Deletion Analysis of 15 exons located outside and inside a “Hot Spot” in the Duchenne Muscular Dystrophy gene in Duchenne’s Muscular dystrophy patients

Introduction. Duchenne and Becker Muscular Dystrophies (DMD/DMB) are X-linked recessive diseases characterized by progressive muscle weakness and wasting, loss of motor skills and death after the second decade of life. Deletions are the most prevalent mutations that affect the dystrophin gene, which spans 79 exons.Objective: Identify deletions on the dystrophin gene in 58 patients affected with DMD.Methods: Through multiplex PCR identify deletions on the dystrophin gene in 58 patients with DMD and observe the frequency of this mutation in our population.Results: We found deletions in 1.72% of patients (1 of 58 persons). Deletions were not the principal cause of disease in our population. It is possible that duplications and point mutations caused this illness in our patients.Conclusions: The frequency of deletions in the 15 exons analyzed from the dystrophin gene was low. The predominant types of mutation in our patients` samples were not deletions as has been observed in the literature worldwide, therefore, it is important to determine other types of mutations as are duplications and point mutations.

HAWAIIAN SKIRT: an F-box gene that regulates organ fusion and growth in arabidopsis

A fast neutron-mutagenized population of Arabidopsis ( Arabidopsis thaliana) Columbia-0 wild-type plants was screened for floral phenotypes and a novel mutant, termed hawaiian skirt ( hws), was identified that failed to shed its reproductive organs. The mutation is the consequence of a 28 bp deletion that introduces a premature amber termination codon into the open reading frame of a putative F-box protein ( At3g61590). The most striking anatomical characteristic of hws plants is seen in flowers where individual sepals are fused along the lower part of their margins. Crossing of the abscission marker, Pro(PGAZAT):beta-glucuronidase, into the mutant reveals that while floral organs are retained it is not the consequence of a failure of abscission zone cells to differentiate. Anatomical analysis indicates that the fusion of sepal margins precludes shedding even though abscission, albeit delayed, does occur. Spatial and temporal characterization, using Pro(HWS):beta-glucuronidase or Pro(HWS):green fluorescent protein fusions, has identified HWS expression to be restricted to the stele and lateral root cap, cotyledonary margins, tip of the stigma, pollen, abscission zones, and developing seeds. Comparative phenotypic analyses performed on the hws mutant, Columbia-0 wild type, and Pro(35S):HWS ectopically expressing lines has revealed that loss of HWS results in greater growth of both aerial and below-ground organs while overexpressing the gene brings about a converse effect. These observations are consistent with HWS playing an important role in regulating plant growth and development.

An intronic growth hormone receptor mutation causing activation of a pseudoexon is associated with a broad spectrum of growth hormone insensitivity phenotypes

Context: Inherited GH insensitivity (GHI) is usually caused by mutations in the GH receptor (GHR). Patients present with short stature associated with high GH and low IGF-I levels and may have midfacial hypoplasia ( typical Laron syndrome facial features). We previously described four mildly affected GHI patients with an intronic mutation in the GHR gene (A.(1) -> G.(1) substitution in intron 6), resulting in the activation of a pseudoexon (6 Psi) and inclusion of 36 amino acids. Objective: The study aimed to analyze the clinical and genetic characteristics of additional GHI patients with the pseudoexon (6 Psi) mutation. Design/Patients: Auxological, biochemical, genetic, and haplotype data from seven patients with severe short stature and biochemical evidence of GHI were assessed. Main Outcome Measures: We assessed genotype-phenotype relationship. Results: One patient belongs to the same extended family, previously reported. She has normal facial features, and her IGF-I levels are in the low-normal range for age. The six unrelated patients, four of whom have typical Laron syndrome facial features, have heights ranging from -3.3 to -6.0 SD and IGF-I levels that vary from normal to undetectable. We hypothesize that the marked difference in biochemical and clinical phenotypes might be caused by variations in the splicing efficiency of the pseudoexon. Conclusions: Activation of the pseudoexon in the GHR gene can lead to a variety of GHI phenotypes. Therefore, screening for the presence of this mutation should be performed in all GHI patients without mutations in the coding exons.

