Large-scale Genotyping and Genetic Mapping in Plasmodium Parasites

The completion of many malaria parasite genomes provides great opportunities for genomewide characterization of gene expression and high-throughput genotyping. Substantial progress in malaria genomics and genotyping has been made recently, particularly the development of various microarray platforms for large-scale characterization of the Plasmodium falciparum genome. Microarray has been used for gene expression analysis, detection of single nucleotide polymorphism (SNP) and copy number variation (CNV), characterization of chromatin modifications, and other applications. Here we discuss some recent advances in genetic mapping and genomic studies of malaria parasites, focusing on the use of high-throughput arrays for the detection of SNP and CNV in the P. falciparum genome. Strategies for genetic mapping of malaria traits are also discussed.     

Development of a high-throughput SNP array for sea cucumber (Apostichopus japonicus) and its application in genomic selection with MCP regularized deep neural networks

High-throughput single nucleotide polymorphism (SNP) genotyping assays are powerful tools for genetic studies and genomic breeding applications for many species. Though large numbers of SNPs have been identified in sea cucumber (Apostichopus japonicus), but, as yet, no high-throughput genotyping platform is available for this species. In this study, we designed and developed a high-throughput 24 K SNP genotyping array named HaishenSNP24K for A. japonicus, based on the multi-objective-local optimization (MOLO) algorithm and HD-Marker genotyping method. The SNP array exhibited a relatively high genotyping call rate (> 96%), genotyping accuracy (>95%) and exhibited highly polymorphic in sea cucumber populations. In addition, we also assessed its application in genomic selection (GS). Deep neural networks (DNN) that can capture the complicated interactions of genes have been proposed as a promising tool in GS for SNP-based genomic prediction of complex traits in animal breeding. To overcome the problem of over-fitting when using the HaishenSNP24K array as high-dimensional DNN input, we developed minmax concave penalty (MCP) regularization for sparse deep neural networks (DNN-MCP) that finds an optimal sparse structure of a DNN by minimizing the square error subject to the non-convex penalty MCP on the parameters (weights and biases). Compared to two linear models, namely RR-GBLUP and Bayes B, and the nonlinear model DNN, DNN-MCP has greatly improved the genomic prediction ability for three quantitative traits (e.g., wet weight, dry weight and survival time) in the sea cucumber population. To the best of our knowledge, this is the first work to develop a high-throughput SNP array for A. japonicus and a new model DNN-MCP for genomic prediction of complex traits in GS. The present results provide evidence that supports the HaishenSNP24K array with DNN-MCP will be valuable for genetic studies and molecular breeding in A. japonicus.     

Molecular surveillance of malaria in the Central Highlands,Vietnam

Vietnam achieved outstanding success against malaria in the last few decades. The mortality and morbidity of malaria in Vietnam have decreased remarkably in recent years, but malaria is still a major public health concern in the country, particularly in the Central Highlands region. In this study, molecular analyses of malaria parasites in the Central Highlands were performed to understand the population structure and genetic diversity of the parasites circulating in the region. Plasmodium falciparum (68.7%) and P. vivax (27.4%) along with mixed infections with P. falciparum/P. vivax (3.9%) were detected in 230 blood samples from patients with malaria. Allele-specific nested-polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of pfmsp-1, pfama-1, pvcsp, and pvmsp-1 revealed complex genetic makeup in P. falciparum and P. vivax populations of Vietnam. Substantial multiplicity of infection (MOI) was also identified, suggesting significant genetic diversity and polymorphism of P. falciparum and P. vivax populations in the Central Highlands of Vietnam. These results provide fundamental insight into the current patterns of dispersion and genetic nature of malaria parasites as well as for the development of malaria elimination strategies in the endemic region.     

Complexity of Infection and Genetic Diversity in Cambodian Plasmodium vivax

BackgroundPlasmodium vivax is the most widely distributed human malaria parasite with 2.9 billion people living in endemic areas. Despite intensive malaria control efforts, the proportion of cases attributed to P. vivax is increasing in many countries. Genetic analyses of the parasite population and its dynamics could provide an assessment of the efficacy of control efforts, but, unfortunately, these studies are limited in P. vivax by the lack of informative markers and high-throughput genotyping methods.Conclusions/SignificanceOur findings demonstrate that this high-throughput genotyping assay is efficient in characterizing P. vivax diversity and can provide valuable insights to assess the efficacy of malaria elimination programs or to monitor the spread of specific parasites.     

