Letteratura scientifica selezionata sul tema "Non-syntenic association"

Rheumatoid arthritis (RA) is a polygenic autoimmune disease primarily affecting the synovial joints. Numerous animal models show similarities to RA in humans; some of them not only mimic the clinical phenotypes but also demonstrate the involvement of homologous genomic regions in RA. This paper compares corresponding non-MHC genomic regions identified in rodent and human genome-wide association studies (GWAS). To date, over 30 non-MHC RA-associated loci have been identified in humans, and over 100 arthritis-associated loci have been identified in rodent models of RA. The genomic regions associated with the disease are designated by the name(s) of the gene having the most frequent and consistent RA-associated SNPs or a function suggesting their involvement in inflammatory or autoimmune processes. Animal studies on rats and mice preferentially have used single sequence length polymorphism (SSLP) markers to identify disease-associated qualitative and quantitative trait loci (QTLs) in the genome of F2 hybrids of arthritis-susceptible and arthritis-resistant rodent strains. Mouse GWAS appear to be far ahead of rat studies, and significantly more mouse QTLs correspond to human RA risk alleles.

Abstract Loss of the entire or part of one copy of chromosome 7 [del(7/7q)] is a recurrent cytogenetic abnormality in MDS. Its strong association with previous exposure to alkylating agents, consistently poor response to therapy and discrete gene expression profile strongly suggest that del(7/7q)-MDS is a distinct disease in the MDS spectrum whose pathogenesis is intimately linked to loss of chr7q genetic material. Understanding the role of chr7q loss in cell biology can provide key insights into the pathogenesis of MDS and leukemogenesis. Narrowing down the responsible region on chr7q presents a challenge that has proved intractable with existing approaches. Chr7q deletions physically mapped in large cohorts of patients are typically very large and dispersed along most of the length of chr7q. Modeling in the mouse is problematic for reasons of synteny. Deletion of the syntenic region of one commonly deleted region (7q22 CDR) failed to demonstrate a phenotype. Several lines of evidence point to haploinsufficiency of chr7q genetic material – rather than a 2-hit model – as the underlying mechanism in del(7q)-MDS. No inactivating mutations of candidate 7q genes have been detected by resequencing the remaining allele and haploinsufficiency of coding and miRNA genes has been strongly linked to the pathogenesis of del(5q)-MDS. Haploinsufficiency can only be assessed through functional studies (and not genomic technologies), but these are currently hindered by the lack of a clearly described del(7q)-associated phenotype. With recent advances in human pluripotent stem cell (hPSC) research and genetic engineering technologies, reverse human genetics in an isogenic setting by disruption of genomic elements into their cognate genomic and cellular context - hitherto unthinkable for the human genome – are now a realistic prospect. To determine the impact of hemizygous chr7q loss on the cellular phenotype, we have generated isogenic del(7q)- and normal hPSCs by engineering deletions spanning variable overlapping regions encompassing the entire length of chr7q using adeno-associated virus (AAV)-mediated gene targeting combined with Cre-lox technology. Specifically, we targeted two inverted loxP sites together with a positive (puro) and a negative (HSVtk) selection marker in a near-telomeric region of chromosome 7q (7q36.3) into the H1 hESC line, as well as a karyotypically normal iPSC line (line 2-12) derived from BMMCs of a patient with del(7q)-MDS. Following transient expression of Cre recombinase and ganciclovir selection, clones were screened by qPCR probing different regions along the length of chr7. Four H1-derived and four 2-12-derived clones were selected following screening of 24 and 34 clones, respectively, and after excluding clones with additional chromosomal abnormalities by karyotyping and the exact extent of their chr7q deletions was mapped by aCGH. We focused our phenotypic characterization on two cellular phenotypes that we recently reported in del(7q)-iPSCs derived from MDS BMMCs: cell proliferation and in vitro hematopoietic differentiation potential. 7 of the 8 clones harboring deletions spanning variable lengths along the entire chr7q had a lower (by ½ log) proliferation rate than their corresponding isogenic parental lines. All these clones also exhibited a markedly reduced differentiation potential along all hematopoietic lineages and almost absent clonogenic capacity in methylcellulose. These cellular phenotypes are highly similar to those we find in our del(7q)-MDS-iPSCs. Notably, one of the eight clones (2-12.Cre-44), harboring a smaller deletion spanning 7q11.21-7q.31.1 retained comparable proliferation and differentiation capacity to that of normal isogenic and non-isogenic hPSCs. In conclusion, our results demonstrate that hemizygous loss of chr7q material recapitulates the cellular phenotypes of impaired proliferation and hematopoietic differentiation that we find in del(7q)-MDS-iPSCs, supporting a haploinsufficiency pathogenesis of del(7q)-MDS. Correlation of the phenotypes with the boundaries of chr7q deletions in our collection of hESC and iPSC clones points to a region spanning 7q31.1-7q36.1 as the critical region in del(7q)-MDS. Further studies in additional clones harboring smaller chr7q deletions will further narrow down the responsible region and guide prioritization of candidate genes. Disclosures: No relevant conflicts of interest to declare.

