Polymorphisms In Target Genes Research Articles

Mycobacterium Abscessus – Diagnostic and Therapeutic Frontiers in Infection Management

Abstract Mycobacterium (M.) abscessus, a highly pathogenic non-tuberculous mycobacterium, is responsible for several clinical manifestations. A very frequent occurrence is proven in patient with various lung diseases. Furthermore, it can result in complications such as skin and soft tissue diseases, central nervous system infections, bacteremia, eye infections, and others. M. abscessus is a clinical contraindication in cystic fibrosis patients awaiting a lung transplant, as it can exacerbate disease progression. Its pathogenicity and the emergence of resistance are influenced by factors including the composition of the cell envelope, rough and smooth M. abscessus morphotypes, efflux pumps, antibiotic-modifying/inactivating enzymes, and genetic polymorphisms in target genes. Management of the infection requires multicomponent therapy due to the high level of resistance. The following antibiotics are recommended according to the guidelines from the year 2017: amikacin, tigecycline, and imipenem with a macrolide. In order to properly manage patients with M. abscessus infection, correct identification of the subspecies as well as determination of resistance is essential. To achieve this goal, molecular-genetic techniques, such as whole-genome sequencing, are becoming increasingly favored in modern clinical practice. In this review, we provide up-to-date information on the issue of infections caused by non-tuberculous M. abscessus. We focus on its characteristics, possible infectious diseases, cystic fibrosis, and resistance, as well as the benefits of whole-genome sequencing.

Susceptibility of Ugandan Plasmodium falciparum Isolates to the Antimalarial Drug Pipeline.

Malaria, especially Plasmodium falciparum infection, remains an enormous problem, and its treatment and control are seriously challenged by drug resistance. New antimalarial drugs are needed. To characterize the Medicines for Malaria Venture pipeline of antimalarials under development, we assessed the ex vivo drug susceptibilities to 19 compounds targeting or potentially impacted by mutations in P. falciparum ABC transporter I family member 1, acetyl-CoA synthetase, cytochrome b, dihydroorotate dehydrogenase, elongation factor 2, lysyl-tRNA synthetase, phenylalanyl-tRNA synthetase, plasmepsin X, prodrug activation and resistance esterase, and V-type H+ ATPase of 998 fresh P. falciparum clinical isolates collected in eastern Uganda from 2015 to 2022. Drug susceptibilities were assessed by 72-h growth inhibition (half-maximum inhibitory concentration [IC50]) assays using SYBR green. Field isolates were highly susceptible to lead antimalarials, with low- to midnanomolar median IC50s, near values previously reported for laboratory strains, for all tested compounds. However, outliers with decreased susceptibilities were identified. Positive correlations between IC50 results were seen for compounds with shared targets. We sequenced genes encoding presumed targets to characterize sequence diversity, search for polymorphisms previously selected with in vitro drug pressure, and determine genotype-phenotype associations. We identified many polymorphisms in target genes, generally in <10% of isolates, but none were those previously selected in vitro with drug pressure, and none were associated with significantly decreased ex vivo drug susceptibility. Overall, Ugandan P. falciparum isolates were highly susceptible to 19 compounds under development as next-generation antimalarials, consistent with a lack of preexisting or novel resistance-conferring mutations in circulating Ugandan parasites. IMPORTANCE Drug resistance necessitates the development of new antimalarial drugs. It is important to assess the activities of compounds under development against parasites now causing disease in Africa, where most malaria cases occur, and to determine if mutations in these parasites may limit the efficacies of new agents. We found that African isolates were generally highly susceptible to the 19 studied lead antimalarials. Sequencing of the presumed drug targets identified multiple mutations in these genes, but these mutations were generally not associated with decreased antimalarial activity. These results offer confidence that the activities of the tested antimalarial compounds now under development will not be limited by preexisting resistance-mediating mutations in African malaria parasites.

