Molecular anatomy of the cytoplasmic domain of bovine growth hormone receptor, a quantitative trait locus

Quantitative trait loci (QTL) studies have indicated growth hormone receptor (GHR) as a candidate gene affecting cattle milk yield and composition. In order to characterize genetic variation at GHR in cattle, we studied European and East African breeds with different histories of selection, and Bos grunniens, Ovis aries, Sus scrofa, Bison bison and Rangifer tarandus as references. We sequenced most of the cytoplasmic domain (900 bp of exon 10), 89 bp of exon 8, including the putative causative mutation for the QTL effect, and 390 bp of intron 8 for comparison. In the cytoplasmic domain, seven synonymous and seven non-synonymous single nucleotide polymorphisms (SNP) were identified in cattle. Three non-synonymous SNPs were found in sheep and one synonymous SNP in yak, while other studied species were monomorphic. Three major haplotypes were observed, one unique to African breeds, one unique to European breeds and one shared. Bison and yak haplotypes are derivatives of the European haplotype lineage. Most of the exon 10 non-synonymous cattle SNPs appear at phylogenetically highly conserved sites. The polymorphisms in exon 10 cluster around a ruminant-specific tyrosine residue, suggesting that this site may act as an additional signalling domain of GHR in ruminants. Alternative explanations for the persistent polymorphism include balancing selection, hitch-hiking, pleiotropic or sexually antagonistic fitness effects or relaxed functional constraints.     

A QTL with major effect on milk yield and composition maps to bovine Chromosome 14

A whole genome scan was undertaken in a granddaughter design comprising 1158 progeny-tested bulls in order to map QTL influencing milk yield and composition. In this paper we report the identification of a locus on the centromeric end of bovine Chromosome (Chr) 14, with major effect on fat and protein percentage as well as milk yield. The genuine nature of this QTL was verified using the grand²-daughter design, that is, by tracing the segregating QTL alleles from heterozygous grandsires to their maternal grandsons and confirming the predicted QTL allele substitution effect. Received: 30 December 1997 / Accepted: 21 February 1998     

Simultaneous mining of linkage and linkage disequilibrium to fine map quantitative trait loci in outbred half-sib pedigrees: revisiting the location of a quantitative trait locus with major effect on milk production on bovine chromosome 14

A maximum-likelihood QTL mapping method that simultaneously exploits linkage and linkage disequilibrium and that is applicable in outbred half-sib pedigrees is described. The method is applied to fine map a QTL with major effect on milk fat content in a 3-cM marker interval on proximal BTA14. This proximal location is confirmed by applying a haplotype-based association method referred to as recombinant ancestral haplotype analysis. The origin of the discrepancy between the QTL position derived in this work and that of a previous analysis is examined and shown to be due to the existence of distinct marker haplotypes associated with QTL alleles having large substitution effects.     

Mapping of a milk production quantitative trait locus to a 420-kb region on bovine chromosome 6

A QTL affecting milk production traits was previously mapped to an interval of 7.5 cM on chromosome 6 in Norwegian dairy cattle. This article aimed to refine this position by increasing the map density in the region by a set of single-nucleotide polymorphisms and analyzing the data with a combined linkage and linkage disequilibrium approach. Through a series of single- and multitrait and single- and multipoint analyses, the QTL was positioned to an interval surrounded by the genes ABCG2 and LAP3. As no recombinations were detected in this interval, physical mapping was required for further refining. By using radiation hybrid mapping as well as BAC clones, the bovine and human comparative maps in the region are resolved, and the QTL is mapped within a distance of 420 kb.     

In an elite cross of maize a major quantitative trait locus controls one-fourth of the genetic variation for grain yield

Quantitative trait loci (QTLs) for grain yield, dry matter content and test weight were identified in an F₂ segregating population derived from a single cross between two elite maize lines (B73 and A7) and testcrossed to two genetically divergent in breds. Most of the QTLs inferred were consistent across locations, indicating that the expression of the genes influencing the traits under investigation was largely independent of the environment. By using two different tester lines we found that QTLs exhibited by one tester may not necessarily be detected with the second one. Only loci with larger effects were consistent across testers, suggesting that interaction with tester alleles may contribute to the identification of QTLs in a specific fashion. Analysis across both testers revealed four significant QTLs for grain yield that explained more than 35% of the phenotypic variation and showed an overall phenotypic effect of more than 2t/ha. The major QTL for grain yield, located in the proximity of the Nucleolus Organiser Region, accounted for 24.5% of the phenotypic variation for grain yield and showed an average effect of allele substitution of approximately 1 t/ha. Marker-assisted introgression of the superior A7 allele at this locus in the B73 genetic background will not differ from qualitative trait introgression and will eventually lead to new lines having superior testcross performance.     

