Allelic Recombination Research Articles

The genomic diversification of the whole Acinetobacter genus: origins, mechanisms, and consequences.

Bacterial genomics has greatly expanded our understanding of microdiversification patterns within a species, but analyses at higher taxonomical levels are necessary to understand and predict the independent rise of pathogens in a genus. We have sampled, sequenced, and assessed the diversity of genomes of validly named and tentative species of the Acinetobacter genus, a clade including major nosocomial pathogens and biotechnologically important species. We inferred a robust global phylogeny and delimited several new putative species. The genus is very ancient and extremely diverse: Genomes of highly divergent species share more orthologs than certain strains within a species. We systematically characterized elements and mechanisms driving genome diversification, such as conjugative elements, insertion sequences, and natural transformation. We found many error-prone polymerases that may play a role in resistance to toxins, antibiotics, and in the generation of genetic variation. Surprisingly, temperate phages, poorly studied in Acinetobacter, were found to account for a significant fraction of most genomes. Accordingly, many genomes encode clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems with some of the largest CRISPR-arrays found so far in bacteria. Integrons are strongly overrepresented in Acinetobacter baumannii, which correlates with its frequent resistance to antibiotics. Our data suggest that A. baumannii arose from an ancient population bottleneck followed by population expansion under strong purifying selection. The outstanding diversification of the species occurred largely by horizontal transfer, including some allelic recombination, at specific hotspots preferentially located close to the replication terminus. Our work sets a quantitative basis to understand the diversification of Acinetobacter into emerging resistant and versatile pathogens.

Transcriptomic landscape of prophase I sunflower male meiocytes.

Meiosis is a form of specialized cell division that generates gametes, allowing recombination of alleles and halving the chromosome number. Arabidopsis and maize are the plant models that have been most extensively studied to determine the genes involved in meiosis. Here we present an RNA-seq study in which gene expression in male meiocytes isolated during prophase I was compared to that in somatic tissues of the sunflower HA89 line. We sampled more than 490 million gene tags from these libraries, assembled them de novo into a sunflower transcriptome. We obtained expression data for 36,304 sunflower genes, of which 19,574 (54%) were differentially expressed (DE) between meiocytes and somatic tissue. We also determined the functional categories and metabolic pathways that are DE in these libraries. As expected, we found large differences between the meiotic and somatic transcriptomes, which is in accordance with previous studies in Arabidopsis and maize. Furthermore, most of the previously implicated meiotic genes were abundantly and DE in meiocytes and a large repertoire of transcription factors (TF) and genes related to silencing are expressed in the sunflower meiocytes. We detected TFs which appear to be exclusively expressed in meiocytes. Our results allow for a better understanding of the conservation and differences in the meiotic transcriptome of plants.

A recombination allele of the lipase gene CEL and its pseudogene CELP encodes a hybrid protein and is a genetic risk factor for chronic pancreatitis

A recombination allele of the lipase gene CEL and its pseudogene CELP encodes a hybrid protein and is a genetic risk factor for chronic pancreatitis

Revertant mutation releases confined lethal mutation, opening Pandora's box: a novel genetic pathogenesis.

When two mutations, one dominant pathogenic and the other “confining” nonsense, coexist in the same allele, theoretically, reversion of the latter may elicit a disease, like the opening of Pandora's box. However, cases of this hypothetical pathogenic mechanism have never been reported. We describe a lethal form of keratitis-ichthyosis-deafness (KID) syndrome caused by the reversion of the GJB2 nonsense mutation p.Tyr136X that would otherwise have confined the effect of another dominant lethal mutation, p.Gly45Glu, in the same allele. The patient's mother had the identical misssense mutation which was confined by the nonsense mutation. The biological relationship between the parents and the child was confirmed by genotyping of 15 short tandem repeat loci. Haplotype analysis using 40 SNPs spanning the >39 kbp region surrounding the GJB2 gene and an extended SNP microarray analysis spanning 83,483 SNPs throughout chromosome 13 in the family showed that an allelic recombination event involving the maternal allele carrying the mutations generated the pathogenic allele unique to the patient, although the possibility of coincidental accumulation of spontaneous point mutations cannot be completely excluded. Previous reports and our mutation screening support that p.Gly45Glu is in complete linkage disequilibrium with p.Tyr136X in the Japanese population. Estimated from statisitics in the literature, there may be approximately 11,000 p.Gly45Glu carriers in the Japanese population who have this second-site confining mutation, which acts as natural genetic protection from the lethal disease. The reversion-triggered onset of the disesase shown in this study is a previously unreported genetic pathogenesis based on Mendelian inheritance.

