Deletion Patterns Research Articles

ATOXIGENIC ASPERGILLUS FLAVUS ISOLATES AS CANDIDATE BIOCONTROL AGENTS IN MAIZE

In Italy, nearly 90% of the maize is produced in five regions in the north of the country (Emilia Romagna, Friuli Venezia Giulia, Lombardy, Piedmont and Veneto). Although contaminations by fumonisins are very fre- quent in these areas, high levels of aflatoxins (AFs) were detected for the first time in 2003, a situation that turned worse in 2012. Aflatoxins are secondary metabolites pro- duced by several members of the genus Aspergillus sec- tion Flavi. Among these, aflatoxin B1 (AFB1) is the most dangerous and has been classified by the International Agency for Research on Cancer as class 1 toxin, due to its confirmed carcinogenic and teratogenic activity in hu- mans. The main responsible of maize AFB1 contamina- tion is Aspergillus flavus, a filamentous fungus with a vegetative incompatibility system that limits hyphal fu- sion and gene flow between individuals belonging to dif- ferent vegetative compatibility groups (VCGs). Isolates are assigned to VCGs with functional vegetative compat- ibility analyses (VCAs) typically using nitrate nonutiliz- ing auxotrophs (nit- mutants). The ability to synthesize AFs varies very much within A. flavus population, with fungal isolates that produce anywhere from over 1000 ppm AF to no toxin at all (atoxigenic isolates). The genes involved in AFs biosynthesis (confirmed 25) are contained within a 65 to 70 kb cluster, and several dis- tinct lesions within this cluster have been described that may each be responsible for atoxigenicity in various iso- lates. The main objectives of this study were: (i) VCAs of A. flavus isolates from northern Italy, (ii) selection of atoxigenic isolates potentially useful as biocontrol agents to reduce AFB1 contamination in maize. A high number (45%) of the isolates tested on autoclaved maize were unable to produce detectable levels of aflatoxins. Nit- mutants were obtained from all isolates within 10 days on chlorate-amended medium. More than 1500 mutant sectors were obtained and phenotyped revealing 72% to be niaD, 21% nirA and 7% cnx. Forty-six VCGs were identified; 24 contained only atoxigenic isolates, and the remaining 22 only aflatoxin producers. Members of the largest atoxigenic VCG were found in four of the five ar- eas sampled and in different years of sampling. Molecu- lar analysis of the genes in the aflatoxin biosynthesis gene cluster showed six deletion patterns. In particular, no deletions were detected in 10 atoxigenic isolates, whereas eight isolated had the entire cluster deleted. The capability of 18 atoxigenic isolates to reduce AFB1 pro- duction by a toxigenic strain was evaluated on maize. Each atoxigenic strain was co-inoculated with a toxi- genic, in the same proportion (1:1) or in a reduced amount (1 atoxigenic : 4 toxigenic). The extent of reduc- tion of AFB1 ranged from 60% to 89%, and the best five isolates, belonging to five different VCGs, showed a percentage of reduction similar to or greater than that achieved by a A. flavus strain registered in the USA as a biocontrol agent. Although through the proportion 1:1 (atoxigenic versus toxigenic) the highest percentage of reduction was achieved, the efficiency, intended as the ability of the inoculum unit of an atoxigenic strain to re- duce AFB1 produced by a toxigenic one, was greater in the 1:4 proportion. This study analysed for the first time the VCG structure of a A. flavus population isolated in the major maize-producing districts of Italy. Several atoxigenic fungal strains were identified as potential bio- control agents for mitigating aflatoxin contamination, thus laying the basis for the development of a reportedly very effective biocontrol strategy in Italy.

