and ZNF Overexpression Are Predictors of Poor Response to Imatinib in Gastrointestinal Stromal Tumors

Oncogenic mutations in gastrointestinal stromal tumors (GISTs) predict prognosis and therapeutic responses to imatinib. In wild-type GISTs, the tumor-initiating events are still unknown, and wild-type GISTs are resistant to imatinib therapy. We performed an association study between copy number alterations (CNAs) identified from array CGH and gene expression analyses results for four wild-type GISTs and an imatinib-resistant PDGFRA D842V mutant GIST, and compared the results to those obtained from 27 GISTs with KIT mutations. All wild-type GISTs had multiple CNAs, and CNAs in 1p and 22q that harbor the SDHB and GSTT1 genes, respectively, correlated well with expression levels of these genes. mRNA expression levels of all SDH gene subunits were significantly lower (P≤0.041), whereas mRNA expression levels of VEGF (P=0.025), IGF1R (P=0.026), and ZNFs (P<0.05) were significantly higher in GISTs with wild-type/PDGFRA D842V mutations than GISTs with KIT mutations. qRT-PCR validation of the GSTT1 results in this cohort and 11 additional malignant GISTs showed a significant increase in the frequency of GSTT1 CN gain and increased mRNA expression of GSTT1 in wild-type/PDGFRA D842V GISTs than KIT-mutant GISTs (P=0.033). Surprisingly, all four malignant GISTs with KIT exon 11 deletion mutations with primary resistance to imatinib had an increased GSTT1 CN and mRNA expression level of GSTT1. Increased mRNA expression of GSTT1 and ZNF could be predictors of a poor response to imatinib. Our integrative approach reveals that for patients with wild-type (or imatinib-resistant) GISTs, attempts to target VEGFRs and IGF1R may be reasonable options.

The Succinate Dehydrogenase Genes of the Silkworm,&lt;i&gt; Bombyx mori&lt;/i&gt;

The succinate sehydrogenase (SDH) encoded by nuclear genome is a conjugating enzyme in the inner mitochondrial membrane. It is a junction for oxidative phosphorylation and the electron transport chain.As a part of complex II on the inner mt membranes, the SDH consists of four subunit proteins (A to D). We searched the genome sequence database of Bombyx mori with the Drosophila melanogaste and Nasonia vitripennis SDH by tBlastn and determined the structure of all putative B. mori genes of sdh by DNAMAN. The main results are as follows: The SdhAgene of B. mori contains 13 exons. The lengths of its cDNA is 2 880 bp at least. The SdhB gene of B. mori contains 1 exon only. The lengths of cDNA is 2 847 bp at least. The SdhC gene contains 4 exons. The lengths of cDNA is 714 bp at least. The SdhD gene contains 4 exons, too. The lenghts of cDNA is 1 976 bp at least.

Paragangliomas: Update on differential diagnostic considerations, composite tumors, and recent genetic developments

Recent developments in molecular genetics have expanded the spectrum of disorders associated with pheochromocytomas (PCCs) and extra-adrenal paragangliomas (PGLs) and have increased the roles of pathologists in helping to guide patient care. At least 30% of these tumors are now known to be hereditary, and germline mutations of at least 10 genes are known to cause the tumors to develop. Genotype-phenotype correlations have been identified, including differences in tumor distribution, catecholamine production, and risk of metastasis, and types of tumors not previously associated with PCC/PGL are now considered in the spectrum of hereditary disease. Important new findings are that mutations of succinate dehydrogenase genes SDHA, SDHB, SDHC, SDHD, and SDHAF2 (collectively "SDHx") are responsible for a large percentage of hereditary PCC/PGL and that SDHB mutations are strongly correlated with extra-adrenal tumor location, metastasis, and poor prognosis. Further, gastrointestinal stromal tumors and renal tumors are now associated with SDHx mutations. A PCC or PGL caused by any of the hereditary susceptibility genes can present as a solitary, apparently sporadic, tumor, and substantial numbers of patients presenting with apparently sporadic tumors harbor occult germline mutations of susceptibility genes. Current roles of pathologists are differential diagnosis of primary tumors and metastases, identification of clues to occult hereditary disease, and triaging of patients for optimal genetic testing by immunohistochemical staining of tumor tissue for the loss of SDHB and SDHA protein. Diagnostic pitfalls are posed by morphological variants of PCC/PGL, unusual anatomic sites of occurrence, and coexisting neuroendocrine tumors of other types in some hereditary syndromes. These pitfalls can be avoided by judicious use of appropriate immunohistochemical stains. Aside from loss of staining for SDHB, criteria for predicting risk of metastasis are still controversial, and "malignancy" is diagnosed only after metastases have occurred. All PCCs/PGLs are considered to pose some risk of metastasis, and long-term follow-up is advised.

