Unknown Genotype Research Articles

Efficient isolation of avian bornaviruses (ABV) from naturally infected psittacine birds and identification of a new ABV genotype from a salmon-crested cockatoo (Cacatua moluccensis)

Avian bornaviruses (ABV) have been discovered in 2008 as the causative agent of proventricular dilatation disease (PDD) in psittacine birds. To date, six ABV genotypes have been described in psittacines. Furthermore, two additional but genetically different ABV genotypes were recognized in non-psittacine birds such as canary birds and wild waterfowl. This remarkable genetic diversity poses a considerable challenge to ABV diagnosis, since polymerase chain reaction (PCR) assays may fail to detect distantly related or as yet unknown genotypes. In this study we investigated the use of virus isolation in cell culture as a strategy for improving ABV diagnosis. We found that the quail fibroblast cell line CEC-32 allows very efficient isolation of ABV from psittacine birds. Isolation of ABV was successful not only from organ samples but also from cloacal and pharyngeal swabs and blood samples collected intra vitam from naturally infected parrots. Importantly, using this experimental approach we managed to isolate a new ABV genotype, termed ABV-7, from a salmon-crested cockatoo (Cacatua moluccensis). Phylogenetic analysis showed that ABV-7 is most closely related to the psittacine genotypes ABV-1, -2, -3, and -4 and clearly distinct from genotypes ABV-5 and -6. Our successful identification of ABV-7 emphasizes the necessity to consider the high genetic diversity when trying to diagnose ABV infections with high reliability and further shows that classical virus isolation may represent a useful diagnostic option, particularly for the detection of new ABV genotypes.

An ensemble-based approach to imputation of moderate-density genotypes for genomic selection with application to Angus cattle

Summary Imputation of moderate-density genotypes from low-density panels is of increasing interest in genomic selection, because it can dramatically reduce genotyping costs. Several imputation software packages have been developed, but they vary in imputation accuracy, and imputed genotypes may be inconsistent among methods. An AdaBoost-like approach is proposed to combine imputation results from several independent software packages, i.e. Beagle(v3.3), IMPUTE(v2.0), fastPHASE(v1.4), AlphaImpute, findhap(v2) and Fimpute(v2), with each package serving as a basic classifier in an ensemble-based system. The ensemble-based method computes weights sequentially for all classifiers, and combines results from component methods via weighted majority 'voting' to determine unknown genotypes. The data included 3078 registered Angus cattle, each genotyped with the Illumina BovineSNP50 BeadChip. SNP genotypes on three chromosomes (BTA1, BTA16 and BTA28) were used to compare imputation accuracy among methods, and the application involved the imputation of 50K genotypes covering 29 chromosomes based on a set of 5K genotypes. Beagle and Fimpute had the greatest accuracy among the six imputation packages, which ranged from 0·8677 to 0·9858. The proposed ensemble method was better than any of these packages, but the sequence of independent classifiers in the voting scheme affected imputation accuracy. The ensemble systems yielding the best imputation accuracies were those that had Beagle as first classifier, followed by one or two methods that utilized pedigree information. A salient feature of the proposed ensemble method is that it can solve imputation inconsistencies among different imputation methods, hence leading to a more reliable system for imputing genotypes relative to independent methods.

PMO-181 Long term follow-up of chronic hepatitis B (HBV) patients treated with pegylated interferon: single centre experience

IntroductionPegylated Interferon (PEG IFN) is NICE approved for the treatment of chronic hepatitis B (HBV) but it is now known that the response is genotype dependent. To analyse the long...

PMO-180 Raised serum immunoglobulins in chronic hepatitis C: incidence and association with genotype, liver fibrosis and sustained viral response

IntroductionSerum Immunoglobulins (Igs) are commonly raised in Chronic Hepatitis C (HCV) but their clinical significance is not fully known. There is also little information on the normalisation of Igs post...

The efficacy of targeted next-generation sequencing for detection of clinically actionable mutations in cancer.