Inferring outcrossing in the homothallic fungus Sclerotinia sclerotiorum using linkage disequilibrium decay.

The occurrence and frequency of outcrossing in homothallic fungal species in nature is an unresolved question. Here we report detection of frequent outcrossing in the homothallic fungus Sclerotinia sclerotiorum. In using multilocus linkage disequilibrium (LD) to infer recombination among microsatellite alleles, high mutation rates confound the estimates of recombination. To distinguish high mutation rates from recombination to infer outcrossing, 8 population samples comprising 268 S. sclerotiorum isolates from widely distributed agricultural fields were genotyped for 12 microsatellite markers, resulting in multiple polymorphic markers on three chromosomes. Each isolate was homokaryotic for the 12 loci. Pairwise LD was estimated using three methods: Fisher's exact test, index of association (IA) and Hedrick's D'. For most of the populations, pairwise LD decayed with increasing physical distance between loci in two of the three chromosomes. Therefore, the observed recombination of alleles cannot be simply attributed to mutation alone. Different recombination rates in various DNA regions (recombination hot/cold spots) and different evolutionary histories of the populations could explain the observed differences in rates of LD decay among the chromosomes and among populations. The majority of the isolates exhibited mycelial incompatibility, minimizing the possibility of heterokaryon formation and mitotic recombination. Thus, the observed high intrachromosomal recombination is due to meiotic recombination, suggesting frequent outcrossing in these populations, supporting the view that homothallism favors universal compatibility of gametes instead of traditionally believed haploid selfing in S. sclerotiorum. Frequent outcrossing facilitates emergence and spread of new traits such as fungicide resistance, increasing difficulties in managing Sclerotinia diseases.

GISH INVESTIGATION OF CROSSOVER EVENTS DURING MEIOSIS OF INTERSPECIFIC HYBRIDS OF LILY

Crossover not only establishes proper segregation of omologous/homoeologous chromosomes during meiosis I, but also promotes genetic diversity though creating new recombination of maternal and paternal alleles, which is essential for introgression breeding. In this paper, crossovers between non-sister chromatids were observed during meiosis of interspecific hybrids of Lilium longiflorum × Asiatic (LA) using genomic in situ hybridization (GISH). Results showed that different types of crossing over events, viz. single, two strand double, three strand double, four strand double crossovers, occurred between non-sister chromatids during meiosis of selected interspecific lily hybrids. From an analysis of a total of 637 pairs of half-bivalents it was evident that a large majority (65%) was two strand single crossovers, 5.5% were two strands double, 3.0% three strand double, 9.3% four strand double and 17.3% were multiple crossing over events. In one specific genotype which showed the existence of a reciprocal translocation, the frequency of multiple crossovers was relatively high, which might alter the map distances of markers on the translocated chromosomes.

Notch1 and Notch2 expression in osteoblast precursors regulates femoral microarchitecture

Notch receptors regulate cell differentiation and function. Notch1 and Notch2 inactivation in osteoblasts and osteocytes increases cancellous bone volume, but the function of Notch signaling in osteoblast precursors is unknown. To inactivate Notch signaling in immature osteoblastic cells, mice homozygous for conditional Notch1 and Notch2 alleles (Notch1loxP/loxP;Notch2loxP/loxP) were crossed with mice where the osterix (Osx) promoter, regulated by a Tet-Off cassette, governs Cre expression (Osx-Cre). Notch1loxP/loxP;Notch2loxP/loxP control and Osx-Cre+/−;Notch1Δ/Δ;Notch2Δ/Δ experimental littermate cohorts were obtained. To prevent the effects of embryonic Osx-Cre expression, doxycycline was administered to pregnant dams, but not to newborns. Recombination of conditional alleles was documented in calvarial DNA extracts from 1month old mice. Notch1 and Notch2 inactivation did not affect femoral microarchitecture at 1month of age. Cancellous bone volume was higher and structure model index was lower in 3 and 6month old Osx-Cre+/−;Notch1Δ/Δ;Notch2Δ/Δ mice than in control littermates and the effect was more pronounced in female mice. One month old Osx-Cre+/−;Notch1Δ/Δ;Notch2Δ/Δ male mice transiently exhibited an increase in osteoblast number and a modest suppression in bone resorption. Osx-Cre+/−;Notch1Δ/Δ;Notch2Δ/Δ female mice displayed a tendency toward increased bone formation at 3months of age, although bone remodeling was suppressed in 6month old Osx-Cre+/−;Notch1Δ/Δ;Notch2Δ/Δ female mice. Notch1 and Notch2 inactivation increased porosity and reduced thickness of cortical bone. These effects were modest and more evident in 3 and 6month old female than in male mice of the same age. In conclusion, Notch1 and Notch2 expression in osteoblast precursors regulates cancellous bone volume and microarchitecture.

