Shifting Paradigm from Gene Expressions to Pathways Reveals Physiological Mechanisms in Blood Pressure Control in Causation.

Genetics for blood pressure (BP) in human and animals has been partitioned into two separate specialties. However, this divide is mechanistically-misleading. BP physiology is mechanistically participated by products of quantitative trait loci (QTLs). The key to unlocking its mechanistic mystery lies in the past with mammalian ancestors before humans existed. By pivoting from effects to causes, physiological mechanisms determining BP by six QTLs have been implicated. Our work relies on congenic knock-in genetics in vivo using rat models, and has reproduced the physiological outcome based on a QTL being molecularly equal to one gene. A gene dose for a QTL is irrelevant to physiological BP controls in causation. Together, QTLs join one another as a group in modularized Mendelian fashion to achieve polygenicity. Mechanistically, QTLs in the same module appear to function in a common pathway. Each is involved in a different step in the pathway toward polygenic hypertension. This work has implicated previously-concealed components of these pathways. This emerging concept is a departure from the human-centric precept that the level of QTL expressions, not physiology, would ultimately determine BP. The modularity/pathway paradigm breaks a unique conceptual ground for unravelling the physiological mechanisms of polygenic and quantitative traits like BP.

Genetics of Normotension Preventing Hypertension Leads to a Novel Physiological Paradigm.

Possessing blood pressure in normal ranges is considered healthy, and does not warrant medical attention for obvious clinical reasons. However, to realize normotension and then maintain it even when confronted with a hypertensive threat must have its biological 'shield of armour'. While sensitivity to hypertension has been widely recognized and studied, inherent mechanisms that enable a physiological resistance to hypertension to occur have received little attention. Recent advances in normotension genetics have produced unexpected insights. A hypertension 'suppressor' likely inhabits the normotensive genome of inbred Lewis rats. This suppressor behaves as a 'master' control capable of functionally abrogating the effects of hypertension-promoting alleles from multiple quantitative trait loci. This conceptual advancement lays the foundation for uncovering an anti-hypertension gene. Discovering its identity will assist our attempts at developing innovative diagnostic and therapeutic strategies for circumventing and treating hypertension. This new domain of suppressing hypertension goes beyond the conventional pharmacological treatments of hypertension before symptoms appear. For this purpose, a valid theoretical basis and framework is needed that can interpret the experimental data and produce testable predictions for authenticating, enriching or amending the normotension paradigm in the future.

Atg8 transfer from Atg7 to Atg3: a distinctive E1-E2 architecture and mechanism in the autophagy pathway.

Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7(NTD)) recruits a unique "flexible region" from Atg3 (Atg3(FR)). The structure of an Atg7(NTD)-Atg3(FR) complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and crosslinking data allow modeling of a full-length, dimeric (Atg7~Atg8-Atg3)(2) complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic cysteine of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distinctive E1~UBL-E2 architecture for enzymes mediating autophagy.

Structural Insight into the Photochemistry of Split Green Fluorescent Proteins: A Unique Role for a His-Tag.

Oligohistidine affinity tags (His-tags) are commonly fused to proteins to aid in their purification via metal affinity chromatography. These His-tags are generally assumed to have minimal impact on the properties of the fusion protein, as they have no propensity to form ordered elements, and are small enough not to significantly affect the solubility or size. Here we report structures of two variants of truncated green fluorescent protein (GFP), i.e., split GFP with a ß-strand removed, that were found to behave differently in the presence of light. In these structures, the N-terminal His-tag and several neighboring residues play a highly unusual structural and functional role in stabilizing the truncated GFP by substituting as a surrogate ß-strand in the groove vacated by the native strand. This finding provides an explanation for the seemingly very different peptide binding and photodissociation properties of split proteins involving ß-strands 10 and 11. We show that these truncated GFPs can bind other non-native sequences, and this promiscuity invites the possibility for rational design of sequences optimized for strand binding and photodissociation, both useful for optogenetic applications.

Retinoblastoma-associated protein 140 as a candidate for a novel etiological gene to hypertension.