Molecular surveillance for anti-malarial drug resistance and genetic diversity of Plasmodium falciparum after chloroquine and sulfadoxine-pyrimethamine withdrawal in Quibdo, Colombia, 2018

BackgroundResistance to anti-malarial drugs is associated with polymorphisms in target genes and surveillance for these molecular markers is important to detect the emergence of mutations associated with drug resistance and signal recovering sensitivity to anti-malarials previously used.MethodsThe presence of polymorphisms in genes associated with Plasmodium falciparum resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated by Sanger sequencing, in 85 P. falciparum day of enrollment samples from a therapeutic efficacy study of artemether–lumefantrine conducted in 2018–2019 in Quibdo, Colombia. Samples were genotyped to assess mutations in pfcrt (codons 72–76), pfdhfr (codons 51, 59, 108, and 164), and pfdhps genes (codons 436, 437, 540, and 581). Further, the genetic diversity of infections using seven neutral microsatellites (NMSs) (C2M34, C3M69, Poly α, TA1, TA109, 2490, and PfPK2) was assessed.ResultsAll isolates carried mutant alleles for pfcrt (K76T and N75E), and for pfdhfr (N51I and S108N), while for pfdhps, mutations were observed only for codon A437G (32/73, 43.8%). Fifty samples (58.8%) showed a complete neutral microsatellites (NMS) profile. The low mean number of alleles (2 ± 0.57) per locus and mean expected heterozygosity (0.17 ± 0.03) showed a reduced genetic diversity. NMS multilocus genotypes (MMG) were built and nine MMG were identified.ConclusionsOverall, these findings confirm the fixation of chloroquine and pyrimethamine-resistant alleles already described in the literature, implying that these drugs are not currently appropriate for use in Colombia. In contrast, mutations in the pfdhps gene were only observed at codon 437, an indication that full resistance to sulfadoxine has not been achieved in Choco. MMGs found matched the clonal lineage E variant 1 previously reported in northwestern Colombia.

Association of CAPN10 haplotype combinations with type 2 diabetes mellitus and metabolic syndrome among Egyptians: pilot study\u2014genotyping of three CAPN10 variants

BackgroundT2DM is a polygenic, metabolic complex and multifactorial disease. Several genes contribute to risk of type 2 diabetes and metabolic syndrome among different populations.ResultsAn relationship between three identified CAPN-10 variants [SNP-43 (rs#3792267), SNP-19 (rs#3842570) and SNP-63 (rs#5030952)] localized on 2q37 and type 2 diabetic patients with and without metabolic syndrome (MS) have been reported in our comparative study (diabetic vs health control individuals). Genotyping of study cohorts was carried out using restriction fragment length polymorphism (RFLP-PCR). Statistical analysis of data reveals that the haplotype combination 111/112 confers a significant risk for type 2 diabetes mellitus (T2DM). Patients with the homozygous haplotype combination of 122/122 are less susceptible to MS when compared to other patients carrying other haplotype combinations. Regarding obesity, a core component in MS, the haplotype combinations 111/121 and 122/122 demonstrate a significant protective role. Furthermore, the haplotype combination 111/111 displays a significant risk for high levels of total cholesterol.ConclusionPresent findings address that these haplotype combinations 111/112, 111/121 and 122/122 of CAPN-10 SNP-43, -19 and -63 constitute unique DNA biomarker fingerprints toward susceptibility and risk for T2DM and MS among Egyptians when compared to other haplotype combinations reported in other populations of different ethnicity. To enhance the power of human evolution control nowadays, mutations and polymorphisms in target genes associated with human diseases should be well understood.

Genetic Profile in Genes Associated with Cardiorespiratory Fitness in Elite Spanish Male Endurance Athletes.

Background: most of the research concerning the influence of genetics on endurance performance has been carried out by investigating target genes separately. However, endurance performance is a complex trait that can stem from the interaction of several genes. The objective of this study was to compare the frequencies of polymorphisms in target genes involving cardiorespiratory functioning in elite endurance athletes vs. non-athlete controls. Methods: genotypic frequencies were determined in 123 elite endurance athletes and in 122 non-athletes. Genotyping of ACE (rs4340), NOS3 (rs2070744 and rs1799983), ADRA2a (rs1800544 and rs553668), ADRB2 (rs1042713 and rs1042714), and BDKRB2 (rs5810761) was performed by polymerase chain reaction. The total genotype score (TGS: from 0 to 100 arbitrary units; a.u.) was calculated from the genotype score in each polymorphism. Results: the mean TGS in non-athletes (47.72 ± 11.29 a.u.) was similar to elite endurance athletes (46.54 ± 11.32 a.u., p = 0.415). The distribution of TGS frequencies were also similar in non-athletes and elite endurance athletes (p = 0.333). There was no TGS cut-off point to discriminate being elite endurance athletes. Conclusions: the genetic profile in the selected genes was similar in elite endurance athletes and in controls, suggesting that the combination of these genes does not determine endurance performance.

