Implementation of dihydropyrimidine dehydrogenase deficiency testing in Europe.

BACKGROUND: The main cause for fluoropyrimidine-related toxicity is deficiency of the metabolizing enzyme dihydropyrimidine dehydrogenase (DPD). In 2020, the European Medicines Agency (EMA) recommended two methods for pre-treatment DPD deficiency testing in clinical practice: phenotyping using endogenous uracil concentration or genotyping for DPYD risk variant alleles. This study assessed the DPD testing implementation status in Europe before (2019) and after (2021) the release of the EMA recommendations. METHODS: The survey was conducted from 16 March 2022 to 31 July 2022. An electronic form with seven closed and three open questions was e-mailed to 251 professionals with DPD testing expertise of 34 European countries. A descriptive analysis was conducted. RESULTS: We received 79 responses (31%) from 23 countries. Following publication of the EMA recommendations, 87% and 75% of the countries reported an increase in the amount of genotype and phenotype testing, respectively. Implementation of novel local guidelines was reported by 21 responders (27%). Countries reporting reimbursement of both tests increased in 2021, and only four (18%) countries reported no coverage for any testing type. In 2019, major implementation drivers were 'retrospective assessment of fluoropyrimidine-related toxicity' (39%), and in 2021, testing was driven by 'publication of guidelines' (40%). Although the major hurdles remained the same after EMA recommendations-'lack of reimbursement' (26%; 2019 versus 15%; 2021) and 'lack of recognizing the clinical relevance by medical oncologists' (25%; 2019 versus 8%; 2021)-the percentage of specialists citing these decreased. Following EMA recommendations, 25% of responders reported no hurdles at all in the adoption of the new testing practice in the clinics. CONCLUSIONS: The EMA recommendations have supported the implementation of DPD deficiency testing in Europe. Key factors for successful implementation were test reimbursement and clear clinical guidelines. Further efforts to improve the oncologists' awareness of the clinical relevance of DPD testing in clinical practice are needed.

An SSP-PCR method for the rapid detection of disease-associated alleles HLA-A*29 and HLA-B*51.

HLA-A*29 and HLA-B*51 are associated with birdshot uveitis and Behçet's disease, respectively, and are used as a diagnostic criterion in patients with suspected disease, requiring their detection in diagnostic laboratories. While commercial tests for individual HLA alleles are available for other disease-associated HLA variants, no similar allele-specific assays are available for HLA-A*29 and HLA-B*51. Here, we report sequence-specific priming-polymerase chain reaction (SSP-PCR) methods for the detection of HLA-A*29 and HLA-B*51 using a single PCR reaction per allele. The assays were tested in 30 and 32 previously HLA-typed samples, respectively, representing >97% of HLA-A alleles and >93% of HLA-B alleles in a European population. A concordance of 100% was observed with previous typing results, validating these methods for use in a diagnostic or research context.

Novel Genetic Variants in Carboxylesterase 1 Predict Severe Early-Onset Capecitabine-Related Toxicity.

An important concern with the anticancer drug capecitabine (Cp), an oral prodrug of 5-fluorouracil, are dose-limiting adverse effects, in particular hand-foot syndrome (HFS) and diarrhea. Here we evaluated the association of genetic variability in all enzymes of the Cp-activation pathway to 5-fluorouracil with Cp-related early-onset toxicity in 144 patients receiving Cp. We identified a haplotype encompassing five variants in the carboxylesterase 1 (CES1) gene region including an expression quantitative trait locus associated with early-onset Cp-toxicity (Haplotype A3: ORadditive = 2.2, 95% CI 1.2-4.0, Padjusted = 0.012; ORrecessive = 10.3, 95% CI 2.1-49.4, Padjusted = 0.0038). Furthermore, the association of two linked cytidine deaminase (CDA) promoter variants (c.1-451C>T: ORdominant = 4.3, 95% CI 1.3-14.2, Padjusted = 0.017; and c.1-92A>G: ORdominant = 4.4, 95% CI 1.3-14.5, Padjusted = 0.015) with Cp-related diarrhea was replicated. This first study identifying an association of genetic variation in CES1 with Cp-related toxicity provides further evidence for the existence of a functional noncoding CES1-variant with a possible regulatory impact.

The impact of ABCC11 polymorphisms on the risk of early-onset fluoropyrimidine toxicity.

