The finding of a reciprocal whole-arm translocation t(X;12)(p10;p10) in association with atypical chronic myeloid leukaemia.

Atypical chronic myeloid leukaemia (aCML) belongs to the myeloproliferative/myelodysplastic category of haematological disease. Main characteristics are marked dysgranulopoiesis, bone marrow dysfunction and the failure to demonstrate the presence of the Philadelphia chromosome or BCR/ABL fusion gene normally associated with CML t(9;22)(q34;q11). It carries a poor prognosis with limited therapeutic options available. Most cases of aCML have one or more karyotypic abnormalities. We highlight a clinical presentation of aCML associated with an acquired reciprocal whole-arm translocation (WAT), t(X;12)(p10;p10), which to our knowledge has not yet been described. We also discuss how such a translocation might lead to tumorigenesis.

Myeloid sarcoma of the small bowel associated with a CBFbeta/MYH11 fusion and inv(16)(p13q22): a case report.

This report describes a case of aleukaemic myeloid sarcoma of the small intestine in a 50-year-old woman presenting with small bowel obstruction. Fluorescence in situ hybridisation analysis of interphase nuclei revealed a split CBFbeta signal, consistent with an underlying inversion of chromosome 16, inv(16)(p13q22). The resultant type A CBFbeta/MYH11 transcript was detected by reverse transcriptase PCR. Immunohistochemistry with the AH107 antibody to the CBFbeta-SMMHC chimeric protein showed strong nuclear staining of the tumour cell nuclei. This represents the first use of this antibody in the diagnosis of this subtype of myeloid sarcoma in the small intestine.

Use of imatinib mesylate in elderly patients in Northern Ireland: evidence of comparable haematological and molecular responses to younger patients.

Advanced age is an indicator of poor prognosis in chronic myeloid leukaemia (CML). Since obtaining its UK licence in 2001, the tyrosine kinase inhibitor imatinib mesylate has effected a paradigm shift in the treatment of CML. We compared survival and molecular response rates in elderly patients to younger patients presenting with CML since the introduction of imatinib. Twenty-five patients aged >60 years were identified. No significant survival difference was found when this group was compared with younger patients. In the elderly group, 53% of those with molecular data (36% of all elderly patients) had a major molecular response as assessed by real time quantitative PCR (RT-PCR). The advent of imatinib therapy appears to have ameliorated much of the negative impact of advancing age on survival in patients with CML.

Does fructan have a functional role in physiological traits? Investigation by quantitative trait locus mapping.

* The role of fructan in growth and drought-stress responses of perennial ryegrass (Lolium perenne) was investigated in an F(2) mapping family that segregates for carbohydrate metabolism. * A quantitative trait locus approach was used to compare the genetic control of traits. * Growth and drought-stress traits were extremely variable within the family. Most traits had high broad-sense heritability. Quantitative trait loci (QTL) were identified for most traits; the maximum number of QTL per trait was four. Between 11% and 75% of total phenotypic variation was explained. Few growth-trait QTL coincided with previously identified fructan QTL. A cluster of drought-trait QTL was close to two previously identified regions of the genome with tiller base fructan QTL in repulsion. * The high sugar parent contributed few alleles that increased 'reserve-driven' growth or performance during drought-stress. Correlation of growth and drought-stress traits with fructan content was low and increasing fructan content per se would not appear to improve drought resistance. Complex patterns of carbohydrate partitioning and metabolism within the cell may explain contradictory relationships between carbohydrate content and growth/stress-resistance traits.

GISH/FISH mapping of genes for freezing tolerance transferred from Festuca pratensis to Lolium multiflorum.

