Integration of Murine and Human Studies for Mapping Periodontitis Susceptibility.

Periodontitis is one of the most common inflammatory human diseases with a strong genetic component. Due to the limited sample size of available periodontitis cohorts and the underlying trait heterogeneity, genome-wide association studies (GWASs) of chronic periodontitis (CP) have largely been unsuccessful in identifying common susceptibility factors. A combination of quantitative trait loci (QTL) mapping in mice with association studies in humans has the potential to discover novel risk loci. To this end, we assessed alveolar bone loss in response to experimental periodontal infection in 25 lines (286 mice) from the Collaborative Cross (CC) mouse population using micro-computed tomography (µCT) analysis. The orthologous human chromosomal regions of the significant QTL were analyzed for association using imputed genotype data (OmniExpress BeadChip arrays) derived from case-control samples of aggressive periodontitis (AgP; 896 cases, 7,104 controls) and chronic periodontitis (CP; 2,746 cases, 1,864 controls) of northwest European and European American descent, respectively. In the mouse genome, QTL mapping revealed 2 significant loci (-log P = 5.3; false discovery rate = 0.06) on chromosomes 1 ( Perio3) and 14 ( Perio4). The mapping resolution ranged from ~1.5 to 3 Mb. Perio3 overlaps with a previously reported QTL associated with residual bone volume in F2 cross and includes the murine gene Ccdc121. Its human orthologue showed previously a nominal significant association with CP in humans. Use of variation data from the genomes of the CC founder strains further refined the QTL and suggested 7 candidate genes ( CAPN8, DUSP23, PCDH17, SNORA17, PCDH9, LECT1, and LECT2). We found no evidence of association of these candidates with the human orthologues. In conclusion, the CC populations enabled mapping of confined QTL that confer susceptibility to alveolar bone loss in mice and larger human phenotype-genotype samples and additional expression data from gingival tissues are likely required to identify true positive signals.

The PF4/PPBP/CXCL5 Gene Cluster Is Associated with Periodontitis.

Periodontitis is a common dysbiotic inflammatory disease with an estimated heritability of 50%. Due to the limited sample size of available periodontitis cohorts and the underlying trait heterogeneity, genome-wide association studies (GWAS) of chronic periodontitis (CP) have been unsuccessful in discovering susceptibility factors. A strategy that combines agnostic GWAS with a well-powered candidate-gene approach has the potential to discover novel loci. We combined RNA-seq data from gingival tissues with quantitative trait loci (QTLs) that were identified in a F2-cross of mice resistant and susceptible to infection with oral bacterial pathogens. Four genes, which were located within the mapped QTLs, showed differential expression. The chromosomal regions across the human orthologous were interrogated for putative periodontitis-associated variants using existing GWAS data from a German case-control sample of aggressive periodontitis (AgP; 651 cases, 4,001 controls), the most severe and early onset form of periodontitis. Two haplotype blocks, one upstream to the coding region of UGT2A1 (rs146712414, P = 9.1 × 10-5; odds ratio [OR], 1.34; 95% confidence interval [CI], 1.16-1.56) and one downstream of the genes PF4/PPBP/CXCL5 (rs1595009, P = 1.3 × 10-4; OR, 1.32; 95% CI, 1.15-1.52), were associated with AgP. The association of rs1595009 was validated in an independent cohort of CP of European Americans (1,961 cases and 1,864 controls; P = 0.03; OR, 1.45; 95% CI, 1.01-1.29). This association was further replicated in another sample of 399 German CP cases (disease onset <60 y of age) and 1,633 controls ( P = 0.03; OR, 1.75; 95% CI, 1.06-2.90). The combined estimates of association from all samples were P = 2.9 × 10-5 (OR, 1.2; 95% CI, 1.1-1.3). This study shows the strength of combining QTL mapping and RNA-Seq data from a mouse model with association studies in human case-control samples to identify genetic risk variants of periodontitis.

