Recombinant Phenotypes Research Articles

Successful ganciclovir treatment of primary cytomegalovirus infection containing the UL97 mutation N510S in an intestinal graft recipient

In solid organ transplantation, human cytomegalovirus (HCMV) is considered to be the most important viral pathogen. We report a case of a CMV R-/D+ small intestine transplant recipient with a primary CMV infection on valganciclovir prophylaxis. Sequencing of the HCMV DNA for drug resistance-associated mutations revealed the UL97 mutation N510S. This mutation has been initially reported to confer ganciclovir resistance. Based on in vitro recombinant phenotyping, this assumption has recently been questioned. Switching the antiviral treatment to an intravenous regimen of ganciclovir eliminated HCMV DNAemia, showing the in vivo efficacy of ganciclovir for the UL97 mutation N510S. Hence, knowledge of drug efficacy is crucial for an adequate choice of antiviral medication, carefully balancing antiviral potency versus the risk of harmful side effects.

Drug Resistance due to Mutation in UL 97 and Certain Other Specific Genes in Cytomegalovirus Strains: An Overview

The study of human cytomegalovirus (HCMV) antiviral drug resistance has enhanced knowledge of the virological targets and the mechanisms of antiviral activity. The currently approved drugs, ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV), target the viral DNA polymerase. The widespread use of ganciclovir (GCV) to treat cytomegalovirus (CMV) infections in immuno-suppressed patients has led to the development of drug resistance.GCV anabolism also requires phosphorylation by the virus-encoded UL97 kinase. GCV resistance mutations have been identified in both genes, while FOS and CDV mutations occur only in the DNA polymerase gene. Confirmation of resistance mutations requires genotypic assays based on sequencing provide more rapid results but are dependent on prior validation by phenotypic methods. Recombinant phenotyping methods performed in a few research laboratories have resolved some of the conflicting results. Treatment options for drug-resistant HCMV infections are complex and have not been subjected to controlled clinical trials, although consensus guidelines have been proposed.

Transgressive Hybrids as Hopeful Monsters

The origin of novelty is a critical subject for evolutionary biologists. Early geneticists speculated about the sudden appearance of new species via special macromutations, epitomized by Goldschmidt’s infamous “hopeful monster”. Although these ideas were easily dismissed by the insights of the Modern Synthesis, a lingering fascination with the possibility of sudden, dramatic change has persisted. Recent work on hybridization and gene exchange suggests an underappreciated mechanism for the sudden appearance of evolutionary novelty that is entirely consistent with the principles of modern population genetics. Genetic recombination in hybrids can produce transgressive phenotypes, “monstrous” phenotypes beyond the range of parental populations. Transgressive phenotypes can be products of epistatic interactions or additive effects of multiple recombined loci. We compare several epistatic and additive models of transgressive segregation in hybrids and find that they are special cases of a general, classic quantitative genetic model. The Dobzhansky-Muller model predicts “hopeless” monsters, sterile and inviable transgressive phenotypes. The Bateson model predicts “hopeful” monsters with fitness greater than either parental population. The complementation model predicts both. Transgressive segregation after hybridization can rapidly produce novel phenotypes by recombining multiple loci simultaneously. Admixed populations will also produce many similar recombinant phenotypes at the same time, increasing the probability that recombinant “hopeful monsters” will establish true-breeding evolutionary lineages. Recombination is not the only (or even most common) process generating evolutionary novelty, but might be the most credible mechanism for sudden appearance of new forms.

Epigenetic functions enriched in transcription factors binding to mouse recombination hotspots

The regulatory mechanism of recombination is a fundamental problem in genomics, with wide applications in genome-wide association studies, birth-defect diseases, molecular evolution, cancer research, etc. In mammalian genomes, recombination events cluster into short genomic regions called "recombination hotspots". Recently, a 13-mer motif enriched in hotspots is identified as a candidate cis-regulatory element of human recombination hotspots; moreover, a zinc finger protein, PRDM9, binds to this motif and is associated with variation of recombination phenotype in human and mouse genomes, thus is a trans-acting regulator of recombination hotspots. However, this pair of cis and trans-regulators covers only a fraction of hotspots, thus other regulators of recombination hotspots remain to be discovered. In this paper, we propose an approach to predicting additional trans-regulators from DNA-binding proteins by comparing their enrichment of binding sites in hotspots. Applying this approach on newly mapped mouse hotspots genome-wide, we confirmed that PRDM9 is a major trans-regulator of hotspots. In addition, a list of top candidate trans-regulators of mouse hotspots is reported. Using GO analysis we observed that the top genes are enriched with function of histone modification, highlighting the epigenetic regulatory mechanisms of recombination hotspots.

