Comparison of metaphase and interphase FISH monitoring of minimal residual disease with MLL gene probe: case study of AML with t(9;11).

The place of FISH in the monitoring of minimal residual disease (MRD) is yet to be fully characterised. Routine bone marrow cytogenetics at diagnosis in a 22 year old patient with acute myeloid leukemia FAB type M5 detected a translocation t(9;11)(p22;q23). We report our investigations to assess residual levels of translocation using a FISH probe designed to detect a gene split by the translocation. We used MLL (Oncor), a probe which spans the MLL gene at 11q23, in both metaphase and interphase preparations. At diagnosis, metaphase FISH showed 3 distinct cell lines-normal with 2 signals, abnormal with 3 signals and abnormal with 2 signals, while interphase FISH showed only 2 cell lines, one with 2 signals (which could be normal or abnormal) and one with 3 signals (split MLL). Following treatment, with the patient in clinical remission, 7 further cytogenetic analyses and 2 further FISH analyses were compared. Our results suggest that monitoring of the t(9;11) by metaphase FISH is feasible and straightforward compared to cytogenetics but interphase FISH may be problematic.     

A second nonrandom translocation, der(16)t(1;16)(q21;q13), in Ewing sarcoma and peripheral neuroectodermal tumor

The majority of Ewing sarcomas and peripheral neuroectodermal tumors (PNET) that have been karyotyped contain a specific translocation, t(11;22)(q23;q11). We report here a second nonrandom translocation, der(16)t(1;16)(q21;q13), in 2 of 20 cases of Ewing sarcoma (seven previously unreported) and 2 of 7 cases of PNET (all previously unreported). All cases with this translocation also contained the t(11;22). Comparison of C-banding patterns in tumor and peripheral lymphocyte karyotypes in one case indicated that the likely breakpoints were 1q21 and 16q13. The presence of this translocation in cell lines will enable further investigation of the molecular events important in the pathogenesis of Ewing sarcoma and PNET.     

ABL/BCR gene variant with two-step mechanism: Unusual localization and rare/novel chromosomal rearrangements in CML patients

Aims: Variant translocations involving 9q, 22q and at least one additional genomic locus occur in 5-10% of the patients with chronic myeloid leukemia (CML). The mechanisms for the formation of these variant translocations are not fully characterized. Here we report CML cases presenting a variant translocation indicating two-step mechanism with rare/novel chromosomal rearrangement. Methods: Karyotype analysis was performed on metaphases obtained through short-term cultures of bone marrow and blood. Detection of BCR-ABL fusion gene was performed using dual-color dual-fusion (D-FISH) and extra signal (ES) translocation probes. BAC-FISH was also carried out. Results: In Patient 1, the third partner chromosome was der(11)(p15) with a 2F2G1R signal pattern, which is an unusual signal pattern with the two-step mechanism. Patients 2 and 3 showed typical positive (2F1G1R) signal pattern. In Patient 2, both the chromosome 22s were involved in variant formation. The second fusion was observed below the BCR gene of the second homologue. In Patient 3 the third chromosome was der(13)(q14). The fourth patient showed a variant pattern with BCR/ABL-ES probe involving der(X)(q13) region. Conclusion: The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. In each case with variants, further studies with FISH, BAC-FISH or more advanced technique such as microarray should be performed. Future studies should be performed to confirm the presence of true breakpoint hot spots and assess their implications in CML with variant Ph.     

Patterns of BCR/ABL Gene Rearrangements in Chronic Myeloid Leukemia with Complex t(9;22) Using Fluorescence In Situ Hybridization (FISH)

Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation t(9;22)(q34;q11.2) which fuses the ABL1 oncogene on chromosome 9 with the BCR gene on chromosome 22. It is the BCR/ABL protein that drives the neoplasm and the ABL/BCR is not necessary for the disease. In the majority of CML cases, the BCR/ABL fusion gene is cytogenetically recognizable as a small derivative chromosome 22(der 22), which is known as the Philadelphia (Ph) chromosome. However, approximately 2-10% of patients with CML involve cryptic or complex variant translocations with deletions on the der(9) and/or der(22) occuring in roughly 10-15% of CML cases. Fluorescence in situ hybridization (FISH) analysis can help identify deletions and complex or cryptic rearrangements. Various BCR/ABL FISH probes are available, which include dual color single fusion, dual color extra signal (ES), dual color dual fusion and tri color dual fusion probes. To test the utility of these probes, six patients diagnosed with CML carrying different complex variant Ph translocations were studied by G-banding and FISH analysis using the BCR/ABL ES, BCR/ABL dual color dual fusion, and BCR/ABL tricolor probes. There are differences among the probes in their ability to detect variant rearrangements, with or without accompanying chromoso me 9 and/or 22 deletions, and low level disease.     

Characterization of cryptic rearrangements, deletion, complex variants of PML, RARA in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by a reciprocal translocation t(15;17)(q22;q21) leading to the disruption of Promyelocytic leukemia (PML) and Retionic Acid Receptor Alpha (RARA) followed by reciprocal PML-RARA fusion in 90% of the cases. Fluorescence in situ hybridization (FISH) has overcome the hurdles of unavailability of abnormal and/or lack of metaphase cells, and detection of cryptic, submicroscopic rearrangements. In the present study, besides diagnostic approach we sought to analyze these cases for identification and characterization of cryptic rearrangements, deletion variants and unknown RARA translocation variants by application of D-FISH and RARA break-apart probe strategy on interphase and metaphase cells in a large series of 200 cases of APL. Forty cases (20%) had atypical PML-RARA and/or RARA variants. D-FISH with PML/RARA probe helped identification of RARA insertion to PML. By application of D-FISH on metaphase cells, we documented that translocation of 15 to 17 leads to 17q deletion which results in loss of reciprocal fusion and/or residual RARA on der(17). Among the complex variants of t(15;17), PML-RARA fusion followed by residual RARA insertion closed to PML-RARA on der(15) was unique and unusual. FISH with break-apart RARA probe on metaphase cells was found to be a very efficient strategy to detect unknown RARA variant translocations like t(11;17)(q23;q21), t(11;17)(q13;q21) and t(2;17)(p21;q21). These findings proved that D-FISH and break-apart probe strategy has potential to detect primary as well as secondary additional aberrations of PML, RARA and other additional loci. The long-term clinical follow-up is essential to evaluate the clinical importance of these findings.     

Meiotic studies of a human male carrier of the common translocation, t(11;22), suggests postzygotic selection rather than preferential 3:1 MI segregation as the cause of liveborn offspring with an unbalanced translocation

The t(11;22)(q23;q11) translocation is the only non-Robertsonian rearrangement for which there are a large number of unrelated families, apparently with the same breakpoints. These families most often have been ascertained through an abnormal child with the karyotype 47,XX or XY, +der(22) t(11;22)(q23;q11). To explain the high incidence of 3:1 segregants, rarely seen in offspring of carriers of other reciprocal translocations, a number of theoretical models have been suggested. We have used both electron microscope analysis of the synaptonemal complex (SC) and dual-color FISH to investigate the meiotic chromosome behavior in a male carrier of the translocation who has the karyotype 46,XY, t(11;22)(q23;q11). Chromosome synapsis, first-meiotic chiasma configuration, and segregation behavior of this translocation have been analyzed directly. Examination of SCs by electron microscopy showed pachytene-cross formation in 49/50 nuclei. Approximately 50% (26/50) revealed a classical fully synapsed quadrivalent. A proportion of these (10/26), however, showed some central asymmetry, suggesting heterologous synapsis. The remaining cells appeared to have incomplete synapsis. FISH analysis showed only quadrivalents in all 100 metaphase I nuclei. The chiasma frequency was increased within the interstitial segments, in comparison with the same region in normal bivalents. All types of segregation category were found in metaphase II nuclei. There was no indication of preferential 3:1 anaphase I segregation. We conclude that the +der(22) constitution in offspring of carriers of t(11;22)(q23;q11) is not likely to be due to meiotic 3:1 segregation being especially common. Rather, the +der(22) constitution is more likely to be the result of postzygotic selection against other unbalanced karyotypes.     

