Evolutionary Changes in Gene Expression,Coding Sequence and Copy-Number at the Cyp6g1 Locus Contribute to Resistance to Multiple Insecticides in Drosophila

Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster–D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species.     

High expression of Cyp6g1, a cytochrome P450 gene, does not necessarily confer DDT resistance in Drosophila melanogaster

Cytochrome P450 monooxygenases, a family of detoxifying enzymes, are thought to confer resistance to various insecticides including DDT. Daborn et al. [Daborn, P., Yen, J.L., Bogwitz, M., Le Goff, G., Feil, et al. 2002. A single p450 allele associated with insecticide resistance in Drosophila. Science 297, 2253-2256.] suggested that the Accord transposable element causes overexpression of a Cyp6g1 allele, which has spread globally and is the basis of DDT resistance in Drosophila melanogaster populations. To determine whether the same phenomenon also operates in other Drosophila strains, we investigated 91-R, 91-C, ry(506), Wisconsin, Canton-SH and Hikone-RH strains. While the LC(50) values for the 91-R and Wisconsin strains are 8348 microg and 447 microg of DDT, respectively, values for the other four strains range between 0.74 to 20.9 microg. As expected, the susceptible ry(506) and 91-C strains have about 16-33-fold lower levels of CYP6G1 mRNA than the resistant 91-R and Wisconsin strains. Surprisingly, CYP6G1 mRNA and protein levels in the Canton-SH and Hikone-RH strains are as high as in the two resistant strains, yet they are as susceptible as the 91-C strain. The susceptible phenotype of the Canton-SH and Hikone-RH strains is not due to mutation in the Cyp6g1 gene; sequence analysis showed that Cyp6g1 alleles of resistant and susceptible strains are very similar and cannot be classified into resistant and susceptible alleles. As observed by others, we also found that only the 5'-upstream DNA of overexpressing alleles of Cyp6g1 has an insertional DNA, which is similar to Accord and Ninja elements. To examine the role of Cyp6g1 in DDT resistance, we substituted the Cyp6g1 allele of the 91-R strain with the allele from the susceptible 91-C strain via recombination and synthesized three recombinant lines. All three lines lacked Accord insertion and showed low expression of Cyp6g1 like the 91-C strain, yet they were as highly resistant as the 91-R strain. We conclude a strain may not have to have Accord insertion in the Cyp6g1 gene and the Cyp6g1 itself may not have to be overexpressed for DDT resistance to occur.     

Human Placental-Specific Epipolymorphism and its Association with Adverse Pregnancy Outcomes

Interindividual variation in DNA-methylation level is widespread in the human genome, despite its critical role in regulating gene expression. The nature of this variation, including its tissue-specific nature, and the role it may play in human phenotypic variation and disease is still poorly characterized. The placenta plays a critical role in regulating fetal growth and development in ways that have lifelong effects on health. To identify genes with a high degree of interindividual DNA methylation variation in the human placenta, we surveyed the human genome using the Illumina GoldenGate Methylation Cancer panel targeting 1505 CpG sites of 807 genes. While many sites show a continuous pattern of methylation levels, WNT2, TUSC3 and EPHB4 were identified to have a polymorphic “on-or-off” pattern of DNA methylation variation at their promoter region which was confirmed by pyrosequencing. Methylation of these genes can be found in 7%–25% of over 100 placentas tested. The methylation state at the promoter of these genes is concordant with mRNA allelic expression. In three informative cases TUSC3 was observed to be methylated on the maternal allele, and it is thus possible this represents a polymorphically imprinted gene. Furthermore, TUSC3 promoter methylation showed evidence for association with preeclampsia. A biological significance of these methylation allelic polymorphisms (MAPs) to human placental diversity is further implied by their placental specificity and absence in mouse. An extended study of blood suggests that MAPs may also be found in other tissues, implicating their utility for tissue-specific association with complex disorders. The identification of such “epipolymorphism” in other tissues and their use in association studies, should improve our understanding of interindividual phenotypic variability and complex disease susceptibility.     

