Sequencing Chromatograms Research Articles

MYD88 somatic mutations in MALT lymphomas

Extranodal marginal zone lymphoma (MZL) of mucosa-associated lymphoid tissue type (MALT lymphoma) is an indolent lymphoma that is found at a number of different extranodal anatomical sites and in total accounts for 7–8% of all non-Hodgkin (Bertoni et al, 2011). Recurrent chromosomal translocations and unbalanced genomic aberrations have been reported in MALT, many of which lead to activation of the nuclear factor (NF)-κB pathway (Bertoni et al, 2011). MYD88 is a central molecule in the cascade that activates NF-κB signalling after stimulation of either Toll-like, interleukin (IL) 1 and IL18 receptors (Ngo et al, 2011). The MYD88 gene is located at chromosome 3p22, a region commonly gained in MALT lymphomas (Rinaldi et al, 2011). MYD88 mutations were first described to occur almost exclusively in the activated B-cell like subtype (39%) of diffuse large B-cell lymphomas (Ngo et al, 2011), which are also characterized by NF-κB activation. Recurrent mutations were also described in 9% of gastric MALT lymphomas (Ngo et al, 2011). The vast majority of mutations, and all those observed in MALT lymphomas, determined a non-synonymous change at amino acid position 265 with a change from leucine to proline (L265P) (Ngo et al, 2011). Very recently, Xu et al (2011) identified the same MYD88 L265P change in 46/51 (90%) lymphoplasmacytic lymphomas (LPL), 87% with an IgM monoclonal protein (Waldenstrom Macroglobulinemia, WM), contrasting with 3/46 (6%) in MZLs, including 1/20 (5%) MALT lymphomas (Xu et al, 2011). These authors suggested that a L265P mutation could be used to differentiate LPL/WM from MZLs (Xu et al, 2011). With the aim of assessing the prevalence of MYD88 L265P mutations in MALT lymphomas, we studied 53 MALT lymphoma cases, previously characterized by genome-wide DNA profiling (Rinaldi et al, 2011). Informed consent was obtained in accordance with the Declaration of Helsinki following the procedures approved by the local ethical committees and institutional review boards of each participating institution. Whole genome-amplification (WGA) was performed as previously described (Mensah et al, 2012). MYD88 exon 5 underwent polymerase chain reaction (PCR) amplification using 5′-AGG GAG GACT GTG GAT GCA GTA CCA A -3′ and 5′-CTG CAG ACG TGT CTG TGA AGT T-3′, as forward and reverse primers (Morin et al, 2011), respectively. PCR products were sequenced as previously described (Mensah et al, 2012), and the sequence chromatograms were viewed with ChromasLite 2.01 (Technelysium Pty Ltd, Brisbane, Qld, Australia) and compared against GenBank NM_002468. Three out of the 53 (6%) of the MALT lymphoma cases revealed non-synonymous MYD88 mutations, all of which were verified using non-WGA DNA (Fig 1). Two cases harboured a T978C mutation causing the reported L265P substitution. Both of these cases presented with orbital adnexa involvement and no additional genomic lesions; one case had bone marrow involvement. One case of salivary gland MALT lymphoma, with trisomies of chromosomes 3 and 18, showed a novel interstitial 27bp deletion (c.1039_1065del) resulting in the loss of amino acids VCDYTNPCT (p.V286_T294del) that precede an internal inhibitory domain (Li et al, 2005; Ngo et al, 2011), which could become disrupted and allow an increased binding of MYD88 to IRAK1/IRAK4. Somatic mutations of MYD88 have been shown to activate down-stream signalling leading to the activation of both NF-κB but also JAK/STAT3 signalling (Ngo et al, 2011), providing a rationale for molecule-targeted pharmaceutical approaches in these patients. In conclusion, our data indicate that MYD88 is deregulated in a small subset of patients with MALT lymphoma. These MYD88 mutation-bearing patients, as well patients affected by LPL/WM (Xu et al, 2011) or by other indolent lymphoid tumours carrying similar mutations (Fabbri et al, 2011; Wang et al, 2011; Yan et al, 2012) will benefit from the development of new therapeutic strategies that target specific pathways irrespective of the lymphoma subtype. This work was supported by Oncosuisse grant OCS-02034-02-2007, Nelia and Amedeo Barletta Foundation (Lausanne, Switzerland) and Italian Association for Cancer Research (AIRC). FB conceived and designed the study. ZML, AR, AC and AAM performed analyses. MP, RDG, GB and EZ collected and characterized MZL samples. ZML, AC and FB interpreted the data and co-wrote the manuscript. All authors read and approved the final manuscript. All authors declare no conflict of interest.

Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via FUSARIUM-ID and Fusarium MLST

We constructed several multilocus DNA sequence datasets to assess the phylogenetic diversity of insecticolous fusaria, especially focusing on those housed at the Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF), and to aid molecular identifications of unknowns via the FUSARIUM-ID and Fusarium MLST online databases and analysis packages. Analyses of a 190-taxon, two-locus dataset, which included 159 isolates from insects, indicated that: (i) insect-associated fusaria were nested within 10 species complexes spanning the phylogenetic breadth of Fusarium, (ii) novel, putatively unnamed insecticolous species were nested within 8/10 species complexes and (iii) Latin binomials could be applied with confidence to only 18/58 phylogenetically distinct fusaria associated with pest insects. Phylogenetic analyses of an 82-taxon, three-locus dataset nearly fully resolved evolutionary relationships among the 10 clades containing insecticolous fusaria. Multilocus typing of isolates within four species complexes identified surprisingly high genetic diversity in that 63/65 of the fusaria typed represented newly discovered haplotypes. The DNA sequence data, together with corrected ABI sequence chromatograms and alignments, have been uploaded to the following websites dedicated to identifying fusaria: FUSARIUM-ID (http://isolate.fusariumdb.org) at Pennsylvania State University’s Department of Plant Pathology and Fusarium MLST (http://www.cbs.knaw.nl/fusarium) at the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center.

Novel splicing-factor mutations in juvenile myelomonocytic leukemia

Novel splicing-factor mutations in juvenile myelomonocytic leukemia

Numts help to reconstruct the demographic history of the ocellated lizard (Lacerta lepida) in a secondary contact zone

In northwestern Iberia, two largely allopatric Lacerta lepida mitochondrial lineages occur, L5 occurring to the south of Douro River and L3 to the north, with a zone of putative secondary contact in the region of the Douro River valley. Cytochrome b sequence chromatograms with polymorphisms at nucleotide sites diagnostic for the two lineages were detected in individuals in the region of the Douro River and further north within the range of L3. We show that these polymorphisms are caused by the presence of four different numts (I-IV) co-occurring with the L3 genome, together with low levels of heteroplasmy. Two of the numts (I and II) are similar to the mitochondrial genome of L5 but are quite divergent from the mitochondrial genome of L3 where they occur. We show that these numts are derived from the mitochondrial genome of L5 and were incorporated in L3 through hybridization at the time of secondary contact between the lineages. The additional incidence of these numts to the north of the putative contact zone is consistent with an earlier postglacial northward range expansion of L5, preceding that of L3. We show that genetic exchange between the lineages responsible for the origin of these numts in L3 after secondary contact occurred prior to, or coincident with, the northward expansion of L3. This study shows that, in the context of phylogeographic analysis, numts can provide evidence for past demographic events and can be useful tools for the reconstruction of complex evolutionary histories.

Genetic diversity of ITS sequences of Bursaphelenchus xylophilus

The sequence variation of internal transcribed spacer (ITS) regions of ribosomal DNA has been routinely used for species identification and species-level phylogeny of the pinewood nematode, Bursaphelenchus xylophilus. In this study, the intraspecies ITS genetic diversity of B. xylophilus was evaluated. Three pinewood nematode isolates from the United States, Japan, and Portugal were used for polymerase chain reaction (PCR) ITS region amplification and sequencing. Multiple peaks were observed in sequencing chromatograms from ITS regions of American and Japanese isolates, suggesting the presence of more than one ribosomal sequence for each isolate. PCR products were further cloned and 10 clones of each isolate were subsequently sequenced. Additionally, the ITS regions of individual nematodes from each isolate were amplified, cloned and sequenced. Among the 3 B. xylophilus isolates analyzed, an intraspecific and intra-isolate molecular variability was found. The intra-isolate ITS molecular diversity in the American isolate was higher than that in the Japanese and Portuguese isolates. However, the level of sequence variation observed within isolates was about the same as that described among ITS repeats within individuals.

Allelic sequence heterozygosity in single Giardia parasites

BackgroundGenetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates?ResultsWe used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient.ConclusionsOur results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.

Influence of PGE2 on Expression of a Functionally-Relevant p53 SNP (Pro72Arg) within Heterozygous Normal Cycling B Cells,

