Interval mapping methods for detecting QTL affecting survival and time-to-event phenotypes

Quantitative trait loci (QTL) are usually searched for using classical interval mapping methods which assume that the trait of interest follows a normal distribution. However, these methods cannot take into account features of most survival data such as a non-normal distribution and the presence of censored data. We propose two new QTL detection approaches which allow the consideration of censored data. One interval mapping method uses a Weibull model (W), which is popular in parametrical modelling of survival traits, and the other uses a Cox model (C), which avoids making any assumption on the trait distribution. Data were simulated following the structure of a published experiment. Using simulated data, we compare W, C and a classical interval mapping method using a Gaussian model on uncensored data (G) or on all data (G'=censored data analysed as though records were uncensored). An adequate mathematical transformation was used for all parametric methods (G, G' and W). When data were not censored, the four methods gave similar results. However, when some data were censored, the power of QTL detection and accuracy of QTL location and of estimation of QTL effects for G decreased considerably with censoring, particularly when censoring was at a fixed date. This decrease with censoring was observed also with G', but it was less severe. Censoring had a negligible effect on results obtained with the W and C methods.     

Alterations in mitochondrial function in a mouse model of hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by varying degrees of ventricular hypertrophy and myofibrillar disarray. Mutations in cardiac contractile proteins cause HCM. However, there is an unexplained wide variability in the clinical phenotype, and it is likely that there are multiple contributing factors. Because mitochondrial dysfunction has been described in heart disease, we tested the hypothesis that mitochondrial dysfunction contributes to the varying HCM phenotypes. Mitochondrial function was assessed in two transgenic models of HCM: mice with a mutant myosin heavy chain gene (MyHC) or with a mutant cardiac troponin T (R92Q) gene. Despite mitochondrial ultrastructural abnormalities in both models, the rate of state 3 respiration was significantly decreased only in the mutant MyHC mice by approximately 23%. Notably, this decrease in state 3 respiration preceded hemodynamic dysfunction. The maximum activity of alpha-ketogutarate dehydrogenase as assayed in isolated disrupted mitochondria was decreased by 28% compared with isolated control mitochondria. In addition, complexes I and IV were decreased in mutant MyHC transgenic mice. Inhibition of beta-adrenergic receptor kinase, which is elevated in mutant MyHC mouse hearts, can prevent mitochondrial respiratory impairment in mutant MyHC mice. Thus our results suggest that mitochondria may contribute to the hemodynamic dysfunction seen in some forms of HCM and offer a plausible mechanism responsible for some of the heterogeneity of the disease phenotypes.     

QTL mapping reveals the genetic architecture of loci affecting pre- and post-zygotic isolating barriers in Louisiana Iris

ABSTRACT: BACKGROUND: Hybridization among Louisiana Irises has been well established and the genetic architecture of reproductive isolation is known to affect the potential for and the directionality of introgression between taxa. Here we use co-dominant markers to identify regions where QTL are located both within and between backcross maps to compare the genetic architecture of reproductive isolation and fitness traits across treatments and years. RESULTS: QTL mapping was used to elucidate the genetic architecture of reproductive isolation between Iris fulva and Iris brevicaulis. Homologous co-dominant EST-SSR markers scored in two backcross populations between I. fulva and I. brevicaulis were used to generate genetic linkage maps. These were used as the framework for mapping QTL associated with variation in 11 phenotypic traits likely responsible for reproductive isolation and fitness. QTL were dispersed throughout the genome, with the exception of one region of a single linkage group (LG) where QTL for flowering time, sterility, and fruit production clustered. In most cases, homologous QTL were not identified in both backcross populations, however, homologous QTL for flowering time, number of growth points per rhizome, number of nodes per inflorescence, and number of flowers per node were identified on several linkage groups. CONCLUSION: Two different traits affecting reproductive isolation, flowering time and sterility, exhibit different genetic architectures, with numerous QTL across the Iris genome controlling flowering time and fewer, less distributed QTL affecting sterility. QTL for traits affecting fitness are largely distributed across the genome with occasional overlap, especially on LG 4, where several QTL increasing fitness and decreasing sterility cluster. Given the distribution and effect direction of QTL affecting reproductive isolation and fitness, we have predicted genomic regions where introgression may be more likely to occur (those regions associated with an increase in fitness and unlinked to loci controlling reproductive isolation) and those that are less likely to exhibit introgression (those regions linked to traits decreasing fitness and reproductive isolation).     

