Brown Norway Alleles Research Articles

Dissecting the role of Folr1 and Folh1 genes in the pathogenesis of metabolic syndrome in spontaneously hypertensive rats.

Increased levels of plasma cysteine predispose to obesity and metabolic disturbances. Our recent genetic analyses in spontaneously hypertensive rats (SHR) revealed mutated Folr1 (folate receptor 1) on chromosome 1 as a quantitative trait gene associated with reduced folate levels, hypercysteinemia and metabolic disturbances. The Folr1 gene is closely linked to the Folh1 (folate hydrolase 1) gene which codes for an enzyme involved in the hydrolysis of dietary polyglutamyl folates in the intestine. In the current study, we obtained evidence that Folh1 mRNA of the BN (Brown Norway) origin is weakly but significantly expressed in the small intestine. Next we analyzed the effects of the Folh1 alleles on folate and sulfur amino acid levels and consecutively on glucose and lipid metabolism using SHR-1 congenic sublines harboring either Folr1 BN and Folh1 SHR alleles or Folr1 SHR and Folh1 BN alleles. Both congenic sublines when compared to SHR controls, exhibited significantly reduced folate clearance and lower plasma cysteine and homocysteine levels which was associated with significantly decreased serum glucose and insulin concentrations and reduced adiposity. These results strongly suggest that, in addition to Folr1, the Folh1 gene also plays an important role in folate and sulfur amino acid levels and affects glucose and lipid metabolism in the rat.

Abstract P5-05-02: A laboratory/machine learning based comparative genetics model accurately predicts breast cancer in a human cohort

Abstract Inherited genetic variants are estimated to account for 30-35% of overall breast cancer risk. A few rare, highly penetrant, genetic determinants of risk in humans have been well defined. However, the actions of many common weakly penetrant breast cancer risk loci remain uncharacterized. Additionally, it is well established that endocrine factors in general, and estrogens in particular, influence breast cancer etiology. We are using the ACI rat model of 17β-estradiol (E2)-induced mammary cancer to parse the contributions of individual genetic risk variants to breast cancer susceptibility in a physiologically relevant context. ACI females develop mammary carcinomas at an incidence approaching 100% when exposed to physiological levels of E2, and these carcinomas share many features with luminal-type breast cancers in humans. In contrast, Brown Norway (BN) rats are highly resistant to E2-induced mammary cancer. Linkage analyses of progeny from intercrosses between susceptible ACI and resistant BN rats led to the identification of multiple quantitative trait loci for E2-induced mammary cancer. One such locus, Estrogen-induced mammary cancer 4 (Emca4), is the focus of the current investigation. We generated a series of novel congenic rat strains which carry BN alleles at distinct regions of interest across the Emca4 locus, introgressed onto the ACI genetic background. Characterization of mammary cancer phenotypes in the congenic strains facilitated fine resolution mapping of the Emca4 locus. These studies revealed that Emca4 is a complex locus harboring at least four interacting genetic determinants of risk, designated Emca4.1 – Emca4.4, and is orthologous to the 8q24.21 breast cancer risk locus in humans. To assess the relevance of the rat genetic data to human populations, novel machine learning methods were employed to generate risk prediction models using data from a human cohort. Genotype data for 76 SNPs located in the regions of the human genome orthologous to Emca4.1 – 4.4 were obtained from the Cancer Genetics Markers of Susceptibility case control population. Models generated from this data set were optimized with novel algorithms to identify a subset of 16 variants that significantly influenced the risk models. The best model distinguished breast cancer cases from controls with a remarkably high degree of accuracy for a model based on genotype (AUC = 0.6, P < 10-11 relative to random guessing). It is worth noting that the predictive power of this model arose from interactions between human SNPs. Our data show that Emca4 is a complex locus containing multiple interacting determinants of risk; variants in the orthologous 8q24.21 breast cancer risk locus in human interact to influence breast cancer risk as predicted by the rat model; and accounting for interactions between variants achieves a predictive power beyond what is observed with individual SNPs. We have demonstrated, for the first time, the ability to develop a multi-component genetic model in rats and test it in a human population. This illustrates the power of the rat model to elucidate the complex mechanisms through which common, weakly penetrant variants influence breast cancer risk in humans. Citation Format: Dennison KL, Chack A, Escanilla NS, Page D, Shull JD. A laboratory/machine learning based comparative genetics model accurately predicts breast cancer in a human cohort [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-05-02.

