Shane Yerga-Woolwine

Congenic Interval Mapping of RNO10 Reveals a Complex Cluster of Closely-Linked Genetic Determinants of Blood Pressure

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.

Augmented Rififylin Is a Risk Factor Linked to Aberrant Cardiomyocyte Function, Short-QT Interval and Hypertension

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 (P<0.001) 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; P<0.01). The congenic strain implanted with electrocardiography transmitters demonstrated shorter-QT intervals and increased heart rate compared with S rats (P<0.01). The average survival of the congenic strain was shorter (134 days) compared with the S rat (175 days; P<0.0007). The critical region was narrowed to <42.5 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 (P<0.01). Frequency of cardiomyocyte beats in the congenic strain (62±9 bpm) was significantly higher than that of the S rat (24±6 bpm; P<0.001). 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.

Association of von Willebrand factor activity with ACE I/D and MTHFR C677T polymorphisms in migraine

Angiotensin-converting enzyme (ACE) insertion (I)/deletion (D) and methylene-tetrahydrofolate reductase (MTHFR) C677T polymorphisms are linked to endothelial dysfunction and to cerebral white matter lesions. Objectives of this study were to determine if ACE and MTHFR gene polymorphisms are associated with von Willebrand factor (vWF) activity, an endothelial dysfunction marker, and with a distinct headache phenotype. We enrolled 64 women (18–50 years old) with International Classification of Headache Disorders, 2nd edn migraine without aura (MoA) and 61 with aura (MA). Genotypic frequencies: ACE DD 35%, ID 42%, II 23%, and MTHFR TT 17%, CT 40%, CC 43%. Those with ACE DD genotype had higher levels of vWF activity (152%) compared with ID and II genotypes. Levels were highest (179%) with combined ACE DD and MTHFR TT genotypes. ACE DD was associated with higher headache frequency, and MTHFR TT was associated with MA. In migraine, vWF activity may be a marker of endothelial-mediated genetic risk for ischaemic conditions.

Refined mapping of blood pressure quantitative trait loci using congenic strains developed from two genetically hypertensive rat models

Previously linkage and substitution mapping were conducted between the Dahl Salt-sensitive (S) rat and the Spontaneously Hypertensive Rat (SHR) to address the hypothesis that genetic contributions to blood pressure (BP) in two genetically hypertensive rat strains are different. Among the BP quantitative trait loci (QTLs) detected, two are located on chromosome 9 within large genomic segments. The goal of the current study was to develop new iterations of congenic substrains, to further resolve both of these BP QTLs on chromosome 9 as independent congenic segments. A total of 10 new congenic substrains were developed and characterized. The newly developed congenic substrains S.SHR(9)x8Ax11A and S.SHR(9)x10Ax1, with introgressed segments of 2.05 and 6.14 Mb, represented the shortest genomic segments. Both of these congenic substrains, S.SHR(9)x8Ax11A and S.SHR(9)x10Ax1 lowered BP of the S rat by 56 mm Hg (P<0.001) and 15 mm Hg (P<0.039), respectively. The BP measurements were corroborated by radiotelemetry. Urinary protein excretion was significantly lowered by SHR alleles within S.SHR(9)x10Ax1 but not by S.SHR(9)x8Ax11A. The shorter of the two congenic segments, 2.05 Mb was further characterized and found to contain a single differentially expressed protein-coding gene, Tomoregulin-2 (Tmeff2). The protein expression of Tmeff2 was higher in the S rat compared with S.SHR(9)x8Ax11A, which also had lower cardiac hypertrophy as measured by echocardiography. Tmeff2 is known to be upregulated in patients from multiple cohorts with cardiac hypertrophy. Taken together, Tmeff2 can be prioritized as a candidate gene for hypertension and associated cardiac hypertrophy in both rats and in humans.

Epistatic genetic determinants of blood pressure and mortality in a salt-sensitive hypertension model.

