Genomic imprinting variations in the mouse type 3 deiodinase gene between tissues and brain regions.

The Dio3 gene, which encodes for the type 3 deiodinase (D3), controls thyroid hormone (TH) availability. The lack of D3 in mice results in tissue overexposure to TH and a broad neuroendocrine phenotype. Dio3 is an imprinted gene, preferentially expressed from the paternally inherited allele in the mouse fetus. However, heterozygous mice with paternal inheritance of the inactivating Dio3 mutation exhibit an attenuated phenotype when compared with that of Dio3 null mice. To investigate this milder phenotype, the allelic expression of Dio3 was evaluated in different mouse tissues. Preferential allelic expression of Dio3 from the paternal allele was observed in fetal tissues and neonatal brain regions, whereas the biallelic Dio3 expression occurred in the developing eye, testes, and cerebellum and in the postnatal brain neocortex, which expresses a larger Dio3 mRNA transcript. The newborn hypothalamus manifests the highest degree of Dio3 expression from the paternal allele, compared with other brain regions, and preferential allelic expression of Dio3 in the brain relaxed in late neonatal life. A methylation analysis of two regulatory regions of the Dio3 imprinted domain revealed modest but significant differences between tissues, but these did not consistently correlate with the observed patterns of Dio3 allelic expression. Deletion of the Dio3 gene and promoter did not result in significant changes in the tissue-specific patterns of Dio3 allelic expression. These results suggest the existence of unidentified epigenetic determinants of tissue-specific Dio3 imprinting. The resulting variation in the Dio3 allelic expression between tissues likely explains the phenotypic variation that results from paternal Dio3 haploinsufficiency.This is the final version of the article. It is available from the Endocrine Society in Molecular Endocrinology here: http://press.endocrine.org/doi/pdf/10.1210/me.2014-1210

Analysis of genetically driven alternative splicing identifies FBXO38 as a novel COPD susceptibility gene

While many disease-associated single nucleotide polymorphisms (SNPs) are associated with gene expression (expression quantitative trait loci, eQTLs), a large proportion of complex disease genome-wide association study (GWAS) variants are of unknown function. Some of these SNPs may contribute to disease by regulating gene splicing. Here, we investigate whether SNPs that are associated with alternative splicing (splice QTL or sQTL) can identify novel functions for existing GWAS variants or suggest new associated variants in chronic obstructive pulmonary disease (COPD). RNA sequencing was performed on whole blood from 376 subjects from the COPDGene Study. Using linear models, we identified 561,060 unique sQTL SNPs associated with 30,333 splice sites corresponding to 6,419 unique genes. Similarly, 708,928 unique eQTL SNPs involving 15,913 genes were detected at 10% FDR. While there is overlap between sQTLs and eQTLs, 55.3% of sQTLs are not eQTLs. Co-localization analysis revealed that 7 out of 21 loci associated with COPD (p</p