Elevation of fumarase attenuates hypertension and can result from a nonsynonymous sequence variation or increased expression depending on rat strain.

The activity of fumarase, an enzyme in the tricarboxylic acid cycle, is lower in Dahl salt-sensitive SS rats compared with SS.13BN rats. SS.13BN rats have a Brown Norway (BN) allele of fumarase and exhibit attenuated hypertension. The SS allele of fumarase differs from the BN allele by a K481E sequence variation. It remains unknown whether higher fumarase activities can attenuate hypertension and whether the mechanism is relevant without the K481E variation. We developed SS-TgFh1 transgenic rats overexpressing fumarase on the background of the SS rat. Hypertension was attenuated in SS-TgFh1 rats. Mean arterial pressure in SS-TgFh1 rats was 20 mmHg lower than transgene-negative SS littermates after 12 days on a 4% NaCl diet. Fumarase overexpression decreased H2O2, while fumarase knockdown increased H2O2 Ectopically expressed BN form of fumarase had higher specific activity than the SS form. However, sequencing of more than a dozen rat strains indicated most rat strains including salt-insensitive Sprague-Dawley (SD) rats had the SS allele of fumarase. Despite that, total fumarase enzyme activity in the renal medulla was still higher in SD rats than in SS rats, which was associated with higher expression of fumarase in SD. H2O2 can suppress the expression of fumarase. Renal medullary interstitial administration of fumarase siRNA in SD rats resulted in higher blood pressure on the high-salt diet. These findings indicate elevation of total fumarase activity attenuates the development of hypertension and can result from a nonsynonymous sequence variation in some rat strains and higher expression in other rat strains.

Validation of six genetic determinants of susceptibility to estrogen-induced mammary cancer in the rat and assessment of their relevance to breast cancer risk in humans.

When treated with 17β-estradiol, female ACI rats (Rattus norvegicus) rapidly develop mammary cancers that share multiple phenotypes with luminal breast cancers. Seven distinct quantitative trait loci that harbor genetic determinants of susceptibility to 17β-estradiol−induced mammary cancer have been mapped in reciprocal intercrosses between susceptible ACI rats and resistant Brown Norway (BN) rats. A panel of unique congenic rat strains has now been generated and characterized to confirm the existence of these quantitative trait loci, designated Emca3 through Emca9, and to quantify their individual effects on susceptibility to 17β-estradiol−induced mammary cancer. Each congenic strain carries BN alleles spanning an individual Emca locus, introgressed onto the ACI genetic background. Data presented herein indicate that BN alleles at Emca3, Emca4, Emca5, Emca6, and Emca9 reduce susceptibility to 17β-estradiol−induced mammary cancer, whereas BN alleles at Emca7 increase susceptibility, thereby confirming the previous interval mapping data. All of these Emca loci are orthologous to regions of the human genome that have been demonstrated in genome-wide association studies to harbor genetic variants that influence breast cancer risk. Moreover, four of the Emca loci are orthologous to loci in humans that have been associated with mammographic breast density, a biomarker of breast cancer risk. This study further establishes the relevance of the ACI and derived congenic rat models of 17β-estradiol−induced mammary cancer for defining the genetic bases of breast cancer susceptibility and elucidating the mechanisms through which 17β-estradiol contributes to breast cancer development.

