Single Nucleotide Polymorphism Mutation Research Articles

A High Frequency of MSH6 G268A Polymorphism and Survival Association in Glioblastoma

ABSTRACTMSH6 (mutS homolog 6), one of the five key mismatch repair (MMR) genes, was found to play an important role in conferring resistance to alkylating agents—temozolomide (TMZ) in malignant glioma. This study aims to investigate whether genetic variations in MSH6 gene are associated with the survival outcomes in patients with malignant glioma. Each exon of the MSH6 gene was sequenced, and single nucleotide polymorphism (SNP) analysis was performed using 74 tumor tissues from glioblastoma multiforme (GBM) patients. Among these patients, 54 patients received radiotherapy plus TMZ treatment; 20 patients had radiotherapy only. The promoter methylation of O6-methylguanine methyltransferase (MGMT) was measured by methylation-specific polymerase chain reaction. Literature mining and related data collection were done with NCBI and PubMed databases. Of the 74 GBM patients, 50% (n = 37) harbored MSH6 G268A polymorphism, and no significant rates of other SNP or gene mutation across MSH6 exons were detected. The median overall survival (OS) was 15.6 months for who harbored the SNP and 12.6 months for SNP-negative patients (log-rank test: p = .324). The median OS for the MGMT promoter methylation group (n = 25) and nonmethylation group (n = 29) of the 54 GBM patients treated with TMZ was 21.3 and 8.9 months, respectively, (p = .002). In conclusion, we identified a high frequency of MSH6 G268A polymorphism in MSH6 gene, which did not have a notable influence on survival for the malignant glioma patients with/without TMZ treatment.

Rhodium interaction with human NRG1 gene of Schizophrenia

Rhodium (II) acetate [Rh2 (O2CCH3)4] couldbe used as an indicator for single nucleotide polymorphism(SNPs) involved in the onset of schizophrenia. Rhodium(Rh1) has affinity to make covalent interactions withneuregulin (NRG1) gene at SNPs mutation. Binding effects ofRh1 has been studied under different molar concentrationsat different time periods. In this study we used Rh1 toevaluate its interaction with NRG1 gene in Schizophrenicpatients of Pakistan. Rh-NRG1 adduct were amplified by PCR and visualized on agarose gel electrophoresis. Herewe show Rh1 binding with NRG1 gene was inhibited withincreasing concentration ranges from 0.5 -3 μM. It has beennoted that upon binding with NRG1 gene Rh1 decreasedthe mobility and intensity of the DNA bands. Noticeably Rh1didn’t inhibit the activity of Mun1 restriction enzyme havingspecific CAAA cleavage site. After the digestion of NRG1gene having SNPs mutation combining with Rh1 proves itscovalent binding only with Guanine or Thymine and notwith Adenine or Cytosine. This is a novel study that showsrhodium can covalently binds with human dsDNA and caninhibit its amplification. The effect of Rh1 to target differentSNPs mutations (normally occurs in genetic diseases suchas schizophrenia) can be identified by using this technique.There are variations between human populations, so a SNPallele that is common in one geographical or ethnic groupmay be much rarer in another, and Rh1 can act as a usefultool to identify SNPs of schizophrenic genes. Keywords - Pakistani Population, Schizophrenia, SingleNucleotide polymorphism (SNP), Neuregulin (NRG1),Rhodium (Rh)

Intestinal Tissues Induce an SNP Mutation in Pseudomonas aeruginosa That Enhances Its Virulence: Possible Role in Anastomotic Leak