A quantitative trait locus involved in maize yield is tightly associated to the r1 gene on the long arm of chromosome 10

We produced and studied for 3?years two synthetic populations of maize differing in their constitution only for the selected alleles present at the red color 1 (r1) locus (R-sc vs. r?Cr). r1 is a regulatory gene conferring anthocyanin pigmentation in different tissues: the R-sc allele confers pigmentation only in the aleurone seed layer, while the r?Cr allele confers pigmentation in several tissues such as root, silk and anther but the seed is colourless. The colourless population (r?Cr/r?Cr) was characterized by improved agronomic features, such as ear weight and plant height, compared with the R-sc/R-sc coloured population. This finding was confirmed by studying single F4 R/r families where the presence of the r?Cr allele conferred positive features, acting as a dominant trait. Quantitative trait locus (QTL) analysis performed using molecular markers on the long arm of chromosome 10 (bin 10.06), where the r1 gene maps, identified a QTL map position for plant height tightly associated to the r1 gene. Thus the r1 gene may represent a major QTL or it could be closely linked to another gene involved in the agronomic performance of the two populations studied.     

An MspI polymorphism at the bovine growth hormone (bGH) gene is linked to a locus affecting milk protein percentage

SSCP analysis of the bovine growth hormone (bGH) gene in Israel Holstein dairy cattle uncovered five intragenic haplotypes, denoted A to E. Of these, Haplotype E differed from the others at six fragments; one of which corresponded to the polymorphic MspI site in intron III, at which haplotype E carried the disabled MspI (-) allele. Haplotype E was observed in a single sire only, carrying haplotype A as the second bGH allele. In 523 daughters of this sire genotyped for the MspI polymorphism, heterozygous (+/-) as compared to homozygous (+/+) daughters, showed a significant increasing effect on protein percentage and kg protein per year; and a decreasing effect (P < 0.10) on milk somatic cell counts (MSSC). None of the daughters were homozygous (-/-), indicating that the frequency of this allele in the general population was essentially zero. Calculated skewness (g1) values for the two daughter groups differed significantly with (+/-) daughters showing negative skewness (in the direction of lower protein percentage), and (+/+) daughters positive skewness (in the direction of higher protein percentage). The direction of skewness in each group is indicative of the presence of a QTL having an increasing effect on milk protein percentage in coupling linkage with the MspI (-) allele in this sire, but at some distance from it. Maximum likelihood estimates of the proportion of recombination (r) between the putative QTL and bGH, and the allele substitution effect at the QTL (d), were r = 0.33, a = 0.07% protein, with standard errors 0.058 and 0.009% protein, respectively.     

Role of the growth hormone (GH) receptor transmembrane domain in receptor predimerization and GH-induced activation

The GH receptor (GHR) mediates GH effects by activating the GHR-associated cytoplasmic tyrosine kinase, Janus kinase 2. Recent studies indicate that GHRs exist as dimers independently of GH binding. Some authors suggest that receptor predimerization is mediated by the transmembrane domain (TMD) and that GH binding initiates signaling by triggering changes in the orientation of the two GHRs within the dimer. In this study, we investigate the role of GHR TMD in GH-independent receptor dimerization and ligand-induced activation. We prepared a GHR mutant, GHR(LDLR), in which the TMD is replaced with the TMD of the human low-density lipoprotein receptor (LDLR). The resultant chimera has a TMD two residues shorter than the native GHR TMD; thus, in addition to possessing a different TMD, the altered GHR(LDLR) TMD helical register may change positions of the GHR extracellular domain (ECD) and intracellular domain relative to the TMD when compared with the wild-type (WT) receptor. When each was coexpressed with an intracellular domain-truncated GHR mutant, GHR(1-274-Myc), both WT GHR and GHR(LDLR) were specifically coprecipitated with GHR(1-274-Myc), indicating that the GHR TMD was not required for GHR heterodimerization with GHR(1-274-Myc). We further examined the contribution of the so-called "dimerization interface," a GHR ECD region that is critical for GH-induced signaling, to receptor predimerization. Coimmunoprecipitation experiments with either WT GHR, a dimerization interface mutant (GHR-H150D), or a control mutant (GHR-T147D) with GHR(1-274-Myc) showed dramatically reduced coprecipitation of GHR-H150D with GHR(1-274-Myc) when compared with WT GHR or GHR-T147K. This result suggests that, in contrast to some recent models, the dimerization interface contributes to GHR predimerization. We also compared WT GHR with GHR(LDLR) and GHR(LDLRDelta4) (a chimera in which the LDLR TMD has an internal deletion of four residues) with regard to response to GH stimulation. Although the chimeras had similar GH dose responses and time courses for signaling as WT GHR, they were markedly less sensitive to inhibition of signaling by a conformation-sensitive GHR ECD monoclonal antibody. Further, the chimeras were much less sensitive to inducible metalloprotease cleavage than was WT GHR, implying that the ECD conformations of the chimera receptors differ from WT GHR. Collectively, our data indicate that the composition and/or length of the TMD affect some aspects of GHR function, but do not affect receptor predimerization or GH-induced GHR activation. Further, they suggest that the GHR ECD-TMD is more flexible than previously thought in terms of the ability to achieve the active conformation in response to GH.     