SNP-based large-scale identification of allele-specific gene expression in human B cells

Polymorphism and variations in gene expression provide the genetic basis for human variation. Allelic variation of gene expression, in particular, may play a crucial role in phenotypic variation and disease susceptibility. To identify genes with allelic expression in human cells, we genotyped genomic DNA and cDNA isolated from 31 immortalized B cell lines from three Centre d'Etude du Polymorphisme Humain (CEPH) families using high-density single-nucleotide polymorphism (SNP) chips containing 13,900 exonic SNPs. We identified seven SNPs in five genes with monoallelic expression, 146 SNPs in 125 genes with allelic imbalance in expression with preferentially higher expression of one allele in a heterozygous individual. The monoallelically expressed genes (ERAP2, MDGA1, LOC644422, SDCCAG3P1 and CLTCL1) were regulated by cis-acting, non-imprinted differential allelic control. In addition, all monoallelic gene expression patterns and allelic imbalances in gene expression in B cells were transmitted from parents to offspring in the pedigree, indicating genetic transmission of allelic gene expression. Furthermore, frequent allele substitution, probably due to RNA editing, was also observed in 21 genes in 23 SNPs as well as in 48 SNPs located in regions containing no known genes. In this study, we demonstrated that allelic gene expression is frequently observed in human B cells, and SNP chips are very useful tools for detecting allelic gene expression. Overall, our data provide a valuable framework for better understanding allelic gene expression in human B cells.     

Population stratification of a common APOBEC gene deletion polymorphism

The APOBEC3 gene family plays a role in innate cellular immunity inhibiting retroviral infection, hepatitis B virus propagation, and the retrotransposition of endogenous elements. We present a detailed sequence and population genetic analysis of a 29.5-kb common human deletion polymorphism that removes the APOBEC3B gene. We developed a PCR-based genotyping assay, characterized 1,277 human diversity samples, and found that the frequency of the deletion allele varies significantly among major continental groups (global F_ST = 0.2843). The deletion is rare in Africans and Europeans (frequency of 0.9% and 6%), more common in East Asians and Amerindians (36.9% and 57.7%), and almost fixed in Oceanic populations (92.9%). Despite a worldwide frequency of 22.5%, analysis of data from the International HapMap Project reveals that no single existing tag single nucleotide polymorphism may serve as a surrogate for the deletion variant, emphasizing that without careful analysis its phenotypic impact may be overlooked in association studies. Application of haplotype-based tests for selection revealed potential pitfalls in the direct application of existing methods to the analysis of genomic structural variation. These data emphasize the importance of directly genotyping structural variation in association studies and of accurately resolving variant breakpoints before proceeding with more detailed population-genetic analysis.     

Microarray analysis of cytochrome P450 mediated insecticide resistance in Drosophila

Insecticide resistance in laboratory selected Drosophila strains has been associated with upregulation of a range of different cytochrome P450s, however in recent field isolates of D. melanogaster resistance to DDT and other compounds is conferred by one P450 gene, Cyp6g1. Using microarray analysis of all Drosophila P450 genes, here we show that different P450 genes such as Cyp12d1 and Cyp6a8 can also be selected using DDT in the laboratory. We also show, however, that a homolog of Cyp6g1 is over-expressed in a field resistant strain of D. simulans. In order to determine why Cyp6g1 is so widely selected in the field we examine the pattern of cross-resistance of both resistant strains and transgenic flies over-expressing Cyp6g1 alone. We show that all three DDT selected P450s can confer resistance to the neonicotinoid imidacloprid but that Cyp6a8 confers no cross-resistance to malathion. Transgenic flies over-expressing Cyp6g1 also show cross-resistance to other neonicotinoids such as acetamiprid and nitenpyram. We suggest that the broad level of cross-resistance shown by Cyp6g1 may have facilitated its selection as a resistance gene in natural Drosophila populations.     