Whole genome HBV deletion profiles and the accumulation of preS deletion mutant during antiviral treatment

BackgroundHepatitis B virus (HBV), because of its error-prone viral polymerase, has a high mutation rate leading to widespread substitutions, deletions, and insertions in the HBV genome. Deletions may significantly change viral biological features complicating the progression of liver diseases. However, the clinical conditions correlating to the accumulation of deleted mutants remain unclear. In this study, we explored HBV deletion patterns and their association with disease status and antiviral treatment by performing whole genome sequencing on samples from 51 hepatitis B patients and by monitoring changes in deletion variants during treatment. Clone sequencing was used to analyze preS regions in another cohort of 52 patients.ResultsAmong the core, preS, and basic core promoter (BCP) deletion hotspots, we identified preS to have the highest frequency and the most complex deletion pattern using whole genome sequencing. Further clone sequencing analysis on preS identified 70 deletions which were classified into 4 types, the most common being preS2. Also, in contrast to the core and BCP regions, most preS deletions were in-frame. Most deletions interrupted viral surface epitopes, and are possibly involved in evading immuno-surveillance. Among various clinical factors examined, logistic regression showed that antiviral medication affected the accumulation of deletion mutants (OR = 6.81, 95% CI = 1.296 ~ 35.817, P = 0.023). In chronic carriers of the virus, and individuals with chronic hepatitis, the deletion rate was significantly higher in the antiviral treatment group (Fisher exact test, P = 0.007). Particularly, preS2 deletions were associated with the usage of nucleos(t)ide analog therapy (Fisher exact test, P = 0.023). Dynamic increases in preS1 or preS2 deletions were also observed in quasispecies from samples taken from patients before and after three months of ADV therapy. In vitro experiments demonstrated that preS2 deletions alone were not responsible for antiviral resistance, implying the coordination between wild type and mutant strains during viral survival and disease development.ConclusionsWe present the HBV deletion distribution patterns and preS deletion substructures in viral genomes that are prevalent in northern China. The accumulation of preS deletion mutants during nucleos(t)ide analog therapy may be due to viral escape from host immuno-surveillance.

Slow DNA Loss in the Gigantic Genomes of Salamanders

Evolutionary changes in genome size result from the combined effects of mutation, natural selection, and genetic drift. Insertion and deletion mutations (indels) directly impact genome size by adding or removing sequences. Most species lose more DNA through small indels (i.e., ∼1–30 bp) than they gain, which can result in genome reduction over time. Because this rate of DNA loss varies across species, small indel dynamics have been suggested to contribute to genome size evolution. Species with extremely large genomes provide interesting test cases for exploring the link between small indels and genome size; however, most large genomes remain relatively unexplored. Here, we examine rates of DNA loss in the tetrapods with the largest genomes—the salamanders. We used low-coverage genomic shotgun sequence data from four salamander species to examine patterns of insertion, deletion, and substitution in neutrally evolving non-long terminal repeat (LTR) retrotransposon sequences. For comparison, we estimated genome-wide DNA loss rates in non-LTR retrotransposon sequences from five other vertebrate genomes: Anolis carolinensis, Danio rerio, Gallus gallus, Homo sapiens, and Xenopus tropicalis. Our results show that salamanders have significantly lower rates of DNA loss than do other vertebrates. More specifically, salamanders experience lower numbers of deletions relative to insertions, and both deletions and insertions are skewed toward smaller sizes. On the basis of these patterns, we conclude that slow DNA loss contributes to genomic gigantism in salamanders. We also identify candidate molecular mechanisms underlying these differences and suggest that natural variation in indel dynamics provides a unique opportunity to study the basis of genome stability.

A novel contiguous gene deletion of AVPR2 and ARHGAP4 genes in male dizygotic twins with nephrogenic diabetes insipidus and intellectual disability

The clinical features of loss of ARHGAP4 function remain unclear despite several reports of different patterns of deletions inactivating different functional regions of the protein. The protein encoded by ARHGAP4 is thought to function as a Rho GTPase activating protein. Characterization of the genetic defect causing X-linked nephrogenic diabetes insipidus (NDI) and intellectual disability in two dizygotic twin brothers revealed a novel contiguous deletion of 17,905 bp encompassing the entire AVPR2 gene and extending into intron 7 of the ARHGAP4 gene. Examination of their mother showed that she was a carrier of this deletion. An attempt was made to distinguish the putative clinical signs of an ARHGAP4 deletion from the well-defined phenotype of X-linked NDI caused by an AVPR2 gene deletion. By reviewing all characterized deletions encompassing ARHGAP4, we reconsider the potential role of ARHGAP4 in cognition.