Rab38 Modulates Proteinuria in Model of Hypertension-Associated Renal Disease

We previously reported that the fawn-hooded hypertensive (FHH) rat is a natural Rab38 knockout, supported by a congenic animal (FHH.BN-Rab38) having less proteinuria than FHH animals. Because these congenic animals contain Brown Norway (BN) alleles for five other named genes; however, a causal role for Rab38 in the FHH phenotype remains uncertain. Here, we used transgenic and knockout models to validate Rab38 and to exclude other genes within the 1.5 Mb congenic region from involvement in causing the FHH phenotype. Transgenic rats homozygous for the wild-type Rab38 BN allele on the FHH background exhibited phenotypic rescue, having 43% lower proteinuria and 75% lower albuminuria than nontransgenic FHH littermates. Conversely, knockout of the Rab38 gene on the FHH.BN-Rab38 congenic line recapitulated a proteinuric phenotype indistinguishable from the FHH strain. In addition, in cultured proximal tubule LLC-PK1 cells, knockdown of Rab38 mRNA significantly decreased endocytosis of colloidal gold-coupled albumin, supporting the hypothesis that Rab38 modulates proteinuria through effects on tubular re-uptake and not by altering glomerular permeability. Taken together, these findings validate Rab38 as a gene having a causal role in determining the phenotype of the FHH rat, which models hypertension-associated renal disease. Furthermore, our data suggest that Rab38 affects urinary protein excretion via effects in the proximal tubule.

Mapping of Three Genetic Determinants of Susceptibility to Estrogen-Induced Mammary Cancer within the Emca8 Locus on Rat Chromosome 5

The ACI rat model of 17β-estradiol (E2)-induced mammary cancer has gained wide use in the study of breast cancer etiology, prevention, and genetics. Emca8, a QTL that determines susceptibility to E2-induced mammary cancer, was previously mapped to rat chromosome 5 (RNO5) in an intercross between resistant Brown Norway (BN) and susceptible ACI rats. In this study, a panel of congenic rat strains, each of which carries BN alleles across a defined segment of RNO5 on the ACI genetic background, was generated and used to map more precisely the Emca8 determinants of mammary cancer susceptibility. Three distinct genetic determinants were localized within Emca8, and two of these were mapped to intervals of less than 15 megabases. Emca8.1 harbors Cdkn2a, Cdkn2b, and other genes and is orthologous to the 9p21 breast cancer locus identified in genome-wide and candidate gene association studies. Emca8.2 harbors Cdkn2c and other genes and is orthologous to the 1p32 locus in humans that is frequently deleted in breast cancers. Both Emca8.1 and Emca8.2 harbor copy number variants that are orthologous to copy number variant regions in humans. Gene expression profiles were defined for mammary tissues from E2-treated ACI and ACI.BN-Emca8 rats to define the impact of Emca8 on gene expression and identify differentially expressed genes residing within Emca8.1 and Emca8.2. This study further illustrates the relevance of the ACI rat model of E2-induced mammary cancer for identifying novel genetic determinants of breast cancer susceptibility and defining the mechanisms through which estrogens contribute to breast cancer development. Cancer Prev Res; 6(1); 59-69. ©2012 AACR.

Abstract 361: Characterization of a 1,45 Mbp Blood Pressure QTL in Rat Chromosome 13.

We previously showed that transfer of a 13.3 Mb segment of the Brown Norway (BN) rat chromosome 13 (from position 65.9 to 79.21 Mb) into the Dahl salt-sensitive (SS) background attenuates the development of hypertension. In order to narrow the region and identify the causative genes and sequence variants, we created and phenotyped 13 overlapping congenic strains spanning the candidate region. We performed sequence analysis and gene expression analysis by rtPCR and whole-genome RNA-seq on kidneys of the congenic carrying the 13.3 Mb BN segment and the SS rat. Seven strains carrying segments of BN chromosome 13 spanning the region from 73.59 to 75.04 Mb (1.45 Mbp region) were protected from the development of hypertension by at least 20 mmHg. Proteinuria was also markedly reduced in these strains. In contrast, blood pressure was high in the six strains carrying the SS allele in this 1.45 Mb segment. NextGen sequencing of this 1.45 Mbp candidate region, which contained five genes, revealed 2,198 sequence variants between SS and BN. Two of these were nonsynonymous SNPs in Astn1 and one variant was located on miR488 . Initial expression analysis of the genes within this region suggests that Astn1 and Rfwd2 are differentially expressed between SS and the protected congenic strain after high salt; Pappa2 was differentially expressed between SS and the congenic carrying the BN allele at both high and low-salt diet. In summary, we have narrowed a region for salt-sensitive hypertension to 1.55 Mbps, containing five candidate genes, each of which may contribute in some way to salt-sensitive hypertension.

