MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice

F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression.     

BRIT1/MCPH1 is a multifunctional DNA damage responsive protein mediating DNA repair-associated chromatin remodeling

Comment on: Peng, G, et al. BRIT1/MCPH1 links chromatin remodelling to DNA damage response. Nat Cell Biol 2009; 11: 865-872.     

MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination

Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB). This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 ^−/− spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob ^−/− meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.     

Meiotic Recombination, Noncoding DNA and Genomic Organization in Caenorhabditis Elegans

The genetic map of each Caenorhabditis elegans chromosome has a central gene cluster (less pronounced on the X chromosome) that contains most of the mutationally defined genes. Many linkage group termini also have clusters, though involving fewer loci. We examine the factors shaping the genetic map by analyzing the rate of recombination and gene density across the genome using the positions of cloned genes and random cDNA clones from the physical map. Each chromosome has a central gene-dense region (more diffuse on the X) with discrete boundaries, flanked by gene-poor regions. Only autosomes have reduced rates of recombination in these gene-dense regions. Cluster boundaries appear discrete also by recombination rate, and the boundaries defined by recombination rate and gene density mostly, but not always, coincide. Terminal clusters have greater gene densities than the adjoining arm but similar recombination rates. Thus, unlike in other species, most exchange in C. elegans occurs in gene-poor regions. The recombination rate across each cluster is constant and similar; and cluster size and gene number per chromosome are independent of the physical size of chromosomes. We propose a model of how this genome organization arose.     

Replication Protein A (RPA1a) Is Required for Meiotic and Somatic DNA Repair But Is Dispensable for DNA Replication and Homologous Recombination in Rice