Fine mapping of an epilepsy modifier gene on mouse Chromosome 19

Mutations in voltage-gated sodium channels are associated with several types of human epilepsy. Variable expressivity and penetrance are common features of inherited epilepsy caused by sodium channel mutations, suggesting that genetic modifiers may influence clinical severity. The mouse model Scn2a ^Q54 has an epilepsy phenotype due to a mutation in Scn2a that results in elevated persistent sodium current. Phenotype severity in Scn2a ^Q54 mice is dependent on the genetic background. Congenic C57BL/6J.Q54 mice have delayed onset and low seizure frequency compared to (C57BL/6J × SJL/J)F1.Q54 mice. Previously, we identified two modifier loci that influence the Scn2a ^Q54 epilepsy phenotype: Moe1 (modifier of epilepsy 1) on Chromosome 11 and Moe2 on Chromosome 19. We have constructed interval-specific congenic strains to further refine the position of Moe2 on Chromosome 19 to a 5-Mb region. Sequencing and expression analyses of genes in the critical interval suggested two potential modifier candidates: (1) voltage-gated potassium channel subunit subfamily V, member 2 (Kcnv2), and (2) SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 2 (Smarca2). Based on its biological role in regulating membrane excitability and the association between ion channel variants and seizures, Kcnv2 is a strong functional candidate for Moe2. Modifier genes affecting the epilepsy phenotype of Scn2a ^Q54 mice may contribute to variable expressivity and penetrance in human epilepsy patients with sodium channel mutations.     

Admixture mapping of an allele affecting interleukin 6 soluble receptor and interleukin 6 levels

Circulating levels of inflammatory markers can predict cardiovascular disease risk. To identify genes influencing the levels of these markers, we genotyped 1,343 single-nucleotide polymorphisms (SNPs) in 1,184 African Americans from the Health, Aging and Body Composition (Health ABC) Study. Using admixture mapping, we found a significant association of interleukin 6 soluble receptor (IL-6 SR) with European ancestry on chromosome 1 (LOD 4.59), in a region that includes the gene for this receptor (IL-6R). Genotyping 19 SNPs showed that the effect is largely explained by an allele at 4% frequency in West Africans and at 35% frequency in European Americans, first described as associated with IL-6 SR in a Japanese cohort. We replicate this association (P<1.0x10-12) and also demonstrate a new association with circulating levels of a different molecule, IL-6 (P<3.4x10-5). After replication in 1,674 European Americans from Health ABC, the combined result is even more significant: P<1.0x10-12 for IL-6 SR, and P<2.0x10-9 for IL-6. These results also serve as an important proof of principle, showing that admixture mapping can not only coarsely localize but can also fine map a phenotypically important variant.     

Identification of Sox8 as a modifier gene in a mouse model of Hirschsprung disease reveals underlying molecular defect

Mice carrying heterozygous mutations in the Sox10 gene display aganglionosis of the colon and represent a model for human Hirschsprung disease. Here, we show that the closely related Sox8 functions as a modifier gene for Sox10-dependent enteric nervous system defects as it increases both penetrance and severity of the defect in Sox10 heterozygous mice despite having no detectable influence on enteric nervous system development on its own. Sox8 exhibits an expression pattern very similar to Sox10 with occurrence in vagal and enteric neural crest cells and later confinement to enteric glia. Loss of Sox8 alleles in Sox10 heterozygous mice impaired colonization of the gut by enteric neural crest cells already at early times. Whereas proliferation, apoptosis, and neuronal differentiation were normal for enteric neural crest cells in the gut of mutant mice, apoptosis was dramatically increased in vagal neural crest cells outside the gut. The defects in enteric nervous system development of mice with Sox10 and Sox8 mutations are therefore likely caused by a reduction of the pool of undifferentiated vagal neural crest cells. Our study suggests that Sox8 and Sox10 are jointly required for the maintenance of these vagal neural crest stem cells.     