Impaired spermatogenesis and GR/GR deletions related to Y chromosome haplogroups in Korean men

Microdeletion of the Azoospermia Factor (AZF) regions in Y chromosome is a well-known genetic cause of male infertility resulting from spermatogenetic impairment. However, the partial deletions of AZFc region related to spermatogenetic impairment are controversial. Therefore, this study was designed to characterize the partial AZFc deletion patterns and their clinical consequences in Korean population.

Losing identity: structural diversity of transposable elements belonging to different classes in the genome of Anopheles gambiae

BackgroundTransposable elements (TEs), both DNA transposons and retrotransposons, are genetic elements with the main characteristic of being able to mobilize and amplify their own representation within genomes, utilizing different mechanisms of transposition. An almost universal feature of TEs in eukaryotic genomes is their inability to transpose by themselves, mainly as the result of sequence degeneration (by either mutations or deletions). Most of the elements are thus either inactive or non-autonomous. Considering that the bulk of some eukaryotic genomes derive from TEs, they have been conceived as “TE graveyards.” It has been shown that once an element has been inactivated, it progressively accumulates mutations and deletions at neutral rates until completely losing its identity or being lost from the host genome; however, it has also been shown that these “neutral sequences” might serve as raw material for domestication by host genomes.ResultsWe have analyzed the sequence structural variations, nucleotide divergence, and pattern of insertions and deletions of several superfamilies of TEs belonging to both class I (long terminal repeats [LTRs] and non-LTRs [NLTRs]) and II in the genome of Anopheles gambiae, aiming at describing the landscape of deterioration of these elements in this particular genome. Our results describe a great diversity in patterns of deterioration, indicating lineage-specific differences including the presence of Solo-LTRs in the LTR lineage, 5′-deleted NLTRs, and several non-autonomous and MITEs in the class II families. Interestingly, we found fragments of NLTRs corresponding to the RT domain, which preserves high identity among them, suggesting a possible remaining genomic role for these domains.ConclusionsWe show here that the TEs in the An. gambiae genome deteriorate in different ways according to the class to which they belong. This diversity certainly has implications not only at the host genomic level but also at the amplification dynamic and evolution of the TE families themselves.

Y chromosome interstitial deletion induced Y-STR allele dropout in AMELY-negative individuals

Short tandem repeat (STR) multiplexes with the amelogenin (AMEL) gene as a gender marker have been used as a routine tool of forensic DNA analysis. It has been reported that AMEL-based gender detection could misidentify a known male as a female due to the dropout of amelogenin Y (AMELY) allele. Other gender markers, such as Y-chromosomal short tandem repeat (Y-STR), may be a substitution of AMEL and help the sex determination. In current study, employing AmpFlSTR® Sinofiler and AmpFlSTR® Y-filer™ PCR Amplification kit, 18 AMELY-negative males were identified. Accordingly, the incidence of the AMELY dropout was 0.227 ‰ (18/79,304) in Chinese population. Sequencing of AMELY allele and analyzing of azoospermia factors region suggested that 3 out of 18 misidentifications were induced by mutations in the primer-binding region of the AMELY, while other 15 sex misidentifications were results of Y chromosome microdeletions with variant lengths. Moreover, variant combination patterns of AMELY dropout and Y-STRs deletions were also observed. Our data suggested that Y-STR locus dropout may indicate more problems, especially in the mixed sample's interpretation. Results of haplogroup prediction showed that seven AMELY dropouts combined with variant Y-STR deletions can be classified as the J2 subdivision, suggesting that some of these Y chromosomes might descend from a common ancestor.