Mechanisms involved in the establishment and maintenance of genomic imprints

Genomic imprinting is a phenomenon that results in parent of origin dependant gene expression. During mammalian gametogenesis, genomic imprints are erased, and reestablished in sperm and oocytes in a sex-dependent manner. Most genomic imprints that are inherited by the embryo from its parents are maintained throughout the life of the individual. Aberrant genomic imprinting may lead to embryonic lethality, developmental anomalies and increased risk of cancer. To better understand the mechanisms of imprinting errors, we explored two potential sources of such errors: a) genetic anomalies that affect DNA methylation and spermatogenesis in mice, and b) the influence of cell culture on imprinting maintenance in human somatic cells. We found that heterozygous mutations in murine Mthfr, Dnmt1, Dnmt3a/3b, Spo11, and Msh5 that are known to affect DNA methylation or meiotic recombination have no effect on imprint establishment of H19 and the intragenic differentially methylated region (IGDMR) in mature sperm. Next, we tested the hypothesis that local chromosomal anomalies such as deletions or translocations may impair imprint establishment using two mouse models, mice with an H19 DMR deletion and carriers of Robertsonian translocations. We found incomplete establishment of H19 DMR methylation in the sperm of heterozygous carriers of a Robertsonian translocation, Rb 8.12, that suggests a mechanistic link between chromosomal anomalies and imprint establishment or maintenance errors. DNA methylation patterns are often poorly conserved through cell culture. This could significantly impact gene expression profiles. To investigate the effect of cell culture on DNA methylation profiles, we analyzed methylation in the differentially methylated domains of 5 imprinted regions: the IGDMR; potassium voltage-gated channel, KQT-like subfamily, member 1, (KCNQ1); small nuclear ribonucleoprotein polypeptide N (SNRPN), mesoderm speRÉSUMÉL'empreinte génomique est un processus biologique où l'expression d'un gène dépend de son origine parentale. Chez les mammifères, au cours de la gamétogenèse, les empreintes génomiques sont supprimées puis rétablies dans les spermatozoïdes et les ovules, en fonction du sexe. La plupart des empreintes génomiques qui sont héritées des parents sont maintenues tout au long de la vie de chaque individu. Une empreinte génomique aberrante peut induire une létalité embryonnaire, des développements anormaux ou augmenter le risque d'apparition de cancer. Afin de mieux comprendre comment se produisent ces erreurs, nous avons étudié deux mécanismes potentiellement impliqués dans l'apparition d'empreintes erronées: a) les anomalies génétiques qui perturbent la méthylation de l'ADN ou la spermatogenèse chez la souris, et b) l'influence de la culture cellulaire sur le maintien de l'empreinte dans les cellules somatiques humaines. Nous avons déterminé que, chez la souris, les mutations à l'état hétérozygote de Mthfr, Dnmt1, Dnmt3a/3b, Spo11 et Msh5, qui sont connues pour altérer la méthylation de l'ADN ou la recombinaison méiotique, n'ont aucun effet sur l'établissement de l'empreinte au niveau du gène H19 et de la région intragenic differentially methylated region (IGDMR) dans le sperme. Nous avons ensuite cherché à déterminer si des anomalies chromosomiques locales comme les délétions ou les translocations pouvaient perturber l'établissement de l'empreinte. Pour tester cette hypothèse, nous avons utilisé deux modèles murins : une lignée de souris portant une délétion au niveau de la région différentiellement méthylées H19 DMR et une lignée de souris portant des translocations robertsoniennes. Nous avons mis en évidence que le sperme de souris hétérozygotes pour la translocation robertsonienne Rb 8.12 présente un défaut dans l'établissement de la m

Genetic association and gene expression analysis of inflammatory genes in cystic fibrosis

Cystic fibrosis (CF) is characterized by a progressive decline in lung function due to airway obstruction, infection, and inflammation. CF patients are particularly susceptible to respiratory infection by a variety of pathogens, and the inflammatory response in CF is dysregulated and prolonged. This thesis identifies and characterizes BPI fold containing family A, member 1 (BPIFA1) and BPIFB1 as putative anti-inflammatory molecules in CF, and explores the CF inflammatory response to rhinovirus infection. BPIFA1 and BPIFB1 are proposed innate immune molecules expressed in the upper airways. We interrogated BPIFA1/BPIFB1 single-nucleotide polymorphisms in data from the North American genome-wide association study (GWAS) for lung disease severity in CF and discovered that the G allele of rs1078761 was associated with reduced lung function in CF patients. Microarray and qPCR gene expression analysis implicated rs1078761 G as being associated with reduced BPIFA1 and BPIFB1 gene expression, suggesting that decreased levels of these genes are detrimental in CF. Functional assays to characterize the role of BPIFA1 and BPIFB1 in CF indicated that these molecules do not have an anti-bacterial role against P. aeruginosa, but do have an immunomodulatory function in CF airway epithelial cells. To further investigate the mechanism of action of BPIFA1 and BPIFB1 during bacterial infection, gene expression was profiled using RNA-Seq in airway epithelial cells stimulated with P. aeruginosa and treated with recombinant BPIFA1 and BPIFB1. Viral infections are now recognized to play an important role in the short and long term health of CF patients. Rhinovirus is emerging as a lead viral pathogen although little is known about the inflammatory response triggered by rhinovirus in the CF lung. To investigate whether CF patients have a dysregulated response to rhinovirus infection, primary airway epithelial cells from CF and healthy control children were infected with rhinovirus and gene expression profiles were assessed by RNA-Seq. Although rhinovirus stimulation resulted significantly altered gene expression, the response to infection was not different in CF patients compared to healthy controls. However, CF cells had significantly higher rhinovirus levels than controls, indicating that CF patients may have a deficient antiviral response allowing for increased rhinovirus replication.Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat

Longer telomere length in COPD patients with α1-antitrypsin deficiency independent of lung function.

Oxidative stress is involved in the pathogenesis of airway obstruction in α1-antitrypsin deficient patients. This may result in a shortening of telomere length, resulting in cellular senescence. To test whether telomere length differs in α1-antitrypsin deficient patients compared with controls, we measured telomere length in DNA from peripheral blood cells of 217 α1-antitrypsin deficient patients and 217 control COPD patients. We also tested for differences in telomere length between DNA from blood and DNA from lung tissue in a subset of 51 controls. We found that telomere length in the blood was significantly longer in α1-antitrypsin deficient COPD patients compared with control COPD patients (p = 1×10(-29)). Telomere length was not related to lung function in α1-antitrypsin deficient patients (p = 0.3122) or in COPD controls (p = 0.1430). Although mean telomere length was significantly shorter in the blood when compared with the lungs (p = 0.0078), telomere length was correlated between the two tissue types (p = 0.0122). Our results indicate that telomere length is better preserved in α1-antitrypsin deficient COPD patients than in non-deficient patients. In addition, measurement of telomere length in the blood may be a suitable surrogate for measurement in the lung

Immunomodulatory function of the cystic fibrosis modifier gene BPIFA1.

BackgroundCystic fibrosis (CF) is characterized by a progressive decline in lung function due to airway obstruction, infection, and inflammation. CF patients are particularly susceptible to respiratory infection by a variety of pathogens, and the inflammatory response in CF is dysregulated and prolonged. BPI fold containing family A, member 1 (BPIFA1) and BPIFB1 are proteins expressed in the upper airways that may have innate immune activity. We previously identified polymorphisms in the BPIFA1/BPIFB1 region associated with CF lung disease severity.MethodsWe evaluated whether the BPIFA1/BPIFB1 associations with lung disease severity replicated in individuals with CF participating in the International CF Gene Modifier Consortium (n = 6,365). Furthermore, we investigated mechanisms by which the BPIFA1 and BPIFB1 proteins may modify lung disease in CF.ResultsThe association of the G allele of rs1078761 with reduced lung function was replicated in an independent cohort of CF patients (p = 0.001, n = 2,921) and in a meta-analysis of the full consortium (p = 2.39x10-5, n = 6,365). Furthermore, we found that rs1078761G which is associated with reduced lung function was also associated with reduced BPIFA1, but not BPIFB1, protein levels in saliva from CF patients. Functional assays indicated that BPIFA1 and BPIFB1 do not have an anti-bacterial role against P. aeruginosa but may have an immunomodulatory function in CF airway epithelial cells. Gene expression profiling using RNAseq identified Rho GTPase signaling pathways to be altered in CF airway epithelial cells in response to treatment with recombinant BPIFA1 and BPIFB1 proteins.ConclusionsBPIFA1 and BPIFB1 have immunomodulatory activity and genetic variation associated with low levels of these proteins may increase CF lung disease severity

Log₁₀ of telomere length in the lung vs. log₁₀ of telomere length in the blood.

<p>Log₁₀ of telomere length in the lung vs. log₁₀ of telomere length in the blood.</p