Human immunodeficiency virus type 1 viral background plays a major role in development of resistance to protease inhibitors.

The observed in vitro and in vivo benefit of combination treatment with anti-human immunodeficiency virus (HIV) agents prompted us to examine the potential of resistance development when two protease inhibitors are used concurrently. Recombinant HIV-1 (NL4-3) proteases containing combined resistance mutations associated with BMS-186318 and A-77003 (or saquinavir) were either inactive or had impaired enzyme activity. Subsequent construction of HIV-1 (NL4-3) proviral clones containing the same mutations yielded viruses that were severely impaired in growth or nonviable, confirming that combination therapy may be advantageous. However, passage of BMS-186318-resistant HIV-1 (RF) in the presence of either saquinavir or SC52151, which represented sequential drug treatment, produced viable viruses resistant to both BMS-186318 and the second compound. The predominant breakthrough virus contained the G48V/A71T/V82A protease mutations. The clone-purified RF (G48V/A71T/V82A) virus, unlike the corresponding defective NL4-3 triple mutant, grew well and displayed cross-resistance to four distinct protease inhibitors. Chimeric virus and in vitro mutagenesis studies indicated that the RF-specific protease sequence, specifically the Ile at residue 10, enabled the NL4-3 strain with the triple mutant to grow. Our results clearly indicate that viral genetic background will play a key role in determining whether cross-resistance variants will arise.

Refining animal models in fracture research: Seeking consensus in optimising both animal welfare and scientific validity for appropriate biomedical use

Background In an attempt to establish some consensus on the proper use and design of experimental animal models in musculoskeletal research, AOVET (the veterinary specialty group of the AO Foundation) in concert with the AO Research Institute (ARI), and the European Academy for the Study of Scientific and Technological Advance, convened a group of musculoskeletal researchers, veterinarians, legal experts, and ethicists to discuss, in a frank and open forum, the use of animals in musculoskeletal research. Methods The group narrowed the field to fracture research. The consensus opinion resulting from this workshop can be summarized as follows: Results & Conclusion Anaesthesia and pain management protocols for research animals should follow standard protocols applied in clinical work for the species involved. This will improve morbidity and mortality outcomes. A database should be established to facilitate selection of anaesthesia and pain management protocols for specific experimental surgical procedures and adopted as an International Standard (IS) according to animal species selected. A list of 10 golden rules and requirements for conduction of animal experiments in musculoskeletal research was drawn up comprising 1) Intelligent study designs to receive appropriate answers; 2) Minimal complication rates (5 to max. 10%); 3) Defined end-points for both welfare and scientific outputs analogous to quality assessment (QA) audit of protocols in GLP studies; 4) Sufficient details for materials and methods applied; 5) Potentially confounding variables (genetic background, seasonal, hormonal, size, histological, and biomechanical differences); 6) Post-operative management with emphasis on analgesia and follow-up examinations; 7) Study protocols to satisfy criteria established for a "justified animal study"; 8) Surgical expertise to conduct surgery on animals; 9) Pilot studies as a critical part of model validation and powering of the definitive study design; 10) Criteria for funding agencies to include requirements related to animal experiments as part of the overall scientific proposal review protocols. Such agencies are also encouraged to seriously consider and adopt the recommendations described here when awarding funds for specific projects. Specific new requirements and mandates related both to improving the welfare and scientific rigour of animal-based research models are urgently needed as part of international harmonization of standards.

Calcitonin receptor plays a physiological role to protect against hypercalcemia in mice

It is well established that calcitonin is a potent inhibitor of bone resorption; however, a physiological role for calcitonin acting through its cognate receptor, the calcitonin receptor (CTR), has not been identified. Data from previous genetically modified animal models have recognized a possible role for calcitonin and the CTR in controlling bone formation; however, interpretation of these data are complicated, in part because of their mixed genetic background. Therefore, to elucidate the physiological role of the CTR in calcium and bone metabolism, we generated a viable global CTR knockout (KO) mouse model using the Cre/loxP system, in which the CTR is globally deleted by >94% but <100%. Global CTRKOs displayed normal serum ultrafiltrable calcium levels and a mild increase in bone formation in males, showing that the CTR plays a modest physiological role in the regulation of bone and calcium homeostasis in the basal state in mice. Furthermore, the peak in serum total calcium after calcitriol [1,25(OH)2D3]-induced hypercalcemia was substantially greater in global CTRKOs compared with controls. These data provide strong evidence for a biological role of the CTR in regulating calcium homeostasis in states of calcium stress.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