Recent Advances in Biomarkers and Regenerative Medicine for Diabetic Neuropathy.

Diabetic neuropathy is one of the most common complications of diabetes. This complication is peripheral neuropathy with predominant sensory impairment, and its symptoms begin with hyperesthesia and pain and gradually become hypoesthesia with the loss of nerve fibers. In some cases, lower limb amputation occurs when hypoalgesia makes it impossible to be aware of trauma or mechanical stimuli. On the other hand, up to 50% of these complications are asymptomatic and tend to delay early detection. Therefore, sensitive and reliable biomarkers for diabetic neuropathy are needed for an early diagnosis of this condition. This review focuses on systemic biomarkers that may be useful at this time. It also describes research on the relationship between target gene polymorphisms and pathological conditions. Finally, we also introduce current information on regenerative therapy, which is expected to be a therapeutic approach when the pathological condition has progressed and nerve degeneration has been completed.

Association between gene polymorphism of folate metabolism and recurrent spontaneous abortion in Asia

To evaluate the association between gene polymorphisms of MTHFR (C677T, A1298C) and MTRR (A66G), and the recurrent spontaneous abortion (RSA) risk in Asia.Related case-control studies were collected, selected, and screened. A meta-analysis was conducted by Stata 12.0 software to assess the association between polymorphisms of target genes and RSA.Altogether 30 studies examining the relationship between genetic polymorphism of folate metabolism and RSA risk were included, among which 20 studies were related to MTHFR C677T, 11 to MTHFR A1298C and 6 to MTRR A66G. The studies suggested that MTHFR C677T polymorphism was closely connected with RSA risk under all models (P < .05). Furthermore according to the subgroup analysis of ethnicity, the correlation between C677T polymorphism and RSA was stronger in north of China when compared with south of China and other Asian countries (P > . 05). For MTHFR A1298C, it was closely related to RSA risk in all gene models except for (AC vs AA) (P < .05). However, when it comes to MTRR A66G, there was no significant correlation between gene A66G polymorphism and RSA risk except for the additive gene model (G vs A) (P < .05).The present evidence shows that the correlation between gene polymorphisms and RSA risk can be found in MTHFR C677T, A1298C (except for heterozygote model) and MTRR A66G (only in additive genotypes), and the detection of the correlated gene polymorphisms mentioned above is of certain guiding significance for preventing RSA and screening high-risk groups.

Replication of GWAS identified miR-137 and its target gene polymorphisms in Schizophrenia of South Indian population and meta-analysis with Psychiatric Genomics Consortium

Schizophrenia is a complex psychiatric disorder involving multiple genes each contributing a small risk. Genome-wide association studies (GWASs) have identified hundreds of risk loci for schizophrenia including miR-137, a miRNA shown to be involved in neuronal development. Several genes regulated by miR-137 were also reported as top risk genes associated with schizophrenia and has been hypothesised that the dysregulation of miR-137 and its target could be involved in the aetiology of schizophrenia. Here, we replicated the four European GWAS hits, miR-137-rs1625579 and three of its validated target gene loci SNPs (ZNF804a-rs1344706, CACNA1C-rs4765905 and TCF4-rs9960767) by genotyping in 2074 samples (schizophrenia cases-1005; controls-1069) from South Indian Population. In this study, only the CACNA1C rs4765905 showed a significant association (OR=1.24, p=0.006). Three SNPs (rs1625579, rs1344706 and rs4765905) showed a consistent direction of effect with previous studies and the polygenic risk score constructed using the weighted sum of these three SNPs showed a significant association with Schizophrenia in this population (OR=3.78, p=0.005). Further, we carried out meta-analysis combining our results with the Psychiatric Genomics Consortium (PGC2) data and observed a considerable increase in GWAS significance.

β3-Adrenoceptor agonists for overactive bladder syndrome: Role of translational pharmacology in a repositioning clinical drug development project.