Gene discovery in animal models may lead to the revelation of therapeutic targets for essential hypertension as well as mechanistic insights into blood pressure (BP) regulation. Our aim was to identify a disease-causing gene for a component of polygenic hypertension contrasting inbred hypertensive Dahl salt-sensitive (DSS) and normotensive Lewis rats. The chromosome segment harboring a quantitative trait locus (QTL), C16QTL, was first isolated from the rat genome via congenic strains. A candidate gene responsible for C16QTL causing a BP difference between DSS and Lewis rats was then identified using molecular analyses combining our independently-conducted total genome and gene-specific sequencings. The retinoblastoma-associated protein 140 (Rap140)/family with sequence similarity 208 member A (Fam208a) is the only candidate gene supported to be C16QTL among three genes in genome block 1 present in the C16QTL-residing interval. A mode of its actions could be to influence the expressions of genes that are downstream in a pathway potentially leading to BP regulation such as that encoding the solute carrier family 7 (cationic amino acid transporter, y+ system) member 12 (Slc7a12), which is specifically expressed in kidneys. Thus, Rap140/Fam208a probably encoding a transcription factor is the strongest candidate for a novel BP QTL that acts via a putative Rap140/Fam208a-Slc7a12-BP pathway. These data implicate a premier physiological role for Rap140/Fam208 beyond development and a first biological function for the Slc7a12 protein in any organism.

Modularization and epistatic hierarchy determine homeostatic actions of multiple blood pressure quantitative trait loci.

Hypertension, the most frequently diagnosed clinical condition world-wide, predisposes individuals to morbidity and mortality, yet its underlying pathological etiologies are poorly understood. So far, a large number of quantitative trait loci (QTLs) have been identified in both humans and animal models, but how they function together in determining overall blood pressure (BP) in physiological settings is unknown. Here, we systematically and comprehensively performed pair-wise comparisons of individual QTLs to create a global picture of their functionality in an inbred rat model. Rather than each of numerous QTLs contributing to infinitesimal BP increments, a modularized pattern arises: two epistatic 'blocks' constitute basic functional 'units' for nearly all QTLs, designated as epistatic module 1 (EM1) and EM2. This modularization dictates the magnitude and scope of BP effects. Any EM1 member can contribute to BP additively to that of EM2, but not to those of the same module. Members of each EM display epistatic hierarchy, which seems to reflect a related functional pathway. Rat homologues of 11 human BP QTLs belong to either EM1 or EM2. Unique insights emerge into the novel genetic mechanism and hierarchy determining BP in the Dahl salt-sensitive SS/Jr (DSS) rat model that implicate a portion of human QTLs. Elucidating the pathways underlying EM1 and EM2 may reveal the genetic regulation of BP.

Modularity/non-cumulativity of quantitative trait loci on blood pressure.

Large numbers of quantitative trait loci (QTLs) for blood pressure (BP) exist and have long been thought to function by accumulating their individual miniscule effects. Recent experimental evidence in the functional biology of BP control has tested this intuitive assumption. A new paradigm has emerged that BP is biologically determined in modularity by multiple QTLs. Functionally, when a master regulator is taken out, distinct epistatic modules organize biological 'blocks' into a genetic architecture, and serve as basic functional cores from which numerous QTLs act together to physiologically formulate BP. An epistatic module refers to the grouping of QTLs that perform their functions epistatically to one another and influence BP as a group. The modularity mechanism framework indicates that BP as a quantitatively-measured trait is not cumulatively determined and implies that the QTLs in the same epistatic module may participate in the same pathway leading to the BP control, and the QTLs from separate epistatic modules may act in divergent but parallel pathways. This mechanistic conceptualization and subsequent validations synergize with anticipated demands from current human epidemiological studies, since the outcome from them primarily implicates single nucleotide polymorphisms with unknown functions. Eventually, functional understandings of the human results have to be realized by their pathogenic directionality and mechanisms biologically controlling BP.

Conserved mammalian modularity of quantitative trait loci revealed human functional orthologs in blood pressure control.

Genome-wide association studies (GWAS) have routinely detected human quantitative trait loci (QTLs) for complex traits. Viewing that most GWAS single nucleotide polymorphisms (SNPs) are found in non-coding regions unrelated to the physiology of a polygenic trait of interest, a vital question to answer is whether or not any of these SNPs can functionally alter the phenotype with which it is associated. The study of blood pressure (BP) is a case in point. Conserved mechanisms in controlling BP by modularity is now unifying differing mammalian orders in that understanding mechanisms in rodents is tantamount to revealing the same in humans, while overcoming experimental limitations imposed by human studies. As a proof of principle, we used BP QTLs from Dahl salt-sensitive rats (DSS) as substitutes to capture distinct human functional orthologs. 3 DSS BP QTLs are located into distinct genome regions and correspond to several human GWAS genes. Each of the QTLs independently exerted a major impact on BP in vivo. BP was functionally changed by normotensive alleles from each of these QTLs, and yet, the human GWAS SNPs do not exist in the rat. They cannot be responsible for physiological alterations in BP caused by these QTLs. These SNPs are genome emblems for QTLs nearby, rather than being QTLs per se, since they only emerged during primate evolution after BP-regulating mechanisms have been established. We then identified specific mutated coding domains that are conserved between rodents and humans and that may implicate different steps of a common pathway or separate pathways.