The prognostic difference of monoallelic versus biallelic deletion of 13q in chronic lymphocytic leukemia

In a recent online paper on del13q14 chronic lymphocytic leukemia (CLL), Garg at al1 state that neither pattern of deletion (mono- versus biallelic) nor the percentage of deleted cells influences risk of progression. Interestingly, in 12 patients who had a repeat fluorescent in situ hybridization (FISH) analysis, an increase in the number of deleted cells was detected. In light of these data, we reviewed our del13q14-CLL patients who were monitored by FISH during their clinical course. Between 2001 and 2009, del13q14 was identified as the sole abnormality in 137 of 352 CLL patients (39%). Baseline FISH analysis was performed at diagnosis or within 12 months (probes: LSI-D13S319 and LSI-Rb1, LSI-ATM, LSI-p53, CEP12; Vysis, Downers Grove, Ill). D13S319 deletion was monoallelic in 121 cases (88%) and biallelic in 16. The median percentage of abnormal nuclei was 50% (range, 15%-96%) and was higher in patients with a biallelic pattern (73% vs 49%; P < .0001, Mann-Whitney test). With a median follow-up of 59 months (range, 12-124 months), the 5-year treatment-free survival rate was 54% (Kaplan-Meier). As a continuous variable, the baseline percentage of abnormal nuclei was related to progression (hazard ratio [HR], 1.02; 95% confidence interval [CI], 1.01-1.04; P = .0002; Cox regression), whereas the pattern of deletion was not significant. Repeat FISH analyses were performed on 100 patients (median interval from baseline to repeat FISH analysis, 36 months; range, 9-97 months). A significant increase in the proportion of del13q14 cells was detected (P < .0001, Wilcoxon matched pairs), and change in the percentage of deleted cells per unit of time (1 month) significantly affected the risk of treatment requirement (HR, 2.03; P = .016; 95% CI, 2.03-3.6; Cox regression). Although del13q14 CLL carries a good prognosis, it is clinically and biologically heterogeneous. Although prognostic impact of baseline FISH profile is controversial, our experience confirms2, 3 that the percentage of deleted cells at diagnosis represents an useful prognostic parameter. Additionally, we advise FISH monitoring for a dynamic outcome estimate and to guide clinical surveillance. No specific funding was disclosed. CONFLICT OF INTEREST DISCLOSURES The authors made no disclosures. Ester M. Orlandi MD*, Paolo Bernasconi Prof*, Cristiana Pascutto PhD*, * Hematology Unit, Department of Hematology-Oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.

Altered Patterns of Fractionation and Exon Deletions in Brassica rapa Support a Two-Step Model of Paleohexaploidy

The genome sequence of the paleohexaploid Brassica rapa shows that fractionation is biased among the three subgenomes and that the least fractionated subgenome has approximately twice as many orthologs as its close (and relatively unduplicated) relative Arabidopsis than had either of the other two subgenomes. One evolutionary scenario is that the two subgenomes with heavy gene losses (I and II) were in the same nucleus for a longer period of time than the third subgenome (III) with the fewest gene losses. This "two-step" hypothesis is essentially the same as that proposed previously for the eudicot paleohexaploidy; however, the more recent nature of the B. rapa paleohexaploidy makes this model more testable. We found that subgenome II suffered recent small deletions within exons more frequently than subgenome I, as would be expected if the genes in subgenome I had already been near maximally fractionated before subgenome III was introduced. We observed that some sequences, before these deletions, were flanked by short direct repeats, a unique signature of intrachromosomal illegitimate recombination. We also found, through simulations, that short--single or two-gene--deletions appear to dominate the fractionation patterns in B. rapa. We conclude that the observed patterns of the triplicated regions in the Brassica genome are best explained by a two-step fractionation model. The triplication and subsequent mode of fractionation could influence the potential to generate morphological diversity--a hallmark of the Brassica genus.

