DM Genes Research Articles

Molecular diagnosis in patients with monogenic diabetes mellitus, and detection of a novel candidate gene

AimWe aimed to investigate molecular genetic basis of monogenic diabetes (DM) and novel responsible candidate genes with targeted Next Generation Sequencing (NGS) and Whole Exome Sequencing (WES). MethodsA hundred cases presenting with clinical findings and a family history of monogenic DM were included in the study. Molecular analysis was performed using an NGS panel including 14 genes. Following targeted NGS, WES was planned in cases in whom no variant was detected. ResultsThirty different disease-causing variants in seven different genes were detected in thirty-five (35 %) cases with targeted NGS approach. Most common pathogenic variant was found in GCK gene in 25 (25 %) cases. Four different variants were detected in 4 (4 %) patients in ABCC8 gene. In 45 of 65 cases; WES analyses were done. A heterozygous c.2635C > T(p.Gln879Ter) variant was detected in IFIH1 gene in a patient with incidental hyperglycemia. In the segregation analysis affected mother was shown to be heterozygous for the same variant. ConclusionMolecular etiology was determined in 35 % cases with the NGS targeted panel. Seventeen novel variants in monogenic DM genes have been identified. A candidate gene determined by WES analysis in a case that could not be diagnosed with NGS panel in this study.

The Epigenetic Link of Perinatal BMI to Maternal Phenotype and Fetal Heart Rate

Obesity is a metabolic stressor associated with a suboptimal physiological milieu including an inflammatory state and a dysregulated stress axis. Perinatal maternal BMI impacts pregnancy and birth outcomes along with child metabolic and neurodevelopmental health that are believed to be mediated by epigenetic mechanisms. Our research examines the impacts of pre-pregnancy BMI in a cohort of healthy pregnancies (n=187); first, by characterizing maternal phenotype throughout pregnancy, including her stress physiology (diurnal salivary cortisol), inflammatory status (blood cytokine levels), perinatal depression (EPDS scores), and the underlying epigenetic signature (DNA methylation) in blood that could mediate these effects. Second, we examine the effects of pre-pregnancy BMI on fetal heart rate measurements as an early marker of programming effects of altered autonomic nervous system and the associated placental DNA methylation with a focus on sex-dimorphic effects. Finally, we explore the impact of increasing the allostatic load of high BMI with stress, which was phenotyped in women early in pregnancy driven by data with 27 variables assessing psychological and physical health, on differential placental DNA methylation. To date: regional analysis revealed about 4500 differentially methylated regions (DMR) overall in response to high BMI. Gene ontology enrichment analysis revealed that DM genes annotated to these DMRs were enriched for biological processes involving inflammation in males and RNA silencing in females. This work highlights that the metabolic stress associated with BMI could leave a molecular fingerprint that mediate other maternal physiological and psychological outcomes, which in turn program the fetal neurodevelopmental outcomes.

Registration of HA‐DM12, HA‐DM13, and HA‐DM14 oilseed sunflower germplasms with resistance to sunflower downy mildew and rust

AbstractDowny mildew (DM) and rust are two major global sunflower (Helianthin annuus L.) diseases causing significant yield losses and reducing seed quality. Host plant resistance mediated by dominant race‐specific genes has been extensively used in sunflower production to control these diseases. However, the considerable variability of the DM and rust pathogens caused by mutation or recombination has changed the dynamics of the diseases, significantly increasing their incidence in recent years. This necessitates the development and release of sunflower germplasm with enhanced levels of disease resistance. Germplasm lines HA‐DM12 (Reg. no. GP‐382, PI 700006), HA‐DM13 (Reg. no. GP‐383, PI 700007), and HA‐DM14 (Reg. no. GP‐384, PI 700008), all with multiple DM and rust resistance, were developed using phenotypic evaluation and marker‐assisted selection. The three lines share a common DM gene (PlArg) and a rust gene (R12) and a combination of different DM genes (Pl8, Pl17, and Pl18, respectively). The triple‐gene pyramids—HA‐DM12 (Pl8Pl8/PlArgPlArg/R12R12), HA‐DM13 (Pl17Pl17/PlArgPlArg/R12R12), and HA‐DM14 (Pl18Pl18/PlArgPlArg/R12R12)—confer comprehensive resistance to all races of DM and rust identified so far and can be used in sunflower breeding programs to develop hybrids with durable resistance to DM and rust.

