A recurrent germline BAP1 mutation and extension of the BAP1 tumor predisposition spectrum to include basal cell carcinoma.

We report four previously undescribed families with germline BRCA1-associated protein-1 gene (BAP1) mutations and expand the clinical phenotype of this tumor syndrome. The tumor spectrum in these families is predominantly uveal malignant melanoma (UMM), cutaneous malignant melanoma (CMM) and mesothelioma, as previously reported for germline BAP1 mutations. However, mutation carriers from three new families, and one previously reported family, developed basal cell carcinoma (BCC), thus suggesting inclusion of BCC in the phenotypic spectrum of the BAP1 tumor syndrome. This notion is supported by the finding of loss of BAP1 protein expression by immunochemistry in two BCCs from individuals with germline BAP1 mutations and no loss of BAP1 staining in 53 of sporadic BCCs consistent with somatic mutations and loss of heterozygosity of the gene in the BCCs occurring in mutation carriers. Lastly, we identify the first reported recurrent mutation in BAP1 (p.R60X), which occurred in three families from two different continents. In two of the families, the mutation was inherited from a common founder but it arose independently in the third family.

Identification of Zeoliarus oppositus (Hemiptera: Cixiidae) as a Vector of 'Candidatus Phytoplasma australiense'.

'Candidatus Phytoplasma australiense' is associated with a number of plant diseases in New Zealand. The only known vector of this pathogen was Zeoliarus atkinsoni, a planthopper considered to be monophagous on New Zealand flax (Phormium spp.). The work carried out shows that Z. oppositus, which is polyphagous, is able to vector 'Ca. P. australiense' to both Coprosma robusta (karamu) and Cordyline australis (New Zealand cabbage tree). Although transmission was achieved to both these species, the disease symptomatology was more evident in C. australis. Two approaches were taken to achieve transmission. First, insects were collected from areas around symptomatic Coprosma plants and caged directly on test plants. Second, insects were collected from grasses and sedges in areas where disease was less evident and were fed on known infected Coprosma plants prior to being caged on test plants. Transmission was achieved using both approaches, although transmission was far greater (30% compared with 4%) from insects that were directly applied. Phytoplasma DNA was detected in 12% of Z. oppositus individuals tested during all the trials. This work identifies a new vector for 'Ca. P. australiense' and contributes to our understanding of the ecology of Cordyline sudden decline and Coprosma lethal decline.

Development of standardized approaches to reporting of minimal residual disease data using a reporting software package designed within the European LeukemiaNet.

Quantitative PCR (qPCR) for detection of fusion transcripts and overexpressed genes is a promising tool for following minimal residual disease (MRD) in patients with hematological malignancies. Its widespread clinical use has to some extent been hampered by differences in data analysis and presentation that complicate multicenter clinical trials. To address these issues, we designed a highly flexible MRD-reporting software program, in which data from various qPCR platforms can be imported, processed, and presented in a uniform manner to generate intuitively understandable reports. The software was tested in a two-step quality control (QC) study; the first step involved eight centers, whose previous experience with the software ranged from none to extensive. The participants received cDNA from consecutive samples from a BCR-ABL+ chronic myeloid leukemia (CML) patient and an acute myeloid leukemia (AML) patient with both CBFß-MYH11 and WT1 target genes, they conducted qPCR on their respective hardware platforms and generated a series of reports with pre-defined features. In step two, five centers used the software to report BCR-ABL+ MRD in a harmonized manner, applying their recently obtained CML international scale conversion factors. The QC study demonstrated that this MRD-reporting software is suitable for efficient handling of qPCR data, generation of MRD reports and harmonization of MRD data.

Sustained major molecular response on interferon alpha-2b in two patients with polycythemia vera.

Quantitative assessment of the JAK2 V617F allele burden during disease evolution and ongoing myelosuppressive treatment is likely to be implemented in the future clinical setting. Interferon alpha has demonstrated efficacy in treatment of both chronic myeloid leukemia and the Philadelphia chromosome negative chronic myeloproliferative disorders. Reductions in the JAK2 V617F allele burden in patients treated with pegylated interferon alpha-2a (Peg-IFN-2a) have been demonstrated, although follow-up was relatively short. We report here the first profound and sustained molecular responses with a JAK2 V617F allele burden below 1.0% in two patients with polycythemia vera treated with interferon alpha-2b (IFN-2b). Discontinuation of IFN-2b in one of the patients was followed by a sustained long-lasting (12 months of follow-up) major molecular response.

NPM1 mutations in therapy-related acute myeloid leukemia with uncharacteristic features.

