Cristina Chauvet

Submegabase resolution of epistatically interacting quantitative trait loci for blood pressure applicable for essential hypertension

Objective Although genetic mapping of quantitative trait loci for blood pressure to large chromosome segments is readily achievable, their final identification confronts formidable hurdles. Restriction of the genes lodging in one quantitative trait locus interval to experimental limitation can facilitate their positional cloning. We previously delineated several quantitative trait loci for blood pressure on chromosome 10 of Dahl salt-sensitive rats, but their chromosome delimitations were either large or not definitive. Methods In this study, we systematically and comprehensively constructed congenic strains with submegabase (Mb) genome resolution and analyzed their blood pressure by telemetry. Results Three quantitative trait loci have been conclusively delimited by three congenic strains, each independently lowering the blood pressure. Their intervals are demarcated by genomic regions between 350 and 910 kilobases (kb) in size. Two of the three quantitative trait loci share an epistatic relationship and are separated from one another by less than 170 kb. Two additional quantitative trait loci for blood pressure were also tentatively delineated and their intervals range from 520 kb to 1.75 Mb. Possible genes dwelling in each quantitative trait locus-interval number between 11 and 17. None of these genes is known to exert a functional impact on blood pressure. Work is underway to find candidate genes with mutations that could be responsible for the blood pressure effect. Conclusion Novel diagnostic, prognostic, preventive and/or therapeutic targets for essential hypertension and hypertension-associated diseases are likely to emerge from the identification of these quantitative trait loci. Potential applications of these quantitative trait loci to humans are suggested from the positive results from several association studies, demonstrating the existence of quantitative trait loci in the broad homologous regions.

α-Kinase 2 is a novel candidate gene for inherited hypertension in Dahl rats.

Objectives The interval harboring a quantitative trait locus for blood pressure (BP), C18QTL3, contains β-2 adrenergic receptor (Adrb2) and neural precursor cell expressed, developmentally downregulated 4-like (Nedd4l) genes. None of the other genes in the C18QTL3-residing interval is known to affect BP. The identification of C18QTL3 might uncover a brand new gene that could prosper into a novel diagnostic and/or therapeutic target for essential hypertension, if neither Adrb2 nor Nedd4l could be upheld as candidate genes. Methods Congenic fine resolution was combined with gene analyses. Results The gene encoding α-kinase 2 (Alpk2) contains a three base-pair deletion and multiple nonconserved mutations in its coding region in Dahl salt-sensitive (DSS) rats. In contrast, the gastrin-releasing peptide gene (Grp) possesses two nonconserved mutations, designated as single nucleotide polymorphisms 1 and 2 (i.e. SNP1 and SNP2), but could not be supported as a candidate gene because the C18S.L14 congenic strain displayed a homozygous DSS genotype at both SNP1 and SNP2. Furthermore, Adrb2 and Nedd4l could not account for the BP-diminishing effect of Lewis alleles in C18S.L14, as their DSS alleles bear functionally identical domains as those of Lewis, and no evidence of differential expression and splicing was evident. No significant nucleotide variations were found in 13 other genes closely linked to Alpk2. Conclusion Alpk2 emerged as a strong candidate gene for C18QTL3. The present study is the first to implicate Alpk2 in the genetics of polygenic hypertension and paves the way for novel gene discovery.

Novel genes as primary triggers for polygenic hypertension.

Objectives The discovery of causative genes leading to hypertension in animal models can reveal new mechanistic insights into blood pressure (BP) regulations. Previously, we isolated segments that harbor BP quantitative trait loci (QTLs) on rat chromosome 10 as defined by congenic strains made from crosses of inbred hypertensive Dahl salt-sensitive (DSS) and normotensive Lewis rats. The aim of the current study was to identify hypertension-causing genes for each QTL. Methods Molecular analysis was performed. Results A systematic and comprehensive molecular analysis divulged particular genes that carry nonconserved mutations. Specifically, the proline rich 11 gene is likely responsible for C10QTL5. C10QTL1 is one of five genes, namely Benzodiazepine receptor associated protein 1, Loc689764, myotubularin related protein 4, protein phosphatase 1E, PP2C domain containing and ring finger protein 43. Loc100363423 with no known function is a candidate for C10QTL3. The ATP-binding cassette, subfamily A (ABC1), member 8a gene is probably responsible for C10QTL2. Conclusions Primary genes initiating polygenic hypertension are those not known to be involved in BP modulation. Novel pathways towards BP homeostasis appear to underlie the functionality of C10QTL5, C10QTL1 and C10QTL3 and C10QTL2. Moreover, these genes may become innovative targets for the diagnosis and therapeutics of essential hypertension.