The most feared complication following intestinal resection is anastomotic leakage. In high risk areas (esophagus/rectum) where neoadjuvant chemoradiation is used, the incidence of anastomotic leaks remains unacceptably high (∼10%) even when performed by specialist surgeons in high volume centers. The aims of this study were to test the hypothesis that anastomotic leakage develops when pathogens colonizing anastomotic sites become in vivo transformed to express a tissue destroying phenotype. We developed a novel model of anastomotic leak in which rats were exposed to pre-operative radiation as in cancer surgery, underwent distal colon resection and then were intestinally inoculated with Pseudomonas aeruginosa, a common colonizer of the radiated intestine. Results demonstrated that intestinal tissues exposed to preoperative radiation developed a significant incidence of anastomotic leak (>60%; p<0.01) when colonized by P. aeruginosa compared to radiated tissues alone (0%). Phenotype analysis comparing the original inoculating strain (MPAO1- termed P1) and the strain retrieved from leaking anastomotic tissues (termed P2) demonstrated that P2 was altered in pyocyanin production and displayed enhanced collagenase activity, high swarming motility, and a destructive phenotype against cultured intestinal epithelial cells (i.e. apoptosis, barrier function, cytolysis). Comparative genotype analysis between P1 and P2 revealed a single nucleotide polymorphism (SNP) mutation in the mexT gene that led to a stop codon resulting in a non-functional truncated protein. Replacement of the mutated mexT gene in P2 with mexT from the original parental strain P1 led to reversion of P2 to the P1 phenotype. No spontaneous transformation was detected during 20 passages in TSB media. Use of a novel virulence suppressing compound PEG/Pi prevented P. aeruginosa transformation to the tissue destructive phenotype and prevented anastomotic leak in rats. This work demonstrates that in vivo transformation of microbial pathogens to a tissue destroying phenotype may have important implications in the pathogenesis of anastomotic leak.

Systematic Functional Characterization of Cytochrome P450 2E1 Promoter Variants in the Chinese Han Population

CYP2E1 promoter polymorphisms can lead to significant interindividual differences in expression of CYP2E1. Using a database of CYP2E1 gene polymorphisms established in 2010, our study aimed to functionally characterize the single nucleotide polymorphisms (SNPs) of the promoter region and corresponding haplotypes in the Chinese Han population. Six novel SNPs and seven haplotypes with a frequency equal to or greater than 0.01 were constructed on a luciferase reporter system on the basis of site-directed mutagenesis. Dual luciferase reporter systems were used to analyze regulatory activity. The constructs including single novel SNP mutations exhibited insignificant change in luciferase activity, whereas, the activity produced by Haplo1(GTTGCTATAT), Haplo2 (CTTGCTATAT) and Haplo7 (GAGCTCACAT), containing a −333T>A polymorphism was significantly greater than for the wild type in Hep G2 cells (p<0.05), being 1.5−, 2.0− and 1.4− times greater respectively. These findings suggest the possibility of significant clinical prediction of adverse drug reaction and the facilitation of personalized medicine.

A novel mouse model of veno-occlusive disease provides strategies to prevent thioguanine-induced hepatic toxicity

ObjectiveThe anti-leukemic drugs, azathioprine and 6-mercaptopurine (6MP), are important in the treatment of inflammatory bowel disease but an alternative faster-acting, less-allergenic thiopurine, 6-thioguanine (6TG), can cause hepatic veno-occlusive disease/sinusoidal obstructive...

Evolution and Association Analysis of Ghd7 in Rice

Plant height, heading date, and yield are the main targets for rice genetic improvement. Ghd7 is a pleiotropic gene that controls the aforementioned traits simultaneously. In this study, a rice germplasm collection of 104 accessions (Oryza sativa) and 3 wild rice varieties (O.rufipogon) was used to analyze the evolution and association of Ghd7 with plant height, heading date, and yield. Among the 104 accessions, 76 single nucleotide polymorphisms (SNPs) and six insertions and deletions were found within a 3932-bp DNA fragment of Ghd7. A higher pairwise π and θ in the promoter indicated a highly diversified promoter of Ghd7. Sixteen haplotypes and 8 types of Ghd7 protein were detected. SNP changes between haplotypes indicated that Ghd7 evolved from two distinct ancestral gene pools, and independent domestication processes were detected in indica and japonica varietals respectively. In addition to the previously reported premature stop mutation in the first exon of Ghd7, which caused phenotypic changes of multiple traits, we found another functional C/T mutation (SNP S_555) by structure-based association analysis. SNP S_555 is located in the promoter and was related to plant height probably by altering gene expression. Moreover, another seven SNP mutations in complete linkage were found to be associated with the number of spikelets per panicle, regardless of the photoperiod. These associations provide the potential for flexibility of Ghd7 application in rice breeding programs.

