Andrew M. Fisher

Centromeric inactivation in a dicentric human Y;21 translocation chromosome

Abstract. A de novo dicentric Y;21 (q11.23;p11) translocation chromosome with one of its two centromeres inactive has provided the opportunity to study the relationship between centromeric inactivation, the organization of alphoid satellite DNA and the distribution of CENP-C. The proband, a male with minor features of Down’s syndrome, had a major cell line with 45 chromosomes including a single copy of the translocation chromosome, and a minor one with 46 chromosomes including two copies of the translocation chromosome and hence effectively trisomic for the long arm of chromosome 21. Centromeric activity as defined by the primary constriction was variable: in most cells with a single copy of the Y;21 chromosome, the Y centromere was inactive. In the cells with two copies, one copy had an active Y centromere (chromosome 21 centromere inactive) and the other had an inactive Y centromere (chromosome 21 centromere active). Three different partial deletions of the Y alphoid array were found in skin fibroblasts and one of these was also present in blood. Clones of single cell origin from fibroblast cultures were analysed both for their primary constriction and to characterise their alphoid array. The results indicate that (1) each clone showed a fixed pattern of centromeric activity; (2) the alphoid array size was stable within a clone; and (3) inactivation of the Y centromere was associated with both full-sized and deleted alphoid arrays. Selected clones were analysed with antibodies to CENP-C, and staining was undetectable at both intact and deleted arrays of the inactive Y centromeres. Thus centromeric inactivation appears to be largely an epigenetic event.

Breakpoints of variant 9;22 translocations in chronic myeloid leukemia locate preferentially in the CG-richest regions of the genome.

From 5% to 10% of 9;22 translocations in chronic myeloid leukemia (CML) are reported to occur in variant form, that is, with the involvement of other regions of the genome in 3‐way or more rearrangements. The literature indicates that the alternative breakpoints are not distributed randomly in the genome but show hotspots. We present data on 289 unpublished cases of CML with variant 9;22 translocations having a total of 342 variant breakpoints, the largest independent series to date. We found that the distribution of breaks was in loose agreement with the literature but that some new hotspots were identified; furthermore, some published hotspots were not fully supported by our data. Moreover, when our 342 variant breakpoints were plotted against profiles of CG heterogeneity in the genome, a significant positive correlation between breakpoint locations and CG composition was observed. In an ancillary study, we compared the frequency of variant t(9;22) with that of variants of t(15;17) associated with acute promyelocytic leukemia (AML M3). We found that the frequency of the former, 9.3%, was significantly higher than that of the latter, 2.6%.

RAPID IN SITU HARVESTING AND CYTOGENETIC ANALYSIS OF PERINATAL TISSUE SAMPLES

Thirty perinatal solid tissue samples were used in a pilot study to test the efficacy of collagenase disaggregation onto coverslips, open system culture under low O2 conditions, pre‐harvest incubation in bromodeoxyuridine (BrdU) and colcemid, and in situ automated harvesting. Following the success of the pilot study, the new method was applied to a further 126 consecutive diagnostic samples making a total of 156. The method reduced average tissue culture times from 17 to 8·7 days (range 2–17), improved success rates from 76 to 88 per cent, and simultaneously increased the resolution of cytogenetic analysis.

Inv(10)(p11.2q21.2), a variant chromosome

Abstract We present 33 families in which a pericentric inversion of chromosome 10 is segregating. In addition, we summarise the data on 32 families in which an apparently identical inv(10) has been reported in the literature. Ascertainment was through prenatal diagnosis or with a normal phenotype in 21/33 families. In the other 12 families, probands were ascertained through a wide variety of referral reasons but in all but one case (a stillbirth), studies of the family showed that the reason for referral was unrelated to the chromosome abnormality. There has been, to our knowledge, no recorded instance of a recombinant chromosome 10 arising from this inversion and no excess of infertility or spontaneous abortion among carriers of either sex. We propose that inv(10)(p11.2q21.2) can be regarded as a variant analogous to the pericentric inversion of chromosome 2(p11q13). We conclude that prenatal chromosome analysis is not justified for inv(10) carriers. In addition, family investigation of carrier status is not warranted in view of the unnecessary concern this may cause parents and other family members.

Incidence of circulating cells positive for GpIb and GpIIIa in infants with Down's syndrome.

Abstract: Blood samples from 43 infants with Downs syndrome and from 67 with a normal karyotype were screened for the presence of cells positive for platelet glycoproteins GpIb and GpIIIa. There was 1 case of fatal leukaemia and 1 case of transient leukaemia among the Downs syndrome patients, both cases had increased numbers of circulating blood cells positive for platelet glycoprotein markers. These cells were present in small numbers in both the Downs and control groups, especially among stillbirths, and there was no difference in the numbers of such cells found in Downs syndrome patients in comparison with the controls.

Haemopoietic growth factors significantly improve the mitotic index and chromosome quality in cytogenetic cultures of myeloid neoplasia

Haemopoietic growth factors stimulate bone marrow cell division, differentiation, and survival in vivo. We have investigated the use of recombinant human haemopoietic growth factors in vitro to improve cytogenetic cultures. Using a combination of granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, stem cell factor, and interleukin‐3, we developed an additive for bone marrow cultures intended to stimulate myeloid cell growth. Sixty‐seven paired parallel cultures were analyzed, of which 50 were abnormal. The growth factor (GF) cultures showed a median four‐ to five‐fold increase in mitotic index (MI) (P < 0.0001). In addition, the chromosome morphology was significantly improved in the GF cultures with a median increase in United Kingdom National External Quality Assessment Scheme quality score of 1.25 points (P < 0.0001). There was no statistically significant difference in the number of abnormal cells between the two culture methods. The combination of higher MI and improved chromosome quality substantially reduces the time required to process a case; furthermore, the GF medium is cheaper than the medium with which it was compared. This method is suitable for both diagnostic and follow‐up cytogenetic analysis of acute and chronic myeloid neoplasia and is particularly useful for poorly cellular marrow samples or blood samples that would be expected to fail on standard culture. The use of this method has enabled substantial improvements in work efficiency in our oncology cytogenetic laboratory and reduced average reporting times from 9.0 days (2004/5) to 7.1 days (2005/6), despite a 6% increase in sample numbers.