Abstract 6000: Distinct HOX gene family DNA methylation profiles in histologically normal skin dependent on dermoscopic pattern of adjacent nevi

Abstract Melanoma may arise within a pre-existing nevus, but commonly forms in the skin adjacent to nevi. We have performed global DNA methylation profiling using the Illumina EPIC array (>800K loci) of 32 dermoscopically ('globular' vs 'non-globular' pattern) and histopathologically classified nevi, together with matching adjacent perilesional skin, and discovered that methylation patterns can accurately decompose the proportion of cell types present in the tissue. In this study, we sought to assess whether there are differences in the DNA methylation profiles of histologically normal skin samples that surround nevi, independent of cellular composition. Among the 739,270 CpG loci included in the analysis, there were a total of 8,200 CpG loci identified as significantly differentially methylated (Q < 0.05) in ‘globular skin’ vs ‘non-globular skin’ adjusted for age, sex, and the estimated relative proportions of epithelial cells, fibroblasts, and melanocytes in each sample. After imposing a more stringent threshold (M value of ≥0.5) a total 816 CpG loci were found to be DM, with 696 loci identified as significantly hypermethylated, and 120 loci significantly hypomethylated (Q < 0.05 and ΔM > 0.5) in ‘globular’ vs ‘non-globular’ skin. Mapping of the DM (Q < 0.05 and |ΔM| < 0.5) CpG loci to their respective genes revealed that eight of the top 20 DM genes, by proportion of differentially methylated loci, were positioned in proximity to suggest regulation of members of the HOX gene family. We next assessed the differential gene expression of the eight DM HOX genes in the perilesional skin as well as the matching adjacent nevus. After adjusting for age, sex, and sample cellular composition, none of the assessed HOX genes demonstrated statistically significant (P ≤ 0.05) differential expression in the nevi, however, ‘non-globular skin’ demonstrated a statistically significant (P < 0.05) increase in the expression of five of the eight HOX genes relative to ‘globular skin’. Importantly, this increased gene expression corresponds to the methylation status of the DM loci. HOX genes are known to be involved in limb and axial development as well as the positioning of dermal fibroblasts. Moreover, melanocytes from different anatomic positions have distinct transcriptional profiles and the anatomical position of melanocytes plays a key role in determining whether genetic alterations will subsequently drive melanoma formation. This positioning of melanoma subtypes (cutaneous vs acral) was recently shown to be related to interaction of specific HOX genes with site-specific oncogenic alterations. The distinct HOX gene family members we have revealed, differ to those related to melanoma development and positioning, as such we propose that these may contribute to the positioning of acquired melanocytic nevi, and in combination with common somatic alterations in BRAF, may contribute to nevus formation. Citation Format: Meghan E. Muse, Helmut Schaider, Harald Oey, H. Peter Soyer, Brock C. Christensen, Mitchell S. Stark. Distinct HOX gene family DNA methylation profiles in histologically normal skin dependent on dermoscopic pattern of adjacent nevi [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6000.

Registration of HA‐DM9, HA‐DM10, and HA‐DM11 oilseed sunflower germplasms with dual resistance to sunflower downy mildew

AbstractDowny mildew (DM), caused by the obligate pathogen Plasmopara halstedii (Farl.) Berl. & de Toni, is a worldwide yield‐limiting disease of sunflower (Helianthus annuus L.). To obtain durable DM resistance in sunflower hybrids, there is a need to stack new resistance genes into the best available germplasm. The DM resistance genes Pl8, Pl17, and Pl18 were recently fine mapped to sunflower chromosomes 13, 4, and 2, respectively. The current study pyramided these genes into a single genotype by marker‐assisted selection. Three oilseed sunflower germplasms, HA‐DM9 (Reg. no. GP‐381, PI 700009), HA‐DM10 (Reg. no. GP‐379, PI 700004), and HA‐DM11 (Reg. no. GP‐380, PI 700005), are dual DM‐resistant germplasm lines developed and have been released by the USDA‐ARS, Sunflower and Plant Biology Research Unit in collaboration with the North Dakota Agricultural Experiment Station in 2021. The three lines, HA‐DM9 (Pl8Pl8/Pl17Pl17), HA‐DM10 (Pl8Pl8/Pl18Pl18), and HA‐DM11 (Pl17Pl17/Pl18Pl18), each stacked two different DM genes, providing broad resistance to all P. halstedii races identified in North America and Europe so far, and can be used in sunflower breeding programs to enhance DM resistance.

Genome-wide association mapping reveals genomic regions frequently associated with lettuce field resistance to downy mildew.