10598 Background: The emergence of next-generation sequencing (NGS) technologies has significantly accelerated the identification of cancer-causing mutations and the development of personalized cancer care. However, the clinical application of these technologies to detect cancer gene mutations has been extremely limited due to the long turn around time, the high cost, and large amount of input DNA required by existing NGS-based tests. Methods: We have assessed the performance of a novel NGS technology that merges multiplex PCR with ion semiconductor sequencing (AmpliSeq, Life Technologies, Inc.) in our clinical diagnostic laboratory. The test interrogates 739 common mutations in 46 cancer genes including many clinically actionable mutations concurrently. First, we studied 12 tumor samples including 4 archived FFPE, 4 blood/bone marrow, and 4 cell line samples with known mutations to evaluate the sensitivity and specificity of the test. We then studied 34 de-identified, archived FFPE tumor samples of unknown genotype to further evaluate the efficacy of the test. Results: We successfully identified all known mutations previously detected by Pyposequencing or Sanger sequencing technologies. Multiple serial dilution studies showed that the test could detect mutations at frequencies as low as 5% with 99% confidence. For the samples of unknown genotype, we detected 23 COSMIC mutations in 16 samples including HRAS, BRAF, MET, TP53 mutations in lung cancer, KRAS, PIK3CA, TP53, APC, BRAF, ERBB2 mutations in colon cancer, TP53 and KRAS mutations in breast cancer, and KIT and PDGFRA mutations in GIST. Analysis of the variant call data showed that a minimum of 100X coverage is required in order to detect mutations at 10% frequency or above; a minimum 300K final library reads are necessary in order to minimize/eliminate amplicon dropout. Conclusions: The targeted NGS test can effectively detect cancer gene mutation with input DNA as low as a few nanograms, turn around time can be as short as two days, and can significantly lower cost compared to traditional Sanger sequencing. Our experience demonstrates that this technology holds great potential for clinical use, including diagnostic and therapeutic applications.

Chemotherapy with erlotinib or chemotherapy alone in advanced NSCLC with acquired resistance to EGFR tyrosine kinase inhibitors (TKI).

7524 Background: EGFR-mutant NSCLC has an oncogene-addicted phenotype that confers sensitivity to EGFR TKIs. Prior data suggests EGFR addiction persists after development of TKI resistance, leading many clinicians to continue TKI along with chemotherapy (chemo); however, this strategy has not been formally evaluated. Methods: An institutional database was reviewed to identify patients (pts) with advanced NSCLC and acquired resistance to EGFR TKIs by Jackman criteria. Pts were included if they subsequently received chemo. Objective response rate (RR) to chemo/erlotinib or chemo alone was assessed by blinded radiographic review and compared by Fisher’s exact test and multivariable logistic regression. Progression-free survival (PFS) and overall survival (OS) from TKI failure (defined as chemo initiation date) were compared by log-rank test and multivariable Cox analysis. Results: 78 pts were eligible (34 chemo/erlotinib, 44 chemo alone). 70 pts (90%) had a documented EGFR mutation and were on TKI for a median of 15 months (range 4-51); in the 8 pts with unknown genotype the median duration on TKI was 11 months (range 5-16). Baseline characteristics were well balanced except more pts received erlotinib as initial TKI in the chemo/erlotinib group. RR was evaluable in 57 pts and was higher with chemo/erlotinib compared to chemo alone (41% vs 18%; OR 0.31, 95% CI 0.09, 1.04; p=0.08). RR adjusted for chemo regimen and time to TKI failure yielded OR 0.20 (95% CI 0.05, 0.78; p=0.02), favoring chemo/erlotinib. Median PFS was 4.4 months in the chemo/erlotinib group and 4.2 months in the chemo alone group (crude HR=0.84, 95% CI 0.52, 1.38; p=0.50; adjusted HR 0.79, 95% CI 0.48, 1.29; p=0.34). There was no significant difference in OS in the crude or adjusted analyses. Conclusions: This is the first study, to our knowledge, to show that continuation of an EGFR TKI with chemo compared to chemo alone significantly increases the RR in pts with advanced NSCLC and acquired TKI resistance, though we did not observe an impact on PFS or OS. Continued concurrent TKI may be a valuable strategy, particularly for pts with symptomatic progression, when a higher response rate may be beneficial. Further study is warranted.

