Mutant Allele Of Gene Research Articles

Role of gene polymorphism in the development of disorders of the lipid profile in individuals exposed to chemicals

Introduction. The prevalence of arterial hypertension and dyslipidemia in the Russian population exceeds 50%. By a number of working industrial factors have been proved to play the negative role in their development. The results of earlier studies indicate to the epigenetic role of toxic substances in relation to various genes. The aim of the study was to establish associations of polymorphic variants of cardiovascular risk genes with disorders of lipid metabolism in workers performing liquidation works in the accumulated environmental risks zone. Materials and methods. Ninety two and 82 employees from Federal Environmental Operator (FEO) and EMERCOM, respectively were studied. Parameters of lipid metabolism and polymorphic variants of APOE Cys130Arg (rs429358) and PPARG Pro12Ala (rs1801282) genes were investigated. Results. In FEO workers, each variant allele of the APOE Cys130Arg gene is associated with impaired LDL-C concentration in an additive manner (OR = 2.69; 95% CI = 1.03–7.08, p=0.04). Carrying either the T/C or C/C variant allele of this polymorphic variant or the C/G or G/G polymorphic variant Pro12Ala of the PPARG gene increases the odds of developing abnormalities in total cholesterol levels by more than 3.5-times. The risk influence of the mutant genotype G/G and allele G on the increase of Apo B concentration was also established. In EMERCOM workers, the presence of both alleles of this polymorphic variant increased the probability of a decrease in HDL-H levels by 5 times. Limitations. Male persons are employees of the FEO and the Ministry of Emergency Situations. Age accounts of 30–50 years. Conclusions. An increased risk of deviations of total cholesterol concentration, proatherogenic cholesterol, and lipoprotein fractions associated with the PPARG Pro12Ala and APOE Cys130Arg genes polymorphisms was found in FEO workers, whereas only the reduced high-density lipoprotein cholesterol risk in carriers of the PPARG Pro12Ala gene mutant allele was found in EMERCOM workers.

Identification of two mutant JASON-RELATED genes associated with unreduced pollen production in potato

Key messageMultiple QTLs control unreduced pollen production in potato. Two major-effect QTLs co-locate with mutant alleles of genes with homology to AtJAS, a known regulator of meiotic spindle orientation.In diploid potato the production of unreduced gametes with a diploid (2n) rather than a haploid (n) number of chromosomes has been widely reported. Besides their evolutionary important role in sexual polyploidisation, unreduced gametes also have a practical value for potato breeding as a bridge between diploid and tetraploid germplasm. Although early articles argued for a monogenic recessive inheritance, the genetic basis of unreduced pollen production in potato has remained elusive. Here, three diploid full-sib populations were genotyped with an amplicon sequencing approach and phenotyped for unreduced pollen production across two growing seasons. We identified two minor-effect and three major-effect QTLs regulating this trait. The two QTLs with the largest effect displayed a recessive inheritance and an additive interaction. Both QTLs co-localised with genes encoding for putative AtJAS homologs, a key regulator of meiosis II spindle orientation in Arabidopsis thaliana. The function of these candidate genes is consistent with the cytological phenotype of mis-oriented metaphase II plates observed in the parental clones. The alleles associated with elevated levels of unreduced pollen showed deleterious mutation events: an exonic transposon insert causing a premature stop, and an amino acid change within a highly conserved domain. Taken together, our findings shed light on the natural variation underlying unreduced pollen production in potato and will facilitate interploidy breeding by enabling marker-assisted selection for this trait.