The epithelial to mesenchymal transition and metastatic progression in carcinoma

Many data have emerged in support of the concept that the promotion of a migratory phenotype in epithelial cells is the result of an EMT, which leads to the loss of differentiation and to the acquisition of several mesenchymal features. Such an event is likely to occur during the metastatic conversion enabling the metastatic cell to invade the connective tissue and disseminate. Even though it cannot be excluded that some cancer cells might constitutively express a metastatic phenotype, the EMT implicated in the tumor progression process would rather be transient, induced in certain cells of the primary tumor by different factors, and reversed when the cells settle down in secondary organs (as schematically represented in Figure 1). However, it is clear that the phenotype that would emerge from the EMT occurring during tumor progression would also be modulated by the genetic background of the cells and must be to some extent different than the ones observed in normal physiological processes, and must also vary from one tumor to another.

Myopic anisometropia : ocular characteristics and aetiological considerations

Anisometropia represents a unique example of ocular development, where the two eyes of an individual, with an identical genetic background and seemingly subject to identical environmental influences, can grow asymmetrically to produce significantly different refractive errors. This review provides an overview of the research examining myopic anisometropia, the ocular characteristics underlying the condition and the potential aetiological factors involved. Various mechanical factors are discussed, including corneal structure, intraocular pressure and forces generated during near work that may contribute to development of anisomyopia. Potential visually guided mechanisms of unequal ocular growth are also explored, including the influence of astigmatism, accommodation, higher-order aberrations and the choroidal response to altered visual experience. The association between binocular vision, ocular dominance and asymmetric refraction is also considered, along with a review of the genetic contribution to the aetiology of myopic anisometropia. Despite a significant amount of research into the biomechanical, structural and optical characteristics of anisometropic eyes, there is still no unifying theory, which adequately explains how two eyes within the same visual system grow to different endpoints.

Modelling Leaf Production and Crop Development in Maize (Zea mays L.) After Tassel Initiation Under Diverse Conditions of Temperature and Photoperiod

Prediction of the initiation, appearance and emergence of leaves is critically important to the success of simulation models of crop canopy development and some aspects of crop ontogeny. Data on leaf number and crop ontogeny were collected on five cultivars of maize differing widely in maturity and genetic background grown under natural and extended photoperiods, and planted on seven sowing dates from October 1993 to March 1994 at Gatton, South-east Queensland. The same temperature coefficients were established for crop ontogeny before silking, and the rates of leaf initiation, leaf tip appearance and full leaf expansion, the base, optimum and maximum temperatures for each being 8, 34 and 40 degrees C. After silking, the base temperature for ontogeny was 0 degrees C, but the optimum and maximum temperatures remained unchanged. The rates of leaf initiation, appearance of leaf tips and full leaf expansion varied in a relatively narrow range across sowing times and photoperiod treatments, with average values of 0.040 leaves (degrees Cd)-1, 0.021 leaves (degrees Cd)-1, and 0.019 leaves (degrees Cd)-1, respectively. The relationships developed in this study provided satisfactory predictions of leaf number and crop ontogeny (tassel initiation to silking, emergence to silking and silking to physiological maturity) when assessed using independent data from Gatton (South eastern Queensland), Katherine and Douglas Daly (Northern Territory), Walkamin (North Queensland) and Kununurra (Western Australia).

Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea)

Background: Cultivated peanut or groundnut (Arachis hypogaea L.) is the fourth most important oilseed crop in the world, grown mainly in tropical, subtropical and warm temperate climates. Due to its origin through a single and recent polyploidization event, followed by successive selection during breeding efforts, cultivated groundnut has a limited genetic background. In such species, microsatellite or simple sequence repeat (SSR) markers are very informative and useful for breeding applications. The low level of polymorphism in cultivated germplasm, however, warrants a need of larger number of polymorphic microsatellite markers for cultivated groundnut. Results: A microsatellite- enriched library was constructed from the genotype TMV2. Sequencing of 720 putative SSR-positive clones from a total of 3,072 provided 490 SSRs. 71.2% of these SSRs were perfect type, 13.1% were imperfect and 15.7% were compound. Among these SSRs, the GT/CA repeat motifs were the most common (37.6%) followed by GA/CT repeat motifs (25.9%). The primer pairs could be designed for a total of 170 SSRs and were optimized initially on two genotypes. 104 (61.2%) primer pairs yielded scorable amplicon and 46 (44.2%) primers showed polymorphism among 32 cultivated groundnut genotypes. The polymorphic SSR markers detected 2 to 5 alleles with an average of 2.44 per locus. The polymorphic information content (PIC) value for these markers varied from 0.12 to 0.75 with an average of 0.46. Based on 112 alleles obtained by 46 markers, a phenogram was constructed to understand the relationships among the 32 genotypes. Majority of the genotypes representing subspecies hypogaea were grouped together in one cluster, while the genotypes belonging to subspecies fastigiata were grouped mainly under two clusters. Conclusion. Newly developed set of 104 markers extends the repertoire of SSR markers for cultivated groundnut. These markers showed a good level of PIC value in cultivated germplasm and therefore would be very useful for germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships in cultivated groundnut as well as related Arachis species.

Genotypic variation in seedling root architectural traits and implications for drought adaptation in wheat (Triticum aestivum L.)

Root system characteristics are of fundamental importance to soil exploration and below-ground resource acquisition. Root architectural traits determine the in situ space-filling properties of a root system or root architecture. The growth angle of root axes is a principal component of root system architecture that has been strongly associated with acquisition efficiency in many crop species. The aims of this study were to examine the extent of genotypic variability for the growth angle and number of seminal roots in 27 current Australian and 3 CIMMYT wheat (Triticum aestivum L.) genotypes, and to quantify using fractal analysis the root system architecture of a subset of wheat genotypes contrasting in drought tolerance and seminal root characteristics. The growth angle and number of seminal roots showed significant genotypic variation among the wheat genotypes with values ranging from 36 to 56 (degrees) and 3 to 5 (plant-1), respectively. Cluster analysis of wheat genotypes based on similarity in their seminal root characteristics resulted in four groups. The group composition reflected to some extent the genetic background and environmental adaptation of genotypes. Wheat cultivars grown widely in the Mediterranean environments of southern and western Australia generally had wider growth angle and lower number of seminal axes. In contrast, cultivars with superior performance on deep clay soils in the northern cropping region, such as SeriM82, Baxter, Babax, and Dharwar Dry exhibited a narrower angle of seminal axes. The wheat genotypes also showed significant variation in fractal dimension (D). The D values calculated for the individual segments of each root system suggested that, compared to the standard cultivar Hartog, the drought-tolerant genotypes adapted to the northern region tended to distribute relatively more roots in the soil volume directly underneath the plant. These findings suggest that wheat root system architecture is closely linked to the angle of seminal root axes at the seedling stage. The implications of genotypic variation in the seminal root characteristics and fractal dimension for specific adaptation to drought environment types are discussed with emphasis on the possible exploitation of root architectural traits in breeding for improved wheat cultivars for water-limited environments.