Cloning of DNA sequences from the white locus of D. melanogaster by a novel and general method

We describe the isolation of a cloned DNA segment carrying unique sequences from the white locus of Drosophila melanogaster. Sequences within the cloned segment are shown to hybridize in situ to the white locus region on the polytene chromosomes of both wild-type strains and strains carrying chromosomal rearrangements whose breakpoints bracket the white locus. We further show that two small deficiency mutations, deleting white locus genetic elements but not those of complementation groups contiguous to white, delete the genomic sequences corresponding to a portion of the cloned segment. The strategy we have employed to isolate this cloned segment exploits the existence of an allele at the white locus containing a copy of a previously cloned transposable, reiterated DNA sequence element. We describe a simple, rapid method for retrieving cloned segments carrying a copy of the transposable element together with contiguous sequences corresponding to this allele. The strategy described is potentially general and we discuss its application to the cloning of the DNA sequences of other genes in Drosophila, including those identified only by genetic analysis and for which no RNA product is known.     

Identification of a Novel Allele of TaCKX6a02 Associated with Grain Size,Filling Rate and Weight of Common Wheat

Cytokinin oxidase (CKX) plays a crucial role in plant growth and development by reversibly inactivating cytokinin (CTK). Twenty-four primer pairs, designed from ESTs of the TaCKX genes family of common wheat, were used to identify their allelic variations associated with grain size, weight, and filling rate in 169 recombinant inbred lines (RIL) derived from Jing 411 × Hongmangchun 21. TaCKX6a02, a member of TaCKX gene family, amplified by primer pair T31–32, showed a close association with grain traits in this RIL population. Statistical analysis indicated that allelic variation of TaCKX6a02 had significant correlation with grain size, weight, and filling rate (GFR; P < 0.001) under varied environments. The TaCKX6a02-D1a allele from Jing411 significantly increased grain size, weight and grain filling rate, compared with TaCKX6a02-D1b from Hongmangchun 21. TaCKX6a02 was located on chromosome 3DS in the interval of Xbarc1119 and Xbarc1162, with a genetic distance of 1.4 cM. The location was further confirmed using Chinese Spring nulli–tetrasomic lines. A major QTL (quantitative trait locus) tightly linked to TaCKX6a02 was detected in the RIL population, explaining 17.1~38.2% of phenotype variations for grain size, weight, GFRmax and GFRmean in different environments. In addition, significant effects of variations of TaCKX6a02 on grain weight and GFR were further validated by association analysis among 102 wheat varieties in two cropping seasons. 12.8~35.1% of phenotypic variations were estimated for these genotypes. A novel 29-bp InDel behind the stop codon was detected by DNA sequence analysis between the two alleles of TaCKX6a02-D1. The gene-specific marker, TKX3D, was designed according to the novel variation, and can be used in marker-assisted selection (MAS) for grain size, weight, and GFR in common wheat.     

Congenic mice reveal genetic epistasis and overlapping disease loci for autoimmune diabetes and listeriosis

The nonobese diabetic (NOD) mouse strain serves as a genomic standard for assessing how allelic variation for insulin-dependent diabetes (Idd) loci affects the development of autoimmune diabetes. We previously demonstrated that C57BL/6 (B6) mice harbor a more diabetogenic allele than NOD mice for the Idd14 locus when introduced onto the NOD genetic background. New congenic NOD mouse strains, harboring smaller B6-derived intervals on chromosome 13, now localize Idd14 to an ~18-Mb interval and reveal a new locus, Idd31. Notably, the B6 allele for Idd31 confers protection against diabetes, but only in the absence of the diabetogenic B6 allele for Idd14, indicating genetic epistasis between these two loci. Moreover, congenic mice that are more susceptible to diabetes are more resistant to Listeria monocytogenes infection. This result co-localizes Idd14 and Listr2, a resistance locus for listeriosis, to the same genomic interval and indicates that congenic NOD mice may also be useful for localizing resistance loci for infectious disease.     