SDHB gene positive metastatic paraganglioma associated with lesions which demonstrate both positive and negative uptake of 18FDG PET and 131MIBG

### Learning Points for Clinicians Paragangliomas are rare caetacholamine secreting neuroendocrine tumours. The diagnosis and management of these tumours is often difficult, particularly as a diagnosis of metastases is often made retrospectively when disease recurrence occurs. In recent years, the role of genotyping has allowed us to better predict which tumours are more likely to be metastatic. Mutations in the SDHB gene have been associated with higher rates of metastatic disease.1 Unfortunately, mutations in this gene can also lower the sensitivity of radiolabeled metaiodobenzylguanidine (MIBG). MIBG scanning is considered the gold standard imaging modality for the diagnosis of recurrent disease but this case illustrates the potential diagnostic pitfalls associated with this imaging modality in cases of SDHB gene mutations. The case demonstrates the importance of genotyping as a diagnostic and predictive tool and the importance of clinical acumen in the face of negative test results. This gentleman underwent a MRI of pelvis in February 2011 for investigation of possible sacroiliitis on a background of known inflammatory bowel disease. This MRI demonstrated a 2.3 × 2.3 cm nodule posterior to the pubic …

Integrative analysis of miRNA and mRNA expression profiles in pheochromocytoma and paraganglioma identifies genotype-specific markers and potentially regulated pathways

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine neoplasias of neural crest origin that can be part of several inherited syndromes. Although their mRNA profiles are known to depend on genetic background, a number of questions related to tumor biology and clinical behavior remain unanswered. As microRNAs (miRNAs) are key players in the modulation of gene expression, their comprehensive analysis could resolve some of these issues. Through characterization of miRNA profiles in 69 frozen tumors with germline mutations in the genes SDHD, SDHB, VHL, RET, NF1, TMEM127, and MAX, we identified miRNA signatures specific to, as well as common among, the genetic groups of PCCs/PGLs. miRNA expression profiles were validated in an independent series of 30 composed of VHL-, SDHB-, SDHD-, and RET-related formalin-fixed paraffin-embedded PCC/PGL samples using quantitative real-time PCR. Upregulation of miR-210 in VHL- and SDHB-related PCCs/PGLs was verified, while miR-137 and miR-382 were confirmed as generally upregulated in PCCs/PGLs (except in MAX-related tumors). Also, we confirmed overexpression of miR-133b as VHL-specific miRNAs, miR-488 and miR-885-5p as RET-specific miRNAs, and miR-183 and miR-96 as SDHB-specific miRNAs. To determine the potential roles miRNAs play in PCC/PGL pathogenesis, we performed bioinformatic integration and pathway analysis using matched mRNA profiling data that indicated a common enrichment of pathways associated with neuronal and neuroendocrine-like differentiation. We demonstrated that miR-183 and/or miR-96 impede NGF-induced differentiation in PC12 cells. Finally, global proteomic analysis in SDHB and MAX tumors allowed us to determine that miRNA regulation occurs primarily through mRNA degradation in PCCs/PGLs, which partially confirmed our miRNA-mRNA integration results.

Testing new susceptibility genes in the cohort of apparently sporadic phaeochromocytoma/paraganglioma patients with clinical characteristics of hereditary syndromes

Phaeochromocytoma (PCC) and paraganglioma (PGL) can occur sporadically or as a part of familial cancer syndromes. Red flags of hereditary syndromes are young age and multifocal tumours. We hypothesized that such patients are candidates for further molecular diagnosis in case of normal results in 'classical' genes. We selected patients with PCC/PGL under the age of 40 and/or with multiple tumours. First, we tested the genes RET, VHL, NF1, SDHB, SDHC and SDHD. Patients without mutations in these genes were tested for mutations in MAX, TMEM127 and SDHAF2. In 153 patients included, mutations were detected in the classical genes in 72 patients (47%) [RET-22 (14%), VHL-13 (9%), NF1-3 (2%), SDHB-13 (9%), SDHC-3 (2%), SDHD-16 (11%), SDHB large deletions- 2 (1%)]. One patient with MAXc.223C>T (p.R75X) mutation was detected. It was a male with bilateral, metachronous phaeochromocytomas diagnosed in 36 and 40 years of age. Remarkably, he showed in the period before the MAX gene was detected, a RET p. Y791F variant. During 10-year follow-up, we did not find any thyroid abnormalities. LOH examination of tumour tissue showed somatic loss of the wild-type allele of MAX. Analysis of the MAX gene should be performed in selected patients, especially those with bilateral adrenal phaeochromocytoma in whom mutations of the classical genes are absent. Our study provides with further support that Y791F RET is a polymorphism.

Characterization of Monilinia fructicola Strains Resistant to Both Propiconazole and Boscalid.