Population-specific differences in gene conversion patterns between human SUZ12 and SUZ12P are indicative of the dynamic nature of interparalog gene conversion

Nonallelic homologous gene conversion (NAHGC) resulting from interparalog recombination without crossover represents an important influence on the evolution of duplicated sequences in the human genome. In 17q11.2, different paralogous sequences mediate large NF1 deletions by nonallelic homologous recombination with crossover (NAHR). Among these paralogs are SUZ12 and its pseudogene SUZ12P which harbour the breakpoints of type-2 (1.2-Mb) NF1 deletions. Such deletions are caused predominantly by mitotic NAHR since somatic mosaicism with normal cells is evident in most patients. Investigating whether SUZ12 and SUZ12P have also been involved in NAHGC, we observed gene conversion tracts between these paralogs in both Africans (AFR) and Europeans (EUR). Since germline type-2 NF1 deletions resulting from meiotic NAHR are very rare, the vast majority of the gene conversion tracts in SUZ12 and SUZ12P are likely to have resulted from mitotic recombination during premeiotic cell divisions of germ cells. A higher number of gene conversion tracts were noted within SUZ12 and SUZ12P in AFR as compared to EUR. Further, the distinctive signature of NAHGC (a high number of SNPs per paralog and a high number of shared SNPs between paralogs), a characteristic of many actively recombining paralogs, was observed in both SUZ12 and SUZ12P but only in AFR and not in EUR. A novel polymorphic 2.3-kb deletion in SUZ12P was identified which exhibited a high allele frequency in EUR. We postulate that this interparalog structural difference, together with low allelic recombination rates, could have caused a reduction in NAHGC between SUZ12 and SUZ12P during human evolution.

Impact on Reindeer (Rangifer tarandus) Genetic Diversity from Two Parallel Population Bottlenecks Founded from a Common Source

Population bottlenecks and founder events reduce genetic diversity through stochastic processes associated with the sampling of alleles at the time of the bottleneck, and the recombination of alleles that are identical by descent. At the same time bottlenecks and founder events can structure populations through the stochastic distortion of allele frequencies. Here we undertake an empirical assessment of the impact of two independent bottlenecks of known size from a known source, and consider inference about evolutionary process in the context of simulations and theoretical expectations. We find a similar level of reduced variation in the parallel bottleneck events, with the greater impact on the population that began with the smaller number of females. The level of diversity remaining was consistent with model predictions, but only if re-growth of the population was essentially exponential and polygeny was minimal at the early stages. There was a high level of differentiation seen compared to the source population and between the two bottlenecked populations, reflecting the stochastic distortion of allele frequencies. We provide empirical support for the theoretical expectations that considerable diversity can remain following a severe bottleneck event, given rapid demographic recovery, and that populations founded from the same source can become quickly differentiated. These processes may be important during the evolution of population genetic structure for species affected by rapid changes in available habitat.