Estimation of genetic variance contributed by a quantitative trait locus: correcting the bias associated with significance tests

The Beavis effect in quantitative trait locus (QTL) mapping describes a phenomenon that the estimated effect size of a statistically significant QTL (measured by the QTL variance) is greater than the true effect size of the QTL if the sample size is not sufficiently large. This is a typical example of the Winners’ curse applied to molecular quantitative genetics. Theoretical evaluation and correction for the Winners’ curse have been studied for interval mapping. However, similar technologies have not been available for current models of QTL mapping and genome-wide association studies where a polygene is often included in the linear mixed models to control the genetic background effect. In this study, we developed the theory of the Beavis effect in a linear mixed model using a truncated noncentral Chi-square distribution. We equated the observed Wald test statistic of a significant QTL to the expectation of a truncated noncentral Chi-square distribution to obtain a bias-corrected estimate of the QTL variance. The results are validated from replicated Monte Carlo simulation experiments. We applied the new method to the grain width (GW) trait of a rice population consisting of 524 homozygous varieties with over 300 k single nucleotide polymorphism markers. Two loci were identified and the estimated QTL heritability were corrected for the Beavis effect. Bias correction for the larger QTL on chromosome 5 (GW5) with an estimated heritability of 12% did not change the QTL heritability due to the extremely large test score and estimated QTL effect. The smaller QTL on chromosome 9 (GW9) had an estimated QTL heritability of 9% reduced to 6% after the bias-correction.     

The F7 gene and clotting factor VII levels: dissection of a human quantitative trait locus

Localization of human quantitative trait loci (QTLs) is now routine. However, identifying their functional DNA variants is still a formidable challenge. We present a complete dissection of a human QTL using novel statistical techniques to infer the most likely functional polymorphisms of a QTL that influence plasma levels of clotting factor VII (FVII), a risk factor for cardiovascular disease. Resequencing of 15 kb in and around the F7 gene identified 49 polymorphisms, which were then genotyped in 398 people. Using a Bayesian quantitative trait nucleotide (BQTN) method, we identified four to seven functional variants that completely account for this QTL. These variants include both rare coding variants and more common, potentially regulatory polymorphisms in intronic and promoter regions.     

A finite locus effect diffusion model for the evolution of a quantitative trait

A diffusion model is constructed for the joint distribution of absolute locus effect sizes and allele frequencies for loci contributing to an additive quantitative trait under selection in a haploid, panmictic population. The model is designed to approximate a discrete model exactly in the limit as both population size and the number of loci affecting the trait tend to infinity. For the case when all loci have the same absolute effect size, formal multiple-timescale asymptotics are used to predict the long-time response of the population trait mean to selection. For the case where loci can take on either of two distinct effect sizes, not necessarily with equal probability, numerical solutions of the system indicate that response to selection of a quantitative trait is insensitive to the variability of the distribution of effect sizes when mutation is negligible.     