Abstract 3234Prostaglandin E2 (PGE2) plays a pivotal role in inflammation. It is produced by COX-2-positive cells of the innate immune system, pre-neoplastic and neoplastic tissue, and activated B cells. p53 is a tumor suppressor with broad effects that include regulation of the cell cycle, DNA repair, and induction of apoptosis. A common single nucleotide polymorphism (SNP) at codon 72, within the proline rich domain of p53, can alter p53 protein structure, phosphorylation state, and function. The p53-72R (arginine) form is significantly more effective than p53-72P (proline) at promoting apoptosis in several cell lineages, but its function in B lymphocytes is unknown. COX-2 and p53 are often found co-expressed. A mechanistic link between the two might help explain why a COX-2/PGE2 axis augments the viability of cycling non-transformed human B lymphocytes (J. Immunol. 176:6736, 2006).This study tested the hypothesis that apoptosis-prone p53-72R-positive B lymphoblasts are preferentially rescued by PGE2. This analysis was aided by our discovery that p53 expression in activated B cells exhibits allelic exclusion. Thus, individual B cells whose DNA is heterozygous (HET) for p53-p72P/R express only p72P mRNA or p72R mRNA, but never both (Haque et al., submitted ASH abstract, 2011). In the present study, single cell RT-PCR of activated B cells bearing p72P/R HET DNA was used to examine whether expression of the p72P or p72R allelic forms is influenced by level of PGE2 exposure.Purified HET quiescent tonsillar B2 cells were activated in vitro by a surrogate for C3dg-coated antigen (anti-IgM:anti-CD21:dextran), IL4 and BAFF. To permit later differentiation of undivided and dividing subsets, cells were pre-labeled with CFSE. To supplement the low levels of PGE2 produced in density-limiting cultures, one set of parallel cultures received exogenous PGE2 (50 nM) on d2 and d4. On d5, harvested cells were analysed by FACS and sorted as one cell per well into 96-well PCR plates containing lysis buffer. RNA was isolated and cDNA prepared, using random hexamer primers. Two rounds of amplification of a sequence comprising p53 exons 2a, 3, and 4 involved primers, Forward 5-cagccagactgccttccg-3 & Reverse 5-gcaagtcacagacttggctg-3, and yielded a 400bp product upon semi-quantitative gel electrophoresis. Sequencing of PCR-amplified p53 cDNA from single cells was performed commercially (Applied Biosystems Big Dye Terminator v3.1 cycle sequencing) with analysis by chromasPro software. Within each experiment, an equivalent number of single sorted cells was analyzed for each cell subset (undivided or divided ± exogenous PGE2).The frequency of single sorted cells yielding p53 amplicons from cultures without added PGE2 was 6.1 ± 6.0 % and 12.8 ± 4.9 % for the undivided and divided subsets, respectively. In contrast, the corresponding frequency from PGE2-pulsed cultures was 8.1 ± 4.9 % and 23.8 ± 8.1 %, respectively. The higher yield of p53+ divided cells upon greater PGE2-exposure is in agreement with quantitative RT-PCR of p53 mRNA isolated from cell pools. PGE2-pulsed cultures had a 2.26 ± 0.15 fold increase p53 mRNA, as compared to non-pulsed cultures (p = 0.001).Sequence chromatograms of PCR-amplified p53 cDNA revealed the identity of the expressed p53 SNP. Consistent with conclusions of p53 allelic exclusion in activated human B cells (Haque et al, submitted ASH abstract), individual B cells of p72P/R heterozygous donors manifest either p72P or p72R, but never both. While the reportedly more pro-apoptotic p72R allele was expressed in 0% (0 of 5) p53+ divided blasts from cultures without supplementary PGE2, the allele was manifest in 62% (13 of 21) of the p53+ divided blasts from PGE2-supplemented cultures. PGE2 did not promote p72R allele representation in the undivided subset. Within p53+ undivided cells, p72R was found a frequency of 50% (4 of 8) and 25% (1 of 4) in PGE2 unsupplemented and supplemented cultures respectively. Thus, the p72R allele is underrepresented in replicating B cell blasts exposed to autocrine PGE2 alone, but expressed comparably to the p72P allele when higher viability-promoting concentrations of PGE2 are present. Taken together, the observations suggest that the p72R allele promotes greater activation-induced B cell apoptosis; PGE2 has a role in dampening p53-driven apoptosis within activated B cells; and the recovery of B cell blasts expressing the p53 p72R allele will be heightened in inflammatory settings. Disclosures:No relevant conflicts of interest to declare.

Evidence of alternative splicing of porcine β‐casein (CSN2)

Figure S1 Electrophoretic migrating profiles. Figure S2 Sequencing chromatograms. Table S1 Oligonucleotide primer sequences and thermal cycling conditions. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Identification of a novel mutation in exon 1 of androgen receptor gene in an azoospermic patient with mild androgen insensitivity syndrome: case report and literature review

To report a case of an azoospermic subject with mild androgen insensitivity syndrome (MAIS) and review the relevant literature. Case report. Academic research hospital. A 49-year-old man with undermasculinized features and a history of cryptorchidism and azoospermia. Hormonal evaluation and genetic testing of the androgen receptor gene (AR). Hormonal levels and sequence chromatogram of the proband and his mother. We found total T in the normal range and high levels of gonadotropins. Karyotype was 46,XY. Genetic testing identified a novel mutation of exon 1 of AR, which resulted in an alanine to serine substitution in the transactivation domain at codon 240 (A240S). Fourteen other mutations of exon 1 of AR have been associated with MAIS to date. The novel mutation A240S of AR is involved in MAIS, a syndrome associated with azoospermia.