Sex‐specific recombination rates and allele frequencies affect the invasion of sexually antagonistic variation on autosomes

The introduction and persistence of novel, sexually antagonistic alleles can depend upon factors that differ between males and females. Understanding the conditions for invasion in a two-locus model can elucidate these processes. For instance, selection can act differently upon the sexes, or sex linkage can facilitate the invasion of genetic variation with opposing fitness effects between the sexes. Two factors that deserve further attention are recombination rates and allele frequencies - both of which can vary substantially between the sexes. We find that sex-specific recombination rates in a two-locus diploid model can affect the invasion outcome of sexually antagonistic alleles and that the sex-averaged recombination rate is not necessarily sufficient to predict invasion. We confirm that the range of permissible recombination rates is smaller in the sex benefitting from invasion and larger in the sex harmed by invasion. However, within the invasion space, male recombination rate can be greater than, equal to or less than female recombination rate in order for a male-benefit, female-detriment allele to invade (and similarly for a female-benefit, male-detriment allele). We further show that a novel, sexually antagonistic allele that is also associated with a lowered recombination rate can invade more easily when present in the double heterozygote genotype. Finally, we find that sexual dimorphism in resident allele frequencies can impact the invasion of new sexually antagonistic alleles at a second locus. Our results suggest that accounting for sex-specific recombination rates and allele frequencies can determine the difference between invasion and non-invasion of novel, sexually antagonistic alleles in a two-locus model.

Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Modulates N-Methyl-d-aspartate (NMDA) Receptor-dependent Intracellular Signaling and NMDA-induced Regulation of Postsynaptic Protein Complexes

The lipoprotein receptor LRP1 is essential in neurons of the central nervous system, as was revealed by the analysis of conditional Lrp1-deficient mouse models. The molecular basis of its neuronal functions, however, is still incompletely understood. Here we show by immunocytochemistry, electron microscopy, and postsynaptic density preparation that LRP1 is located postsynaptically. Basal and NMDA-induced phosphorylation of the transcription factor cAMP-response element-binding protein (CREB) as well as NMDA target gene transcription are reduced in LRP1-deficient neurons. In control neurons, NMDA promotes γ-secretase-dependent release of the LRP1 intracellular domain (LRP1-ICD). However, pull-down and chromatin immunoprecipitation (ChIP) assays showed no direct interaction between the LRP1-ICD and either CREB or target gene promoters. On the other hand, NMDA-induced degradation of the postsynaptic scaffold protein PSD-95 was impaired in the absence of LRP1, whereas its ubiquitination was increased, indicating that LRP1 influences the composition of postsynaptic protein complexes. Accordingly, NMDA-induced internalization of the AMPA receptor subunit GluA1 was impaired in LRP1-deficient neurons. These results show a role of LRP1 in the regulation and turnover of synaptic proteins, which may contribute to the reduced dendritic branching and to the neurological phenotype observed in the absence of LRP1.

Residual recombination in Neurospora crassa spo11 deletion homozygotes occurs during meiosis

Spo11 is considered responsible for initiation of meiotic recombination in higher organisms, but previous analysis using spo11 (RIP) mutants suggests that the his-3 region of Neurospora crassa experiences spo11-independent recombination. However, despite possessing several stop codons, it is conceivable that the mutants are not completely null. Also, since lack of spo11 interferes with chromosomal pairing and proper segregation at Meiosis I, spores can be partially diploid for a period after meiosis. Thus, it is possible that the recombination observed could be an abnormal event, occurring during the period of aneuploidy rather than during meiosis. To test the former hypothesis, we generated spo11 deletion homozygotes. Using crosses heteroallelic for his-3 mutations, we showed that His(+) progeny are generated in spo11 deletion homozygotes at a frequency at least as high as in wild type and, as in the spo11 (RIP) mutants, local crossing over is not reduced. To test the latter hypothesis, we utilised mutations in either end of a histone H1-GFP fusion gene, inserted between the recombination hotspot cog and his-3, in which GFP(+) spores arise as a result of recombination in a cross between the two GFP alleles. In a control cross homozygous for spo11 (+), the frequency at which GFP(+) spores arise is comparable to the frequency of His(+) spores and glowing nuclei first appear during prophase, prior to metaphase I, as expected for a product of meiotic recombination. Similarly in spo11 deletion homozygotes, GFP(+) spores arise at high frequency and glowing nuclei are first seen before metaphase, indicating that allelic recombination occurs during meiosis in the absence of spo11. We have therefore shown that spo11 is not essential for either his-3 allelic recombination or crossing over in the vicinity of his-3, and that spo11-independent allelic recombination is meiotic, indicating that there is a spo11-independent mechanism for initiation of recombination in Neurospora.

