Immunoglobulin Heavy Chain (IgH) Gene Somatic Hypermutation Status Using Homoduplex-Separated RT-PCR Products.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4813-4813
Author(s):  
Melissa A. Melan ◽  
Kenneth A. Foon ◽  
Stanley M. Marks ◽  
Jeffrey A. Kant
Keyword(s):  
Somatic Hypermutation ◽  
Lymphocytic Leukemia ◽  
Rt Pcr ◽  
Mutation Status ◽  
Percent Identity ◽  
Pcr Product ◽  
Pcr Products ◽  
Reverse Primer ◽  
Almost All ◽  
Blast Program

Abstract Assessment of IgH somatic hypermutation status has been shown to be a valuable indicator for judging the prognosis of patients with chronic lymphocytic leukemia (CLL). Our laboratory has developed a streamlined method to improve the rate of successful evaluation of IgH mutation status. Six individual PCR reactions are first performed using random hexamer-generated cDNA as template. These reactions have identical reaction parameters, use a common JH reverse primer and one of six VH class-specific forward primers within Framework 1. PCR products are separated on acrylamide or MetaPhor® agarose gels following formamide denaturation. In almost all cases, a single homoduplex band is resolved indicating a class-specific clonal product. The homoduplex band is excised from the gel, eluted and directly sequenced. Mutation analysis is performed using the NCBI Ig BLAST program with percent identity determined for the region from the beginning of CDR1 to the end of Framework 3. To date, less than 10% of cases analyzed have not yielded a clearcut clonal PCR product using this approach

scholarly journals Molecular Analysis of Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in Compost and Composted Materials

1999 ◽  
Vol 65 (2) ◽  
pp. 396-403 ◽  
Author(s):  
George A. Kowalchuk ◽  
Zinaida S. Naoumenko ◽  
Piet J. L. Derikx ◽  
Andreas Felske ◽  
John R. Stephen ◽  
...  
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Ammonia Oxidizer ◽  
Ammonia Oxidizing Bacteria ◽  
Nitrogen Transformations ◽  
Rt Pcr ◽  
Gene Encoding ◽  
Specific Pcr ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
Almost All

ABSTRACT Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class Proteobacteriain a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms.

scholarly journals Mutation Status and Immunoglobulin Gene Rearrangements in Patients from Northwest and Central Region of Spain with Chronic Lymphocytic Leukemia

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
I. González-Gascón y Marín ◽  
J. A. Hernández ◽  
A. Martín ◽  
M. Alcoceba ◽  
M. E. Sarasquete ◽  
...  
Keyword(s):  
Overall Survival ◽  
Chronic Lymphocytic Leukemia ◽  
Central Region ◽  
Lymphocytic Leukemia ◽  
Gene Rearrangements ◽  
Immunoglobulin Gene ◽  
Mutation Status ◽  
Almost All ◽  
Time To First Treatment ◽  
Immunoglobulin Gene Rearrangements

The aim of this study was to investigate the frequency and mutation status of the immunoglobulin heavy variable chain (IGHV) in a cohort of 224 patients from northwest and central region of Spain diagnosed with chronic lymphocytic leukemia (CLL), and to correlate it with cytogenetic abnormalities, overall survival (OS) and time to first treatment (TTFT). 125 patients had mutated IGHV, while 99 had unmutated IGHV. The most frequently used IGHV family was IGHV3, followed by IGHV1 and IGHV4. The regions IGHV3-30, IGHV1-69, IGHV3-23, and IGHV4-34 were the most commonly used. Only 3.1% of the patients belonged to the subfamily IGHV3-21 and we failed to demonstrate a worse clinical outcome in this subgroup. The IGHV4 family appeared more frequently with mutated pattern, similar to IGHV3-23 and IGHV3-74. By contrast, IGHV1-69 was expressed at a higher frequency in unmutated CLL patients. All the cases from IGHV3-11 and almost all from IGHV5-51 subfamily belonged to the group of unmutated CLL.

scholarly journals AtypicalCTSKTranscripts andARNTTranscription Read-Through intoCTSK