β3-Adrenoceptor agonists were originally considered as a promising drug class for the treatment of obesity and/or type 2 diabetes. When these development efforts failed, they were repositioned for the treatment of the overactive bladder syndrome. Based on the example of the β3-adrenoceptor agonist mirabegron, but also taking into consideration evidence obtained with ritobegron and solabegron, we discuss challenges facing a translational pharmacology program accompanying clinical drug development for a first-in-class molecule. Challenges included generic ones such as ligand selectivity, species differences and drug target gene polymorphisms. Challenges that are more specific included changing concepts of the underlying pathophysiology of the target condition while clinical development was under way; moreover, a paucity of public domain tools for the study of the drug target and aspects of receptor agonists as drugs had to be addressed. Nonetheless, a successful first-in-class launch was accomplished. Looking back at this translational pharmacology program, we conclude that a specifically tailored and highly flexible approach is required. However, several of the lessons learned may also be applicable to translational pharmacology programs in other indications.

Polymorphisms in the TNF-α, TNFR1 gene and risk of rheumatoid arthritis in Chinese Han population.

Recent advances have highlighted a major genetic contribution to the pathogenesis of rheumatoid arthritis (RA).The aim of this study was to investigate whether polymorphisms of TNF-α (rs1800630, rs1800629) and TNFR1 (rs767455) were associated with susceptibility to and clinical outcome of RA in Chinese Han population. The target gene polymorphisms were genotyped in 256 patients with RA and 331 healthy controls using a high resolution melting (HRM) method. ESR, CRP, RF anti-CCP and anti-GPI level were also assayed and compared in genotypes of each polymorphism. Significant difference was observed in the genotype distributions and allele frequencies of TNF-α rs1800629 (P = 0.001, P < 0.001, respectively) between patients with RA and controls. There is no evidence to suggest an association between genotypes of the 3 SNPs according to age, gender, disease duration, DAS28 and serum level of autoantibodies. This study identifies a potentially important role for TNF-α rs1800629 polymorphisms in the susceptibility to RA.However, further studies in larger cohorts are required.

Polimorfismos en los genes dihidrofolatorreductasa (dhfr) y dihidropteroatosintetasa (dhps) y modelamiento estructural del gen dhps en aislamientos colombianos de Toxoplasma gondii

There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates.This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii .Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced.One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations.The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.

[Gene polymorphisms in the dihydrofolate reductase ( dhfr ) and dihydropteroate synthase ( dhps ) genes and structural modelling of the dhps gene in Colombian isolates of Toxoplasma gondii].

There are no reports describing polymorphisms in target genes of anti- Toxoplasma drugs in South American isolates. This study sought to perform cloning and sequencing of the dihydrofolate reductase ( dhfr ) and dihydropteroate-synthase ( dhps ) genes of the reference Rh strain and two Colombian isolates of Toxoplasma gondii . Two isolates were obtained from the cerebrospinal fluid of HIV-infected patients with cerebral toxoplasmosis. A DNA extraction technique and PCR assay for the dhfr and dhps genes were standardized, and the products of amplification were cloned into Escherichia coli and sequenced. One polymorphism (A « G) was found at position 235 of exon 2 in the dhps gene. In addition, two polymorphisms (G « C) at positions 259 and 260 and one polymorphism (T « G) at position 371 within exon 4 of the dhps gene were detected. In this last exon, a bioinformatic analysis revealed a non-synonymous polymorphism in the coding region that could lead to the substitution of Glu (CAA or CAG) for His (encoded by codons AAU or AAC). A structural model of the T. gondii DHPS protein was calculated, and the results revealed modifications in secondary structure due to mutations. The methods described in this study can be used as a tool to search for polymorphisms in samples from patients with different clinical manifestations of toxoplasmosis and to examine their relationship with the therapeutic response.

Genes and beans: pharmacogenomics of renal transplant

Advances in the management of patients after solid organ transplantation have led to dramatic decreases in rates of acute rejection, but long-term graft and patient survival have remained unchanged. Individualized therapy after transplant will ideally provide adequate immunosuppression while limiting the adverse effects of drug therapy that significantly impact graft survival. Therapeutic drug monitoring represents the best approximation of individualized drug therapy in transplant at this time; however, obtaining pharmacogenomic data in transplant patients has the potential to enhance our current practice. Polymorphisms of target genes that impact pharmacokinetics have been identified for most immunosuppressants, including tacrolimus, cyclosporine, mycophenolate, azathioprine and sirolimus. In the future, pre-emptive assessment of a patient's genetic profile may inform drug selection and provide information on specific doses that will improve efficacy and limit toxicity.