GWAS identified 63 QTLs for resistance to downy mildew. Though QTLs were distributed across all chromosomes, the genomic regions frequently associated with resistance were located on chromosomes 4 and 5. Lettuce downy mildew is one of the most economically important diseases of cultivated lettuce worldwide. We have applied the genome-wide association mapping (GWAS) approach to detect QTLs for field resistance to downy mildew in the panel of 496 accessions tested in 21 field experiments. The analysis identified 131 significant marker-trait associations that could be grouped into 63 QTLs. At least 51 QTLs were novel, while remaining 12 QTLs overlapped with previously described QTLs for lettuce field resistance to downy mildew. Unlike race-specific, dominant Dm genes that mostly cluster on three out of nine lettuce chromosomes, QTLs (qDMR loci) for polygenic resistance are randomly distributed across all nine chromosomes. The genomic regions frequently associated with lettuce field resistance to downy mildew are located on chromosomes 4 and 5 and could be used for detailed study of the mechanism of polygenic resistance. The most resistant accessions identified in the current study (cvs. Auburn, Grand Rapids, Romabella, PI 226514, and PI 249536) are being incorporated into our breeding program. Markers closely linked to the resistance QTLs could be potentially used for marker-assisted selection, or in combination with other markers in the genome, for a combined genomic and marker-assisted selection. Up to date this is the most comprehensive study of QTLs for field resistance to downy mildew and the first study that uses GWAS for mapping disease resistance loci in lettuce.

Intronic Architecture Links DNA Methylation to Gene Expression and Helps Drive Subtype-Specific Transcriptional Landscapes in DNMT3A- and IDH1/2-Mutant Acute Myeloid Leukemias (AML)

Mutations in DNMT3A and IDH1/2 are each found in ~20% of AML patients. 10-15% of AMLs carry mutations in both genes (herein, double mutants), resulting in a unique methylation landscape and upregulation of a signaling signature. In murine models, the presence of both mutations results in greater leukemogenic potential. However, the specific mechanism through which DNA methylation (DNAme) drives gene expression programs in double mutants remains unclear. We hypothesized that the link between DNAme and gene expression would be explained by more than simple proximity, and that the genomic architecture of the affected genes would play a key role.To test this, we first performed an unbiased correlation analysis of gene expression with DNAme at all CpG sites (mCs) located within the same topologically associated domain (TAD). We identified 406 genes with significant (FDR> 5% and absolute rho > 0.5) expression-methylation correlations with mCs proximal to the respective genes (herein the E-M gene set). In addition, another 2,088 genes (the L E-M set) were identified with long-range correlations (>2Kb from the gene body) with mCs in the respective TAD (median distance = 451 Kb). As a set, the E-M genes significantly overlapped (P < 10 -2) with genes identified as either differentially expressed (DE; n=890) or differentially methylated (DM; n= 4,006) between IDH1/2 and DNMT3A mutant AMLs. Notably, a simple overlap analysis of DE and DM genes showed no significant overlap between them, thus demonstrating that correlation analysis performed better in bridging the epigenome with the transcriptome. DAVID and Gene Set Enrichment Analysis on the genes ranked by correlation strength revealed that signaling, fructose and lipid metabolism pathways are enriched in the E-M gene set (FDR < 5%) but not in the L E-M set. Analysis of transcription factor (TF) binding profiles did not reveal a common set of TF(s) binding to the mCs proximal to the genes of the identified pathways. Thus, we hypothesized that the E-M genes have other structural characteristics in common that drive regulation through DNAme, for which we focused on their genomic architecture. This analysis revealed that introns of genes in both the E-M and L E-M sets are significantly denser in Mammalian Interspersed Repeats (MIR) than expected by random chance (P < 10 -2). Additionally, E-M genes were significantly sparser in endogenous retroviruses (ERVL) and primate-specific Alu elements. mCs with significant correlations were also enriched at MIR and depleted from Alu elements (P < 10 -2), thus creating a regulatory network between mCs and genes with MIR sequences as the common denominator. Genome-wide, CpGs within retrotransposons that were differentially methylated among the three AML subtypes were enriched at enhancer regions or coding genes, particularly the E-M genes. Furthermore, the Dnmt3a knock-out (KO) or Idh2 R140Q knock-in mouse models display the same architectural biases at genes correlated with DNAme as the E-M genes identified in the human samples. Next, we sought to put our findings in the context of normal hematopoiesis and found that genes upregulated during normal hematopoietic differentiation are significantly denser in MIR elements and sparser of Alu elements than expected (P < 10 -2). Alignment of the leukemic samples within normal differentiation trajectories revealed that double mutants resembled differentiated cell types more closely, while DNMT3A and IDH1/2 single mutants resembled hematopoietic stem cells. The E-M and L E-M sets significantly overlapped (P < 10 -2) with those genes upregulated during myeloid but not erythroid or lymphoid differentiation, demonstrating that genes regulated by DNAme are at the core of the biology of these AMLs.In summary, our integrative work sheds light on a novel mechanism in which epigenetic modifications can regulate gene expression through MIR sequences within introns of hematopoietic-relevant genes and we posit that overlapping CpG dinucleotides may act as recruiters or substrates of DNMT3A and/or TET proteins. This mechanism seems to also be active in normal hematopoiesis and thus, is hijacked by leukemic cells. Therefore, our findings identify retrotransposons as a missing link in the understanding of epigenetic regulation of gene expression, reveal a previously uncharacterized role for these elements in leukemogenesis, and point to different cells of origin for each AML subtype. DisclosuresNo relevant conflicts of interest to declare.