Sequence characteristics and phylogenetic implications of the nrDNA internal transcribed spacers (ITS) in the genus Nymphaea with focus on some Indian representatives

Nymphaea, an aquatic perennial herb with exceptionally beautiful flowers and floating leaves, is well represented globally. Out of ten species reported from India, the internal transcribed spacers (ITS) region of nrDNA was investigated in seven species of Nymphaea viz. N. alba var. rubra, N. caerulea, N. × marliacea, N. nouchali, N. pubescens, N. rubra and N. tetragona. Barring N. pubescens, whereby double peaks detected in the sequencing chromatograms may be due to random mutations occurring in some of the ITS paralogues, the additional signals detected for N. alba var. rubra and N. rubra are probably influenced by recent hybridization and introgression. Our study on sequence characteristics of ITS 1 and ITS 2 revealed high G + C content (ITS 1, 45.5–48.4%; ITS 2, 50.2–51.5%) and sequence divergence. Percentage of sequence divergence based on substitution and substitution plus indels is 44.15 and 57.19, respectively, for the ITS 1; 29.74 and 47.96% were recorded for the ITS 2. Although highly variable, conserved motifs within the ITS 1 and ITS 2 region of Nymphaea were identified and are found to be common throughout the order Nymphaeales. Sequence analysis of the ITS 1 and ITS 2 failed to detect any variation between two morphotypes of N. nouchali, namely N. nouchali JD 06 and N. nouchali JD 07, differing in flower color and found at the same geographical location. However, on comparison with another specimen of N. nouchali found at a different location, they showed considerable variation in nucleotide composition. Complemented by sequence data retrieved from GenBank, phylogenetic tree reconstruction of the genus Nymphaea based on neighbor-joining, maximum parsimony, maximum likelihood and Bayesian inference methods is presented and discussed.

Analysis of GJB2 gene coding sequence in patients with nonsyndromic hearing loss

To analyze the coding sequence of GJB2 gene in six pedigrees with nonsyndromic hearing loss in order to find deafness-causing mutations in the GJB2 gene, and to explore the inherent pattern of deafness-causing mutations in the GJB2 gene. Genomic DNA was extracted from peripheral blood for the probands and their family members. Coding sequence of the GJB2 gene was amplified by polymerase chain reaction, sequence variations were determined by DNA sequencing. Amplified fragments with overlapping peaks on sequencing chromatogram were sequenced by TA cloning in order to determine whether the mutations originated from the same allele. Mutations in the GJB2 gene were found in 4 out of the 6 pedigrees with nonsyndromic hearing loss. Four types of mutations were detected in the GJB2 gene, which were 235delC, 299-300delAT, 79G>A+341A>G, and 109G>A. Compound heterozygous polymorphisms 79G>A and 341A>G, and mutations 109G>A and 235delC had deafness-causing effects. Heterogeneous mutations of the GJB2 gene are frequently seen in patients with nonsyndromic hearing loss. Sometimes, polymorphisms may cause deafness when they are combined. Environmental factors and other genes may contribute to hearing loss caused by the GJB2 gene mutations.

A Bioinformatics Workflow for Variant Peptide Detection in Shotgun Proteomics

Shotgun proteomics data analysis usually relies on database search. However, commonly used protein sequence databases do not contain information on protein variants and thus prevent variant peptides and proteins from been identified. Including known coding variations into protein sequence databases could help alleviate this problem. Based on our recently published human Cancer Proteome Variation Database, we have created a protein sequence database that comprehensively annotates thousands of cancer-related coding variants collected in the Cancer Proteome Variation Database as well as noncancer-specific ones from the Single Nucleotide Polymorphism Database (dbSNP). Using this database, we then developed a data analysis workflow for variant peptide identification in shotgun proteomics. The high risk of false positive variant identifications was addressed by a modified false discovery rate estimation method. Analysis of colorectal cancer cell lines SW480, RKO, and HCT-116 revealed a total of 81 peptides that contain either noncancer-specific or cancer-related variations. Twenty-three out of 26 variants randomly selected from the 81 were confirmed by genomic sequencing. We further applied the workflow on data sets from three individual colorectal tumor specimens. A total of 204 distinct variant peptides were detected, and five carried known cancer-related mutations. Each individual showed a specific pattern of cancer-related mutations, suggesting potential use of this type of information for personalized medicine. Compatibility of the workflow has been tested with four popular database search engines including Sequest, Mascot, X!Tandem, and MyriMatch. In summary, we have developed a workflow that effectively uses existing genomic data to enable variant peptide detection in proteomics.