Influence of the Photorhabdus luminescens phosphomannose isomerase gene, manA, on mannose utilization, exopolysaccharide structure, and biofilm formation

Extracellular polysaccharide (EPS) is produced by diverse bacterial pathogens and fulfills assorted roles, including providing a structural matrix for biofilm formation and more specific functions in virulence, such as protection against immune defenses. We report here the first investigation of some of the genes important for biofilm formation in Photorhabdus luminescens and demonstrate the key role of the phosphomannose isomerase gene, manA, in the structure of functional EPS. Phenotypic analyses of a manA-deficient mutant showed the importance of EPS in motility, insect virulence, and biofilm formation on abiotic surfaces as well as the requirement of this gene for the use of mannose as the sole carbon source. Conversely, this defect had no apparent impact on symbiosis with the heterorhabditid nematode vector. A more detailed analysis of biofilm formation revealed that the manA mutant was able to attach to surfaces with the same efficiency as that of the wild-type strain but could not develop the more extended biofilm matrix structures. A compositional analysis of P. luminescens EPS reveals how the manA mutation has a major effect on the formation of a complete, branched EPS.

Profiling dehydrin gene sequence and physiological parameters in drought tolerant and susceptible spring wheat cultivars.

Physiological and yield traits such as stomatal conductance (mmol m-2s-1), Leaf relative water content (RWC %) and grain yield per plant were studied in a separate experiment. Results revealed that five out of sixteen cultivars viz. Anmol, Moomal, Sarsabz, Bhitai and Pavan, appeared to be relatively more drought tolerant. Based on morphophysiological results, studies were continued to look at these cultivars for drought tolerance at molecular level. Initially, four well recognized primers for dehydrin genes (DHNs) responsible for drought induction in T. durum L., T. aestivum L. and O. sativa L. were used for profiling gene sequence of sixteen wheat cultivars. The primers amplified the DHN genes variably like Primer WDHN13 (T. aestivum L.) amplified the DHN gene in only seven cultivars whereas primer TdDHN15 (T. durum L.) amplified all the sixteen cultivars with even different DNA banding patterns some showing second weaker DNA bands. Third primer TdDHN16 (T. durum L.) has shown entirely different PCR amplification prototype, specially showing two strong DNA bands while fourth primer RAB16C (O. sativa L.) failed to amplify DHN gene in any of the cultivars. Examination of DNA sequences revealed several interesting features. First, it identified the two exon/one intron structure of this gene (complete sequences were not shown), a feature not previously described in the two database cDNA sequences available from T. aestivum L. (gi|21850). Secondly, the analysis identified several single nucleotide polymorphisms (SNPs), positions in gene sequence. Although complete gene sequence was not obtained for all the cultivars, yet there were a total of 38 variable positions in exonic (coding region) sequence, from a total gene length of 453 nucleotides. Matrix of SNP shows these 37 positions with individual sequence at positions given for each of the 14 cultivars (sequence of two cultivars was not obtained) included in this analysis. It demonstrated a considerable diversity for this gene with only three cultivars i.e. TJ-83, Marvi and TD-1 being similar to the consensus sequence. All other cultivars showed a unique combination of SNPs. In order to prove a functional link between these polymorphisms and drought tolerance in wheat, it would be necessary to conduct a more detailed study involving directed mutation of this gene and DHN gene expression.

Mutation in TERMINAL FLOWER1 reverses the photoperiodic requirement for flowering in the wild strawberry, Fragaria vesca

Photoperiodic flowering has been extensively studied in the annual short-day and long-day plants rice and Arabidopsis while less is known about the control of flowering in perennials. In the perennial wild strawberry, Fragaria vesca L. (Rosaceae), short-day and perpetual flowering long-day accessions occur. Genetic analyses showed that differences in their flowering responses are caused by a single gene, the SEASONAL FLOWERING LOCUS which may encode the F. vesca homolog of TERMINAL FLOWER1 (FvTFL1). We show through high-resolution mapping and transgenic approaches that FvTFL1 is the basis of this change in flowering behavior and demonstrate that FvTFL1 acts as a photoperiodically regulated repressor. In short-day F. vesca, long photoperiods activate FvTFL1 mRNA expression and short days suppress it, promoting flower induction. These seasonal cycles in FvTFL1 mRNA level confer seasonal cycling of vegetative and reproductive development. Mutations in FvTFL1 prevent LD suppression of flowering, and the early flowering that then occurs under LD is dependent on the F. vesca homolog of FLOWERING LOCUS T. This photoperiodic response mechanism differs from those described in model annual plants. We suggest that this mechanism controls flowering within the perennial growth cycle in F. vesca, and demonstrate that a change in a single gene reverses the photoperiodic requirements for flowering.

Pathogenic LRRK2 mutations do not alter gene expression in cell model systems or human brain tissue

Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.