Frameshift mutations of the nucleophosmin gene (NPM1) were recently reported as a frequently occurring abnormality in patients with de novo acute myeloid leukemia (AML). To evaluate the frequency of NPM1 mutations in patients with therapy-related myelodysplasia (t-MDS) and therapy-related AML (t-AML), and their possible association to type of previous therapy and to other gene mutations, 140 patients with t-MDS or t-AML were analyzed for mutations of NPM1. NPM1 mutations were observed in 7 of 51 patients presenting as overt t-AML, as compared to only 3 of 89 patients presenting as t-MDS (P=0.037). The mutations were not related to any specific type of previous therapy, but they were significantly associated with a normal karyotype and mutations of FLT3 (P=0.0002 for both comparisons). Only 1 of 10 patients with NPM1 mutations presented chromosome aberrations characteristic of therapy-related disease, and 7q-/-7, the most frequent abnormalities of t-MDS/t-AML, were not observed (P=0.002). This raises the question whether some of the cases presenting NPM1 mutations were in fact cases of de novo leukemia. The close association to class I mutations and the inverse association to class II mutations suggest mutations of NPM1 as representing a class II mutation-like abnormality in AML.

Genetics of therapy-related myelodysplasia and acute myeloid leukemia.

Myelodysplasia (MDS) and acute myeloid leukemia (AML) are heterogeneous, closely associated diseases arising de novo or following chemotherapy with alkylating agents, topoisomerase II inhibitors, or after radiotherapy. Whereas de novo MDS and AML are almost always subclassified according to cytogenetic characteristics, therapy-related MDS (t-MDS) and therapy-related AML (t-AML) are often considered as separate entities and are not subdivided. Alternative genetic pathways were previously proposed in t-MDS and t-AML based on cytogenetic characteristics. An increasing number of gene mutations are now observed to cluster differently in these pathways with an identical pattern in de novo and in t-MDS and t-AML. An association is observed between activating mutations of genes in the tyrosine kinase RAS-BRAF signal-transduction pathway (Class I mutations) and inactivating mutations of genes encoding hematopoietic transcription factors (Class II mutations). Point mutations of AML1 and RAS seem to cooperate and predispose to progression from t-MDS to t-AML. Recently, critical genetic effects underlying 5q-/-5 and 7q-/-7 have been proposed. Their association and cooperation with point mutations of p53 and AML1, respectively, extend the scenario of cooperating genetic abnormalities in MDS and AML. As de novo and t-MDS and t-AML are biologically identical diseases, they ought to be subclassified and treated similarly.

Development of real-time PCR and hybridization methods for detection and identification of thermophilic Campylobacter spp. in pig faecal samples.

AIMS: To develop a real-time (rt) PCR for species differentiation of thermophilic Campylobacter and to develop a method for assessing co-colonization of pigs by Campylobacter spp. METHODS AND RESULTS: The specificity of a developed 5' nuclease rt-PCR for species-specific identification of Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and of a hipO gene nucleotide probe for detection of C. jejuni by colony-blot hybridization were determined by testing a total of 75 reference strains of Campylobacter spp. and related organisms. The rt-PCR method allowed species-specific detection of Campylobacter spp. in naturally infected pig faecal samples after an enrichment step, whereas the hybridization approach enhanced the specific isolation of C. jejuni (present in minority to C. coli) from pigs. CONCLUSIONS: The rt-PCR was specific for Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis and the colony-blot hybridization approach provided an effective tool for isolation of C. jejuni from pig faecal samples typically dominated by C. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Species differentiation between thermophilic Campylobacter is difficult by phenotypic methods and the developed rt-PCR provides an easy and fast method for such differentiation. Detection of C. jejuni by colony hybridization may increase the isolation rate of this species from pig faeces.

Chicken juice, a food-based model system suitable to study survival of Campylobacter jejuni.

AIMS: The purpose of this study was to develop a food-based model system that resembles the environment that Campylobacter jejuni experiences on raw poultry products and use this model system to investigate growth and survival of the bacterium. METHODS AND RESULTS: Chicken juice was collected from frozen chickens and subsequently cleared by centrifugation and subjected to sterile filtration. At low temperatures (5 and 10 degrees C) C. jejuni NCTC11168 remained viable in chicken juice for a remarkably longer period of time than in the reference medium BHI. When exposed to heat stress (48 degrees C) C. jejuni NCTC11168 also showed increased viability in chicken juice compared with the reference medium. Furthermore, agar plates made with chicken juice supported growth of four clinical isolates of C. jejuni and a C. jejuni strain obtained from chicken at both 37 and 42 degrees C. CONCLUSIONS: Our work shows that minimal processed and sterilized chicken juice is an ideal environment for survival of C. jejuni and that it is useful as a food-based model system. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed model system may contribute to the understanding of C. jejuni viability on poultry products and can be instrumental in the development of alternative preservation strategies.