Abstract Common genetic variation associated with fetal hemoglobin (HbF) level and β-hemoglobin disorder clinical severity marks an erythroid enhancer within the BCL11A gene. The 12 kb intronic enhancer contains three ~1 kb erythroid DNase I hypersensitive sites (DHSs), termed +55, +58, and +62. Here we utilized a human adult-stage erythroid cell line to show by CRISPR-Cas9 mediated targeted deletion that the composite enhancer is required both for BCL11A expression and HbF repression. Because deletion of the entire enhancer is currently too inefficient to consider for a gene editing approach to hemoglobin disorders, we sought to define the critical features of the enhancer in its natural genomic context. We designed and synthesized a tiling pooled guide RNA (gRNA) library to conduct saturating mutagenesis of the enhancer sequences in situ using the CRISPR-Cas9 gene editing platform. The gRNAs direct Cas9 cleavage and non-homologous end-joining repair at discrete sites throughout the enhancer. By comparing the representation of lentiviral gRNA integrants in high and low HbF pools of the adult erythroid cells, we generated a functional map approaching nucleotide resolution of sequences within the enhancer influencing BCL11A regulation. We observed several discrete enhancer regions required for maximal expression. The largest effect was observed by producing mutations within a narrow functional core of the +58 DHS. These sequences include a GATA1 motif conserved among vertebrates located within a primate-specific context. This region constitutes an Achilles Heel for functional inactivation of the enhancer. We also identified rare genetic variants within the +58 DHS core in individuals with sickle cell disease that are associated with HbF level, independent of all known associations of common genetic variants. In parallel, we performed a similar saturating CRISPR mutagenesis screen of the corresponding murine Bcl11a enhancer. To our surprise, despite low-resolution evidence of conservation by primary sequence homology, syntenic genomic position, and shared chromatin signature, the mouse enhancer sequence determinants of BCL11A expression showed substantial functional divergence. The +58 orthologous sequences were dispensable whereas the +62 orthologous sequences were critically required in murine adult erythroid cells. These results were validated by producing targeted deletions in mouse and human adult erythroid cell lines. Furthermore we subjected cells to individual gRNAs to correlate individual nucleotide disruptions with loss of BCL11A expression. To substantiate the tissue-restricted effect of the enhancer mutations, we generated transgenic mice with deletion of the Bcl11a enhancer and found these sequences were dispensable for expression in developing neurons and B-lymphocytes (unlike conventional Bcl11a knockout) but essential for appropriate hemoglobin switching in vivo. We showed that in primary CD34+ hematopoietic stem and progenitor derived human erythroid precursors that delivery of an individual gRNA and Cas9 is sufficient to produce robust reinduction of HbF. These results validate the BCL11A erythroid enhancer as a promising therapeutic target. Our findings define the most favorable regions for generation of indel mutations in the BCL11A erythroid enhancer as a therapeutic genome editing strategy for HbF reinduction for the β-hemoglobin disorders. Disclosures Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Zhang:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Horizon Discovery: Membership on an entity's Board of Directors or advisory committees. Orkin:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Pfizer: Research Funding; Sangamo Biosciences: Consultancy.