Genetic risk factors for glucocorticoid-induced osteonecrosis: A meta-analysis

Glucocorticoid-induced osteonecrosis is a common and severe adverse event. We conducted a meta-analysis to investigate whether polymorphisms in target genes were associated with the risk of corticosteroid-induced osteonecrosis. Published literature from PubMed and EMBASE were searched for eligible publications. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using a fixed- or random-effects model. There were 23 articles with 35 genes described the relationship between polymorphisms and glucocorticoid-induced osteonecrosis. Meta-analyses were carried out for those SNPs with three or more eligible studies, which included four SNPs located in three genes (PAI-1, MTHFR, ABCB1). The meta-analysis revealed that the PAI-1 4G allele was associated with an increased risk of osteonecrosis compared with the 5G allele (combined studies: OR=1.932, 95% CI=1.145–3.261). The OR for the 4G/4G vs. 5G/5G genotype of PAI-1 was 3.217 (95% CI 1.667–6.209 with combined studies), The relative risk of osteonecrosis was increased in the 4G allele vs. 5G/5G and 4G/4G genotype vs. 5G allele, with odds ratios of 2.304 (95% CI=1.235–4.299) and 2.307 (95% CI=1.527–3.485) in combined studies, respectively. The ABCB1 C3435T genotype distributions available confirmed that the C allele increased osteonecrosis risk compared with the T allele (OR 1.668, 95% CI=1.214–2.293) and TT genotype (OR 2.946, 95% CI=1.422–6.101). There was no evidence for significant association between MTHFR C677T and ABCB1 G2677T/A polymorphisms and risk of osteonecrosis. Results of this meta-analysis indicate that the PAI-1 4G/5G and ABCB1 C3435T polymorphisms may be risk factors for osteonecrosis.

Comparison of Real-Time PCR Assays for Detection of Pathogenic Leptospira spp. in Blood and Identification of Variations in Target Sequences

Leptospirosis is considered an underdiagnosed disease. Although several PCR-based methods are currently in use, there is little information on their comparability. In this study, four quantitative real-time PCR (qPCR) assays (SYBR green and TaqMan chemistries) targeting the secY, lfb1, and lipL32 genes were evaluated as diagnostic assays. In our hands, these assays can detect between 10(2) and 10(3) bacteria/ml of pure culture, whole-blood, plasma, and serum samples. In three independent experiments, we found a slightly higher sensitivity of the PCR assays in plasma than in whole blood and serum. We also evaluated the specificity of the PCR assays on reference Leptospira strains, including newly described Leptospira species, and clinical isolates. No amplification was detected for DNA obtained from saprophytic or intermediate Leptospira species. However, among the pathogens, we identified sequence polymorphisms in target genes that result in primer and probe mismatches and affect qPCR assay performance. In conclusion, most of these assays are sensitive and specific tools for routine diagnosis of leptospirosis. However, it is important to continually evaluate and, if necessary, modify the primers and/or probes used to ensure effective detection of the circulating Leptospira isolates.

Pharmacogenomic association study on the role of drug metabolizing, drug transporters and drug target gene polymorphisms in drug-resistant epilepsy in a north Indian population