In 2011 and 2012, significant brown rot disease caused by Monilinia fructicola was observed in a peach orchard in Spartanburg County, SC, despite preharvest fungicide applications of demethylation inhibitor (DMI), quinone outside inhibitor (QoI), and succinate dehydrogenase inhibitor (SDHI) fungicides. All 22 isolates obtained in 2011 from this orchard were sensitive to the QoI fungicide, azoxystrobin, and the methyl benzimidazole carbamate (MBC) fungicide, thiophanate-methyl. Five were resistant to the DMI fungicide, propiconazole, and were selected, together with five propiconazole-sensitive isolates, for further investigations. One of the 10 isolates was resistant to propiconazole but sensitive to the SDHI fungicide, boscalid (EC50 = 0.42 μg/ml), 3 were resistant to propiconazole with intermediate sensitivity to boscalid (EC50 0.72 to 2.1 μg/ml); 2 were sensitive to propiconazole with intermediate sensitivity to boscalid; 3 were sensitive to propiconazole but resistant to boscalid (EC50 ≥ 2.1 μg/ml); and 1 (isolate MD22) was resistant to both propiconazole and boscalid. Disease incidence on detached fruit treated with formulated propiconazole or boscalid was significantly higher for MD22 compared to a sensitive control isolate. Continued monitoring of fungicide resistance in the same orchard in 2012 revealed an increase of isolates resistant to propiconazole from 22.7% in 2011 to 34.7%, and an increase of isolates resistant to both propiconazole and boscalid from 4.5% in 2011 to 18.4%. Propiconazole resistance was always associated with the presence of the 'Mona' mobile element located upstream of the sterol 14α-demethylase (MfCYP51) gene. To investigate whether mutations in the subunits of the succinate dehydrogenase enzyme were involved in boscalid resistance, significant portions of the M. fructicola SdhA, SdhB, SdhC, and SdhD genes were cloned and analyzed for 2 sensitive, 2 boscalid-resistant, and 6 dual-resistant isolates. Although sequence variation was found among the isolates, no single change correlated with resistance. Interestingly, analysis of isolates collected from orchards in 2001 and 2002, prior to the registration of boscalid, revealed a range of sensitivities to boscalid (EC50 0.03 to 3.46 μg/ml) including boscalid-resistant isolates. The presence of boscalid-resistant isolates in the baseline population was unexpected and requires further investigation.

Abstract 5301: Integrated array CGH and expression profiling revealed candidate biomarkers in gastrointestinal stromal tumors.

Abstract Oncogenic KIT and PDGFRA mutations play an important role in the development of some, but not all, gastrointestinal stromal tumors (GISTs). Other chromosomal alterations are involved in the clinical progression of these cancers, but these have not been well defined. We studied the association between copy number alterations (CNAs) and gene expression in 32 GISTs, including 4 wild type tumors, using array CGH and gene expression analyses. All tumors exhibited multiple CNAs and significant numbers of novel recurrent regions were identified in wild type GISTs. Among them, a significant correlation with the corresponding expression between wild type and mutant GISTs was observed in 1p and 22q, which harbor SDHB and GSTT1 genes, respectively. All 7 GISTs with copy number losses on 1p36.33-p11.2 showed loss of heterozygosity in at least one of 3 microsatellite markers in the SDHB gene. Loss of SDH complex activity in GISTs can result in increased level of HIF1, which activates the transcription of VEGF and IGF2. In this study, VEGF (2.31-fold increase, P=0.025) and IGF1R (2.76-fold increase, P=0.062) expression levels were higher in wild type/PDGFRA D842V GISTs compared to KIT-mutant GISTs. As constituents of the MAPK cascade, mRNA levels of BRAF (0.50-fold increase, P=0.001) and its downstream effector, MYC (2.21-fold increase, P=0.017) were also increased in wild type/PDGFRA D842V GISTs. For GSTT1, qRT-PCR on all 32 GISTs in this cohort and 22 additional malignant GISTs showed significantly frequent gains of GSTT1 copy number in wild type/PDGFRA D842V GISTs compared to KIT-mutant GISTs (91.7% vs. 54.8%; P=0.03). Gains of GSTT1 copy number correlated positively with mRNA expression by qRT-PCR (r=0.705, P &amp;lt;0.001). Unexpectedly, all four patients with malignant GISTs in the small intestine and KIT exon 11 deletion mutations showing primary resistance to imatinib showed increased copy numbers and mRNA expression of GSTT1. Our integrative approach reveals that for the patients with wild type (or imatinib-resistant) GISTs, attempts to target IGF1R and VEGFR2 would seem to be reasonable options. Moreover, copy number analysis of GSTT1 may be an effective tool to predict imatinib response, and hence help select the best drug and an optimal dose. Further large-scale and well-designed clinical studies are warranted to validate our findings. Citation Format: Guhyun Kang, Eui Jin Lee, Shin Woo Kang, Kee-Taek Jang, Jeeyun Lee, Joon Oh Park, Cheol Keun Park, Tae Sung Sohn, Sung Kim, In-Gu Do, Kyoung-Mee Kim, Christopher L. Corless. Integrated array CGH and expression profiling revealed candidate biomarkers in gastrointestinal stromal tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5301. doi:10.1158/1538-7445.AM2013-5301