Replication protein A (RPA), a highly conserved single-stranded DNA-binding protein in eukaryotes, is a stable complex comprising three subunits termed RPA1, RPA2, and RPA3. RPA is required for multiple processes in DNA metabolism such as replication, repair, and homologous recombination in yeast (Saccharomyces cerevisiae) and human. Most eukaryotic organisms, including fungi, insects, and vertebrates, have only a single RPA gene that encodes each RPA subunit. Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), however, possess multiple copies of an RPA gene. Rice has three paralogs each of RPA1 and RPA2, and one for RPA3. Previous studies have established their biochemical interactions in vitro and in vivo, but little is known about their exact function in rice. We examined the function of OsRPA1a in rice using a T-DNA insertional mutant. The osrpa1a mutants had a normal phenotype during vegetative growth but were sterile at the reproductive stage. Cytological examination confirmed that no embryo sac formed in female meiocytes and that abnormal chromosomal fragmentation occurred in male meiocytes after anaphase I. Compared with wild type, the osrpa1a mutant showed no visible defects in mitosis and chromosome pairing and synapsis during meiosis. In addition, the osrpa1a mutant was hypersensitive to ultraviolet-C irradiation and the DNA-damaging agents mitomycin C and methyl methanesulfonate. Thus, our data suggest that OsRPA1a plays an essential role in DNA repair but may not participate in, or at least is dispensable for, DNA replication and homologous recombination in rice.In a population of organisms, it is crucial to maintain the integrity of genome among individuals as well as shuffle genetic information at the population level. To maintain such genetic integrity, cells have evolved elaborate mechanisms such as base excision repair (BER; Hegde et al., 2008), nucleotide excision repair (NER; Shuck et al., 2008), homologous recombination (HR; Li and Heyer, 2008) repair, and nonhomologous end joining (Weterings and Chen, 2008) pathways to repair diverse types of DNA damage. To allow for variation, however, organisms utilize meiosis to shuffle genetic material so as to increase genetic diversity in populations and in the species.DNA double-strand break (DSB) repair is particularly important in maintaining the integrity of genome among individuals and shuffling genetic information among population, because DSBs are generated not only in meiotic cells but also from the action of certain endogenous or exogenous DNA-damaging agents and during repair of other kinds of DNA lesions by NER or BER (West et al., 2004; Bleuyard et al., 2006). The past decade has witnessed an explosion in understanding of this complex process by using yeast (Saccharomyces cerevisiae) as a model organism (Aylon and Kupiec, 2004). Cells can repair DSBs by the relatively inaccurate process of rejoining the two broken ends directly (i.e. nonhomologous end joining) or much more accurately by HR (Bleuyard et al., 2006; Wyman and Kanaar, 2006). These two pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type (Shrivastav et al., 2008). According to the current general model for meiotic DSB repair (Bishop and Zickler, 2004; Ma, 2006; San Filippo et al., 2008), when DSBs occur the MRN complex (composed of Mre11, Rad50, and NBS1) resects the DSBs to generate 5′→3′ single-stranded DNA (ssDNA) ends. Subsequently, the replication protein A (RPA) protein complex binds to the ssDNA ends to protect them from attack by endogenous exonucleases; then, in concert with catalysis by Rad52, Rad55, and Rad57, the recombinase Rad51 displaces RPA, resulting in the generation of a Rad51 nucleoprotein filament that in turn catalyzes the search and invasion into the recombination partner with the help of proteins belonging to the RAD52 epistasis group to form a D loop that accompanies DNA synthesis. Thereafter, at least two competing mechanisms may come into play. One is the DSB repair pathway, in which the capture of the second DSB end and additional DNA synthesis result in an intermediate that harbors two Holliday junctions. The subsequent resolution of Holliday junctions results in the formation of crossovers. Alternatively, in the synthesis-dependent strand annealing pathway, the D loop dissociates and the invading single strand with newly synthesized DNA reanneals with the other DSB end, followed by gap-filling DNA synthesis and ligation, forming only noncrossover products (Ma, 2006; San Filippo et al., 2008).RPA is comprised of three subunits of RPA1, 2, and 3, alternatively termed as RPA70, 32, and 14, respectively, according to their apparent M_rs (Wold, 1997; Iftode et al., 1999). RPA is an essential protein in various DNA metabolism pathways such as DNA replication, repair, and HR (Wold, 1997; Iftode et al., 1999). In these pathways, the most basic function of RPA is binding to ssDNA to protect it from exonucleases, and its general roles in DNA metabolism depend on its interactions with other proteins in various pathways (Wold, 1997; Iftode et al., 1999). For example, in human NER pathway, RPA binds to damaged DNA and interacts with xeroderma pigmentosum damage-recognition protein, XPA, in the damage recognition step, and then the endonucleases XPG and ERCC1/XPF are recruited to the RPA-XPA-damaged DNA complex in the excision step (He et al., 1995). Interactions of RPA with those proteins are critical in this process (Wold, 1997; Iftode et al., 1999). A great deal of protein dynamics research has indicated that the interactions between RPA and other DNA-metabolism proteins are choreographed on the ssDNA to recruit the required protein present at the proper time (Fanning et al., 2006).Human, animals, and fungi have single copy for each subunit of RPA (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi). Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), however, have multiple genes for most RPA subunits (Ishibashi et al., 2006; Shultz et al., 2007). Most of them have not unveiled exact function up to now. To elucidate the molecular basis of meiosis in rice, we performed a large-scale screen for sterile mutants using our T-DNA insertion mutant library (Wu et al., 2003). Previously, we reported the cloning of OsPAIR3, a novel gene required for homologous chromosome pairing and synapsis in rice (Yuan et al., 2009). Here we report the characterization of another sterile mutant with a T-DNA insertion in OsRPA1a. Our results indicate that OsRPA1a is essential for DNA repair but may play redundant roles in DNA replication and recombination in rice.     

The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex

BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.     

SLX-1 Is Required for Maintaining Genomic Integrity and Promoting Meiotic Noncrossovers in the Caenorhabditis elegans Germline

Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644) mutants. SLX-1 shows a HIM-18/SLX-4-dependent endonuclease activity toward replication forks, 5'-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs) along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs.     