Abstract Abstract 115 Extra copies of chromosome 21 (polysomy 21) is the most common somatic aneuploidy in B-cell acute lymphoblastic leukemia (B-ALL), including >90% of cases with high hyperdiploidy. In addition, children with Down syndrome (DS) have a 20-fold increased risk of developing B-ALL, of which ∼60% harbor CRLF2 rearrangements. To examine these associations within genetically defined models, we investigated B-lineage phenotypes in Ts1Rhr mice, which harbor triplication of 31 genes syntenic with the DS critical region (DSCR) on human chr.21. Murine pro-B cell (B220+CD43+) development proceeds sequentially through “Hardy fractions” defined by cell surface phenotype: A (CD24−BP-1−), B (CD24+BP-1−) and then C (CD24+BP-1+). Compared with otherwise isogenic wild-type littermates, Ts1Rhr bone marrow harbored decreased percentages of Hardy fraction B and C cells, indicating that DSCR triplication is sufficient to disrupt the Hardy A-to-B transition. Of note, the same phenotype was reported in human DS fetal liver B-cells, which have a block between the pre-pro- and pro-B cell stages (analogous to Hardy A-to-B). To determine whether DSCR triplication affects B-cell proliferation in vitro, we analyzed colony formation and serial replating in methylcellulose cultures. Ts1Rhr bone marrow (B6/FVB background) formed 2–3-fold more B-cell colonies in early passages compared to bone marrow from wild-type littermates. While wild-type B-cells could not serially replate beyond 4 passages, Ts1Rhr B-cells displayed indefinite serial replating (>10 passages). Ts1Rhr mice do not spontaneously develop leukemia, so we utilized two mouse models to determine whether DSCR triplication cooperates with leukemogenic oncogenes in vivo. First, we generated Eμ-CRLF2 F232C mice, which express the constitutively active CRLF2 mutant solely within B-cells. Like Ts1Rhr B-cells, (but not CRLF2 F232C B-cells) Ts1Rhr/CRLF2 F232C cells had indefinite serial replating potential. In contrast with Ts1Rhr B-cells, Ts1Rhr/CRLF2 F232C B-cells also engrafted into NOD.Scid.IL2Rγ−/− mice and caused fatal and serially transplantable B-ALL. Second, we retrovirally transduced BCR-ABL1 into unselected bone marrow from wild-type and Ts1Rhr mice and transplanted into irradiated wild-type recipients. Transplantation of transduced Ts1Rhr cells (106, 105, or 104) caused fatal B-ALL in recipient mice with shorter latency and increased penetrance compared to recipients of the same number of transduced wild-type cells. By Poisson calculation, the number of B-ALL initiating cells in transduced Ts1Rhr bone marrow was ∼4-fold higher than in wild-type animals (1:60 vs 1:244, P=0.0107). Strikingly, transplantation of individual Hardy A, B, and C fractions after sorting and BCR-ABL1 transduction demonstrated that the increased leukemia-initiating capacity almost completely resides in the Ts1Rhr Hardy B fraction; i.e., the same subset suppressed during Ts1Rhr B-cell differentiation. To define transcriptional determinants of these phenotypes, we performed RNAseq of Ts1Rhr and wild-type B cells in methylcellulose culture (n=3 biologic replicates per genotype). As expected, Ts1Rhr colonies had ∼1.5-fold higher RNA abundance of expressed DSCR genes. We defined a Ts1Rhr signature of the top 200 genes (false discovery rate (FDR) <0.25) differentially expressed compared with wild-type cells. Importantly, this Ts1Rhr signature was significantly enriched (P=0.02) in a published gene expression dataset of DS-ALL compared with non-DS-ALL (Hertzberg et al., Blood 2009). Query of >2,300 signatures in the Molecular Signatures Database (MSigDB) C2 Chemical and Genetic Perturbations with the Ts1Rhr signature identified enrichment in multiple gene sets of polycomb repressor complex (PRC2) targets and H3K27 trimethylation. Most notably, SUZ12 targets within human embryonic stem cells were more highly expressed in Ts1Rhr cells (P=1.2×10−6, FDR=0.003) and the same SUZ12 signature was enriched in patients with DS-ALL compared to non-DS-ALL (P=0.007). In summary, DSCR triplication directly suppresses precursor B-cell differentiation and promotes B-cell transformation both in vitro and by cooperating with proliferative alterations such as CRLF2 activation and BCR-ABL1 in vivo. Pharmacologic modulation of H3K27me3 effectors may overcome the pro-leukemogenic effects of polysomy 21. Disclosures: No relevant conflicts of interest to declare.

