The Genetics of Human Schistosomiasis Infection Intensity and Liver Disease: A Review.

Schistosomiasis remains the fourth most prevalent parasitic disease affecting over 200 million people worldwide. Control efforts have focussed on the disruption of the life cycle targeting the parasite, vector and human host. Parasite burdens are highly skewed, and the majority of eggs are shed into the environment by a minority of the infected population. Most morbidity results from hepatic fibrosis leading to portal hypertension and is not well-correlated with worm burden. Genetics as well as environmental factors may play a role in these skewed distributions and understanding the genetic risk factors for intensity of infection and morbidity may help improve control measures. In this review, we focus on how genetic factors may influence parasite load, hepatic fibrosis and portal hypertension. We found 28 studies on the genetics of human infection and 20 studies on the genetics of pathology in humans. S. mansoni and S. haematobium infection intensity have been showed to be controlled by a major quantitative trait locus SM1, on chromosome 5q31-q33 containing several genes involved in the Th2 immune response, and three other loci of smaller effect on chromosomes 1, 6, and 7. The most common pathology associated with schistosomiasis is hepatic and portal vein fibroses and the SM2 quantitative trait locus on chromosome six has been linked to intensity of fibrosis. Although there has been an emphasis on Th2 cytokines in candidate gene studies, we found that four of the five QTL regions contain Th17 pathway genes that have been included in schistosomiasis studies: IL17B and IL12B in SM1, IL17A and IL17F in 6p21-q2, IL6R in 1p21-q23 and IL22RA2 in SM2. The Th17 pathway is known to be involved in response to schistosome infection and hepatic fibrosis but variants in this pathway have not been tested for any effect on the regulation of these phenotypes. These should be priorities for future studies.

TIGERi: modeling and visualizing the responses to perturbation of a transcription factor network.

BACKGROUND: Transcription factor (TF) networks play a key role in controlling the transfer of genetic information from gene to mRNA. Much progress has been made on understanding and reverse-engineering TF network topologies using a range of experimental and theoretical methodologies. Less work has focused on using these models to examine how TF networks respond to changes in the cellular environment. METHODS: In this paper, we have developed a simple, pragmatic methodology, TIGERi (Transcription-factor-activity Illustrator for Global Explanation of Regulatory interaction), to model the response of an inferred TF network to changes in cellular environment. The methodology was tested using publicly available data comparing gene expression profiles of a mouse p38α (Mapk14) knock-out line to the original wild-type. RESULTS: Using the model, we have examined changes in the TF network resulting from the presence or absence of p38α. A part of this network was confirmed by experimental work in the original paper. Additional relationships were identified by our analysis, for example between p38α and HNF3, and between p38α and SOX9, and these are strongly supported by published evidence. FXR and MYC were also discovered in our analysis as two novel links of p38α. To provide a computational methodology to the biomedical communities that has more user-friendly interface, we also developed a standalone GUI (graphical user interface) software for TIGERi and it is freely available at https://github.com/namshik/tigeri/ . CONCLUSIONS: We therefore believe that our computational approach can identify new members of networks and new interactions between members that are supported by published data but have not been integrated into the existing network models. Moreover, ones who want to analyze their own data with TIGERi could use the software without any command line experience. This work could therefore accelerate researches in transcriptional gene regulation in higher eukaryotes.

A simple procedure for directly obtaining haplotype sequences of diploid genomes.

