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DEFINING A RAT BLOOD PRESSURE QUANTITATIVE TRAIT LOCUS TO A <81.8KB CONGENIC SEGMENT: COMPREHENSIVE SEQUENCING AND RENAL TRANSCRIPTOME ANALYSIS.

ABSTRACT: Evidence from multiple linkage and genome-wide association studies suggest that human chromosome 2 (HSA2) contains alleles that influence blood pressure (BP). Homologous to a large segment of HSA2 is rat chromosome 9 (RNO9), to which a BP quantitative trait locus (QTL) was previously mapped. The objective of the current study was to further resolve this BP QTL. Eleven congenic strains with introgressed segments spanning <81.8kb to <1.33Mb were developed by introgressing genomic segments of RNO9 from the Dahl salt-resistant (R) rat onto the genome of the Dahl salt-sensitive (S) rat and tested for BP. The congenic strain with the shortest introgressed segment spanning <81.8kb significantly lowered BP of the hypertensive S rat by 25 mm Hg and significantly increased its mean survival by 45 days. In contrast, two other congenic strains had increased BP compared with the S. We focused on the <81.8kb congenic strain which represents the shortest genomic segment to which a BP QTL has been definitively mapped to date in any species. Sequencing of this entire region in both S and R rats detected 563 variants. The region did not contain any known or predicted rat protein coding genes. Further, a whole genome renal transcriptome analysis between S and the <81.8kb S.R congenic strain revealed alterations in several critical genes implicated in renal homeostasis. Taken together, our results provide the basis for future studies to examine the relationship between the candidate variants within the QTL region and the renal differentially expressed genes as potential causal mechanisms for BP regulation.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE22515 | GEO | 2010/07/15

SECONDARY ACCESSION(S): PRJNA128475

REPOSITORIES: GEO

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Project description:Evidence from multiple linkage and genome-wide association studies suggest that human chromosome 2 (HSA2) contains alleles that influence blood pressure (BP). Homologous to a large segment of HSA2 is rat chromosome 9 (RNO9), to which a BP quantitative trait locus (QTL) was previously mapped. The objective of the current study was to further resolve this BP QTL. Eleven congenic strains with introgressed segments spanning <81.8kb to <1.33Mb were developed by introgressing genomic segments of RNO9 from the Dahl salt-resistant (R) rat onto the genome of the Dahl salt-sensitive (S) rat and tested for BP. The congenic strain with the shortest introgressed segment spanning <81.8kb significantly lowered BP of the hypertensive S rat by 25 mm Hg and significantly increased its mean survival by 45 days. In contrast, two other congenic strains had increased BP compared with the S. We focused on the <81.8kb congenic strain which represents the shortest genomic segment to which a BP QTL has been definitively mapped to date in any species. Sequencing of this entire region in both S and R rats detected 563 variants. The region did not contain any known or predicted rat protein coding genes. Further, a whole genome renal transcriptome analysis between S and the <81.8kb S.R congenic strain revealed alterations in several critical genes implicated in renal homeostasis. Taken together, our results provide the basis for future studies to examine the relationship between the candidate variants within the QTL region and the renal differentially expressed genes as potential causal mechanisms for BP regulation. Six male S control and 6 male congenic S.R(9)x3x2Bx1x8 rats born on the same day were selected, weaned at 30 days of age, and caged with 1 congenic and 1 S rat per cage. They were raised on a High-salt (2%) diet (Harlan Teklad diet TD 7034; Harlan–Sprague-Dawley) and sacrificed at 53 days of age and total RNAs were isolated from the kidney. The isolated RNA from two animals were pooled together and considered as one biological sample. Three such RNA samples from S and congenic rats were used for the cRNA preparation. cRNA was prepared and fragmented as suggested by Affymetrix technical manual, and simultaneously hybridized (15 µg adjusted cRNA for each chip) to Rat Expression Array 230 2.0 (3' IVT Expression Analysis). Statistical analyses of the microarray data were performed using R statistical package (version 2.8.1).