Genetic mapping of sulfur assimilation genes reveals a QTL for onion bulb pungency

Onion exhibits wide genetic and environmental variation in bioactive organosulfur compounds that impart pungency and health benefits. A PCR-based molecular marker map that included candidate genes for sulfur assimilation was used to identify genomic regions affecting pungency in the cross 'W202A' × 'Texas Grano 438'. Linkage mapping revealed that genes encoding plastidic ferredoxin-sulfite reductase (SiR) and plastidic ATP sulfurylase (ATPS) are closely linked (1–2 cM) on chromosome 3. Inbred F₃ families derived from the F₂ population used to construct the genetic map were grown in replicated trials in two environments and bulb pungency was evaluated as pyruvic acid or lachrymatory factor. Broad-sense heritability of pungency was estimated to be 0.78–0.80. QTL analysis revealed significant associations of both pungency and bulb soluble solids content with marker intervals on chromosomes 3 and 5, which have previously been reported to condition pleiotropic effects on bulb carbohydrate composition. Highly significant associations (LOD 3.7–8.7) were observed between ATPS and SiR Loci and bulb pungency but not with bulb solids content. This association was confirmed in two larger, independently derived F₂ families from the same cross. Single-locus models suggested that the partially dominant locus associated with these candidate genes controls 30–50% of genetic variation in pungency in these pedigrees. These markers may provide a practical means to select for lower pungency without correlated selection for lowered solids.     

Genetic mapping of a mouse modifier gene that can prevent ALS onset

Mutations in the cytoplasmic Cu/Zn superoxide dismutase (SOD1) gene on human chromosome 21q22.1 cause 10-20% of familial amyotrophic lateral sclerosis (ALS) cases. The expression of the ALS phenotype in mice carrying the murine G86R SOD1 mutation is highly dependent upon the mouse genetic background. This is similar to the phenotypic variation observed in ALS patients containing identical SOD1 mutations. In the FVB/N background, mice expressing mG86R SOD1 develop an ALS phenotype at approximately 100 days. However, when these mice were bred into a mixed background of C57Bl6/129Sv, the onset of the ALS phenotype was delayed (143 days to >2 years). Using 129 polymorphic autosomal markers in a whole genome scan, we have identified a major genetic modifier locus with a maximum lod score of 5.07 on mouse chromosome 13 between D13mit36 and D13mit76. This 5- to 8-cM interval contains the spinal muscular atrophy (SMA)-associated gene Smn (survival motor neuron) and seven copies of Naip (neuronal apoptosis inhibitory protein), suggesting a potential link between SMA and ALS.     

SNP mapping of QTL affecting growth and fatness on chicken GGA1

An F2 chicken population was established from a crossbreeding between a Xinghua line and a White Recessive Rock line. A total of 502 F2 chickens in 17 full-sib families from six hatches was obtained, and phenotypic data of 488 individuals were available for analysis. A total of 46 SNP on GGA1 was initially selected based on the average physical distance using the dbSNP database of NCBI. After the polymorphism levels in all F0 individuals (26 individuals) and part of the F1 individuals (22 individuals) were verified, 30 informative SNP were potentially available to genotype all F2 individuals. The linkage map was constructed using Cri-Map. Interval mapping QTL analyses were carried out. QTL for body weight (BW) of 35 d and 42 d, 49 d and 70 d were identified on GGA1 at 351–353 cM and 360 cM, respectively. QTL for abdominal fat weight was on GGA1 at 205 cM, and for abdominal fat rate at 221 cM. Two novel QTL for fat thickness under skin and fat width were detected at 265 cM and 72 cM, respectively.     