ANALYSIS OF GENETIC DIVERSITY AND STRUCTURE OF SOME HISTORIC OLIVE (OLEA EUROPAEA L.) GERMPLASM IN THE UNITED STATES USING MICROSATELLITE MARKERS

Fifteen microsatellite simple-sequence repeat (SSR) loci were used to genotype 58 accessions of cultivated olive, Olea europaea L. ssp. europaea var. europaea, from two collections of 19th to 20th century plantings in Arizona and California, in order to examine genetic diversity and structure within and among collections. These collections are Lotusland Gardens in Santa Barbara, CA, and the Arboretum of the University of Arizona, Tucson. Genotypes in the olive collection at the USDA-ARS National Clonal Germplasm Repository for Fruit & Nut Crops (NCGR) in Davis, California were compared with the historic collections to potentially establish cultivar identity. Mean number of alleles per locus was 5.0 for Lotusland and 3.5 for the University of Arizona. Mean observed heterozygosity per collection was 0.650 for Lotusland and 0.598 for the University of Arizona. Up to 90% of the samples per collection were found to be synonymous with named, primarily Spanish or Italian, cultivars, yet these historic collections also contain significant genetic diversity, including genotypes and alleles not present in the NCGR germplasm collection. The cluster analysis placed the unknown genotypes within clades containing cultivars from the Eastern Mediterranean or from throughout the Mediterranean region, and in some cases produced relatively distinct branches.

Sample Size Estimation for Future Therapeutic Trials in SCAs (S12.004)

Objective: To evaluate disease progression and determine validity of clinical tools for therapeutic trials in a cohort of dominant ataxias. Background Autosomal dominant cerebellar ataxias (SCAs) are genetically heterogeneous with more than 30 loci involved. Cerebellar degeneration is accompanied by changes in the brainstem, basal ganglia, cerebral cortex, spinal cord and peripheral nervous system. Design/Methods: 162 patients with SCAs, including SCAs with polyglutamine expansions (25 SCA1, 35 SCA2, 58 SCA3, 5 SCA6, 10 SCA7) and other SCAs (1 SCA5, 1 SCA14, 2 SCA21,1 SCA25, 2 SCA28, 22 unknown genotypes) were followed prospectively for 3 years in the national French Referral Network including 7 centers. The outcome measures were the quantitative composite cerebellar functional score with writing (CCFSw) and the scale for the assessment and rating of ataxia (SARA). Results: Disease worsened in patients with SCA1, 2, 3 mutations and remained stable in patients with SCA6, SCA7 or other SCA mutations with both the CCFSw and the SARA. CCFSw progression was faster in SCA3 patients with Parkinsonism and/or dystonia at baseline (p=.003) while SARA progression in SCA2 patients depend of the gender, the age at onset and at inclusion, the number of repeats of the normal SCA2 allele, and the posterior column dysfunction. A two-arm trial with SARA as the outcome measure would require 57 SCA2, 70 SCA1 and 75 SCA3 patients per group to detect a 50% reduction in disease progression (power 80%, alpha 0.05). Conclusions: Disease progressed faster in SCAs with polyglutamine expansions in SCA1, 2 and 3 than the other groups. Both outcome measures are suitable for therapeutic trials; SARA requires fewer patients to attain the same power, but CCFSw needs less stratification. We demonstrate that the choice of clinical outcome measure is critical for reliable evaluation of progression in neurodegenerative diseases. Disclosure: Dr. Tezenas Du Montcel has nothing to disclose. Dr. Charles Ignatiew has nothing to disclose. Dr. Goizet has received research support from Genzyme Corporation. Dr. Marelli has nothing to disclose. Dr. Anheim has nothing to disclose. Dr. Guyant-Marchal has nothing to disclose. Dr. Le Bayon has nothing to disclose. Dr. Vandenberghe has nothing to disclose. Dr. Tchikviladze has nothing to disclose. Dr. Devos has received personal compensation for activities with Novartis and Aguettant. Dr. Le Ber has nothing to disclose. Dr. N9Guyen has nothing to disclose. Dr. Cazeneuve has nothing to disclose. Dr. Tallaksen has nothing to disclose. Dr. Brice has nothing to disclose. Dr. Durr has nothing to disclose.