Host susceptibility to periodontitis: mapping murine genomic regions.

Host susceptibility to periodontal infection is controlled by genetic factors. As a step toward identifying and cloning these factors, we generated an A/J x BALB/cJ F2 mouse resource population. A genome-wide search for Quantitative Trait Loci (QTL) associated with periodontitis was performed. We aimed to quantify the phenotypic response of the progenies to periodontitis by microCT analysis, to perform a genome-wide search for QTL associated with periodontitis, and, finally, to suggest candidate genes for periodontitis. We were able to produce 408 F2 mice. All mice were co-infected with Porphyromonas gingivalis and Fusobacterium nucleatum bacteria. Six weeks following infection, alveolar bone loss was quantified by computerized tomography (microCT) technology. We found normal distribution of the phenotype, with 2 highly significant QTL on chromosomes 5 and 3. A third significant QTL was found on chromosome 1. Candidate genes were suggested, such as Toll-like receptors (TLR) 1 and 6, chemokines, and bone-remodeling genes (enamelin, ameloblastin, and amelotin). This report shows that periodontitis in mice is a polygenic trait with highly significant mapped QTL.

Identification of quantitative trait loci affecting resistance to gastrointestinal parasites in a double backcross population of Red Maasai and Dorper sheep.

A genome-wide scan for quantitative trait loci (QTL) affecting gastrointestinal nematode resistance in sheep was completed using a double backcross population derived from Red Maasai and Dorper ewes bred to F(1) rams. This design provided an opportunity to map potentially unique genetic variation associated with a parasite-tolerant breed like Red Maasai, a breed developed to survive East African grazing conditions. Parasite indicator phenotypes (blood packed cell volume - PCV and faecal egg count - FEC) were collected on a weekly basis from 1064 lambs during a single 3-month post-weaning grazing challenge on infected pastures. The averages of last measurements for FEC (AVFEC) and PCV (AVPCV), along with decline in PCV from challenge start to end (PCVD), were used to select lambs (N = 371) for genotyping that represented the tails (10% threshold) of the phenotypic distributions. Marker genotypes for 172 microsatellite loci covering 25 of 26 autosomes (1560.7 cm) were scored and corrected by Genoprob prior to qxpak analysis that included Box-Cox transformed AVFEC and arcsine transformed PCV statistics. Significant QTL for AVFEC and AVPCV were detected on four chromosomes, and this included a novel AVFEC QTL on chromosome 6 that would have remained undetected without Box-Cox transformation methods. The most significant P-values for AVFEC, AVPCV and PCVD overlapped the same marker interval on chromosome 22, suggesting the potential for a single causative mutation, which remains unknown. In all cases, the favourable QTL allele was always contributed from Red Maasai, providing support for the idea that future marker-assisted selection for genetic improvement of production in East Africa will rely on markers in linkage disequilibrium with these QTL.

Quantitative trait loci for resistance to Heligmosomoides bakeri and associated immunological and pathological traits in mice: comparison of loci on chromosomes 5, 8 and 11 in F2 and F6/7 inter-cross lines of mice.

A comparison of F2 and F6/7 inter-cross lines of mice, derived from CBA and SWR parental strains, has provided strong evidence for several previously undetected quantitative trait loci (QTL) for resistance to Heligmosomoides bakeri. Five QTL affecting average faecal egg counts and/or worm burdens in week 6 were detected on mouse chromosomes 5 (Hbnr9 and Hbnr10), 8 (Hbnr11) and 11 (Hbnr13 and Hbnr14). Three QTL for faecal egg counts in weeks 4 and 6 were found on both chromosomes 5 (Hbnr9) and 11 (Hbnr13 and Hbnr14). Two QTL for the mucosal mast cell protease 1 (MCPT1) response were located on chromosomes 8 (Hbnr11) and 11 (Hbnr13), two for the IgG1 antibody response to adult worms on chromosomes 5 (Hbnr10) and 8 (Hbnr11), two for PCV in week 6 on chromosomes 5 (Hbnr9) and 11 (Hbnr13), and two for the granulomatous response on chromosome 8 (Hbnr12) and 11 (Hbnr15). Our data emphasize that the control of resistance to H. bakeri is multigenic, and regulated by genes within QTL regions that have a complex range of hierarchical relationships.