Xrs2, a DNA Repair Gene of Saccharomyces Cerevisiae, Is Needed for Meiotic Recombination

The XRS2 gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that XRS2 also encodes an essential meiotic function. Spore inviability of xrs2 strains is rescued by a spo13 mutation, but meiotic recombination (both gene conversion and crossing over) is highly depressed in spo13 xrs2 diploids. The xrs2 mutation suppresses spore inviability of a spo13 rad52 strain suggesting that XRS2 acts prior to RAD52 in the meiotic recombination pathway. In agreement with the genetic data, meiosis-specific double-strand breaks at the ARG4 meiotic recombination hotspot are not detected in xrs2 strains. Despite its effects on meiotic recombination, the xrs2 mutation does not prevent mitotic recombination events, including homologous integration of linear DNA, mating-type switching and radiation-induced gene conversion. Moreover, xrs2 strains display a mitotic hyper-rec phenotype. Haploid xrs2 cells fail to carry out G2-repair of gamma-induced lesions, whereas xrs2 diploids are able to perform some diploid-specific repair of these lesions. Meiotic and mitotic phenotypes of xrs2 cells are very similar to those of rad50 cells suggesting that XRS2 is involved in homologous recombination in a way analogous to that of RAD50.     

MCPH1/BRIT1 cooperates with E2F1 in the activation of checkpoint, DNA repair and apoptosis

Microcephalin (MCPH1) has a crucial role in the DNA damage response by promoting the expression of Checkpoint kinase 1 (CHK1) and Breast cancer susceptibility gene 1 (BRCA1); however, the mechanism of this regulation remains unclear. Here, we show that MCPH1 regulates CHK1 and BRCA1 through the interaction with E2F1 on the promoters of both genes. MCPH1 also regulates other E2F target genes involved in DNA repair and apoptosis such as RAD51, DDB2, TOPBP1, p73 and caspases. MCPH1 interacts with E2F1 on the p73 promoter, and regulates p73 induction and E2F1-induced apoptosis as a result of DNA damage. MCPH1 forms oligomers through the second and third BRCT domains. An MCPH1 mutant containing only its oligomerization domain has a dominant-negative role by blocking MCPH1 binding to E2F1. It also inhibits p73 induction in DNA damage and E2F1-dependent apoptosis. Taken together, MCPH1 cooperates with E2F1 to regulate genes involved in DNA repair, checkpoint and apoptosis, and might participate in the maintenance of genomic integrity.     

BRIT1/MCPH1: A Guardian of Genome and an Enemy of Tumors

The DNA of every cell is constantly exposed to insult mediated by endogenous and environmental factors that induced damage in its structure. To react to these attacks and maintain the integrity of the genome, eukaryotic cells are equipped with sophisticated mechanisms to detect, signal the presence of and repair DNA damage. The cellular response to DNA damage is a critical event for maintaining genomic stability and limiting neoplastic transformation. BRIT1, a newly identified protein, forms specific irradiation-induced nuclear foci. Our recent investigation demonstrates that BRIT1 functions as a proximal factor in the DNA damage checkpoints that control multiple damage sensors and early mediators. BRIT1 is also implicated in cell cycle checkpoints, controlling and regulating other important molecules and thus affecting the timing of mitosis. Depletion of BRIT1 abolishes the DNA damage response and results in centrosomal abnormalities and chromosomal aberrations. Moreover, aberrantly reduced expression of BRIT1 in human carcinomas implicates this protein in cancer initiation and progression. Together, the findings identify BRIT1 as a potential tumor suppressor. Fully elucidating the function of this intriguing protein may lead to new therapeutic approaches for the improved cancer treatment.     

CENTRAL REGION COMPONENT1, a Novel Synaptonemal Complex Component,Is Essential for Meiotic Recombination Initiation in Rice

In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated CENTRAL REGION COMPONENT1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID RECEPTOR-INTERACTING PROTEIN13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13.     

