European recommendations and quality assurance for cytogenomic analysis of haematological neoplasms.

Cytogenomic investigations of haematological neoplasms, including chromosome banding analysis, fluorescence in situ hybridisation (FISH) and microarray analyses have become increasingly important in the clinical management of patients with haematological neoplasms. The widespread implementation of these techniques in genetic diagnostics has highlighted the need for guidance on the essential criteria to follow when providing cytogenomic testing, regardless of choice of methodology. These recommendations provide an updated, practical and easily available document that will assist laboratories in the choice of testing and methodology enabling them to operate within acceptable standards and maintain a quality service.

FISH detection of chromosome 14q32/IgH translocations: evaluation in follicular lymphoma.

A FISH strategy capable of detecting chromosome 14q32 rearrangements involving the IgH locus, including in interphase nuclei, was developed using Ig variable and constant region cosmids from the extremities of the locus in a dual hybridization approach, using signal splitting as evidence of rearrangement. The large size of the locus (1.3 Mb) and the propensity for internal deletion due to physiological VDJ recombination and isotype switching complicate analysis of this locus. We used the Ig10 cosmid, which hybridizes to C epsilon and C alpha2 at the 3' end of the constant region, in order to minimize deletion and/or splitting of the constant region probe. Cos Ig10 and the IgV18 VH probes were compared with a specific IgH-BCL2 FISH dual hybridization approach in follicular lymphoma (FL). Both were capable of detecting the t(14;18) in interphase nuclei, including in cases with no apparent abnormality by classic karyotype analysis, although the sensitivity of the IgH approach was slightly lower. We have also successfully applied these probes to whole cell cytospin preparations, rendering analysis of cryopreserved material possible, although interpretation should be limited to frequent events, particularly following cell manipulation. Analysis of flow cytometric sorted bone marrow fractions from three FL patients by FISH and FICTION showed that the t(14;18) was present in a much lower proportion of CD34 positive than negative cells but that the higher level of background hybridization limits use of these techniques for the reliable quantification of rare events.

Simultaneous detection of MYC, BVR1, and PVT1 translocations in lymphoid malignancies by fluorescence in situ hybridization.

The rapid detection of chromosome band 8q24 rearrangements, including classical translocations involving MYC and variant 3' translocations, is important for the accurate diagnosis and appropriate treatment of lymphoid malignancies. We have identified and characterized a CEPH YAC, 934e1, which extends from at least 190 kbp upstream to over 280 kbp downstream to MYC, allowing detection of classical t(8; 14)(q24;q32) and variant t(8;22)(q24;q11) and t(8;14)(q24;q11), extending distal to PVT1 and therefore, by extrapolation, to BVR1. This YAC also allowed clarification of complex chromosome 8 abnormalities and the identification of translocations in interphase nuclei. A second CEPH YAC, 904c3, previously shown to contain the PVT1 locus but not MYC, allowed distinction between translocations occurring centromeric and telomeric to MYC. Use of the 934e1 YAC will aid classification of a variety of lymphoid proliferations and further characterization of rearranged cases with the 904c3 YAC will simplify mapping of their diverse breakpoints.

A chromosome 14q11/TCR alpha/delta specific yeast artificial chromosome improves the detection rate and characterization of chromosome abnormalities in T-lymphoproliferative disorders.

The rate of detection of chromosome abnormalities in T-cell proliferations is lower than that observed in B-cell malignancies. The former frequently involve the TCR alpha/delta locus at chromosome band 14q11. We have identified a YAC encompassing 70% of the TCR alpha/delta locus, which has been used as a fluorescence in situ hybridization probe to detect chromosome rearrangements involving 14q11, both at metaphase and within interphase nuclei, in patients with a variety of T-lymphoproliferative disorders. Its use allowed detection of previously unsuspected TCR alpha/delta rearrangements in 4/13 (30%) immature T-lineage acute leukemias, including two t(10;14) and 2 minor inversion 14s. It also clarified interpretation of complex chromosome 14 abnormalities in mature T-cell proliferations (T-prolymphocytic leukemia and ataxia telangiectasia). Use of this probe will aid the detection and characterization of abnormalities involving the TCR alpha/delta locus, particularly in cases with normal or complex karyotypes and in those proliferations for which mitoses are difficult to obtain.

Absence of the XIST gene from late-replicating isodicentric X chromosomes in leukaemia.