AZFb microdeletions and oligozoospermia—which mechanisms?

To characterize the deletion patterns and its breakpoints in oligozoospermic patients presenting AZFb and AZFc microdeletions and to understand the recombination mechanisms underlying these microdeletions. Case report. Genetics Department of Faculty of Medicine of Porto, Porto, Portugal. Two men with severe oligozoospermia and two men with nonobstructive azoospermia identified as having different AZFb+c deletion patterns via Y chromosome microdeletion analysis. Definition of microdeletions and the fine characterization of the respective breakpoints by sequence-tagged sites (STS) polymerase chain reaction (PCR) and single-nucleotide variant (SNV) PCR. Study of the fine structure of the Y-chromosome and discussion of the putative mechanisms involved in each microdeletion pattern. From the four patients studied, three deletion patterns were identified: IR4/distal-P2 (25%; 1 of 4), P5/proximal-P1 (50%; 2 of 4), and P5/distal-P1 (25%; 1 of 4). Although severe oligozoospermia is normally associated with AZFc, a complete AZFb deletion was found in one case. Analysis of these patients has revealed a new putative region that may be involved in spermatogenesis conservation.

LOH-profiling by SNP-mapping in a case of multifocal head and neck cancer

To introduce an approach for the detection of putative genetic host factors that predispose patients to develop head and neck squamous cell carcinomas (HNSCC). HNSCC most often result from the accumulation of somatic gene alterations found in tumor cells. A cancer-predisposing genetic background must be expected in individuals who develop multiple cancers, starting at an unexpectedly young age or with little carcinogen exposure. Genome-wide loss of heterozygosity (LOH) profiling by single nucleotide polymorphism microarray mapping was performed in a patient with a remarkable history of multifocal HNSCC. Regions of genomic deletions in germline DNA were identified on several chromosomes with a remarkable size between 1.6 Mb and 8.1 Mb (mega base-pair). No LOH was detected at the genomic location of the tumor suppressor gene P53. Specific patterns of germline DNA deletions may be responsible for susceptibility to HNSCC and should be further analyzed.

Enamel malformations associated with a defined dentin sialophosphoprotein mutation in two families

Wang S‐K, Chan H‐C, Rajderkar S, Milkovich RN, Uston KA, Kim J‐W, Simmer JP, Hu JC‐C. Enamel malformations associated with a defined dentin sialophosphoprotein mutation in two families. Eur J Oral Sci 2011; 119 (Suppl. 1): 158–167. © 2011 Eur J Oral SciDentin sialophosphoprotein (DSPP) mutations cause dentin dysplasia type II (DD‐II) and dentinogenesis imperfecta types II and III (DGI‐II and DGI‐III, respectively). We identified two kindreds with DGI‐II who exhibited vertical bands of hypoplastic enamel. Both families had a previously reported DSPP mutation that segregated with the disease phenotype. Oral photographs and dental radiographs of four affected and one unaffected participant in one family and of the proband in the second family were used to document the dental phenotypes. We aligned the 33 unique allelic DSPP sequences showing variable patterns of insertions and deletions (indels), generated a merged dentin phosphoprotein (DPP) sequence that includes sequences from all DSPP length haplotypes, and mapped the known DSPP mutations in this context. Analyses of the DSPP sequence changes and their probable effects on protein expression, as well as published findings of the dental phenotype in Dspp null mice, support the hypothesis that all DSPP mutations cause pathology through dominant‐negative effects. Noting that Dspp is transiently expressed by mouse pre‐ameloblasts during formation of the dentino–enamel junction, we hypothesize that DSPP dominant‐negative effects potentially cause cellular pathology in pre‐ameloblasts that, in turn, causes enamel defects. We conclude that enamel defects can be part of the dental phenotype caused by DSPP mutations, although DSPP is not critical for dental enamel formation.