Identification of SNP Mutations in DREB1, HKT1, and WRKY1 Genes Involved in Drought and Salt Stress Tolerance in Durum Wheat (Triticum turgidum L. var durum)

Tolerance mechanisms to salinity and drought stress are quite complex. Plants have developed a complex and elaborate signaling network that ensures their adaptation to this stress. For example, salinity tolerance is thought to be due to three main factors: Na(+) exclusion, tolerance to Na(+) in the tissues and osmotic tolerance. Recently, many transcription factors for tolerance to salt and drought stresses have been identified. In this study, multialignments of conserved domains in DREB1, WRKY1 transcription factors (TFs), and HKT-1 have been utilized to design specific primers in order to identify functional single nucleotide polymorphisms (SNPs). These primers have been used to probe on several genotypes of durum wheat that are differentially tolerant to salt and drought stress; they were grown in increasing concentrations of NaCl. The selected portions have been analyzed using high-resolution melting curve (HRM) technology that currently represents one of the most recent and powerful tools for detecting SNP and INDEL mutations. Analyzing the amplification profiles, observed in the resulting melting curves, samples corresponding to different treatment conditions were selected, sequenced, and aligned with the homolog sequences present in gene databases to identify and characterize potential SNP and INDEL mutations. The PCR amplicons, containing single and double SNPs, produced distinctive HRM profiles. By sequencing the polymerase chain reaction (PCR) products, several SNPs have been identified and validated. All the discovered mutations were able to generate changes in amino acid sequences of the corresponding proteins. Most of the identified SNPs were found in salt and drought tolerant durum wheat genotypes. These varieties are of great value for durum wheat breeding works.

Out-of-Africa, the peopling of continents and islands: tracing uniparental gene trees across the map

Genetic relationships between human groups were first studied by comparisons of relative allele frequency at multiple loci. Geographical study of detailed, highly resolved trees of single, non-recombining uniparental loci (mitochondrial DNA: mtDNA and Y chromosome/non-recombining Y: NRY), following specific lineages rather than populations, then revolutionized knowledge of the peopling of the world, although, curiously, the use of geographically highly specific mutations that protect against malaria, found on individual autosomal globin genes, were first in single-locus phylogeography. mtDNA, with its high single nucleotide polymorphism (SNP) mutation rates and relative ease of dating, led the way and gave stronger proof of the recent near replacement of all human species by anatomically modern humans (AMH). AMH left Africa via a single southern exit about 70 000 years ago and rapidly spread around the Indian Ocean towards the Antipodes, long before a small branch left a South Asian colony, earlier on the trail, to populate Europe. The worldwide skeleton phylogeny of mtDNA is fully resolved, but a regional analysis will continue to illuminate subsequent migrations. NRY with a lower SNP mutation rate still has a dating problem relating to use the of single tandem repeats (STRs), but has validated mtDNA results and with more geographical specificity and genomic size, as with the autosomal human genome, has much more detail to offer for the future.