PP4 Is Essential for Germinal Center Formation and Class Switch Recombination in Mice

PP4 is a serine/threonine phosphatase required for immunoglobulin (Ig) VDJ recombination and pro-B/pre-B cell development in mice. To elucidate the role of PP4 in mature B cells, we ablated the catalytic subunit of murine PP4 in vivo utilizing the CD23 promoter and cre-loxP recombination and generated CD23^crePP4^F/F mice. The development of follicular and marginal zone B cells was unaffected in these mutants, but the proliferation of mature PP4-deficient B cells stimulated by in vitro treatment with either anti-IgM antibody (Ab) or LPS was partially impaired. Interestingly, the induction of CD80 and CD86 expression on these stimulated B cells was normal. Basal levels of serum Igs of all isotypes were strongly reduced in CD23^crePP4^F/F mice, and their B cells showed a reduced efficiency of class switch recombination (CSR) in vitro upon stimulation by LPS or LPS plus IL-4. When CD23^crePP4^F/F mice were challenged with either the T cell-dependent antigen TNP-KLH or the T cell-independent antigen TNP-Ficoll, or by H1N1 virus infection, the mutant animals failed to form germinal centers (GCs) in the spleen and the draining mediastinal lymph nodes, and did not efficiently mount antigen-specific humoral responses. In the resting state, PP4-deficient B cells exhibited pre-existing DNA fragmentation. Upon stimulation by DNA-damaging drug etoposide in vitro, mutant B cells showed increased cleavage of caspase 3. In addition, the mutant B cells displayed impaired CD40-mediated MAPK activation, abnormal IgM-mediated NF-κB activation, and reduced S phase entry upon IgM/CD40-stimulation. Taken together, our results establish a novel role for PP4 in CSR, and reveal crucial functions for PP4 in the maintenance of genomic stability, GC formation, and B cell-mediated immune responses.     

Spontaneous Mitotic Recombination and Evidence for an X-Ray-Inducible System for the Repair of DNA Damage in DROSOPHILA MELANOGASTER

Spontaneous mitotic recombination in the left arm of chromosome 3 was examined in both unirradiated control flies and sibs irradiated early in development by determining the sizes and frequencies of multiple-wing-hair (mwh) clones in the wing blade of heterozygous mwh/+ flies. Approximately 16% of the spontaneous mwh clones arise from events generating cells with normal division rates. The remaining 84% result from events generating cells with an average cell division rate one-third that of the surrounding cells; these are thought to result from events that generate aneuploid cells. Such clones probably arise from a failure correctly to repair spontaneous DNA damage. The frequency of spontaneous events late in development decreases significantly after irradiation as much as 150 hours earlier in development. The suppression of spontaneous events decreases with a longer period of time between irradiation and the final cell divisions in the wing blade. These results suggest the existence of a repair system for DNA damage in Drosophila that is induced by irradiation. The decrease in effect with time following irradiation could result from slow degradation or dilution by subsequent cell growth and division.     

Pds1p Is Required for Meiotic Recombination and Prophase I Progression in Saccharomyces cerevisiae

Sister-chromatid separation at the metaphase–anaphase transition is regulated by a proteolytic cascade. Destruction of the securin Pds1p liberates the Esp1p separase, which ultimately targets the mitotic cohesin Mcd1p/Scc1p for destruction. Pds1p stabilization by the spindle or DNA damage checkpoints prevents sister-chromatid separation while mutants lacking PDS1 (pds1Δ) are temperature sensitive for growth due to elevated chromosome loss. This report examined the role of the budding yeast Pds1p in meiotic progression using genetic, cytological, and biochemical assays. Similar to its mitotic function, Pds1p destruction is required for metaphase I–anaphase I transition. However, even at the permissive temperature for growth, pds1Δ mutants arrest with prophase I spindle and nuclear characteristics. This arrest was partially suppressed by preventing recombination initiation or by inactivating a subset of recombination checkpoint components. Further studies revealed that Pds1p is required for recombination in both double-strand-break formation and synaptonemal complex assembly. Although deleting PDS1 did not affect the degradation of the meiotic cohesin Rec8p, Mcd1p was precociously destroyed as cells entered the meiotic program. This role is meiosis specific as Mcd1p destruction is not altered in vegetative pds1Δ cultures. These results define a previously undescribed role for Pds1p in cohesin maintenance, recombination, and meiotic progression.     

