Mutations impliquées dans la progression du cancer épithélial de l'ovaire

Le cancer épithélial de l’ovaire (CEO) est le cancer gynécologique le plus létal. Plus de 70% des patientes diagnostiquées avec une tumeur de stade avancé rechutent suite aux traitements chimiothérapeutiques de première ligne, la survie à cinq ans étant ainsi très faible. Afin de mieux comprendre l’évolution de la maladie, nous avons recherché de nouveaux gènes, responsables de l’initiation et de la progression du CEO. Précédemment, des lignées cellulaires ont été dérivées à partir de la tumeur primaire et récurrente et/ou d’ascites de trois patientes. Le séquençage de l’ARN de ces lignées par la technologie de séquençage de nouvelle génération (TSNG) nous a permis d’identifier des mutations ponctuelles qui pourraient nous indiquer des gènes dérégulés dans le CEO. La TSNG est un bon outil qui permet d’identifier et de cribler à grande échelle des mutations. Nous avons sélectionné PLEC1, SCRIB, NCOR2, SEMA6C, IKBKB, GLCE et ITGAE comme gènes candidats présentant des mutations dans nos lignées et ayant une relation fonctionnelle avérée avec le cancer. Étant donné que la TSNG est une technique à taux de fiabilité limité, nous avons validé ces mutations par séquençage Sanger. Ensuite, nous avons étudié l’effet de ces mutations sur la structure protéique et l’expression de PLEC1, de SCRIB et de SEMA6C. Seules certaines mutations dans les gènes PLEC1, SCRIB et SEMA6C ont pu être confirmées. PLEC1 et SCRIB sont deux protéines d’échafaudage dont la mutation, rapportée dans plusieurs cancers, pourrait induire des changements de leurs conformations et affecter leurs interactions et leurs fonctions. Les conséquences de ces mutations sur la tumorigenèse de l’ovaire devront être étudiées.

Searching for novel gene functions in yeast : identification of thousands of novel molecular interactions by protein-fragment complementation assay followed by automated gene function prediction and high-throughput lipidomics

La compréhension de processus biologiques complexes requiert des approches expérimentales et informatiques sophistiquées. Les récents progrès dans le domaine des stratégies génomiques fonctionnelles mettent dorénavant à notre disposition de puissants outils de collecte de données sur l’interconnectivité des gènes, des protéines et des petites molécules, dans le but d’étudier les principes organisationnels de leurs réseaux cellulaires. L’intégration de ces connaissances au sein d’un cadre de référence en biologie systémique permettrait la prédiction de nouvelles fonctions de gènes qui demeurent non caractérisées à ce jour. Afin de réaliser de telles prédictions à l’échelle génomique chez la levure Saccharomyces cerevisiae, nous avons développé une stratégie innovatrice qui combine le criblage interactomique à haut débit des interactions protéines-protéines, la prédiction de la fonction des gènes in silico ainsi que la validation de ces prédictions avec la lipidomique à haut débit. D’abord, nous avons exécuté un dépistage à grande échelle des interactions protéines-protéines à l’aide de la complémentation de fragments protéiques. Cette méthode a permis de déceler des interactions in vivo entre les protéines exprimées par leurs promoteurs naturels. De plus, aucun biais lié aux interactions des membranes n’a pu être mis en évidence avec cette méthode, comparativement aux autres techniques existantes qui décèlent les interactions protéines-protéines. Conséquemment, nous avons découvert plusieurs nouvelles interactions et nous avons augmenté la couverture d’un interactome d’homéostasie lipidique dont la compréhension demeure encore incomplète à ce jour. Par la suite, nous avons appliqué un algorithme d’apprentissage afin d’identifier huit gènes non caractérisés ayant un rôle potentiel dans le métabolisme des lipides. Finalement, nous avons étudié si ces gènes et un groupe de régulateurs transcriptionnels distincts, non préalablement impliqués avec les lipides, avaient un rôle dans l’homéostasie des lipides. Dans ce but, nous avons analysé les lipidomes des délétions mutantes de gènes sélectionnés. Afin d’examiner une grande quantité de souches, nous avons développé une plateforme à haut débit pour le criblage lipidomique à contenu élevé des bibliothèques de levures mutantes. Cette plateforme consiste en la spectrométrie de masse à haute resolution Orbitrap et en un cadre de traitement des données dédié et supportant le phénotypage des lipides de centaines de mutations de Saccharomyces cerevisiae. Les méthodes expérimentales en lipidomiques ont confirmé les prédictions fonctionnelles en démontrant certaines différences au sein des phénotypes métaboliques lipidiques des délétions mutantes ayant une absence des gènes YBR141C et YJR015W, connus pour leur implication dans le métabolisme des lipides. Une altération du phénotype lipidique a également été observé pour une délétion mutante du facteur de transcription KAR4 qui n’avait pas été auparavant lié au métabolisme lipidique. Tous ces résultats démontrent qu’un processus qui intègre l’acquisition de nouvelles interactions moléculaires, la prédiction informatique des fonctions des gènes et une plateforme lipidomique innovatrice à haut débit , constitue un ajout important aux méthodologies existantes en biologie systémique. Les développements en méthodologies génomiques fonctionnelles et en technologies lipidomiques fournissent donc de nouveaux moyens pour étudier les réseaux biologiques des eucaryotes supérieurs, incluant les mammifères. Par conséquent, le stratégie présenté ici détient un potentiel d’application au sein d’organismes plus complexes.