Quantitative trait loci (QTL) analysis for rice grain width and fine mapping of an identified QTL allele gw-5 in a recombination hotspot region on chromosome 5

Rice grain width and shape play a crucial role in determining grain quality and yield. The genetic basis of rice grain width was dissected into six additive quantitative trait loci (QTL) and 11 pairs of epistatic QTL using an F(7) recombinant inbred line (RIL) population derived from a single cross between Asominori (japonica) and IR24 (indica). QTL by environment interactions were evaluated in four environments. Chromosome segment substitution lines (CSSLs) harboring the six additive effect QTL were used to evaluate gene action across eight environments. A major, stable QTL, qGW-5, consistently decreased rice grain width in both the Asominori/IR24 RIL and CSSL populations with the genetic background Asominori. By investigating the distorted segregation of phenotypic values of rice grain width and genotypes of molecular markers in BC(4)F(2) and BC(4)F(3) populations, qGW-5 was dissected into a single recessive gene, gw-5, which controlled both grain width and length-width ratio. gw-5 was narrowed down to a 49.7-kb genomic region with high recombination frequencies on chromosome 5 using 6781 BC(4)F(2) individuals and 10 newly developed simple sequence repeat markers. Our results provide a basis for map-based cloning of the gw-5 gene and for marker-aided gene/QTL pyramiding in rice quality breeding.     

Comparative proteomics profiling of a phospholamban mutant mouse model of dilated cardiomyopathy reveals progressive intracellular stress responses

Defective mobilization of Ca2+ by cardiomyocytes can lead to cardiac insufficiency, but the causative mechanisms leading to congestive heart failure (HF) remain unclear. In the present study we performed exhaustive global proteomics surveys of cardiac ventricle isolated from a mouse model of cardiomyopathy overexpressing a phospholamban mutant, R9C (PLN-R9C), and exhibiting impaired Ca2+ handling and death at 24 weeks and compared them with normal control littermates. The relative expression patterns of 6190 high confidence proteins were monitored by shotgun tandem mass spectrometry at 8, 16, and 24 weeks of disease progression. Significant differential abundance of 593 proteins was detected. These proteins mapped to select biological pathways such as endoplasmic reticulum stress response, cytoskeletal remodeling, and apoptosis and included known biomarkers of HF (e.g. brain natriuretic peptide/atrial natriuretic factor and angiotensin-converting enzyme) and other indicators of presymptomatic functional impairment. These altered proteomic profiles were concordant with cognate mRNA patterns recorded in parallel using high density mRNA microarrays, and top candidates were validated by RT-PCR and Western blotting. Mapping of our highest ranked proteins against a human diseased explant and to available data sets indicated that many of these proteins could serve as markers of disease. Indeed we showed that several of these proteins are detectable in mouse and human plasma and display differential abundance in the plasma of diseased mice and affected patients. These results offer a systems-wide perspective of the dynamic maladaptions associated with impaired Ca2+ homeostasis that perturb myocyte function and ultimately converge to cause HF.     

Colivelin prolongs survival of an ALS model mouse

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease for which there is no sufficiently effective therapy. We have reported in our earlier study that intracerebroventricular (i.c.v.) injection of activity-dependent neurotrophic factor (ADNF) improves motor performance of G93A-SOD1 transgenic mice without significant prolongation in survival. Here, we found that i.c.v. injection of a synthetic hybrid peptide named Colivelin composed of ADNF and AGA-(C8R)HNG17, a potent derivative of Humanin that is a bioactive peptide with anti-Alzheimer's disease activity, dose-dependently improved motor performance and prolonged survival of ALS mice. Histological analysis, performed at the age of 120 days, demonstrated increased motoneuronal survival in spinal cords of Colivelin-treated mice as compared with saline- or ADNF-treated mice, indicating that Colivelin is a promising neurotrophic peptide for treatment of ALS.     