Emanuel syndrome due to unusual segregation of paternal origin

Emanuel syndrome is an inherited chromosomal abnormality resulting from 3:1 meiotic segregation from parental balanced translocation carrier t(11;22)(q23;q11), mostly of maternal origin. It is characterized by mental retardation, microcephaly, preauricular tag or sinus, ear anomalies, cleft or high arched palate, micrognathia, congenital heart diseases, kidney abnormalities, structural brain anomalies and genital anomalies in male. Here in, we describe a female patient with supernumerary der(22) syndrome (Emanuel syndrome) due to balanced translocation carrier father t(11;22) (q23;q11). She was mentally and physically disabled and had most of the craniofacial dysmorphism of this syndrome. Our patient had cleft palate, maldeveloped corpus callosum and hind brain with normal internal organs. Additionally, arachnodactyly, hyperextensibility of hand joints, abnormal deep palmar and finger creases, extra finger creases and bilateral talipus were evident and not previously described with this syndrome. Cytogenetic analysis and FISH documented that the patient had both translocation chromosomes plus an additional copy of der(22) with karyotyping: 47,XX,t(11; 22)(q23;q11),+der(22)t(11;22)(q23;q11). We postulated that this rare chromosomal complement can arise from; 2:2 segregation in the first meiotic division of the balanced translocation father followed by non-disjunction at meiosis II in the balanced spermatocyte.     

Primitive neuroectodermal tumor of the kidney. A report of two cases diagnosed by fine needle aspiration cytology

BACKGROUND: Primitive neurocetodermal tumors (PNETs) constitute a family of neoplasms of presumed neuroectrodermal origin most often presenting as bone or soft tissue masses. There are very few reported cases of PNET of the kidney and none diagnosed by fine needle aspiration cytology (FNAC), to the best of our knowledge, in the world literature. We present two cases of renal PNET diagnosed on cytology. CASES: Two patients with renal masses were diagnosed as having PNET on FNAC. Cytologically the tumors showed a dispersed population of malignant small round cells with focal rosette formation and perivascular arrangement of tumor cells. Immunohistochemistry on the cell blocks in both cases showed strong membrane positivity for CD99 (MIC2). Cytogenetic studies in both cases showed the characteristic t(11;22)(q24;q12) translocation, with additional chromosomal abnormalities in case 2. CONCLUSION: PNET of the kidney is a distinct entity and can be diagnosed on fine needle aspiration smears and confirmed with immunohistochemistry and cytogenetic studies. A diagnosis of PNET must be included in the differential diagnosis of renal masses in adolescents and young adults.     

Two cases of partial trisomy 10q syndrome due to a familial 10;20 translocation

We describe an eleven day-old boy and his first degree double cousin who both have distal trisomy 10q syndrome. Their cytogenetic analysis using GTG-banding showed an unbalanced translocation 46, XY, -20, +der(20), t(10;20)(q22.3, p11) mat and 46, XX, -20, +der(20), t(10;20)(q22.3, p11) mat. The translocation was confirmed by FISH. We have found balanced translocation t(10;20)(q22.3; p11) with cytogenetic and FISH studies in the mothers and maternal grandfather of these children. Our cases had typical craniofacial and visceral anomalies of this syndrome. However case 1 had an agenesia of corpus callosum which was not previously described and case 2 had hypertrophied cardiomyopathy and cliteromegaly which were previously described as rare anomalies for this syndrome.     

Infertile couples with Robertsonian translocations: preimplantation genetic analysis of embryos reveals chaotic cleavage divisions