Polymorphic substitutions in folate cycle genes as predictors of hyperhomocysteinemia in children

Introduction. Mutant alleles of genes of folate cycle enzymes can lead to the significant deterioration of its function and varying severity of pathology. Several defects in these genes lead to severe hyperhomocysteinemia, the most common form of which is a deficiency of cystathionine beta-synthase B. 
 Aim: to establish polymorphic substitutions in the genes of folate cycle enzymes that contribute to the formation of hyperhomocysteinemia in children.
 Materials and methods. Two hundred seventy one children aged of 13–18 years were examined. The analysis of genetic polymorphisms of the folate cycle was carried out using a molecular genetic method. Quantitative determination of the blood homocysteine and folic acid level was performed by chemiluminescent immunoassay on microparticles. Statistical data processing was carried out using Statistica 6.1 application programs (StatSoft Inc., USA). 
 Results. The frequency of the T allele of the MTHFR 677 gene was revealed to be higher in adolescents of the main group compared with the control (p = 0.043). The frequency of the homozygous genotype 66 AA of the MTRR gene in children of the comparison group was significantly higher (p = 0.049), however, the heterozygous genotype 66 AG of the MTRR gene was significantly more often detected in adolescents of the main group (p = 0.008). The average concentrations of homocysteine in children of the main group were 11.6 mmol/L, in adolescents of the control group 9.3 mmol/L (p = 0.021). Hyperhomocysteinemia in children of the main group was detected in 217 (80.1%) adolescents, and in 57 (49.6%) children of the control group (p < 0.001). The baseline serum folate level was determined in the children of the main group. The average amount of vitamin B9 in the blood of children of the main group was 3.7 ng/ml, and in 145 (53.5%) children this indicator was significantly reduced.
 Conclusion. Low levels of folic acid contribute to an increase in homocysteine in blood plasma. Taking vitamin B9 and vitamin folate complexes significantly reduces the level of homocysteine in blood plasma (p < 0.001).

Phase 1/1b first-in-human study of IDRX-42, a novel oral tyrosine kinase inhibitor (TKI), in patients with metastatic and/or unresectable gastrointestinal stromal tumors (GISTs).

TPS483 Background: The majority of GISTs are driven by constitutively activated kinases, either KIT or platelet-derived growth factor-alpha ( PDGFRA), and respond to TKI treatment (eg, imatinib, sunitinib, regorafenib, ripretinib). Most GISTs develop heterogeneous resistance to TKI during treatment, usually driven by secondary mutations in the primary kinase oncogene ( KIT or PDGFRA). Novel therapies are needed to prevent emergence of resistant subclones or to treat TKI-resistant GISTs. IDRX-42 (formerly M4205) is a potent, highly selective, orally administered small-molecule TKI targeting disease-specific oncogenic drivers and a range of clinically relevant resistance mutations of KIT. Nonclinical studies of IDRX-42 have demonstrated robust antitumor activity in patient and cell line-derived GIST xenograft models, including models with relevant KIT mutations and TKI resistance mutations (De Sutter et al AACR 2022). Methods: IDRX-42-001 is a phase 1/1b, first-in-human, open-label, multicenter study to evaluate single-agent IDRX-42 in participants with metastatic and/or surgically unresectable GISTs. Phase 1 dose escalation utilizes a 3+3 design to evaluate safety, tolerability, PK, and preliminary antitumor activity of IDRX-42 to establish the MTD and RP2D. Phase 1b will consist of exploratory cohorts with selected lines of prior therapy to assess the antitumor activity of IDRX-42 and further characterize its safety profile at RP2D. Eligible participants are ≥18 yr old with ECOG PS 0–1, documented pathogenic mutation in KIT or any PDGFRA mutation other than exon 18 mutations, and ≥1 measurable lesion per mRECIST v1.1 by investigator assessment. Phase 1 dose-escalation participants must have documented progression on at least imatinib. For phase 1b exploratory cohorts, cohort 1 participants must have progressed on imatinib only (2nd line); cohort 2 participants must have progressed on imatinib and sunitinib (3rd line) or progressed on imatinib, sunitinib, and an additional agent (ie, regorafenib or ripretinib; 4th line); cohort 3 participants must have progressed on imatinib, sunitinib, regorafenib, and ripretinib (≥5th line). IDRX-42 will be initiated at 120 mg PO QD in 28-d cycles until disease progression, unacceptable toxicity, or other reason. Primary endpoints are to determine the nature, incidence, & severity of IDRX-42 TEAEs and DLTs (phase 1), and to evaluate safety and determine ORR per investigator assessed mRECIST v1.1 (phase 1b). Secondary efficacy endpoints include PK parameters, DoR, PFS, and time to response related to IDRX-42. Exploratory endpoints will evaluate potential predictive and pharmacodynamic markers using ctDNA, as well as changes in KIT, PDGFRA, and other relevant gene mutant allele fractions and their correlation with clinical outcomes. Enrollment is ongoing (NCT05489237). Clinical trial information: NCT05489237 .