Cardiac pathways distinguish two epistatic modules enacting BP quantitative trait loci and candidate gene analysis

Animal models emulating essential hypertension are an informative means by which to elucidate the physiological mechanisms and gene–gene interactions underlying blood pressure (BP) regulation. We have localized earlier quantitative trait loci (QTLs) for BP on Chromosome (Chr) 2 of Dahl salt-sensitive (DSS) rats, but their chromosome delineations were too large for gene identification. To advance toward positional cloning of these QTLs, we constructed congenic strains that systematically dissect a Chr 2 segment with no overlaps. BP and cardiac functions were measured by telemetry and echocardiography. Six QTLs were delimited, each independently influencing BP. The intervals lodging two of them harbor 10–15 genes and undefined loci. These six QTLs can be grouped into two epistatic modules distinguishable by cardiac pathways/cascades. None of the genes known to exert physiological effects on BP in the segments harboring the six QTLs are leading candidates, as their protein products are the same in DSS rats and similar to those in their Milan normotensive counterparts. Specifically, the lack of an amino-acid alteration, coupled with a lack of difference in the α1-Na-K-ATPase activity, excluded ATPase, Na+/K+-transporting, α-1 polypeptide as a candidate gene for C2QTL6. The identification of the six QTLs will likely develop into a novel diagnostic and/or therapeutic target for essential hypertension and hypertension-associated diseases.

Normotension in Lewis and Dahl salt-resistant rats is governed by different genes

Objectives Inbred rodent models simulating essential hypertension and normotension are useful tools in discovering genes controlling blood pressure (BP) homeostasis. An analysis of a F2 population made from crosses of hypertensive Dahl salt-sensitive (DSS) and normotensive Lewis rats did not detect a BP quantitative trait locus (QTL) on chromosome 7 (Chr 7). However, false negativity could not be excluded. If a BP QTL could be proven to exist, what gene(s) may be responsible for this QTL. Methods We first constructed reciprocal congenic strains for a Chr 7 segment and determined functional domains of prominent candidate genes. Results A congenic strain made in the DSS rat background exhibited a BP effect, indicating that a BP QTL, C7QTL, inhabits Chr 7. Contrarily, a congenic strain constructed in the Lewis rat background did not change BP, demonstrating a dependence of C7QTL on the DSS rats environment. Among the candidate genes, tachykinin 2 (Tac2), neurexophilin 4 (Nxph4) and retinol dehydrogenase 2 (Rdh2) bear nonsynonymous changes comparing DSS and Lewis rats, but are the same comparing DSS and Dahl salt-resistant (DSR) rats. In contrast, the Lewis alleles of 11-beta-hydroxylase (Cyp11b1), aldosterone synthase (Cyp11b2) and Cytochrome P-450 11B3 (Cyp11b3) are identical to those of DSS rats, but different from those of DSR rats. Conclusion Thus, the failure to detect a linkage between a Chr 7 segment and BP in F2(DSS × Lewis) can be attributed to false negativity. Tac2, Nxph4 and Rdh2 are priority candidate genes for C7QTL. Lewis and DSR rats are both normotensive, but their underlying genetic determinants are different.