Preimplantation genetic diagnosis (PGD) may provide a feasible option for some Robertsonian translocation carriers who experience severe difficulty in achieving a normal pregnancy. We report on five PGD cycles for two such couples, 45,XY,der(13;14)(q10:q10) and 45,XX,der(13;21)(q10;q10), carried out by biopsy of two cells from day 3 post-insemination embryos generated by in vitro fertilisation. Locus-specific YAC probes for chromosomes 13, 14 and 21 were used to detect the chromosomes involved in the translocation using multicolour FISH. Three embryos transfers were carried out (two single embryo transfers and one double transfer) but no clinical pregnancies were established. In two cycles no embryos were transferred as all those biopsied were chromosomally abnormal. Combined results from both couples show 13% (6/45) of embryos analysed were normal for the translocation chromosomes and 87% (39/45) were chromosomally abnormal; these were categorised as 36% aneuploid or aneuploid mosaic and 51% chaotic where the chromosome constitution varied randomly from cell to cell. This suggests two factors may be acting to reduce fertility in these couples; the aneuploid segregation of the parental Robertsonian translocation and also a post-zygotic factor leading to uncontrolled chromosome distribution in early cleavage stages in an exceptionally high proportion of embryos. Received: 24 September 1997 / Accepted: 22 October 1997     

Cytogenetic and molecular characterization of eight new reciprocal translocations in the pig species. Estimation of their incidence in French populations

Eight new cases of reciprocal translocation in the domestic pig are described. All the rearrangements were highlighted using GTG banding techniques. Chromosome painting experiments were also carried out to confirm the proposed hypotheses and to accurately locate the breakpoints. Three translocations, rcp(4;6)(q21;p14), rcp(2;6)(p17;q27) and rcp(5;17)(p12;q13) were found in boars siring small litters (8.3 and 7.4 piglets born alive per litter, on average, for translocations 2/6 and 5/17, respectively). The remaining five, rcp(5;8)(p12;q21), rcp(15;17)(q24;q21), rcp(7;8)(q24;p21), rcp(5;8)(p11;p23) and rcp(3;15)(q27;q13) were identified in young boars controlled before entering reproduction. A decrease in prolificacy of 22% was estimated for the 3/15 translocation after reproduction of the boar carrier. A parental origin by inheritance of the translocation was established for the (5;8)(p11;p23) translocation. The overall incidence of reciprocal translocations in the French pig populations over the 2000/2001 period was estimated (0.34%).     

Automated fluorescent in situ hybridization (FISH) analysis of t(9;22)(q34;q11) in interphase nuclei.

BACKGROUND: For chronic myeloid leukemia, the FISH detection of t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes is an alternative method to bone marrow karyotyping for monitoring treatment. With automation, several drawbacks of manual analysis may be circumvented. In this article, the capabilities of a commercially available automated image acquisition and analysis system were determined by detecting t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes. METHODS: Three peripheral blood samples of normal adults, 21 samples of CML patients, and one sample of a t(9;22)(q34;q11) positive cell-line were used. RESULTS: Single nuclei with correctly detected signals amounted to 99.6% of nuclei analyzed after exclusion of overlapping nuclei and nuclei with incorrect signal detection. A cut-off value of 0.84 mum was defined to discriminate between translocation positive and negative nuclei based on the shortest distance between signals. Using this value, the false positive rate of the automated analysis for negative samples was 7.0%, whereas that of the manual analysis was 5.8%. Automated and manual results showed strong correlation (R(2) = 0.985), the mean difference of results was only 3.7%. CONCLUSIONS: A reliable and objective automated analysis of large numbers of cells is possible, avoiding interobserver variability and producing statistically more accurate results than manual evaluation.     

Fine mapping of the constitutional translocation t(11;22)(q23;q11)