The biology of cytomegalovirus drug resistance

This review assesses recently published data on cytomegalovirus (CMV) antiviral drug resistance. Resistance is typically encountered after prolonged ganciclovir treatment for posttransplant primary CMV infection and is diagnosed by the detection of characteristic mutations in the viral UL97 kinase and UL54 DNA polymerase genes in clinical specimens. One of seven canonical UL97 mutations is detected in most cases of ganciclovir resistance, but many viral sequence variants of unknown relevance are being reported after drug exposure in vitro and in vivo. Rapid technical advances in recombinant phenotyping have shown that many of these variants confer no detectable drug resistance, whereas some unusual resistance mutations are newly confirmed. All currently marketed CMV antiviral drugs, including foscarnet and cidofovir, target the viral DNA polymerase, and cross-resistance may result from some UL54 mutations. To decrease cross-resistance and toxicity, there is an ongoing effort to develop anti-CMV drugs with different resistance pathways and alternative targets, such as the UL97 kinase or UL56-UL89 terminase enzymes. An increasing volume of information correlating CMV genotypes and drug susceptibility phenotypes is becoming available. This will improve the interpretation of sequence-based assays currently used for clinical diagnosis and guide the development of new antiviral drugs.

The Genetic Basis of Human Cytomegalovirus Resistance and Current Trends in Antiviral Resistance Analysis

Infections due to resistant human cytomegalovirus (CMV) are an emerging problem, particularly in immunocompromised hosts. When managing such patients, clinicians should be aware of the possibility of developing CMV antiviral resistance, especially while on prolonged therapy or if severe immunosuppression is present. CMV resistance to current antiviral agents is mediated by alterations in either the UL97 kinase or DNA polymerase, encoded by the UL97 and UL54 genes, respectively. UL97 mutations are capable of conferring resistance to ganciclovir, while UL54 mutations can impart resistance to ganciclovir, cidofovir, and foscarnet. If treatment failure is suspected to be due to antiviral resistance, CMV resistance analysis should be obtained. Phenotypic resistance assays performed on clinical isolates measure antiviral susceptibilities directly, but are laborious and time-consuming. Therefore, genotypic resistance analysis has become the more common means of diagnosing CMV resistance. Mutations in UL97 or UL54 may be clinically associated with resistance, but their effect on antiviral susceptibility must be confirmed by marker transfer techniques such as recombinant phenotyping.

Recombinant Phenotyping of Cytomegalovirus UL54 Mutations That Emerged during Cell Passages in the Presence of either Ganciclovir or Foscarnet

Selection of human cytomegalovirus variants in the presence of ganciclovir or foscarnet led to 18 DNA polymerase mutations, 14 of which had not been previously studied. Using bacterial artificial chromosome technology, each of these mutations was individually transferred into the genome of a reference strain. Following reconstitution of infectious viral stocks, each mutant was assessed for its drug susceptibility and growth kinetics in cell culture. Computer-assisted three-dimensional (3D) modeling of the polymerase was also used to position each of the mutations in one of four proposed structural domains and to predict their influence on structural stability of the protein. Among the 10 DNA polymerase mutations selected with ganciclovir, 7 (P488R, C539R, L545S, V787L, V812L, P829S, and L862F) were associated with ganciclovir resistance, whereas 2 (F595I and V946L) conferred only foscarnet resistance. Among the eight mutations selected with foscarnet, only two (T552N and S585A) conferred foscarnet resistance, whereas four (N408D, K500N, L802V, and L957F) had an impact on ganciclovir susceptibility. Surprisingly, the combination of mutations, some of which were not associated with resistance for a specific antiviral, resulted in increasing resistance effects. 3D modeling suggested that none of the mutated residues were directly involved in the polymerase catalytic site but rather had an influence on drug susceptibility by modifying the structural flexibility of the protein. Our study significantly adds to the number of DNA polymerase mutations conferring in vitro drug resistance and emphasizes the point that evaluation of individual mutations may not accurately reflect the phenotype conferred by multiple mutations.