BACKGROUND: Almost all genome sequencing projects neglect the fact that diploid organisms contain two genome copies and consequently what is published is a composite of the two. This means that the relationship between alternate alleles at two or more linked loci is lost. We have developed a simplified method of directly obtaining the haploid sequences of each genome copy from an individual organism. RESULTS: The diploid sequences of three groups of cattle samples were obtained using a simple sample preparation procedure requiring only a microscope and a haemocytometer. Samples were: 1) lymphocytes from a single Angus steer; 2) sperm cells from an Angus bull; 3) lymphocytes from East African Zebu (EAZ) cattle collected and processed in a field laboratory in Eastern Kenya. Haploid sequence from a fosmid library prepared from lymphocytes of an EAZ cow was used for comparison. Cells were serially diluted to a concentration of one cell per microlitre by counting with a haemocytometer at each dilution. One microlitre samples, each potentially containing a single cell, were lysed and divided into six aliquots (except for the sperm samples which were not divided into aliquots). Each aliquot was amplified with phi29 polymerase and sequenced. Contigs were obtained by mapping to the bovine UMD3.1 reference genome assembly and scaffolds were assembled by joining adjacent contigs that were within a threshold distance of each other. Scaffolds that appeared to contain artefacts of CNV or repeats were filtered out leaving scaffolds with an N50 length of 27-133 kb and a 88-98 % genome coverage. SNP haplotypes were assembled with the Single Individual Haplotyper program to generate an N50 size of 97-201 kb but only ~27-68 % genome coverage. This method can be used in any laboratory with no special equipment at only slightly higher costs than conventional diploid genome sequencing. A substantial body of software for analysis and workflow management was written and is available as supplementary data. CONCLUSIONS: We have developed a set of laboratory protocols and software tools that will enable any laboratory to obtain haplotype sequences at only modestly greater cost than traditional mixed diploid sequences.

Multiorgan dysfunction caused by travel-associated African trypanosomiasis.

We describe a case of multiorgan dysfunction secondary to Trypanosoma brucei rhodesiense infection acquired on safari in Zambia. This case was one of several recently reported to ProMED-mail in persons who had traveled to this region. Trypanosomiasis remains rare in travelers but should be considered in febrile patients who have returned from trypanosomiasis-endemic areas of Africa.

A comprehensive genetic analysis of candidate genes regulating response to Trypanosoma congolense infection in mice.

BACKGROUND: African trypanosomes are protozoan parasites that cause "sleeping sickness" in humans and a similar disease in livestock. Trypanosomes also infect laboratory mice and three major quantitative trait loci (QTL) that regulate survival time after infection with T. congolense have been identified in two independent crosses between susceptible A/J and BALB/c mice, and the resistant C57BL/6. These were designated Tir1, Tir2 and Tir3 for Trypanosoma infection response, and range in size from 0.9-12 cM. PRINCIPAL FINDINGS: Mapping loci regulating survival time after T. congolense infection in an additional cross revealed that susceptible C3H/HeJ mice have alleles that reduce survival time after infection at Tir1 and Tir3 QTL, but not at Tir2. Next-generation resequencing of a 6.2 Mbp region of mouse chromosome 17, which includes Tir1, identified 1,632 common single nucleotide polymorphisms (SNP) including a probably damaging non-synonymous SNP in Pram1 (PML-RAR alpha-regulated adaptor molecule 1), which was the most plausible candidate QTL gene in Tir1. Genome-wide comparative genomic hybridisation identified 12 loci with copy number variants (CNV) that correlate with differential gene expression, including Cd244 (natural killer cell receptor 2B4), which lies close to the peak of Tir3c and has gene expression that correlates with CNV and phenotype, making it a strong candidate QTL gene at this locus. CONCLUSIONS: By systematically combining next-generation DNA capture and sequencing, array-based comparative genomic hybridisation (aCGH), gene expression data and SNP annotation we have developed a strategy that can generate a short list of polymorphisms in candidate QTL genes that can be functionally tested.

Genotype and expression analysis of two inbred mouse strains and two derived congenic strains suggest that most gene expression is trans regulated and sensitive to genetic background.

BACKGROUND: Differences in gene expression may be caused by nearby DNA polymorphisms (cis regulation) or by interactions of gene control regions with polymorphic transcription factors (trans regulation). Trans acting loci are much harder to detect than cis acting loci and their effects are much more sensitive to genetic background. RESULTS: To quantify cis and trans regulation we correlated haplotype data with gene expression in two inbred mouse strains and two derived congenic lines. Upstream haplotype differences between the parental strains suggested that 30-43% of differentially expressed genes were differentially expressed because of cis haplotype differences. These cis regulated genes displayed consistent and relatively tissue-independent differential expression. We independently estimated from the congenic mice that 71-85% of genes were trans regulated. Cis regulated genes were associated with low p values (p < 0.005) for differential expression, whereas trans regulated genes were associated with values 0.005 < p < 0.05. The genes differentially expressed between congenics and controls were not a subset of those that were differentially expressed between the founder lines, showing that these were dependent on genetic background. For example, the cholesterol synthesis pathway was strongly differentially expressed in the congenic mice by indirect trans regulation but this was not observable in the parental mice. CONCLUSIONS: The evidence that most gene regulation is trans and strongly influenced by genetic background, suggests that pathways that are modified by an allelic variant, may only exhibit differential expression in the specific genetic backgrounds in which they were identified. This has significant implications for the interpretation of any QTL mapping study.