The first backcross breeding programme for the transfer of freezing-tolerance genes from winter hardy Festuca pratensis to winter-sensitive Lolium multiflorum is described. A partly fertile, triploid F(1) hybrid F. pratensis (2n=2x=14) x L. multiflorum (2n=4x=28) was employed initially, and after two backcrosses to L. multiflorum (2x) a total of 242 backcross two (BC(2)) plants were generated. Genomic in situ hybridisation (GISH) was performed on 61 BC(2) plants selected for their good growth and winter survival characters in the spring following one Polish winter (2000-2001). Among the winter survivors, diploid chromosome numbers were present in 80% of plants. An appropriate single Festuca introgression in an otherwise undisturbed Lolium genome could provide increased freezing tolerance without compromise to the good growth and plant vigour found in Lolium. Among all the diploids, a total of 20 individuals were identified, each with a single F. pratensis chromosome segment. Another diploid plant contained 13 Lolium chromosomes and a large metacentric F. pratensis chromosome, identified as chromosome 4, with two large distal Lolium introgressions on each chromosome arm. Three of the diploid BC(2), including the genotype with Festuca chromosome 4 DNA sequences, were found to have freezing tolerance in excess of that of L. multiflorum, and in one case in excess of the F. pratensis used as control. A detailed cytological analysis combining GISH and fluorescence in situ hybridisation analyses with rDNA probes revealed that the other two freezing-tolerant genotypes carried a Festuca chromosome segment at the same terminal location on the non-satellite arm of Lolium chromosome 2.

A changing climate for grassland research.

Here, we review the current genetic approaches for grass improvement and their potential for the enhanced breeding of new varieties appropriate for a sustainable agriculture in a changing global climate. These generally out-breeding, perennial, self-incompatible species present unique challenges and opportunities for genetic analysis. We emphasise their distinctiveness from model species and from the in-breeding, annual cereals. We describe the modern genetic approaches appropriate for their analysis, including association mapping. Sustainability traits discussed here include stress resistance (drought, cold and pathogeneses) and favourable agronomic characters (nutrient use efficiency, carbohydrate content, fatty acid content, winter survival, flowering time and biomass yield). Global warming will predictably affect temperature-sensitive traits such as vernalisation, and these traits are under investigation. Grass biomass utilisation for carbon-neutral energy generation may contribute to reduced atmospheric carbon emissions. Because the wider potential outcomes of climate change are unpredictable, breeders must be reactive to events and have a range of well-characterised germplasm available for new applications.

Introgression-mapping of genes for drought resistance transferred from Festuca arundinacea var. glaucescens into Lolium multiflorum.

Procedures for the transfer of genes for drought resistance from Festuca glaucescens (2n=4x=28) into Lolium multiflorum (2n=2x=14) are described. Following the initial hybridisation of a synthetic autotetraploid of L. multiflorum (2n=4x=28) with F. glaucescens, the F1 hybrid was backcrossed twice onto diploid L. multiflorum (2n=2x=14) to produce a diploid Lolium genotype with a single F. glaucescens introgression located distally on the nucleolar organiser region arm of chromosome 3. The transmission of F. glaucescens-derived amplified fragment length polymorphisms and a sequence-tagged-site (STS) marker was monitored throughout the breeding programme. Those genotypes of a mapping population of backcross 3 that survived combined severe drought and heat stress all contained the F. glaucescens-derived markers. The STS marker provided a prototype for a PCR-based system for high-throughput screening during cultivar development for the presence of the F. glaucescens-derived genes for drought resistance. The frequency of intergeneric recombination between L. multiflorum and F. glaucescens is described. During the initial stages of the breeding programme, preferential intraspecific chromosome pairing between Lolium homologues and Festuca homoeologues dominated with low frequencies of intergeneric chromosome associations. However, these increased in the backcross 1 due to the absence of opportunities for intraspecific chromosome pairing between homoeologous Festuca chromosomes following the loss of half of the Festuca chromosomes. Once transferred to Lolium, F. glaucescens sequences recombined with Lolium at high frequencies, thereby enabling the loss of potentially deleterious gene combinations that might reduce the forage quality of Lolium.

The prognostic value of FISH as an adjunct to conventional cytogenetics for the detection of cryptic gene rearrangements on chromosome 16. A retrospective investigation of 13 patients from Northern Ireland diagnosed with M4Eo acute myeloid leukaemia.