Knockdown of parathyroid hormone related protein in smooth muscle cells alters renal hemodynamics but not blood pressure

Parathyroid hormone-related protein (PTHrP) belongs to vasoactive factors that regulate blood pressure and renal hemodynamics both by reducing vascular tone and raising renin release. PTHrP is expressed in systemic and renal vasculature. Here, we wanted to assess the contribution of vascular smooth muscle cell endogenous PTHrP to the regulation of cardiovascular and renal functions. We generated a mouse strain (SMA-CreERT2/PTHrPL2/L2 or premutant PTHrPSM-/-), which allows temporally controlled, smooth muscle-targeted PTHrP knockdown in adult mice. Tamoxifen treatment induced efficient recombination of PTHrP-floxed alleles and decreased PTHrP expression in vascular and visceral smooth muscle cells of PTHrPSM-/- mice. Blood pressure remained unchanged in PTHrPSM-/- mice, but plasma renin concentration and creatinine clearance were reduced. Renal hemodynamics were further analyzed during clearance measurements in anesthetized mice. Conditional knockdown of PTHrP decreased renal plasma flow and glomerular filtration rate with concomitant reduction in filtration fraction. Similar measurements were repeated during acute saline volume expansion. Saline volume expansion induced a rise in renal plasma flow and reduced filtration fraction; both were blunted in PTHrPSM-/- mice leading to impaired diuresis. These findings show that endogenous vascular smooth muscle PTHrP controls renal hemodynamics under basal conditions, and it is an essential factor in renal vasodilation elicited by saline volume expansion.

Copy number variation-based polymorphism in a new pseudoautosomal region 3 (PAR3) of a human X-chromosome-transposed region (XTR) in the Y chromosome

A 3.5-Mb region of the X chromosome underwent duplication and transposition to the Y chromosome ~5-6Mya. This X-transposed-region (XTR) originated at Xq21.3 and was inserted at Yp11.2. The two locations have 98.78% homology and a high concentration of tandem repeats. In whole-genome scans of ten large families with dyslexic members, we identified transposed blocks comprising >102kb of the Yp11.2 region in its homologous region at Xq21.3 in three females from three different families. Although recombination is known to be limited only to the pseudoautosomal regions (PARs) of the X and Y chromosomes, we report allelic unequal recombination between the XTR region Yp11.2 and Xq21.3, indicating the presence of a new PAR, which we named PAR3. This PAR3 region was also found in 2% of the general population. An additional layer of justification could be provided from six other dyslexic cases which harbored duplications and deletions in the same Xq21.3 and Yp11.2 regions through allelic unequal recombination.

Trans-Lesion DNA Polymerases May Be Involved in Yeast Meiosis.

Trans-lesion DNA polymerases (TLSPs) enable bypass of DNA lesions during replication and are also induced under stress conditions. Being only weakly dependent on their template during replication, TLSPs introduce mutations into DNA. The low processivity of these enzymes ensures that they fall off their template after a few bases are synthesized and are then replaced by the more accurate replicative polymerase. We find that the three TLSPs of budding yeast Saccharomyces cerevisiae Rev1, PolZeta (Rev3 and Rev7), and Rad30 are induced during meiosis at a time when DNA double-strand breaks (DSBs) are formed and homologous chromosomes recombine. Strains deleted for one or any combination of the three TLSPs undergo normal meiosis. However, in the triple-deletion mutant, there is a reduction in both allelic and ectopic recombination. We suggest that trans-lesion polymerases are involved in the processing of meiotic double-strand breaks that lead to mutations. In support of this notion, we report significant yeast two-hybrid (Y2H) associations in meiosis-arrested cells between the TLSPs and DSB proteins Rev1-Spo11, Rev1-Mei4, and Rev7-Rec114, as well as between Rev1 and Rad30. We suggest that the involvement of TLSPs in processing of meiotic DSBs could be responsible for the considerably higher frequency of mutations reported during meiosis compared with that found in mitotically dividing cells, and therefore may contribute to faster evolutionary divergence than previously assumed.