2005 ◽  
Vol 6 (5-6) ◽  
pp. 268-276
Author(s):  
Fabienne S. Giraudeau ◽  
Jean-Philippe Walhin ◽  
Paul R. Murdock ◽  
Nigel K. Spurr ◽  
Ian C. Gray
Keyword(s):  
Negative Impact ◽  
Cathepsin K ◽  
Chromosome 1 ◽  
Rt Pcr ◽  
Transcription Start ◽  
Aryl Hydrocarbon ◽  
Pcr Product ◽  
Close Proximity ◽  
Pcr Products ◽  
Human Chromosome 1

The aryl hydrocarbon receptor nuclear translocator (ARNT) and cathepsin K (CTSK) genes lie in a tandem head-to-tail arrangement on human chromosome 1. The two genes are in extremely close proximity; the usualCTSKtranscription start site is less than 1.4 kb downstream of the end of the longest reportedARNTtranscript. By generating an RT-PCR product that overlaps both the 3′ end ofARNTand the 5′ end ofCTSK, we show thatARNTtranscripts may extend through theARNT–CTSKintergenic region and progress into theCTSKgene. Furthermore, by using quantitative RT-PCR from several tissues to detect theARNTexpression signature inCTSKintrons, we show thatARNTtranscripts can read through intoCTSKas far asCTSKintron 3, extending approximately 3.7 kb downstream of the end of the longest previously describedARNTmRNA. Given thatARNTandCTSKare expressed in an overlapping range of tissues,ARNTread-through may have a negative impact onCTSKtranscript levels by interfering withCTSKexpression. We also present evidence for novelCTSKtranscripts following sequence analysis ofCTSK-derived ESTs and RT-PCR products. These transcripts show alternate 5′ splicing and or 5′ extension and are sometimes initiated from a cryptic alternative promoter which is upstream of the knownCTSKpromoter and possibly in the 3′ UTR ofARNT.

scholarly journals First Report of Apium virus Y on Cilantro, Celery, and Parsley in California

Plant Disease ◽  
2008 ◽  
Vol 92 (8) ◽  
pp. 1254-1254 ◽  
Author(s):  
T. Tian ◽  
H.-Y. Liu ◽  
S. T. Koike
Keyword(s):  
Mosaic Virus ◽  
Consensus Sequence ◽  
Amino Acid Sequences ◽  
Apium Graveolens ◽  
Petroselinum Crispum ◽  
Rt Pcr ◽  
First Report ◽  
Virus Particles ◽  
Pcr Products ◽  
Reverse Primer

Recently, Apium virus Y (ApVY) was detected in field-grown cilantro (Coriandrum sativum), celery (Apium graveolens), and parsley (Petroselinum crispum) in California. In 2003, cilantro plants growing in three different fields in California (Monterey, San Joaquin, and San Luis Obispo counties) expressed symptoms of mosaic, vein clearing, and stunting. When plant sap was examined by transmission electron microscopy, flexuous, rod-shaped virus particles were observed. Total RNA was extracted from the symptomatic cilantro plants and used as a template in reverse transcription (RT)-PCR using universal potyvirus primers according to Chen et al. (1). The RT-PCR product was cloned into pGEM-T (Promega, Madison, WI) and the insert of 1,713 bp was sequenced (GenBank Accession No. EU515125). Nucleotide sequences from clones derived from three different infected cilantro plants were 89 to 97% identical to ApVY sequences encoding partial sequence of polyprotein in GenBank (Accession Nos. AY049716, EU127499, AF207594, AF203529, and EU255632). In 2007, celery plants showing necrotic line patterns and necrotic lesions on lower leaves and petioles were observed in several fields in two coastal counties in California (Monterey and Santa Clara counties). Flexuous, rod-shaped virus particles were also observed in the sap of those plants. ELISA for Cucumber mosaic virus and RT-PCR for Celery mosaic virus were negative. ApVY specific primers were designed on the basis of a consensus sequence of ApVY identified from cilantro in 2003; reverse primer 5′-GGCTCTTGCTATAGACAAATAGT-3′ and forward primer 5′-GAAGACCAAGCCAATGTGTGTA-3′. The sequence of RT-PCR products (GenBank Accession No. EU515126) amplified from infected celery had 90 to 98% nucleotide identity to ApVY. When the deduced amino acid sequences of NIb and CP regions from both cilantro and celery were used for comparison, they showed 95 to 99% identity with the known ApVY GenBank sequences mentioned above. More than 10 asymptomatic parsley plants growing in fields adjacent to the infected celery were also tested for ApVY and found to be infected. ApVY was previously identified in three Apiaceae weeds in Australia (2) and in celery in New Zealand (3). To our knowledge, this is the first report of ApVY on cilantro, celery, and parsley in California. References: (1) J. Chen et al. Arch. Virol. 146:757, 2001. (2) J. Moran et al. Arch. Virol. 147:1855, 2002. (3) J. Tang et al. Plant Dis. 91:1682, 2007.