Symbiotic Regulatory Genes Controlling Nodule Development in Pisum sativum L.

Analyses of natural variation and the use of mutagenesis and molecular-biological approaches have revealed 50 symbiotic regulatory genes in pea (Pisum sativum L.). Studies of genomic synteny using model legumes, such as Medicago truncatula Gaertn. and Lotus japonicus (Regel) K. Larsen, have identified the sequences of 15 symbiotic regulatory genes in pea. These genes encode receptor kinases, an ion channel, a calcium/calmodulin-dependent protein kinase, transcription factors, a metal transporter, and an enzyme. This review summarizes and describes mutant alleles, their phenotypic manifestations, and the functions of all identified symbiotic regulatory genes in pea. Some examples of gene interactions are also given. In the review, all mutant alleles in genes with identified sequences are designated and still-unidentified symbiotic regulatory genes of great interest are considered. The identification of these genes will help elucidate additional components involved in infection thread growth, nodule primordium development, bacteroid differentiation and maintenance, and the autoregulation of nodulation. The significance of symbiotic mutants of pea as extremely fruitful genetic models for studying nodule development and for comparative cell biology studies of legume nodules is clearly demonstrated. Finally, it is noted that many more sequences of symbiotic regulatory genes remain to be identified. Transcriptomics approaches and genome-wide sequencing could help address this challenge.

Development of a Pharmacogenetic Lab-on-Chip Assay Based on the In-Check Technology to Screen for Genetic Variations Associated to Adverse Drug Reactions to Common Chemotherapeutic Agents.

Background: Antineoplastic agents represent the most common class of drugs causing Adverse Drug Reactions (ADRs). Mutant alleles of genes coding for drug-metabolizing enzymes are the best studied individual risk factors for these ADRs. Although the correlation between genetic polymorphisms and ADRs is well-known, pharmacogenetic tests are limited to centralized laboratories with expensive or dedicated instrumentation used by specialized personnel. Nowadays, DNA chips have overcome the major limitations in terms of sensibility, specificity or small molecular detection, allowing the simultaneous detection of several genetic polymorphisms with time and costs-effective advantages. In this work, we describe the design of a novel silicon-based lab-on-chip assay able to perform low-density and high-resolution multi-assay analysis (amplification and hybridization reactions) on the In-Check platform. Methods: The novel lab-on-chip was used to screen 17 allelic variants of three genes associated with adverse reactions to common chemotherapeutic agents: DPYD (Dihydropyrimidine dehydrogenase), MTHFR (5,10-Methylenetetrahydrofolate reductase) and TPMT (Thiopurine S-methyltransferase). Results: Inter- and intra assay variability were performed to assess the specificity and sensibility of the chip. Linear regression was used to assess the optimal hybridization temperature set at 52 °C (R2 ≈ 0.97). Limit of detection was 50 nM. Conclusions: The high performance in terms of sensibility and specificity of this lab-on-chip supports its further translation to clinical diagnostics, where it may effectively promote precision medicine.

Mouse Models of Inherited Retinal Degeneration with Photoreceptor Cell Loss.