A major quantitative trait locus co-localizing with cholecystokinin type A receptor gene influences poor pancreatic proliferation in a spontaneously diabetogenic rat

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type, non-insulin-dependent diabetes mellitus (NIDDM) in humans. The OLETF rat has poor capacity for pancreatic proliferation, which may be the critical pathogenetic event in NIDDM development. Our investigation was designed to identify quantitative trait loci (QTLs) responsible for poor pancreatic proliferation by examining compensatory proliferation of the pancreatic remnant after partial pancreatectomy and performing a genome-wide scan in an F₂ intercross obtained by mating the OLETF and the Fischer-344 (F344) rats. We identified a highly significant QTL on rat Chromosome 14 with a maximum lod score of 16.7, which accounts for 55% of the total variance. The QTL co-localizes with the gene encoding cholecystokinin type A receptor (CCKAR) which is likely to mediate the trophic effect of cholecystokinin on pancreas and is defective in the OLETF rat. Received: 3 March 1998 / Accepted: 18 June 1998     

Detection of a quantitative trait locus associated with resistance to Ascaris suum infection in pigs

Helminths almost invariably have an over-dispersed distribution in the host population. Human and animal studies have provided evidence suggesting that a large part of this variation is due to host genetic factors. Recently, the heritability for roundworm (Ascaris suum) infection levels in pigs was estimated to be 0.45. We used single nucleotide polymorphism markers to perform a whole-genome scan on 195 pigs experimentally infected with A. suum. A putative quantitative trait locus for worm burden on chromosome 4 covering 2.5 Mbp was identified by measured genotype analysis, although none of the SNPs reached genome-wide significance. To validate the putative quantitative trait locus, we genotyped two of the SNPs within the region in unrelated, informative animals exposed to experimental or natural infections and from which we had worm counts and/or faecal egg counts; the validation studies showed that one of the SNPs (TXNIP) was associated with total worm burden (P < 0.001) and adult worm burden(P < 0.0001), whereas the other SNP (ARNT) was associated with adult worm burden (P < 0.025) in these populations. We were thus able to confirm the existence of the quantitative trait locus on chromosome 4.This is to our knowledge the first report of a quantitative trait locus associated with helminth burden in pigs.     

Mapping of a milk production quantitative trait locus to a 1.056 Mb region on bovine chromosome 5 in the Fleckvieh dual purpose cattle breed

Background

In a previous study in the Fleckvieh dual purpose cattle breed, we mapped a quantitative trait locus (QTL) affecting milk yield (MY1), milk protein yield (PY1) and milk fat yield (FY1) during first lactation to the distal part of bovine chromosome 5 (BTA5), but the confidence interval was too large for positional cloning of the causal gene. Our objective here was to refine the position of this QTL and to define the candidate region for high-throughput sequencing.

Methods

In addition to those previously studied, new Fleckvieh families were genotyped, in order to increase the number of recombination events. Twelve new microsatellites and 240 SNP markers covering the most likely QTL region on BTA5 were analysed. Based on haplotype analysis performed in this complex pedigree, families segregating for the low frequency allele of this QTL (minor allele) were selected. Single- and multiple-QTL analyses using combined linkage and linkage disequilibrium methods were performed.

Results

Single nucleotide polymorphism haplotype analyses on representative family sires and their ancestors revealed that the haplotype carrying the minor QTL allele is rare and most probably originates from a unique ancestor in the mapping population. Analyses of different subsets of families, created according to the results of haplotype analysis and availability of SNP and microsatellite data, refined the previously detected QTL affecting MY1 and PY1 to a region ranging from 117.962 Mb to 119.018 Mb (1.056 Mb) on BTA5. However, the possibility of a second QTL affecting only PY1 at 122.115 Mb was not ruled out.

Conclusion

This study demonstrates that targeting families segregating for a less frequent QTL allele is a useful method. It improves the mapping resolution of the QTL, which is due to the division of the mapping population based on the results of the haplotype analysis and to the increased frequency of the minor allele in the families. Consequently, we succeeded in refining the region containing the previously detected QTL to 1 Mb on BTA5. This candidate region contains 27 genes with unknown or partially known function(s) and is small enough for high-throughput sequencing, which will allow future detailed analyses of candidate genes.     

Structural basis of the signal transduction via transmembrane domain of the human growth hormone receptor

Background

Prior studies of the human growth hormone receptor (GHR) revealed a distinct role of spatial rearrangements of its dimeric transmembrane domain in signal transduction across membrane. Detailed structural information obtained in the present study allowed elucidating the bases of such rearrangement and provided novel insights into receptor functioning.