Polymorphism of DMA (DM a Chain) gene in IPB-D2 chicken

The DM gene group is a non-classical MHC Class II gene group that plays a role in the process of presenting antigens by producing DM protein. The DMA gene, which is a group of DM genes, has an a chain that helps present exogenous antigens to CD4 + via MHC class II molecules. IPB-D2 chickens are the result of selection from IPB-D1 chickens which have disease-resistant trait. The aim of this study was to determine genetic polymorphism of DMA genes in IPB-D2 chickens. In this study, blood samples were collected from 21 weeks old chickens that are raised in the field laboratory of the Faculty of Animal Science IPB University. The PCR was used to amplify 601 bp fragments for DMA gen locus. SNP identification used direct sequencing method and analyzed using MegaX and Bioedit. The results found 4 SNPs, namely base position c.2328 G>A on exon 3, c.2c612 G>A, c.2686 G>A, and c.2710 G>A on exon 4. All SNPs are polymorphic and in Hardy-Weinberg equilibrium. The highest allele frequency for each SNP is allele A (0.5745; 0.5851; 0.9362), except at SNP c.2612 G>A (0.4575).

Identification of Major Quantitative Trait Loci Controlling Field Resistance to Downy Mildew in Cultivated Lettuce (Lactuca sativa).

Lettuce downy mildew, caused by Bremia lactucae Regel, is the most economically important foliar disease of lettuce (Lactuca sativa L.). The deployment of resistant cultivars carrying dominant resistance genes (Dm genes) plays a crucial role in integrated downy mildew disease management; however, high variability in pathogen populations leads to the defeat of plant resistance conferred by Dm genes. Some lettuce cultivars exhibit field resistance that is only manifested in adult plants. Two populations of recombinant inbred lines (RILs), originating from crosses between the field resistant cultivars Grand Rapids and Iceberg and susceptible cultivars Salinas and PI491224, were evaluated for downy mildew resistance under field conditions. In all, 160 RILs from the Iceberg × PI491224 and 88 RILs from the Grand Rapids × Salinas populations were genotyped using genotyping by sequencing, which generated 906 and 746 high-quality markers, respectively, that were used for quantitative trait locus (QTL) analysis. We found a QTL in chromosome 4 that is present in both Grand Rapids × Salinas and Iceberg × PI491224 populations that has a major effect on field resistance. We also found two additional significant QTLs in chromosomes 2 and 5 in the Iceberg × PI491224 RIL population. Marker-assisted gene pyramiding of multiple Dm genes in combination with QTLs for field resistance provide the opportunity to develop cultivars with more durable resistance to B. lactucae.

Identification and mapping of new genes for resistance to downy mildew in lettuce

Key messageEleven new major resistance genes for lettuce downy mildew were introgressed from wild Lactuca species and mapped to small regions in the lettuce genome.Downy mildew, caused by the oomycete pathogen Bremia lactucae Regel, is the most important disease of lettuce (Lactuca sativa L.). The most effective method to control this disease is by using resistant cultivars expressing dominant resistance genes (Dm genes). In order to counter changes in pathogen virulence, multiple resistance genes have been introgressed from wild species by repeated backcrosses to cultivated lettuce, resulting in numerous near-isogenic lines (NILs) only differing for small chromosome regions that are associated with resistance. Low-pass, whole genome sequencing of 11 NILs was used to identify the chromosome segments introgressed from the wild donor species. This located the candidate chromosomal positions for resistance genes as well as additional segments. F2 segregating populations derived from these NILs were used to genetically map the resistance genes to one or two loci in the lettuce reference genome. Precise knowledge of the location of new Dm genes provides the foundation for marker-assisted selection to breed cultivars with multiple genes for resistance to downy mildew.

Morphological and genetic diversity of Slovene lettuce landrace ‘Ljubljanska ledenka’ (Lactuca sativa L.)