Molecular genetic cause of X-linked retinitis pigmentosa in a Czech family

Editor, A branch of a large eight generation Caucasian Czech family diagnosed with presumable X-linked retinitis pigmentosa (XLRP) was investigated (Fig. 1). This study adhered to the tenets of the Declaration of Helsinki. Men were severely affected in this family with early onset of night blindness and myopia, usually at the beginning of the second decade, and progressive loss of visual function resulting in blindness in the fourth decade. Women were either asymptomatic or developed symptoms with later onset (Otradovec et al. 1979). There is no evidence of male to male transmission. After obtaining informed consent, blood samples were drawn and DNA extracted. Polymorphic microsatellite markers located on the X chromosome (Table 1) were amplified using fluorescently labelled oligonucleotides and analysed on an ABI-377 DNA sequencer (Applied Biosystems, Darmstadt, Germany) using GeneScan software (Applied Biosystems). Haplotype analysis indicated that the disease segregates with markers encompassing both the RPGR and RP2 genes as RPGR lies between markers DXS1214 and DXS1068 and RP2 between DXS1055 and DXS991 (Fig. 1). The chromosomal region between the most proximal and most distal marker used in this study encompassed approximately 200 known protein-coding genes (http://www.ensembl.org). Investigated branch of an eight generation Czech family with X-linked retinitis pigmentosa. The affected haplotype is shown as a black bar and encompasses the RPGR and RP2 genes located between DXS1214–DXS1068 and DXS1055–DXS991, respectively. Sequencing of the RP2 gene, as previously described (Hardcastle et al. 1999), did not reveal any pathogenic changes. ORF15 of the RPGR gene (Vervoort et al. 2000) was then amplified and bidirectionally sequenced in four overlapping fragments using the ABI Prism® BigDye™ dGTP Terminator Ready Reaction Cycle Sequencing Kit (Applied Biosystems). A pathogenic mutation, c.2426_2427 delAG (g.ORF15+673_674delAG), leading to p.Glu809fs (ORF15Glu224fs) was identified in all affected males and three female obligate carriers (Fig. 2). The change was also detected in one other female family member (individual IV:6) and was absent in females IV:2 and IV:4 (1, 2). This mutation has been observed previously in different XLRP patient populations (http://rpgr.hgu.mrc.ac.uk), including one Japanese XLRP families (Jin et al. 2006). Interestingly, no other sequence variants were identified in the highly polymorphic RPGR ORF15 in our family. Sequence chromatogram showing the c.2426_2427delAG mutation in ORF15 of the RPGR gene in an obligate carrier III:1 (A) and an affected male IV:5 (B). The mutation was not detected in an unaffected female IV:4 (C). Reference sequence NM_001034853 was used. All three affected men were documented to have bone spicules on fundus examination. There were marked interindividual differences between IV:1 and IV:5 despite similar age; IV:5 was much more severely affected with night blindness, whereas IV:1 reported no symptoms. II:3 was registered blind in the fourth decade. Examined female carriers (III:1, III:5, IV:6) had some peripheral pigmentary changes but not classic bone spicules. RPRG mutations are the most frequent cause of XLRP (Vervoort et al. 2000; http://rpgr.hgu.mrc.ac.uk). The pedigree we have investigated here represents the first Czech family with an identified molecular genetic cause of RP. Although in Western European countries the prevalence and mutational spectrum of various RP types has been extensively studied, little is known about this heterogeneous group of disorders and their molecular genetic cause in the restructured countries of Central and Eastern Europe. To the best of our knowledge, prior to this study, only one other report describes a pathogenic mutation causing XLRP in a family of Bulgarian Gypsy origin (Chakarova et al. 2006). Here, we would like to propose the necessity for service offering molecular genetic testing for RP patients, and also for patients with other inherited ocular disorders, in all European countries. This should be supported by continuous education in the field of ophthalmic genetics among medical practitioners which would lead to improved counselling. However, as it is technically challenging to establish mutation analysis for every gene causing inherited ocular disorders in every country, we suggest that where X-linked inheritance cannot be excluded, the RPGR gene is screened for mutations using already established diagnostic services. This work was in part supported by the Czech Ministry of Education, Youth and Sports research project 0021620806/20610011.

Diversity gradients and phylogeographic patterns in Santiria trimera (Burseraceae), a widespread African tree typical of mature rainforests