Inherited retinal degeneration (RD) leads to the impairment or loss of vision in millions of individuals worldwide, most frequently due to the loss of photoreceptor (PR) cells. Animal models, particularly the laboratory mouse, have been used to understand the pathogenic mechanisms that underlie PR cell loss and to explore therapies that may prevent, delay, or reverse RD. Here, we reviewed entries in the Mouse Genome Informatics and PubMed databases to compile a comprehensive list of monogenic mouse models in which PR cell loss is demonstrated. The progression of PR cell loss with postnatal age was documented in mutant alleles of genes grouped by biological function. As anticipated, a wide range in the onset and rate of cell loss was observed among the reported models. The analysis underscored relationships between RD genes and ciliary function, transcription-coupled DNA damage repair, and cellular chloride homeostasis. Comparing the mouse gene list to human RD genes identified in the RetNet database revealed that mouse models are available for 40% of the known human diseases, suggesting opportunities for future research. This work may provide insight into the molecular players and pathways through which PR degenerative disease occurs and may be useful for planning translational studies.

Vasoactive genes as molecular and genetic predictors of severe preeclampsia

Aim. To analyze an incidence rate of polymorphisms and combinations of genes AGT-704T> C, AGT-521C> T, AGTR1-1166A> C, AGTR2-1675G> A, еNO3-786T> C, еNOS3-894G> C, ADD1-1378G> T, CYP11B2 -304C> T, GNB3-825C> T and to assess their association with a risk of severe pre-eclampsia. Materials and methods. The study included women of early fertile age (20-35 years old) with spontaneous singleton pregnancy, no bad habits (smoking, alcohol or drug use), no extragenital diseases and no family (mother or sister) or an individual history of pre-eclampsia and with body mass index in the first trimester of pregnancy less than 35 kg/m2. The study group consisted of 100 patients with severe preeclampsia and the control group included 100 women with uncomplicated pregnancy. Genotyping was carried out by method of polymerase chain reaction. Data analysis included compliance with the Hardy - Weinberg law, Cramer's V criterion, χ2 test, odds ratio (OR) and its 95% confidence interval (CI). To assess a distribution of stated polymorphisms of genes and their alleles a general (χ2 test, df=2) and multiplicative (χ2 test, df=1) inheritance models were used. Results and discussion. Patients with severe pre-eclampsia had statistically significantly higher incidence rate of mutant homozygous genotypes AGTR1-1166CC (χ2=5,54; p=0,05) and еNO3-786CC (χ2=23,05; p=1,0E-5). A significant association between a carrier of the mutant homozygous genotype eNO3-786СC (χ2=19,780; p=0,000) and severe pre-eclampsia (OR 45,07, 95% CI 2,68-759,30) was found out. Combinations of mutant alleles of potentially predictive polymorphisms of vasoactive genes in women with preeclampsia were recorded 7,7 times more often (23% vs 3%; χ2=17,683, p=0,000; average Cramer's V link) which led to a significant association link with a risk of a disease complication (OR 9,658, 95% CI 2,795-33,367). Conclusion. A synergistic interaction between polymorphic loci in severe preeclampsia was established. The mutant homozygous genotype eNO3-786СC as well as a combination of at least two mutant alleles of genes - candidates for arterial hypertension or their combination with the mutant gene ADD1-1378TT or GNB3-825TT can claim a role of molecular and genetic predictors of severe preeclampsia.

Identification of Novel Alleles Conferring Superior Production of Rose Flavor Phenylethyl Acetate Using Polygenic Analysis in Yeast.