A set of accessions of lettuce landrace ‘Ljubljanska ledenka’ (Lactuca sativa L.) was characterized by morphological and molecular markers and for resistance to Bremia lactucae, with the aim of assessing the variability of the collection, exploring the genetic structure and potentially identifying the characters responsible for differentiation of the accessions. Wide phenotypic variation was observed among 51 accessions screened for 26 morphological and phenological traits. UPGMA cluster analysis and principal component analysis based on phenotypic data enabled the studied accessions to be divided into four clusters. The most important character by which the largest two clusters were differentiated was anthocyanin coloration. The clustering pattern of the AFLP dendrogram where two major clusters were identified was similar but not identical to the pattern of the phenotypic dendrogram. The Mantel test showed a high correlation between the phenotypic and the molecular data obtained (r = 0.67). In spite of the weak genetic differentiation between the accessions, STRUCTURE analysis based on AFLP data provided a clear indication for the existence of sub-groups within the two clusters. Thirty-four accessions were screened for resistance to 12 races of B. lactucae. The results show that the accessions very frequently express various reaction patterns of race specificity. Expression of race-specificity was not uniform across the set of accessions and at least 11 different reaction patterns were recorded, indicating that different race-specific resistance factors (R-factors) or genes (Dm genes) could be expected. This conclusion is supported by the results obtained in morphological and molecular studies, showing that lettuce landrace ‘Ljubljanska ledenka’ is not genetically uniform and is represented by a variety of genotypes.

Genome-wide screening of DNA methylation in bovine blastocysts with different kinetics of development

Background The timing of the first cell divisions may predict the developmental potential of an embryo, including its ability to establish pregnancy. Besides differences related to metabolism, stress, and survival, embryos with different speeds of development present distinct patterns of gene expression, mainly related to energy and lipid metabolism. As gene expression is regulated by epigenetic factors, and that includes DNA methylation patterns, in this study we compared the global DNA methylation profile of embryos with different kinetics of development in order to identify general pathways and regions that are most influenced by this phenotype. For this purpose, bovine embryos were in vitro produced using sexed semen (female), classified as fast (four or more cells) or slow (two cells) at 40 hpi and cultured until blastocyst stage, when they were analyzed.ResultsGenome-wide DNA methylation analysis identified 11,584 differently methylated regions (DMRs) (7976 hypermethylated regions in fast and 3608 hypermethylated regions in slow embryos). Fast embryos presented more regions classified as hypermethylated distributed throughout the genome, as in introns, exons, promoters, and repeat elements while in slow embryos, hypermethylated regions were more present in CpG islands. DMRs were clustered by means of biological processes, and the most affected pathways were related to cell survival/differentiation and energy/lipid metabolism. Transcripts profiles from DM genes connected with these pathways were also assessed, and the most part disclosed changes in relative quantitation.ConclusionThe kinetics of the first cleavages influences the DNA methylation and expression profiles of genes related to metabolism and differentiation pathways and may affect embryo viability.

Resistance to Downy Mildew in Lettuce 'La Brillante' is Conferred by Dm50 Gene and Multiple QTL.

Many cultivars of lettuce (Lactuca sativa L.) are susceptible to downy mildew, a nearly globally ubiquitous disease caused by Bremia lactucae. We previously determined that Batavia type cultivar 'La Brillante' has a high level of field resistance to the disease in California. Testing of a mapping population developed from a cross between 'Salinas 88' and La Brillante in multiple field and laboratory experiments revealed that at least five loci conferred resistance in La Brillante. The presence of a new dominant resistance gene (designated Dm50) that confers complete resistance to specific isolates was detected in laboratory tests of seedlings inoculated with multiple diverse isolates. Dm50 is located in the major resistance cluster on linkage group 2 that contains at least eight major, dominant Dm genes conferring resistance to downy mildew. However, this Dm gene is ineffective against the isolates of B. lactucae prevalent in the field in California and the Netherlands. A quantitative trait locus (QTL) located at the Dm50 chromosomal region (qDM2.2) was detected, though, when the amount of disease was evaluated a month before plants reached harvest maturity. Four additional QTL for resistance to B. lactucae were identified on linkage groups 4 (qDM4.1 and qDM4.2), 7 (qDM7.1), and 9 (qDM9.2). The largest effect was associated with qDM7.1 (up to 32.9% of the total phenotypic variance) that determined resistance in multiple field experiments. Markers identified in the present study will facilitate introduction of these resistance loci into commercial cultivars of lettuce.

Transcriptomic and Epigenomic Reponses During Human Adaptation to High‐Altitude Hypoxia