Candidate gene studies on cardiovascular traits

Cardiovascular diseases (CVD) are a major cause of death and disability in Western countries and a growing health problem in the developing world. The genetic component of both coronary heart disease (CHD) and ischemic stroke events has been established in twin studies, and the traits predisposing to CVD, such as hypertension, dyslipidemias, obesity, diabetes, and smoking behavior, are all partly hereditary. Better understanding of the pathophysiology of CVD-related traits could help to target disease prevention and clinical treatment to individuals at an especially high disease risk and provide novel pharmaceutical interventions. This thesis aimed to clarify the genetic background of CVD at a population level using large Nordic population cohorts and a candidate gene approach. The first study concentrated on the allelic diversity of the thrombomodulin (THBD) gene in two Finnish cohorts, FINRISK-92 and FINRISK-97. The results from this study implied that THBD variants do not substantially contribute to CVD risk. In the second study, three other candidate genes were added to the analyses. The study investigated the epistatic effects of coagulation factor V (F5), intercellular adhesion molecule -1 (ICAM1), protein C (PROC), and THBD in the same FINRISK cohorts. The results were encouraging; we were able to identify several single SNPs and SNP combinations associating with CVD and mortality. Interestingly, THBD variants appeared in the associating SNP combinations despite the negative results from Study I, suggesting that THBD contributes to CVD through gene-gene interactions. In the third study, upstream transcription factor -1 (USF1) was analyzed in a cohort of Swedish men. USF1 was associated with metabolic syndrome, characterized by accumulation of different CVD risk factors. A putative protective and a putative risk variant were identified. A direct association with CVD was not observed. The longitudinal nature of the study also clarified the effect of USF1 variants on CVD risk factors followed in four examinations throughout adulthood. The three studies provided valuable information on the study of complex traits, highlighting the use of large study samples, the importance of replication, and the full coverage of the major allelic variants of the target genes to assure reliable findings. Although the genetic basis of coronary heart disease and ischemic stroke remains unknown, single genetic findings may facilitate the recognition of high-risk subgroups.

LKB1 tumor suppression in Peutz-Jeghers Syndrome

Kasvainten, ajatellaan syntyvän yksittäisen solun perimän mutaatioista, jonka seurauksena tuon solun kasvu häiriintyy. Ruoansulatuskanavan polyyppien syntyä käytetään usein mallina siitä, miten nämä epiteelisoluun kerääntyvät mutaatiot aiheuttavat asteittain pahenevan kasvuhäiriön. Peutz–Jeghersin oireyhtymä (PJS) on perinnöllinen polypoosisyndrooma, jossa oireita aiheuttavat erityisesti maha-suolikanavan hamartomatoottiset polyypit. Noin puolella PJS potilaista havaitaan mutaatioita LKB1 kasvunrajoite geenissä. Hiirille joilta toinen Lkb1 alleeli on poistettu (Lkb1+/-) kehittyy PJS-tyypin maha-suolikanavan polyyppeja, joissa on epiteelin liikakasvun lisäksi merkittävä sileälihaskomponentti, aivan kuten PJS polyypeissa. Kuten myös muissa ruoansulatuskanavan polypooseissa, sekä PJS että hiirten polyypeissa Cyclo-oxygenaasi-2:n (COX-2) määrä on usein kohonnut. PJS-polyyppien kehittymisen molekulaarinen mekanismi on kuitenkin selvittämättä. Koska vain osa PJS potilaista kantaa LKB1 mutaatioita, mutaatiot jossakin toisessa lokuksessa saattaisivat selittää osan PJS tapauksista. Jotta PJS:n geneettinen tausta selviäisi, seulottiin kolmen LKB1:n kanssa interaktoivan proteiinin (BRG1, STRADα ja MO25α) geenit PJS potilaista joilla ei ole havaittu LKB1 mutaatioita. Yhdessäkään tutkituista geeneistä ei havaittu tautia aiheuttavia mutaatioita. Näiden kolmen geenin pois sulkeminen, ja uusien menetelmien ansiosta kasvanut havaittujen Lkb1 mutaatioden määrä viittaavat LKB1:n olevan useimpien PJS tapausten taustalla. COX-2:n estäjien käyttö on tehokkaasti vähentänyt polyyppien määrää familiaarisessa adenomatoottisessa polypoosissa. Tästä johtuen COX-2:n eston tehokkuutta tutkittiin PJS polypoosissa. PJS-tyypin polypoosin havaittin pienenevän merkittävästi Lkb1+/- hiirissä, joilta oli lisäksi poistettu toinen tai molemmat COX-2:n alleeleista. Lisäksi farmakologinen COX-2:n esto Celecoxib:lla vähensi polypoosia tehokkaasti. Näin ollen COX-2:n eston tehokkuutta tutkittiin seuraavaksi PJS potilaissa. Kuuden kuukauden Celecoxib hoidon jälkeen polypoosin havaittiin vähentyneen merkittävästi osalla potilaista (2/6). Nämä tulokset osoittavat COX-2:n roolin PJS-polyyppien kehityksessä, ja viittaavat COX-2:n eston vähentävän polypoosia. Kasvunrajoitegeenin klassisen määritelmän mukaan kasvaimen kehitys vaatii perinnöllisen mutaation lisäksi geenin toisenkin alleelin mutaation, mutta PJS-polyyppien häiriintyneestä epiteelistä ei kuitenkaan systemaattisesti löydy toista LKB1:n mutaatiota. Havainto johti tutkimukseen, jossa selvitettiin voisiko LKB1:n kasvun rajoitus välittyäkin epäsuorasti tukikudokseksi ajatelluista sileälihassoluista. Tätä tutkittiin kehittämällä poistogeeninen hiirimalli jossa Lkb1 on mutatoitunut vain sileälihassoluissa. Näille hiirille kehittyi polyyppeja, jotka ovat kaikin tavoin PJS-polyyppien kaltaisia. Lkb1:n menettäneiden solujen havaittiin tuottavan vähemmän transformoivaa kasvutekijä beetaa (TGFß), joka aiheutti solujen välisen viestinnän heikentymisen ja mahdollisesti viereisten epiteelisolujen liikakasvun. Vastaava häiriö havaittiin myös PJS-potilaiden polyypeissa, mikä viittaa siihen, että potilaillakin sileälihassolujen häiriö on polyyppien taustalla. Havainto suuntaa täten hoitokohteiden etsintää ja osoittaa että LKB1 toimii kasvunrajoittajana epätyypillisellä tavalla pitäen naapurisolujen kasvun kurissa.