Sa1001 Raised Serum Immunoglobulins in Chronic Hepatitis C: Incidence and Association With Genotype, Liver Fibrosis & Sustained Viral Response

Introduction Serum Immunoglobulins (Igs) are commonly raised in Chronic Hepatitis C (HCV) but their clinical significance is not fully known. There is also little information on the normalisation of Igs post HCV treatment. We aimed to assess (1) the incidence of raised Igs in HCV patients, (2) the association between the most commonly raised Ig [Immunoglobulin (IgG)] and genotype [G] and liver fibrosis and (3) the association between normalisation of IgG in those achieving sustained viral response (SVR). Methods Demographics, genotype, pre-and post-treatment Igs, Ishak liver fibrosis scores (F) and SVR of all patients undergoing treatment for HCV since 2006 was collected. Data from G2, G4, G6 and unknown genotype patients were not included in the analyses. Results 295 (n) patients were treated in the study period {Genotype 1 [G1] 71, Genotype 3 [G3] 205, males 181 (mean age 45.4); females 114 (mean age 41.3)}. 217/295 (73%) patients had raised pre-treatment Igs-either alone or in combination. Raised pre-treatment IgG, IgM and IgA were seen in 32%, 22% and 11% of G1 and in 56%, 16% and 8% of G3 patients respectively. A significant association between viral genotype and raised pre-treatment level was seen only with IgG (p=0.0009) and not with IgA (p=0.46) or IgM (p=0.20). In G1, 43% of patients with advanced fibrosis (F>4) had raised pre-treatment IgG compared to 29% of patients with F≤4 [non-significant (NS) association, p=0.66]. However in G3 advanced fibrosis (F>4) was significantly associated with raised pre-treatment IgG [33/41 (80%) with F>4 vs 70/130 (54%) with F≤4, p=0.0031] suggesting that pre-treatment IgG can be a good predictor of advanced fibrosis in G3. Overall SVR was achieved in 34% in G1 and 65% in G3. In those who achieved SVR, normalisation of raised IgG was seen more in G3 than in G1 [52% vs 44%, NS association, p=0.72]. Conclusion Our study confirms: (1) Presence of raised serum immunoglobulins, particularly that of IgG is common in both G1 and G3 patients. (2) Significant association between raised pre-treatment IgG and advanced fibrosis is seen in G3 but not in G1. In G3, pre-treatment IgG level can be good predictor of advanced fibrosis. (3) Post-SVR normalisation of IgG is seen more in G3 than in G1. Competing interests None declared.

Hepatitis B virus with a proposed genotype I was found in Sichuan Province, China

To date, eight hepatitis B virus (HBV) genotypes, A-H, have been designated, and two additional genotypes, I and J, have also been proposed. A serological survey targeting children in difficult-to-reach vaccination areas was carried out in remote counties of Sichuan Province, China. HBV genotypes and serotypes were also determined from HBsAg-positive serum samples by direct sequencing. Phylogenetic analysis showed two strains isolated from the Yi ethnic children clustered with the proposed genotype I. The pairwise genome genetic distance was 7.5% between genotypes I and C, and ranged from 8.4% to 15.2% between genotype I and other genotypes, except genotype C. Grouping Scan analyses of the two strains revealed apparent recombination events between an unknown genotype and genotype C. Two out of four HBV strains isolated from the Yi ethnic children were confirmed to be genotype I, suggesting widespread circulation and common infection with genotype I HBV in the local Yi population. High prevalence of HBsAg and low hepatitis B vaccination coverage indicated that additional efforts are needed to control HBV infection in those areas.