Abstract Maturity Onset Diabetes of the Young type 3 (MODY3), linked to mutations in the transcription factor HNF1A, is the most prevalent form of monogenic diabetes mellitus. HNF1alpha-deficiency leads to defective insulin secretion via a molecular mechanism that is still not completely understood. Moreover, in MODY3 patients the severity of insulin secretion can be extremely variable even in the same kindred, indicating that modifier genes may control the onset of the disease. With the use of a mouse model for HNF1alpha-deficiency, we show here that specific genetic backgrounds (C3H and CBA) carry a powerful genetic suppressor of diabetes. A genome scan analysis led to the identification of a major suppressor locus on chromosome 3 (Moda1). Moda1 locus contains 11 genes with non-synonymous SNPs that significantly interacts with other loci on chromosomes 4, 11 and 18. Mechanistically, the absence of HNF1alpha in diabetic-prone (sensitive) strains leads to postnatal defective islets growth that is remarkably restored in resistant strains. Our findings are relevant to human genetics since Moda1 is syntenic with a human locus identified by genome wide association studies of fasting glycemia in patients. Most importantly, our results show that a single genetic locus can completely suppress diabetes in Hnf1a-deficiency.

Genome-wide association studies (GWAS) have detected associations of genetic elements on human chromosome 2 with hypertension but lack the ability to discern whether these associations are/are not causal factors for hypertension. Using rat genetic models of hypertension, we have identified that factors linked to the inheritance of hypertension map to rat chromosome 9, which is homologous to human chromosome 2. Here we report that by applying high resolution mapping approaches, we have prioritized two regions on rat chromosome 9, one spanning < 788kb and a second region spanning < 81.8kb, as genomic segments containing novel inherited genetic elements controlling blood pressure. The <788kb region contains 3 protein coding genes, Trmp8 , Spp2 and Arl4c , which are also associated with human hypertension. Of these, there was only one nonsynonymous polymorphism within the gene Spp2 between the Dahl S and S.SHR strains used to map this locus. The expression of Spp2 was significantly higher in the S.SHR rat compared with S. We therefore prioritize Spp2 as a positional candidate for the <788kb region. The <81.8kb region contains no known/predicted protein-coding genes. We hypothesized that polymorphisms within gene regulatory elements underlie the <81.8kb locus. By combining the locations of the relevant polymorphisms with promoters predicted by the Proscan promoter prediction software, 5 regions were prioritized for further analysis. Alleles from the normotensive R rat from 1 out of these 5 regions had a 5.14 fold higher luciferase activity compared with that of the hypertensive S rat alleles (p<0.05). This region maps from chr9:80880396 to chr9:80882643 (Rnor5.0) on the rat genome. The downstream target for this promoter activity is unknown. Interestingly, within 110,419bp downstream of this region, there is a predicted long non-coding RNA in the mouse (AK079660) and in humans ( DIRC4) . By RT-PCR using rat kidney, we detected a novel rat transcript that is potentially a rat homologue of the mouse and human predictions. Collectively, these data point to conserved syntenic regions in rats and humans that contain novel promoter sequence variants and variants within the conserved gene, Spp2 , as potential quantitative trait nucleotides for blood pressure regulation.

ABSTRACT Human genetics influence a range of pathological and clinical phenotypes in respiratory infections; however, the contributions of disease modifiers remain underappreciated. We exploited the Collaborative Cross (CC) mouse genetic-reference population to map genetic modifiers that affect the severity of Pseudomonas aeruginosa lung infection. Screening for P. aeruginosa respiratory infection in a cohort of 39 CC lines exhibits distinct disease phenotypes ranging from complete resistance to lethal disease. Based on major changes in the survival times, a quantitative-trait locus (QTL) was mapped on murine chromosome 3 to the genomic interval of Mb 110.4 to 120.5. Within this locus, composed of 31 protein-coding genes, two candidate genes, namely, dihydropyrimidine dehydrogenase (Dpyd) and sphingosine-1-phosphate receptor 1 (S1pr1), were identified according to the level of genome-wide significance and disease gene prioritization. Functional validation of the S1pr1 gene by pharmacological targeting in C57BL/6NCrl mice confirmed its relevance in P. aeruginosa pathophysiology. However, in a cohort of Canadian patients with cystic fibrosis (CF) disease, regional genetic-association analysis of the syntenic human locus on chromosome 1 (Mb 97.0 to 105.0) identified two single-nucleotide polymorphisms (rs10875080 and rs11582736) annotated to the Dpyd gene that were significantly associated with age at first P. aeruginosa infection. Thus, there is evidence that both genes might be implicated in this disease. Our results demonstrate that the discovery of murine modifier loci may generate information that is relevant to human disease progression. IMPORTANCE Respiratory infection caused by P. aeruginosa is one of the most critical health burdens worldwide. People affected by P. aeruginosa infection include patients with a weakened immune system, such as those with cystic fibrosis (CF) genetic disease or non-CF bronchiectasis. Disease outcomes range from fatal pneumonia to chronic life-threatening infection and inflammation leading to the progressive deterioration of pulmonary function. The development of these respiratory infections is mediated by multiple causes. However, the genetic factors underlying infection susceptibility are poorly known and difficult to predict. Our study employed novel approaches and improved mouse disease models to identify genetic modifiers that affect the severity of P. aeruginosa lung infection. We identified candidate genes to enhance our understanding of P. aeruginosa infection in humans and provide a proof of concept that could be exploited for other human pathologies mediated by bacterial infection.