Project description:Although the evidence for a genetic predisposition to human essential hypertension is compelling, the genetic control of blood pressure (BP) is poorly understood. The Dahl salt-sensitive (S) rat is a model for studying the genetic component of BP. Using this model we previously reported the identification of 16 different genomic regions that contain one or more BP quantitative trait loci (QTLs). The proximal region of rat chromosome 1 contains multiple BP QTLs. Of these, we have localized the BP QTL1b region to a 13.5cM (20Mb) region. Interestingly, five additional independent studies in rats and four independent studies in humans have reported genetic linkage for BP control by regions homologous to QTL1b. To view the overall renal transcriptional topography of the positional candidate genes for this QTL, we sought a comparative gene expression profiling between a congenic strain containing QTL1b and control S rats by employing: (1) a saturated QTL1b interval specific oligonucleotide array, and (2) a whole genome cDNA microarray representing 20,465 unique genes that are positioned outside the QTL. Results indicated that 19 out of the 231 positional candidate genes for this QTL are differentially expressed between the two strains tested. Surprisingly, over 1,500 genes outside of QTL1b were differentially expressed between the two rat strains. Integrating the results from the two approaches revealed at least one complex network of transcriptional control initiated by the positional candidate Nr2f2. This network appears to account for the majority of gene expression differences occurring outside of the QTL interval. Further substitution mapping is currently underway to test the validity of each of these differentially expressed positional candidate genes. These results demonstrate the importance of using a saturated oligonucleotide array for identifying and prioritizing differentially expressed positional candidate genes of a BP QTL. Keywords: : rat, hypertension, genetics, polygenic trait, microarray, gene expression

Project description:Although the evidence for a genetic predisposition to human essential hypertension is compelling, the genetic control of blood pressure (BP) is poorly understood. The Dahl salt-sensitive (S) rat is a model for studying the genetic component of BP. Using this model we previously reported the identification of 16 different genomic regions that contain one or more BP quantitative trait loci (QTLs). The proximal region of rat chromosome 1 contains multiple BP QTLs. Of these, we have localized the BP QTL1b region to a 13.5cM (20Mb) region. Interestingly, five additional independent studies in rats and four independent studies in humans have reported genetic linkage for BP control by regions homologous to QTL1b. To view the overall renal transcriptional topography of the positional candidate genes for this QTL, we sought a comparative gene expression profiling between a congenic strain containing QTL1b and control S rats by employing: (1) a saturated QTL1b interval specific oligonucleotide array, and (2) a whole genome cDNA microarray representing 20,465 unique genes that are positioned outside the QTL. Results indicated that 19 out of the 231 positional candidate genes for this QTL are differentially expressed between the two strains tested. Surprisingly, over 1,500 genes outside of QTL1b were differentially expressed between the two rat strains. Integrating the results from the two approaches revealed at least one complex network of transcriptional control initiated by the positional candidate Nr2f2. This network appears to account for the majority of gene expression differences occurring outside of the QTL interval. Further substitution mapping is currently underway to test the validity of each of these differentially expressed positional candidate genes. These results demonstrate the importance of using a saturated oligonucleotide array for identifying and prioritizing differentially expressed positional candidate genes of a BP QTL. Pairs of Cy5 and Cy3 labeled targets were co-hybridized onto either the oligonucleotide microarray or a custom TIGR rat cDNA array consisting of 26,401 probe elements representing 20,465 unique non-QTL1b genes. A âflip-dyeâ design was used as the experimental method of choice to account for potential dye-bias labeling effects. Seven âflip dyeâ normalized files are submitted for the oligonucleotide array and twelve individual hybridizations are submitted for the cDNA array.