A missense variant (c.1637C>T, T546M) in ABCC11 encoding the MRP8 (multidrug resistance protein 8), a transporter of 5-fluorodeoxyuridine monophosphate, has been associated with an increased risk of 5-fluorouracil-related severe leukopenia. To validate this association, we investigated the impact of the ABCC11 variants c.1637C>T, c.538G>A and c.395+1087C>T on the risk of early-onset fluoropyrimidine-related toxicity in 514 cancer patients. The ABCC11 variant c.1637C>T was strongly associated with severe leukopenia in patients carrying risk variants in DPYD, encoding the key fluoropyrimidine-metabolizing enzyme dihydropyrimidine dehydrogenase (odds ratio (OR): 71.0; 95% confidence interval (CI): 2.5-2004.8; Pc.1637C>T*DPYD=0.013). In contrast, in patients without DPYD risk variants, no association with leukopenia (OR: 0.95; 95% CI: 0.34-2.6) or overall fluoropyrimidine-related toxicity (OR: 1.02; 95% CI: 0.5-2.1) was observed. Our study thus suggests that c.1637C>T affects fluoropyrimidine toxicity to leukocytes particularly in patients with high drug exposure, for example, because of reduced fluoropyrimidine catabolism.

High prevalence of hereditary thrombotic thrombocytopenic purpura in central Norway: from clinical observation to evidence.

UNLABELLED: Essentials The population prevalence of hereditary thrombotic thrombocytopenic purpura (TTP) is unknown. We studied the prevalence of hereditary TTP and population frequencies of two ADAMTS-13 mutations. A high frequency of hereditary TTP related to ADAMTS-13 mutation c.4143_4144dupA was found. Vicinity of ABO blood group and ADAMTS-13 loci may facilitate screening of ADAMTS-13 mutations. SUMMARY: Background Hereditary thrombotic thrombocytopenic purpura (TTP) caused by ADAMTS-13 mutations is a rare, but serious condition. The prevalence is unknown, but it seems to be high in Norway. Objectives To identify all patients with hereditary TTP in central Norway and to investigate the prevalence of hereditary TTP and the population frequencies of two common ADAMTS-13 mutations. Patients/Methods Patients were identified in a cross-sectional study within the Central Norway Health Region by means of three different search strategies. Frequencies of ADAMTS-13 mutations, c.4143_4144dupA and c.3178 C>T (p.R1060W), were investigated in a population-based cohort (500 alleles) and in healthy blood donors (2104 alleles) by taking advantage of the close neighborhood of the ADAMTS-13 and ABO blood group gene loci. The observed prevalence of hereditary TTP was compared with the rates of ADAMTS-13 mutation carriers in different geographical regions. Results We identified 11 families with hereditary TTP in central Norway during the 10-year study period. The prevalence of hereditary TTP in central Norway was 16.7 × 10(-6) persons. The most prevalent mutation was c.4143_4144dupA, accounting for two-thirds of disease causing alleles among patients and having an allelic frequency of 0.33% in the central, 0.10% in the western, and 0.04% in the southeastern Norwegian population. The allelic frequency of c.3178 C>T (p.R1060W) in the population was even higher (0.3-1%), but this mutation was infrequent among patients, with no homozygous cases. Conclusions We found a high prevalence of hereditary TTP in central Norway and an apparently different penetrance of ADAMTS-13 mutations.

Allopurinol hypersensitivity is primarily mediated by dose-dependent oxypurinol-specific T cell response.

BACKGROUND: Allopurinol is a main cause of severe cutaneous adverse reactions (SCAR). How allopurinol induces hypersensitivity remains unknown. Pre-disposing factors are the presence of the HLA-B*58:01 allele, renal failure and possibly the dose taken. OBJECTIVE: Using an in vitro model, we sought to decipher the relationship among allopurinol metabolism, HLA-B*58:01 phenotype and drug concentrations in stimulating drug-specific T cells. METHODS: Lymphocyte transformation test (LTT) results of patients who had developed allopurinol hypersensitivity were analysed. We generated allopurinol or oxypurinol-specific T cell lines (ALP/OXP-TCLs) from allopurinol naïve HLA-B*58:01(+) and HLA-B*58:01(-) individuals using various drug concentrations. Their reactivity patterns were analysed by flow cytometry and (51) Cr release assay. RESULTS: Allopurinol allergic patients are primarily sensitized to oxypurinol in a dose-dependent manner. TCL induction data show that both the presence of HLA-B*58:01 allele and high concentration of drug are important for the generation of drug-specific T cells. The predominance of oxypurinol-specific lymphocyte response in allopurinol allergic patients can be explained by the rapid conversion of allopurinol to oxypurinol in vivo rather than to its intrinsic immunogenicity. OXP-TCLs do not recognize allopurinol and vice versa. Finally, functional avidity of ALP/OXP-TCL is dependent on both the induction dose and HLA-B*58:01 status. CONCLUSIONS AND CLINICAL RELEVANCE: This study establishes the important synergistic role of drug concentration and HLA-B*58:01 allele in the allopurinol or oxypurinol-specific T cell responses. Despite the prevailing dogma that Type B adverse drug reactions are dose independent, allopurinol hypersensitivity is primarily driven by oxypurinol-specific T cell response in a dose-dependent manner, particular in the presence of HLA-B*58:01 allele.

