Development of Congenic Lines and Application of Physical Mapping Strategies for the Dissection of Blood Pressure Quantitative Trait Loci in the Stroke-Prone Spontaneously Hypersensitive Rat

Negrin Deus, Cervantes Domingo (2000) Development of Congenic Lines and Application of Physical Mapping Strategies for the Dissection of Blood Pressure Quantitative Trait Loci in the Stroke-Prone Spontaneously Hypersensitive Rat. PhD thesis, University of Glasgow.

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Abstract

Human essential hypertension is a complex, multifactorial, quantitative trait under polygenic control. Several strategies have been developed over the last decade to dissect genetic determinants of hypertension. Of these, the most successful have been studies identifying rare Mendelian syndromes in which a single gene mutation causes high blood pressure (BP). The attempts to identify multiple genes, each having a small contribution to the common polygenic form of hypertension, have been less successful. Experimental models of genetic hypertension have been used to develop paradigms for the study of human essential hypertension in order to remove some of the complexity inherent in studying human subjects. Several laboratories, using diverse crosses between hypertensive and normotensive strains, identified several quantitative trait loci (QTLs) for BP regulation. The strategy used to identify BP QTLs is known as a genome scan and involves the determination of the BP in a large segregating F2 population derived by crossing contrasting inbred rat strains, and the genotyping of a large panel of polymorphic microsatellite markers with a thorough coverage of the entire rat genome. The next step is the production of congenic strains and substrains to confirm the existence of the BP QTLs and to narrow down the chromosomal region of interest. The investigations reported in this thesis incorporate the use and validation of a "speed" congenic strategy to dissect two BP QTLs identified previously on rat chromosome 2. We produced 4 congenic strains through introgression of various segments of chromosome 2 from the WKYGla strain into the recipient SHRSPGla strain, and vice versa. Transfer of the region of rat chromosome 2 containing both BP QTLs from WKYGla into an SHRSPGla genetic background lowered both baseline and salt-loaded systolic BP by ~20 and ~40 mmHg in male congenic rats compared with the SHRSP parental strain (F=53.4, p<0.005; F=28.0, p<0.0005, respectively). In contrast, control animals for stowaway heterozygosity presented no deviation from the BP values recorded for the SHRSPcia, indicating that if such heterozygosity exists, its effect on BP is negligible. Reciprocal congenic strain in which one QTL was transferred from SHRSPGla onto the WKYoia background resulted in statistically significant but smaller BP increase. This implicated region contains different candidate genes including the Na+-K+ATPase a1 isoform (Atplal), natriuretic peptide receptor A/Guanylate cyclase A (Gca), angiotensin II receptor type IB (Agtrib), and calcium/calmodulin-dependent protein kinase II delta subunit (Camk2d). Sequencing analysis showed no differences in the coding regions of the Atplal gene between the WKYGla strain and the published sequence. Two transitions were found between WKYGla and SHRSPGla resulting in silent mutations. Therefore, the Atplal gene was not supported as a candidate gene for the BP QTL on rat chromosome 2. Radiation hybrid mapping was performed along with fluorescence in situ hybridisation of rat chromosome 5 due to the discrepancies between our genetic map and other genetic maps of rat chromosome 5. We successfully constructed a radiation hybrid map of rat chromosome 5 using 35 microsatellite markers covering a genetic distance of 78 cM, corresponding to a physical distance of approximately 1,304 cR (17 cR/cM) and comparable to reports from other laboratories. The Anf microsatellite marker was mapped between D5Rat48 and D5Rat47 located at the telomeric end of rat chromosome 5. Fluorescence in situ hybridisation confirmed that the Anf gene is localised to the 5q36.3, which corresponds to the telomeric end of the chromosome 5. Two different physical mapping methods have therefore given identical results and are in agreement with genetic maps published by other groups. We also produced a high resolution radiation hybrid map of the telomeric end of rat chromosome 2 between markers D2MH6 and D2Mghl2. The physical to genetic distance conversion gave us an estimate of 20.8 cM to 31.2 cM for this region and facilitated fine mapping of the two BP QTLs on rat chromosome 2. We constructed congenic substrain SP.WKY.Gla2c* where a small segment of approximately 20 cM was transferred from the normotensive WKYGla strain into the hypertensive SHRSPGla- Phenotyping of the congenic substrain is currently ongoing and will determine if the BP QTL was successfully trapped. Additionally, we produced a high resolution radiation hybrid map of this segment, which will help in the identification of the gene(s) involved in this BP QTL. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Anna Dominiczak
Keywords: Biochemistry, Physiology
Date of Award: 2000
Depositing User: Enlighten Team
Unique ID: glathesis:2000-76130
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 16:37
Last Modified: 19 Nov 2019 16:37
URI: http://theses.gla.ac.uk/id/eprint/76130

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