Alterations in Fibronectin Type III Domain Containing 1 Protein Gene Are Associated with Hypertension

Multiple quantitative trait loci (QTLs) for blood pressure (BP) have been detected in rat models of human polygenic hypertension. Great challenges confronting us include molecular identifications of individual QTLs. We first defined the chromosome region harboring C1QTL1 to a segment of 1.9 megabases that carries 9 genes. Among them, we identified the gene encoding the fibronectin type III domain containing 1 protein (Fndc1)/activator of G protein signaling 8 (Ags8) to be the strongest candidate for C1QTL1, since numerous non-synonymous mutations are found. Moreover, the 5’ Fndc1/Ags8 putative promoter contains numerous mutations that can account for its differential expression in kidneys and the heart, prominent organs in modulating BP, although the Fndc1/Ags8 protein was not detectable in these organs under our experimental conditions. This work has provided the premier evidence that Fndc1/Ags8 is a novel and strongest candidate gene for C1QTL1 without completely excluding other 8 genes in the C1QTL1-residing interval. If proven true by future in vivo function studies such as single-gene Fndc1/Ags8 congenics, transgenesis or targeted-gene modifications, it might represent a part of the BP genetic architecture that operates in the upstream position distant from the end-phase physiology of BP control, since it activates a Gbetagamma component in a signaling pathway. Its functional role could validate the concept that a QTL in itself can influence BP ‘indirectly’ by regulating other genes downstream in a pathway. The elucidation of the mechanisms initiated by Fndc/Ags8 variations will reveal novel insights into the BP modulation via a regulatory hierarchy.

Combining distinctive and novel loci doubles BP reduction, reverses diastolic dysfunction and mitigates LV hypertrophy.

Objectives: Diastolic dysfunction often represents the onset of diastolic heart failure (DHF). We previously showed in principle that diastolic function in Dahl salt-sensitive rats (DSS) can be genetically determined by quantitative trait loci (QTLs) that also modulate blood pressure (BP). Methods: We analyzed cardiac phenotypes of four ‘single’ congenic strains by echocardiography, in which a specific DSS chromosome segment was replaced by its normotensive Lewis homologue. Results: Two of the strains permanently lowered BP, and but attenuated diastolic dysfunction only in rats at 10 weeks of age, not at 15 weeks fed on a 2% NaCl diet starting from 8 weeks of age. We then combined multiple QTLs by integrating several ‘single’ congenic strains. As a result, BP was greatly reduced. Cardiac dysfunction and LV hypertrophy were continuously improved from 10 to 15 weeks, although the degree and timing of the improvement varied among different congenic combinations. Conclusion: Distinct QTLs exist that simultaneously modulate BP and diastolic function. These QTLs, in combination, synergistically lowered BP and permanently alleviated or reversed diastolic dysfunction. The genes that are contained in the congenic strains affecting diastolic function are not known for their specific influence on BP. Novel long-term strategies of prognosis, diagnosis and therapy for hypertensive DHF appear from this work.

Two candidate genes for two quantitative trait loci epistatically attenuate hypertension in a novel pathway.

Objectives: Multiple quantitative trait loci (QTLs) for blood pressure (BP) have been detected in rat models of human polygenic hypertension. They influence BP physiologically via epistatic modules. Little is known about the causal genes and virtually nothing is known on modularized mechanisms governing their regulatory connections. Methods and results: Two genes responsible for two individual BP QTLs on rat Chromosome 18 have been identified that belong to the same epistatic module. Treacher Collins-Franceschetti syndrome 1 (Tcof1) gene is the only function candidate for C18QTL3. Haloacid dehalogenase like hydrolase domain containing 2 (Hdhd2), although a gene of previously unknown function, is C18QTL4, and encodes a newly identified phosphatase. The current work has provided the premier evidence that Hdhd2/C18QTL4 and Tcof1/C18QTL3 may be involved in polygenic hypertension. Hdhd2/C18QTL4 can regulate the function of Tcof1/C18QTL3 via de-phosphorylation, and, for the first time, furbishes a molecular mechanism in support of a genetically epistatic hierarchy between two BP QTLs, and thus authenticates the epistasis-common pathway paradigm. Conclusion: The pathway initiated by Hdhd2/C18QTL4 upstream of Tcof1/C18QTL3 reveals novel mechanistic insights into BP modulations. Their discovery might yield innovative therapeutic targets and diagnostic tools predicated on a novel BP cause and mechanism that is determined by a regulatory hierarchy. Optimizing the de-phosphorylation capability and its downstream target could be antihypertensive. The conceptual paradigm of an order and regulatory hierarchy may help unravel genetic and molecular relationships among certain human BP QTLs.