Variation in human recombination rates and its genetic determinants.

BackgroundDespite the fundamental role of crossing-over in the pairing and segregation of chromosomes during human meiosis, the rates and placements of events vary markedly among individuals. Characterizing this variation and identifying its determinants are essential steps in our understanding of the human recombination process and its evolution.Study Design/ResultsUsing three large sets of European-American pedigrees, we examined variation in five recombination phenotypes that capture distinct aspects of crossing-over patterns. We found that the mean recombination rate in males and females and the historical hotspot usage are significantly heritable and are uncorrelated with one another. We then conducted a genome-wide association study in order to identify loci that influence them. We replicated associations of RNF212 with the mean rate in males and in females as well as the association of Inversion 17q21.31 with the female mean rate. We also replicated the association of PRDM9 with historical hotspot usage, finding that it explains most of the genetic variance in this phenotype. In addition, we identified a set of new candidate regions for further validation.SignificanceThese findings suggest that variation at broad and fine scales is largely separable and that, beyond three known loci, there is no evidence for common variation with large effects on recombination phenotypes.

Recombination phenotypes of the NCI-60 collection of human cancer cells

BackgroundThe NCI-60 is a collection of tumor cell lines derived from a variety of human adult cancer tissue types and is commonly used for genetic analysis and screening of potential chemotherapeutic agents. We wanted to understand the contributions of specific mechanisms of genomic instability to the etiology of cancers represented by the NCI-60.ResultsWe screened the NCI-60 for dysregulated homologous recombination by using the gene cluster instability (GCI) assay we pioneered, and for defects in base excision repair by sensitivity to 5-hydroxymethyl-2'-deoxyuridine (hmdUrd). We identified subsets of the NCI-60 lines that either displayed the characteristic molecular signature of GCI or were sensitive to hmdUrd. With the exception of the NCI-H23 lung cancer line, these phenotypes were not found to overlap. None of the lines examined in either subset exhibited significant changes in the frequency of sister chromatid exchanges (SCE), neither did any of the lines in either subset exhibit microsatellite instability (MSI) indicative of defects in DNA mismatch repair.ConclusionsGene cluster instability, sensitivity to hmdUrd and sister chromatid exchange are mechanistically distinct phenomena. Genomic instability in the NCI-60 appears to involve only one mechanism of instability for each individual cell line.

Phenotypic diversity of cytomegalovirus DNA polymerase gene variants observed after antiviral therapy

Background Cytomegalovirus UL54 DNA polymerase mutations observed in clinical specimens are of diagnostic significance if confirmed to affect antiviral drug susceptibility. Objectives Validate an updated recombinant phenotyping method to determine the degree of drug resistance conferred by previously uncharacterized UL54 sequence variants, in comparison with known resistance-related mutations. Study design Bacterial artificial chromosome clones of viral DNA were mutagenized by recombination, transfected to produce live virus and phenotyped by standardized reporter-based yield reduction assays. Results Sixteen recombinant viruses were constructed, representing baseline sequences, known resistance-related mutations and amino acid changes of unproven significance from clinical specimens. Phenotypes of baseline strains and known mutants were comparable to results from prior methods and helped to resolve some published inconsistencies. Mutations F412L, F412S, L545W were newly confirmed to confer ganciclovir and cidofovir resistance, while Q578H conferred ganciclovir and foscarnet resistance with borderline cidofovir resistance. Some foscarnet-resistant mutants were appreciably growth-retarded. Conclusions Results add to known exonuclease domain mutations that confer ganciclovir-cidofovir cross-resistance, polymerase domain mutations that confer foscarnet resistance with variably decreased ganciclovir/cidofovir susceptibility, and increase the list of sequence variants with no measurable impact on drug susceptibility. The phenotypic diversity of similar UL54 genotypic variants complicates the interpretation of genotypic resistance testing. Technical improvements are facilitating the phenotyping of remaining unknown sequence variants.