Deletion of /t, d/ and the Acquisition of Linguistic Variation by Second Language Learners of English

This study investigated second language (L2) learners’ acquisition of English /t, d/ deletion patterns in word‐final consonant clusters, (a) focusing on how constraints such as grammatical conditioning and phonological environment affect deletion of /t, d/ in L2 acquisition and (b) determining the extent to which these L2 learners had acquired native‐speaker‐like patterns of deletion in ways that may be similar to patterns attested for first and second language acquisition in previous research. Seven native speakers of Mandarin Chinese, all graduate students at a Midwestern university in the United States, participated in the study. Findings indicate that the participants had not yet acquired targetlike use of /–t, –d/ deletion patterns for all constraints. Following the linguistic environment, possibly due to the influence of sonority, appeared to be the easiest constraint to acquire. Other linguistic constraints, such as preceding linguistic environment and grammatical conditioning, appeared to be more difficult to acquire, probably due to the effects of first language transfer.

Investigation of tumor suppressor genes apart from VHL on 3p by deletion mapping in sporadic clear cell renal cell carcinoma (cRCC)

ObjectivesTo investigate the most recurrent deletion loci on 3p12-p26 by deletion mapping studies by PCR-LOH and BAC array-FISH in sporadic conventional renal cell carcinoma (cRCC) and further, to evaluate the their clinicopathologic significance in cRCC. Comparative allelotyping studies in cRCC and major epithelial carcinomas (MEC) such as lung, breast, and bladder tumors were also carried out to investigate the specificity of the targeted loci in cRCC. Subjects and methodsA total of 40 c-RCC patients were enrolled in this study, categorized in to 2 groups: group I comprises of patients of stages I and II and group II includes patients at stages III and IV. Loss of heterozygosity (LOH) studies were performed by PCR using 15 microsatellite markers of region 3p12-p26 on paired normal-tumor tissues. The recurrent LOH loci found in 27 cRCC tumors were further validated by BAC array-FISH using 23 serially mapped BAC clones. Simultaneously, the allelic deletion status of fragile histidine triad (FHIT) gene was studied by FISH in cRCC and major epithelial carcinoma (MEC) tumors. The numerical aberrations of chromosome 3 were also studied using the centromere enumeration probe (CEP) probe for chromosome 3 to validate the observed allelic losses by BAC array-FISH in cRCC as well as MECs. ResultsOur study revealed 3 affected regions of LOH on 3p in cRCC: 3p12.2-p14.1, 3p14.2-p21.1, and 3p24.2-p26.1 in both group I (stages I and II) and group II (stage III and IV). Comparative allelotyping studies revealed that except for LOH loci D3S2406 (20%), D3S1766 (14%), and D3S1560 (20%), remaining affected loci revealed retention of heterozygosity (ROH) in breast carcinomas. Lung and bladder tumors revealed ROH at all affected LOH loci. FISH with FHIT gene probe revealed deletions in cRCC (88%), breast (30%), and lung tumors (10%). FHIT gene deletions frequency was almost equal in both groups I and II (>70%), whereas a locus 3p13 (D3S2454) revealed the highest LOH in group II (83%) patients in comparison to group I (16%). BAC array-FISH studies in cRCC identified 15 recurrent deletion loci at crucial regions, 3p12.2, 3p14.2, 3p21.3, and 3p24.2-p26 with long continuous deletion of 3p14.1-p26.1 exclusively in patients of stages III and IV. Validation of LOH loci in breast carcinomas by BAC array-FISH with BAC clones mapped at these loci revealed comparatively lower deletion frequency for RP11-59E22 (3p12.2) (30%), RP11-759B7(3p21.1) (12%), and RP11-57D6 (3p25.2, proximal to VHL) (15%) than cRCC. ConclusionMolecular cytogenetic studies by BAC array-FISH was found to be more sensitive over LOH. Deletion patterns on 3p explored that deletion of FHIT and flanking loci may occur as an initiating event followed by deletions at 3p12.2, 3p21.31–3p21.32, and 3p24.2–3p26.1 in the initial stage of development of disease, while continuous large deletions of 3p21.3–3p26.1 and 3p14.1–3p26.1 occur as progressive deletion due to genetic instability. Lack of VHL along with flanking loci in 50% cRCC patients that included both groups I and II supported the hypothesis of both VHL dependent and VHL independent pathways in cRCC tumorigenesis. Comparative allelotyping studies in cRCC and MECs indicated association of specific targeted loci including VHL in cRCC. Further expansion of these studies with characterization of the genes at targeted loci and correlation with clinical outcome will explore the prognostic significance and also provide an insight into the mechanisms of tumor suppressive pathways in genitourinary cancers such as CRCC.