Gender-specific elimination of continuous-infusional 5-fluorouracil in patients with gastrointestinal malignancies: results from a prospective population pharmacokinetic study.

BACKGROUND: This study was initiated to assess the quantitative impact of patient anthropometrics and dihydropyrimidine dehydrogenase (DPYD) mutations on the pharmacokinetics (PK) of 5-fluorouracil (5FU) and to explore limited sampling strategies of 5FU. PATIENTS AND METHODS: We included 32 patients with gastrointestinal malignancies, receiving 46-h continuous-infusional 5FU and performed PK-sampling at baseline, 15, 30, 45 min, 1 and 2 h after the start of infusion and at the end of infusion, for 2 subsequent cycles. Plasma concentrations of 5FU, 5-fluorodihydrouracil (5FUH2), uracil (U) and 5,6-dihydrouracil (UH2) were determined using LC-MS/MS and submitted to population PK analysis using nonlinear mixed-effects modeling. Broad genotyping of DPYD was performed, and the potential impact of the DPYD genotype on the elimination of 5FU was assessed. Limited sampling strategies were evaluated for their accuracy to predict steady-state concentrations of 5FU (CSS(5FU)), using data simulations based on the final PK-model. RESULTS: The area-under-the concentration-time curve of 5FU (AUC(5FU)) was found to be <20 mg h/L in 33 occasions (58 %), between 20 and 30 mg h/L in 17 occasions (30 %) and >30 mg h/L in 7 occasions (12 %). Men had a 26 % higher elimination of 5FU and a 18 % higher apparent elimination of 5FUH2. Accordingly, women had a higher AUC(5FU) compared to men (22 vs. 18 mg h/L, p = 0.04). No DPYD risk variants were found, and the DPYD variants detected (c.496A>G, c.1601G>A, c.1627A>G) were not significantly associated with the elimination of 5FU. Individual baseline UH(2)/U ratio was significantly associated with AUC(5FU) (R = -0.49, p < 0.001). Limited sampling strategies with time-points <3 h after the start of infusion were not adequate to predict CSS(5FU). Female gender was the only predictor of nausea/emesis in the multivariate model. CONCLUSIONS: Gender-specific elimination of 5FU is supported by the present data and may partly explain the gender-specific association between DPYD risk variants and 5FU-specific toxicity.

Eutrophication causes speciation reversal in whitefish adaptive radiations.

Species diversity can be lost through two different but potentially interacting extinction processes: demographic decline and speciation reversal through introgressive hybridization. To investigate the relative contribution of these processes, we analysed historical and contemporary data of replicate whitefish radiations from 17 pre-alpine European lakes and reconstructed changes in genetic species differentiation through time using historical samples. Here we provide evidence that species diversity evolved in response to ecological opportunity, and that eutrophication, by diminishing this opportunity, has driven extinctions through speciation reversal and demographic decline. Across the radiations, the magnitude of eutrophication explains the pattern of species loss and levels of genetic and functional distinctiveness among remaining species. We argue that extinction by speciation reversal may be more widespread than currently appreciated. Preventing such extinctions will require that conservation efforts not only target existing species but identify and protect the ecological and evolutionary processes that generate and maintain species.

Patterns of morphological changes and hybridization between sympatric whitefish morphs (Coregonus spp.) in a Swiss lake: a role for eutrophication?

