A prevalence-based transmission model for the study of the epidemiology and control of soil-transmitted helminthiasis.

Much effort has been devoted by the World Health Organization (WHO) to eliminate soil-transmitted helminth (STH) infections by 2030 using mass drug administration targeted at particular risk groups alongside the availability to access water, sanitation and hygiene services. The targets set by the WHO for the control of helminth infections are typically defined in terms of the prevalence of infection, whereas the standard formulation of STH transmission models typically describe dynamic changes in the mean-worm burden. We develop a prevalence-based deterministic model to investigate the transmission dynamics of soil-transmitted helminthiasis in humans, subject to continuous exposure to infection over time. We analytically determine local stability criteria for all equilibria and find bifurcation points. Our model predicts that STH infection will either be eliminated (if the initial prevalence value, y(0), is sufficiently small) or remain endemic (if y(0) is sufficiently large), with the two stable points of endemic infection and parasite eradication separated by a transmission breakpoint. Two special cases of the model are analysed: (1) the distribution of the STH parasites in the host population is highly aggregated following a negative binomial distribution, and (2) no density-dependent effects act on the parasite population. We find that disease extinction is always possible for Case (1), but it is not so for Case (2) if y(0) is sufficiently large. However, by introducing stochastic perturbation into the deterministic model, we discover that chance effects can lead to outcomes not predicted by the deterministic model alone, with outcomes highly dependent on the degree of worm clumping, k. Specifically, we show that if the reproduction number and clumping are sufficiently bounded, then stochasticity will cause the parasite to die out. It follows that control of soil-transmitted helminths will be more difficult if the worm distribution tends towards clumping.

What is the impact of acquired immunity on the transmission of schistosomiasis and the efficacy of current and planned mass drug administration programmes?

Schistosomiasis causes severe morbidity in many countries with endemic infection with the schistosome digenean parasites in Africa and Asia. To control and eliminate the disease resulting from infection, regular mass drug administration (MDA) is used, with a focus on school-aged children (SAC; 5-14 years of age). In some high transmission settings, the World Health Organization (WHO) also recommends the inclusion of at-risk adults in MDA treatment programmes. The question of whether ecology (age-dependant exposure) or immunity (resistance to reinfection), or some combination of both, determines the form of observed convex age-intensity profile is still unresolved, but there is a growing body of evidence that the human hosts acquire some partial level of immunity after a long period of repeated exposure to infection. In the majority of past research modelling schistosome transmission and the impact of MDA programmes, the effect of acquired immunity has not been taken into account. Past work has been based on the assumption that age-related contact rates generate convex horizontal age-intensity profiles. In this paper, we use an individual based stochastic model of transmission and MDA impact to explore the effect of acquired immunity in defined MDA programmes. Compared with scenarios with no immunity, we find that acquired immunity makes the MDA programme less effective with a slower decrease in the prevalence of infection. Therefore, the time to achieve morbidity control and elimination as a public health problem is longer than predicted by models with just age-related exposure and no build-up of immunity. The level of impact depends on the baseline prevalence prior to treatment (the magnitude of the basic reproductive number R0) and the treatment frequency, among other factors. We find that immunity has a larger impact within moderate to high transmission settings such that it is very unlikely to achieve morbidity and transmission control employing current MDA programmes.

Deworming women of reproductive age during adolescence and pregnancy: what is the impact on morbidity from soil-transmitted helminths infection?

BACKGROUND: Soil-transmitted helminths (STHs) are a major cause of poor health in low- and middle-income countries. In particular, hookworm is known to cause anaemia in children and women of reproductive age (WRA). One goal of the World Health Organization's (WHO) 2030 roadmap for neglected tropical diseases is to reduce STH-related morbidity in WRA. As a minimal intervention, the WHO recommends deworming adolescent girls annually during human papilloma virus vaccination programmes and WRA during pregnancy and lactation. These routine interventions are low cost and can be implemented even by the most basic health services in endemic countries. In this study we use a cohort model to investigate the potential impact on STH-related morbidity in WRA. RESULTS: Annual deworming treatment of adolescent girls reduces the prevalence of moderate- and heavy-intensity infections in this age group by up to 60% in moderate transmission settings and by 12-27% in high transmission settings. Treatment of WRA during pregnancy and lactation on its own has a small (< 20%) but significant effect on morbidity although it does not lead to the achievement of the morbidity target (< 2% moderate- to high-intensity infections) in this age group. However, depending on the age-intensity profile of infection, which may vary geographically, and assumptions on the density-dependence of egg production by fertilised female worms, continued school-based treatment may be able to reduce the force of infection acting on WRA, both through an indirect effect on the overall population-based force of infection and via reducing the burden of infection as children age and move into the WRA age classes. As a result, morbidity in WRA may be eliminated. CONCLUSION: While deworming during pregnancy and lactation does not lead to the achievement of the morbidity target in WRA and its efficacy may vary by setting, it is still expected to be beneficial for maternity and child health. Monitoring of any WRA-based intervention is recommended to evaluate its effectiveness.