Biphasic survival analysis of trypanotolerance QTL in mice.

A marker-assisted introgression (MAI) experiment was conducted to transfer trypanotolerance quantitative trait loci (QTL) from a donor mouse strain, C57BL/6, into a recipient mouse strain, A/J. The objective was to assess the effect of three previously identified chromosomal regions on mouse chromosomes 1 (MMU1), 5 (MMU5) and 17 (MMU17) in different genetic backgrounds on the survival pattern following infection with Trypanosoma congolense. An exploratory data analysis revealed a biphasic pattern of time to death, with highly distinct early and late mortality phases. In this paper, we present survival analysis methods that account for the biphasic mortality pattern and results of reanalyzing the data from the MAI experiment. The analysis with a Weibull mixture model confirmed the biphasic pattern of time to death. Mortality phase, an unobserved variable, appears to be an important factor influencing survival time and is modeled as a binary outcome variable using logistic regression analysis. Accounting for this biphasic pattern in the analysis reveals that a previously observed sex effect on average survival is rather an effect on proportion of mice in the two mortality phases. The C57BL/6 (donor) QTL alleles on MMU1 and MMU17 act dominantly in the late mortality phase while the A/J (recipient) QTL allele on MMU17 acts dominantly in the early mortality phase. From this study, we found clear evidence for a biphasic survival pattern and provided models for its analysis. These models can also be used when studying defense mechanisms against other pathogens. Finally, these approaches provide further information on the nature of gene actions.

The antiplasmodial activity of spermine alkaloids isolated from Albizia gummifera.

In the present study the methanolic extract of Albizia gummifera was fractionated into various fractions. These fractions were tested against choroquine sensitive (NF54) and resistant (ENT30) strains of Plasmodium falciparum. All other fractions apart from the alkaloidal fraction showed low activity with IC 50 above 3 microg/ml. The alkaloidal fraction exhibited strong activity against NF54 and ENT30 with IC 50 of 0.16+/-0.05 and 0.99+/-0.06 microg/ml, respectively. Five known spermine alkaloids were isolated from the alkaloidal fraction. These alkaloids exhibited activities against NF54 and ENT30 with IC 50 ranging from 0.09+/-0.02 to 0.91+/-0.10 microg/ml. Four of the alkaloids were further evaluated for in vivo activity against rodent malaria parasite Plasmodium berghei. The alkaloids showed percentage chemosuppression of parasitaemia in mice ranging from 43 to 72%. The use of the extracts A. gummifera for treatment of malaria in traditional medicine seems to have a scientific basis.

Pathology of Tnf-deficient mice infected with Plasmodium chabaudi adami 408XZ.

Tumor necrosis factor alpha (Tnf) plays a pleiotropic role in murine malaria. Some investigations have correlated Tnf with hypothermia, hyperlactatemia, hypoglycemia, and a suppression of the erythropoietic response, although others have not. In this study, we have evaluated parasitemia, survival rate and several pathological features in C57BL/6JTnf(-/-) and C57BL/6JTnf(+/+) mice after infection with Plasmodium chabaudi adami 408XZ. Compared to the C57BL/6JTnf(+/+) mice, C57BL/6JTnf(-/-) mice showed increased parasitemia and decreased survival rate, whereas blood glucose, blood lactate and body weight were not significantly different. However, C57BL/6JTnf(-/-) mice suffered significantly more from severe anemia and hypothermia than C57BL/6JTnf(+/+) mice. These results suggest that Tnf is an important mediator of parasite control, but not of anemia development. We hypothesize that the high mortality observed in the Tnf knock-out mice is due to increased anemia and pathology as a direct result of increased levels of parasitemia.