Although genetic determinants protecting against the development of elevated blood pressure (BP) are well investigated, less is known regarding their impact on longevity. We concomitantly assessed genomic regions of rat chromosomes 3 and 7 (RNO3 and RNO7) carrying genetic determinants of BP without known epistasis, for their independent and combinatorial effects on BP and the presence of genetic determinants of survival using Dahl salt-sensitive (S) strains carrying congenic segments from Dahl salt-resistant (R) rats. Although congenic and bicongenic S.R strains carried independent BP quantitative trait loci within the RNO3 and RNO7 congenic regions, only the RNO3 allele(s) independently affected survival. The bicongenic S.R strain showed epistasis between R-rat RNO3 and RNO7 alleles for BP under salt-loading conditions, with less-than-additive effects observed on a 2% NaCl diet and greater-than-additive effects observed after prolonged feeding on a 4% NaCl diet. These RNO3 and RNO7 congenic region alleles had more-than-additive effects on survival. Increased survival of bicongenic compared with RNO3 congenic rats was attributable, in part, to maintaining lower BP despite chronic exposure to an increased dietary salt (4% NaCl) intake, with both strains showing delays in reaching highest BP. R-rat RNO3 alleles were also associated with superior systolic function, with the S.R bicongenic strain showing epistasis between R-rat RNO3 and RNO7 alleles leading to compensatory hypertrophy. Whether these alleles affect survival by additional actions within other BP-regulating tissues/organs remains unexplored. This is the first report of simultaneous detection of independent and epistatic loci dictating, in part, longevity in a hypertensive rat strain.

Defining a rat blood pressure quantitative trait locus to a <81.8 kb congenic segment: comprehensive sequencing and renal transcriptome analysis

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 <81.8 kb to <1.33 Mb 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 <81.8 kb significantly lowered BP of the hypertensive S rat by 25 mmHg 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 <81.8 kb congenic strain, which represents the shortest genomic segment to which a BP QTL has been 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. Furthermore, a whole genome renal transcriptome analysis between S and the <81.8 kb 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.

Cross-Talk of Expression Quantitative Trait Loci Within 2 Interacting Blood Pressure Quantitative Trait Loci

Genetic dissection of the S rat genome has provided strong evidence for the presence of 2 interacting blood pressure quantitative trait loci (QTLs), termed QTL1 and QTL2, on rat chromosome 5. However, the identities of the underlying interacting genetic factors remain unknown. Further experiments targeted to identify the interacting genetic factors by the substitution mapping approach alone are difficult because of the interdependency of natural recombinations to occur at the 2 QTLs. We hypothesized that the interacting genetic factors underlying these 2 QTLs may interact at the level of gene transcription and thereby represent expression QTLs or eQTLs. To detect these interacting expression QTLs, a custom QTL chip containing the annotated genes within QTL1 and QTL2 was developed and used to conduct a transcriptional profiling study of S and 2 congenic strains that retain either 1 or both of the QTLs. The results uncovered an interaction between 2 transcription factor genes, Dmrta2 and Nfia. Furthermore, the “biological signature” elicited by these 2 transcription factors was differential between the congenic strain that retained Lewis alleles at both QTL1 and QTL2 compared with the congenic strain that retained Lewis alleles at QTL1 alone. A network of transcription factors potentially affecting blood pressure could be traced, lending support to our hypothesis.

Blood pressure and proteinuria effects of multiple quantitative trait loci on rat chromosome 9 that differentiate the spontaneously hypertensive rat from the Dahl salt-sensitive rat

A blood pressure (BP) quantitative trait locus (QTL) was previously located within 117 kb on rat chromosome 9 (RNO9) using hypertensive Dahl salt-sensitive and normotensive Dahl salt-resistant rats. An independent study between two hypertensive rat strains, the Dahl salt-sensitive rat and the spontaneously hypertensive rat (SHR), also detected a QTL encompassing this 117 kb region. Dahl salt-sensitive alleles in both of these studies were associated with increased BP. To map SHR alleles that decrease BP in the Dahl salt-sensitive rat, a panel of eight congenic strains introgressing SHR alleles onto the Dahl salt-sensitive genetic background were constructed and characterized. S.SHR(9)x3B, S.SHR(9)x3A and S.SHR(9)x2B, the congenic regions of which span a portion or all of the 1 logarithm of odds (LOD) interval identified by linkage analysis, did not significantly alter BP. However, S.SHR(9), S.SHR(9)x4A, S.SHR(9)x7A, S.SHR(9)x8A and S.SHR(9)x10A, the introgressed segments of which extend distal to the 1 LOD interval, significantly reduced BP. The shortest genomic segment, BP QTL1, to which this BP-lowering effect can be traced is the differential segment of S.SHR(9)x4A and S.SHR(9)x2B, to which an urinary protein excretion QTL also maps. However, the introgressed segment of S.SHR(9)x10A, located outside of this QTL1 region, represented a second BP QTL (BP QTL2) having no detectable effects on urinary protein excretion. In summary, the data suggest that there are multiple RNO9 alleles of the SHR that lower BP of the Dahl salt-sensitive rat with or without detectable effects on urinary protein excretion and that only one of these BP QTLs, QTL1, overlaps with the 117 kb BP QTL region identified using Dahl salt-sensitive and Dahl salt-resistant rats.