Clinical chemistry of congenic mice with quantitative trait loci for predicted responses to Trypanosoma congolense infection.

Trypanosoma congolense is a protozoan parasite that causes severe diseases in livestock. Three major quantative trait loci (QTL), Tir1, Tir2, and Tir3, control the survival time of mice after infection with T. congolense. Congenic mice carrying the C57BL/6 resistance alleles on the A/J background were developed for each of these loci. The congenic mice were used to physically map the regions containing the QTL gene(s) and to investigate the physiological effect of each locus. Clinical chemistry data for infected A/J, C57BL/6, and BALB/c mice were obtained for 15 analytes at five time points. Congenic mice were assessed for survival, parasitemia, and anemia as well as seven clinical-chemical analytes. The survival times were significantly increased in the Tir1 and Tir2 mice but not Tir3 congenic mice. The survival time of the parental inbred mice correlated negatively with parasitemia but positively with alanine aminotransferase activities in serum, suggesting that inflammatory reactions in the liver had a beneficial effect possibly associated with reduced parasitemia. However, there was no difference in parasitemia or liver enzyme activities of Tir1 and Tir2 congenic mice relative to their controls, showing that survival, parasitemia, and degree of liver damage are not associated with each other, despite the correlation in the parental lines. These data suggest that the congenic loci affect survival but do not affect control of parasite number. They may therefore act by limiting the pathological consequences of T. congolense infection.

Mechanisms controlling anaemia in Trypanosoma congolense infected mice.

BACKGROUND: Trypanosoma congolense are extracellular protozoan parasites of the blood stream of artiodactyls and are one of the main constraints on cattle production in Africa. In cattle, anaemia is the key feature of disease and persists after parasitaemia has declined to low or undetectable levels, but treatment to clear the parasites usually resolves the anaemia. METHODOLOGY/PRINCIPAL FINDINGS: The progress of anaemia after Trypanosoma congolense infection was followed in three mouse strains. Anaemia developed rapidly in all three strains until the peak of the first wave of parasitaemia. This was followed by a second phase, characterized by slower progress to severe anaemia in C57BL/6, by slow recovery in surviving A/J and a rapid recovery in BALB/c. There was no association between parasitaemia and severity of anaemia. Furthermore, functional T lymphocytes are not required for the induction of anaemia, since suppression of T cell activity with Cyclosporin A had neither an effect on the course of infection nor on anaemia. Expression of genes involved in erythropoiesis and iron metabolism was followed in spleen, liver and kidney tissues in the three strains of mice using microarrays. There was no evidence for a response to erythropoietin, consistent with anaemia of chronic disease, which is erythropoietin insensitive. However, the expression of transcription factors and genes involved in erythropoiesis and haemolysis did correlate with the expression of the inflammatory cytokines Il6 and Ifng. CONCLUSIONS/SIGNIFICANCE: The innate immune response appears to be the major contributor to the inflammation associated with anaemia since suppression of T cells with CsA had no observable effect. Several transcription factors regulating haematopoiesis, Tal1, Gata1, Zfpm1 and Klf1 were expressed at consistently lower levels in C57BL/6 mice suggesting that these mice have a lower haematopoietic capacity and therefore less ability to recover from haemolysis induced anaemia after infection.

Strong position-dependent effects of sequence mismatches on signal ratios measured using long oligonucleotide microarrays.