Role of the Protein Tyrosine Kinase 7 gene in human neural tube defects

Les anomalies du tube neural (ATN) sont des anomalies développementales où le tube neural reste ouvert (1-2/1000 naissances). Afin de prévenir cette maladie, une connaissance accrue des processus moléculaires est nécessaire. L’étiologie des ATN est complexe et implique des facteurs génétiques et environnementaux. La supplémentation en acide folique est reconnue pour diminuer les risques de développer une ATN de 50-70% et cette diminution varie en fonction du début de la supplémentation et de l’origine démographique. Les gènes impliqués dans les ATN sont largement inconnus. Les études génétiques sur les ATN chez l’humain se sont concentrées sur les gènes de la voie métabolique des folates du à leur rôle protecteur dans les ATN et les gènes candidats inférés des souris modèles. Ces derniers ont montré une forte association entre la voie non-canonique Wnt/polarité cellulaire planaire (PCP) et les ATN. Le gène Protein Tyrosine Kinase 7 est un membre de cette voie qui cause l’ATN sévère de la craniorachischisis chez les souris mutantes. Ptk7 interagit génétiquement avec Vangl2 (un autre gène de la voie PCP), où les doubles hétérozygotes montrent une spina bifida. Ces données font de PTK7 comme un excellent candidat pour les ATN chez l’humain. Nous avons re-séquencé la région codante et les jonctions intron-exon de ce gène dans une cohorte de 473 patients atteints de plusieurs types d’ATN. Nous avons identifié 6 mutations rares (fréquence allélique <1%) faux-sens présentes chez 1.1% de notre cohorte, dont 3 sont absentes dans les bases de données publiques. Une variante, p.Gly348Ser, a agi comme un allèle hypermorphique lorsqu'elle est surexprimée dans le modèle de poisson zèbre. Nos résultats impliquent la mutation de PTK7 comme un facteur de risque pour les ATN et supporte l'idée d'un rôle pathogène de la signalisation PCP dans ces malformations.

Deletion Analysis of 15 exons located outside and inside a “Hot Spot” in the Duchenne Muscular Dystrophy gene in Duchenne’s Muscular dystrophy patients

Introduction. Duchenne and Becker Muscular Dystrophies (DMD/DMB) are X-linked recessive diseases characterized by progressive muscle weakness and wasting, loss of motor skills and death after the second decade of life. Deletions are the most prevalent mutations that affect the dystrophin gene, which spans 79 exons.Objective: Identify deletions on the dystrophin gene in 58 patients affected with DMD.Methods: Through multiplex PCR identify deletions on the dystrophin gene in 58 patients with DMD and observe the frequency of this mutation in our population.Results: We found deletions in 1.72% of patients (1 of 58 persons). Deletions were not the principal cause of disease in our population. It is possible that duplications and point mutations caused this illness in our patients.Conclusions: The frequency of deletions in the 15 exons analyzed from the dystrophin gene was low. The predominant types of mutation in our patients` samples were not deletions as has been observed in the literature worldwide, therefore, it is important to determine other types of mutations as are duplications and point mutations.