Genotyping of single nucleotide polymorphisms in cytokine genes using real-time PCR allelic discrimination technology

Single nucleotide polymorphisms (SNPs), particularly those within regulatory regions of genes that code for cytokines often impact expression levels and can be disease modifiers. Investigating associations between cytokine genotype and disease outcome provides valuable insight into disease etiology and potential therapeutic intervention. Traditionally, genotyping for cytokine SNPs has been conducted using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), a low throughput technique not amenable for use in large-scale cytokine SNP association studies. Recently, Taqman real-time PCR chemistry has been adapted for use in allelic discrimination assays. The present study validated the accuracy and utility of real-time PCR technology for a number of commonly studied cytokine polymorphisms known to influence chronic inflammatory diseases. We show that this technique is amenable to high-throughput genotyping and overcomes many of the problematic features associated with PCR-RFLP including post-PCR manipulation, non-standardized assay conditions, manual allelic identification and false allelic identification due to incomplete enzyme digestion. The real-time PCR assays are highly accurate with an error rate in the present study of <1% and concordance rate with PCR-RFLP genotyping of 99.4%. The public databases of cytokine polymorphisms and validated genotyping assays highlighted in the present study will greatly benefit this important field of research.     

Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence

Background

The malaria parasite Plasmodium falciparum exhibits abundant genetic diversity, and this diversity is key to its success as a pathogen. Previous efforts to study genetic diversity in P. falciparum have begun to elucidate the demographic history of the species, as well as patterns of population structure and patterns of linkage disequilibrium within its genome. Such studies will be greatly enhanced by new genomic tools and recent large-scale efforts to map genomic variation. To that end, we have developed a high throughput single nucleotide polymorphism (SNP) genotyping platform for P. falciparum.

Results

Using an Affymetrix 3,000 SNP assay array, we found roughly half the assays (1,638) yielded high quality, 100% accurate genotyping calls for both major and minor SNP alleles. Genotype data from 76 global isolates confirm significant genetic differentiation among continental populations and varying levels of SNP diversity and linkage disequilibrium according to geographic location and local epidemiological factors. We further discovered that nonsynonymous and silent (synonymous or noncoding) SNPs differ with respect to within-population diversity, inter-population differentiation, and the degree to which allele frequencies are correlated between populations.

Conclusions

The distinct population profile of nonsynonymous variants indicates that natural selection has a significant influence on genomic diversity in P. falciparum, and that many of these changes may reflect functional variants deserving of follow-up study. Our analysis demonstrates the potential for new high-throughput genotyping technologies to enhance studies of population structure, natural selection, and ultimately enable genome-wide association studies in P. falciparum to find genes underlying key phenotypic traits.     

Deletion of CDY1b copy of Y chromosome CDY1 gene is a risk factor of male infertility in Tunisian men

The relationship between male infertility and microdeletions in the Y chromosome that remove multiple genes varies among countries and populations. The aim of this study was to investigate the different types of Chromodomain protein, Y-linked 1 (CDY1) gene deletions and their effect on male infertility and spermatogenesis in Tunisian men. A total of 241 infertile men with different spermatogenic impairments and 115 fertile men were included in this study. We determined the prevalence of CDY1a and CDY1b copy deletions by PCR-RFLP using PvuII as restriction endonuclease. Results: Among the 356 Tunisian individuals, 93.25% had the two copies (CDY1a and CDY1b) of CDY gene (91.2% in infertile patients and 97.3% in fertile men). We also found that deletion of CDY1b was significantly more frequent in infertile patients (azoo/oligospermic and normospermic) than in fertile men (7% vs 1.7% respectively; p value = 0.02). However, deletion of CDY1a copy was very rare, and was detected in only one fertile man and four normospermic infertile patients. Our findings showed that deletion of CDY1b copy gene is a significant risk factor for male infertility independent of sperm concentration, whereas deletion of CDY1a gene seems to have no effect on fertility in the Tunisian population.     