Analysis of Non-Expressed IGK Locus Rearrangements in Chronic Lymphocytic Leukemia Indicates a Role for Secondary Rearrangements in Shaping the Expressed Immunoglobulin Repertoire.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 972-972 ◽  
Author(s):  
Chrysoula Belessi ◽  
Kostas Stamatopoulos ◽  
Katerina Hatzi ◽  
Anastasia Hadzidimitriou ◽  
Fotini Marantidou ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Light Chain ◽  
Somatic Hypermutation ◽  
Lymphocytic Leukemia ◽  
Receptor Editing ◽  
Rt Pcr ◽  
Stop Codons ◽  
Immunoglobulin Kappa ◽  
Immunoglobulin Repertoire ◽  
Dna Pcr

Abstract Immunoglobulin kappa (IGK) locus rearrangements were analyzed in 164 κ-light chain (LC) expressing chronic lymphocytic leukemia (CLL) cases and 95 λ-LC expressing CLL cases. In κ-CLL, 151 IGKV-J transcripts were successfully amplified by RT-PCR; 147/151 were in frame; four out-of-frame IGKV-J transcripts were heavily mutated and contained one or more stop codons, presumably introduced by somatic hypermutation; in all four cases the corresponding expressed transcript was also mutated. DNA-PCR identified 24/164 κ-CLL cases (15%) with double IGKV-J rearrangements; 9/24 non-expressed IGKV-J rearrangements were in-frame; 3/9 were mutated. The most frequently used IGKV genes in expressed IGKV-J rearrangements were: 3–20, 1-39/1D-39, 1–5 and 4-1; the IGKV4-1 gene was by far the most frequent in non-expressed IGKV-J rearrangements (9/24 cases, 33%). In λ-CLL, a total of 65 IGKV-J rearrangements were amplified in 59/95 cases (62.0%); six cases had two different rearrangements. IGVK4-1 was the most frequently used gene (14/65 sequences, 21.5%), followed by IGKV1-16 (8 cases,12.3%), 1-33/1D-33 and 2-30 (7 cases each, 10.8%). IGKV-J transcripts were detected by RT-PCR in 10/59 λ-CLL cases with IGKV-J rearrangements, of which four were in-frame and six out-of-frame. Seven IGKV-J rearrangements in λ-CLL had less than 100% homology to germline; 3/7 were in-frame; 6/7 patients had mutated IGHV and 5/7 had mutated IGLV genes. In κ-CLL, biased usage of the IGKJ1/IGKJ2 genes was observed both in expressed (69%) and non-expressed rearrangements (78%); in contrast, in λ-CLL, downstream IGKJ (IGKJ3-5) usage was observed in 32/59 sequences (53%). Nonproductive rearrangements involving the kappa deleting element (KDE) that render the IGK locus inactive were also analyzed. In κ-CLL, 22/147 cases (15%) carried IGKV to KDE rearrangements, while 24/147 cases (16.5%) carried IGKJ-C- INTRON (JKI) to KDE rearrangements. In λ-CLL, IGKV-KDE rearrangements were amplified in 55/94 cases (59%); JKI-KDE rearrangements were amplified in 52/94 cases (56%). IGKV1D-43 was the most frequent gene in IGKV-KDE joints in κ-CLL (4/22 cases); in contrast, the most frequent genes in IGKV-KDE joints in λ-CLL were IGKV3-20 and IGKV2-30 (9/55 and 7/55 cases, respectively). In conclusion, the present study confirms IGK locus rearrangements in the vast majority of λ-LC expressing CLL cases. Differential usage of IGKJ genes along with significant IGKV repertoire differences in both IGKV-J and IGKV-KDE rearrangements between κ- and λ-CLL allude to prolonged IGK locus recombination before CLL clonogenic cells became λ-producers. The inactivation of productive and potentially functional IGKV-J joints by secondary rearrangements indicates a role for receptor editing in shaping the expressed IG repertoire in CLL. Finally, the identification of mutated, non-expressed IGKV-J rearrangements both in κ- and λ-CLL might be considered as evidence for secondary rearrangements occurring after the onset of somatic hypermutation, at least in a proportion of cases.