Spatial scales in human movement between reservoirs of infection.

The life cycle of parasitic organisms that are the cause of much morbidity in humans often depend on reservoirs of infection for transmission into their hosts. Understanding the daily, monthly and yearly movement patterns of individuals between reservoirs is therefore of great importance to implementers of control policies seeking to eliminate various parasitic diseases as a public health problem. This is due to the fact that the underlying spatial extent of the reservoir of infection, which drives transmission, can be strongly affected by inputs from external sources, i.e., individuals who are not spatially attributed to the region defined by the reservoir itself can still migrate and contribute to it. In order to study the importance of these effects, we build and examine a novel theoretical model of human movement between spatially-distributed focal points for infection clustered into regions defined as 'reservoirs of infection'. Using our model, we vary the spatial scale of human moment defined around focal points and explicitly calculate how varying this definition can influence the temporal stability of the effective transmission dynamics - an effect which should strongly influence how control measures, e.g., mass drug administration (MDA), define evaluation units (EUs). Considering the helminth parasites as our main example, by varying the spatial scale of human movement, we demonstrate that a critical scale exists around infectious focal points at which the migration rate into their associated reservoir can be neglected for practical purposes. This scale varies by species and geographic region, but is generalisable as a concept to infectious reservoirs of varying spatial extents and shapes. Our model is designed to be applicable to a very general pattern of infectious disease transmission modified by the migration of infected individuals between clustered communities. In particular, it may be readily used to study the spatial structure of hosts for macroparasites with temporally stationary distributions of infectious focal point locations over the timescales of interest, which is viable for the soil-transmitted helminths and schistosomes. Additional developments will be necessary to consider diseases with moving reservoirs, such as vector-born filarial worm diseases.

Stochastic challenges to interrupting helminth transmission.

Predicting the effect of different programmes designed to control both the morbidity induced by helminth infections and parasite transmission is greatly facilitated by the use of mathematical models of transmission and control impact. In such models, it is essential to account for the many sources of uncertainty - natural, or otherwise - to ensure robustness in prediction and to accurately depict variation around an expected outcome. In this paper, we investigate how well the standard deterministic models match the predictions made using individual-based stochastic simulations. We also explore how well concepts which derive from deterministic models, such as 'breakpoints' in transmission, apply in the stochastic world. Employing an individual-based stochastic model framework we also investigate how transmission and control are affected by the migration of infected people into a defined community. To give our study focus we consider the control of soil-transmitted helminths (STH) by mass drug administration (MDA), though our methodology is readily applicable to the other helminth species such as the schistosome parasites and the filarial worms. We show it is possible to theoretically define a 'stochastic breakpoint' where much noise surrounds the expected deterministic breakpoint. We also discuss the concept of the 'interruption of transmission' independent of the 'breakpoint' concept where analyses of model behaviour illustrate the current limitations of deterministic models to account for the 'fade-out' or transmission extinction behaviour in simulations. Our analysis of migration confirms a relationship between the critical infected human migration rate scale (i.e., order of magnitude) per unit of time and the death rate of infective stages that are released into the free-living environment. This relationship is shown to determine the likelihood that control activities aim at chemotherapeutic treatment of the human host will eliminate transmission. The development of a new stochastic simulation code for STH in the form of a publicly-available open-source python package which includes features to incorporate many population stratifications, different control interventions including mass drug administration (with defined frequency, coverage levels and compliance patterns) and inter-village human migration is also described.