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.     

Joint efficacy of the three biomarkers SNCA,GYPB and HBG1 for atrial fibrillation and stroke: Analysis via the support vector machine neural network

Atrial fibrillation (AF) is the most common type of persistent arrhythmia. Although its incidence has been increasing, the pathogenesis of AF in stroke remains unclear. In this study, a total of 30 participants were recruited, including 10 controls, 10 patients with AF and 10 patients with AF and stroke (AF + STROKE). Differentially expressed genes (DEGs) were identified, and functional annotation of DEGs, comparative toxicogenomic database analysis associated with cardiovascular diseases, and predictions of miRNAs of hub genes were performed. Using RT‐qPCR, biological process and support vector machine neural networks, numerous DEGs were found to be related to AF. HBG1, SNCA and GYPB were found to be upregulated in the AF group. Higher expression of hub genes in AF and AF + STROKE groups was detected via RT‐PCR. Upon training the biological process neural network of SNCA and GYPB for HBG1, only small differences were detected. Based on the support vector machine, the predicted value of SNCA and GYPB for HBG1 was 0.9893. Expression of the hub genes of HBG1, SNCA and GYPB might therefore be significantly correlated to AF. These genes are involved in the incidence of AF complicated by stroke, and may serve as targets for early diagnosis and treatment.     

Yeast DEL assay detects clastogens

Chromosomal rearrangements, including DNA deletions are involved in carcinogenesis. The deletion (DEL) assay scoring for DNA deletions in the yeast Saccharomyces cerevisiae is able to detect a wide range of carcinogens. Among approximately 60 compounds of known carcinogenic activity, the DEL assay detected 86% correctly whereas the Ames Salmonella assay detected only 30% correctly [R.J. Brennan, R.H. Schiestl, Detecting carcinogens with the yeast DEL assay, Methods Mol. Biol. 262 (2004) 111-124]. Since the DEL assay is highly inducible by DNA double strand breaks, this study examined the utility of the DEL assay for detecting clastogens. Ten model compounds, with varied mechanisms of genotoxicity, were examined for their effect on the frequency of DNA deletions with the DEL assay. The compounds tested were: actinomycin D, camptothecin, methotrexate and 5-fluorodeoxyuridine, which are anticancer agents, noscapine and furosemide are therapeutics, acridine, methyl acrylate and resorcinol are industrial chemicals and diazinon is an insecticide. The in vitro micronucleus assay (IVMN) in CHO cells, a commonly used tool for detection of clastogens, was performed on the same compounds and the results of the two assays were compared. The results of our study show that there is 70% concordance in the presence of metabolic activation (rat liver S9) and 80% concordance in the absence of metabolic activation between the DEL assay and the standard in vitro micronucleus assay. The lack of cytotoxicity observed for four of the ten compounds examined indicates limited diffusion of lipophilic compounds across the yeast cell wall. Thus, the development of a more permeable yeast tester strain is expected to greatly improve concordance of the DEL assay with the IVMN assay. The yeast DEL assay is inexpensive, amenable to automation and requires less expertise to perform than the IVMN. Thus, it has a strong potential as a robust, fast and economical screen for detecting clastogens in vitro.     

Copy Number Variants in the Kallikrein Gene Cluster

The kallikrein gene family (KLK1-KLK15) is the largest contiguous group of protease genes within the human genome and is associated with both risk and outcome of cancer and other diseases. We searched for copy number variants in all KLK genes using quantitative PCR analysis and analysis of inheritance patterns of single nucleotide polymorphisms. Two deletions were identified: one 2235-bp deletion in KLK9 present in 1.2% of alleles, and one 3394-bp deletion in KLK15 present in 4.0% of alleles. Each deletion eliminated one complete exon and created out-of-frame coding that eliminated the catalytic triad of the resulting truncated gene product, which therefore likely is a non-functional protein. Deletion breakpoints identified by DNA sequencing located the KLK9 deletion breakpoint to a long interspersed element (LINE) repeated sequence, while the deletion in KLK15 is located in a single copy sequence. To search for an association between each deletion and risk of prostate cancer (PC), we analyzed a cohort of 667 biopsied men (266 PC cases and 401 men with no evidence of PC at biopsy) using short deletion-specific PCR assays. There was no association between evidence of PC in this cohort and the presence of either gene deletion. Haplotyping revealed a single origin of each deletion, with most recent common ancestor estimates of 3000-8000 and 6000-14 000 years for the deletions in KLK9 and KLK15, respectively. The presence of the deletions on the same haplotypes in 1000 Genomes data of both European and African populations indicate an early origin of both deletions. The old age in combination with homozygous presence of loss-of-function variants suggests that some kallikrein-related peptidases have non-essential functions.     