Xenografts of highly resistant leukemia recapitulate the clonal composition of the leukemogenic compartment

AbstractClonal evolution of the leukemogenic compartment may contribute to alter the therapeutic response in acute lymphoblastic leukemia (ALL). Using xenotransplantation of primary leukemia cells, we evaluated the phenotypic and genetic composition of de novo resistant very high risk precursor B-cell ALL, a subgroup defined by the persistence of minimal residual disease despite intensive chemotherapy. Analysis of copy number alterations (CNAs) showed that the xenografted leukemia, even when reconstituted from 100 cells, remained highly related to the diagnostic sample, with minor changes in CNAs, mostly deletions, emerging in most cases in the first passage into mice. At the single-cell level, the pattern of monoallelic and biallelic deletions of the CDKN2A locus revealed distinct leukemia subpopulations, which were reproducibly tracked in xenografts. In most very high risk ALL cases, the predominant diagnostic clones were reconstituted in xenografts, as shown by multiplex polymerase chain reaction analysis of immunoglobulin and T-cell receptor loci. In other cases, the pattern in CNAs and immunoglobulin and T-cell receptor rearrangement was less concordant in xenografts, suggesting the outgrowth of subclones. These results unequivocally demonstrate the existence of clonally closely related but distinct subsets of leukemia initiating cells in ALL, which has important implications for drug development and preclinical disease modeling.

Exon‐skipping events in candidates for clinical trials of morpholino

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out-of-frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in-frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino-mediated exon skipping, which changes out-of-frame deletions to in-frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon-skipping event in cells of DMD patients before the clinical trial. Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon-skipping event was analyzed on reverse transcription-polymerase chain reaction. Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51-skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon-skipping patterns, which are total or partial exon 51-skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out-of-frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51-skipping event. An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.

Brand and stock-keeping-unit (SKU) assortments, assortment changes and category sales

This study examines how brand and stock-keeping-unit (SKU) assortments affect category sales across a large number of categories over two time periods in which brand and/or SKU assortments were changed in two retail outlets. It uses store range records and sales data from a retailer who primarily added or deleted entire brands in one outlet and primarily added or deleted SKUs within brands in a secondoutlet. Categories with a greater number of SKUs achieved relatively higher sales revenue. Categories which increased assortments also increased sales. A partitioning of categories into groups undergoing distinct patterns of assortment additions and deletions reveals that categories that underwent additions only, especially brand additions, experienced the highest levels of sales increase.

Abstract 5330: Leukemia initiating cells are frequent and oligoclonal in de novo resistant ALL