895 HEPATITIS C VIRUS SPECIFIC T LYMPHOCYTES RESPONSE INDUCED BY DENDRITIC CELL LOADED WITH NOVEL IDENTIFIED HLA-A2 RESTRICTED HEPATITIS C VIRUS EPITOPE PEPTIDES

3 ) is a collaboration of nine cohort studies from Australia, Europe and North America. Data on HCV seroconversion among IDUs between 1986 and 2009 were included. Inclusion criteria were: history of IDU, documented anti-HCV seroconversion within a two-year period and minimum of two therapy-naive HCV RNA assessments following the estimated date of seroconversion. We estimated the proportion with spontaneous clearance during the first two years following infection and identified factors associated with clearance using logistic regression. Results: Among 517 with incident HCV infection (173 females), HCV genotype (G) prevalence was: G1, 49%; G2, 6%; G3, 30%; G4, 1%; G6, 1%; mixed genotypes, 2% and unknown genotypes, 11%. The prevalence of IL28B genotypes was 50% CC, 37% CT and 12% TT (n = 406). Spontaneous clearance was observed in 26% (136 of 517). Spontaneous clearance was higher among females (38% vs. 21% males, P< 0.001) and among those with favorable CC IL28B genotype (37% vs. 24% CT/TT, P = 0.005). Among females with IL28B (n = 131), the proportion with spontaneous clearance was 58% in those with favorable CC genotypes as compared to 27% in those with unfavorable CT/TT genotypes (P = 0.002). In unadjusted analysis, factors associated with spontaneous clearance included female sex, Aboriginal ethnicity, no recent IDU and favorable CC IL28B genotype (vs. CT/TT). In adjusted analysis, female sex (AOR 2.66; 95% CI: 1.69, 4.18; P< 0.001) and favorable CC IL28B genotype (vs. CT/TT: AOR 2.03; 1.30, 3.19; P = 0.002) were independently associated with spontaneous clearance. The interaction between female sex and IL28B was assessed, but not significant. Conclusions: Female sex and IL28B genotype are independently associated with spontaneous clearance during acute HCV. Delayed therapeutic intervention during acute HCV infection could be recommended for females with favorable IL28B genotypes to allow time for spontaneous clearance.

Molecular diversity and genetic relationship of pomegranate (Punica granatum L.) genotypes using microsatellite markers

In the present research, we used microsatellite markers to study the genetic diversity and phylogenetic analysis among 75 pomegranate genotypes from Iran, Japan, Turkmenistan, Russia, Italy and USA. Ten out of 25 primer pairs showed 12 polymorphic loci and revealed 38 alleles with an average of 3.1667 alleles per locus and the mean of polymorphic information content (PIC) was calculated as 0.358. Comparing the group of genotypes from different countries revealed that the Iranian genotypes have the highest genetic diversity. The analysis of molecular variance (AMOVA) showed that the high level of genetic diversity occurred within groups and low level of that obtained among them. According to the results, most of the Russian accessions clustered with the Iranian genotypes and some of the American genotypes were grouped with Turkmenistan accessions. Also unknown genotypes showed the close relationships to Turkmenistan and Iranian ones. The results could show the existence of the common ancestors among the genotypes and probably confirms the hypothesis that pomegranate spread from central Asia, especially parts of Iran and neighboring areas such as Turkmenistan to other regions of the world.