Biological and Clinical Relevance of Surrogate Markers of IgVH Mutational Status in B-Cell Chronic Lymphocytic Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1062-1062
Author(s):  
Fortunato Morabito ◽  
Marta Lionetti ◽  
Giovanna Cutrona ◽  
Katia Todoerti ◽  
Serena Matis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Candidate Genes ◽  
Lymphocytic Leukemia ◽  
Surrogate Markers ◽  
Rt Pcr ◽  
Mutation Status ◽  
Expression Levels ◽  
Cell Chronic Lymphocytic Leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease; some patients have a rapidly progressing disease and others exhibit an indolent course and survive for many years without treatment. Mutation status of IgVH genes utilized by CLL cells represents a very reliable predictor of clinical outcome in B-CLL, but its analysis is expensive and beyond the capacities of most diagnostic laboratories. To identify surrogate markers we performed a gene expression profiling analysis of CD19+ purified cells from 80 B-CLL untreated patients in Binet stage A, by means of Affymetrix GeneChip® HGU133A arrays. The comparison of 46 IgVH-unmutated versus 34 mutated samples using the Prediction Analysis of Microarrays software identified 78 differentially expressed probes, specific for 59 well-characterized genes. Specifically, 43 genes had a higher and 16 genes a lower average expression in the IgVH unmutated group. These genes are involved in cellular functions, including cell cycle regulation (SEPT7, SEPT10, CDK2AP1), cell proliferation (SLAMF1, LDOC1), apoptosis (CD63, IFT57, P2RX1, RNF130, TNFRSF1B), cell adhesion (CNTNAP2, C1orf38, PCDH9), immune response (ZAP70, IFI44), signal transduction (AKAP13, RASGRP1, USP6NL, TGFBR3, AKAP12), lipid metabolism and fatty-acid degradation (FADS3, LPL, LASS6), cell-cell signalling (FCRL2), phospholipid biosynthetic process (AYTL2), regulation of circadian rhythm (EGR3, CRY1, OPN3), DNA-dependent regulation of transcription (MYBL1, NR4A2, NRIP1, ZBTB20), muscle development (VAMP5, SRI, DMD). The expression signature identified in the proprietary database was then validated by means of a meta-analysis of a publicly available gene expression dataset of 100 B-CLL (Haslinger et al., 2005), showing classification accuracy measures leading to a global classification rate of 82.93% of the test set and thus suggesting the strength of the identified expression signature. The expression levels of 11 genes (LPL, ZBTB20, ZAP70, CRY1, COBLL1, SEPT10, LDOC1, TNFRSF1B, DMD, SRI, NRIP1) were confirmed by means of quantitative real-time PCR (Q-RT-PCR) in a subset of 40 CLL patients. The prognostic impact for Time To Treatment (TTT) of the 59 candidate genes of our classifier model was investigated in 77 patients. Forty-nine (36.4%) of these received treatment after a median follow up of 4 years. As expected, patients with unmutated IgVH genes had a risk of therapy requirement that was about 3 times higher (HR: 3.1,95% C.I. 1.6–5.8, p<0.0001) than those with mutated IgVH. Based on microarray expression levels, 43/59 genes significantly predicted TTT with a HR ranging from 1.5 (LPL gene) to 4.2 (SRI gene) (value for ZAP-70 = HR: 1.9, 95% C.I. 1.0–3.4, p=0.039). The same analysis performed in the panel of the 11 genes validated by Q-RT-PCR revealed 4 candidate genes which significantly predicted TTT. Specifically, Cox univariate analysis confirmed ZAP-70 as a predictor of disease outcome and underscored the prognostic role of the LPL, TNFRSF1B and CRY1 genes. The predictive power of the novel putative surrogate markers for the IgVH mutation status is now being further validated at protein expression level.