Characterization of recombinant influenza B viruses with key neuraminidase inhibitor resistance mutations

Objectives and methods: An influenza B virus plasmid-based rescue system was used to introduce site-specific mutations, previously observed in neuraminidase (NA) inhibitor-resistant viruses, into the NA protein of six recombinant viruses. Three mutations observed only among in vitro selected zanamivir-resistant influenza A mutants were introduced into the B/Beijing/1/87 virus NA protein, to change residue E116 to glycine, alanine or aspartic acid. Residue E116 was also mutated to valine, a mutation found in the clinic among oseltamivir-resistant viruses. An arginine to lysine change at position 291 (292 N2 numbering) mimicked that seen frequently in influenza A N2 clinical isolates resistant to oseltamivir. Similarly, an arginine to lysine change at position 149 (152 in N2 numbering) was made to reproduce the change found in the only reported zanamivir-resistant clinical isolate of influenza B virus. In vitro selection and prolonged treatment in the clinic leads to resistance pathways that require compensatory mutations in the haemagglutinin gene, but these appear not to be important for mutants isolated from immunocompetent patients. The reverse genetics system was therefore used to generate mutants containing only the NA mutation. Results and conclusions: With the exception of a virus containing the E116G mutation, mutant viruses were attenuated to different levels in comparison with wild-type virus. This attenuation was a result of altered NA activity or stability depending on the introduced mutation. Mutant viruses displayed increased resistance to zanamivir, oseltamivir and peramivir, with certain viruses displaying cross-resistance to all three drugs.

Identification of mutations conferring insecticide-insensitive AChE in the cotton-melon aphid, Aphis gossypii Glover

We have identified two mutations in the ace1 gene of Aphis gossypii that are associated with insensitivity of acetylcholinesterase (AChE) to carbamate and organophosphate insecticides. The first of these, S431F (equivalent to F331 in Torpedo californica), is associated with insensitivity to the carbamate insecticide pirimicarb in a range of A. gossypii clones. The S431F mutation is also found in the peach-potato aphid, Myzus persicae (Sulzer), and a rapid RFLP diagnostic allows the identification of individuals of both aphid species with a resistant genotype. This diagnostic further revealed the presence of S431 in several other pirimicarb-susceptible aphid species. The serine at this position in the wild-type enzyme has only been reported for aphids and provides a molecular explanation of why pirimicarb has a specific aphicidal action. A less specific insensitivity to a wide range of carbamates and organophosphates is associated with a second mutation, A302S (A201 in T. californica).

Influence of mutations affecting gonadotropin production or responsiveness on expression of inhibin subunit mRNA and protein in the mouse ovary

Measurement of inhibins A and B in the serum of normal cyclic rodents has implicated FSH in the regulation of these peptides within the ovary. To extend these observations we have used a panel of mutant mice carrying mutations which affect either the production of, or the ability to respond to, FSH and LH. As a consequence, the females are infertile and show different degrees of follicular development. The aim of this study was to measure inhibin gene transcription in the ovaries of these mutant females together with inhibin protein levels in ovaries and serum and to relate these to follicular development within the ovary. Comparison was made with a pool of normal/heterozygous females. In hpg females where lack of GnRH production results in the absence of gonadotropin synthesis, in FSHbeta knockout (FSHbetaKO) females where disruption of the gene encoding FSHbeta results in the absence of FSH production, and in FSH receptor knockout (FSHRKO) females which are unable to respond to circulating FSH, follicular development remains at the pre-antral stage in these three mutants. Only in the hpg females were common inhibin alpha subunit mRNA levels significantly lower than normal. In these three mutants, however, mRNA levels for both the betaA and betaB subunits were extremely low compared with normal mice. At the protein level, neither inhibin A nor B was detected in the serum of these three mutants; however inhibin B, albeit at very low levels, was detectable within the ovaries. These observations confirm a major role for FSH in the control of transcription of the RA and betaB genes but suggest that the constitutive transcription of the alpha subunit is less dependent on FSH. In contrast, in LH receptor knockout (LuRKO) female mice inhibin betaA subunit mRNA levels were similar to those measured in normal/heterozygous females but levels of inhibin alpha and betaB subunit mRNAs were significantly higher than in the normal group. This was reflected in significantly higher inhibin B protein levels in ovaries and serum. An inability to respond to LH combined with high circulating levels of FSH leads to a high proportion of antral follicles in LuRKO females, with granulosa cells constituting the major cell type within the ovary. The high percentage of antral granulosa cells is likely to account for the significantly higher levels of inhibin B production in these ovaries.