Mannose-Binding Lectin 2 Polymorphisms Do Not Influence Frequency or Type of Infection in Adults with Chemotherapy Induced Neutropaenia

BackgroundMannose-binding Lectin protein (MBL) has been suggested to be relevant in the defence against infections in immunosuppressed individuals. In a Swedish adult cohort immunosuppressed from both the underlying disease and from iatrogenic treatments for their underlying disease we investigated the role of MBL in susceptibility to infection.MethodsIn this cross sectional, prospective study, blood samples obtained from 96 neutropaenic febrile episodes, representing 82 individuals were analysed for single nucleotide polymorphism (SNP) in the MBL2 gene. Concurrent measurement of plasma MBL protein concentrations was also performed for observation of acute response during febrile episodes.FindingsNo association was observed between MBL2 genotype or plasma MBL concentrations, and the type or frequency of infection. Adding to the literature, we found no evidence that viral infections or co-infections with virus and bacteria would be predisposed by MBL deficiency. We further saw no correlation between MBL2 genotype and the risk of fever. However, fever duration in febrile neutropaenic episodes was negatively associated with MBL2 SNP mutations (p<0.05). Patients with MBL2 SNP mutations presented a median febrile duration of 1.8 days compared with 3 days amongst patients with wildtype MBL2 genotype.InterpretationWe found no clear association between infection, or infection type to MBL2 genotypes or plasma MBL concentration, and add to the reports casting doubts on the benefit of recombinant MBL replacement therapy use during iatrogenic neutropaenia.

Rhodium Interaction with Human NRG1 Gene of Schizophrenia

Abstract - Rhodium (II) acetate [Rh2 (O2CCH3)4] could be used as an indicator for single nucleotide polymorphism (SNPs) involved in the onset of schizophrenia. Rhodium (Rh1) has affinity to make covalent interactions with neuregulin (NRG1) gene at SNPs mutation. Binding effects of Rh1 has been studied under different molar concentrations at different time periods. In this study we used Rh1 to evaluate its interaction with NRG1 gene in Schizophrenic patients of Pakistan. Rh-NRG1 adduct were amplified by PCR and visualized on agarose gel electrophoresis. Here we show Rh1 binding with NRG1 gene was inhibited with increasing concentration ranges from 0.5 -3µM. It has been noted that upon binding with NRG1 gene Rh1 decreased the mobility and intensity of the DNA bands. Noticeably Rh1 didn’t inhibit the activity of Mun1 restriction enzyme having specific CAAA cleavage site. After the digestion of NRG1 gene having SNPs mutation combining with Rh1 proves its covalent binding only with Guanine or Thymine and not with Adenine or Cytosine. This is a novel study that shows rhodium can covalently binds with human dsDNA and can inhibit its amplification. The effect of Rh1 to target different SNPs mutations (normally occurs in genetic diseases such as schizophrenia) can be identified by using this technique. There are variations between human populations, so a SNP allele that is common in one geographical or ethnic group may be much rarer in another, and Rh1 can act as a useful tool to identify SNPs of schizophrenic genes.

Analysis of TLR4 and TLR2 polymorphisms in inflammatory bowel disease in a Guangxi Zhuang population

To study the polymorphisms of toll-like receptor 4 (TLR4) gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp and susceptibility to inflammatory bowel disease (IBD) in the Zhuang population from Guangxi, China. A case-control study was performed from February 2007 to October 2011 which included 146 Zhuang patients with IBD in the experimental group and 164 healthy Zhuang subjects who acted as the control group. All patients and healthy subjects were from the Guangxi Zhuang Autonomous Region of China. Genomic DNA was extracted from intestinal tissue by the phenol chloroform method. TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp were amplified by polymerase chain reaction (PCR), and then detected by PCR-restriction fragment length polymorphism (RFLP). The TLR4 gene Asp299Gly was digested using Nco I restriction enzyme, and a single band of 249 bp was observed which showed that it was a wild type (AA). The TLR4 gene Thr399Ile was digested using Hinf Irestriction enzyme and only the wild type (CC) was detected. In addition, the TLR2 gene Arg677Trp was digested using Aci I restriction enzyme and only the wild type (CC) was detected. The TLR2 gene Arg753Gln was digested using Pst I restriction enzyme. Only the wild type (GG) as a single band of 254 bp was observed during RFLP. Overall, no heterozygous or homozygous single nucleotide polymorphism mutations were found in patients with Crohn's disease and ulcerative colitis both in the TLR4 gene Asp299Gly, Thr399Ile and the TLR2 gene Arg677Trp, Arg753Gln in the Zhuang population from the Guangxi Zhuang Autonomous Region of China. The TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp polymorphisms may not be associated with IBD in the Zhuang population from the Guangxi Zhuang Autonomous Region of China.