Apolipoprotein E Gene Associations in Age-related Macular Degeneration: The Melbourne Collaborative Cohort Study

The apolipoprotein E gene (APOE) has been found to be associated with age-related macular degeneration (AMD). Reported associations have been questioned, as they are opposite those for Alzheimer's disease and cardiovascular disease. The authors examined associations between APOE genotype and AMD using a case-control study (2,287 cases and 2,287 controls individually matched on age, sex, and country of origin) nested within Melbourne Collaborative Cohort Study participants aged 48-86 years at AMD detection. The odds ratio for early AMD among participants with ε2-containing genotypes (ε2ε2/ε2ε3/ε2ε4) was 1.32 (95% confidence interval (CI): 1.11, 1.58; P = 0.002) versus persons with genotype ε3ε3. Associations with early AMD varied by smoking status; ε2-containing genotypes were positively associated with early AMD for never and previous smokers (never smokers: odds ratio (OR) = 1.40, 95% CI: 1.12, 1.76 (P = 0.003); previous smokers: OR = 1.39, 95% CI: 1.00, 1.93 (P = 0.05)) but not for current smokers (OR = 0.66, 95% CI: 0.34, 1.30 (P = 0.2; interaction P = 0.05). The ε4-containing genotype group (ε3ε4/ε4ε4) had an inverse association with early AMD among current smokers only (OR = 0.41, 95% CI: 0.22, 0.77 (P = 0.005)). These results highlight the importance of stratifying by smoking status in elderly populations. Smokers who survive to old age may be more likely to possess unknown genotypes which modify exposure-disease associations.

Ovine prion protein genotype frequencies in northwestern China

Anti-scrapie breeding programs have been initiated to screen for scrapie-resistant sheep based on ovine prion protein gene (PRNP) genotypes at codons 136, 154 and 171 in many countries, especially European Union member states. However, investigation of sheep PRNP genotypes is limited in China, despite the large number of sheep breeds. We analyzed 432 sheep of five different breeds from farms in northwestern China, using PCR-single-strand conformational polymorphism analysis (PCR-SSCP); the corresponding haplotypes of different PRNP alleles were cloned. PRNP allele genotyping was done by amplification refractory mutation system-PCR (ARMS-PCR), according to the haplotype clones of each PRNP allele. The validity of ARMS-PCR was checked by PCR-SSCP. Another 325 unknown PRNP genotypes of other sheep breeds were analyzed according to the established ARMS-PCR. Genotype frequencies of 757 sheep were analyzed with these two methods to evaluate susceptibility to scrapie in northwestern China. Relevant mutations were also detected at other sites. Both methods were effective for ovine PRNP allele genotyping, and the results of the analysis completely coincided. Scrapie-resistant genotypes were found to be uncommon, indicating a high risk for ovine scrapie in northwest China. In addition to codons 136, 154 and 171, we found numerous new mutations; nearly half of them were previously unreported. These sheep populations have a high degree of polymorphism at the PRNP locus.

Microsatellite marker-based identification of mother plants for the reliable propagation of olive (Olea europaea L.) cultivars in Australia

SummaryOlive production in Australia has continued to increase in recent years, however there remains a high degree of confusion on the genetic identities of the cultivars being grown. In the present study, seven microsatellite (simple sequence repeat; SSR) loci were used to identify a set of 53 olive tree samples from different sources. The microsatellite DNA profiles of all 53 tree samples, including seven unknown trees, were compared with the SSR profiles of 14 reference olive cultivars. A total of 60 fragments (alleles), averaging 8.57 alleles per microsatellite locus, were amplified. High average values were found for the observed heterozygosity, the expected heterozygosity, and the polymorphic information content (0.73, 0.74, and 0.72, respectively). While all seven microsatellite markers proved useful for characterisation and identification purposes, a combination of three SSR primer pairs (DCA9, DCA18, and EM030) was sufficient to distinguish all 53 olive samples. The microsatellite allelic profiles allowed the 53 tree samples to be grouped into 23 genotypes. The allelic profiles of 14 of these genotypes matched with their reference cultivars, while the genetic identities of the remaining nine genotypes could not be confirmed. Some of these unknown genotypes may have been derived from feral olive trees, or were due to mislabelling and/or planting errors among Australian olive cultivars. Our results confirm the usefulness of microsatellite markers as a tool for cultivar differentiation and identification, and indicate the need for reliable identification of mother plants for commercial propagation.