Functional Captures of Multiple Human Quantitative Trait Loci Regulating Blood Pressure with the Use of Orthologs in Genetically Defined Rat Models.

BACKGROUND: Most signals from human genome-wide association studies (GWAS) for blood pressure (BP) are single-nucleotide polymorphisms (SNPs). It was unknown if such SNPs can functionally affect BP. Because BP is similar between humans and rodents, unraveling basic mechanisms from rodents can reveal the same BP-modulating mechanisms in humans originating from their common ancestors while overcoming limitations in human epidemiology. METHODS: For the first time, we used quantitative trait loci (QTLs) from Dahl salt-sensitive (DSS) rats as functional surrogates to capture human BP QTLs. RESULTS: A total of 107 human GWAS genes may be classified into 2 common pathways of hypertension pathogeneses. Among them, 4 DSS BP QTLs correspond to 4 human GWAS genes. Each of them independently showed a major impact on BP in vivo and thus functional redundancy. BP was altered by each of these 4 QTLs, but human GWAS SNPs marking these QTLs do not exist in the rat. They cannot be responsible for physiological changes in BP caused by these QTLs and are genome signposts marking positions of the QTLs nearby, rather than being QTLs themselves. These SNPs appeared during primate evolution, independently of BP regulation. Because the functional dosage of QTLs, not their gene dose, determined hypertension pathogenesis, a role for the noncoding GWAS SNPs in BP via regulating gene expressions can be discounted. CONCLUSIONS: The human QTLs may function in a common pathway, with each involved in a different step in the pathway leading to BP control. These results may be conceptually paradigm shifting.

Biological convergence of three human and animal model quantitative trait loci for blood pressure.

OBJECTIVES: Blood pressure (BP) is comparable among different mammalian orders, despite their evolution divergence. Because of it, fundamental mechanisms should connect humans and rodents by their shared BP physiology. We hypothesized that similar quantitative trait loci (QTLs) function in both humans and rodents in controlling BP. METHODS: We utilized inbred hypertensive Dahl salt-sensitive rats (DSS) as a functional proxy to evaluate the relevance of human genome-wide association studies (GWAS) genes in BP regulation. RESULTS: First, three DSS BP QTLs functionally captured three specific human GWAS genes. Each QTL has a major biological impact, not a miniscule effect, on BP, in causation by function. Second, noncoding single-nucleotide polymorphisms (SNPs) found in GWAS are by products of primate evolution, instead of mechanistic drivers in regulating BP, because their absence did not impact on BP of mammals. Third, a missense mutation, rather than a noncoding GWAS SNP marking it nearby, is the priority functional basis for a given QTL. Depleting such a noncoding GWAS SNP had no impact, whereas eliminating the muscarinic cholinergic receptor 3 (M3R) signaling decreased BP. Finally, epistatic modularity biologically organizes multiple QTLs with redundant functions, and is the genetic mechanism that modulates the BP homeostasis when QTLs function collectively. CONCLUSIONS: Two pathogenic pathways of hypertension biologically unify mechanisms of BP regulations for humans and their functional surrogates. The mechanism-based biology for the M3R-mediated pathway in raising BP has established M3R as a novel pathogenesis-driven target for antihypertension therapies.

Functional Dosage of Muscarinic Cholinergic Receptor 3 Signalling, Not the Gene Dose, Determines Its Hypertension Pathogenesis.

BACKGROUND: Multiple quantitative trait loci for blood pressure (BP) have been localized throughout human and rodent genomes. Few of them have been functionally identified especially in humans, and little is known about their pathogenic directionality when identified. We focused on Chrm3 encoding the muscarinic cholinergic receptor 3 (M3R) as the causal gene for C17QTL1 in the Dahl salt-sensitive rat model. METHODS AND RESULTS: Congenic knock-ins, gene-specific knockout, and ex vivo and in vivo function studies were applied in the Dahl salt-sensitive rat model of polygenic hypertension. A Chrm3 missense T1667C mutation in the last intracellular domain functionally correlated with a rise in BP increased the M3R signalling and resensitization, and adrenal epinephrogenesis. Gene targeting that abolished the M3R function without affecting any of noncoding Chrm3 variants caused a decrease in BP, indicating that the M3R-mediated signalling promotes hypertension. In contrast, removing 8 amino acids from the M3R first extracellular loop had no effect on BP. CONCLUSIONS: The M3R-specialized signalling constitutes a new pathway of hypertension pathogenesis within the context of a polygenic and quantitative trait. Increased epinephrine in the circulation and secreted from the adrenal glands are suggestive of a molecular mechanism partially mediating M3R to promote hypertension. The structure-function relationships for various M3R domains in their effects on BP pave the way for identifying missense mutations that impact functions on BP as potential diagnostic targets.