Little is known about early genomic responses in human adaptation to high‐altitude hypoxia. At 5260 m (PB=406 Torr) we studied transcriptomic and epigenomic responses of 21 young, healthy men and women during 16 days, and again on return after 7 days in normoxia. Physiological responses showed marked improvement in performance at day 16 compared to day 1, with better SaO2%, submaximal work capacity and cognitive function, and complete protection from altitude illness. Most of these improvements were maintained after 7 days in normoxia. Accompanying these changes, 6350 genes were differentially expressed (DE, q&lt;=0.01) from sea level (SL) on day 7/16, with 3557 (48%/52% up/down) only on day 7 (early), 1418 at both day 7 &amp; 16 (40%/60% up/down), and on day 16 (late) 1375 (44%/56% up/down). Differential methylation (DM, q&lt;0.01) was also observed genome‐wide, and followed a similar time pattern. The DE and DM genes include many known hypoxia‐responsive genes (HIF1α, HIF2α, VEGF, HMOX, etc.). Surprisingly, 1608 genes were DM from SL to day 16, and maintained their DM status after 7 days in normoxia, making these genes prime candidates for control of the onset and retention of physiological adjustments to hypoxia. These data provide a unique resource for future exploration of the molecular and cellular mechanisms controlling human adaptation to hypoxia. Funding for AltitudeOmics was provided, in part, by US DoD W81XWH‐11‐2‐0040 (RCR).

Specific in planta recognition of two GKLR proteins of the downy mildew Bremia lactucae revealed in a large effector screen in lettuce.

Breeding lettuce (Lactuca sativa) for resistance to the downy mildew pathogen Bremia lactucae is mainly achieved by introgression of dominant downy mildew resistance (Dm) genes. New Bremia races quickly render Dm genes ineffective, possibly by mutation of recognized host-translocated effectors or by suppression of effector-triggered immunity. We have previously identified 34 potential RXLR(-like) effector proteins of B. lactucae that were here tested for specific recognition within a collection of 129 B. lactucae-resistant Lactuca lines. Two effectors triggered a hypersensitive response: BLG01 in 52 lines, predominantly L. saligna, and BLG03 in two L. sativa lines containing Dm2 resistance. The N-terminal sequences of BLG01 and BLG03, containing the signal peptide and GKLR variant of the RXLR translocation motif, are not required for in planta recognition but function in effector delivery. The locus responsible for BLG01 recognition maps to the bottom of lettuce chromosome 9, whereas recognition of BLG03 maps in the RGC2 cluster on chromosome 2. Lactuca lines that recognize the BLG effectors are not resistant to Bremia isolate Bl:24 that expresses both BLG genes, suggesting that Bl:24 can suppress the triggered immune responses. In contrast, lettuce segregants displaying Dm2-mediated resistance to Bremia isolate Bl:5 are responsive to BLG03, suggesting that BLG03 is a candidate Avr2 protein.

The non-classical class II genes of the chicken, DMA and DMB, are similar to those of mammals, but a second DMB gene (DMB1) is differentially expressed with unusual regulatory and structural features.

Event Abstract Back to Event The non-classical class II genes of the chicken, DMA and DMB, are similar to those of mammals, but a second DMB gene (DMB1) is differentially expressed with unusual regulatory and structural features. Aimee Parker1*, Karen Staines2, Colin Butter2 and Jim Kaufman1, 3 1 University of Cambridge, Department of Pathology, United Kingdom 2 Institute for Animal Health, Avian Viral Immunology, United Kingdom 3 University of Cambridge, Department of Veterinary Medicine, United Kingdom Although smaller, simpler and rearranged, the chicken major histocompatibility complex (MHC) contains most of the core antigen presentation genes found in the mammalian MHC. Strong associations of chicken MHC haplotypes with resistance to pathogens and response to vaccines are attributed to single dominantly- expressed MHC class I (BF) and class II (BL) molecules. The single dominant class I is the result of co-evolution with the closely-linked antigen processing genes TAP and tapasin, but the situation for class II is less clear. To facilitate investigation of whether single dominant class II expression could result from similar co-evolution with associated antigen presentation molecules, we characterised the chicken class II DM region. We show that chickens have three DM genes in their MHC, a single alpha chain gene DMA, and, unusually, two beta chain genes DMB1 and DMB2, of which DMB2 is dominantly-expressed. The chicken DM genes encode proteins with high structural and sequence homology to those described in mammals. However, the two beta chains differ substantially at the amino acid level and several features of DMB1 suggest an unusual function. We find DMB1 and DMB2 differentially expressed in tissues at the RNA and protein level, with DMB1 strongly expressed in intestinal epithelial cells, suggestive of a role in gut immunity. In pursuing the functional properties of the DMs and their interaction with the classical class II molecules, we aim to enhance our understanding of chicken MHC associations with immune responses and, more generally, illuminate mechanisms underlying class II evolution. Keywords: MHC, DM, antigen processing and presentation, Chickens, intestinal immunity, evolution Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Parker A, Staines K, Butter C and Kaufman J (2013). The non-classical class II genes of the chicken, DMA and DMB, are similar to those of mammals, but a second DMB gene (DMB1) is differentially expressed with unusual regulatory and structural features.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01174 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Aimee Parker, University of Cambridge, Department of Pathology, Cambridge, Cambridgeshire, CB2 1QP, United Kingdom, ap509@cam.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Aimee Parker Karen Staines Colin Butter Jim Kaufman Google Aimee Parker Karen Staines Colin Butter Jim Kaufman Google Scholar Aimee Parker Karen Staines Colin Butter Jim Kaufman PubMed Aimee Parker Karen Staines Colin Butter Jim Kaufman Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Organisation and diversity of the class II DM region of the chicken MHC