20 SNPs as supplementary markers in kinship testing

Single Nucleotide Polymorphisms (SNPs) are having an increasingly role in Forensic Genetics due to very low SNP mutation rates and the possibility to multiplex a great number of loci. The purpose of this study was to evaluate the use of 20 autosomal SNPs as additional markers in the resolution of kinship casework where the alleged father was not available for testing and close relatives were used instead. A total of six caseworks which included alleged paternal grandparents, alleged uncles or alleged brothers were studied. All individuals studied in these cases were typed before with 17 autosomal STRs using Identifiler(Plus) ® and Powerplex 16 ® systems. Twenty SNPs were typed using SNaPshot ® methodology with two 10-plex, previously shown useful in paternity testing. LRs were calculated with “ Familias” using South Portugal STR and SNP frequency databases. This study confirms that even as few as 20 autosomal SNP loci can be very useful in kinship analysis as a complement to standard methodologies. Moreover, SNaPshot ® methodology can be easily implemented in any Forensic Laboratory.

Functional effect of grapevine 1-deoxy-D-xylulose 5-phosphate synthase substitution K284N on Muscat flavour formation

Grape berries of Muscat cultivars (Vitis vinifera L.) contain high levels of monoterpenols and exhibit a distinct aroma related to this composition of volatiles. A structural gene of the plastidial methyl-erythritol-phosphate (MEP) pathway, 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS), was recently suggested as a candidate gene for this trait, having been co-localized with a major quantitative trait locus for linalool, nerol, and geraniol concentrations in berries. In addition, a structured association study discovered a putative causal single nucleotide polymorphism (SNP) responsible for the substitution of a lysine with an asparagine at position 284 of the VvDXS protein, and this SNP was significantly associated with Muscat-flavoured varieties. The significance of this nucleotide difference was investigated by comparing the monoterpene profiles with the expression of VvDXS alleles throughout berry development in Moscato Bianco, a cultivar heterozygous for the SNP mutation. Although correlation was detected between the VvDXS transcript profile and the accumulation of free monoterpenol odorants, the modulation of VvDXS expression during berry development appears to be independent of nucleotide variation in the coding sequence. In order to assess how the non-synonymous mutation may enhance Muscat flavour, an in vitro characterization of enzyme isoforms was performed followed by in vivo overexpression of each VvDXS allele in tobacco. The results showed that the amino acid non-neutral substitution influences the enzyme kinetics by increasing the catalytic efficiency and also dramatically affects monoterpene levels in transgenic lines. These findings confirm a functional effect of the VvDXS gene polymorphism and may pave the way for metabolic engineering of terpenoid contents in grapevine.

First Report of ‘Candidatus Liberibacter solanacearum’ Naturally Infecting Tomatoes in the State of Mexico, Mexico