Translocation t(11;22)(q23;q11) is the most common constitutional reciprocal translocation in man. Balanced carriers are phenotypically normal, except for decreased fertility, an increased spontaneous abortion rate and a possible predisposition to breast cancer in some families. Here, we report the high resolution mapping of the t(11;22)(q23;q11) breakpoint. We have localised the breakpoint, by using fluorescence in situ hybidisation (FISH) walking, to a region between D11S1340 and WI-8564 on chromosome 11, and D22S134 and D22S264 on chromosome 22. We report the isolation of a bacterial artificial chromosome (BAC) clone spanning the breakpoint in 11q23. We have narrowed down the breakpoint to an 80-kb sequenced region on chromosome 11 and FISH analysis has revealed a variation of the breakpoint position between patients. In 22q11, we have sequenced two BACs (BAC2280L11 and BAC41C4) apparently mapping to the region; these contain low copy repeats (LCRs). Southern blot analysis with probes from BAC2280L11 has revealed different patterns between normal controls and translocation carriers, indicating that sequences similar/identical to these probes flank the translocation breakpoint. The occurrence of LCRs has previously been associated with genomic instability and "unclonable" regions. Hence, the presence of such repeats renders standard translocation breakpoint cloning techniques ineffective. Thus, we have used high resolution fiber-FISH to study this region in normal and translocation cases by using probes from 22q11, LCRs and 11q23. We demonstrate that the LCR containing the gap in 22q11 is probably substantially larger than the previous estimates of 100 kb. Using fiber-FISH, we have localised the breakpoint in 22q11 to approximately 20-40 kb from the centromeric border of the LCR (i.e. the telomeric end of AC006547) and have confirmed the breakpoint position on 11q23.     

A Palindrome-Mediated Recurrent Translocation with 3:1 Meiotic Nondisjunction: The t(8;22)(q24.13;q11.21)

Palindrome-mediated genomic instability has been associated with chromosomal translocations, including the recurrent t(11;22)(q23;q11). We report a syndrome characterized by extremity anomalies, mild dysmorphia, and intellectual impairment caused by 3:1 meiotic segregation of a previously unrecognized recurrent palindrome-mediated rearrangement, the t(8;22)(q24.13;q11.21). There are at least ten prior reports of this translocation, and nearly identical PATRR8 and PATRR22 breakpoints were validated in several of these published cases. PCR analysis of sperm DNA from healthy males indicates that the t(8;22) arises de novo during gametogenesis in some, but not all, individuals. Furthermore, demonstration that de novo PATRR8-to-PATRR11 translocations occur in sperm suggests that palindrome-mediated translocation is a universal mechanism producing chromosomal rearrangements.     

Characterization of an interstitial deletion del(13)(q22q32) using microdissection and sequential FISH and G-banding

The objective of this study was to delineate a chromosome 13 abnormality and establish its clinical correlation by using molecular cytogenetics procedures. A newborn boy presented with clinical findings, including mild symmetric intrauterine growth retardation (IUGR), small ears with thickened helices, a scalp lesion, short fifth fingers, missing toes, and talipes equinovarus. Routine G-banding of cultured peripheral blood cells revealed that the patient had one abnormal and shortened chromosome 13, but uncertainty remained as to whether the abnormality was the result of an interstitial deletion or a translocation. Thirteen copies of G-banded abnormal chromosomes 13 were isolated with microdissection and amplified with PCR using degenerate oligonucleotide primers. Fluorescence in situ hybridization (FISH) of the PCR product to normal metaphases showed one pair of acrocentrics hybridized, more or less uniformly, along the length of the long arm with an unhybridized gap in the distal region, indicative of an interstitial deletion. Sequential FISH and G-banding of the same chromosome preparations conclusively demonstrated that the deleted segment was 13q22-q32. Four cases of del(13)(q22q32) have been previously reported. The common findings in all five cases, including the present one, are psychomotor and growth retardation, as well as hand and foot anomalies.     

X-inactivation patterns in lymphocytes and skin fibroblasts of three cases of X-autosome translocations with abnormal phenotypes

Summary X-inactivation patterns were studied by replication analyses both in lymphocytes and skin fibroblasts of two patients carrying balanced X-autosome translocations, t(X;10)-(pter;q11) and t(X;17)(q11;q11), and one patient with an unbalanced translocation t(X;22)(p21;q11). Preferential late replication of the normal X chromosome was found in lymphocytes of both patients carrying balanced translocations and in skin fibroblasts of the patient carrying the translocation t(X;17). However, skin fibroblasts of the patient with a translocation t(X;10) showed preferential late replication of the abnormal der(X) chromosome with no spreading of late replication to the autosomal segment. In the case of unbalanced translocation t(X;22) there was preferential late replication of the der(X) chromosome both in lymphocytes and skin fibroblasts. The abnormal phenotype of the patients is discussed in relation to the observed X-inactivation patterns and the variability of the patterns in different tissues.