Abstract Background Comparative genomics studies are central in identifying the coding and non-coding elements associated with complex traits, and the functional annotation of genomes is a critical step to decipher the genotype-to-phenotype relationships in livestock animals. As part of the Functional Annotation of Animal Genomes (FAANG) action, the FR-AgENCODE project aimed to create reference functional maps of domesticated animals by profiling the landscape of transcription (RNA-seq), chromatin accessibility (ATAC-seq) and conformation (Hi-C) in species representing ruminants (cattle, goat), monogastrics (pig) and birds (chicken), using three target samples related to metabolism (liver) and immunity (CD4+ and CD8+ T cells). Results RNA-seq assays considerably extended the available catalog of annotated transcripts and identified differentially expressed genes with unknown function, including new syntenic lncRNAs. ATAC-seq highlighted an enrichment for transcription factor binding sites in differentially accessible regions of the chromatin. Comparative analyses revealed a core set of conserved regulatory regions across species. Topologically associating domains (TADs) and epigenetic A/B compartments annotated from Hi-C data were consistent with RNA-seq and ATAC-seq data. Multi-species comparisons showed that conserved TAD boundaries had stronger insulation properties than species-specific ones and that the genomic distribution of orthologous genes in A/B compartments was significantly conserved across species. Conclusions We report the first multi-species and multi-assay genome annotation results obtained by a FAANG project. Beyond the generation of reference annotations and the confirmation of previous findings on model animals, the integrative analysis of data from multiple assays and species sheds a new light on the multi-scale selective pressure shaping genome organization from birds to mammals. Overall, these results emphasize the value of FAANG for research on domesticated animals and reinforces the importance of future meta-analyses of the reference datasets being generated by this community on different species.

Variations in gene expression have long been hypothesised to be the major cause of individual differences. An initial focus of this research thesis is to elucidate the genetic regulatory architecture of gene expression. Expression quantitative trait locus (eQTL) mapping analyses have been performed on expression levels of over 22,000 mRNAs from three tissues of a panel of recombinant inbred mice. These analyses are "single-locus" where "linkage" (i.e. significant correlation) between an expression trait and a putative eQTL is considered independently of other loci. Major conclusions from these analyses are: 1. Gene expression is mainly influenced by genetic (sequence) variations that act in trans rather than in cis; 2. Subsets of genes are controlled by master regulators that influence multiple genes; 3. Gene expression is a polygenic trait with multiple regulators. Single-locus mapping analyses are not designed for detecting multiple regulators of gene expression, and so observation of multiple-linkages (i.e. one expression trait mapped to multiple eQTLs) formed the basis of the second objective of this research project: to investigate the relationship between multiple-linkages and genotype pattern-association. A locus-pair is said to have associated genotype patterns if they have similar inheritance pattern across a panel of individuals, and these are attributed to one of fours sources: 1. linkage disequilibrium between loci located on the same chromosome; 2. non-syntenic association; 3. random association; 4. un-associated. To understand the validity of multiple-linkages observed in single-locus mapping studies, a newly developed method, bqtl.twolocus, is applied to confirm two-locus effects for a total of 898 out of 1,233 multiple-linkages identified from the three studies mentioned above as well as from seven publicly available eQTL-mapping studies. Combining these results with information of genotype pattern-association, a subset of 478 multiple-linkages has been deduced for which there is high confidence to be real.