Genetic relatedness among some wild cherry (Prunus subgenus Cerasus) genotypes native to Iran assayed by morphological traits and random amplified polymorphic DNA analysis

Subgenus Cerasus species are useful genetic resources for cherry breeding programs. A total of 17 morphological traits together with 19 random amplified polymorphic DNA (RAPD) primers were used to study 39 accessions including 34 wild Cerasus subgenus genotypes belonging to Prunus avium L., P. cerasus L., P. mahaleb L., P. microcarpa Boiss., P. incana Pall., and P. brachypetala Boiss. species, along with an unknown wild Cerasus sample, two advanced cherry cultivars (‘Lambert’ and ‘Bulgar’), and two rootstocks (‘Colt’ and ‘Gisela 6’). Genotypes were separated into different groups according to their species and collection sites using cluster analysis performed by Ward’s clustering method based on morphological data. Nineteen RAPD primers from 60 screened produced 304 polymorphic reproducible bands (98.15% polymorphism). According to the similarity matrix, the lowest similarity was obtained between P. avium and P. microcarpa samples. A dendrogram was prepared by the unweighted pair-group method with arithmetic average (UPGMA), and the accessions were separated according to their species and geographic origin. In both morphological and molecular results, the advanced cultivars and rootstocks were separated from wild genotypes, and the unknown genotype was grouped with P. mahaleb accessions. Grouping by morphological characteristics was compared with the results of RAPD analysis, with no significant correlations between morphological and molecular data being found. This is the first report of molecular (RAPD) genetic diversity study in wild Cerasus subgenus genotypes from Iran, and the results demonstrate the high potential of RAPD analysis for discrimination of Cerasus subgenus genotypes.

Which compound to select in lead optimization? Prospectively validated proteochemometric models guide preclinical development.

In quite a few diseases, drug resistance due to target variability poses a serious problem in pharmacotherapy. This is certainly true for HIV, and hence, it is often unknown which drug is best to use or to develop against an individual HIV strain. In this work we applied ‘proteochemometric’ modeling of HIV Non-Nucleoside Reverse Transcriptase (NNRTI) inhibitors to support preclinical development by predicting compound performance on multiple mutants in the lead selection stage. Proteochemometric models are based on both small molecule and target properties and can thus capture multi-target activity relationships simultaneously, the targets in this case being a set of 14 HIV Reverse Transcriptase (RT) mutants. We validated our model by experimentally confirming model predictions for 317 untested compound – mutant pairs, with a prediction error comparable with assay variability (RMSE 0.62). Furthermore, dependent on the similarity of a new mutant to the training set, we could predict with high accuracy which compound will be most effective on a sequence with a previously unknown genotype. Hence, our models allow the evaluation of compound performance on untested sequences and the selection of the most promising leads for further preclinical research. The modeling concept is likely to be applicable also to other target families with genetic variability like other viruses or bacteria, or with similar orthologs like GPCRs.