New insights into the history of the African rainforest can be gathered from the phylogeographic structures of their constituent species, but few studies have been performed in this ecosystem. We studied the phylogeographic structure of Santiria trimera, a primate- and bird-dispersed, dioecious tree typical of mature African rainforests. We sequenced three chloroplast DNA (cpDNA) regions (trnL-F, rbcL, and rpl36-infA-rps8) in 377 individuals from 42 populations. Sequence chromatograms regularly displayed double peaks of unequal heights. Cloning of PCR products and sequencing of outgroup taxa led to assigning the taller peak in ambiguous sequence positions to cpDNA. A total of 14 polymorphic cpDNA sites and 12 haplotypes were detected. Populations from three distinct biogeographic regions, namely, Upper Guinea, Lower Guinea, and the volcanic island of São Tomé, did not share any haplotype, indicating allopatric divergence. In Lower Guinea, Gabonese forests had high diversity mainly from the sympatry of two genetically divergent morphotypes, whereas forests of eastern Cameroon were less diversified. The two haplotypes of the morphotype without stilt roots were distributed north and south of the Ogooué River, suggesting refuges on both sides of the river bed. The divergence between Upper and Lower Guinean rainforests is explained by the discontinuity of forest between those regions throughout most of the Quaternary. The distribution of rare endemic haplotypes concurred with proposed Pleistocene rainforest refuges in west and southwest Cameroon. Overall, phylogeographic structure is consistent with the biogeographic hypotheses largely based on patterns of species diversity.

Determination of organophosphate and carbamate resistance allele frequency in diamondback moth populations by quantitative sequencing and inhibition tests

A quantitative sequencing (QS) protocol was established for predicting the frequencies of the A298S and G324A mutations in the diamondback moth ( Plutella xylostella) type-1 acetylcholinesterase (AChE) locus, putatively involved in organophosphate (OP) and carbamate (CB) insecticide resistance. The nucleotide resistant signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were highly correlated with resistance allele frequencies ( r 2 > 0.987). QS analysis of 15 representative regional field populations of DBM in Korea revealed that the allele frequencies of both A298S and G324A were over 70% in most field populations, implying the prevalent state of these resistance-associated mutations. In the AChE inhibition assay, all populations showed reduced sensitivity to paraoxon, DDVP, carbaryl, and carbofuran, supporting the notion that DBM resistance to OPs and CBs is widespread in Korea.

Do the SLC25A38 or ALAS2 Genes Play a Role In the Phenotypic Expression of Ringed Sideroblasts In Acquired Myelodysplastic Syndrome?

AbstractAbstract 4954 Background.The SLC25A38 gene has recently been identified to play a role in the pathogenesis of congenital sideroblastic anemia (CSA). The erythroid specific mitochondrial carrier family protein SLC25A38 is important for the biosynthesis of heme. The ALAS2 gene is also frequently mutated in CSA. Refractory anemia with ringed sideroblasts (RARS) is an acquired myelodysplastic condition characterized by lineage dysplasia with an excess number of ringed sideroblasts in the marrow. A genetic cause for the expression of ringed sideroblasts in RARS or other myleodysplasias has not been clearly determined. We examine whether loss of function mutations in SLC25A38 or ALAS2 genes are associated with acquired myelodysplastic syndromes (MDS) with ringed sideroblasts. Methods.Participants had to have adequate banked DNA or bone marrow from diagnosis and meet WHO 2001 criteria of MDS and a high percentage of ringed sideroblasts. Medical records were retrospectively examined for patient demographics and outcomes. All diagnostic bone marrows were reviewed to confirm the diagnosis and the percentage of ringed sideroblasts and blasts was recorded. Cytogenetic findings were also recorded. DNA was extracted as needed by standard techniques. Coding exons and exon/intron boundaries of SLC25A38/ALAS2 were PCR-amplified using primers designed with Primer3 (http://frodo.wi.mit.edu/) from each affected individual. These products were then sequenced using the ABI 3130 xl electrophoresis instrument (Applied Biosystems). Sequence chromatograms were interpreted using the MutationSurveyor program from SoftGenetics, Inc., with gene annotations from GenBank looking for mutations predicted to result in loss of function. Results.12 samples were identified from patients diagnosed between 2003 and 2008. The diagnosis by WHO 2001 classification of myelodysplastic syndrome was refractory anemia with ringed sideroblasts [RARS] (n=7), refractory cytopenia with multilineage dysplasia (RCMD-RS] (n=4) and refractory anemia with ringed sideroblasts with thrombocytosis [RARS-T] (n=1). The median age at diagnosis was 82 years (range 58–94 years). Participants were males (n=9) and females (n=3). The clinical status was as follows: Remission (n = 1), Active Disease (n = 7), and Unknown (n = 4). Treatment provided to patients included transfusion supportive care only (n =2), erythropoietin (n= 3) and MDS directed drug therapy (n=1) and unknown (n=5). For those with blood counts available at diagnosis, the median white blood cell count was 8 × 109/L (range 4.7–10.9), the hemoglobin was 94 g/L (range 85–112) and the platelets were 327 × 109/L (range 3–951). The absolute neutrophil count was 5.3 × 109/L (range 3.1–7) and the absolute lymphocyte count was 1.9 × 109/L (range 0.7 – 2.2). The median percentage ringed sideroblast count in the diagnostic bone marrow was 51% (range 15–81%). Conventional G-banding cytogenetics showed a definitive abnormality in only one of the 12 patients. Mutations predicted to result in complete loss of function occurred in 0/12 in the SLC25A38 gene and 0/12 in the ALAS2 gene. One previously unreported variant of unknown significance at SLC25A38 E03 (cDNA position #239C>G; 80T>R) was identified in homozygous form in a patient with RARS and normal cytogenetics. Conclusions.Variations in the coding regions of SLC25A38 or ALAS2 genes were not obviously associated with mutations predicted to result in complete loss of function in this cohort of patients with acquired myelodysplastic syndromes with ringed sideroblasts. We did identify one unique variant in homozygous form but its significance is uncertain. We plan to expand this study to confirm these findings. Whilst loss of complete function of these genes does not appear to be associated with acquired ringed sideroblasts, we have not ruled out a contribution of functional mutations resulting in reduced expression of these genes. Further examination of this question may clarify the physiological understanding of ringed sideroblasts in acquired MDS, which in turn may represent a target for therapy. Disclosures:No relevant conflicts of interest to declare.