Individual adherence to mass drug administration in neglected tropical disease control: A probability model conditional on past behaviour.

We present a general framework which describes the systematic (binary) scenario of individuals either taking treatment or not for any reason, over the course of mass drug administration (MDA)-which we refer to as 'adherence' and 'non-adherence'. The probability models developed can be informed by observed adherence behaviour as well as employed to explore how different patterns influence the impact of MDA programmes, by the use of mathematical models of transmission and control. We demonstrate the interpretative value of the developed probability model employing a dataset collected in the TUMIKIA project, a randomised trial of deworming strategies to control soil-transmitted helminths (STH) by MDA conducted in coastal Kenya. We stratify our analysis by age and sex, although the framework which we introduce here may be readily adapted to accommodate other stratifications. Our findings include the detection of specific patterns of non-adherence in all age groups to varying extents. This is particularly apparent in men of ages 30+. We then demonstrate the use of the probability model in stochastic individual-based simulations by running two example forecasts for the elimination of STH transmission employing MDA within the TUMIKIA trial setting with different adherence patterns. This suggested a substantial reduction in the probability of elimination (between 23-43%) when comparing observed adherence patterns with an assumption of independence, with important implications for programmes. The results here demonstrate the considerable impact and utility of considering non-adherence on the success of MDA programmes to control neglected tropical diseases (NTDs).

Forecasting the effectiveness of the DeWorm3 trial in interrupting the transmission of soil-transmitted helminths in three study sites in Benin, India and Malawi.

BACKGROUND: The DeWorm3 project is an ongoing cluster-randomised trial assessing the feasibility of interrupting the transmission of soil-transmitted helminths (STH) through mass drug administration (MDA) using study sites in India, Malawi and Benin. In this article, we describe an approach which uses a combination of statistical and mathematical methods to forecast the outcome of the trial with respect to its stated goal of reducing the prevalence of infection to below 2%. METHODS: Our approach is first to define the local patterns of transmission within each study site, which is achieved by statistical inference of key epidemiological parameters using the baseline epidemiological measures of age-related prevalence and intensity of STH infection which have been collected by the DeWorm3 trials team. We use these inferred parameters to calibrate an individual-based stochastic simulation of the trial at the cluster and study site level, which is subsequently run to forecast the future prevalence of STH infections. The simulator takes into account both the uncertainties in parameter estimation and the variability inherent in epidemiological and demographic processes in the simulator. We interpret the forecast results from our simulation with reference to the stated goal of the DeWorm3 trial, to achieve a target of [Formula: see text] prevalence at a point 24 months post-cessation of MDA. RESULTS: Simulated output predicts that the two arms will be distinguishable from each other in all three country sites at the study end point. In India and Malawi, measured prevalence in the intervention arm is below the threshold with a high probability (90% and 95%, respectively), but in Benin the heterogeneity between clusters prevents the arm prevalence from being reduced below the threshold value. At the level of individual study arms within each site, heterogeneity among clusters leads to a very low probability of achieving complete elimination in an intervention arm, yielding a post-study scenario with widespread elimination but a few 'hot spot' areas of persisting STH transmission. CONCLUSIONS: Our results suggest that geographical heterogeneities in transmission intensity and worm aggregation have a large impact on the effect of MDA. It is important to accurately assess cluster-level, or even smaller scale, heterogeneities in factors which influence transmission and aggregation for a clearer perspective on projecting the outcomes of MDA control of STH and other neglected tropical diseases.

The impact of mass drug administration on Schistosoma haematobium infection: what is required to achieve morbidity control and elimination?