ABSTRACTFlavor compound metabolism is one of the last areas in metabolism where multiple genes encoding biosynthetic enzymes are still unknown. A major challenge is the involvement of side activities of enzymes having their main function in other areas of metabolism. We have applied pooled-segregant whole-genome sequence analysis to identify novel Saccharomyces cerevisiae genes affecting production of phenylethyl acetate (2-PEAc). This is a desirable flavor compound of major importance in alcoholic beverages imparting rose- and honey-like aromas, with production of high 2-PEAc levels considered a superior trait. Four quantitative trait loci (QTLs) responsible for high 2-PEAc production were identified, with two loci each showing linkage to the genomes of the BTC.1D and ER18 parents. The first two loci were investigated further. The causative genes were identified by reciprocal allele swapping into both parents using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. The superior allele of the first major causative gene, FAS2, was dominant and contained two unique single nucleotide polymorphisms (SNPs) responsible for high 2-PEAc production that were not present in other sequenced yeast strains. FAS2 encodes the alpha subunit of the fatty acid synthetase complex. Surprisingly, the second causative gene was a mutant allele of TOR1, a gene involved in nitrogen regulation. Exchange of both superior alleles in the ER18 parent strain increased 2-PEAc production 70%, nearly to the same level as in the best superior segregant. Our results show that polygenic analysis combined with CRISPR/Cas9-mediated allele exchange is a powerful tool for identification of genes encoding missing metabolic enzymes and for development of industrial yeast strains generating novel flavor profiles in alcoholic beverages.

Pharmacogenetic technology in psychiatric practice

Pharmacogenetic tests allow to predict speed and features of a metabolism of antipsychotics depending on activity of fermental systems involved in its metabolism, proteins carriers. They are directed as on an assessment of efficiency of therapy by antipsychotics, and studying of shipping and predictive risk of formation of side effects depending on a genotype. The studied material Tests of a buccal epithelium. Techniques Release of nucleinic acids, extraction of metabolites, PCR-analysis, release of recombinant proteins, reconstruction of fermental activity, mass and spectrometer analysis. Methods of diagnostics and additional researches include сomplex clinical inspection – objective survey, collecting of anamnestic data, analysis of experience of treatment by psychotropic medicines. Additional criteria of inclusion in research group: age from 18 to 35 years, lack of secondary resistance, absence of any associated diseases, Belarusians on a nationality, not relatives each other, with hospitalization prospect not less than 4 weeks, signed the informed consent to participation in project. Neurophysiological inspection – visual and computer EEG. Results Sixty-nine patients with schizophrenia were examined. Genomic DNA was used further for identification of mutant alleles of genes of CYP2C9 (*2/*3), CYP2C19 (*2/*3), CYP1A2, CYP2D6. Personal medical recommendations were made to all of patients. The dosage of a neuroleptic was changed depending on result of genotyping. Eighty-five percent of patients received good therapeutic effect in form of a full reduction of psychotic symptoms. Preliminary results show efficiency and prospects of pharmacogenetic technologies in psychiatric practice.

Modification of immune response genes in various types of chronic lymphocytic leukemia

Genetic factors not only contribute to the development of chronic lymphocytic leukemia (CLL), but also can affect the nature of the pathological process. The study included 30 patients with CLL with indolent and progressive course of the disease. The patients were tested on prevalence of genetic polymorphisms in the genes of the innate immune response. Nineteen polymorphic loci in 14 genes were tested. It was found that with indolent course of CLL, mutant alleles of genes of tumor necrosis factor (TNF) (G-308A), Toll-like receptor (tolllike receptor - TLR) TLR9 (T-1237C) and TLR2 (Arg753Gln) (OR: 4.70, p = 0.05 and OR: 8.33, p = 0.03 and OR: 5.50, p = 0.05, respectively) statistically, were encountered much more frequently . In contrast, combination of normal homozygotes of these genes greatly increased the risk of progressive form of the disease. Presence of TNF gene polymorphism in the polymorphic locus -308, TLR2-753, TLR9-1237 can be associated with the nature of the clinical course of CLL to encourage use these markers as early additional diagnostic and prognostic criteria for unfavorable form of leukemic process in order to optimize chemotherapy.