In January 2011, tomato (Solanum lycopersicum) plants exhibiting stunting, yellow mosaic, short, chlorotic leaves, aborted flowers, and reduced-size fruits, symptoms similar to those exhibited by plants infected by 'Candidatus Liberibacter solanacearum' (2), were observed in approximately 5% of tomato plants in greenhouses in Jocotitlan in the State of Mexico, Mexico. Occasional plant recovery was also observed. Tomato plants in this facility were previously shown to be infected by Mexican papita viroid (MPVd), Pepino mosaic virus (PepMV), and aster yellows phytoplasma. Eight symptomatic leaf samples (designated MX11-01 to MX11-08) were collected and screened against selected tomato viruses and pospiviroids by reverse transcription (RT)-PCR using purified plant RNA or for 'Ca. L. solanacearum' by PCR using purified plant DNA. As expected, both PepMV and MPVd were detected in these samples. However, two 'Ca. L. solanacearum'-specific PCR products (1,168 and 669 bp) were also amplified in two samples (MX11-02 and MX11-05) using primers OA2 (2) and OI2c (1) or CL514F/CL514R (3), respectively. Each 'Ca. L. solanacearum'-specific PCR product was gel purified with Geneclean (Q-Biogene, Carlsbad, CA) and cloned into pCR2.1 using TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced (Functional Biosciences, Madison, WI). Sequences of 16S rRNA (1,168 bp) in both isolates (GenBank Accession Nos. JF811596 and JF811597) were identical. However, the 669-bp 50S rRNA sequences in these two isolates (GenBank Accession Nos. JF811598 and JF811599) contained two single nucleotide polymorphism (SNP) mutations. BLASTn searches showed that both 16S rRNA and 50S gene sequences in MX11-05 were identical to the 'Ca. L. solanacearum' previously identified on potato in Chihuahua (GenBank Accession Nos. FJ829811 and FJ829812) and Saltillo (GenBank Accession Nos. FJ498806 or FJ498807) in eastern Mexico. These 'Ca. L. solanacearum' isolates were recently classified as the "b" haplotype (4). Alignment analysis of the 'Ca. L. solanacearum' 16S rRNA sequences also revealed the conserved SNP mutations (g.212T > G and g.581T > C) in MX11-02 and MX11-05 as previously identified for other "b" haplotype isolates (4). 'Ca. L. solanacearum' was first identified in greenhouse tomatoes in 2008 in New Zealand (2). It has also been identified in greenhouse and field tomatoes in the United States. 'Ca. L. solanacearum' was previously reported to infect field tomatoes in Sinaloa, Mexico (3), which was recently considered as the "a" haplotype (4). To our knowledge, this is the first report of 'Ca. L. solanacearum' naturally infecting tomatoes in Jocotitlan in the State of Mexico, Mexico. The greenhouse tomato 'Ca. L. solanacearum' may be transmitted from infected solanaceous plants by potato psyllids (Bactericera cockerelli), which were observed in this facility.

Single nucleotide polymorphisms in the Melanocortin 1 Receptor gene are linked with lightness of fibre colour in Peruvian Alpaca (Vicugna pacos)

Melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) (MC1R) is a gene-controlling melanogenesis in mammals. However, it is not well characterized in alpacas and its association with colour is not known. The aim of this study was to look for polymorphisms in the MC1R gene in Peruvian Huacaya alpacas and to analyse the relationship between MC1R single nucleotide polymorphisms (SNPs) and the variations in the instrumental measurement of colour of alpaca fibre. Sixty alpaca fibre samples from black, brown, cream and white animals (15 for each colour) were used to extract DNA from hair bulbs. Colour was measured with a spectrophotometer to obtain quantitative values (CieL*a*b*). Sixteen samples, four of each colour group, were sequenced. Eighteen SNP mutations, 10 not previously described, were found in these 16 sequences. Three of them were chosen (c.82A>G, c.865C>T, c.901C>T) to analyse genotypes by PCR-RFLP in the other 44 fibre samples and to determine the association of mutations with instrumental colour. These three polymorphisms showed association with fibre lightness (P < 0.05), although there was no correlation with colour groups.