Simultaneous mapping of epistatic QTL in chickens reveals clusters of QTL pairs with similar genetic effects on growth

We used simultaneous mapping of interacting quantitative trait locus (QTL) pairs to study various growth traits in a chicken F2 intercross. The method was shown to increase the number of detected QTLs by 30 % compared with a traditional method detecting QTLs by their marginal genetic effects. Epistasis was shown to be an important contributor to the genetic variance of growth, with the largest impact on early growth (before 6 weeks of age). There is also evidence for a discrete set of interacting loci involved in early growth, supporting the previous findings of different genetic regulation of early and late growth in chicken. The genotype-phenotype relationship was evaluated for all interacting QTL pairs and 17 of the 21 evaluated QTL pairs could be assigned to one of four clusters in which the pairs in a cluster have very similar genetic effects on growth. The genetic effects of the pairs indicate commonly occurring dominance-by-dominance, heterosis and multiplicative interactions. The results from this study clearly illustrate the increase in power obtained by using this novel method for simultaneous detection of epistatic QTL, and also how visualization of genotype-phenotype relationships for epistatic QTL pairs provides new insights to biological mechanisms underlying complex traits.     

Multistep ion channel remodeling and lethal arrhythmia precede heart failure in a mouse model of inherited dilated cardiomyopathy

Background

Patients with inherited dilated cardiomyopathy (DCM) frequently die with severe heart failure (HF) or die suddenly with arrhythmias, although these symptoms are not always observed at birth. It remains unclear how and when HF and arrhythmogenic changes develop in these DCM mutation carriers. In order to address this issue, properties of the myocardium and underlying gene expressions were studied using a knock-in mouse model of human inherited DCM caused by a deletion mutation ΔK210 in cardiac troponinT.

Methodology/Principal Findings

By 1 month, DCM mice had already enlarged hearts, but showed no symptoms of HF and a much lower mortality than at 2 months or later. At around 2 months, some would die suddenly with no clear symptoms of HF, whereas at 3 months, many of the survivors showed evident symptoms of HF. In isolated left ventricular myocardium (LV) from 2 month-mice, spontaneous activity frequently occurred and action potential duration (APD) was prolonged. Transient outward (I_to) and ultrarapid delayed rectifier K⁺ (I_Kur) currents were significantly reduced in DCM myocytes. Correspondingly, down-regulation of Kv4.2, Kv1.5 and KChIP2 was evident in mRNA and protein levels. In LVs at 3-months, more frequent spontaneous activity, greater prolongation of APD and further down-regulation in above K⁺ channels were observed. At 1 month, in contrast, infrequent spontaneous activity and down-regulation of Kv4.2, but not Kv1.5 or KChIP2, were observed.

Conclusions/Significance

Our results suggest that at least three steps of electrical remodeling occur in the hearts of DCM model mice, and that the combined down-regulation of Kv4.2, Kv1.5 and KChIP2 prior to the onset of HF may play an important role in the premature sudden death in this DCM model. DCM mice at 1 month or before, on the contrary, are associated with low risk of death in spite of inborn disorder and enlarged heart.     

Gender and aging in a transgenic mouse model of hypertrophic cardiomyopathy

Mutations in the cardiac myosin heavy chain (MHC) can cause familial hypertrophic cardiomyopathy (FHC). A transgenic mouse model has been developed in which a missense (R403Q) allele and an actin-binding deletion in the alpha-MHC are expressed in the heart. We used an isovolumic left heart preparation to study the contractile characteristics of hearts from transgenic (TG) mice and their wild-type (WT) littermates. Both male and female TG mice developed left ventricular (LV) hypertrophy at 4 mo of age. LV hypertrophy was accompanied by LV diastolic dysfunction, but LV systolic function was normal and supranormal in the young TG females and males, respectively. At 10 mo of age, the females continued to present with LV concentric hypertrophy, whereas the males began to display LV dilation. In female TG mice at 10 mo of age, impaired LV diastolic function persisted without evidence of systolic dysfunction. In contrast, in 10-mo-old male TG mice, LV diastolic function worsened and systolic performance was impaired. Diminished coronary flow was observed in both 10-mo-old TG groups. These types of changes may contribute to the functional decompensation typically seen in hypertrophic cardiomyopathy. Collectively, these results further underscore the potential utility of this transgenic mouse model in elucidating pathogenesis of FHC.     