Genetic variation in resistance to repeated infections with Heligmosomoides polygyrus bakeri, in inbred mouse strains selected for the mouse genome project.

Since the publication of the mouse genome, attention has focused on the strains that were selected for sequencing. In this paper we report the results of experiments that characterized the response to infection with the murine gastrointestinal nematode Heligmosomoides polygyrus of eight new strains (A/J, C57BL/6, C3H, DBA/2, BALB/c, NIH, SJL and 129/J), in addition to the well-characterized CBA (poor responder) and SWR (strong responder) as our controls. We employed the repeated infection protocol (consisting of 7 superimposed doses of 125L3 each administered at weekly intervals, faecal egg counts in weeks 2, 4 and 6 and assessment of worm burdens in week 6) that was used successfully to identify quantitative trait loci for genes involved in resistance to H. polygyrus. SWR, SJL and NIH mice performed indistinguishably and are confirmed as strong responder strains to H. polygyrus. CBA, C3H and A/J mice all tolerated heavy infections and are assessed as poor responders. In contrast, DBA/2, 129/J and BALB/c mice performed variably between experiments, some tolerating heavy worm burdens comparable to those in poor responders, and some showing evidence of resistance, although only in one experiment with female 129/J females and one with female BALB/c was the pattern and extent of worm loss much like that in SWR mice. Because the genetic relationships between six of the strains exploited in this study are now well-understood, our results should enable analysis through single nucleotide polymorphisms and thereby provide more insight into the role of the genes that control resistance to H. polygyrus.

TNF as a malaria candidate gene: polymorphism-screening and family-based association analysis of mild malaria attack and parasitemia in Burkina Faso.

We have previously obtained strong evidence for linkage of mild malaria attack to the MHC region, with a peak close to the tumor necrosis factor (TNF) gene. We screened, for polymorphisms, the entire TNF gene in the same sample of 34 families comprising 197 individuals living in a Plasmodium falciparum endemic area and we found 17 polymorphisms. In a longitudinal study, we investigated whether the 11 most frequent and informative polymorphisms were associated with mild malaria attack and maximum parasitemia, which was the highest parasitemia in each individual over 2 years. Mild malaria attack and maximum parasitemia were positively correlated. Transmission disequilibrium tests showed nominal evidence for association between TNF-1031, TNF-308, TNF851 and TNF1304 polymorphisms, and mild malaria attack on the one hand, and between TNF-238, TNF851 and TNF1304 polymorphisms, and maximum parasitemia on the other hand. After accounting for multiple tests, we confirmed the association of TNF-238 with maximum parasitemia and the association of TNF1304 and TNF851 with maximum parasitemia and mild malaria attack. The association tests with mild malaria attack suggest a moderate effect of TNF-308 polymorphism. In conclusion, our study suggests that several TNF variants may be part of the genetic determinants for maximum parasitemia and/or mild malaria attack.

TNF-alpha mediates the development of anaemia in a murine Trypanosoma brucei rhodesiense infection, but not the anaemia associated with a murine Trypanosoma congolense infection.

Development of anaemia in inflammatory diseases is cytokine-mediated. Specifically, the levels of tumour necrosis factor-alpha (TNF-alpha), produced by activated macrophages, are correlated with severity of disease and anaemia in infections and chronic disease. In African trypanosomiasis, anaemia develops very early in infection around the time when parasites become detectable in the blood. Since the anaemia persists after the first waves of parasitaemia when low numbers of trypanosomes are circulating in the blood, it is generally assumed that anaemia is not directly induced by a parasite factor, but might be cytokine-mediated, as in other cases of anaemia accompanying inflammation. To clarify the role of TNF-alpha in the development of anaemia, blood parameters of wild type (TNF-alpha+/+), TNF-alpha-null (TNF-alpha-/-) and TNF-alpha-hemizygous (TNF-alpha-/+) trypanotolerant mice were compared during infections with the cattle parasite Trypanosoma congolense. No differences in PCV, erythrocyte numbers or haemoglobin were observed between TNF-alpha-deficient and wild type mice, suggesting that the decrease in erythrocytes was not mediated by TNF-alpha. Erythropoetin (EPO) levels increased during infection and no significant differences in EPO levels were observed between the three mouse strains. In contrast, during an infection with the human pathogen Trypanosoma brucei rhodesiense, the number of red blood cells in TNF-alpha-deficient mice remained significantly higher than in the wild type mice. These data suggest that more than one mechanism promotes the development of anaemia associated with trypanosomiasis.