Recombinant Phenotyping of Cytomegalovirus Sequence Variants Detected After 200 or 100 Days of Valganciclovir Prophylaxis

In a phase III controlled trial IMproved Protection Against Cytomegalovirus in Transplantation (IMPACT) comparing 200 with 100 days of valganciclovir prophylaxis in 318 cytomegalovirus D+/R- kidney transplant recipients, an equal number of patients (n=3 per arm) had known ganciclovir resistance mutations detected during viral breakthrough. In addition, many other viral sequence variants were observed that were of unknown significance for ganciclovir resistance. Recombinant phenotyping was performed to determine whether the previously uncharacterized genotypic changes affected ganciclovir susceptibility, especially in those receiving the longer duration of prophylaxis. Sequences encoding individual amino acid substitutions in the UL97 kinase or UL54 DNA polymerase gene were transferred by recombination into a cloned cytomegalovirus laboratory strain, followed by reporter-based yield reduction phenotypic assay of the resulting virus for ganciclovir susceptibility. Twenty-six uncharacterized amino acid substitutions were detected, 2 in UL97 and 24 in UL54. All 10 substitutions in the 200-day arm and 9 of 17 substitutions in the 100-day arm (prioritized based on location and conservation) were selected for phenotyping; one substitution was detected in both subsets. Results were generated for nine of ten 200-day and eight of nine 100-day substitutions, with no substitution demonstrating a significant reduction in ganciclovir susceptibility. The two remaining amino acid substitutions, both in UL54, were not evaluated because of poor viral viability. Phenotypic evaluation of previously uncharacterized viral genotypes in the 200-day valganciclovir prophylaxis group showed no evidence of an increased incidence of genotypic ganciclovir resistance when compared with those in the 100-day prophylaxis group.

Effects of varying gene targeting parameters on processing of recombination intermediates by ERCC1–XPF

The ERCC1–XPF structure-specific endonuclease is necessary for correct processing of homologous recombination intermediates requiring the removal of end-blocking nonhomologies. We previously showed that targeting the endogenous CHO APRT locus with plasmids designed to generate such intermediates revealed defective recombination phenotypes in ERCC1 deficient cells, including suppression of targeted insertion and vector correction recombinants and the generation of a novel class of aberrant recombinants through a deletogenic mechanism. In the present study, we examined some of the mechanistic features of ERCC1–XPF in processing recombination intermediates by varying gene targeting parameters. These included altering the distance between the double-strand break (DSB) in the targeting vector and the inactivating mutation in the APRT target gene, and changing the position of the target gene mutation relative to the DSB to result in target mutations that were either upstream or downstream from the DSB. Increasing the distance from the DSB in the targeting vector to the chromosomal target gene mutation resulted in an ERCC1 dependent decrease in the efficiency of gene targeting from intermediates presenting lengthy end-blocking nonhomologies. This decrease was accompanied by a shift in the distribution of recombinant classes away from target gene conversions to targeted insertions in both wild-type and ERCC1 deficient cells, and a dramatic increase in the proportion of aberrant recombinants in ERCC1 deficient cells. Changing the position of the target gene mutation relative to the DSB in the plasmid also altered the distribution of targeted insertion subclasses recovered in wild-type cells, consistent with two-ended strand invasion followed by resolution into crossover-type products and vector integration. Our results confirm expectations from studies of Rad10–Rad1 in budding yeast that ERCC1–XPF activity affects conversion tract length, and provide evidence for the mechanism of generation of the novel, aberrant recombinant class first described in our previous study.