Distribution,prevalence and host specificity of avian malaria parasites across the breeding range of the migratory lark sparrow (Chondestes grammacus)

The lark sparrow (Chondestes grammacus) is a ground-nesting passerine that breeds across much of the central North American steppe and sand barrens. Through genotyping and sequencing of avian malaria parasites we examined levels of malaria prevalence and determined the distribution of Haemoproteus and Plasmodium lineages across the breeding range of the lark sparrow. Analysis of 365 birds collected from five breeding locations revealed relatively high levels of malaria prevalence in adults (80 %) and juveniles (46 %), with infections being primarily of Haemoproteus (91 % of sequenced samples). Levels of genetic diversity and genetic structure of malaria parasites with respect to the avian host populations revealed distinct patterns for Haemoproteus and Plasmodium, most likely as a result of their distinct life histories, host specificity, and transmission vectors. With the exception of one common Haemoproteus haplotype detected in all populations, all other haplotypes were either population-specific or shared by two to three populations. A hierarchical analysis of molecular variance of Haemoproteus sequences revealed that 15–18 % of the genetic variation can be explained by differences among host populations/locations (p < 0.001). In contrast to the regional patterns of genetic differentiation detected for the lark sparrow populations, Haemoproteus parasites showed high levels of population-specific variation and no significant differences among regions, which suggests that the population dynamics of the parasites may be driven by evolutionary processes operating at small spatial scales (e.g., at the level of host populations). These results highlight the potential effects of host population structure on the demographic and evolutionary dynamics of parasites.     

An Assessment of Heavy Ion Irradiation Mutagenesis for Reverse Genetics in Wheat (Triticum aestivum L.)

Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed.     

The RAD52 gene is not required for the function of the DEL1 mutator gene in Saccharomyces cerevisiae

Summary The DEL1 mutator in Saccharomyces cerevisiae leads to the formation of deletions adjacent to itself (Liebman et al. 1979). Here we show that the frequency of these DEL1-promoted deletions is not altered by the presence of the recombination-deficient mutation, rad52-1. This indicates that generalized recombination is not required for the formation of deletions in DEL1 yeast strains.     

Development of TaqMan allelic discrimination based genotyping of large DNA deletions

The high prevalence of genetic diseases resulting from gross deletions has highlighted a need for a quick, simple, and reliable method of genotyping these mutations. Here, we developed a novel strategy for applying TaqMan allelic discrimination to accurately genotype 3 different large deletions in a high-throughput manner. Allelic discrimination has previously been used to genotype frame shift and point mutations, and small insertions or deletions six base pairs in length, but not large deletions. The assays designed here recognize a 2502 base pair deletion in the Nebulin (NEB) gene that results in Nemaline Myopathy, a 308,769 base pair deletion in the Gap Junction Protein, beta 6 (GJB6) gene that causes Hearing Loss, and a 6433 base pair deletion in the Mucolipin 1 (MCOLN1) gene responsible for causing Mucolipidosis IV Disease. This methodology may also be successfully applied to high throughput genotyping of other large deletions.     