Internet-accessible DNA sequence database for identifying fusaria from human and animal infections.

Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated with human or animal mycoses encountered in clinical microbiology laboratories. The database comprises partial sequences from three nuclear genes: translation elongation factor 1α (EF-1α), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2). These three gene fragments can be amplified by PCR and sequenced using primers that are conserved across the phylogenetic breadth of Fusarium. Phylogenetic analyses of the combined data set reveal that, with the exception of two monotypic lineages, all clinically relevant fusaria are nested in one of eight variously sized and strongly supported species complexes. The monophyletic lineages have been named informally to facilitate communication of an isolate's clade membership and genetic diversity. To identify isolates to the species included within the database, partial DNA sequence data from one or more of the three genes can be used as a BLAST query against the database which is Web accessible at FUSARIUM-ID (http://isolate.fusariumdb.org) and the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center (http://www.cbs.knaw.nl/fusarium). Alternatively, isolates can be identified via phylogenetic analysis by adding sequences of unknowns to the DNA sequence alignment, which can be downloaded from the two aforementioned websites. The utility of this database should increase significantly as members of the clinical microbiology community deposit in internationally accessible culture collections (e.g., CBS-KNAW or the Fusarium Research Center) cultures of novel mycosis-associated fusaria, along with associated, corrected sequence chromatograms and data, so that the sequence results can be verified and isolates are made available for future study.

Establishment of Quantitative Sequencing and Filter Contact Vial Bioassay for Monitoring Pyrethroid Resistance in the Common Bed Bug, Cimex lectularius

Two point mutations (V419L and L925I) in the voltage-sensitive sodium channel α-subunit gene have been identified in deltamethrin-resistant bed bugs. A quantitative sequencing (QS) protocol was developed to establish a population-based genotyping method as a molecular resistance-monitoring tool based on the frequency of the two mutations. The nucleotide signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were shown to highly correlate with resistance allele frequencies (r2 > 0.9928). As determined by QS, neither mutation was found in a bed bug population collected in 1993. Populations collected in recent years (2007–2009), however, exhibited completely or nearly saturating L925I mutation frequencies and highly variable frequencies of the V419L mutation. In addition to QS, the filter contact vial bioassay (FCVB) method was established and used to determine the baseline susceptibility and resistance of bed bugs to deltamethrin and λ-cyhalothrin. A pyrethroid-resistant strain showed >9,375- and 6,990-fold resistance to deltamethrin and λ-cyhalothrin, respectively. Resistance allele frequencies in different bed bug populations predicted by QS correlated well with the FCVB results, confirming the roles of the two mutations in pyrethroid resistance. Taken together, employment of QS in conjunction with FCVB should greatly facilitate the detection and monitoring of pyrethroid-resistant bed bugs in the field. The advantages of FCVB as an on-site resistance-monitoring tool are discussed.

Establishment of Quantitative Sequencing and Filter Contact Vial Bioassay for Monitoring Pyrethroid Resistance in the Common Bed Bug, <I>Cimex lectularius</I>

Two point mutations (V419L and L925I) in the voltage-sensitive sodium channel alpha-subunit gene have been identified in deltamethrin-resistant bed bugs. A quantitative sequencing (QS) protocol was developed to establish a population-based genotyping method as a molecular resistance-monitoring tool based on the frequency of the two mutations. The nucleotide signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were shown to highly correlate with resistance allele frequencies (r2 > 0.9928). As determined by QS, neither mutation was found in a bed bug population collected in 1993. Populations collected in recent years (2007-2009), however, exhibited completely or nearly saturating L925I mutation frequencies and highly variable frequencies of the V419L mutation. In addition to QS, the filter contact vial bioassay (FCVB) method was established and used to determine the baseline susceptibility and resistance of bed bugs to deltamethrin and lambda-cyhalothrin. A pyrethroid-resistant strain showed >9,375- and 6,990-fold resistance to deltamethrin and lambda-cyhalothrin, respectively. Resistance allele frequencies in different bed bug populations predicted by QS correlated well with the FCVB results, confirming the roles of the two mutations in pyrethroid resistance. Taken together, employment of QS in conjunction with FCVB should greatly facilitate the detection and monitoring of pyrethroid-resistant bed bugs in the field. The advantages of FCVB as an on-site resistance-monitoring tool are discussed.