Whitefish, genus Coregonus, show exceptional levels of phenotypic diversity with sympatric morphs occurring in numerous postglacial lakes in the northern hemisphere. Here, we studied the effects of human-induced eutrophication on sympatric whitefish morphs in the Swiss lake, Lake Thun. In particular, we addressed the questions whether eutrophication (i) induced hybridization between two ecologically divergent summer-spawning morphs through a loss of environmental heterogeneity, and (ii) induced rapid adaptive morphological changes through changes in the food web structure. Genetic analysis based on 11 microsatellite loci of 282 spawners revealed that the pelagic and the benthic morph represent highly distinct gene pools occurring at different relative proportions on all seven known spawning sites. Gill raker counts, a highly heritable trait, showed nearly discrete distributions for the two morphs. Multilocus genotypes characteristic of the pelagic morph had more gill rakers than genotypes characteristic of benthic morph. Using Bayesian methods, we found indications of recent but limited introgressive hybridization. Comparisons with historical gill raker data yielded median evolutionary rates of 0.24 haldanes and median selection intensities of 0.27 for this trait in both morphs for 1948-2004 suggesting rapid evolution through directional selection at this trait. However, phenotypic plasticity as an alternative explanation for this phenotypic change cannot be discarded. We hypothesize that both the temporal shifts in mean gill raker counts and the recent hybridization reflect responses to changes in the trophic state of the lake induced by pollution in the 1960s, which created novel selection pressures with respect to feeding niches and spawning site preferences.

Divergence along a steep ecological gradient in lake whitefish (Coregonus sp.).

To understand mechanisms structuring diversity in young adaptive radiations, quantitative and unbiased information about genetic and phenotypic diversity is much needed. Here, we present the first in-depth investigation of whitefish diversity in a Swiss lake, with continuous spawning habitat sampling in both time and space. Our results show a clear cline like pattern in genetics and morphology of populations sampled along an ecological depth gradient in Lake Neuchâtel. Divergent natural selection appears to be involved in shaping this cline given that trait specific P(ST)-values are significantly higher than F(ST)-values when comparing populations caught at different depths. These differences also tend to increase with increasing differences in depth, indicating adaptive divergence along a depth gradient, which persists despite considerable gene flow between adjacent demes. It however remains unclear, whether the observed pattern is a result of currently stable selection-gene flow balance, incipient speciation, or reverse speciation due to anthropogenic habitat alteration causing two formerly divergent species to collapse into a single gene pool.

How normal is abnormal? Discrimination between deformations and natural variation in gonad morphology of European whitefish Coregonus lavaretus.

The gonad morphology of whitefish Coregonus lavaretus collected in Lake Thun, Switzerland, and two neighbouring lakes was assessed in order to differentiate between 'normal' and 'abnormal' character states of gonad morphology, which had been previously described in C. lavaretus from Lake Thun (constrictions, asymmetries, aplasia, compartmentations, fusions and hermaphroditism). In total, 4668 fish were collected and analysed using two complementary sampling schemes: (1) monthly samples of catches by the commercial fishermen and (2) samples of ripe spawners of all known 33 spawning sites of the three lakes. Considerable variation in gonad morphology in C. lavaretus populations of all lakes was found. Notably, all deviation types were observed in fish of all three lakes. Asymmetries and constrictions were frequent in all three lakes and showed systematic differences in frequency between the two sampling strategies. This indicates that asymmetries and constrictions represent to a large extent natural variation in gonad morphology of C. lavaretus and are also prone to considerable measurement error. In contrast, aplasia, fusions, compartmentations and hermaphroditism occurred predominantly in one C. lavaretus form of Lake Thun and in particular in populations spawning at great depths. This suggests that these deviation types are probably reliable indicators for gonad deformations and supports the interpretation that Lake Thun harbours a unique case of deformed gonads in C. lavaretus of yet unknown origin.

Phylogeography of bleaks Alburnus spp. (Cyprinidae) in Italy, based on cytochrome b data.

Sequence variation of a fragment of the mitochondrial DNA encoding for the cytochrome b gene was used to reconstruct the phylogeography of the two species of bleaks occurring in Italy: the alborella Alburnus arborella in northern Italy and the vulturino Alburnus albidus in southern Italy. The study includes four populations of the alborella and 14 populations of the vulturino. A total of 57 haplotypes were identified; these could not be sorted into two reciprocally monophyletic clusters. Multiple phylogenetic methods and nested clade phylogeographical analysis consistently retrieved three well-supported clades, two of which contained both Northern and Southern Italian haplotypes. A third clade is limited to southern Italy. This clade is tentatively assigned to the vulturino. The placement in the same clade of northern and southern Italian haplotypes is explained in light of the introductions of fishes operated from northern to central and southern Italy. The origin of the vulturino dates back to the last two million years. This divergence time estimate identifies the Pleistocene confluences between adjacent river basins along the Adriatic slope of the Italian peninsula and their subsequent isolation as the cause that triggered the diversification of the genus in the area. The existence of a clade endemic to southern Italy supports the recognition of the area as a new peri-Mediterranean ichthyogeographic district, the borders of which correspond to the northern and southern edges of the vulturino range.