BACKGROUND: Schistosomiasis remains an endemic parasitic disease causing much morbidity and, in some cases, mortality. The World Health Organization (WHO) has outlined strategies and goals to combat the burden of disease caused by schistosomiasis. The first goal is morbidity control, which is defined by achieving less than 5% prevalence of heavy intensity infection in school-aged children (SAC). The second goal is elimination as a public health problem (EPHP), achieved when the prevalence of heavy intensity infection in SAC is reduced to less than 1%. Mass drug administration (MDA) of praziquantel is the main strategy for control. However, there is limited availability of praziquantel, particularly in Africa where there is high prevalence of infection. It is therefore important to explore whether the WHO goals can be achieved using the current guidelines for treatment based on targeting SAC and, in some cases, adults. Previous modelling work has largely focused on Schistosoma mansoni, which in advance cases can cause liver and spleen enlargement. There has been much less modelling of the transmission of Schistosoma haematobium, which in severe cases can cause kidney damage and bladder cancer. This lack of modelling has largely been driven by limited data availability and challenges in interpreting these data. RESULTS: In this paper, using an individual-based stochastic model and age-intensity profiles of S. haematobium from two different communities, we calculate the probability of achieving the morbidity and EPHP goals within 15 years of treatment under the current WHO treatment guidelines. We find that targeting SAC only can achieve the morbidity goal for all transmission settings, regardless of the burden of infection in adults. The EPHP goal can be achieved in low transmission settings, but in some moderate to high settings community-wide treatment is needed. CONCLUSIONS: We show that the key determinants of achieving the WHO goals are the precise form of the age-intensity of infection profile and the baseline SAC prevalence. Additionally, we find that the higher the burden of infection in adults, the higher the chances that adults need to be included in the treatment programme to achieve EPHP.

Patterns of individual non-treatment during multiple rounds of mass drug administration for control of soil-transmitted helminths in the TUMIKIA trial, Kenya: a secondary longitudinal analysis.

BACKGROUND: Few studies have been done of patterns of treatment during mass drug administration (MDA) to control neglected tropical diseases. We used routinely collected individual-level treatment records that had been collated for the Tuangamize Minyoo Kenya Imarisha Afya (Swahili for Eradicate Worms in Kenya for Better Health [TUMIKIA]) trial, done in coastal Kenya from 2015 to 2017. In this analysis we estimate the extent of and factors associated with the same individuals not being treated over multiple rounds of MDA, which we term systematic non-treatment. METHODS: We linked the baseline population of the TUMIKIA trial randomly assigned to receive biannual community-wide MDA for soil-transmitted helminthiasis to longitudinal records on receipt of treatment in any of the four treatment rounds of the study. We fitted logistic regression models to estimate the association of non-treatment in a given round with non-treatment in the previous round, controlling for identified predictors of non-treatment. We also used multinomial logistic regression to identify factors associated with part or no treatment versus complete treatment. FINDINGS: 36 327 participants were included in our analysis: 16 236 children aged 2-14 years and 20 091 adults aged 15 years or older. The odds of having no treatment recorded was higher if a participant was not treated during the previous round of MDA (adjusted odds ratio [OR] 3·60, 95% CI 3·08-4·20 for children and 5·58, 5·01-6·21 for adults). For children, school attendance and rural residence reduced the odds of receiving part or no treatment, whereas odds were increased by least poor socioeconomic status and living in an urban or periurban household. Women had higher odds than men of receiving part or no treatment. However, when those with pregnancy or childbirth in the previous 2 weeks were excluded, women became more likely to receive complete treatment. Adults aged 20-25 years were the age group with the highest odds of receiving part (OR 1·41, 95% CI 1·22-1·63) or no treatment (OR 1·81, 95% CI 1·53-2·14). INTERPRETATION: Non-treatment was associated with specific sociodemographic groups and characteristics and did not occcur at random. This finding has important implications for MDA programme effectiveness, the relevance of which will intensify as disease prevalence decreases and infections become increasingly clustered. FUNDING: Bill & Melinda Gates Foundation, Joint Global Health Trials Scheme of the Medical Research Council, UK Department for International Development, Wellcome Trust, Children's Investment Fund Foundation, and London Centre for Neglected Tropical Diseases.

The 'breakpoint' of soil-transmitted helminths with infected human migration.

Building on past research, we here develop an analytic framework for describing the dynamics of the transmission of soil-transmitted helminth (STH) parasitic infections near the transmission breakpoint and equilibria of endemic infection and disease extinction, while allowing for perturbations in the infectious reservoir of the parasite within a defined location. This perturbation provides a model for the effect of infected human movement between villages with differing degrees of parasite control induced by mass drug administration (MDA). Analysing the dynamical behaviour around the unstable equilibrium, known as the transmission 'breakpoint', we illustrate how slowly-varying the dynamics are and develop an understanding of how discrete 'pulses' in the release of transmission stages (eggs or larvae, depending on the species of STH), due to infected human migration between villages, can lead to perturbations in the deterministic transmission dynamics. Such perturbations are found to have the potential to undermine targets for parasite elimination as a result of MDA and/or improvements in water and sanitation provision. We extend our analysis by developing a simple stochastic model and analytically investigate the uncertainty this induces in the dynamics. Where appropriate, all analytical results are supported by numerical analyses.