M4Eo acute myeloid leukaemia (AML) patients with the typical chromosome 16 abnormalities have a favourable prognosis. These subtle 16q22 gene rearrangements can be difficult to detect by conventional cytogenetic methods and if missed could lead to the incorrect assignment of prognostic group and hence subsequent treatment strategies. We retrospectively studied 13 patients diagnosed with M4Eo AML for such chromosome 16 abnormalities comparing conventional cytogenetic (G-banding) and molecular (FISH) methods. G-banded analysis detected only 2 patients with definite chromosome 16 abnormalities whereas FISH detected 4 patients, one with the typical inversion and three with the typical chromosome 16 translocation. FISH analysis also confirmed a false +ve G-banded result in one patient and a false -ve G-banded result in another patient. Finally, FISH confirmed a deletion of one chromosome 16 homologue in another patient indicating a poor prognosis. The overall survival of patients with the typical 16q22 rearrangements (n=4) was also significantly better (P=0.007) than patients with normal chromosome 16 homologues or having other numerical and/or structural abnormalities (n=9). This set of data shows that FISH is a more accurate method for the detection of cryptic 16q22 gene rearrangements and because of the prognostic implications has become a mandatory test along with conventional cytogenetics for all newly diagnosed M4Eo AML patients in Northern Ireland.

Androgenesis from Festuca pratensis x Lolium multiflorum amphidiploid cultivars in order to select and stabilize rare gene combinations for grass breeding.

Androgenesis using amphidiploid cultivars of Festuca pratensis x Lolium multiflorum as parents, overcame earlier problems that gave rise to widespread plant sterility amongst androgenic Festulolium populations. Two Festuca pratensis x Lolium multiflorum (2n = 4x = 28) cultivars, Sulino and Felopa, were highly amenable to androgenesis and 10% of plants, including some novel androgenic genotypes, had sufficient fertility to produce progeny and further generations. The genomes of amphidiploid cultivars, which represent the F8 generation, were the result of considerable intergeneric chromosome recombination. Moreover, during cultivar development, natural and breeders' selection pressures had led to the assembly of gene combinations that conferred good growth characters and fertility with the removal of putative deleterious gene combinations. Over 80% of the androgenic plants derived from the amphidiploid F. pratensis x L. multiflorum (2n = 4x = 28) had 14 chromosomes and were likely to be dihaploids with a single genome of Lolium and of Festuca. In contrast, hybrids of F. pratensis x L. multiflorum (2n = 2x = 14) found naturally are invariably sterile. Structural reorganization within the genomes of the androgenic Festulolium plants had restored fertility in genotypes expected to contain the haploid genome of Lolium and Festuca. This provided opportunities for their future incorporation in breeding programmes and the development of fertile diploid Lolium-Festuca hybrids. Amongst the androgenic plants, Festulolium genotypes were recovered that conferred excellent drought resistance or freezing tolerance and were thought to be highly suitable for entry into plant breeding programmes.

Fluorescence in situ hybridisation detection of erbB2 amplification in breast cancer fine needle aspirates.

AIM: To develop a method for the detection of amplification of the erbB2 oncogene in breast cancer fine needle aspirates using fluorescence in situ hybridisation (FISH) and to compare amplification with immunohistochemical detection of the erbB2 protein. METHODS: A digoxigenin labelled probe to the erbB2 gene was hybridised to 15 aspirates prepared from operative breast cancer specimens. A chromosome 17 centromere probe was also hybridised to the aspirates either separately or in combination with the erbB2 probe. The aspirates were scored for erbB2 amplification and chromosome 17 centromere number. Subsequently, paraffin wax embedded sections of the tumours were stained with the antibody CB11 and scored for the presence of membrane staining. RESULTS: Three of the 15 tumour aspirates showed high level amplification of erbB2 detected by FISH. These three tumours also showed chromosome 17 polysomy and diffuse membrane staining by immunohistochemistry. CONCLUSIONS: FISH can be used to detect erbB2 amplification in fine needle aspirates and results correlate with conventional immunohistochemical staining. Difficulties were encountered in the visualisation of the signals in non-amplified cases without the use of specialised digital imaging.

De novo direct duplication 2 (p12-->p21) with paternally inherited pericentric inversion 2p11.2 2q12.2.

We report a 4-year-old girl with a previously undescribed de novo duplication of 2p12->2p21 on the same homologue as a paternally inherited pericentric inversion of region 2p11.2-->2q12.2, resulting in dysmorphic features, cardiac abnormality, cleft palate, respiratory problems, severe growth retardation and developmental delay. This case raises an important question--did the paternal pericentric inversion influence the occurrence of the de novo duplication?