Genetic analysis of potential postglacial watershed crossings in Central Europe by the bullhead (Cottus gobio L.).

Natural colonizations across watersheds have been frequently proposed to explain the present distributions of many freshwater fish species. However, detailed studies of such potential watershed crossings are still missing. Here, we investigated potential postglacial watershed crossings of the widely distributed European bullhead (Cottus gobio L.) in two different areas along the Rhine-Rhône watershed using detailed genetic analysis. The main advantage of studying bullheads vs. other freshwater fish species is that their distribution has been lightly influenced by human activities and as such, interpretations of colonization history are not confounded by artificial transplantations. The genetic analyses of eight microsatellite loci revealed strong genetic similarities between populations of both sides of the Rhine-Rhône watershed in the Lake Geneva area, giving strong evidence for a natural watershed crossing of bullheads from the upper Rhine drainage into the Rhône drainage in the Lake Geneva area likely facilitated by the retreat of the glaciers after the last glacial maximum some 20,000 years ago. Populations from the Lake Geneva basin were genetically more similar to populations from across the watershed in the upper Rhine drainage than to populations further downstream in the lower Rhône. In contrast, populations from Belfort, an area, which was not covered by ice during the last glacial maximum, showed strong genetic differentiation between populations of the upper Rhine and Rhône drainages. Based on our results on the bullhead, we propose that glacial retreat may have eased the dispersal of numerous European freshwater fish species across several geological boundaries.

Novel mutation in OTC gene causes neonatal death in twin brothers.

Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of the urea cycle. OTC locus is located in the short arm of X-chromosome. Authors report a case of a woman who gave birth to monozygotic male twins who later died because of severe neonatal-onset hyperammonaemic encephalopathy caused by a novel mutation of OTC gene. Post-mortem liver biopsy was taken from the second twin; afterwards, blood was drawn from the mother for examination. DNA sequence data showed that the mother was a carrier of the same novel mutation that was previously detected in the case of her son. In OTC deficiency, detection of female carriers is important for genetic counselling and eventual prenatal diagnosis.

Estimation of effective population size and detection of a recent population decline coinciding with habitat fragmentation in a ground beetle.

We assess the impact of habitat fragmentation on the effective size (N(e)) of local populations of the flightless ground beetle Carabus violaceus in a small (<25 ha) and a large (>80 ha) forest fragment separated by a highway. N(e) was estimated based on the temporal variation of allele frequencies at 13 microsatellite loci using two different methods. In the smaller fragment, N(e) estimates ranged between 59 and a few hundred, whereas values between 190 and positive infinity were estimated for the larger fragment. In both samples, we detected a signal of population decline, which was stronger in the small fragment. The estimated time of onset of this N(e) reduction was consistent with the hypothesis that recent road constructions have divided a continuous population into several isolated subpopulations. In the small fragment, N(e) of the local population may be so small that its long-term persistence is endangered.

Recent habitat fragmentation due to roads can lead to significant genetic differentiation in an abundant flightless ground beetle.

Although habitat fragmentation is suspected to pose a major threat to biodiversity, its impact on abundant invertebrate species has been little investigated. We assessed the genetic population structure of the flightless ground beetle Abax parallelepipedus in a forest fragmented by two main roads and a highway using five microsatellite loci. We detected low levels of genetic differentiation, which was concordant with the high population densities of 632-1707 individuals/ha estimated with a mark-recapture method. A Mantel test detected a highly significant increase of pairwise F(ST)-values with the number of roads between sampling locations. As expected, the most pronounced effect of the isolation due to roads was observed in the sample from the smallest fragment (highway exit loop), which was differentiated significantly from most other locations. However, no signs of a recent bottleneck or a loss of genetic variability were detected in this population, indicating a still relatively large effective population size (N(e)). Computer simulations confirmed that the observed F(ST)-values were indeed compatible with a N(e) of a few hundred individuals in this fragment, assuming strong or absolute isolation since the construction of the roads. We discuss the implications of our findings for the conservation of abundant but poorly dispersing species in fragmented habitats.