Reversible phase variation in the phnE gene, which is required for phosphonate metabolism in Escherichia coli K-12

It is known that Escherichia coli K-12 is cryptic (Phn(-)) for utilization of methyl phosphonate (MePn) and that Phn(+) variants can be selected for growth on MePn as the sole P source. Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter. Here we show that Phn(+) variants are present at the surprisingly high frequency of >10(-2) in K-12 strains. Amplified-fragment length polymorphism analysis was used to monitor instability in phnE in various strains growing under different conditions. This revealed that, once selection for growth on MePn is removed, Phn(+) revertants reappear and accumulate at high levels through reinsertion of the 8-bp repeat element sequence. It appears that, in K-12, phnE contains a high-frequency reversible gene switch, producing phase variation which either allows ("on" form) or blocks ("off" form) MePn utilization. The switch can also block usage of other metabolizable alkyl phosphonates, including the naturally occurring 2-aminoethylphosphonate. All K-12 strains, obtained from collections, appear in the "off" form even when bearing mutations in mutS, mutD, or dnaQ which are known to enhance slip strand events between repetitive sequences. The ability to inactivate the phnE gene appears to be unique to K-12 strains since the B strain is naturally Phn(+) and lacks the inactivating 8-bp insertion in phnE, as do important pathogenic strains for which genome sequences are known and also strains isolated recently from environmental sources.

Attenuating mutations in the influenza virus genome which may increase the safety of vaccine production

Influenza virus epidemics occur on an annual basis and cause severe disease in the very young and old. The vaccine administered to high-risk groups is generated by amplifying reassortant viruses, with chronologically relevant viral surface antigens, in eggs. Every 20 years or so, influenza pandemics occur causing widespread fatality in all age groups. These viruses display novel viral surface antigens acquired from a zoonotic source, and vaccination against them poses new issues since production of large amounts of a respiratory virus containing novel surface antigens could be dangerous for those involved in manufacture. To minimise risks, it is advisable to use a virus whose genetic backbone is highly attenuated in man. Traditionally, the A/PR/8/34 strain of virus is used, however, the genetic basis of its attenuation is unclear. Cold-adapted (CA) strains of the influenza virus are all based on the H2N2 subtype, itself a virus with pandemic potential, and again the genetic basis of temperature sensitivity is not yet established. Reverse genetics technology allows us to engineer designer influenza viruses to order. Using this technology, we have been investigating mutations in several different gene segments to effectively attenuate potential vaccine strains allowing the safe production of vaccine to protect against the next pandemic. (C) 2003 Elsevier B.V. All rights reserved.