Abstract 4378: Defining the role of mammary stem cells and progenitor cells in the development of estrogen-induced mammary cancer in the rat

Abstract Substantial data implicate both estrogens and progestins in the etiology of breast cancer. However, the mechanisms through which these steroid hormones elicit their effects on breast cancer risk have not been elucidated. The ACI rat serves as a unique model for investigating the mechanisms through which estrogens and progestins contribute to breast cancer development and for defining the genetic bases of inherited risk of breast cancer. When treated with physiologic levels of 17β-estradiol (E2), female ACI rats develop mammary cancer at an incidence that approaches 100%. Interestingly, rapid development of mammary cancer in E2 treated ACI rats is dependent upon progesterone. The mammary cancers that develop in E2 treated ACI rats are estrogen dependent, frequently exhibit non-random patterns of genome instability, and are invasive. Because each of these phenotypes is also commonly exhibited by breast cancers in humans, the ACI rat appears to be highly relevant to the study of breast cancer etiology. Whereas female ACI rats are highly susceptible to E2-induced mammary cancer, female Brown Norway (BN) rats are resistant. Genetic studies performed by our laboratory localized a total of six quantitative trait loci (QTL), designated Emca3 through Emca9, that account for a large fraction of inherited susceptibility in F2 progeny generated in intercrosses between ACI and BN rats. The purpose of this study is to test the hypothesis that ACI and BN rats exhibit differences in the responsiveness of their mammary stem cell (MaSCs) and/or progenitor cell (MaPCs) populations to E2 and/or progesterone. By using a combination of markers CD31, CD45, CD24, CD29 and fluorescence-activated cell sorting, we have identified four distinct populations of mammary cells. To quantify the numbers of MaSC and MaPC in these populations, we are evaluating the ability of the basal (Lin-CD24+CD29high) and luminal (Lin-CD24+CD29low) cell populations to form colonies when cultured in vitro and to generate mamamry gland outgrowths when transplanted into the interscapular fat pad of recipient syngenaic or (ACIxBN)F1 rats. Any observed differeces in MaSC and/or MaPC number or function will be evaluated further using a set of congenic rat strains that harbor BN alleles across an individual Emca locus on the ACI genetic background. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4378. doi:10.1158/1538-7445.AM2011-4378

Genomic Analysis of Left Ventricular Remodeling

The left ventricle (LV) of the heart remodels in response to hemodynamic load through the processes of physiological or pathophysiological hypertrophy.1–3 Despite extensive study of the molecular basis of cardiac hypertrophy and LV remodeling,1,4 the mainstay of medical treatment of maladaptive remodeling remains based on therapies initially devised for treating hypertension. The limited progress in the development of specific therapies for LV remodeling has led some to refer to “the impossible task of developing a new treatment for heart failure” and is the reason why, in part, device therapies have received such recent attention.5–8 To gain a better understanding of the biology of maladaptive LV remodeling, with a view to identifying therapeutic targets, new paradigms and experimental approaches are needed, and genomics provides one such methodology. Genomics is the study of genome-scale data sets, at the DNA or RNA level, and when combined with studies of physiological traits or disease phenotypes, genomics can be used to infer molecular insights. Unlike single-gene studies, which are the favored hypothesis-driven design,4 genomic approaches examine variation in up to gigabases of sequences to find statistical associations between transcripts and traits in a hypothesis-free system. These hypothesis-free studies have often been criticized as unfocused, but following the successes of genome-wide association studies (GWAS) and the recent achievements of integrated genetic and genomic analyses, a new era of genomic experimentation presents itself.9–14 It is notable that GWAS and integrated genomic studies in humans often identify regions associated with disease rather than specific genes and that effect sizes are small; nevertheless, these data remain highly informative.15–17 In this article, we review the genomic analysis of LV remodeling in the context of the recent past, the state of the art, and the imminent use of next-generation sequencing. A glossary of terms is …

Restoration of Vascular Relaxation in Cerebral Arteries of Congenic Dahl Rats Receiving the Brown Norway (BN) Renin Gene