Abstract Xenotransplantation of primary leukemia cells constitutes an attractive strategy to model disease for translational research, but the consequences of selective pressure on leukemia cells need to be clarified. Here we evaluate the phenotypic and genetic stability of primograft samples that were established by xenotransplantation of ALL cells from a group of patients with very high-risk precursor B-cell ALL, characterized by the persistence of minimal residual disease (VHR-ALL by MRD) after an intensive induction and consolidation therapy. Phenotypic properties remained unchanged, with concordant immunophenotypes in 9-10/10 leukemia-associated markers after up to 5 passages in Nod/scidIL2Rgammanull (NSG) mice and with stable drug response profiles in vitro on bone marrow stroma co-cultures. In 4/6 cases, 100 unsorted ALL cells were sufficient to reconstitute the leukemia. From up to 5 recurrent copy number abnormalities (CNA) detected in diagnostic samples, most were maintained in primografts. Changes in CNAs upon transplantation in NSG mice were few with 0-2 new lesions per case, mostly occurring during the first xenotransplantation. At the single cell level, the pattern of deletions in the CDKN2A/B locus revealed distinct subsets of ALL cells, which could reproducibly be tracked in primografts. These results provide unequivocal data for the existence of clonally closely related but distinct subsets of leukemia initiating cells in ALL, which has important implications for the use of this model for translational research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5330. doi:10.1158/1538-7445.AM2011-5330

Phonological Deletion in Text Massages: A Cognitive View

The aim of the present study is to represent the evidence of various correspondences between phonology and cognitive linguistics and to show the advantages of cognitive grammar than generative grammar. The article evaluates the hypothesis holding that connectionist theories (e.g. cognitive grammar) don’t have any directionality in their formulations due to their cross-dimensional correspondences and this can be their exclusive feature. This research can be considered as a theoretical and descriptive study. The samples were chosen from among English abbreviations, specifically short messages. In prominence view, arranging data is determinant in ordering things and events in the mind. This point of view is based on “figure and ground” that can be used in phonology as well. The rich- get richer and the poor- get poorer’ is catchphrases frequently used in economy and I use them in my paper. We can use the proverbs as principles in phonological deletion. The results also refer to the principle, according to which vowels are expected to undergo reduction in unstressed positions. Corroborating the “bathtub effect”(a principle in psychology) in consonants deletions was another emerged result. After assessing the samples, the hypothesis was approved and it was shown that having a cognitive approach to various linguistic modules causes some exclusive and attractive features which the generative grammar theories don’t have them. Also, it was revealed that a cognitive principle affects the pattern of deletions in text messages.

Occurrence of RD149 and RD152 deletions in Mycobacterium tuberculosis strains from Pakistan

Central Asian Strain 1 (CAS1) is the predominant Mycobacterium tuberculosis genotype in Pakistan. The occurrence of deletions in regions of differences (RDs) among CAS1 and other predominant genogroups in the country were investigated. Using stratified random sampling, 235 M. tuberculosis (185 pulmonary; 50 extrapulmonary) strains were selected from 926 previously spoligotyped isolates, including 171 CAS strains (133 CAS1 (ST26), 38 CAS subfamily), 8 Beijing isolates, 47 isolates belonging to other previously defined ("Other") clusters, and 9 previously undefined Unique isolates. Commonly reported RD deletions, RD1, RD750, RD207, RD149, RD152, RD105, RD150, RD142 and RD181, were investigated using a PCR-based method. Deletions in RDs 750, 149 and 152 were identified among CAS strains, and in RDs 207, 149, 152, 105, 150, 142 and 181 in Beijing isolates. CAS1 strains showed more frequent RD149 deletions compared with CAS subfamily strains (p = 0.036), and more frequent RD152 deletions compared with "Other" clusters (p = 0.003). RD149 and RD152 deletions were more frequent in Beijing isolates compared with CAS1 strains (p < 0.001). Concurrent RD149 and RD152 deletions were more frequent in CAS1 compared with "Other" clusters (p < 0.001) and in Beijing strains compared with CAS1 (p < 0.001). No significant difference was detected in RD deletion patterns between pulmonary and extra pulmonary isolates. Higher frequencies of RD149 and RD152 deletions and of concurrent RD149 and RD152 deletions were found in CAS1 and Beijing strains compared with CAS subfamilies, "Other" clusters and Unique strains. No association between these deletions and disease presentation, pulmonary or extrapulmonary tuberculosis, was observed.