RNAi: a potential new class of therapeutic for human genetic disease

Dominant negative genetic disorders, in which a mutant allele of a gene causes disease in the presence of a second, normal copy, have been challenging since there is no cure and treatments are only to alleviate the symptoms. Current therapies involving pharmacological and biological drugs are not suitable to target mutant genes selectively due to structural indifference of the normal variant of their targets from the disease-causing mutant ones. In instances when the target contains single nucleotide polymorphism (SNP), whether it is an enzyme or structural or receptor protein are not ideal for treatment using conventional drugs due to their lack of selectivity. Therefore, there is a need to develop new approaches to accelerate targeting these previously inaccessible targets by classical therapeutics. Although there is a cooling trend by the pharmaceutical industry for the potential of RNA interference (RNAi), RNAi and other RNA targeting drugs (antisense, ribozyme, etc.) still hold their promise as the only drugs that provide an opportunity to target genes with SNP mutations found in dominant negative disorders, genes specific to pathogenic tumor cells, and genes that are critical for mediating the pathology of various other diseases. Because of its exquisite specificity and potency, RNAi has attracted a considerable interest as a new class of therapeutic for genetic diseases including amyotrophic lateral sclerosis, Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), spinocerebellar ataxia, dominant muscular dystrophies, and cancer. In this review, progress and challenges in developing RNAi therapeutics for genetic diseases will be discussed.

Functional effects of ERAAP polymorphisms linked to ankylosing spondylitis (100.18)

Abstract Optimal peptide loading on the MHC I molecule is essential for antigen presentation to CD8+ cytotoxic T lymphocytes. Endoplasmic Reticulum Aminopeptidase associated with Antigen Processing (ERAAP) is responsible for N-terminal trimming of peptides to optimal length for stable loading and presentation through MHC I. In humans, ERAAP is also known to play a role in pro-inflammatory cytokine receptor cleavage as well as regulating blood pressure and angiogenesis. Recently, a single nucleotide polymorphism (SNP) linkage analysis study has identified ERAAP as being associated with increased risk of the autoimmune inflammatory disorder, ankylosing spondylitis (AS). Here, we show that SNP mutation of ERAAP alters its ability to trim peptides. Transfection of ERAAP-/- cells with ERAAP molecules incorporating each of the SNPs identified in the linkage analysis show a hierarchy of reduced function. More pronounced functional phenotypes are revealed with additional mutations suggesting a cumulative effect. Although the mechanism for the effect of SNP mutation on ERAAP function is unclear, it appears that they cause a dramatic effect on peptide trimming.

HRM technology for the identification and characterization of INDEL and SNPs mutations in genes involved in drought and salt tolerance of durum wheat

WRKY transcription factors are one of the largest families of transcriptional regulators and form an integral part of signalling webs which modulate many plant processes, such as abiotic stress tolerance. In the present paper, an innovative method has been applied to identify novel WRKY-1 alleles involved in the responses to salt and drought stresses in Triticum durum. This technique involves scanning for sequencing variations in cDNA-derived PCR amplicons, using high-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM represents a novel advance in detection of single-nucleotide polymorphisms (SNPs) by measuring temperature-induced strand separation of short PCR amplicons. The use of this approach is still limited in the field of plant biology. Here, HRM analysis has been applied to the discovery and genotyping of durum wheat SNPs. Specific primers have been designed, starting at multi-alignment of WRKY-1-conserved portions. The PCR amplicons, containing single SNPs, produce distinctive HRM profiles, and by sequencing the PCR products identified, SNPs have been characterized and validated. The results showed that all the revealed SNPs are located on salt-tolerant varieties, confirming their value in breeding activities.

The association between intragenic SNP haplotypes and mutations of the beta globin gene in a Turkish population

Identification of the beta globin gene mutation-related haplotypes is of interest for the delineation of the clinical heterogeneity as well as understanding of the origin and spreading of the beta globin gene mutations. We screened the whole beta globin gene in 197 Turkish patients by direct sequencing and performed Haploview analyses for beta globin gene haplotyping using five common intragenic SNPs; rs713040, rs10768683, rs7480526, rs7946748, and rs1609812. We found 25 different beta globin gene point mutations by sequencing. A Turkish type of inv/del mutation by MLPA and Gap-PCR was also detected with additional studies. The seven most common mutations with higher frequency of 5% were IVS-I-110 (G>A) (35.6%), Hb S(10.6%), IVS-I-6 (T>C) (7.4%), IVS-I-1 (G>A) (6.9%), IVS-II-1 (G>A) (6.9%), Cod8(−AA) (6%), IVS-II-745 (C>G) (5.1%) and accounted for 78.7% of all mutations. We identified seven different haplotypes (Haplotype I–VII) using five intragenic single nucleotide polymorphisms (SNPs) genotyped by sequencing of the beta globin gene. The association between the mutations and the haplotypes was defined for 16 different mutations. We suggest that haplotyping by these five intragenic SNPs will provide useful information about the origin of the mutations and gene flow among as well as the explanation of the clinical heterogeneity.