Endemic Opisthorchis Felineus Infection Limits Changes of Individual with Mutant Cyp1a1 and Gstm1 Genes to be Effectively Involved in Oil Field Working in the North of Siberia

More than 90% of the population of the north of Siberia (Russian Federation) inhabiting near oil field sites is infected with the en- demic helminth of Opisthorchis felineus. We demonstrated that the oil industry workers with the length of service at oil fields more than ten years do not have homozygous mutant alleles of CYP1A1 and GSTM1 genes of xenobiotic biotransformation system. Moreover, opisthorchiasis infection induces among the workers with homozygous mutant alleles of the genes significant increase in the frequency of cytogenetical abnormal cells as well as the deterioration of their health. It has been found that the Opisthorchis felineus infected workers with mutant alleles of the genes generally fail to work effectively and are retired. The habit of smoking among the workers also significantly increased the frequency of micronucleated cells.

Mak5 and Ebp2 Act Together on Early Pre-60S Particles and Their Reduced Functionality Bypasses the Requirement for the Essential Pre-60S Factor Nsa1

Ribosomes are the molecular machines that translate mRNAs into proteins. The synthesis of ribosomes is therefore a fundamental cellular process and consists in the ordered assembly of 79 ribosomal proteins (r-proteins) and four ribosomal RNAs (rRNAs) into a small 40S and a large 60S ribosomal subunit that form the translating 80S ribosomes. Most of our knowledge concerning this dynamic multi-step process comes from studies with the yeast Saccharomyces cerevisiae, which have shown that assembly and maturation of pre-ribosomal particles, as they travel from the nucleolus to the cytoplasm, relies on a multitude (>200) of biogenesis factors. Amongst these are many energy-consuming enzymes, including 19 ATP-dependent RNA helicases and three AAA-ATPases. We have previously shown that the AAA-ATPase Rix7 promotes the release of the essential biogenesis factor Nsa1 from late nucleolar pre-60S particles. Here we show that mutant alleles of genes encoding the DEAD-box RNA helicase Mak5, the C/D-box snoRNP component Nop1 and the rRNA-binding protein Nop4 bypass the requirement for Nsa1. Interestingly, dominant-negative alleles of RIX7 retain their phenotype in the absence of Nsa1, suggesting that Rix7 may have additional nuclear substrates besides Nsa1. Mak5 is associated with the Nsa1 pre-60S particle and synthetic lethal screens with mak5 alleles identified the r-protein Rpl14 and the 60S biogenesis factors Ebp2, Nop16 and Rpf1, which are genetically linked amongst each other. We propose that these ’Mak5 cluster’ factors orchestrate the structural arrangement of a eukaryote-specific 60S subunit surface composed of Rpl6, Rpl14 and Rpl16 and rRNA expansion segments ES7L and ES39L. Finally, over-expression of Rix7 negatively affects growth of mak5 and ebp2 mutant cells both in the absence and presence of Nsa1, suggesting that Rix7, at least when excessively abundant, may act on structurally defective pre-60S subunits and may subject these to degradation.

21-Hydroxylase gene mutant allele CYP21A2∗15 strongly linked to the resistant HLA haplotype B∗14:02-DRB1∗01:02 in chronic Chagas disease

We previously reported protective haplotype HLA-B*14:02-DRB1*01:02 against chronic Chagas disease in Bolivia. The V281L mutant allele of the 21-Hydroxylase gene, CYP21A2*15, is reported to be located in the Class III region of the Human leukocyte antigen region and linked to the haplotype HLA-B*14:02-DRB1*01:02. The mutant allele might play a primary role in the pathogenesis of chronic Chagas disease in the associated HLA region. We analyzed the frequency of this allele in the same subjects for the previous one. The statistical analysis showed a significant association of the CYP21A2*15 with resistance to severe chronic Chagas disease (OR=0.207273; Pv=0.0041). However, there is no significant tendency of the mutant gene contribution to the resistance after the elimination of the HLA-B*14:02-DRB1*01:02 linked mutants (OR=0.38; Pv=0.1533). Although the frequency of the CYP21A2*15 was small, we found no primary contribution of this mutation to the protection against chronic Chagas disease.