The mechanism of X-inactivation in man is thought to involve a specific cis-acting locus within the X-inactivation centre at Xq13 (1,2). The XIST gene (X inactive specific transcript) at Xq13 is ubiquitously expressed only from the inactive X and as such may be involved in or influenced by the X-inactivation process (3,4). We have localised the breakpoints on two acquired isodicentric X chromosomes associated with leukaemia to a 450 kilobase region of DNA within Xq13, which result in deletion of the XIST gene. We have demonstrated that these chromosomes remain inactive and that there is no evidence of XIST expression from the remaining intact X chromosomes. The data suggest that XIST is not required for the maintenance of X-inactivation on these somatically rearranged X chromosomes.

Characterisation of molecular DNA rearrangements within the Xq12-q13.1 region, in three patients with X-linked hypohidrotic ectodermal dysplasia (EDA).

A panel of somatic cell hybrids and X-linked hypohidrotic ectodermal dysplasia (EDA) patient-derived cell lines, containing different rearranged X chromosomes, have been used to refine the physical map of the Xq12-q13.1 region. The patient-derived material included genomic DNA from an EDA male (EDA family 1015) with an interstitial deletion, and a cell line GM0705A, obtained from an isolated female patient with a de novo balanced (X;9) translocation, and the somatic hybrid, AnLy, derived from this cell line. This map subdivides the region into at least 6 mapping-intervals. DNA probes from DXS732 and DXS453, identified as the closest flanking marker loci to the EDA locus, were used to identify homologous Yeast Artificial Chromosome (YAC) clones. Two of the DXS732-specific YACs were shown by fluorescent in situ hybridisation (FISH) analysis to bridge the (X;9) translocation breakpoint. These two YACs were also screened against the ICRF human X chromosome cosmid library and identified 36 cosmid clones. Direct cosmid-cosmid hybridisation analysis placed subsets of these clones within four different cosmid contigs. Mapping of anchor clones from each contig, against the mapping panel, localised all these contigs within the Xq12-q13.1 region. One cosmid, ICRFc104C03.184, identified potential junctional-fragments in several restriction digests of AnLy hybrid DNA. This was confirmed by FISH analysis of the GM0705A cell line with total cosmid ICRFc104C03.184, in which both chromosomal elements of the (X;9) translocation were identified. A single-copy probe pC03.184E2, derived from this cosmid, also identified the der(9)-derived junctional fragment when hybridised against AnLy DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of three de novo derivative chromosomes 16 by "reverse chromosome painting" and molecular analysis.

We have analyzed three de novo chromosome 16 rearrangements--two with a 16p+ chromosome and one a 16q+--none of which could be fully characterized by conventional cytogenetics. In each case, flow karyotypes have been produced, and the aberrant chromosome has been isolated by flow sorting. The origin of the additional material has been ascertained by amplifying and labeling the DNA of the abnormal chromosome by degenerate-oligonucleotide-primer-PCR and hybridizing it in situ to normal metaphase spreads (reverse chromosome painting). Both 16p+ chromosomes contain more than 30 Mb of DNA from the short arm of chromosome 9(9p21.2-pter), while the 16q+ contains approximately 9 Mb of DNA from 2q37. The breakpoints on chromosome 16 have been localized in each case; the two breakpoints on the short arm are at different points within the terminal band, 16p13.3. The breakpoint on the long arm of chromosome 16 is very close to (within 230 kb of) the 16q telomere. Determination of the regions of monosomy and trisomy allowed the observed phenotypes to be compared with other reported cases involving aneuploidy for these regions.

Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16.

We have completed a long-range restriction map of the terminal region of the short arm of human chromosome 16 (16p13.3) by physically linking a distal genetic locus (alpha-globin) with two recently isolated probes to telomere-associated repeats (TelBam3.4 and TelBam-11). Comparison of 47 chromosomes has revealed major polymorphic length variation in this region: we have identified three alleles in which the alpha-globin genes lie 170 kb, 350 kb, or 430 kb from the telemere. The two most common alleles contain different terminal segments, starting 145 kb distal to the alpha-globin genes. Beyond this boundary these alleles are nonhomologous, yet each contains sequences related to other (different) chromosome termini. This chromosome size polymorphism has probably arisen by occasional exchanges between the subtelomeric regions of nonhomologous chromosomes; analogous length variation is likely to be present at other human telomeres.