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.

Distinct genomic replacements from Lewis correct diastolic dysfunction, attenuate hypertension, and reduce left ventricular hypertrophy in Dahl salt-sensitive rats.

BACKGROUND: Hypertension and diastolic heart failure are two common cardiovascular diseases that inflict heavy morbidity and mortality, yet relatively little is understood about their pathophysiology. The identification of quantitative trait loci for blood pressure is important in unveiling the causes of polygenic hypertension. Although Dahl salt-sensitive strain is also an excellent model for the study of diastolic heart failure, virtually nothing is known about the quantitative trait loci determining diastolic heart failure. Diastolic dysfunction often represents the onset of diastolic heart failure. METHODS: We first characterized the cardiac phenotype of Dahl salt-sensitive strain and normotensive Lewis control rats by echocardiography to ascertain diastolic function. We then analyzed corresponding features of four newly developed and two existing congenic strains, each of which carries a specific chromosome substitution of Dahl salt-sensitive strain by its Lewis homologue and each lowering blood pressure. RESULTS: Dahl salt-sensitive strain displayed diastolic dysfunction that was rectified in two of six congenic strains, designated as positive congenic strains, which represent the first rodent models exhibiting functional normalization of diastolic dysfunction caused by naturally occurring genetic variants. The two positive congenic strains also showed a reduction in left ventricular mass. In contrast, four of six congenic strains did not change diastolic function despite their blood pressure-lowering effects. CONCLUSION: Genes present in the replaced chromosome segments of the two positive congenic strains are not commonly known to affect blood pressure, diastolic function or left ventricular mass. Consequently, novel prognostic, diagnostic and therapeutic strategies for hypertensive diastolic heart failure likely emerge from this work.

Sexual dimorphism on hypertension of quantitative trait loci entrapped in Dahl congenic rats.

Although it is well-known that quantitative trait loci (QTLs) influence blood pressure (BP) in male Dahl salt-sensitive rats (DSS), few studies have been carried out to ascertain the BP effect of these QTLs in females. In the current work, we analyzed BP of seven selected congenic strains constructed in the DSS background. One QTL, C8QTL2, exhibited similar effects on systolic (SAP), diastolic (DAP), and mean arterial (MAP) pressures in females as previously shown in males. In contrast, six QTLs that previously demonstrated influences on SAP, DAP, and MAP in males did not have effects in females. These male-specific QTLs are likely regulated differently in males than in females and emphasize the necessity of identifying female-specific QTLs for diagnosing and treating hypertension in women. Current findings may have implications in genetic research of essential hypertension, and association and linkage analyses should be performed in separate genders. Men and women may possess distinctive as well as shared genetic determinants for SAP, DAP, and MAP. The data on a single gene or marker might be pooled from both genders only when evidence favors the sex-independence in a study.

Novel Pathogenesis of Hypertension and Diastolic Dysfunction Caused by M3R (Muscarinic Cholinergic 3 Receptor) Signaling.

Multiple quantitative trait loci for blood pressure (BP) are localized in humans and rodent models. Model studies have not only produced human quantitative trait loci homologues but also provided unforeseen mechanistic insights into the function modality of quantitative trait loci actions. Presently, congenic knockins, gene-specific knockout, and in vitro and in vivo function studies were used in a rat model of polygenic hypertension, DSS (Dahl salt sensitive) rats. One gene previously unknown in regulating BP was detected with 1 structural mutation(s) for each of 2 quantitative trait loci classified into 2 separate epistatic modules 1 and 3. C17QTL1 in epistatic module 2 was identified to be the gene Chrm3 encoding the M3R (muscarinic cholinergic 3 receptor), since a single function-enhancing M3RT556M conversion correlated with elevated BP. To definitively prove that the enhanced M3R function is responsible for BP changes by the DSS alleles of C17QTL1, we generated a Chrm3 gene-specific rat knockout. We observed a reduction in BP without tachycardia in both sexes, regardless of the amount of dietary salt, and an improvement in diastolic and kidney dysfunctions. All occurred in spite of a significant reduction in M3R-dependent vasodilation. The previously seen sexual dimorphism for C17QTL1 on BP disappeared in the absence of M3R. A Chrm3-coding variation increased M3R signaling, correlating with higher BP. Removing the M3R signaling led to a decrease in BP and improvements in cardiac and renal malfunctions. A novel pathogenic pathway accounted for a portion of polygenic hypertension and has implications in applying new diagnostic and therapeutic uses against hypertension and diastolic dysfunction.