Development of a DNA Microarray for the Simultaneous Detection and Genotyping of Noroviruses

2008 ◽  
Vol 71 (7) ◽  
pp. 1434-1441 ◽  
Author(s):  
FRANCO PAGOTTO ◽  
NATHALIE CORNEAU ◽  
KIRSTEN MATTISON ◽  
SABAH BIDAWID
Keyword(s):  
Molecular Epidemiology ◽  
Molecular Characterization ◽  
Simultaneous Detection ◽  
Oligonucleotide Array ◽  
Rt Pcr ◽  
Major Drawback ◽  
Pcr Product ◽  
Reaction Specificity ◽  
Pcr Products

Current methods for detecting and genotyping noroviruses focus on the use of reverse transcriptase (RT)–mediated PCR. A major drawback of this approach is that short target RT-PCR products do not always encompass sequences that can be compared among research laboratories, resulting in difficulties for molecular epidemiology. We describe the use of a microarray-based system for simultaneous detection and molecular characterization of noroviruses. The protocol generates a 917-bp RT-PCR product that encompasses two major regions currently used for detection and analysis of norovirus genomes. The PCR products are then hybridized to an oligonucleotide array (NoroChip) based on 50-mer features, which allows for both confirmation of reaction specificity and molecular characterization of the amplified genome. Parallel sequence analyses of amplicons revealed that our microarray data were robust in separating genogroups I and II, and further subtyping to the cluster level was possible. This approach, combining detection and characterization, overcomes the need for expensive and time-consuming sequence analysis of amplified genome targets for molecular epidemiology.

scholarly journals VH CDR3-Focused Somatic Hypermutation in CLL IGHV-IGHD-IGHJ Gene Rearrangements with 100% IGHV Germline Identity

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4277-4277 ◽  
Author(s):  
Katerina Gemenetzi ◽  
Andreas Agathangelidis ◽  
Fotis Psomopoulos ◽  
Kostas Pasentsis ◽  
Evdoxia Koravou ◽  
...  
Keyword(s):  
Research Funding ◽  
Somatic Hypermutation ◽  
Lymphocytic Leukemia ◽  
Gene Rearrangements ◽  
Rt Pcr ◽  
Bioinformatics Pipeline ◽  
Sample Mean ◽  
Mean Frequency ◽  
High Data ◽  
Ighv Gene

Classification of patients with chronic lymphocytic leukemia (CLL) based on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the sequence of the rearranged IGHV gene excluding the VH CDR3. This is mostly due to the difficulty in discriminating actual SHM from random nucleotides added between the recombined IGHV, IGHD and IGHJ genes. Hence, this approach may underestimate the true impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions i.e. the VH CDR3. Relevant to mention in this respect, studies from our group in CLL with mutated IGHV genes (M-CLL), particularly subset #4, have revealed considerable intra-VH CDR3 diversity attributed to ongoing SHM. Prompted by these findings, here we investigated whether SHM may also be present in cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3), focusing on two well characterized stereotyped subsets i.e. subset #1 (IGHV clan I/IGHD6-19/IGHJ4) and subset #6 (IGHV1-69/IGHD3-16/IGHJ3). These subsets carry germline-encoded amino acid (aa) motifs within the VH CDR3, namely QWL and YDYVWGSY, originating from the IGHD6-19 and IGHD3-16 gene, respectively. However, in both subsets, cases exist with variations in these motifs that could potentially represent SHM. The present study included 12 subset #1 and 5 subset #6 patients with clonotypic IGHV genes lacking any SHM (100% germline identity). IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified by subgroup-specific leader primers and a high-fidelity polymerase in order to ensure high data quality. RT-PCR products were subjected to paired-end NGS on the MiSeq platform. Sequence annotation was performed with IMGT/HighV-QUEST and metadata analysis was undertaken using an in-house purpose-built bioinformatics pipeline. Rearrangements with the same IGHV gene and identical VH CDR3 aa sequences were defined as clonotypes. Overall, we obtained 1,570,668 productive reads with V-region identity 99-100%; of these, 1,232,958 (mean: 102,746, range: 20,796-242,519) concerned subset #1 while 337,710 (mean: 67,542, range: 50,403-79,683) concerned subset #6. On average, 64.4% (range: 1.7-77.5%) of subset #1 reads and 49.2% (range: 0.7-70%) of subset #6 reads corresponded to rearrangements with IGHV genes lacking any SHM (100% germline identity). Clonotype computation revealed 1,831 and 1,048 unique clonotypes for subset #1 and #6, respectively. Subset #1 displayed a mean of 157 distinct clonotypes per sample (range: 74-267), with the dominant clonotype having a mean frequency of 96.9% (range: 96-98.2%). Of note, 44 clonotypes were shared between different patients (albeit at varying frequencies), including the dominant clonotype of 11/12 cases, which was present in 2-6 additional subset #1 patients. Subset #6 cases carried a higher number of distinct clonotypes per sample (mean: 219, range: 189-243) while the dominant clonotype had a mean frequency of 95.6% (range: 94.5-96.5%). Shared clonotypes (n=30) were identified also in subset #6 and the dominant clonotype of each subset #6 case was present in 3-5 additional subset #6 patients. Focusing on the VH CDR3, in particular the IGHD-encoded part, the following observations were made: (1) in both subsets, extensive intra-VH CDR3 variation was detected at certain positions within the IGHD gene; (2) in most cases, the observed aa substitutions were conservative i.e. concerned aa sharing similar physicochemical properties. Particularly noteworthy in this respect were the observations in subset #6 that: (i) the valine residue (V) in the D-derived YDYVWGSY motif was very frequently mutated to another aliphatic residue (A, I, L); (ii) in cases were the predominant clonotype carried I (also in the Sanger-derived sequence), several minor clonotypes carried the germline-encoded V, compelling evidence that the observed substitution concerned true SHM. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, very likely attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. While the prognostic/predictive relevance of this observation is beyond the scope of the present work, our findings highlight the possible need to reappraise definitions ('semantics') regarding SHM status in CLL. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Chatzidimitriou:Janssen: Honoraria.