Population genetics of immune-related multilocus copy number variation in Native Americans.

While multiallelic copy number variation (mCNV) loci are a major component of genomic variation, quantifying the individual copy number of a locus and defining genotypes is challenging. Few methods exist to study how mCNV genetic diversity is apportioned within and between populations (i.e. to define the population genetic structure of mCNV). These inferences are critical in populations with a small effective size, such as Amerindians, that may not fit the Hardy-Weinberg model due to inbreeding, assortative mating, population subdivision, natural selection or a combination of these evolutionary factors. We propose a likelihood-based method that simultaneously infers mCNV allele frequencies and the population structure parameter f, which quantifies the departure of homozygosity from the Hardy-Weinberg expectation. This method is implemented in the freely available software CNVice, which also infers individual genotypes using information from both the population and from trios, if available. We studied the population genetics of five immune-related mCNV loci associated with complex diseases (beta-defensins, CCL3L1/CCL4L1, FCGR3A, FCGR3B and FCGR2C) in 12 traditional Native American populations and found that the population structure parameters inferred for these mCNVs are comparable to but lower than those for single nucleotide polymorphisms studied in the same populations.

Timing, rates and spectra of human germline mutation.

Germline mutations are a driving force behind genome evolution and genetic disease. We investigated genome-wide mutation rates and spectra in multi-sibling families. The mutation rate increased with paternal age in all families, but the number of additional mutations per year differed by more than twofold between families. Meta-analysis of 6,570 mutations showed that germline methylation influences mutation rates. In contrast to somatic mutations, we found remarkable consistency in germline mutation spectra between the sexes and at different paternal ages. In parental germ line, 3.8% of mutations were mosaic, resulting in 1.3% of mutations being shared by siblings. The number of these shared mutations varied significantly between families. Our data suggest that the mutation rate per cell division is higher during both early embryogenesis and differentiation of primordial germ cells but is reduced substantially during post-pubertal spermatogenesis. These findings have important consequences for the recurrence risks of disorders caused by de novo mutations.

Haptoglobin (HP) and Haptoglobin-related protein (HPR) copy number variation, natural selection, and trypanosomiasis.

Haptoglobin, coded by the HP gene, is a plasma protein that acts as a scavenger for free heme, and haptoglobin-related protein (coded by the HPR gene) forms part of the trypanolytic factor TLF-1, together with apolipoprotein L1 (ApoL1). We analyse the polymorphic small intragenic duplication of the HP gene, with alleles Hp1 and Hp2, in 52 populations, and find no evidence for natural selection either from extended haplotype analysis or from correlation with pathogen richness matrices. Using fiber-FISH, the paralog ratio test, and array-CGH data, we also confirm that the HPR gene is copy number variable, with duplication of the whole HPR gene at polymorphic frequencies in west and central Africa, up to an allele frequency of 15 %. The geographical distribution of the HPR duplication allele overlaps the region where the pathogen causing chronic human African trypanosomiasis, Trypanosoma brucei gambiense, is endemic. The HPR duplication has occurred on one SNP haplotype, but there is no strong evidence of extended homozygosity, a characteristic of recent natural selection. The HPR duplication shows a slight, non-significant undertransmission to human African trypanosomiasis-affected children of unaffected parents in the Democratic Republic of Congo. However, taken together with alleles of APOL1, there is an overall significant undertransmission of putative protective alleles to human African trypanosomiasis-affected children.

Automated design of paralogue ratio test assays for the accurate and rapid typing of copy number variation.