Brucella melitensis 16M: characterisation of the galE gene and mouse immunisation studies with a galE deficient mutant

The galE gene of Streptomyces lividans was used to probe a cosmid library harbouring Brucella melitensis 16M DNA and the nucleotide sequence of a 2.5 kb ClaI fragment which hybridised was determined. An open reading frame encoding a predicted polypeptide with significant homology to UDP-galactose-4-epimerases of Brucella arbortus strain 2308 and other bacterial species was identified. DNA sequences flanking the B. melitensis galE gene shared no identity with other gal genes and, as for B. abortus, were located adjacent to a mazG homologue. A plasmid which encoded the B. melitensis galE open reading frame complemented a galE mutation in Salmonella typhimurium LB5010, as shown by the restoration of smooth lipopolysaccharide (LPS) biosynthesis, sensitivity to phage P22 infection and restoration of UDP-galactose-4-epimerase activity. The galE gene on the B. melitensis 16M chromosome was disrupted by insertional inactivation and these mutants lacked UDP-galactose-4-epimerase activity but no discernible differences in LPS structure between parent and the mutants were observed. One B. melitensis 16M galE mutant, Bm92, was assessed for virulence in CD-1 and BALB/c mice and displayed similar kinetics of invasion and persistence in tissues compared with the parent bacterial strain. CD-1 mice immunised with B. melitensis 16M galE were protected against B. melitensis 16M challenge. Crown Copyright (C) 1999 Published by Elsevier Science B.V.

Expression of the efflux pump genes cmeB, cmeF and the porin gene porA in multiple-antibiotic-resistant Campylobacter jejuni

Aims: In Escherichia coli, increased expression of efflux pumps and/or decreased expression of porins can confer multiple antibiotic resistance (MAR), causing resistance to at least three unrelated classes of antibiotics, detergents and dyes. It was hypothesized that in Campylobacter jejuni, the efflux systems CmeABC, CmeDEF and the major outer membrane porin protein, MOMP (encoded by porA) could confer MAR. Methods: The expression of cmeB, cmeF and porA in 32 MAR C. jejuni isolated from humans or poultry was determined by comparative (C)-reverse transcriptase (RT)-PCR and denaturing DHPLC. A further 13 ethidium bromide-resistant isolates and three control strains were also investigated. Accumulation of ciprofloxacin carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) was also determined for all strains. Results: Although resistance to ethidium bromide has been associated with MAR, expression of all three genes was similar in the ethidium bromide-resistant isolates. These data indicate that CmeB, CmeF and MOMP play no role in resistance to this agent in C. jejuni. Six MAR isolates over-expressed cmeB, 3/32 over-expressed cmeB and cmeF. No isolates over-expressed cmeF alone. Expression of porA was similar in all isolates. All nine isolates that over-expressed cmeB contained a mutation in cmeR, substituting glycine 86 with alanine. All cmeB over-expressing isolates also accumulated low concentrations of ciprofloxacin, which were restored to wild-type levels in the presence of CCCP. Conclusions: These data indicate that over-expression of cmeB is associated with MAR in isolates of C. jejuni. However, as cmeB was over-expressed by only one-third (9/32) of MAR isolates, these data also indicate other mechanisms of MAR in C. jejuni.

Genome dynamics explain the evolution of flowering time CCT domain gene families in the Poaceae

Numerous CCT domain genes are known to control flowering in plants. They belong to the CONSTANS-like (COL) and PREUDORESPONSE REGULATOR (PRR) gene families, which in addition to a CCT domain possess B-box or response-regulator domains, respectively. Ghd7 is the most recently identified COL gene to have a proven role in the control of flowering time in the Poaceae. However, as it lacks B-box domains, its inclusion within the COL gene family, technically, is incorrect. Here, we show Ghd7 belongs to a larger family of previously uncharacterized Poaceae genes which possess just a single CCT domain, termed here CCT MOTIF FAMILY (CMF) genes. We molecularly describe the CMF (and related COL and PRR) gene families in four sequenced Poaceae species, as well as in the draft genome assembly of barley (Hordeum vulgare). Genetic mapping of the ten barley CMF genes identified, as well as twelve previously unmapped HvCOL and HvPRR genes, finds the majority map to colinear positions relative to their Poaceae orthologues. Combined inter-/intra-species comparative and phylogenetic analysis of CMF, COL and PRR gene families indicates they evolved prior to the monocot/dicot divergence ~200 mya, with Poaceae CMF evolution described as the interplay between whole genome duplication in the ancestral cereal, and subsequent clade-specific mutation, deletion and duplication events. Given the proven role of CMF genes in the modulation of cereals flowering, the molecular, phylogenetic and comparative analysis of the Poaceae CMF, COL and PRR gene families presented here provides the foundation from which functional investigation can be undertaken.