GENETIC RELATIONSHIPS AMONG PISTACHIO SPECIES USING AFLP MARKERS

This study addresses the phylogenetic relationship between Pistacia species by amplified fragment length polymorphism (AFLP). A total of 44 accessions belonging to P. vera, P. eurycarpa, P. khinjuk, all subspecies of P. atlantica (atlantica, mutica, kurdica and cabulica), three unknown genotypes and three accessions proposed to be hybrids from P. eurycarpa × P. atlantica was the plant material used in this study. The accessions were from Iran, Turkey, USA and Syria. Six AFLP primer combinations produced a total of 475 fragments, with an average of 79.16 fragments per primer pair, of which, 336 bands were polymorphic. Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on jaccards similarity cofficient matrix and also average similarity of each species. According to the results, two main clusters were developed and P. vera, P. eurycarpa, P. atlantica (subsp. atlantica, kurdica, mutica, cabulica) and the hybrid genotypes were located in the first main cluster. P. khinjuk accessions from Iran and USA localized in second main cluster. The hybrid accessions located between eurycarpa and atlantica species and their hybrid nature between these to species was confirmed. One of the unknown accession clustered with the hybrid ones and the two other were grouped closely with P. khinjuk. According to this study, the closest species to P. vera was Eurycarpa group, followed by P. atlantica. UPGMA analysis separated P. atlantica subsp. mutica and cabulica from P. atlantica and P. eurycarpa. Subspecies mutica and cabulica were two closest genotypes, hence P. atlantica subsp. mutica could be classified as a distinct species as P. mutica and the cabulica as a subspecies of P. mutica. This study revealed that P. eurycarpa is a synonym for P. atlantica subsp. kurdica and should be considered distinct from P. atlantica, however P. atlantica showed a closer genetic similarity to P. eurycarpa than the other species.

Chemotypical variation in Vanilla planifolia Jack. (Orchidaceae) from the Puebla-Veracruz Totonacapan region

One of the threats in the diversity loss of the primary gene pool of Vanilla planifolia is the lack of information on existing level of polymorphism in cultivated germplasm, and the different expressions of this polymorphism. For this reason, it is proposed to study the chemical polymorphism of the four phytochemicals that define the vanilla aroma quality in fruits (vanillin, vanillic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid) by HPLC analysis (High Performance Liquid Chromatography) of 25 collections of unknown genotype, grown in the region Totonacapan Puebla-Veracruz, Mexico. The results identified a selection process, domestication in fruit aroma of vanilla, during which increased the participation of vanillin and reduced the presence of three minor compounds (vanillic acid, p-hydroxybenzaldehyde and p-hydroxybenzoic acid) in the global aroma. We distinguished a total of six chemotypes of V. planifolia in the Totonacapan region, some chemotypes with wild aromatic characteristics (low participation of vanillin) related to the material less cultivated in the region and domesticated chemotypes with high participation of vanillin, for the most cultivated material. The results show that the diversification of the chemotypes of V. planifolia is not related to environmental variation. The data indicate that in the possible center of origin of vanilla, there is phytochemical polymorphism, which indirectly suggests the existence of genetic polymorphism, essential for the design of a breeding program for optimizing the use and conservation of diversity of the primary gene pool of Vanilla planifolia.

KRAS, NRAS, PIK3CA Exon 20, and BRAF Genotypes in Synchronous and Metachronous Primary Colorectal Cancers : Diagnostic and Therapeutic Implications

Targeted therapy of advanced colorectal carcinoma (CRC) necessitates KRAS genotyping. Because we were interested in diagnostic and therapeutic consequences, we studied the KRAS, NRAS, PIK3CA exon 20, and BRAF genotypes in synchronous and metachronous primary CRCs; in addition, we studied their available metastases. We studied 21 patients with 43 synchronous and 2 metachronous adenocarcinomas of the colorectum (n = 20) and stomach (n = 1). Five patients had liver metastases and one had a distant lymph node metastasis. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed by Sanger sequencing and pyrosequencing. Fifty-seven different neoplastic lesions were genotyped, showing 18 (31.6%) KRAS, 2 (3.5%) NRAS, and 7 (12.3%) BRAF mutations, distributed among 10 (47.6%), 1 (4.8%), and 5 (23.8%) of the patients. An identical genotype of all synchronous primary CRCs was found only in 7 (35%) of the patients; the remainder had dissimilar genotypes in various combinations. Interestingly, a single patient had an unknown KRAS genotype (c.37_39dupGGC). Six patients with 13 primary carcinomas had distant metastases. In three of these patients, the metastasis shared the genotype only with one of the primary tumors, because the other primary tumors had another genotype. Synchronous and metachronous primary CRCs of the same patient have variable KRAS, NRAS, and BRAF genotypes. When metastases occur in these patients, the genotype has diagnostic and therapeutic implications and should be determined from the simultaneous or metachronous distant metastases.