Leukaemoid monocytosis in M4 AML following chemotherapy and G-CSF.

We describe a patient with M4 AML treated with standard chemotherapy followed by G-CSF who developed marked monocytosis on day 8 of G-CSF therapy. Fourteen days after discontinuation of G-CSF therapy his monocyte counts returned to normal levels and a marrow aspirate showed a reduction in blast cells. He received further chemotherapy without G-CSF and without any recurrence of the raised leucocyte count but failed to achieve full remission. Although this G-CSF-driven leucocytosis was alarming it did not appear to have adversely affected the patient's prognosis.

Intergenomic translocations and the genomic composition of Avena maroccana Gdgr. revealed by FISH.

Fluorescence in situ hybridization (FISH) using total genomic DNA from putative diploid progenitors was used to confirm the presence of the A and C genomes in Avena maroccana. These results confirm cytological data that intergenomic translocations are present in A. maroccana.

Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization.

Genomic in situ hybridization (GISH) was used to identify Festuca chromatin in mitotic chromosomes of Lolium multiflorum (Lm) × Festuca pratensis (Fp) hybrids and hybrid derivatives. In two inverse autoallotriploids LmLmFp and LmFpFp, in situ hybridization was able to discriminate between the Lolium and Festuca chromosomes. In a third triploid hybrid produced by crossing an amphiploid of L. multiflorum × F. pratensis (2n=4x=28) with L. multiflorum (2n=2x=14), the technique identified chromosomes with interspecific recombination. Also, in an introgressed line of L. multiflorum which was homozygous for the recessive sid (senescence induced degradation) allele from F. pratensis, a pair of chromosome segments carrying the sid gene could be identified, indicating the suitability of GISH in showing the presence and location of introgressed genes. By screening backcross progeny for the presence of critical alien segments and the absence of other segments the reconstitution of the genome of the recipient species can be accelerated.

Cytogenetic investigations in a family with ataxia telangiectasia.

Cytogenetic findings on a family with ataxia telangiectasia (A-T) in which three of four sibs were affected are described. The affected individuals had approximately twice the level of spontaneous chromosome breakage of a normal control, while the parents and the normal sib had no significant increase. Lymphocytes from all three A-T homozygotes showed specific stable chromosomal rearrangements involving chromosomes 7 and 14. All of these abnormalities involved breakage at the usual four sites associated with A-T (7p14, 7q35, 14q12, and 14q32). Two rearrangements detected in the eldest and most severely affected patient were clones, one of which [t(14;14)(p11;q12)] is not commonly found in A-T cells. No chromosomal rearrangements were encountered in lymphocytes from the control, the parents, or the normal sib. Lymphocytes from the A-T patients also were found to be 7-11 times more sensitive to the induction of chromatid aberrations by X-irradiation than control cells. Lymphocytes from the parents and normal sib showed a moderately increased frequency of X-ray induced aberrations compared with that of the control.

The use of a triploid hybrid for introgression in Lolium species.

Triploid hybrids of Lolium multiflorum (4x) x L. perenne (2x) behaved cytologically as autotriploids but the segregation of isozyme variants did not always agree with the expected trisomic ratios. The overall effect of these deviations from expectation was a greater proportion than expected of diploid progeny from the cross L. multiflorum (2x) x triploid hybrid which did not include any of the L. perenne alleles at the three marker isozyme loci. The possible mechanisms for these aberrant segregation ratios are discussed together with the advantages of the crossing scheme to accelerate the recovery of the genotype of the recurrent parent in a backcrossing programme to transfer characters from one species to another.

Chromosome location of two isozyme loci in Lolium perenne using primary trisomics.

Primary trisomics were used to locate the structural loci coding for particular forms of the dimeric enzymes phosphoglucoisomerase and glutamate oxaloacetate transaminase in Lolium perenne. The polymorphy of these loci enabled triallelic trisomics to be produced. Each locus could thus be directly assigned to a particular chromosome without the need to examine segregant progeny. The loci for GOT/3 and PGI/2 were found to be located on chromosomes 2 and 6 respectively.