scholarly journals First Report of Potato spindle tuber viroid in Cape Gooseberry (Physalis peruviana) from Turkey and Germany

Plant Disease ◽  
2009 ◽  
Vol 93 (3) ◽  
pp. 316-316 ◽  
Author(s):  
J. Th. J. Verhoeven ◽  
M. Botermans ◽  
J. W. Roenhorst ◽  
J. Westerhof ◽  
E. T. M. Meekes
Keyword(s):  
Potato Spindle Tuber Viroid ◽  
Rt Pcr ◽  
Physalis Peruviana ◽  
Source Of Infection ◽  
Pcr Product ◽  
Pcr Products ◽  
Mother Plants ◽  
Tomato Chlorotic Dwarf Viroid ◽  
Plant Pathol ◽  
Cape Gooseberry

Since the recent identification of Potato spindle tuber viroid (PSTVd) in vegetatively propagated ornamental plant species (4), many growers have asked to have their mother plants tested for this viroid. In December of 2007, a grower from Turkey submitted cuttings of cape gooseberry (Physalis peruviana) to be tested for PSTVd. Initial testing by real-time reverse transcription (RT)-PCR according to Boonham et al. (1) indicated the presence of either Mexican papita viroid, PSTVd, or Tomato chlorotic dwarf viroid in four samples. To identify the viroid(s) present, isolated RNA from these samples was used for RT-PCR (2), and products of the expected full genome size for the three viroids were amplified from each sample. One of the PCR products was sequenced (GenBank Accession No. EU862230) and analysis of the 357 nt sequence indicated it was most related to PSTVd sequences belonging to the so-called ‘Oceanian’ strain of the viroid (3), with 99.7% identity to GenBank Accession No. AY962324. Therefore, the viroid was identified as PSTVd. Pathogenicity of this PSTVd genotype was demonstrated when 4 weeks after mechanical inoculation with sap extracts seedlings of tomato cv. Money-maker showed the expected viroid symptoms of chlorosis and stunting, and the presence of the viroid in these plants was confirmed by RT-PCR (2). In March of 2008, by use of RT-PCR (2) and sequencing of the PCR product (GenBank Accession No. EU862231), PSTVd was identified in young seedlings of P. peruviana from a German grower. The German isolate differed at only three nucleotide positions from the Turkish isolate. The identification of PSTVd in young seedlings indicates that seeds had been source of infection, whereas in the case of the PSTVd infected cuttings from Turkey, the infection originated from infected mother plants. To our knowledge, these are the first reports of PSTVd in P. peruviana. Although infected P. peruviana plants did not show symptoms, they might act as sources of inoculum for crops like potato and tomato, which may suffer serious damage. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) J. Th. J. Verhoeven et al. Plant Pathol. 57:399, 2008.