Comparison of pathology in susceptible A/J and resistant C57BL/6J mice after infection with different sub-strains of Plasmodium chabaudi.

Susceptible A/J and more resistant C57BL/6J mice were infected with Plasmodium chabaudi chabaudi 54X, P.c. chabaudi AS and Plasmodium chabaudi adami 408XZ. As expected, most C57BL/6J mice survived the infections with the different isolates. But in contrast to previous observations, not all A/J mice succumbed to infection: just over 50% of A/J mice survived infections with P.c. chabaudi 54X, while 80% survived P.c. chabaudi AS. The more virulent parasite, P.c. adami 408XZ, was able to kill all A/J mice and 20% of C57BL/6J mice after an intravenous infection with 10(5) pRBC. A detailed study of four parameters of pathology (body weight, body temperature, blood glucose and RBC counts) in both mouse strains after a P.c. adami 408XZ infection showed similar patterns to those previously reported after infection with P.c. chabaudi AS. These data suggest that environmental factors as well as parasite polymorphisms might influence the severity of malaria between susceptible and resistant mice.

Chasing the genes that control resistance to gastrointestinal nematodes.

The host-protective immune response to infection with gastrointestinal (GI) nematodes involves a range of interacting processes that begin with recognition of the parasite's antigens and culminate in an inflammatory reaction in the intestinal mucosa. Precisely which immune effectors are responsible for the loss of specific worms is still not known although many candidate effectors have been proposed. However, it is now clear that many different genes regulate the response and that differences between hosts (fast or strong versus slow or weak responses) can be explained by allelic variation in crucial genes associated with the gene cascade that accompanies the immune response and/or genes encoding constitutively expressed receptor/signalling molecules. Major histocompatibility complex (MHC) genes have been recognized for some time as decisive in controlling immunity, and evidence that non-MHC genes are equally, if not more important in this respect has also been available for two decades. Nevertheless, whilst the former have been mapped in mice, only two candidate loci have been proposed for non-MHC genes and relatively little is known about their roles. Now, with the availability of microsatellite markers, it is possible to exploit linkage mapping techniques to identify quantitative trait loci (QTL) responsible for resistance to GI nematodes. Four QTL for resistance to Heligmosomoides polygyrus, and additional QTL affecting faecal egg production by the worms and the accompanying immune responses, have been identified. Fine mapping and eventually the identification of the genes (and their alleles) underlying QTL for resistance/susceptibility will permit informed searches for homologues in domestic animals, and human beings, through comparative genomic maps. This information in turn will facilitate targeted breeding to improve resistance in domestic animals and, in human beings, focused application of treatment and control strategies for GI nematodes.

Susceptibility of tumour necrosis factor-alpha genetically deficient mice to Trypanosoma congolense infection.

The TNF-alpha gene on mouse chromosome MMU17 is among the candidates for the trypanosomosis resistance QTL Tir1. Tir1 has the largest effect of those loci so far detected which influence degree of resistance to murine trypanosomosis caused by Trypanosoma congolense infection. We therefore studied the survival to 180 days after challenge with T. congolense of mice that were homozygous and hemizygous with respect to a disruption of the TNF-alpha gene on a > 99% C57BL/6 (resistant) background. We also examined the responses of TNF-alpha hemizygous mice produced by crossing the deletion line with mice of the C57BL/6J strain, and with mice of the susceptible A/J strain. Mice lacking a functional TNF-alpha gene were shown to be highly susceptible to challenge with T. congolense with a median survival time of 37 days. This was comparable to 71 days for control wild-type mice, and 61 and 111 days for mice of the susceptible A/J and resistant C57BL/6J strains, respectively. In mice of the deletion line, the C57BL/6 TNF-alpha allele tended to be dominant to the TNF knockout in terms of resistance. We conclude that TNF-alpha plays an important role in resistance to the effects of T. congolense infection in mice.