BACKGROUND:In epilepsy, in spite of the best possible medications and treatment protocols, approximately one-third of the patients do not respond adequately to anti-epileptic drugs. Such interindividual variations in drug response are believed to result from genetic variations in candidate genes belonging to multiple pathways.MATERIALS AND METHODS:In the present pharmacogenetic analysis, a total of 402 epilepsy patients were enrolled. Of them, 128 were diagnosed as multiple drug-resistant epilepsy and 274 patients were diagnosed as having drug-responsive epilepsy. We selected a total of 10 candidate gene polymorphisms belonging to three major classes, namely drug transporters, drug metabolizers and drug targets. These genetic polymorphism included CYP2C9 c.430C>T (*2 variant), CYP2C9 c.1075 A>C (*3 variant), ABCB1 c.3435C>T, ABCB1c.1236C>T, ABCB1c.2677G>T/A, SCN1A c.3184 A> G, SCN2A c.56G>A (p.R19K), GABRA1c.IVS11 + 15 A>G and GABRG2 c.588C>T. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods, and each genotype was confirmed via direct DNA sequencing. The relationship between various genetic polymorphisms and responsiveness was examined using binary logistic regression by SPSS statistical analysis software.RESULTS:CYP2C9 c.1075 A>C polymorphism showed a marginal significant difference between drug resistance and drug-responsive patients for the AC genotype (Odds ratio [OR] = 0.57, 95% confidence interval [CI] = 0.32–1.00; P = 0.05). In drug transporter, ABCB1c.2677G>T/A polymorphism, allele A was associated with drug-resistant phenotype in epilepsy patients (P = 0.03, OR = 0.31, 95% CI = 0.10-0.93). Similarly, the variant allele frequency of SCN2A c.56 G>A single nucleotide polymorphism was significantly higher in drug-resistant patients (P = 0.03; OR = 1.62, 95% CI = 1.03, 2.56). We also observed a significant difference at the genotype as well as allele frequencies of GABRA1c.IVS11 + 15 A > G polymorphism in drug-resistant patients for homozygous GG genotype (P = 0.03, OR = 1.84, 95% CI = 1.05–3.23) and G allele (P = 0.02, OR = 1.43, 95% CI = 1.05–1.95).CONCLUSIONS:Our results showed that pharmacogenetic variants have important roles in epilepsy at different levels. It may be noted that multi-factorial diseases like epilepsy are also regulated by various other factors that may also be considered in the future.

EcoTILLING in Capsicum species: searching for new virus resistances

BackgroundThe EcoTILLING technique allows polymorphisms in target genes of natural populations to be quickly analysed or identified and facilitates the screening of genebank collections for desired traits. We have developed an EcoTILLING platform to exploit Capsicum genetic resources. A perfect example of the utility of this EcoTILLING platform is its application in searching for new virus-resistant alleles in Capsicum genus. Mutations in translation initiation factors (eIF4E, eIF(iso)4E, eIF4G and eIF(iso)4G) break the cycle of several RNA viruses without affecting the plant life cycle, which makes these genes potential targets to screen for resistant germplasm.ResultsWe developed and assayed a cDNA-based EcoTILLING platform with 233 cultivated accessions of the genus Capsicum. High variability in the coding sequences of the eIF4E and eIF(iso)4E genes was detected using the cDNA platform. After sequencing, 36 nucleotide changes were detected in the CDS of eIF4E and 26 in eIF(iso)4E. A total of 21 eIF4E haplotypes and 15 eIF(iso)4E haplotypes were identified. To evaluate the functional relevance of this variability, 31 possible eIF4E/eIF(iso)4E combinations were tested against Potato virus Y. The results showed that five new eIF4E variants (pvr210, pvr211, pvr212, pvr213 and pvr214) were related to PVY-resistance responses.ConclusionsEcoTILLING was optimised in different Capsicum species to detect allelic variants of target genes. This work is the first to use cDNA instead of genomic DNA in EcoTILLING. This approach avoids intronic sequence problems and reduces the number of reactions. A high level of polymorphism has been identified for initiation factors, showing the high genetic variability present in our collection and its potential use for other traits, such as genes related to biotic or abiotic stresses, quality or production. Moreover, the new eIF4E and eIF(iso)4E alleles are an excellent collection for searching for new resistance against other RNA viruses.

Phenotypic Variation for Diastatic Power, β‐Amylase Activity, and β‐Amylase Thermostability vs. Allelic Variation at the Bmy1 Locus in a Sample of North American Barley Germplasm

Malting quality data were collected on malts from three barley (Hordeum vulgare L.) breeding program trials. We tried to identify causal polymorphisms in the Bmy1 intron III and coding regions for use in marker‐assisted selection. Abundant malting quality variation exists in the spring barley germplasm despite the parents having identical Bmy1 intron III and coding regions. After complete Bmy1 sequencing, no polymorphisms associated with malting quality phenotypes, indicating the genetic basis for the observed variation resides outside Bmy1. Complete allele sequencing identified one winter barley parent that had a novel Bmy1 allele (Sd1a) based on amino acid substitutions that are candidates as causative agents for the phenotypic variation. Marker‐assisted selection against the Sd1a allele could be effective in improving diastatic power (DP). The Sd1a allele is associated with low DP and is present in only three of the 51 lines, presumably due to preceding generations being selected for high DP. Selection for DP has subsequently eliminated the Sd1a allele from this breeding program. This research shows the importance of having complete allele sequences and knowledge of functional polymorphisms in target genes before using marker‐assisted selection.