In silico identification and three-dimensional modelling of the missense mutation in ADAMTS2 in a sheep flock with dermatosparaxis

Background Dermatosparaxis (Ehlers–Danlos syndrome in humans) is characterized by extreme fragility of the skin. It is due to the lack of mature collagen caused by a failure in the enzymatic processing of procollagen I. We investigated the condition in a commercial sheep flock. Hypothesis/Objectives Mutations in the ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2) locus, are involved in the development of dermatosparaxis in humans, cattle and the dorper sheep breed; consequently, this locus was investigated in the flock. Animals A single affected lamb, its dam, the dam of a second affected lamb and the rams in the flock were studied. Methods DNA was purified from blood, PCR primers were used to detect parts of the ADAMS2 gene and nucleotide sequencing was performed using Sanger's procedure. Skin samples were examined using standard histology procedures. Results A missense mutation was identified in the catalytic domain of ADAMTS2. The mutation is predicted to cause the substitution in the mature ADAMTS2 of a valine molecule by a methionine molecule (V15M) affecting the catalytic domain of the enzyme. Both the ‘sorting intolerant from tolerant’ (SIFT) and the PolyPhen-2 methodologies predicted a damaging effect for the mutation. Three-dimensional modelling suggested that this mutation may alter the stability of the protein folding or distort the structure, causing the protein to malfunction. Conclusions and clinical importance Detection of the mutation responsible for the pathology allowed us to remove the heterozygote ram, thus preventing additional cases in the flock.

Genetic variant rs17225178 in the ARNT2 gene is associated with Asperger Syndrome

Background Autism Spectrum Conditions (ASC) are neurodevelopmental conditions characterized by difficulties in communication and social interaction, alongside unusually repetitive behaviours and narrow interests. Asperger Syndrome (AS) is one subgroup of ASC and differs from classic autism in that in AS there is no language or general cognitive delay. Genetic, epigenetic and environmental factors are implicated in ASC and genes involved in neural connectivity and neurodevelopment are good candidates for studying the susceptibility to ASC. The aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) gene encodes a transcription factor involved in neurodevelopmental processes, neuronal connectivity and cellular responses to hypoxia. A mutation in this gene has been identified in individuals with ASC and single nucleotide polymorphisms (SNPs) have been nominally associated with AS and autistic traits in previous studies. Methods In this study, we tested 34 SNPs in ARNT2 for association with AS in 118 cases and 412 controls of Caucasian origin. P values were adjusted for multiple comparisons, and linkage disequilibrium (LD) among the SNPs analysed was calculated in our sample. Finally, SNP annotation allowed functional and structural analyses of the genetic variants in ARNT2. We tested the replicability of our result using the genome-wide association studies (GWAS) database of the Psychiatric Genomics Consortium (PGC). Results We report statistically significant association of rs17225178 with AS. This SNP modifies transcription factor binding sites and regions that regulate the chromatin state in neural cell lines. It is also included in a LD block in our sample, alongside other genetic variants that alter chromatin regulatory regions in neural cells. Conclusions These findings demonstrate that rs17225178 in the ARNT2 gene is associated with AS and support previous studies that pointed out an involvement of this gene in the predisposition to ASC.

Characterisation of chicken riboflavin carrier protein gene structure and promoter regulation by estrogen

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2),which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Association of genetic variants in the adiponectin encoding gene (ADIPOQ) with type 2 diabetes in Japanese Brazilians

Aim: The objective of this study is to assess the contribution of ADIPOQ variants to type 2 diabetes in Japanese Brazilians. Methods: We genotyped 200 patients with diabetes mellitus (100 male and 100 female, aged 55.0 years [47.5-64.0 years]) and 200 control subjects with normal glucose tolerant (NGT) (72 male and 128 female, aged 52.0 years [43.5-64.5 years]). Results: Whereas each polymorphism studied (T45G, G276T, and A349G) was not significantly associated with type 2 diabetes mellitus, the haplotype GGA was overrepresented in our diabetic population (9.3% against 3.1% in NGT individuals, P=.0003). Also, this haplotype was associated with decreased levels of adiponectin. We also identified three mutations in exon 3: I164T, R221S, and H241P, but, owing to the low frequencies of them, associations with type 2 diabetes could not be evaluated. The subjects carrying the R221S mutation had plasma adiponectin levels lower than those without the mutation (2.10 mu g/ml [1.35-2.55 mu g/ml] vs. 6.68 mu g/ml [3.90-11.23 mu g/ml], P=.015). Similarly, the I164T mutation carriers had mean plasma adiponectin levels lower than those noncarriers (3.73 mu g/ml [3.10-4.35 mu g/ml] vs. 6.68 mu g/ml [3.90-11.23 mu g/ml]), but this difference was not significant (P=.17). Conclusions: We identified in the ADIPOQ gene a risk haplotype for type 2 diabetes in the Japanese Brazilian population. (C) 2010 Elsevier Inc. All rights reserved.