Interaction between Ustilago Maydis Rec2 and Rad51 Genes in DNA Repair and Mitotic Recombination

A gene encoding a Ustilago maydis Rad51 orthologue has been isolated. rad51-1, a mutant constructed by disrupting the gene, was as sensitive to killing by ultraviolet light and γ radiation as the rec2-1 mutant and slightly more sensitive to killing by methyl methanesulfonate. There was no suppression of killing by ultraviolet light when a rec2-1 strain was transformed with a multicopy plasmid containing RAD51, nor was there suppression when rad51-1 was transformed with a multicopy plasmid containing REC2. Recombination proficiency as measured by a gap repair assay was diminished in both rec2-1 and rad51-1 strains. In rec2-1 the frequency of recombination was decreased, but the spectrum of events was similar to that observed in wild type, while in rad51-1 the frequency as well as the spectrum of recombination events were different. Studies with the rec2-1 rad51-1 double mutant indicated that there was epistasis in the action of REC2 and RAD51 in certain repair and recombination functions, but some measure of independent action in other functions.     

Evidence for Mitotic Recombination in W(ei)/+ Heterozygous Mice

A number of alleles at coat color loci of the house mouse give rise to areas of wild-type pigmentation on the coats of otherwise mutant animals. Such unstable alleles include both recessive and dominant mutations. Among the latter are several alleles at the W locus. In this report, phenotypic reversions of the Wei allele at the W locus were studied Mice heterozygous in repulsion for both Wei and buff (bf) [i.e. Wei+/+bf] were examined for the occurrence of phenotypic reversion events. Buff (bf) is a recessive mutation, which lies 21 cM from W on the telomeric side of chromosome 5 and is responsible for the khaki colored coat of nonagouti buff homozygotes (a/a; bf/bf). Two kinds of fully pigmented reversion spots were recovered on the coats of a/a; Wei+/+bf mice: either solid black or khaki colored. Furthermore phenotypic reversions of Wei/+ were enhanced significantly following X-irradiation of 9.25-day-old Wei/+ embryos (P less than 0.04). These observations are consistent with the suggestion of a role for mitotic recombination in the origin of these phenotypic reversions. In addition these results rise the intriguing possibility that some W mutations may enhance mitotic recombination in the house mouse.     

Visualization of Shared Genomic Regions and Meiotic Recombination in High-Density SNP Data

Background

A fundamental goal of single nucleotide polymorphism (SNP) genotyping is to determine the sharing of alleles between individuals across genomic loci. Such analyses have diverse applications in defining the relatedness of individuals (including unexpected relationships in nominally unrelated individuals, or consanguinity within pedigrees), analyzing meiotic crossovers, and identifying a broad range of chromosomal anomalies such as hemizygous deletions and uniparental disomy, and analyzing population structure.

Principal Findings

We present SNPduo, a command-line and web accessible tool for analyzing and visualizing the relatedness of any two individuals using identity by state. Using identity by state does not require prior knowledge of allele frequencies or pedigree information, and is more computationally tractable and is less affected by population stratification than calculating identity by descent probabilities. The web implementation visualizes shared genomic regions, and generates UCSC viewable tracks. The command-line version requires pedigree information for compatibility with existing software and determining specified relationships even though pedigrees are not required for IBS calculation, generates no visual output, is written in portable C++, and is well-suited to analyzing large datasets. We demonstrate how the SNPduo web tool identifies meiotic crossover positions in siblings, and confirm our findings by visualizing meiotic recombination in synthetic three-generation pedigrees. We applied SNPduo to 210 nominally unrelated Phase I / II HapMap samples and, consistent with previous findings, identified six undeclared pairs of related individuals. We further analyzed identity by state in 2,883 individuals from multiplex families with autism and identified a series of anomalies including related parents, an individual with mosaic loss of chromosome 18, an individual with maternal heterodisomy of chromosome 16, and unexplained replicate samples.

Conclusions

SNPduo provides the ability to explore and visualize SNP data to characterize the relatedness between individuals. It is compatible with, but distinct from, other established analysis software such as PLINK, and performs favorably in benchmarking studies for the analyses of genetic relatedness.