The Dahl Salt Sensitive (SS) rat exhibits impaired regulation of the renin‐angiotensin system (RAS), chronically low levels of angiotensin II, and impaired vascular relaxation in response to vasodilator stimuli. This impaired vascular relaxation is likely due to chronic exposure of the animals to low levels of ANG II. The present study investigates responses to vasodilator stimuli in two different congenic strains in which a portion of the BN chromosome 13 was inserted onto the SS genetic background. Congenic strain SS.BN‐(D13rat88‐D13rat105) contains a 2.9 Mbp portion of chromosome 13 which excludes the BN renin gene, and would not be expected to restore normal regulation of the RAS. Congenic strain SS.BN‐(D13hmgc41‐D13hmgc23) contains a 2.0 Mbp adjacent region that includes the BN renin gene, which should restore normal ANG II levels in animals fed low salt (LS) diet. Males from each congenic strain were fed a LS diet (0.4% NaCl) after weaning. At the age of 10–14 weeks, middle cerebral arteries (MCA) were isolated and cannulated and dilator responses to acetylcholine (10−10M–10−5M) and reduced PO2 were assessed using television microscopy. MCA from the congenic strain containing the BN allele for renin dilated in response to acetylcholine and reduced PO2 while the MCA from the congenic strain containing the SS renin allele did not. These results suggest that the presence of a normally functioning renin gene from the BN rat in the SS genetic background restores normal vasodilator responses in the MCA. [NIH #HL‐29587; #HL‐65289, and #HL‐72920].

Identification of Mutated Srebf1 as a QTL Influencing Risk for Hepatic Steatosis in the Spontaneously Hypertensive Rat

Approximately 30% of patients with hypertension have hepatic steatosis, and it has recently been proposed that fatty liver be considered a feature of the metabolic syndrome. Obesity, diet, and level of physical activity are likely factors modulating risk for hepatic steatosis, however genetic factors could also influence susceptibility or resistance to fatty liver in hypertensive or normotensive subjects. In genetic studies in spontaneously hypertensive rats (SHRs) and Brown Norway (BN) rats, we discovered that a variant form of sterol regulatory element binding transcription factor 1 (Srebf1 gene, SREBP-1 protein) underlies a quantitative trait locus (QTL) influencing hepatic cholesterol levels in response to a high cholesterol diet. Compared with the BN allele of Srebf1, the SHR allele of Srebf1 includes variants in the promoter and coding regions that are linked to hepatic deficiency of SREBP-1 mRNA and protein, reduced expression of the SREBP-1 target gene stearoyl-CoA desaturase 1, reduced promoter activity for SREBP-1c, and relative protection from dietary induced accumulation of liver cholesterol. Genetic correction of reduced SREBP-1 activity by derivation of congenic and transgenic strains of SHR increased hepatic cholesterol levels, thereby confirming Srebf1 as a QTL influencing hepatic lipid metabolism in the rat. The Srebf1 variant regulating hepatic cholesterol did not appear to affect blood pressure. These findings (1) are consistent with the results of association studies indicating that common polymorphisms affecting SREBP-1 may influence cholesterol synthesis in humans and (2) indicate that variation in Srebf1 may influence risk for hepatic steatosis.

Nonrandom pattern of chromosome aberrations in 17β‐estradiol‐induced rat mammary tumors: Indications of distinct pathways for tumor development

Estrogens play an important role in breast cancer etiology and the ACI rat provides a novel animal model for defining the mechanisms through which estrogens contribute to mammary cancer development. In crossing experiments between the susceptible ACI strain and two resistant strains, COP (Copenhagen) and BN (Brown Norway), several quantitative trait loci (QTL) that affect development of 17beta-estradiol (E2)-induced mammary tumors have been defined. Using comparative genomic hybridization (CGH), we have analyzed cytogenetic aberrations in E2-induced mammary cancers and have found clear patterns of nonrandom chromosomal involvement. Approximately two thirds of the tumors exhibited copy number changes. Losses of rat chromosome 5 (RNO5) and RNO20 were particularly common, and it was found that these two aberrations often occurred together. A third recurrent aberration involving proximal gain and distal loss in RNO6 probably defined a distinct subgroup of tumors, since it never occurred in combination with RNO5 loss. Interestingly, QTL with powerful effects on mammary cancer development have been mapped to RNO5 and RNO6. These findings suggest that there were at least two genetic pathways to tumor formation in this rat model of E2-induced mammary cancer. By performing CGH on mammary tumors from ACI rats, F1 rats from crosses between the ACI and COP or BN strains and ACI.BN-Emca8 congenic rats, which carry the BN allele of the Emca8 QTL on RNO5 on the ACI genetic background, we were able to determine that the constitution of the germ line influences the pattern of chromosomal aberrations.