DNA variation in a 13-Mb region including the F9 gene: inferring the genealogical history and causal role of a hemophilia B mutation (IVS 5+13 A -> G)

About 5.5% of all UK hemophilia B patients have the base substitution IVS 5+13 A-->G as the only change in their factor (F)IX gene (F9). This generates a novel donor splice site which fits the consensus better than the normal intron 5 donor splice. Use of the novel splice site should result in a missense mutation followed by the abnormal addition of four amino acids to the patients' FIX. In order to explain the prevalence of this mutation, its genealogical history is examined. Analysis of restriction fragment length polymorphism in the 21 reference UK individuals (from different families) with the above mutation showed identical haplotypes in 19 while two differed from the rest and from each other. In order to investigate the history of the mutation and to verify that it had occurred independently more than once, the sequence variation in 1.5-kb segments scattered over a 13-Mb region including F9 was examined in 18 patients and 15 controls. This variation was then analyzed with a recently developed Bayesian approach that reconstructs the genealogy of the gene investigated while providing evidence of independent mutations that contribute disconnected branches to the genealogical tree. The method also provides minimum estimates of the age of the mutation inherited by the members of coherent trees. This revealed that 17 or 18 mutant genes descend from a founder who probably lived 450 years ago, while one patient carries an independent mutation. The independent recurrence of the IVS5+13 A-->G mutation strongly supports the conclusion that it is the cause of these patients' mild hemophilia.

Dietary, lifestyle and clinico-pathological factors associated with APC mutations and promoter methylation in colorectal cancers from the EPIC-Norfolk Study

The tumour suppressor APC is the most commonly altered gene in colorectal cancer (CRC). Genetic and epigenetic alterations of APC may therefore be associated with dietary and lifestyle risk factors for CRC. Analysis of APC mutations in the extended mutation cluster region (codons 1276-1556) and APC promoter 1A methylation was performed on 185 archival CRC samples collected from participants of the European Prospective Investigation into Cancer (EPIC)-Norfolk Study, with the aim of relating these to high quality seven-day dietary and lifestyle data collected prospectively. Truncating APC mutations (APC+) and promoter 1A methylation (PM+) were identified in 43% and 23% of CRCs analysed, respectively. Distal CRCs were more likely than proximal CRCs to be APC+ or PM+ (P = 0.04). APC+ CRCs were more likely to be moderately/well differentiated and microsatellite stable than APC- CRCs (P = 0.05 and 0.03). APC+ CRC cases consumed more alcohol than their counterparts (P = 0.01) and PM+ CRC cases consumed lower levels of folate and fibre (P = 0.01 and 0.004). APC+ or PM+ CRC cases consumedhigher levels of processed meat and iron from red meat and red meat products (P=0.007 and 0.006). Specifically, CRC cases harbouring GC to AT transition mutations consumed higher levels of processed meat (35 versus 24 g/day, P = 0.04) and iron from red meat and red meat products (0.8 versus 0.6 mg/day, P = 0.05). In a logistic regression model adjusted for age, sex and cigarette smoking status, each 19g/day (1SD) increment increase in processed meat consumption was associated with cases with GC to AT mutations (OR 1.68, 95% CI 1.03-2.75). In conclusion, APC+ and PM+ CRCs may be influenced by diet and GC to AT mutations in APC are associated with processed meat consumption, suggesting a mechanistic link with dietary alkylating agents, such as N-nitroso compounds.

Mutation of toxB and a truncated version of the efa-1 gene in Escherichia coli O157 : H7 influences the expression and secretion of locus of enterocyte effacement-encoded proteins but not intestinal colonization in calves or sheep

Enterohemorrhagic Escherichia coli (EHEC) strains comprise a broad group of bacteria, some of which cause attaching and effacing (AE) lesions and enteritis in humans and animals. Non-O157:H7 EHEC strains contain the gene efa-1 (referred to in previous publications as efa1), which influences adherence to cultured epithelial cells. An almost identical gene in enteropathogenic E. coli (lifA) mediates the inhibition of lymphocyte proliferation and proinflammatory cytokine synthesis. We have shown previously that significantly lower numbers of EHEC 05 and 0111 efa-1 mutants are shed in feces following experimental infection in calves and that these mutants exhibit reduced adherence to intestinal epithelia compared with isogenic wild-type strains. E. coli O157:H7 strains lack efa-1 but encode a homolog on the pO157 plasmid (toxB/l7095) and contain a truncated version of the efa-1 gene (efa-1'/z4332 in O island 122 of the EDL933 chromosome). Here we report that E. coli O157:H7 toxB and efa-1' single and double mutants exhibit reduced adherence to cultured epithelial cells and show reduced expression and secretion of proteins encoded by the locus of enterocyte effacement (LEE), which plays a key role in the host-cell interactions of EHEC. The activity of LEE1, LEE4, and LEE5 promoters was not significantly altered in E. coli O157:H7 strains harboring toxB or efa-1' mutations, indicating that the effect on the expression of LEE-encoded secreted proteins occurs at a posttranscriptional level. Despite affecting type III secretion, mutation of toxB and efa-1' did not significantly affect the course of fecal shedding of E. coli O157:H7 following experimental inoculation of 10- to 14-day-old calves or 6-week-old sheep. Mutation of tir caused a significant reduction in fecal shedding of E. coli O157:H7 in calves, indicating that the formation of AE lesions is important for colonization of the bovine intestine.