Prognostic Significance of Mutations In Isocitrate Dehydrogenase (IDH) Enzyme Isoforms 1 and 2 and Single Nucleotide Polymorphisms (SNP) In IDH1, In Patients with Acute Myeloid Leukemia Treated with High Dose Cytarabine and Idarubicin Induction

AbstractAbstract 2706 BackgroundIDH1 and IDH2 gene mutations have been identified as novel, recurring molecular aberrations among patients with normal karyotype acute myeloid leukemia (AML). The potential impact of these mutations as well as an IDH1 single nucleotide polymorphism (SNP) on the outcome of the patients is being actively investigated. Materials and MethodsAmong 358 patients with AML treated from October 2004 to February 2010 on 4 consecutive protocols using high dose ara-C plus idarubicin induction therapy (IA alone, IA plus tipifarnib [IAT], IA plus sorafenib [IAS], and IA plus vorinostat [IAV]), pre-treatment samples were available for 170 patients [median age 53 years, (range, 17 – 73); 96% ≤ 65 years] for testing for IDH1R132, IDH2R172 and IDH2R140 mutations. All patients received an induction course of therapy followed by up to 5 reduced-dose consolidation cycles followed by maintenance therapy with T, S, or V for up to a year; patients treated with IA had no maintenance. We examined whether presence of mutations in either gene or the codon 105 SNP in IDH1 was associated with pre-treatment characteristics or outcome. We also sought whether treatment with any of the 4 regimens had an impact on the outcome of patients with IDH aberrations. ResultsOverall, IDH1 and IDH2 mutations were present in 12 (7%) and 24 (14%) patients, respectively, and IDH1G105 SNP in 24 (14%). Overall, 52 (30%) patients had IDH gene aberrations; 2 patients had concomitant IDH1R132 mutation and IDH1G105 SNP, 3 patients had IDH2R140 mutation and IDH1G105 SNP, 1 patient IDH2R172 mutation and IDH1G105 SNP, and 1 patient had IDH1R132 mutation, IDH1G105 SNP, and IDH2R140 mutation. There was a strong association with normal karyotype with 11 of 12 (92%) of IDH1 mutated, 18 of 24 (75%) of IDH2 mutated, and 18 of 24 (75%) of IDH1 SNP being diploid. There was no association between any of the aberrations and patient age, sex, therapy-related vs. de novo AML, presenting WBC, peripheral blood blasts, or FAB subtype. IDH1 mutation was associated with a higher presenting platelet count (median 99 vs. 50 × 109/L in IDH1 wild-type [WT], p<0.05) and IDH1 SNP with a higher platelet count (median 68 vs. 46 × 109/L in IDH1WT, p<0.04) and a lower bone marrow blast percentage (median 39% vs. 54% in IDH1WT, p<0.03). 12 of 52 patients with IDH aberrations also had FLT3-ITD compared to 22 of 128 patients with IDH-WT (p=NS). There was no association with achievement of complete response (CR), remission duration, overall and event-free survival and any the IDH aberrations. Furthermore, there was no association with a higher CR rate or survival among any of the 4 different regimens for the 52 patients with aberrant IDH gene. ConclusionsIDH aberrations including mutations and SNP occur in about 30% of younger patients with AML, mostly with diploid karyotype. IDH1 mutations and SNP are associated with a higher presenting platelet count and SNP with lower bone marrow blasts. Using high-dose ara-C based induction regimens, we did not detect an association with outcome for any of the aberrations. Disclosures:No relevant conflicts of interest to declare.