Fine mapping of a distal chromosome 4 QTL affecting growth and muscle mass in a chicken advanced intercross line

In our previous research, QTL analysis in an F₂ cross between the inbred New Hampshire (NHI) and White Leghorn (WL77) lines revealed a growth QTL in the distal part of chromosome 4. To physically reduce the chromosomal interval and the number of potential candidate genes, we performed fine mapping using individuals of generations F₁₀, F₁₁ and F₁₂ in an advanced intercross line that had been established from the initial F₂ mapping population. Using nine single nucleotide polymorphism (SNP) markers within the QTL region for an association analysis with several growth traits from hatch to 20 weeks and body composition traits at 20 weeks, we could reduce the confidence interval from 26.9 to 3.4 Mb. Within the fine mapped region, markers rs14490774, rs314961352 and rs318175270 were in full linkage disequilibrium (D′ = 1.0) and showed the strongest effect on growth and muscle mass (LOD ≥ 4.00). This reduced region contains 30 genes, compared to 292 genes in the original region. Chicken 60 K and 600 K SNP chips combined with DNA sequencing of the parental lines were used to call mutations in the reduced region. In the narrowed‐down region, 489 sequence variants were detected between NHI and WL77. The most deleterious variants are a missense variant in ADGRA3 (SIFT = 0.02) and a frameshift deletion in the functional unknown gene ENSGALG00000014401 in NHI chicken. In addition, five synonymous variants were discovered in genes PPARGC1A, ADGRA3, PACRGL, SLIT2 and FAM184B. In our study, the confidence interval and the number of potential genes could be reduced 8‐ and 10‐ fold respectively. Further research will focus on functional effects of mutant genes.     

Quantitative trait locus (QTL) mapping reveals a role for unstudied genes in Aspergillus virulence

Infections caused by the fungus Aspergillus are a major cause of morbidity and mortality in immunocompromised populations. To identify genes required for virulence that could be used as targets for novel treatments, we mapped quantitative trait loci (QTL) affecting virulence in the progeny of a cross between two strains of A. nidulans (FGSC strains A4 and A91). We genotyped 61 progeny at 739 single nucleotide polymorphisms (SNP) spread throughout the genome, and constructed a linkage map that was largely consistent with the genomic sequence, with the exception of one potential inversion of ～527 kb on Chromosome V. The estimated genome size was 3705 cM and the average intermarker spacing was 5.0 cM. The average ratio of physical distance to genetic distance was 8.1 kb/cM, which is similar to previous estimates, and variation in recombination rate was significantly positively correlated with GC content, a pattern seen in other taxa. To map QTL affecting virulence, we measured the ability of each progeny strain to kill model hosts, larvae of the wax moth Galleria mellonella. We detected three QTL affecting in vivo virulence that were distinct from QTL affecting in vitro growth, and mapped the virulence QTL to regions containing 7-24 genes, excluding genes with no sequence variation between the parental strains and genes with only synonymous SNPs. None of the genes in our QTL target regions have been previously associated with virulence in Aspergillus, and almost half of these genes are currently annotated as "hypothetical". This study is the first to map QTL affecting the virulence of a fungal pathogen in an animal host, and our results illustrate the power of this approach to identify a short list of unknown genes for further investigation.     

Fine mapping and candidate gene analysis of a dravet syndrome modifier locus on mouse chromosome 11

Mammalian Genome - Pathogenic variants in SCN1A result in a spectrum of phenotypes ranging from mild febrile seizures to Dravet syndrome, a severe infant-onset epileptic encephalopathy. Individuals...