Cytomegalovirus UL97 Mutations Affecting Cyclopropavir and Ganciclovir Susceptibility

Among the 7 most common UL97 mutations encountered in ganciclovir-resistant clinical cytomegalovirus isolates, the associated cyclopropavir cross-resistance varies from insignificant (L595S) to substantial (M460I and H520Q) as determined by recombinant phenotyping. Mutations M460I and H520Q were preferentially selected in vitro under cyclopropavir and conferred 12- to 20-fold increases in 50% effective concentration (EC(50)) values, while M460V, C592G, A594V, and C603W conferred 3- to 5-fold increases. Uncommon mutations M460T and C603R increased cyclopropavir EC(50)s by 8- to 10-fold.

LIFE HISTORY VARIATION IN AN ARTIFICIALLY SELECTED POPULATION OF DROSOPHILA MELANOGASTER: PLEIOTROPY, SUPERFLIES, AND AGE-SPECIFIC ADAPTATION

We measured age-specific fecundity and survival in recombinant inbred lines of Drosophila melanogaster that were derived from an artificial selection experiment for delayed reproduction. Age at peak oviposition is highly heritable (h(2) (B) = 0.55). We find three qualitative categories of peak oviposition: early-, midlife-, and bimodal. Genetic correlations between life span and early fecundity are not significantly different from zero, but correlations with midlife fecundity are positive and statistically significant. Long-lived genotypes exhibit a midlife fecundity peak. There is no evidence for a shift of reproductive effort from early to later stages. The existence of qualitatively recombinant phenotypes, including "superflies" that exhibit both enhanced survival and high levels of early fecundity, argues against the widespread idea that life history evolution in Drosophila is dominated by negative pleiotropy. There is clear evidence of age-specific adaptation in the timing of oviposition.

Antiviral Drug Resistance of Human Cytomegalovirus

The study of human cytomegalovirus (HCMV) antiviral drug resistance has enhanced knowledge of the virological targets and the mechanisms of antiviral activity. The currently approved drugs, ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV), target the viral DNA polymerase. GCV anabolism also requires phosphorylation by the virus-encoded UL97 kinase. GCV resistance mutations have been identified in both genes, while FOS and CDV mutations occur only in the DNA polymerase gene. Confirmation of resistance mutations requires phenotypic analysis; however, phenotypic assays are too time-consuming for diagnostic purposes. Genotypic assays based on sequencing provide more rapid results but are dependent on prior validation by phenotypic methods. Reports from many laboratories have produced an evolving list of confirmed resistance mutations, although differences in interpretation have led to some confusion. Recombinant phenotyping methods performed in a few research laboratories have resolved some of the conflicting results. Treatment options for drug-resistant HCMV infections are complex and have not been subjected to controlled clinical trials, although consensus guidelines have been proposed. This review summarizes the virological and clinical data pertaining to HCMV antiviral drug resistance.

Depletion of Werner helicase results in mitotic hyperrecombination and pleiotropic homologous and nonhomologous recombination phenotypes

Werner syndrome (WS) is a rare, segmental progeroid syndrome caused by defects in the WRN gene, which encodes a RecQ helicase. WRN has roles in many aspects of DNA metabolism including DNA repair and recombination. In this study, we exploited two different recombination assays previously used to describe a role for the structure-specific endonuclease ERCC1-XPF in mitotic and targeted homologous recombination. We constructed Chinese hamster ovary (CHO) cell lines isogenic with the cell lines used in these previous studies by depleting WRN using shRNA vectors. When intrachromosomal, mitotic recombination was assayed in WRN-depleted CHO cells, a hyperrecombination phenotype was observed, and a small number of aberrant recombinants were generated. Targeted homologous recombination was also examined in WRN-depleted CHO cells using a plasmid–chromosome targeting assay. In these experiments, loss of WRN resulted in a significant decrease in nonhomologous integration events and ablation of recombinants that required random integration of the corrected targeting vector. Aberrant recombinants were also recovered, but only from WRN-depleted cells. The pleiotropic recombination phenotypes conferred by WRN depletion, reflected in distinct homologous and nonhomologous recombination pathways, suggest a role for WRN in processing specific types of homologous recombination intermediates as well as an important function in nonhomologous recombination.