Fraser Syndrome: Two millennia of cryptophthalmos from Pliny the Elder to FRAS, FREM and GRIP: A historical perspective

From its first description in antiquity, the place of cryptophthalmos in the history of medical genetics is briefly set out until, in the twentieth century, this rare constellation of multiple congenital malformations of which cryptophthalmos is the most striking, even though not obligatory, component, was identified as an inherited autosomal recessive condition. It was given the name of Fraser syndrome and mutant alleles of the genes FRAS1, FREM2 and GRIP1 were identified as being responsible for a proportion of cases. In the remainder of cases, it may be supposed that mutant alleles of other genes, as yet unidentified, are responsible. In general, this association of multiple disparate malformations in an autosomal recessive condition may be expected to throw light on important aspects of gene action in embryogenesis. An aspect of medical genetics, which has become important with respect to the condition, is antenatal diagnosis with the prospect of abortion of affected fetuses.

The p150Glued CAP-Gly Domain Regulates Initiation of Retrograde Transport at Synaptic Termini

p150(Glued) is the major subunit of dynactin, a complex that functions with dynein in minus-end-directed microtubule transport. Mutations within the p150(Glued) CAP-Gly microtubule-binding domain cause neurodegenerative diseases through an unclear mechanism. A p150(Glued) motor neuron degenerative disease-associated mutation introduced into the Drosophila Glued locus generates a partial loss-of-function allele (Gl(G38S)) with impaired neurotransmitter release and adult-onset locomotor dysfunction. Disruption of the p150(Glued) CAP-Gly domain in neurons causes a specific disruption of vesicle trafficking atterminal boutons (TBs), the distal-most ends of synapses. Gl(G38S) larvae accumulate endosomes along with dynein and kinesin motor proteins within swollen TBs, and genetic analyses show that kinesin and p150(Glued) function cooperatively at TBs to coordinate transport. Therefore, the p150(Glued) CAP-Gly domain regulates dynein-mediated retrograde transport at synaptic termini, and this function of dynactin is disrupted by a mutation that causes motorneuron disease.

Abstract A46: A new transgenic mouse model of prostate cancer that displays rapid progression from prostate intraepithelial neoplasia to invasive carcinoma.

Abstract Understanding the molecular, biochemical, and cellular mechanisms that underpin prostate cancer progression will reveal new opportunities for targeted therapies. Our NCI-supported Program Project on “Signaling and Progression in Prostate Cancer” is a team of basic and clinician scientists that share the overall goal of defining how prostate cancer cells subvert signal transduction pathways during progression to androgen independence. Projects and cores within our program are developing new model systems intended to recapitulate human disease progression. Core B in the program (Transgenic Models and Animal Imaging) has developed a new transgenic mouse model by combining mutant alleles of genes known to have important roles in human prostate cancer. The model is based on the phosphatase and tensin homolog (Pten) tumor suppressor, which is mutated in approximately 30% of primary prostate cancers. Prostate-specific deletion of Pten in mice has been shown by multiple groups to cause prostatic intraepithelial neoplasia (PIN), which slowly progresses to invasive cancer. This suggests that Pten loss allows the initiation of cancer, but that progression is subject to restraint mechanisms. We hypothesized that a restraint mechanism might depend on signaling via the TGFβ pathway, owing to the prominent role that TGFβ plays in growth control. We show that conditional deletion of Pten and Tgfbr2, the type II receptor for TGFβ, in prostate epithelium results in large, poorly differentiated prostate tumors with 100% penetrance by 12 weeks of age. Mice with Pten and Tgfbr2 deletions acquire invasive adenocarcinoma and bladder obstruction and most die by 15 weeks of age. Androgen depletion by castration does not provide a survival benefit for these mice. Pten loss causes an increase in expression of the cyclin-dependent kinase inhibitor p27, which likely restrains progression in this setting. However, when both Pten and Tgfbr2 are deleted, p27 levels are reduced and cyclin D levels increase. Our results suggest that Tgfbr2 is an important tumor suppressor in prostate cancer and that its loss allows rapid progression when combined with loss of the Pten tumor suppressor. Disruption of the TGFβ-mediated restraint provides a rapid, penetrant, and physiologically relevant preclinical model that will be helpful for analyzing restraint- and progression-related gene expression and signal transduction changes in Pten-dependent tumorigenesis. Citation Format: Glen Bjerke, Chun-Song Yang, Henry Frierson, Bryce Paschal, David Wotton. A new transgenic mouse model of prostate cancer that displays rapid progression from prostate intraepithelial neoplasia to invasive carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A46.