Characterising co-infections with Plasmodium spp., Mansonella perstans or Loa loa in asymptomatic children,adults and elderly people living on Bioko Island using nucleic acids extracted from malaria rapid diagnostic tests

BackgroundRegular and comprehensive epidemiological surveys of the filarial nematodes Mansonella perstans and Loa loa in children, adolescents and adults living across Bioko Island, Equatorial Guinea are lacking. We aimed to demonstrate that blood retained on malaria rapid diagnostic tests, commonly deployed for malaria surveys, could be used as a source of nucleic acids for molecular based detection of M. perstans and L. loa. We wanted to determine the positivity rate and distribution of filarial nematodes across different age groups and geographical areas as well as to understand level of co-infections with malaria in an asymptomatic population.MethodologyM. perstans, L. loa and Plasmodium spp. parasites were monitored by qPCR in a cross-sectional study using DNA extracted from a subset malaria rapid diagnostic tests (mRDTs) collected during the annual malaria indicator survey conducted on Bioko Island in 2018.Principal findingsWe identified DNA specific for the two filarial nematodes investigated among 8.2% (263) of the 3214 RDTs screened. Positivity rates of M. perstans and L. loa were 6.6% and 1.5%, respectively. M. perstans infection were more prominent in male (10.5%) compared to female (3.9%) survey participants. M. perstans parasite density and positivity rate was higher among older people and the population living in rural areas. The socio-economic status of participants strongly influenced the infection rate with people belonging to the lowest socio-economic quintile more than 3 and 5 times more likely to be L. loa and M. perstans infected, respectively. No increased risk of being co-infected with Plasmodium spp. parasites was observed among the different age groups.Conclusions/SignificanceWe found otherwise asymptomatic individuals were infected with M. perstans and L. loa. Our study demonstrates that employing mRDTs probed with blood for malaria testing represents a promising, future tool to preserve and ship NAs at room temperature to laboratories for molecular, high-throughput diagnosis and genotyping of blood-dwelling nematode filarial infections. Using this approach, asymptomatic populations can be reached and surveyed for infectious diseases beyond malaria.     

Population genetics of GYPB and association study between GYPB*S/s polymorphism and susceptibility to P. falciparum infection in the Brazilian Amazon

Background

Merozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil.

Methods

Two groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection.

Results

GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity.

Conclusion

Epidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is associated with the GPB S+ variant in this population.     

Yeast DEL assay detects protection against radiation-induced cytotoxicity and genotoxicity: adaptation of a microtiter plate version

The DEL assay in yeast detects DNA deletions that are inducible by many carcinogens. Here we use the colorimetric agent MTS to adapt the yeast DEL assay for microwell plate measurement of ionizing radiation-induced cell killing and DNA deletions. Using the microwell-based DEL assay, cell killing and genotoxic DNA deletions both increased with radiation dose between 0 and 2000 Gy. We used the microwell-based DEL assay to assess the effectiveness of varying concentrations of five different radioprotectors, N-acetyl-l-cysteine, l-ascorbic acid, DMSO, Tempol and Amifostine, and one radiosensitizer, 5-bromo-2-deoxyuridine. The microwell format of the DEL assay was able to successfully detect protection against and sensitization to both radiation-induced cytotoxicity and genotoxicity. Such radioprotection and sensitization detected by the microwell-based DEL assay was validated and compared with similar measurements made using the traditional agar-based assay format. The yeast DEL assay in microwell format is an effective tool for rapidly detecting chemical protectors and sensitizers to ionizing radiation and is automatable for chemical high-throughput screening purposes.     

Autoantibodies against red blood cell antigens are common in a malaria endemic area

Plasmodium falciparum malaria can cause severe anemia. Even after treatment, hematocrit can decrease. The role of autoantibodies against erythrocytes is not clearly elucidated and how common they are, or what they are directed against, is still largely unknown.We have investigated antibodies against erythrocytes in healthy adult men living in a highly malaria endemic area in Uganda. We found antibodies in more than half of the individuals, which is significantly more than in a non-endemic area (Sweden). Some of the Ugandan samples had a broad reactivity where it was not possible to determine the exact target of the autoantibodies, but we also found specific antibodies directed against erythrocyte surface antigens known to be of importance for merozoite invasion such as glycophorin A (anti-En^a, anti-M) and glycophorin B (anti-U, anti-S). In addition, several autoantibodies had partial specificities against glycophorin C and the blood group systems Rh, Diego (located on Band 3), Duffy (located on ACKR1), and Cromer (located on CD55), all of which have been described to be important for malaria and therefore of interest for understanding how autoantibodies could potentially stop parasites from entering the erythrocyte.In conclusion, specific autoantibodies against erythrocytes are common in a malaria endemic area.