In mammals, the DM molecules are encoded by the major histocompatibility complex (MHC) and execute key functions in the class II antigen presentation pathway. Here, we characterised three DM genes in the MHC B region of the chicken (Gallus gallus): B-DMA, B-DMB1 and B-DMB2. They encode one class II DM α chain and two β chains, exhibiting motifs of chicken class II molecules as well as specificities of mammal DM proteins. We also studied the expression pattern of those three chicken B-DM genes; they are expressed in immune related tissues. Thus we provide the comprehensive description of the genomic sequence of a class II α gene in the chicken and a valuable description of DM genes in a non-mammalian vertebrate, reinforcing the hypothesis of the existence of DM genes in the primordial MHC, as suggested by previous studies in mammals. We were also able to reconstruct 124 haplotypes corresponding to the 8.8kb B-DM region, in accordance with the 212 SNPs identified in 146 individuals representing a wide range of experimental, commercial, and local breeds from Europe, Asia and Africa, and three wild species of fowl. We also discovered a repeat inside the B-DMA second intron, making possible the design and the typing of a new marker for the chicken MHC, linked to the class II region. Therefore this study not only describes three DM genes in the chicken, it also provides an overview of MHC diversity in the chicken.

Abstract 246: Genome-wide screening for dominant modifiers in Drosophila identified new genes involved in BCR-ABL signaling and chronic myeloid leukemia (CML) progression

Abstract Despite the role of Bcr-Abl in the pathogenesis of CML is well established, the mechanisms responsible for CML progression remain unknown. The aim of the study was to perform a genome-wide screening to identify new genes and pathways leading to CML progression. We performed the genetic screening using our set-up model of human p210 Bcr-Abl (hBcr-Abl) transgenic Drosophila melanogaster (Dm) in which the tissue specific expression of hBcr-Abl is able to induce a severe eye phenotype or the formation of melanotic tumors, when expressed into the fly lymph gland, the Dm hematopoietic system. The whole fly genome containing 14.000 genes was analyzed using 278 fly stocks carrying well characterized chromosome deletions. The resulting progeny was screened using eye and lymph gland as first and second read-out system respectively. Data obtained from both screens were analyzed using the Gene Ontology software. These results were compared with gene expression signatures of CML from Microarray data. As final point, the identified genes were validated analyzing either BM or PB samples from CML patients and healthy donors. The analysis of eye/lymph gland-phenotypes in the progeny obtained from screen crosses, shows a first group of flies (38%) displaying a more aggressive phenotype since they lack genes encoding for hBcr-Abl negative regulators and a second group (32%) showing a mild phenotype due to the absence of genes involved in the oncogenic signalling. 42% of the 4000 Dm genes mapping in these regions displayed a known human counterpart. Gene Ontology profiles of these genes included oncogenes, tumor suppressors and genes involved in the regulation of transcription, signal transduction, proliferation, differentiation, apoptosis and splicing. Moreover, a computational comparison of our results in fly with gene expression signatures of CML from Microarray data, showed only a partial overlap. Interestingly, the 72% of identified genes was not known to be involved in human leukaemia. However, further confirmation of our findings comes from the validation in human samples in which 1250 genes were found to be associated with CML; among these genes, we found not only an alteration of their expression profiles in CML patients compared to the healthy donors, but also protein alterations such as expression of different splicing forms or misplaced proteins, suggesting that Dm screening is a valid approach to identify not only differentially expressed genes but also specific pathways and genes otherwise altered by hBcr-Abl. In conclusion, the identification of these genes allows identifying of the changes occurring in CML at the genomic level and gives deeper insights into the molecular basis of the disease; moreover this study points to specific gene pathways that might represent new targets for therapy in CML in order to prevent or overcome resistance and progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 246.

Genome-Wide Screening for Dominant Modifiers in Drosophila Identified New Cluster of Genes Involved in BCR-ABL Signalling and CML Progression.