BACKGROUND: Microarrays are an important and widely used tool. Applications include capturing genomic DNA for high-throughput sequencing in addition to the traditional monitoring of gene expression and identifying DNA copy number variations. Sequence mismatches between probe and target strands are known to affect the stability of the probe-target duplex, and hence the strength of the observed signals from microarrays. RESULTS: We describe a large-scale investigation of microarray hybridisations to murine probes with known sequence mismatches, demonstrating that the effect of mismatches is strongly position-dependent and for small numbers of sequence mismatches is correlated with the maximum length of perfectly matched probe-target duplex. Length of perfect match explained 43% of the variance in log2 signal ratios between probes with one and two mismatches. The correlation with maximum length of perfect match does not conform to expectations based on considering the effect of mismatches purely in terms of reducing the binding energy. However, it can be explained qualitatively by considering the entropic contribution to duplex stability from configurations of differing perfect match length. CONCLUSION: The results of this study have implications in terms of array design and analysis. They highlight the significant effect that short sequence mismatches can have upon microarray hybridisation intensities even for long oligonucleotide probes. All microarray data presented in this study are available from the GEO database 1, under accession number [GEO: GSE9669]

Leishmania donovani and cutaneous leishmaniasis, Sri Lanka.

To investigate the relationship of cutaneous leishmaniasis isolates from Sri Lanka to known species, we performed DNA sequencing and microsatellite analyses. We identified Leishmania donovani as the agent of Sri Lanka cutaneous leishmaniasis and showed that these parasites are closely related to those causing visceral leishmaniasis in the Indian subcontinent.

A molecular phylogeny of the nightjars (Aves: Caprimulgidae) suggests extensive conservation of primitive morphological traits across multiple lineages.

We report a molecular re-assessment of the classification of the nightjars which draws conclusions that are strongly at odds with the traditional, morphology-based classifications. We used maximum likelihood and Bayesian methods to compare the cytochrome b gene for 14 species from seven of the 15 genera of the Caprimulgidae and partial cytochrome b sequence data was available for a further seven species including three further genera. We found that within the Caprimulgidae there were four geographically isolated clades with bootstrap support greater than 70%. One of these clades contained just Chordeiles species, the remaining three clades each contained a mixture of genera including Caprimulgus sp. A clade of exclusively South American nightjars included the genera Caprimulgus, Uropsalis, Eleopthreptus and Hydropsalis. A clade of African and Eurasian birds included Caprimulgus and Macrodipteryx. Phalaenoptilus nuttallii and Caprimulgus vociferous formed a clade of North American birds. Two ecological factors appear to make morphological classification potentially misleading: first, the apparent retention of primitive anti-predator and foraging-related traits across genetically divergent groups; second, rapid divergence in other traits, especially those related to mating, which generate high levels of morphological divergence between species that are genetically very similar. The cytochrome b data suggests that the genus Caprimulgus is not monophyletic and is restricted to Africa and Eurasia and that Caprimulgus species from outside this area have been misclassified as a consequence of retention of primitive adaptations for crepuscular/nocturnal living. Some other genera also appear to have little support from the cytochrome b data.

Male sex pheromones and the phylogeographic structure of the Lutzomyia longipalpis species complex (Diptera: Psychodidae) from Brazil and Venezuela.

Lutzomyia longipalpis, a sibling complex, is the main vector of Leishmania chagasi/infantum. Discriminating between siblings is important as they may differ in vectorial capacity. Lutzomyia longipalpis populations display distinct male sex pheromone chemotypes. We investigated the phylogeographic pattern of variation at microsatellite loci from 11 populations from Brazil and Venezuela related to their male pheromone. Temporal genetic differentiation was mostly not significant at the same site. Spatial genetic differentiation was, however, strong, although there was only a weak relationship between genetic differentiation and the geographic distance separating the samples (r2 < 0.10); geographic separation explained a much greater (54-97%) percentage of the genetic differences among populations when samples with the same pheromone type were analyzed separately. A cluster analysis showed five groups: Lu. cruzi (Brazil) and Lu. pseudolongipalpis (Venezuela) as separate species, two (mostly 9-methyl-germacrene-B) Venezuelan and Brazilian groups, and a very distinct cluster of Brazilian cembrene populations.