Impact of Three Ampicillin Dosage Regimens on Selection of Ampicillin Resistance in Enterobacteriaceae and Excretion of blaTEM Genes in Swine Feces

The aim of this study was to assess the impact of three ampicillin dosage regimens on ampicillin resistance among Enterobacteriaceae recovered from swine feces by use of phenotypic and genotypic approaches. Phenotypically, ampicillin resistance was determined from the percentage of resistant Enterobacteriaceae and MICs of Escherichia coli isolates. The pool of ampicillin resistance genes was also monitored by quantification of bla_TEM genes, which code for the most frequently produced β-lactamases in gram-negative bacteria, using a newly developed real-time PCR assay. Ampicillin was administered intramuscularly and orally to fed or fasted pigs for 7 days at 20 mg/kg of body weight. The average percentage of resistant Enterobacteriaceae before treatment was between 2.5% and 12%, and bla_TEM gene quantities were below 10⁷ copies/g of feces. By days 4 and 7, the percentage of resistant Enterobacteriaceae exceeded 50% in all treated groups, with some highly resistant strains (MIC of >256 μg/ml). In the control group, bla_TEM gene quantities fluctuated between 10⁴ and 10⁶ copies/g of feces, whereas they fluctuated between 10⁶ to 10⁸ and 10⁷ to 10⁹ copies/g of feces for the intramuscular and oral routes, respectively. Whereas phenotypic evaluations did not discriminate among the three ampicillin dosage regimens, bla_TEM gene quantification was able to differentiate between the effects of two routes of ampicillin administration. Our results suggest that fecal bla_TEM gene quantification provides a sensitive tool to evaluate the impact of ampicillin administration on the selection of ampicillin resistance in the digestive microflora and its dissemination in the environment.     

The genetic basis of pyrethroid and DDT resistance inter-relationships in Aedes aegypti I. Isolation of DDT and pyrethroid resistance factors

DDT resistance in adults of the BKPM3 strain of Aedes aegypti, homozygous for the pyrethroid resistance gene R ^py , was found to be due to factors on both chromosomes II and III. No evidence was found to suggest the presence of factors conferring resistance to permethrin on chromosome II and consequently that DDT resistance factor(s) on this chromosome are related to permethrin resistance. Further confirmation that R ^py is the major gene controlling pyrethroid resistance was obtained and also evidence that it is closely linked or allelic to the chromosome III DDT resistance factor.     

Cis-acting mutation and duplication: History of molecular evolution in a P450 haplotype responsible for insecticide resistance in Culex quinquefasciatus

A cytochrome P450 gene, Cyp9m10, is more than 200-fold overexpressed in a pyrethroid resistant strain of Culex quinquefasciatus, JPal-per. The haplotype of this strain contains two copies of Cyp9m10 resulted from recent tandem duplication. In this study, we discovered and isolated a Cyp9m10 haplotype closely related to this duplicated Cyp9m10 haplotype from JHB, a strain used for the recent genome project for this mosquito species. The isolated haplotype (JHB-NIID-B haplotype) shared the same insertion of a transposable element upstream of the coding region with JPal-per strain but not duplicated. The JHB-NIID-B haplotype was considered to have diverged from the JPal-per lineage just before the duplication event. Cyp9m10 was moderately overexpressed in larvae with the JHB-NIID-B haplotype. The overexpressions in JHB-NIID-B and JPal-per haplotypes were developmentally regulated in similar pattern indicating both haplotypes share a common cis-acting mutation responsible for the overexpressions. The isolated moderately overexpressed haplotype conferred resistance, however, its efficacy was relatively small. We hypothesized that the first cis-acting mutation modified the consequence of the subsequent duplication in JPal-per lineage to confer stronger phenotypic effect than that if it occurred before the first cis-acting mutation.     

Genome-wide association study identifies novel loci associated with resistance to bovine tuberculosis

Tuberculosis (TB) caused by Mycobacterium bovis is a re-emerging disease of livestock that is of major economic importance worldwide, as well as being a zoonotic risk. There is significant heritability for host resistance to bovine TB (bTB) in dairy cattle. To identify resistance loci for bTB, we undertook a genome-wide association study in female Holstein–Friesian cattle with 592 cases and 559 age-matched controls from case herds. Cases and controls were categorised into distinct phenotypes: skin test and lesion positive vs skin test negative on multiple occasions, respectively. These animals were genotyped with the Illumina BovineHD 700K BeadChip. Genome-wide rapid association using linear and logistic mixed models and regression (GRAMMAR), regional heritability mapping (RHM) and haplotype-sharing analysis identified two novel resistance loci that attained chromosome-wise significance, protein tyrosine phosphatase receptor T (PTPRT; P=4.8 × 10⁻⁷) and myosin IIIB (MYO3B; P=5.4 × 10⁻⁶). We estimated that 21% of the phenotypic variance in TB resistance could be explained by all of the informative single-nucleotide polymorphisms, of which the region encompassing the PTPRT gene accounted for 6.2% of the variance and a further 3.6% was associated with a putative copy number variant in MYO3B. The results from this study add to our understanding of variation in host control of infection and suggest that genetic marker-based selection for resistance to bTB has the potential to make a significant contribution to bTB control.     