Insights into the molecular genetics of celiac disease : Applying family- and population-based strategies

Celiac disease, or gluten intolerance, is triggered by dietary glutens in genetically susceptible individuals and it affects approximately 1% of the Caucasian population. The best known genetic risk factors for celiac disease are HLA DQ2 and DQ8 heterodimers, which are necessary for the development of the disease. However, they alone are not sufficient for disease induction, other risk factors are required. This thesis investigated genetic factors for celiac disease, concentrating on susceptibility loci on chromosomes 5q31-q33, 19p13 and 2q12 previously reported in genome-wide linkage and association studies. In addition, a novel genotyping method for the detection of HLA DQ2 and DQ8 coding haplotypes was validated. This study was conducted using Finnish and Hungarian family materials, and Finnish, Hungarian and Italian case-control materials. Genetic linkage and association were analysed in these materials using candidate gene and fine-mapping approaches. The results confirmed linkage to celiac disease on the chromosomal regions 5q31-q33 and 19p13. Fine-mapping on chromosome 5q31-q33 revealed several modest associations in the region, and highlighted the need for further investigations to locate the causal risk variants. The MYO9B gene on chromosome 19p13 showed evidence for linkage and association particularly with dermatitis herpetiformis, the skin manifestation of celiac disease. This implies a potential difference in the genetic background of the intestinal and skin forms of the disease, although studies on larger samplesets are required. The IL18RAP locus on chromosome 2q12, shown to be associated with celiac disease in a previous genome-wide association study and a subsequent follow-up, showed association in the Hungarian population in this study. The expression of IL18RAP was further investigated in small intestinal tissue and in peripheral blood mononuclear cells. The results showed that IL18RAP is expressed in the relevant tissues. Two putative isoforms of IL18RAP were detected by Western blot analysis, and the results suggested that the ratios and total levels of these isoforms may contribute to the aetiology of celiac disease. A novel genotyping method for celiac disease-associated HLA haplotypes was also validated in this thesis. The method utilises single-nucleotide polymorphisms tagging these HLA haplotypes with high sensitivity and specificity. Our results suggest that this method is transferable between populations, and it is suitable for large-scale analysis. In conclusion, this doctorate study provides an insight into the roles of the 5q31-q33, MYO9B, IL18RAP and HLA loci in the susceptibility to celiac disease in the Finnish, Hungarian and Italian populations, highlighting the need for further studies at these genetic loci and examination of the function of the candidate genes.

