Positional identification of variants of Adamts16 linked to inherited hypertension.

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.

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.

Genetic linkage of urinary albumin excretion in Dahl salt-sensitive rats: influence of dietary salt and confirmation using congenic strains.

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.

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.

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.

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.

Congenic mapping of a blood pressure QTL region on rat chromosome 10 using the Dahl salt-sensitive rat with introgressed alleles from the Milan normotensive strain.

Multiple blood pressure (BP) quantitative trait loci (QTLs) are reported on rat chromosome 10 (RNO10). Of these, QTLs detected by contrasting the genome of the hypertensive Dahl salt-sensitive (S) rat with two different relatively normotensive strains, Lewis (LEW) and the Milan normotensive strain (MNS), are reported. Because the deduced QTL regions of both S vs. LEW and S vs. MNS comparisons are within large genomic segments encompassing more than 2 cM, there was a need to further localize these QTLs and determine whether the QTLs are unique to specific strain comparisons. Previously, the S.MNS QTL1 was mapped to less than 2.6 cM as a differential segment between two congenic strains. In this study, multiple congenic strains spanning the projected interval were studied. The BP effect of each strain was interpreted as the net effect of alleles introgressed within that congenic strain. The results suggest that the MNS alleles within the previously proposed differential segment (D10Rat27-D10Rat24) do not independently lower BP of the S rat. However, another congenic strain, S.MNS(10) x 9, containing introgressed MNS alleles that are outside of the previously proposed differential segment is of interest because (1) it demonstrated a BP-lowering effect, (2) it is contained within a single congenic strain and is not based on the observed effect of a differential segment, and, more importantly, (3) it overlaps with the previously identified S.LEW BP QTL region. Identification of the same QTL affecting BP in multiple rat strains will provide further support for the QTL's involvement and importance in human essential hypertension.

Closely linked non-additive blood pressure quantitative trait loci.

There is enough evidence through linkage and substitution mapping to indicate that rat chromosome 1 harbors multiple blood pressure (BP) quantitative trait loci (QTLs). Of these, BP QTL1b was previously reported from our laboratory using congenic strains derived by introgressing normotensive alleles from the LEW rat onto the genetic background of the hypertensive Dahl salt-sensitive (S) rat. The region spanned by QTL1b is quite large (20.92 Mb), thus requiring further mapping with improved resolution so as to facilitate systematic identification of the underlying genetic determinant(s). Using congenic strains containing the LEW rat chromosomal segments on the Dahl salt-sensitive (S) rat background, further iterations of congenic substrains were constructed and characterized. Collective data obtained from this new iteration of congenic substrains provided evidence for further fragmentation of QTL1b with improved resolution. At least two separate genetic determinants of blood pressure underlie QTL1b. These are within 7.40 Mb and 7.31 Mb and are known as the QTL1b1 region and the QTL1b2 region, respectively. A genetic interaction was detected between the two BP QTLs. Interestingly, five of the previously reported differentially expressed genes located within the newly mapped QTL1b1 region remained differentially expressed. The congenic strain S.LEW(D1Mco36-D1Mco101), which harbors the QTL1b1 region alone but not the QTL1b2 region, serves as a genetic tool for further dissection of the QTL1b1 region and validation of Nr2f2 as a positional candidate gene. Overall, this study represents an intermediary yet obligatory progression towards the identification of genetic elements controlling BP.

Fine-mapping and comprehensive transcript analysis reveals nonsynonymous variants within a novel 1.17 Mb blood pressure QTL region on rat chromosome 10.

The presence of blood pressure (BP) quantitative trait loci (QTL) on rat chromosome 10 has been clearly demonstrated by linkage analysis and substitution mapping. Using congenic strains containing the LEW rat chromosomal segments on the Dahl salt-sensitive (S) rat background, further iterations of congenic substrains were constructed and characterized to fine-map a chromosome 10 region (QTL1) linked to blood pressure. Comparison of seven congenic substrains refined QTL1 to a 1.17 Mb segment flanked by D10Mco88 and D10Mco89, which are located at 71,513,116 and 72,684,774 bp, respectively. The newly defined QTL1, containing 18 genes, is captured in its entirety within a single congenic substrain. A thorough transcript analysis revealed that 3 of these 18 genes, Ccl5, Ddx52, and RGD1559577, had nonsynonymous allelic variations between the S rat and the LEW rat. None of the detected transcripts within the newly defined QTL1 are implicated directly in BP control in humans or model organisms. Therefore, the present work defines a novel blood pressure QTL with three potential quantitative trait nucleotides.

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.

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.