Decision criteria for rational selection of homogeneous genotyping platforms for pharmacogenomics testing in clinical diagnostics

Genotyping is crucial for the identification of genetic markers underlying the development of neoplastic diseases and for determining individual variations in response to specific drugs. Technologies which can accurately identify genetic polymorphisms will dramatically affect routine diagnostic processes and future therapeutic developments in personalized medicine. However, such methods need to fulfill the principles of analytical validation to determine their suitability to assess nucleotide polymorphisms in target genes. This article reviews recent developments in homogeneous technologies for the genotyping of single nucleotide polymorphisms. Here, homogeneous methods essentially refer to "single-tube" assays performed in a liquid phase. For the appropriate choice of any method, several criteria must be considered: 1) detection of known genetic variations; 2) analytical performance including specificity, sensitivity and robustness of the method; 3) availability of large platforms and required equipment; 4) suitability of platforms and tests for routine diagnostics; 5) suitability for high throughput implementation. This review is intended to provide the reader with an understanding of these various technologies for pharmacogenomic testing in the routine clinical laboratory. A brief overview is provided on the available technologies for the detection of known mutations, a specific description of the homogeneous platforms currently employed in genotyping analysis, and considerations regarding the proper assessment of the analytical performance of these methods. Based on the criteria proposed here, potential users may evaluate advantages and limitations of the various analytical platforms and identify the most appropriate platform according to their specific setting and diagnostic needs.

May the istotype influence the response to pemetrexed in patients with non small cell lung cancer? Preliminary data of our clinical experience

19115 Background: Patients with relapsed or refractory NSCLC can benefit from pemetrexed, a new-generation antifolate, approved for the treatment of mesothelioma and non-small cell lung cancer (NSCLC).The aim of this retrospective analysis was to evaluate activity and tolerability of pemetrexed in pre-treated patients with relapsed or refractory NSCLC, focusing on potential correlations between antitumor activity of pemetrexed and the different histological subtypes. Methods: Between November 2005 and December 2006, 19 patients received pemetrexed at a dose of 500 mg/m2 on day 1, every 3 weeks, with folic acid and vitamin B12 supplementation. The patients (14 M and 5 F) had a median age of 64.4 years (range 43 to 79), ECOG PS/pts: 0/7 1/8 2/4. Four patients had stage III B, 15 stage IV. Median number of metastatic sites was 2 (range 1–3). Distribution of tumors by histology was: squamous carcinoma 5/19, adenosquamous carcinoma 1/19, adenocarcinoma 7/19, bronchiolar-alveolar adenocarcinoma (BAC) 2/19, large cell carcinoma 1/19, undifferentiated 3/19. Pemetrexed was employed as second-line treatment in 10 patients and as further lines in 9 patients. Results: Rapid disease precluded treatment in one patient, 18 patients were evaluable. Objective response rate (RR) was 45%, including 1 CR and 7 PR, all obtained in patients with adenocarcinoma (RR, adenocarcinoma 45% vs. non-adenocarcinoma 0%, p=0.004, Fisher exact test). The median time to disease progression was 4.3 months (range 2–10) and overall survival was 9.2 months (range 2–17). Pemetrexed was well tolerated and the main side effects were grade 1–2 anemia, asthenia and vomiting, observed in 8 patients. Conclusions: The results of our small experience may suggest an increased sensibility of adenocarcinomas to pemetrexed, including BAC. A plausible hypothesis that may explain these findings may be related to a peculiar genetic expression or to the presence of polymorphisms in target genes. Further studies will be conducted to identify genetic markers of sensitivity to this drug and the possible correlations with the different adenocarcinoma subtypes. No significant financial relationships to disclose.