Stopping the stutter: Improvements in sequence quality from regions with mononucleotide repeats can increase the usefulness of non–coding regions for DNA barcoding

DNA barcoding is now well established in animals based on sequences from a 648 base pair (bp) portion of the mito chondrial coding gene cytochrome oxidase 1 (COl) (Hebert & al., 2003). In contrast, it has proved more difficult to identify a suitable DNA barcoding locus (or loci) for land plants (reviewed by Hollingsworth, 2008). The generally low substitution rate of plant mitochondrial DNA (e.g., Fazekas & al., 2008) has led to investigations into the relative performance and information content of different loci from the plastid genome as alternative DNA barcodes for plants (e.g., Chase & al., 2007; Kress & Er ickson, 2007; Fazekas & al., 2008; Lahaye & al., 2008; CBOL Plant Working Group, 2009; Ford & al., 2009; Hollingsworth & al, 2009). At the third International Barcode of Life Conference in Mexico City (November 2009), the Consortium for the Barcode of Life (CBOL) announced that the standard core DNA bar code for land plants would consist of a two locus combination comprising portions of the protein-coding plastid genes rbcL and matK, to be supplemented with additional loci as required. In making this recommendation, two potential problems were recognized. Firstly, successful amplification and sequencing of the matK barcoding region can be difficult in some taxa with existing primers, and further primer and protocol development is required for this locus. Secondly, the rbcL+matK barcode will not lead to 100% species discrimination in many plant groups, and additional loci beyond this core-barcode will be needed to increase levels of species discrimination in these cases. In recognition of these problems, an 18-month review period on the performance of the rbcL + matK barcode has been established (completion due in mid-2011). During this review period, the executive committee of CBOL recommended that the plant barcoding community continue to collect data from other strongly performing candidate barcoding loci such as the non-coding plastid intergenic spacer trnH-psbA and the internal transcribed spacers (ITS) of nuclear ribosomal DNA. One concern that has been raised regarding the use of non coding plastid regions such as trnH-psbA as DNA barcodes is the presence of microsatellite repeats which can make it dif ficult to obtain bi-directional sequences in some samples (Faze kas & al, 2008; CBOL Plant Working Group, 2009; Devey & al, 2009). Verification of the sequenced strand through bidi rectional sequencing is desirable for the maintenance of data quality standards, as recommended by the CBOL Database Working Group for sequences destined to receive annotation with the reserved keyword 'Barcode' by the INSDC (DDBJ, EMBL & GenBank). However, PCR amplicons derived from regions containing microsatellites can produce poor qual ity sequence chromatograms. Slippage of the polymerase at microsatellite regions during PCR results in a 'stutter' effect observed in the chromatogram, with decreasing sequence qual ity associated with increasing repeat length. The reduction in sequence quality from regions with mononucleotide runs with less than ten repeats is generally moderate and ambigu ous base calls by the software are usually few, requiring little editing by hand. However, as the repeat number increases, the number of ambiguous bases increases disproportionately. This results in a longer amount of time required for editing (with a corresponding reduction in confidence of the true sequence) to the point where sequence data cannot be used at all past the repeat. The net effect is often a contig with overlapping data only at the repeat, and a shortened read length due to missing data at the ends (Fig. 1). In more extreme cases, where mul tiple mononucleotide repeats occur within a given amplicon, disruption of forward and reverse sequencing reads at differ ent mononucleotide runs can lead to only partial sequences of the region with no overlap, thus preventing the construction of a sequence contig. The problem caused by mononucleotide repeats in non-coding regions was noted by the CBOL Plant Working Group (2009) when evaluating the attributes of differ ent barcoding loci for plants, and this contributed towards the selection of an entirely coding core plant barcode. Fixing the 'mononucleotide repeat problem' was considered less tractable than the challenge of improving primer universality for matK. Recently, however, an evaluation of PCR methods and DNA polymerases, focusing on the reduction of the stutter ef fects resulting from mononucleotide repeats, has demonstrated that the use of particular polymerases can reduce the effect of slipped strand mispairing in PCR (Fazekas & al., 2010). This recent study tested the performance of different PCR profiles, reaction chemistries and polymerases on sequences from the trnH-psbA spacer from 25 plant samples. These samples were specifically selected because they contain mononucleotide re peats which had previously resulted in ambiguous base calls and low-quality sequence traces with conventional PCR and