Genetic mapping of Eutr1, a locus controlling E2-induced pyometritis in the Brown Norway rat, to RNO5

In certain rat strains, chronic estrogen administration can lead to pyometritis, an inflammation of the uterus accompanied by infection and the accumulation of intraluminal pus. In this article, we report that the Brown Norway (BN) rat is highly susceptible to pyometritis induced by 17beta-estradiol (E2). The susceptibility of the BN rat to E2-induced pyometritis appears to segregate as a recessive trait in crosses to the resistant August x Copenhagen Irish (ACI) strain. In a (BN x ACI)F(2) population, we find strong evidence for a major genetic determinant of susceptibility to E2-induced pyometritis on rat chromosome 5 (RNO5). Our data are most consistent with a model in which the BN allele of this locus, designated Eutr1 (Estrogen-induced uterine response 1), acts in an incompletely dominant manner to control E2-induced pyometritis. Furthermore, we have confirmed the contribution of Eutr1 to E2-induced uterine pyometritis using an RNO5 congenic rat strain. In addition to Eutr1, we obtained evidence suggestive of linkage for five additional loci on RNO2, 4, 11, 17, and X that control susceptibility to E2-induced pyometritis in the (BN x ACI)F(2) population.

A congenic strain of rat for investigation of control of estrogen-induced growth.

A congenic strain of rat for investigation of control of estrogen-induced growth.

Identification of two susceptibility loci for vascular fragility in the Brown Norway rat.

A trait of vascular fragility, characterized by the formation of abrupt defects within the elastic laminae of the abdominal aorta, has been identified in Brown Norway (BN) rats. These lesions are greatly exacerbated in F(1) rats from a BN x New Zealand genetically hypertensive (GH) intercross, implying that the genetic background provided by the GH rat influences lesion severity. The F(2) progeny of the BN x GH intercross were used to identify susceptibility loci for the lesions as well as exacerbating loci. Two major quantitative trait loci (QTLs) for number of internal elastic lamina lesions were identified on rat chromosomes 5 and 10, with the maximum "log of the odds ratio" (LOD) scores at D5Rat119 (LOD 5.0) and at D10Mit2 (LOD 4.5), respectively, together contributing 33.5% to the genetic variance. Further analysis revealed that the chromosome 10 locus exhibits a dominant mode of inheritance, with BN alleles being associated with increased lesion number (P < 0.0002) compared with GH homozygotes. This locus was in epistasis to a modifier locus on rat chromosome 2 at D2Mit14 (LOD score 2.12). A second major locus was identified on chromosome 5, exhibiting a semidominant mode of inheritance, again with the BN allele being significantly associated with increased lesion number (P < 0.0001). Furthermore, a locus influencing lesion severity was identified on chromosome 3 wherein GH alleles associated with increased severity. This is the first study to identify susceptibility loci for vascular elastic tissue fragility.

Quantitative trait loci for estrogen-dependent pituitary tumor growth in the rat.

Growth control is of fundamental importance to biology in general and of critical importance to cancer research in particular. Tumors develop when control of the normal growth process is lost. The rat pituitary is a model system for control of estrogen-dependent growth. Chronic estrogen treatment induces uncontrolled growth in the pituitaries of Fischer 344 (F344) rats, but not of Brown Norway (BN) rats. We have identified five quantitative trait loci (QTL) for estrogen-dependent pituitary mass (Edpm) in an F2 intercross of F344 and BN. These QTL reside on rat Chromosomes (Chrs) 2, 3, 5, and 9 and explain a total of 55% of the genetic variance in the F2. We have also detected suggestive evidence for a QTL on rat Chr 14. For Edpm2-1, Edpm2-2, Edpm3, and Edpm5, the F344 allele corresponds with increased pituitary mass, as expected. Surprisingly, for Edpm9 and the suggested QTL on Chr 14, the BN allele corresponds with increased pituitary mass. We also find evidence for interaction (epistasis) between Edpm3 and Edpm9 and between Edpm5 and the suggested QTL on Chr 14.