Association of the beta2-adrenergic receptor gene with essential hypertension in the non-Han Chinese Yi minority human population.

OBJECTIVE: The human beta2-adrenergic receptor (ADRB2) gene is a candidate for contributing to the pathophysiology of essential hypertension. The aims of the present study were to investigate the associations of differing single nucleotide polymorphisms (SNPs) and haplotypes of the ADRB2 gene promoter and coding regions with essential hypertension in genetically homogeneous Hani and Yi minority groups that are non-Han Chinese. METHODS: Four SNPs in the regulatory and seven SNPs in the coding region were genotyped in 271 essential hypertension individuals and 267 controls, and eight haplotypes in the regulatory and five haplotypes in the coding region were determined and tested for association using the likelihood test statistic. RESULTS: There were significant associations of essential hypertension with separate SNPs located in both the regulatory and coding regions in the Yi minority group. In contrast, no associations of essential hypertension were detected with any of single SNPs in the Hani minority group. There is a significant difference in haplotype frequency distributions between the hypertensive participants and the controls in two groups (P < 10). CONCLUSION: The results indicate that variants at the ADRB2 locus may play a role in the pathophysiology of hypertension specifically in the Yi minority group.

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.

Severe hypertension caused by alleles from normotensive Lewis for a quantitative trait locus on chromosome 2.

Pursuing fully a suggestion from linkage analysis that there might be a quantitative trait locus (QTL) for blood pressure (BP) in a chromosome (Chr) 2 region of the Dahl salt-sensitive rat (DSS), four congenic strains were made by replacing various fragments of DSS Chr 2 with those of Lewis (LEW). Consequently, a BP QTL was localized to a segment of around 3 cM or near 3 Mb on Chr 2 by comparative congenics. The BP-augmenting alleles of this QTL originated from the LEW rat, a normotensive strain compared with DSS. The dissection of a QTL with such a paradoxical effect illustrated the power of congenics in unearthing a gene hidden in the context of the whole animal system, presumably by interactions with other genes. The locus for the angiotensin II receptor AT-1B (Agtr1b) is not supported as a candidate gene for the QTL because a congenic strain harboring it did not have an effect on BP. There are approximately 19 known and unknown genes present in the QTL interval. Among them, no standout candidate genes are reputed to affect BP. Thus the QTL will likely represent a novel gene for BP regulation.

Complete and overlapping congenics proving the existence of a quantitative trait locus for blood pressure on Dahl rat chromosome 17.

Linkage studies suggested that a quantitative trait locus (QTL) for blood pressure (BP) was present in a region on chromosome 17 (Chr 17) of Dahl salt-sensitive (DSS) rats. A subsequent congenic strain targeting this QTL, however, could not confirm it. These conflicting results called into question the validity of localization of a QTL by linkage followed by the use of a congenic strain made with an incomplete chromosome coverage. To resolve this issue, we constructed five new congenic strains, designated C17S.L1 to C17S.L5, that completely spanned the +/-2 LOD confidence interval supposedly containing the QTL. Each congenic strain was made by replacing a segment of the DSS rat by that of the normotensive Lewis (LEW) rat. The only section to be LL homozygous is the region on Chr 17 specified in a congenic strain, as evidenced by a total genome scan. The results showed that BPs of C17S.L1 and C17S.L2 were lower (P < 0.04) than that of DSS rats. In contrast, BPs of C17S.L3, C17S.L4, and C17S.L5 were not different (P > 0.6) from that of DSS rats. Consequently, a BP QTL must be located in an interval of approximately 15 cM shared between C17S.L1 and C17S.L2 and unique to them both, as opposed to C17S.L3, C17S.L4, and C17S.L5. The present study illustrates the importance of thorough chromosome coverage, the necessity for a genome-wide screening, and the use of "negative" controls in physically mapping a QTL by congenic strains.