Abstract 2186Poster Board II-163Despite the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukaemia (CML) is well established, the mechanisms responsible for CML progression remain largely unknown. The aim of the study was to perform a genome-wide screening to identify new genes and pathways leading to CML progression. We performed a genome-wide genetic screening using our set-up model of human p210 Bcr-Abl transgenic Drosophila melanogaster (Dm) in which the expression of hBcr-Abl in a tissue specific manner is able to induce a severe eye-glazed phenotype or the formation of melanotic tumors, (clusters of hemocytes) when expressed into the fly lymph gland which represent the Dm hematopoietic system. A wide modifier screening of the whole fly genome containing approx 14.000 genes was performed using 278 fly stocks commercially available and carrying well characterized chromosome deletions. The resulting progeny was screened using the eye phenotype as first read-out system. Furthermore each deletion responsible for phenotype changes was analyzed either by expressing it into lymph gland as second read-out system, in order to analyze their function into a haematopoietic background and to exclude genes involved in eye development, such as genes able to modify the eye phenotype even without being directly involved in Bcr-Abl oncogenic signalling (false positives). Data obtained from primary and secondary screen were first analyzed using the Gene Ontology software. These results were compared with gene expression signatures of CML from Microarray data. As final point, the identified candidate genes were tested and validated analyzing either BM or PB samples from CML patients and healthy donors. 14.000 Dm genes were analyzed for their capability to genetically interact with hBcr-Abl in the fly model. The analysis of eye/lymph gland-phenotypes in the progeny obtained from screening crosses, shows a first group of flies (38%) displaying a more aggressive phenotype since they lack genes encoding for hBcr-Abl negative regulators and a second group (32%) showing a mild phenotype due to the absence of genes involved in the oncogenic signalling. We found that 42% of the 4000 Dm genes mapping in the deleted regions able to modify Bcr-Abl phenotype, displayed a known human counterpart. GeneOntology profiles of these genes included oncogenes, tumor suppression genes and human genes encoding proteins involved in the regulation of transcriptions, signal transduction, proliferation and cell growth, differentiation, apoptosis and splicing processes. Moreover, a computational comparison of our results with gene expression signatures of CML from Microarray data, showed only a partially overlap between genes identified in fly screen and genes obtained from Microarray analysis. The 72% of identified genes in fact was not known to be involved in human leukaemia. However, further confirmation of our findings into fly comes from the validation in human samples in which 1250 genes were found to be significantly associated with human CML; among these genes, we found not only an alteration of their expression profiles in CML patients with respect to the healthy donors, but also protein alterations, such as expression of different splicing forms or misplaced proteins, suggesting that Dm screening is a valid approach able to identify not only differentially expressed genes but also specific pathways and genes otherwise altered by hBcr-Abl. In conclusion, the identification of these genes allows identifying of the changes occurring in CML at the genomic level and gives deeper insights into the molecular basis of the disease; moreover this study point to specific gene pathways that might represent new targets for therapy in CML in order to prevent or overcome resistance and progression Disclosures:No relevant conflicts of interest to declare.

Molecular cloning and sexually dimorphic expression of DMRT4 gene in Oreochromis aureus

The DM-domain gene family has at least eight members with conserved DNA-binding DM-domain, which encodes putative transcription factors related to the sexual regulator Dsx of Drosophila and Mab-3 of C. elegans. Although some of the DM genes are involved in sexual development, the function of most of these genes remains unclear. In this study, rapid amplification cDNA ends (RACE) was used for the isolation of DMRT4 full-length cDNA from the ovary of the blue tilapia Oreochromis aureus. The full-length of DMRT4 cDNA was 1,571 bp, containing the 148 bp 5'-untranslated region, 193 bp 3'-untranslated region and 1,230 bp open reading frame. The deduced amino acid sequence of the open reading frame (ORF) encoded a protein of 409 amino acids with a theoretical pI of 8.492 and a calculated molecular weight of 44.12 kDa. One conserved functional domain, DM-domain was identified in blue tilapia DMRT4. The DMRT4 full-length gene obtained from the blood was 1,741 bp, containing a 156 bp intron. Phylogenetic analysis indicated that the amino acid sequences encoded by DMRT4 genes from different species had a high degree of sequence identity as revealed in phylogenetic tree constructed. Real-time quantitative Reverse-Transcription Polymerase Chain Reaction (RT-PCR) was used to analyze the expression patterns of DMRT4 in different developmental stages and different tissues in Oreochromis aureus. DMRT4 mRNA was detected from early gastrulae stage during embryonic development, and maintained a considerable high level until 1 day post hatching. With the increase of age, enhanced DMRT4 mRNA was observed in ovary and brain. After 15 and 30 days, fries treated with 17beta-estradiol had a significant increase in DMRT4 mRNA levels compared with the control fries (P < 0.05). DMRT4 was found to be expressed in the ovary and endbrain, thalamencephalon, pituitary, not detected in the liver, kidney, spleen, heart and muscle of adult fish. These results showed that the DMRT4 gene have potentially important roles in gonadal development and may have contributed to the functional endocrine axis. Our study provides fundamental understanding to the structure and functions of DMRT4 protein and the mechanisms of sex control in Oreochromis aureus.