Validating The Prognostic Significance Of FCRL2 In Predicting IGHV Mutation Status, Clinical Disease Progression, and Survival In CLL

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4140-4140 ◽  
Author(s):  
Fu Jun Li ◽  
Alan B Cantor ◽  
Mikhail A. Shakhmatov ◽  
Ran Li ◽  
Xiao-Jie Yan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Disease Progression ◽  
Therapeutic Potential ◽  
Somatic Hypermutation ◽  
Prognostic Significance ◽  
Variable Region ◽  
Lymphocytic Leukemia ◽  
University Of Alabama ◽  
Region Gene ◽  
Mutation Status

Abstract B cell chronic lymphocytic leukemia (CLL) can be stratified into indolent and aggressive subtypes according to the degree of somatic hypermutation evident in the immunoglobulin heavy chain variable region gene (IGHV). Despite some discordance with IGHV mutation status, both CD38 and ZAP-70 are routinely analyzed in clinical practice and are considered useful prognostic factors for segregating patients with aggressive-unmutated CLL (U-CLL). Members of the Fc receptor-like (FCRL1-5) family have immunoregulatory function and are preferentially expressed by B cells. Their distinct distribution pattern among subsets in healthy donors suggests that the FCRLs may also have useful diagnostic and/or therapeutic potential in B lineage malignancies. Previous work employing a receptor-specific monoclonal antibody (mAb-7F2) and an indirect staining method demonstrated that FCRL2 is particularly upregulated on the surface of indolent-mutated CLL (M-CLL) cells. Among 107 CLL samples, 52 U-CLL and 55 M-CLL, FCRL2 overlapped with IGHV status with 94.4% concordance, relative to 76.6% for CD38 (cutoff of 30%), and 80.4% for ZAP-70 according to the T/B ratio (cutoff of 4.0) or 74.8% by the % method (cutoff of 20%). Because several years have passed since our initial analysis using this reagent, we updated the clinical information to reassess the relationship among these prognostic parameters with FCRL2 staining and to further determine the usefulness of this biomarker in CLL. Similar to IGHV status [Hazard ratio (HR)=5.03], FCRL2 could also, but more powerfully, predict survival (HR=8.58). Both these features were superior to CD38 (HR=1.29) and ZAP-70 by either the T/B ratio (HR=1.42) or % (HR=1.04) approaches. The median survival for FCRL2 positive cases was 26.2 years and for FCRL2 negative donors was 12.7 years. With regard to disease progression, FCRL2 positive cases had a median treatment free interval of 13.5 years, whereas for FCRL2 negative donors it was only 3.2 years. Given these promising results, we pursued optimizing FCRL2 detection and assayability by flow cytometry to validate its clinical utility for diagnostic and prognostic purposes in CLL. A screen of FCRL2-specific mAbs led to the identification of a novel subclone termed 3E11, which by epitope mapping reacted with a site discrete from that of 7F2. The binding kinetics of these two mAbs also differed. According to surface plasmon resonance studies using an FCRL2-Fc recombinant protein, 3E11 possessed ∼2.5X better nanomolar affinity and a slower dissociation rate than 7F2. The superiority of 3E11 was also evidenced by its relatively stronger surface staining reactivity in flow cytometry analyses. To achieve a one-step staining strategy, 3E11 was directly conjugated with PE. Use of this PE-labeled reagent demonstrated brighter and more selective FCRL2 reactivity on M-CLL versus U-CLL (N=30) cells using either the post-Ficoll or whole blood-lysis preparation methods. Importantly, its reactivity was also stable over time (Concordance Correlation Coefficient of 0.9). As a promising CLL biomarker capable of selectively detecting the MT-CLL subtype and predicting disease progression and survival, the current findings demonstrate the practicability and reliability of a newfound mAb for validating the prognostic value of FCRL2 in CLL. Disclosures: LI: University of Alabama at Birmingham: Filed Patent, Filed Patent Patents & Royalties. Davis:NIH an University of Alabama at Birmingham: Patent Filed, Patent Filed Patents & Royalties, Research Funding.

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