Fumarate Hydratase and Succinate Dehydrogenase in Neoplasia

Germline mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell cancer (HLRCC). FH is a nuclear encoded enzyme which functions in the Krebs tricarboxylic acid cycle, and homozygous mutation in FH lead to severe developmental defects. Both uterine and cutaneous leiomyomas are components of the HLRCC phenotype. Most of these tumours show loss of the wild-type allele and, also, the mutations reduce FH enzyme activity, which indicate that FH is a tumour suppressor gene. The renal cell cancers associated with HLRCC are of rare papillary type 2 histology. Other genes involved in the Krebs cycle, which are also implicated in neoplasia are 3 of the 4 subunits encoding succinate dehydrogenase (SDH); mutations in SHDB, SDHC, and SDHD predispose to paraganglioma and phaeochromocytoma. Although uterine leiomyomas (or fibroids) are very common, the estimations of affected women ranging from 25% to 77%, not much is known about their genetic background. Cytogenetic studies have revealed that rearrangements involving chromosomes 6, 7, 12 and 14 are most commonly seen in fibroids. Deletions on the long arm of chromosome 7 have been reported to be involved in about 17 to 34 % of leiomyomas and the small commonly deleted region on 7q22 suggests that there might be an underlying tumour suppressor gene in that region. The purpose of this study was to investigate the genetic mechanisms behind the development of tumours associated with HLRCC, both renal cell cancer and uterine fibroids. Firstly, a database search at the Finnish cancer registry was conducted in order to identify new families with early-onset RCC and to test if the family history was compatible with HLRCC. Secondly, sporadic uterine fibroids were tested for deletions on 7q in order to define the minimal deleted 7q-region, followed by mutation analysis of the candidate genes. Thirdly, oligonucleotide chips were utilised to study the global gene expression profiles of uterine fibroids in order to test whether 7q-deletions and FH mutations significantly affected fibroid biology. In the screen for early-onset RCC, 214 families were identified. Subsequently, the pedigrees were constructed and clinical data obtained. One of the index cases (RCC at the age of 28) had a mother who had been diagnosed with a heart tumour, which in further investigation turned out to be a paraganglioma. This lead to an alternative hypothesis that SDH, instead of FH, could be involved. SDHA, SDHB, SDHC and SDHD were sequenced from these individuals; a germline SDHB R27X mutation was detected with loss of the wild-type allele in both tumours. These results suggest that germline mutations in the SDHB gene predispose to early-onset RCC establishing a novel form of hereditary RCC. This has immediate clinical implications in the surveillance of patients suffering from early-onset RCC and phaeochromocytoma/paraganglioma. For the studies on sporadic uterine fibroids, a set of 166 fibroids from 51 individuals were collected. The 7q LOH mapping defined a commonly deleted region of about 3.2 mega bases in 11 of the 166 tumours. The deletion was consistent with previously reported allelotyping studies of leiomyomas and it therefore suggested the presence of a tumour suppressor gene in the deleted region. Furthermore, the high-resolution aCGH-chip analysis refined the deleted region to only 2.79Mb. When combined with previous data, the commonly deleted region was only 2.3Mb. The mutation screening of the known genes within the commonly deleted region did not reveal pathogenic mutations, however. The expression microarray analysis revealed that FH-deficient fibroids, both sporadic and familial, had their distinct gene expression profile as they formed their own group in the unsupervised clustering. On the other hand, the presence or absence of 7q-deletions did not significantly alter the global gene expression pattern of fibroids, suggesting that these two groups do not have different biological backgrounds. Multiple differentially expressed genes were identified between FH wild-type and FH-mutant fibroids, and the most significant increase was seen in the expression of carbohydrate metabolism-related and hypoxia inducible factor (HIF) target genes.