Use of PB-Cre4 Mice for Mosaic Gene Deletion

Transgene expression from short promoters in transgenic animals can lead to unwanted transgene expression patterns, often as a byproduct of random integration of the expression cassette into the host genome. Here I demonstrate that the often used PB-Cre4 line (also referred to as “Probasin-Cre”), although expressing exclusively in the male prostate epithelium when transmitted through male mice, can lead to recombination of loxP-flanked alleles in a large variety of tissues when transmitted through female mice. This aberrant Cre activity due to Cre expression in the oocytes leads to different outcomes for maternally or paternally transmitted loxP-flanked alleles: Maternally inherited loxP-flanked alleles undergo recombination very efficiently, making female PB-Cre4 mice an efficient monoallelic “Cre deleter line”. However, paternally inherited loxP-flanked alleles are inefficiently recombined by maternal PB-Cre4, giving rise to mosaic expression patterns in the offspring. This mosaic recombination is difficult to detect with standard genotyping approaches of many mouse lines and should therefore caution researchers using PB-Cre4 to use additional approaches to exclude the presence of recombined alleles. However, mosaic recombination should also be useful in transgenic “knockout” approaches for mosaic gene deletion experiments.

The generation of oxidative stress-induced rearrangements in Saccharomyces cerevisiae mtDNA is dependent on the Nuc1 (EndoG/ExoG) nuclease and is enhanced by inactivation of the MRX complex

Oxidative stress is known to enhance the frequency of two major types of alterations in the mitochondrial genome of Saccharomyces cerevisiae: point mutations and large deletions resulting in the generation of respiration-deficient petite rhō mutants. We investigated the effect of antimycin A, a well-known agent inducing oxidative stress, on the stability of mtDNA. We show that antimycin enhances exclusively the generation of respiration-deficient petite mutants and this is accompanied by a significant increase in the level of reactive oxygen species (ROS) and in a marked drop of cellular ATP. Whole mitochondrial genome sequencing revealed that mtDNAs of antimycin-induced petite mutants are deleted for most of the wild-type sequence and usually contain one of the active origins of mtDNA replication: ori1, ori2 ori3 or ori5. We show that the frequency of antimycin-induced rhō mutants is significantly elevated in mutants deleted either for the RAD50 or XRS2 gene, both encoding the components of the MRX complex, which is known to be involved in the repair of double strand breaks (DSBs) in DNA. Furthermore, enhanced frequency of rhō mutants in cultures of antimycin-treated cells lacking Rad50 was further increased by the simultaneous absence of the Ogg1 glycosylase, an important enzyme functioning in mtBER. We demonstrate also that rad50Δ and xrs2Δ deletion mutants display a considerable reduction in the frequency of allelic mitochondrial recombination, suggesting that it is the deficiency in homologous recombination which is responsible for enhanced rearrangements of mtDNA in antimycin-treated cells of these mutants. Finally, we show that the generation of large-scale mtDNA deletions induced by antimycin is markedly decreased in a nuc1Δ mutant lacking the activity of the Nuc1 nuclease, an ortholog of the mammalian mitochondrial nucleases EndoG and ExoG. This result indicates that the nuclease plays an important role in processing of oxidative stress-induced lesions in the mitochondrial genome.

Genome resequencing reveals multiscale geographic structure and extensive linkage disequilibrium in the forest treePopulus trichocarpa

• Plant population genomics informs evolutionary biology, breeding, conservation and bioenergy feedstock development. For example, the detection of reliable phenotype-genotype associations and molecular signatures of selection requires a detailed knowledge about genome-wide patterns of allele frequency variation, linkage disequilibrium and recombination. • We resequenced 16 genomes of the model tree Populus trichocarpa and genotyped 120 trees from 10 subpopulations using 29,213 single-nucleotide polymorphisms. • Significant geographic differentiation was present at multiple spatial scales, and range-wide latitudinal allele frequency gradients were strikingly common across the genome. The decay of linkage disequilibrium with physical distance was slower than expected from previous studies in Populus, with r(2) dropping below 0.2 within 3-6 kb. Consistent with this, estimates of recent effective population size from linkage disequilibrium (N(e) ≈ 4000-6000) were remarkably low relative to the large census sizes of P. trichocarpa stands. Fine-scale rates of recombination varied widely across the genome, but were largely predictable on the basis of DNA sequence and methylation features. • Our results suggest that genetic drift has played a significant role in the recent evolutionary history of P. trichocarpa. Most importantly, the extensive linkage disequilibrium detected suggests that genome-wide association studies and genomic selection in undomesticated populations may be more feasible in Populus than previously assumed.