Fine mapping of trypanosomiasis resistance loci in murine advanced intercross lines.

We have previously reported the results of genome-wide searches in two murine F(2) populations for QTLs that influence survival following Trypanosoma congolense infection. Three loci, Tir1, Tir2, and Tir3, were identified and mapped to mouse Chromosomes (Chrs) 17, 5, and 1 respectively, with confidence intervals (CIs) in the range 10-40 cM. The size of these CIs is to a large degree the consequence of limited numbers of recombination events in small chromosomal regions in F(2) populations. A number of population designs have been proposed to increase recombination levels in crosses, one of which is the advanced intercross line (AIL). Here we report fine mapping of Tir1, Tir2, and Tir3 in G6 populations of two independent murine AILs created by crossing the C57BL/6J strain with the A/J and BALB/cJ strains, respectively. Data were analyzed by two methods that gave equally informative and similar results. The three QTLs were confirmed in the A/J x C57BL/6J AIL and in the combined data set, but Tir2 was apparently lost from the BALB/cJ x C57BL/6J AIL. The reduction in CIs for the Tir loci ranged from 2.5 to more than ten-fold in G6 populations by comparison with CIs obtained previously in the equivalent F(2) generations. Mapping in the AILs also resolved the Tir3 locus into three trypanosomiasis resistance QTLs, revealing a degree of complexity not evident in extensive studies at the F(2) level.

Strategies to optimize marker-assisted introgression of multiple unlinked QTL.

To optimize designs to implement marker-assisted introgression programs aiming to introgress three unlinked quantitative trait loci (QTL), the present paper studies different alternatives versus a traditional backcross or intercross phase. Four alternative backcross strategies appear to be more advantageous by having 50% less genotyping load than a traditional backcross strategy tracking all three QTL at a time through a single line. A multiplication phase following the selection of homozygous animals at the three QTL as an intercross alternative allows doubling of the number of homozygous animals in a mouse model compared with the first intercross generation. Within the same model, a second intercross alternative with individuals carrying all three QTL at the first intercross results in a 12-fold increase in the number of homozygous animals obtained in the first intercross generation. The same ranges of decrease are observed in the number of animals to be genotyped and the number of genotypings when targeting a fixed number of homozygous animals. An option, with two lines each carrying two QTL through the backcross phase and coupled with the second intercross alternative, appears to be the best introgression alternative. This option requires 76% fewer genotypings, 68% fewer animals to be genotyped, and costs 75% less than an option in which all three QTL are introgressed through a single line.

Fine mapping of quantitative trait loci using advanced intercross lines of mice and positional cloning of the corresponding genes.

High-resolution mapping of quantitative trait loci (QTLs) is an essential step towards positional cloning and identification of the corresponding genes. Most QTL detection and mapping studies in mice have been carried out using F2 intercross and backcross populations. As a consequence of the limited number of recombination events in small chromosomal regions, this has generally permitted mapping to only relatively large confidence intervals of 20 to 40 cM. A number of population designs have been proposed to increase recombination level in crosses. This includes advanced intercross lines (AIL) described by Darvasi and Soller [Genomics. 1995; 141: 1199-1207]. In this report demonstration of the utility of the AIL approach for fine mapping of QTL, which previously had been mapped with 95% confidence interval to 20 to 40 cM in a F2 intercross, will be presented. The methodological approaches to go from the fine-mapped QTL to the identification of the actual genes and mutations are discussed.