Molecular classification of familial colorectal and endometrial carcinoma

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common known clearly hereditary cause of colorectal and endometrial cancer (CRC and EC). Dominantly inherited mutations in one of the known mismatch repair (MMR) genes predispose to HNPCC. Defective MMR leads to an accumulation of mutations especially in repeat tracts, presenting microsatellite instability. HNPCC is clinically a very heterogeneous disease. The age at onset varies and the target tissue may vary. In addition, families that fulfill the diagnostic criteria for HNPCC but fail to show any predisposing mutation in MMR genes exist. Our aim was to evaluate the genetic background of familial CRC and EC. We performed comprehensive molecular and DNA copy number analyses of CRCs fulfilling the diagnostic criteria for HNPCC. We studied the role of five pathways (MMR, Wnt, p53, CIN, PI3K/AKT) and divided the tumors into two groups, one with MMR gene germline mutations and the other without. We observed that MMR proficient familial CRC consist of two molecularly distinct groups that differ from MMR deficient tumors. Group A shows paucity of common molecular and chromosomal alterations characteristic of colorectal carcinogenesis. Group B shows molecular features similar to classical microsatellite stable tumors with gross chromosomal alterations. Our finding of a unique tumor profile in group A suggests the involvement of novel predisposing genes and pathways in colorectal cancer cohorts not linked to MMR gene defects. We investigated the genetic background of familial ECs. Among 22 families with clustering of EC, two (9%) were due to MMR gene germline mutations. The remaining familial site-specific ECs are largely comparable with HNPCC associated ECs, the main difference between these groups being MMR proficiency vs. deficiency. We studied the role of PI3K/AKT pathway in familial ECs as well and observed that PIK3CA amplifications are characteristic of familial site-specific EC without MMR gene germline mutations. Most of the high-level amplifications occurred in tumors with stable microsatellites, suggesting that these tumors are more likely associated with chromosomal rather than microsatellite instability and MMR defect. The existence of site-specific endometrial carcinoma as a separate entity remains equivocal until predisposing genes are identified. It is possible that no single highly penetrant gene for this proposed syndrome exists, it may, for example be due to a combination of multiple low penetrance genes. Despite advances in deciphering the molecular genetic background of HNPCC, it is poorly understood why certain organs are more susceptible than others to cancer development. We found that important determinants of the HNPCC tumor spectrum are, in addition to different predisposing germline mutations, organ specific target genes and different instability profiles, loss of heterozygosity at MLH1 locus, and MLH1 promoter methylation. This study provided more precise molecular classification of families with CRC and EC. Our observations on familial CRC and EC are likely to have broader significance that extends to sporadic CRC and EC as well.

Phosphine Resistance in the Rust Red Flour Beetle, Tribolium castaneum (Coleoptera: Tenebrionidae): Inheritance, Gene Interactions and Fitness Costs

The recent emergence of heritable high level resistance to phosphine in stored grain pests is a serious concern among major grain growing countries around the world. Here we describe the genetics of phosphine resistance in the rust red flour beetle Tribolium castaneum (Herbst), a pest of stored grain as well as a genetic model organism. We investigated three field collected strains of T. castaneum viz., susceptible (QTC4), weakly resistant (QTC1012) and strongly resistant (QTC931) to phosphine. The dose-mortality responses of their test- and inter-cross progeny revealed that most resistance was conferred by a single major resistance gene in the weakly (3.2x) resistant strain. This gene was also found in the strongly resistant (431x) strain, together with a second major resistance gene and additional minor factors. The second major gene by itself confers only 12-206x resistance, suggesting that a strong synergistic epistatic interaction between the genes is responsible for the high level of resistance (431x) observed in the strongly resistant strain. Phosphine resistance is not sex linked and is inherited as an incompletely recessive, autosomal trait. The analysis of the phenotypic fitness response of a population derived from a single pair inter-strain cross between the susceptible and strongly resistant strains indicated the changes in the level of response in the strong resistance phenotype; however this effect was not consistent and apparently masked by the genetic background of the weakly resistant strain. The results from this work will inform phosphine resistance management strategies and provide a basis for the identification of the resistance genes.