Project description:Through substitution mapping studies, we previously identified that a <330kb region from a rat strain with no renal pathology (the Lewis rat), which when introgressed onto the genetic background of a rat with renal disease (the Dahl Salt-sensitive (S) rat), caused an increase rather than the expected decrease in proteinuria. The purpose of this study was to prioritize a candidate gene and further delineate the mechanism underlying the observed increased in proteinuria. A higher level of proteinuria independent of dietary salt was observed in the congenic rat at a very young age (50-52 day old). The critical congenic segment was further mapped to <42.5kb containing a single candidate gene, rififylin. Rififylin was expressed 1.59 fold higher in the congenic strain compared with S. Overexpression of rififylin is known to delay recycling of endosomes. Renal transcriptome analysis indicated that Atp1a1 one of the most highly differentially expressed genes. Atp1a1 was 5.33 fold higher in the congenic strain compared with S. The protein product of Atp1a1, the alpha subunit of Na+K+ATPase, was also significantly higher in the endosomes of proximal tubules from the congenic strain compared with S. To determine whether the higher amounts of this protein in the endosomes is due to a delay in recycling of endosomes caused by the overexpression of rififylin in the congenic strain, recycling of exogenously labeled-transferrin by single cell cultures of proximal tubules was monitored by confocal microscopy. Recycling of transferrin was significantly delayed in the congenic strain compared with S. These results suggest that impaired endosomal recycling in the proximal tubules from the congenic strain caused by the overexpression of rififylin is a novel molecular mechanism linked to the observed increase in proteinuria of the congenic strain. Three male S control and 3 male congenic S.LEW(10)x12x2x3x5 rats born on the same day were selected, weaned at 30 days of age, and caged with 1 congenic and 1 S rat per cage. They were raised on a low-salt (0.3%) diet (Harlan Teklad diet TD 7034; Harlan–Sprague-Dawley) and sacrificed at 53 days of age and total RNAs were isolated from the kidney. The isolated RNA from each animal was used for the cRNA preparation. cRNA was prepared and fragmented as suggested by Affymetrix technical manual, and simultaneously hybridized (15 µg adjusted cRNA for each chip) to Rat Expression Array 230 2.0 (3' IVT Expression Analysis). Statistical analyses of the microarray data were performed with BH adjustment using R statistical package (version 2.8.1).

Project description:The study sought to investigate differences in onset and progression of renal disease in the Dahl salt-sensitive (S), S.SHR(2) congenic, and spontaneously hypertensive rat (SHR) using a time-course. The data clearly demonstrates that the locus on chromosome 2 has a major and sustained ability to attenuate renal damage. As early as week 4, significant interstitial changes were observed between the S and the congenic which preceded any significant difference in proteinuria. Gene expression profiling was performed at week 4, 12, and 20 using kidney from the S and S.SHR(2) congenic to: (1) identify expression differences of positional candidates within the QTL region; (2) correlate temporal gene expression changes between the S and congenic with degree of renal damage; and (3) identify biochemical pathways potentially involved in the attenuated renal damaged observed in the congenic. Gene pathway analysis (IngenuityÂ® Systems) performed at week 4, 12, and 20 revealed that pathways involved in cellular assembly and organization, cellular movement, and immune response were controlled differently between the S and congenic. Considering all the data, the chromosome 2 congenic appears to attenuate renal damage primarily through an altered fibrotic response. Experiment Overall Design: DNA microarray analysis was performed using Affymetrix GenechipÂ® Rat Genome 230 2.0 array at three timepoints. The chip contains 31,000 probe sets from more than 30,000 transcripts on one array. Three male S and three male S.SHR(2) congenic were selected at random from each group test for proteinruia at week 4, 12 and 20. Kidney was cut into ~ 0.5cM size cubes, suspended in RNAlater (Ambion, Austin, TX) and stored overnight at 4ÂºC. RNA was extracted using TrizolÂ® reagent (Invitrogen, Carlsbad, CA) and purified using Mini RNeasy kit (Qiagen,Valencia, CA ) according to manufacturerâs protocols. RNA quality was assessed by an OD260/280 ratio > 2.0 and visually by ethidium bromide staining on an agarose gel. Experiment Overall Design: Biotinylated cRNA was synthesized from 10 ug of total RNA using the One-Cycle Target Labeling Kit (Affymetrix, Santa Clara, CA) as directed by the user manual. cRNA quality for each sample was assessed by hybridization of 5ug adjusted kidney cRNA to Affymetrix GenechipÂ® Test3 array. Subsequently, 15ug adjusted kidney cRNA was hybridized to the GenechipÂ® Rat 230 2.0 array. Hybridized chips were automatically washed, stained and scanned at the Medical University of Ohio Bioinformatics & Proteomics/Genomics Program gene array facility using Affymetrix equipment.

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DEFINING A RAT BLOOD PRESSURE QUANTITATIVE TRAIT LOCUS TO A <81.8KB CONGENIC SEGMENT: COMPREHENSIVE SEQUENCING AND RENAL TRANSCRIPTOME ANALYSIS.

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