Ultra-rapid DNA analysis using HyBeacon probes and direct PCR amplification from saliva.

We describe a novel probe technology, termed HyBeacons, which provides a new homogeneous method for fluorescence-based sequence detection and allele discrimination. Employing a single nucleotide polymorphism located in the N-acetyltransferase 2 gene as a model system, we demonstrate the utility of HyBeacon probes for rapid and reliable sequence analysis. We also demonstrate that homozygous and heterozygous samples may be accurately identified using a single HyBeacon oligonucleotide. Polymorphic DNA sequences were detected and differentiated by real-time PCR and melt peak methodologies, without performing extraction of genomic DNA prior to target amplification. Employing a combination of homogeneous HyBeacon analysis, the rapid thermal cycling conditions of the LightCycler and direct amplification from saliva, allowed samples to be genotyped within 30 min. Such rapid non-invasive diagnostic technologies may permit 'point-of-care' genetic testing to be performed in hospitals and doctor's surgeries.

Presence of clone-specific markers at birth in children with acute lymphoblastic leukaemia.

Recent studies have suggested that development of childhood acute lymphoblastic leukaemia may often be initiated in utero. To provide further evidence of an prenatal origin of childhood leukaemia, we conducted a molecular biological investigation of nine children with B-precursor acute lymphoblastic leukaemia carrying the chromosomal translocation t(12;21), the most common subtype of all childhood acute lymphoblastic leukaemia. Specifically, for each child we identified the non-constitutive chromosomal sequences made up by the t(12;21) fusion gene. From these, leukaemia clone-specific DNA primers were constructed and applied in nested polymerase chain reaction analyses of DNA extracted from the patients' Guthrie cards obtained at birth. Leukaemia clone-specific fusion gene regions were demonstrated in Guthrie card DNA of three patients, age 2 years 11 months, 3 years 4 months, and 5 years 8 months at leukaemia diagnosis. Our findings are consistent with previous observations, and thus provide further evidence that the development of t(12;21) B-precursor acute lymphoblastic leukaemia may be initiated in utero. Review of the current literature moreover indicates that age at leukaemia may be inversely correlated with the burden of cells with leukaemia clonal markers, i.e. leukaemia predisposed cells at birth, and that certain types of childhood acute lymphoblastic leukaemia develop as a multiple step process involving both pre- and postnatal genetic events.

Phormium Yellow Leaf Phytoplasma Is Associated with Strawberry Lethal Yellows Disease in New Zealand.

A yellows disease of strawberry plants was identified in propagation beds in New Zealand. Affected plants were flatter to the ground, showed purpling of older leaves, reduced leaf size, yellowing of younger leaves, and sometimes plant death. A phytoplasma was observed in the phloem of affected plants. The 16S rRNA gene of the phytoplasma was amplified by polymerase chain reaction from symptomatic plants and from one asymptomatic plant, but not from 36 other asymptomatic plants. Nucleotide sequence analysis of the 16S rRNA gene showed that the phytoplasma is closely related or identical to the phytoplasma associated with the yellow leaf disease of New Zealand flax (Phormium tenax).

Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma.

Phormium yellow leaf (PYL) phytoplasma causes a lethal disease of the monocotyledon, New Zealand flax (Phormium tenax). The 16S rRNA genes of PYL phytoplasma were amplified from infected flax by PCR and cloned, and the nucleotide sequences were determined. DNA sequencing and Southern hybridization analysis of genomic DNA indicated the presence of two copies of the 16S rRNA gene. The two 16S rRNA genes exhibited sequence heterogeneity in 4 nucleotide positions and could be distinguished by the restriction enzymes BpmI and BsrI. This is the first record in which sequence heterogeneity in the 16S rRNA genes of a phytoplasma has been determined by sequence analysis. A phylogenetic tree based on 16S rRNA gene sequences showed that PYL phytoplasma is most closely related to the stolbur and German grapevine yellows phytoplasmas, which form the stolbur subgroup of the aster yellows group. This phylogenetic position of PYL phytoplasma was supported by 16S/23S spacer region sequence data.

Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block.