Interaction between axons and specific populations of surrounding cells is indispensable for collateral formation in the mammillary system.

BackgroundAn essential phenomenon during brain development is the extension of long collateral branches by axons. How the local cellular environment contributes to the initial sprouting of these branches in specific points of an axonal shaft remains unclear.Methodology/Principal FindingsThe principal mammillary tract (pm) is a landmark axonal bundle connecting ventral diencephalon to brainstem (through the mammillotegmental tract, mtg). Late in development, the axons of the principal mammillary tract sprout collateral branches at a very specific point forming a large bundle whose target is the thalamus. Inspection of this model showed a number of distinct, identified cell populations originated in the dorsal and the ventral diencephalon and migrating during development to arrange themselves into several discrete groups around the branching point. Further analysis of this system in several mouse lines carrying mutant alleles of genes expressed in defined subpopulations (including Pax6, Foxb1, Lrp6 and Gbx2) together with the use of an unambiguous genetic marker of mammillary axons revealed: 1) a specific group of Pax6-expressing cells in close apposition with the prospective branching point is indispensable to elicit axonal branching in this system; and 2) cooperation of transcription factors Foxb1 and Pax6 to differentially regulate navigation and fasciculation of distinct branches of the principal mammillary tract.Conclusions/SignificanceOur results define for the first time a model system where interaction of the axonal shaft with a specific group of surrounding cells is essential to promote branching. Additionally, we provide insight on the cooperative transcriptional regulation necessary to promote and organize an intricate axonal tree.

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference

RNA interference (RNAi) technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs) are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G) alleles of human Presenilin1 gene (PSEN1). This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimer's disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2-Thiouridine chemical modification introduced at the 3′-end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide.

Abrupt telomere losses and reduced end-resection can explain accelerated senescence of Smc5/6 mutants lacking telomerase

The highly conserved Structural Maintenance of Chromosome (SMC) proteins are crucial for the formation of three essential complexes involved in high fidelity chromosome transmission during cell division. Recently, the Smc5/6 complex has been reported to be important for telomere maintenance in yeast and also in cancerous human ALT cells, where it could function in a homologous recombination-based (HR) telomere maintenance pathway. Here, we investigate the possible roles of the budding yeast Smc5/6 complex in maintaining appropriate chromosome end-structures allowing cell survival in absence of telomerase. The results show that cells harbouring mutant alleles of genes encoding Smc5/6-complex proteins rapidly stop growing after telomerase loss. Furthermore, this telomerase-induced growth arrest is much more pronounced as compared to cultures with a functional Smc5/6-complex. Bulk telomere sequence loss is not increased in the mutant cells and the evidence suggests that Smc5/6 slows senescence through a partially HR-independent pathway. We propose that in yeast, the Smc5/6-complex is required for efficient and timely termination of DNA replication and repair at telomeres to avoid stochastic telomere loss during cell division. Consistent with this hypothesis, sequencing of telomeres from telomerase-positive smc5/6 mutant cells revealed a higher frequency of telomere breakage events. Finally, the results also show that on dysfunctional telomeres, the generation of 3′-single stranded DNA is impaired, suggesting that the complex may also participate in the formation of single-stranded overhangs which are thought to be the substrates for telomere repeat replenishment in the absence of telomerase.