MOTIVATION: Genomic copy number variation (CNV) can influence susceptibility to common diseases. High-throughput measurement of gene copy number on large numbers of samples is a challenging, yet critical, stage in confirming observations from sequencing or array Comparative Genome Hybridization (CGH). The paralogue ratio test (PRT) is a simple, cost-effective method of accurately determining copy number by quantifying the amplification ratio between a target and reference amplicon. PRT has been successfully applied to several studies analyzing common CNV. However, its use has not been widespread because of difficulties in assay design. RESULTS: We present PRTPrimer (www.prtprimer.org) software for automated PRT assay design. In addition to stand-alone software, the web site includes a database of pre-designed assays for the human genome at an average spacing of 6 kb and a web interface for custom assay design. Other reference genomes can also be analyzed through local installation of the software. The usefulness of PRTPrimer was tested within known CNV, and showed reproducible quantification. This software and database provide assays that can rapidly genotype CNV, cost-effectively, on a large number of samples and will enable the widespread adoption of PRT. AVAILABILITY: PRTPrimer is available in two forms: a Perl script (version 5.14 and higher) that can be run from the command line on Linux systems and as a service on the PRTPrimer web site (www.prtprimer.org).

ß-defensin genomic copy number is associated with HIV load and immune reconstitution in sub-saharan Africans.

AIDS, caused by the retrovirus human immunodeficiency virus (HIV), is the leading cause of death of economically active people (age, 15-59 years) in sub-Saharan Africa. The host genetic variability of immune response to HIV and immune reconstitution following initiation of highly active antiretroviral therapy (HAART) is poorly understood. Here we focused on copy number variation of the ß-defensin genes, which have been shown to have anti-HIV activity, and are important chemoattractants for Th17 lymphocytes via the chemokine receptor CCR6. We determined ß-defensin gene copy number for 1002 Ethiopian and Tanzanian patients. We show that higher ß-defensin copy number variation is associated with increased HIV load prior to HAART (P=.005) and poor immune reconstitution following initiation of HAART (P=.003). We suggest a model where variable amounts of ß-defensin expression by mucosal cells, due to gene copy number variation, alters the efficacy of recruitment of Th17 lymphocytes to the site of infection, altering the dynamics of infection.

Evolutionary history of copy-number-variable locus for the low-affinity Fcγ receptor: mutation rate, autoimmune disease, and the legacy of helminth infection.

Both sequence variation and copy-number variation (CNV) of the genes encoding receptors for immunoglobulin G (Fcγ receptors) have been genetically and functionally associated with a number of autoimmune diseases. However, the molecular nature and evolutionary context of this variation is unknown. Here, we describe the structure of the CNV, estimate its mutation rate and diversity, and place it in the context of the known functional alloantigen variation of these genes. Deletion of Fcγ receptor IIIB, associated with systemic lupus erythematosus, is a result of independent nonallelic homologous recombination events with a frequency of approximately 0.1%. We also show that pathogen diversity, in particular helminth diversity, has played a critical role in shaping the functional variation at these genes both between mammalian species and between human populations. Positively selected amino acids are involved in the interaction with IgG and include some amino acids that are known polymorphic alloantigens in humans. This supports a genetic contribution to the hygiene hypothesis, which states that past evolution in the context of helminth diversity has left humans with an array of susceptibility alleles for autoimmune disease in the context of a helminth-free environment. This approach shows the link between pathogens and autoimmune disease at the genetic level and provides a strategy for interrogating the genetic variation underlying autoimmune-disease risk and infectious-disease susceptibility.

A worldwide analysis of beta-defensin copy number variation suggests recent selection of a high-expressing DEFB103 gene copy in East Asia.

Beta-defensins are a family of multifunctional genes with roles in defense against pathogens, reproduction, and pigmentation. In humans, six beta-defensin genes are clustered in a repeated region which is copy-number variable (CNV) as a block, with a diploid copy number between 1 and 12. The role in host defense makes the evolutionary history of this CNV particularly interesting, because morbidity due to infectious disease is likely to have been an important selective force in human evolution, and to have varied between geographical locations. Here, we show CNV of the beta-defensin region in chimpanzees, and identify a beta-defensin block in the human lineage that contains rapidly evolving noncoding regulatory sequences. We also show that variation at one of these rapidly evolving sequences affects expression levels and cytokine responsiveness of DEFB103, a key inhibitor of influenza virus fusion at the cell surface. A worldwide analysis of beta-defensin CNV in 67 populations shows an unusually high frequency of high-DEFB103-expressing copies in East Asia, the geographical origin of historical and modern influenza epidemics, possibly as a result of selection for increased resistance to influenza in this region.