The city-fox phenomenon: genetic consequences of a recent colonization of urban habitat.

The red fox (Vulpes vulpes) is one of the best-documented examples of a species that has successfully occupied cities and their suburbs during the last century. The city of Zurich (Switzerland) was colonized by red foxes 15 years ago and the number of recorded individuals has increased steadily since then. Here, we assessed the hypothesis that the fox population within the city of Zurich is isolated from adjacent rural fox populations against the alternative hypothesis that urban habitat acts as a constant sink for rural dispersers. We examined 11 microsatellite loci in 128 foxes from two urban areas, separated by the main river crossing the city, and three adjacent rural areas from the region of Zurich. Mean observed heterozygosity across individuals and the number of detected alleles were lower for foxes collected within the city as compared with their rural conspecifics. Genetic differentiation was significantly lower between rural than between rural and urban populations, and highest value of pairwise FST was recorded between the two urban areas. Our results indicate that the two urban areas were independently founded by a small number of individuals from adjacent rural areas resulting in genetic drift and genetic differentiation between rural and urban fox populations. Population admixture and immigration analysis revealed that urban-rural gene flow was higher than expected from FST statistics. In the five to seven generations since colonization, fox density has dramatically increased. Currently observed levels of migration between urban and rural populations will probably erode genetic differentiation over time.

Inbreeding and developmental stability in three-spined sticklebacks (Gasterosteus aculeatus L.).

Fluctuating asymmetry, small non-directional departures from perfect symmetry in bilateral traits, results from the inability of individuals to buffer development against genetic and environmental perturbations. Fluctuating asymmetry is a widely used measure of developmental stability, and developmental stability has been hypothesised to be inversely related to heterozygosity. We compared male three-spined sticklebacks (Gasterosteus aculeatus L.) that had been inbred for one generation to outbred control males with respect to the asymmetry of a set of bilateral morphometric traits. Inbred fish developed significantly more asymmetric pectoral fins than their outbred counterparts, whereas neither the magnitude of asymmetry for pelvic spines nor for gill covers significantly responded to the treatment. Our results conform to a pattern of heterogeneity amongst traits in their tendency to develop asymmetrically in response to stress.

Mesobuthus gibbosus (Scorpiones: Buthidae) on the island of Rhodes -- hybridization between Ulysses' stowaways and native scorpions?

Allozyme data suggest that the Rhodes population of Mesobuthus gibbosus is a hybrid population of recent origin. Namely, it is a mixture between an autochthonous population and an artificially introduced population probably from the Greek mainland. All samples were mainly composed of F1 hybrid genotypes and genotypes either fixed for autochthonous or introduced alleles. Back-cross hybrid genotypes were very rare. Mitochondrial DNA analysis, in contrast, revealed only one group of closely related haplotypes that are unique for the Rhodes populations, thus suggesting asymmetric introgression of the two marker classes.

Genetic analysis of sneaking and egg-thievery in a natural population of the three-spined stickleback (Gasterosteus aculeatus L.).

Among the fish species that show exclusive male parental care, the three-spined stickleback represents one of the most intensively studied species with regard to reproductive behaviour. In this species, the most common 'parasitic' male tactics in relation to male reproductive behaviour are sneaking and egg thievery, which are often collectively referred to as nest-raiding. However, little is known about the genetic consequences of sneaking and egg thievery in natural populations. Here we assessed the frequency of sneaking and egg-stealing in a natural population, male traits that are associated with the victims of sneaking, and the impact of sneaking and egg-stealing on the reproductive success of nesting males as deduced from the number of offspring in their nests. Fourteen nest-guarding males and a random sample of about 100 eggs/fry of each nest from a natural freshwater population of three-spined sticklebacks were analysed at three microsatellite loci. The analysis revealed a high frequency of genetically successful nest raiding (sneaking or egg thievery), i.e. more than half (57%) of the 14 nests contained offspring (1-94%) which were unrelated to the guardian male. Three of the 14 nests (21%) contained progeny of sneaking males and four of the nests (28%) contained offspring which were unrelated to the guardian male and which probably originated from egg-stealing events. Victims of sneaking were significantly smaller than other guardian males. Moreover, reproductive success correlated positively with male body size.