P4‐095: Association between variants in newly discovered Alzheimer's susceptibility genes and longitudinal cognitive performance in late middle‐aged adults: The WRAP cohort

known “AD genes’” regions, AD subjects have greater rate and total length of duplications than controls. We did not find evidence for common CNV association to AD using logistic regression with numerous covariates. In chr15q11 20.2M-22.6Mwe found large (>1⁄4500 kbp) duplications are overrepresented by AD cases (47 cases, 4 controls, OR1⁄43.4). This 15q11 region is upstream of the region where deletions are associated with either PraderWilli or Angelman syndromes. The presence of abundant segmental duplications in this region makes it a hot spot for Non Allelic Homologous Recombinations (NAHRs). We were able to identify potential pairs of segmental duplications that might have facilitated some of the recurrent duplications. Conclusions: Duplications in the chr15q11 region are associated with increased risk of late onset AD in the Caucasian population.

Abstract 4417: Induction of colonic cre-recombinase expression in carbonic anhydrase1-cre transgenic mice by dextran sulfate sodium

Abstract We previously reported the development of C57BL/6-Tg(Car1-cre)5Flt transgenic mice (CAC) with carbonic anhydrase-1 promoter/enhancer driven expression of cre-recombinase (Cre) in approximately 10 percent of colon epithelial cells. Here we report a novel observation in these mice that expands their utility for colorectal cancer research. Our original goal was to use CAC-ApcΔ580/+ mice, with conditional deletion of one Apc allele from colonic epithelial cells, to determine the mechanism of the interaction between Apc allele deletion and dextran sulfate sodium (DSS)-induced colitis on carcinogenesis. To detect cells with recombined floxed alleles, CAC-ApcΔ580/+ mice were crossed with B6.129S4-Gt(ROSA)26Sortm1Sor/J mice (Rosa26R), which have a beta-galactosidase (Bgal) transgene preceded by a floxed stop codon. We administered 0%, 0.65%, 1.35%, or 2% DSS in drinking water for 5 days to 8 wk old mice. Mice were killed and tissues harvested at 2 or 10 days after DSS was withdrawn. Histologic sections of colon were scored for ulceration and regeneration. Immunohistochemical labeling for Bgal was used as a reporter of CAC expression and recombination of floxed alleles. All levels of DSS caused ulcerative colitis, with severity increasing in a dose dependent manner. With time, ulceration was repaired by regeneration and hyperplasia of remaining epithelial cells. Surprisingly, while only 10 percent of colon crypts express Cre in control mice, nearly 100% of regenerative foci and many adjacent hyperplastic crypts were positive for Bgal after DSS exposure. After 2-3 epithelial replacement cycles (10 d post-DSS), all 3 doses significantly increased Bgal labeling (0.65% DSS-18.4%±.06, 1.35% DSS-17.7%± .04, 2% DSS-18.2%±.06) relative to controls (10.2%±.04). Similar results were seen in CAC-Rosa26R mice with normal Apc alleles, suggesting that the induction of the CAC transgene is directly attributable to DSS. Despite similar allelic recombination throughout the proximal and distal colon after DSS treatment, 10 wk old CAC-ApcΔ580/+ mice treated with 2% DSS for 5 days, developed more tumors at 15 wks of age in the distal colon (74%) than in the proximal colon (26%). This suggests that Apc allele deletion differentially influences cancer risk in proximal versus distal colon, which is consistent with observations of higher Apc mutation rates in human distal colorectal tumors. In conclusion, we report that administration of DSS at doses as low as 0.65% for 5 days to CAC mice causes mild ulcerative colitis that induces transgene expression and recombination of floxed alleles throughout the colon. This model provides a novel tool to differentiate the impact of gene by environment interaction in colon epithelial cells following colitis and offers an innovative approach to discover the mechanisms responsible for disparate pathways to colorectal carcinogenesis in the proximal versus distal colon. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4417. doi:1538-7445.AM2012-4417