White clover mosaic virus strain O (WClMV-O), species of the Potexvirus genus, contains a set of three partially overlapping genes (the triple gene block) that encodes nonvirion proteins of 26 kDa, 13 kDa, and 7 kDa. These proteins are necessary for cell-to-cell movement in plants but not for replication. The WClMV-O 13-kDa gene was mutated (to 13*) in a region of the gene that is conserved in all viruses known to possess triple-gene-block proteins. All 10 13* transgenic lines of Nicotiana benthamiana designed to express the mutated movement protein were shown to be resistant to systemic infection by WClMV-O at 1 microgram of WClMV virions per ml, whereas all plants from susceptible control lines became systemically infected. Of the 13* transgenic lines, 3 selected for their abundant seed supply were shown to be resistant to systemic infection when challenged by inoculation with three different WClMV strains (O, M, and J) or with WClMV-O RNA at 10 micrograms/ml. Most plants were also resistant to systemic infection at inoculum concentrations up to 250 micrograms of WClMV virions per ml. In addition, the three 13* transgenic plant lines were found to be resistant to systemic infection with two other members of the Potexvirus group, potato virus X and narcissus mosaic virus, and the Carlavirus potato virus S but not to be resistant to tobacco mosaic virus of the Tobamovirus group. These results indicate that virus resistance can be engineered into transgenic plants by expression of dominant negative mutant forms of triple-gene-block movement proteins.

The coat protein of white clover mosaic potexvirus has a role in facilitating cell-to-cell transport in plants.

Functions of the coat protein of white clover mosaic potexvirus (WCIMV) were investigated using C-terminal deletion mutants. Whereas plants inoculated with RNA transcripts of a full-length wild-type clone of WCIMV produced typical infections, plants inoculated with transcripts of each mutant did not produce symptoms, and viral RNA species were not detected by Northern analysis. The mutants were able to replicate in protoplasts, although, relative to the wild-type RNA profile, the level of genomic RNA, but not subgenomic RNA, was reduced. These results indicate a role for the coat protein in efficient cell-to-cell transport in plants. Virus-like particles were detected in protoplast extracts inoculated with transcripts of a mutant in which the coat protein was truncated by 31 amino acids. This result suggests that the lack of detectable transport in plants was not due solely to a failure of the mutants to form virus particles. Possible roles for the coat protein in transport and replication are discussed. A 6-kDa open reading frame, internal to the coat protein gene, was shown by mutational analysis not to be essential for replication or transport.

Triple gene block proteins of white clover mosaic potexvirus are required for transport.

The functions of the protein products encoded by a block of three overlapping genes (the triple gene block) of white clover mosaic potexvirus (WCIMV) have been determined. Mutations were introduced into each of the triple gene block open reading frames and in vitro RNA transcripts assayed in plants and protoplasts. None of the mutants was able to induce symptoms or spread in four systemic hosts and one local lesion host, but all were able to produce progeny genomic RNA, subgenomic RNA, coat protein, and virions in inoculated protoplasts, indicating that all the triple gene block proteins are involved in cell-to-cell spread. Based on observed homologies between the triple gene block proteins of the potex-, carla-, furo-, and hordeivirus groups and Nicotiana velutina mosaic virus, and the demonstrated transport function of the WCIMV and barley stripe mosaic virus triple gene block proteins, these proteins are proposed to constitute a new class of transport proteins.

Nucleotide sequence of the geminivirus chloris striate mosaic virus.

The genome of chloris striate mosaic virus (CSMV) comprises a single circular DNA as determined by analyses on virion single-stranded (ss) DNA and virus-specific covalently closed circular (ccc) DNA isolated from infected plants. The nucleotide sequence of CSMV DNA was determined from cccDNA and the data were accommodated into one DNA circle of 2750 nucleotides. Comparison of the nucleotide sequence with those of maize streak virus (MSV), wheat dwarf virus (WDV), and digitaria streak virus (DSV) showed 49, 47, and 48% DNA homology, respectively. The sequence has four potential open reading frames for proteins of greater than 10,000 mol wt, two in the viral (+) sense and two in the complementary (-) sense. Three of these potential coding regions have homologous counterparts, by comparison of the amino acid sequences, among the open reading frames reported for MSV, WDV, and DSV. CSMV encapasidates primer molecules able to prime the synthesis in vitro of a complementary strand to virion DNA, initiating this reaction at one site on the genome. The CSMV primer comprising approximately 88 nucleotides was located within the smaller of two intergenic or noncoding regions.

Cassava latent virus infections mediated by the Ti plasmid of Agrobacterium tumefaciens containing either monomeric or dimeric viral DNA.

Plant infections with cassava latent virus (CLV) were mediated by the Ti plasmid of Agrobacterium tumefaciens containing either monomeric or dimeric copies of the virus genome. The CLV DNAs caused typical symptoms when they were inoculated in Agrobacterium strains C58, LBA4404 and a virE mutant A1026, but not other Agrobacterium strains with mutations in other vir loci or an E. coli polA strain. Virus-specific DNA forms characteristic of normal CLV infections were found after such infection. Characterization of progeny CLV DNA from selected plants identified several infectious mutants. These were found to be small insertions and/or deletions in the coat protein gene of DNA 1 and in the intergenic region of DNA 2.