Determination of beta-defensin genomic copy number in different populations: a comparison of three methods.

BACKGROUND: There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and ß-defensins and Crohn's disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with disease. At present, real-time quantitative PCR (QPCR) is the most commonly used method to determine gene copy number, however the Paralogue Ratio Test (PRT) is being used in more and more laboratories. FINDINGS: In this study we compare a Pyrosequencing-based Paralogue Ratio Test (PPRT) for determining beta-defensin gene copy number with two currently used methods for gene copy number determination, QPCR and triplex PRT by typing five different cohorts (UK, Danish, Portuguese, Ghanaian and Czech) of DNA from a total of 576 healthy individuals. We found a systematic measurement bias between DNA cohorts revealed by QPCR, but not by the PRT-based methods. Using PRT, copy number ranged from 2 to 9 copies, with a modal copy number of 4 in all populations. CONCLUSIONS: QPCR is very sensitive to quality of the template DNA, generating systematic biases that could produce false-positive or negative disease associations. Both triplex PRT and PPRT do not show this systematic bias, and type copy number within the correct range, although triplex PRT appears to be a more precise and accurate method to type beta-defensin copy number.

Age-related accumulation of mutations supports a replication-dependent mechanism of spontaneous mutation at tandem repeat DNA Loci in mice.

Expanded simple tandem repeat (ESTR) loci belong to the class of highly unstable loci in the mouse genome. The mechanisms underlying the very high spontaneous instability at these loci still remain poorly understood. Using single-molecule polymerase chain reaction, here we have compared the pattern of mutation accumulation in tissues with different proliferation capacities in male mice of age 12, 26, 48, and 96 weeks. In the nonproliferating brain, we did not observe any measurable age-related accumulation of ESTR mutations. In contrast, a highly elevated frequency of ESTR mutation was detected in the sperm samples taken from old mice; similar changes were also observed in the bone marrow tissue. The spectra of ESTR mutations accumulated in all tissues of young and old mice did not significantly differ. Taken together, these data clearly imply that spontaneous ESTR mutations occur almost exclusively in replication-proficient cells. To gain further insights into the mechanisms of ESTR mutation, we developed a stochastic model of age-related mutation accumulation. The observed spectra of ESTR mutants accumulated in the brain and sperm were fairly accurately approximated assuming the values of ESTR mutation rate, ranging from 0.01 to 0.04 per cell division. As these estimates dramatically exceed those for protein-coding genes and microsatellite loci, our data therefore suggest that ESTRs represent one of the most unstable loci in the mammalian genome. The results of our study also imply that ESTR loci can be regarded as a class of expanded microsatellites, with the mechanism of spontaneous mutation most probably attributed to replication slippage.

The effects of in utero irradiation on mutation induction and transgenerational instability in mice.

Epidemiological evidence suggests that the deleterious effects of prenatal irradiation can manifest during childhood, resulting in an increased risk of leukaemia and solid cancers after birth. However, the mechanisms underlying the long-term effects of foetal irradiation remain poorly understood. This study was designed to analyse the impact of in utero irradiation on mutation rates at expanded simple tandem repeat (ESTR) DNA loci in directly exposed mice and their first-generation (F(1)) offspring. ESTR mutation frequencies in the germline and somatic tissues of male and female mice irradiated at 12 days of gestation remained highly elevated during adulthood, which was mainly attributed to a significant increase in the frequency of singleton mutations. The prevalence of singleton mutations in directly exposed mice suggests that foetal irradiation results in genomic instability manifested both in utero and during adulthood. The frequency of ESTR mutation in the F(1) offspring of prenatally irradiated male mice was equally elevated across all tissues, which suggests that foetal exposure results in transgenerational genomic instability. In contrast, maternal in utero exposure did not affect the F(1) stability. Our data imply that the passive erasure of epigenetic marks in the maternal genome can diminish the transgenerational effects of foetal irradiation and therefore provide important clues to the still unknown mechanisms of radiation-induced genomic instability. The results of this study offer a plausible explanation for the effects of in utero irradiation on the risk of leukaemia and solid cancers after birth.