Recombinant Phenotyping of Cytomegalovirus UL97 Kinase Sequence Variants for Ganciclovir Resistance

A strain of human cytomegalovirus, T2211, modified from standard laboratory strain AD169 to contain a secreted alkaline phosphatase reporter gene for rapid viral quantitation, was cloned as a bacterial artificial chromosome, BA1, and then mutagenized to create recombinant viruses containing viral UL97 kinase sequence variants found in clinical specimens after ganciclovir treatment, but with no phenotypic data to determine their role in drug resistance. Seven control strains and 14 other recombinant strains were phenotyped for ganciclovir resistance and compared with similar strains created using prior technology to show a good concordance of findings. Sequence changes V466M, H469Y, A478V, N510S, A588V, K599R, L600I, G623S, T659I, and V665I were found to confer no significant ganciclovir resistance, while mutations L405P, M460T, A594E, and C603R conferred 3- to 9-fold increases in ganciclovir 50% inhibitory concentrations. Different mutations at codons 594 (A594V, A594E) and 603 (C603W, C603S) conferred varied amounts of ganciclovir resistance. Advances in recombinant phenotyping make it easier to show that many uncharacterized UL97 sequence variants do not confer ganciclovir resistance, but some are newly confirmed as resistance associated, including one (L405P) which is outside the codon range where such mutations are usually found. This information should improve the interpretation of genotypic data generated by diagnostic laboratories.

Cdk1 Targets Srs2 to Complete Synthesis-Dependent Strand Annealing and to Promote Recombinational Repair

Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB) repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA–independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair.

HIV-1 V3 envelope deep sequencing for clinical plasma specimens failing in phenotypic tropism assays

BackgroundHIV-1 infected patients for whom standard gp160 phenotypic tropism testing failed are currently excluded from co-receptor antagonist treatment. To provide patients with maximal treatment options, massively parallel sequencing of the envelope V3 domain, in combination with tropism prediction tools, was evaluated as an alternative tropism determination strategy. Plasma samples from twelve HIV-1 infected individuals with failing phenotyping results were available. The samples were submitted to massive parallel sequencing and to confirmatory recombinant phenotyping using a fraction of the gp120 domain.ResultsA cut-off for sequence reads interpretation of 5 to10 times the sequencing error rate (0.2%) was implemented. On average, each sample contained 7 different V3 haplotypes. V3 haplotypes were submitted to tropism prediction algorithms, and 4/14 samples returned with presence of a dual/mixed (D/M) tropic virus, respectively at 3%, 10%, 11%, and 95% of the viral quasispecies. V3 tropism prediction was confirmed by gp120 phenotyping, except for two out of 4 D/M predicted viruses (with 3 and 95%) which were phenotypically R5-tropic. In the first case, the result was discordant due to the limit of detection for the phenotyping technology, while in the latter case the prediction algorithms were not computing the viral tropism correctly.ConclusionsAlthough only demonstrated on a limited set of samples, the potential of the combined use of "deep sequencing + prediction algorithms" in cases where routine gp160 phenotype testing cannot be employed was illustrated. While good concordance was observed between gp120 phenotyping and prediction of R5-tropic virus, the results suggest that accurate prediction of X4-tropic virus would require further algorithm development.

Genetic Analysis of Variation in Human Meiotic Recombination

The number of recombination events per meiosis varies extensively among individuals. This recombination phenotype differs between female and male, and also among individuals of each gender. In this study, we used high-density SNP genotypes of over 2,300 individuals and their offspring in two datasets to characterize recombination landscape and to map the genetic variants that contribute to variation in recombination phenotypes. We found six genetic loci that are associated with recombination phenotypes. Two of these (RNF212 and an inversion on chromosome 17q21.31) were previously reported in the Icelandic population, and this is the first replication in any other population. Of the four newly identified loci (KIAA1462, PDZK1, UGCG, NUB1), results from expression studies provide support for their roles in meiosis. Each of the variants that we identified explains only a small fraction of the individual variation in recombination. Notably, we found different sequence variants associated with female and male recombination phenotypes, suggesting that they are regulated by different genes. Characterization of genetic variants that influence natural variation in meiotic recombination will lead to a better understanding of normal meiotic events as well as of non-disjunction, the primary cause of pregnancy loss.