Pyrethroid Resistance in an Anopheles funestus Population from Uganda

Background

The susceptibility status of Anopheles funestus to insecticides remains largely unknown in most parts of Africa because of the difficulty in rearing field-caught mosquitoes of this malaria vector. Here we report the susceptibility status of the An. funestus population from Tororo district in Uganda and a preliminary characterisation of the putative resistance mechanisms involved.

Methodology/Principal Findings

A new forced egg laying technique used in this study significantly increased the numbers of field-caught females laying eggs and generated more than 4000 F₁ adults. WHO bioassays indicated that An. funestus in Tororo is resistant to pyrethroids (62% mortality after 1 h exposure to 0.75% permethrin and 28% mortality to 0.05% deltamethrin). Suspected DDT resistance was also observed with 82% mortality. However this population is fully susceptible to bendiocarb (carbamate), malathion (organophosphate) and dieldrin with 100% mortality observed after exposure to each of these insecticides. Sequencing of a fragment of the sodium channel gene containing the 1014 codon conferring pyrethroid/DDT resistance in An. gambiae did not detect the L1014F kdr mutation but a correlation between haplotypes and resistance phenotype was observed indicating that mutations in other exons may be conferring the knockdown resistance in this species. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA compared to a susceptible strain of Anopheles gambiae. RT-PCR further confirmed the involvement of P450s with a 12-fold over-expression of CYP6P9b in the Tororo population compared to the fully susceptible laboratory colony FANG.

Conclusion

This study represents the first report of pyrethroid/DDT resistance in An. funestus from East Africa. With resistance already reported in southern and West Africa, this indicates that resistance in An. funestus may be more widespread than previously assumed and therefore this should be taken into account for the implementation and management of vector control programs in Africa.     

Lifelong reduction of LDL-cholesterol related to a common variant in the LDL-receptor gene decreases the risk of coronary artery disease--a Mendelian Randomisation study

Background

Rare mutations of the low-density lipoprotein receptor gene (LDLR) cause familial hypercholesterolemia, which increases the risk for coronary artery disease (CAD). Less is known about the implications of common genetic variation in the LDLR gene regarding the variability of cholesterol levels and risk of CAD.

Methods

Imputed genotype data at the LDLR locus on 1 644 individuals of a population-based sample were explored for association with LDL-C level. Replication of association with LDL-C level was sought for the most significant single nucleotide polymorphism (SNP) within the LDLR gene in three European samples comprising 6 642 adults and 533 children. Association of this SNP with CAD was examined in six case-control studies involving more than 15 000 individuals.

Findings

Each copy of the minor T allele of SNP rs2228671 within LDLR (frequency 11%) was related to a decrease of LDL-C levels by 0.19 mmol/L (95% confidence interval (CI) [0.13–0.24] mmol/L, p = 1.5×10⁻¹⁰). This association with LDL-C was uniformly found in children, men, and women of all samples studied. In parallel, the T allele of rs2228671 was associated with a significantly lower risk of CAD (Odds Ratio per copy of the T allele: 0.82, 95% CI [0.76–0.89], p = 2.1×10⁻⁷). Adjustment for LDL-C levels by logistic regression or Mendelian Randomisation models abolished the significant association between rs2228671 with CAD completely, indicating a functional link between the genetic variant at the LDLR gene locus, change in LDL-C and risk of CAD.

Conclusion

A common variant at the LDLR gene locus affects LDL-C levels and, thereby, the risk for CAD.