Abstract-Using congenic strains of the Dahl salt-sensitive (S) rat introgressed with genomic segments from the normotensive Lewis rat, a blood pressure quantitative trait locus was previously mapped within 104 kb on chromosome 10. The goal of the current study was to conduct extensive phenotypic studies and to further fine-map this locus. At 14 weeks of age, the blood pressure of the congenic rats fed a low-salt diet was significantly higher by 47 mm Hg (PϽ0.001) compared with that of the S rat. A time-course study showed that the blood pressure effect was significant from very young ages of 50 to 52 days (13 mm Hg; PϽ0.01). The congenic strain implanted with electrocardiography transmitters demonstrated shorter-QT intervals and increased heart rate compared with S rats (PϽ0.01). The average survival of the congenic strain was shorter (134 days) compared with the S rat (175 days; PϽ0.0007). The critical region was narrowed to Ͻ42.5 kb containing 171 variants and a single gene, rififylin. Both the mRNA and protein levels of rififylin were significantly higher in the hearts of the congenic strain. Overexpression of rififylin is known to delay endocytic recycling. Endocytic recycling of fluorescently labeled holotransferrin from cardiomyocytes of the congenic strain was slower than that of S rats (PϽ0.01). Frequency of cardiomyocyte beats in the congenic strain (62Ϯ9 bpm) was significantly higher than that of the S rat (24Ϯ6 bpm; PϽ0.001). Taken together, our study provides evidence to suggest that early perturbations in endocytic recycling caused by the overexpression of Rffl is a novel physiological mechanism potentially underlying the development of hypertension.
Previously, we reported a linkage analysis for urinary albumin excretion (UAE) from a backcross population derived from the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR) raised on a low-salt diet. The present study sought to examine the effect of salt loading on the observation of UAE quantitative trait loci (QTL) using a F1(S x SHR) x S backcross population (n = 228) raised on a 2% NaCl diet. Parental strain data demonstrated that S rats have significantly higher blood pressure (BP) and UAE compared with either F1(S x SHR) or SHR at 8 wk of age, and this difference was exacerbated by 12 wk of age in response to a high-salt diet (2% NaCl). Genome scans done at 8, 12, and 16 wk of age yielded eight QTL for UAE. At week 8 (low salt), QTL for UAE were observed on rat chromosomes (RNO) 1, 2, 6, 8, 9, 11, 13, and 19. Week 8 linkage analysis confirmed previous linkage data and provided a baseline to examine the effect of salt loading at subsequent time points. At weeks 12 and 16 (after salt- loading), QTL for UAE were observed on RNO1, -6, -8, -9, and -13. Surprisingly, UAE QTL were no longer observed on RNO2, -11, and -19 after salt loading, suggesting that these QTL are attenuated by increased salt intake. The effects of UAE QTL on RNO2, -6, -9, -11, and -13 were examined using congenic strains whereby the SHR alleles at each QTL were placed on the S background. These congenic strains demonstrated large and significant effects on UAE compared with the S rat, proving that QTL for UAE reside on these chromosomes.
A previously reported blood pressure (BP) quantitative trait locus on rat Chromosome 1 was isolated in a short congenic segment spanning 804.6 kb. The 804.6 kb region contained only two genes, LOC306664 and LOC306665. LOC306664 is predicted to translate into A Disintegrin-like and Metalloproteinase with Thrombospondin Motifs-16 (Adamts16). LOC306665 is a novel gene. All predicted exons of both LOC306664 and LOC306665 were sequenced. Non-synonymous variants were identified in only one of these genes, LOC306664. These variants were naturally existing polymorphisms among inbred, outbred and wild rats. The full-length rat transcript of Adamts16 was detected in multiple tissues. Similar to ADAMTS16 in humans, expression of Adamts16 was prominent in the kidney. Renal transcriptome analysis suggested that a network of genes related to BP was differential between congenic and S rats. These genes were also differentially expressed between kidney cell lines with or without knock-down of Adamts16. Adamts16 is conserved between rats and humans. It is a candidate gene within the homologous region on human Chromosome 5, which is linked to systolic and diastolic BP in the Quebec Family Study. Multiple variants, including an Ala to Pro variant in codon 90 (rs2086310) of human ADAMTS16, were associated with human resting systolic BP (SBP). Replication study in GenNet confirmed the association of two variants of ADAMTS16 with SBP, including rs2086310. Overall, our report represents a high resolution positional cloning and translational study for Adamts16 as a candidate gene controlling BP.
Abstract-Genetic dissection of the rat genome for identifying alleles that cause abnormalities in blood pressure (BP) resulted in the mapping of a significant number of BP quantitative trait loci (QTLs). In this study we mapped at least one such BP QTL on rat chromosome 10 (RNO10) as being within the introgressed segment of a S.LEW congenic strain S.LEWx12x2x3x8 spanning 1.34 Mb from 70 725 437 bp to 72 063 232 bp. BP of 3 congenic strains that span shorter segments of this region was additionally examined. Results obtained indicate that LEW alleles that comprise a 375-kb introgressed segment of the congenic strain S.LEWx12x2x3x5 (70 725 437 bp to 71 100 513 bp) increase BP. The magnitude of change in BP exhibited by the 2 strains, S.LEWx12x2x3x8 and S.LEWx12x2x3x5, is the net phenotypic effect of the underlying genetic determinants of BP. In this respect, the current data are superior to previous QTL localization of BP QTL1, which was hypothesized based on differential congenic segments. Genetic dissection using these 2 congenic strains as tools is expected to facilitate further dissection of the regions. Meanwhile, differential congenic segments were used to predict and thereby prioritize regions for candidate gene analysis. Using this approach, 2 distinct regions of 401 kb and 409 kb within S.LEWx12x2x3x8 and a 104 kb region within S.LEWx12x2x3x5 were prioritized for further consideration. Because all of these genetic elements are located within a 1.06-Mb region of RNO10, our study has revealed a remarkable insight into a genomic module comprising very closely-linked, opposing genetic determinants of BP. espite the description of a number of blood pressure (BP) quantitative trait loci (QTLs) in humans, rats, and mice, identities of the underlying genetic determinants conferring susceptibility to hypertension in any species remain largely unknown, [1][2][3][4] with the exception of rat 11--hydroxylase 5,6 and CD36. 7 Linkage analysis and substitution mapping using various rat strain comparisons provide conclusive evidence for the existence of multiple genetic determinants of BP on rat chromosome 10 (RNO10). 8 -32 Located on this chromosome are genes coding for angiotensin-I converting enzyme, nerve growth factor receptor, skeletal myosin heavy polypeptide 3, nitric oxide synthase-2, ATPase-Na ϩ /K ϩ transporting- 2 polypeptide, nicotiniccholinergic receptor-beta polypeptide 1, and protein kinaselysine deficient 4, all of which are appealing candidates for causally controlling BP. However, fine-mapping and/or DNA sequencing has not provided evidence for most of these genes as candidates for BP QTLs, implicating that the identities of the genetic determinants of BP on RNO10 remain elusive. 8 -31 By replacing progressively shorter segments of RNO 10 of the hypertensive Dahl Salt-sensitive (S) rat with corresponding segments from the Lewis (LEW) rat genome, we have previously fine-mapped a BP quantitative trait locus (S.LEW BP QTL1) to a 1.17-Mb region containing 18 genes. 32 None of these genes have any kn...
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