Detection of mutations in Salmonella enterica gyrA, gyrB, parC and parE genes by denaturing high performance liquid chromatography (DHPLC) using standard HPLC instrumentation

Aims: Quinolone antibiotics are the agents of choice for treating systemic Salmonella infections. Resistance to quinolones is usually mediated by mutations in the DNA gyrase gene gyrA. Here we report the evaluation of standard HPLC equipment for the detection of mutations (single nucleotide polymorphisms; SNPs) in gyrA, gyrB, parC and parE by denaturing high performance liquid chromatography (DHPLC). Methods: A panel of Salmonella strains was assembled which comprised those with known different mutations in gyrA (n = 8) and fluoroquinolone-susceptible and -resistant strains (n = 50) that had not been tested for mutations in gyrA. Additionally, antibiotic-susceptible strains of serotypes other than Salmonella enterica serovar Typhimurium strains were examined for serotype-specific mutations in gyrB (n = 4), parC (n = 6) and parE (n = 1). Wild-type (WT) control DNA was prepared from Salmonella Typhimurium NCTC 74. The DNA of respective strains was amplified by PCR using Optimase (R) proofreading DNA polymerase. Duplex DNA samples were analysed using an Agilent A1100 HPLC system with a Varian Helix (TM) DNA column. Sequencing was used to validate mutations detected by DHPLC in the strains with unknown mutations. Results: Using this HPLC system, mutations in gyrA, gyrB, parC and parE were readily detected by comparison with control chromatograms. Sequencing confirmed the gyrA predicted mutations as detected by DHPLC in the unknown strains and also confirmed serotype-associated sequence changes in non-Typhimurium serotypes. Conclusions: The results demonstrated that a non-specialist standard HPLC machine fitted with a generally available column can be used to detect SNPs in gyrA, gyrB, parC and parE genes by DHPLC. Wider applications should be possible.

Dominant β-catenin mutations cause intellectual disability with recognizable syndromic features

The recent identification of multiple dominant mutations in the gene encoding β-catenin in both humans and mice has enabled exploration of the molecular and cellular basis of β-catenin function in cognitive impairment. In humans, β-catenin mutations that cause a spectrum of neurodevelopmental disorders have been identified. We identified de novo β-catenin mutations in patients with intellectual disability, carefully characterized their phenotypes, and were able to define a recognizable intellectual disability syndrome. In parallel, characterization of a chemically mutagenized mouse line that displays features similar to those of human patients with β-catenin mutations enabled us to investigate the consequences of β-catenin dysfunction through development and into adulthood. The mouse mutant, designated batface (Bfc), carries a Thr653Lys substitution in the C-terminal armadillo repeat of β-catenin and displayed a reduced affinity for membrane-associated cadherins. In association with this decreased cadherin interaction, we found that the mutation results in decreased intrahemispheric connections, with deficits in dendritic branching, long-term potentiation, and cognitive function. Our study provides in vivo evidence that dominant mutations in β-catenin underlie losses in its adhesion-related functions, which leads to severe consequences, including intellectual disability, childhood hypotonia, progressive spasticity of lower limbs, and abnormal craniofacial features in adults

Alterations in predicted regulatory and coding regions of the sterol 14α-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae

The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops is increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivities of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to sub-inhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting PbCYP51 evolution is on-going and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed.