Kavita S. Reddy

A FISH Study of Variant Philadelphia Rearrangements

A total of 39 variant Philadelphia (Ph) translocations were studied by fluorescence in situ hybridization (FISH) using MBCR/ABL, mBCR/ABL, or DBCR/ABL probes. Seven cases did not have a BCR/ABL fusion signal. Of a total of 32 fusion-positive cases, 5 were simple variants involving chromosome 22 and another chromosome apart from chromosome 9; 23 were complex variants involving chromosomes 22, 9, and a third chromosome (18 cases), or 22, 9, and two other chromosomes (4 cases). Masked Ph rearrangements were detected in 4 cases. One case was a Ph chromosome mimic. Fluorescence in situ hybridization has become a widely used method for studying Ph rearrangements. The latest probe that is being used is the DBCR/ABL (double reciprocal BCR/ABL signals). The expected pattern for this probe is one green ABL signal (1G) on the normal 9, one red BCR signal (1R) on the normal 22, and two fusion signals, BCR/ABL and ABL/BCR (2F), on a derivative 22 and a derivative 9, respectively. Deviant patterns from 1G1R2F, and sometimes 1G1R2F, were indicative of a variant, as long as there was a fusion signal. However, in interphase analysis, it is not possible to visualize a variant rearrangement, and when a deviant pattern involving at least one fusion signal is observed, the following possibilities should be contemplated. The different patterns observed in fifteen Ph variants are described. The patterns observed in variants studied with the DBCR/ABL probe were 2G2R1F (40%), 1G1R2F (20%), 1G1R1F (20%), 1G2R1F (13.3%), and 2G1R1F (6.66%). A single mechanism is involved in the formation of each of these patterns. A 2G2R1F, FISH pattern in 6 cases appears to involve a single concerted event of simultaneous breaks on the participating chromosomes followed by mismatched joining. The three cases with 1G1R2F most probably arose by two sequential rearrangements. The 1G1R1F pattern suggests that either the BCR and ABL breakpoints are different, or there are deletions at the breakpoints, because residual signals are not observed. Two independent events appear to be involved in 1G2R1F with a reverse cryptic 9,22 rearrangement as the first event. In one case of 2G1R1F, the plausible explanation is an insertion of ABL next to BCR and either a simultaneous or a sequential translocation with another chromosome.

Subtle overlapping deletions in the terminal region of chromosome 6q24.2-q26: three cases studied using FISH.

Interstitial deletions in the terminal region of chromosome 6 are rare. We describe three new cases with subtle interstitial deletions in the q24-q26 region of the long arm of chromosome 6. The karyotypes were analyzed at a 550 band level. Patient1 is a 9-month-old boy with an interstitial deletion, del(6)(q24.2q25.1), developmental delay, low birth weight, hypotonia, heart murmur, respiratory distress, craniofacial and genital anomalies. This is the first report of a case with deletion del(6)(q24.2q25.1). Patient 2 is a 17-year-old young man with an interstitial deletion del(6)(q25.1q25.3), developmental delay, short stature, mental retardation, autism, head, face, chest, hand and feet anomalies and a history of seizures. For the first time autism was described as a manifestation in 6q deletions. Patient 3 is baby boy with a de novo interstitial deletion, del(6)(q25.1q26), anomalies of the brain, genital organs, limbs and feet. This is the first report of a case with deletion, del(6)(q25.1q26). In all three patients, fluorescence in situ hybridization (FISH) using chromosome 6 painting probe ruled out an insertion. The ESR (6q25.1) and TBP (6q27) probes were used to confirm the breakpoints. Since TBP signal is present in all cases, it confirmed an interstitial deletion proximal to this probe. Patient 1 has a deletion of the ESR locus; Patient 2 and 3 have signals for the ESR locus on both chromosomes 6. Therefore the deletion in Patients 2 and 3 are between ESR and TBP loci distal to that of Patient 1. FISH validated the deletion breakpoints assessed by conventional cytogenetics.

Microdeletion of chromosome sub-band 2q37.3 in two patients with abnormal situs viscerum.

We report on two cases of microdeletion of chromosome sub-band 2q37.3 with abnormal situs viscerum. The first patient had dextrocardia, duodenal and jejunal atresia, and an abdominal hernia. The liver was in the left upper quadrant, stomach in the right upper quadrant. In contrast anema the ascending colon was in the left, and descending colon on the right, with an area of atresia in the mid-jejunum. The second patient had malrotation and malposition of large and small bowel, with most of the bowels positioned above the liver and spleen. There was incomplete rotation of the cecum. The right kidney was malrotated and mal-positioned. The finding of 2q37.3 deletion in both patients implies that a locus or loci involved in the development of normal body situs lies within this chromosome region. Molecular cytogenetic evaluation for a possible 2q37.3 deletion should be considered in patients with abnormal situs viscerum.

Intrachromosomal triplication of 2q11.2-q21 in a severely malformed infant: case report and review of triplications and their possible mechanism.

A female fetus with brain malformations, multicystic kidneys, absence of the right thumb, and a posterior cleft of palate was delivered at 32 weeks of gestation. Cytogenetic studies including FISH showed a novel intrachromosomal triplication of the proximal long arm of chromosome 2 (q11.2-q21), resulting in tetrasomy for this segment. The middle repeat was inverted. At least 11 patients with intrachromosomal triplications have been reported, mostly involving chromosome 15q. The mechanism involved in formation of these rearrangements is compatible with U-type exchange events among three chromatids.

A Philadelphia-Negative Chronic Myeloid Leukemia with a BCR/ABL Fusion Gene on Chromosome 9

A 40-year-old man had chronic myeloid leukemia (CML) and an apparently normal karyotype. Fluorescence in situ hybridization with a BCR/ABL1-S probe, which is formatted to display a BCR/ABL fusion signal on chromosome 22, gave a positive fusion signal on a chromosome 9. Therefore this patient has a BCR/ABL fusion gene on chromosome 9. The BCR/ABL1-D probe, formatted to display a fluorescent signal for both the reciprocal products of a 9/22 rearrangement, gave a positive fusion signal on the derivatives 9 and 22. These findings favor either a cryptic reciprocal exchange between BCR and ABL loci or the reversal of a Philadelphia translocation. An insertion of BCR next to ABL is ruled out. The reverse-transcriptase polymerase chain reaction provided molecular evidence that a typical CML chimeric product resulting from a fusion of BCR exon 2 with C-ABL exon II, a2b2, is present.

Intrachromosomal triplications: molecular cytogenetic and clinical studies

A newborn boy had meconium aspiration syndrome, hypospadias, a supernumerary digit on the left hand, hyperbilirubinemia, a fractured right clavicle, osteopenia, liver calcification, and mild pulmonary hyperplasia. Cytogenetic studies showed a chromosome 13 with additional material in 33% of the metaphases. The add(13) was considered to be a probable duplication of 13q12q22. The 13 paint probe hybridized to the add(13) from end to end. Fluorescence in situ hybridization (FISH) studies using retinoblastoma probe (RB)‐1 that maps to 13q14 and D13S585 that maps to 13q32‐q33 gave one signal for RB and three signals for D13S585. The pattern of the three signals from the 13q32q33 region and the G‐banding pattern was best explained as a triplication of 13q22q33, with an inverted middle repeat resulting in tetrasomy for this segment. Mosaicism was confirmed by FISH using a D13S585 probe on a buccal smear. Three triplications detected in our laboratory were compared – 13q22q33, 15q11q13, and 2q11.2q21. FISH was critical in identifying triplications 13q22q33 and 15q11q13. The hybridization pattern also indicated an inverted middle repeat. We conclude that intrachromosomal triplications may be more prevalent than previously assumed and they probably share a common mechanism in their formation. When the G‐bands do not correspond exactly to a duplication or to a tandem triplication, an important consideration is that the majority of triplications have an inverted middle repeat. Triplications can be mistaken for duplications. Therefore, in assessing duplications, FISH confirmation is recommended.

Jumping Translocations Involving Chromosome 1q in a Patient with Crohn Disease and Acute Monocytic Leukemia: A Review of the Literature on Jumping Translocations in Hematological Malignancies and Crohn Disease

A 36-year-old man with a 10-year history of Crohn disease (CD) presented with gross hematuria and blasts in his peripheral blood. A chromosome analysis revealed one normal cell and 33 abnormal cells. The stem line was 47,XY,+8. The multiple side lines also had a jumping translocation between chromosome 1q31-32 and 4, 8, 10, 17, and 18 terminal regions. A cytogenetic, morphologic, and immunophenotypic analysis of a bone marrow aspirate and biopsy demonstrated acute myeloid leukemia of monocytic lineage, AML-M5b. In this paper are reviewed (a) the unusual and rare phenomenon of jumping translocations in hematological malignancies and (b) leukemia in CD.

c-myc amplification in a preleukemia patient with trisomy 4 and double minutes: review of the unique coexistence of these two chromosome abnormalities in acute myelogenous leukemia.

Cytogenetic analysis of the bone marrow from a woman with preleukemia showed an aberrant clone with trisomy 4, double minutes, and a translocation t(8;9)(q21;q34). Fluorescence in situ hybridization (FISH) demonstrated that the double minutes were c-myc amplifications. A review of six cases in the literature and the present case with trisomy 4 and double minutes showed a preponderance of females and that the patients were mostly elderly. The acute myelogenous leukemia (AML) in these patients was either FAB subtype M2 or M4. In two out of seven cases, the double minutes were c-myc amplicons. The patients responded to treatment and there was karyotypic normalization during remission. There was no strong evidence of exposure to genotoxic agents.

Mosaic tetrasomy 8q: Inverted duplication of 8q23.3qter in an analphoid marker

We observed an analphoid marker chromosome stable through cell division in a 16-year-old girl with developmental delay, short stature, limb contractures, and ovaries containing multiple cysts. She also developed myasthenia gravis at 15 years. The marker chromosome, present in 75% of metaphases (and in 90% of transformed lymphoblastoid cells), was C-band negative, and had no pan alpha-satellite sequences detectable by fluorescence in situ hybridization (FISH). The 8q origin of the marker was determined by use of subtelomeric probes and was confirmed by chromosome 8 painting probes. The marker was shown to be an inversion duplication of 8q when subtelomeric, telomeric, and c-myc FISH probes hybridized to both ends of the marker. The karyotype was 47,XX,+inv dup(8)(qter--> q23.3::q23.3-->[neocen]-->qter), resulting in tetrasomy for 8q23.3qter. The parents had normal karyotypes. Centromeric proteins CENP-C and CENP-E were present, but alpha associated centromere protein CENP-B was absent at a position defining a neocentromere.

Segmental amplification of 11q23 region identified by flourescence in situ hybridization in four patients with myeloid disorders: a review

Four cases with a segmental amplification of 11q23 region were detected by FISH. The amplification was either contiguous amplification on chromosome 11, or multiple markers involving the 11q23 region. The markers were derivative chromosomes, or isochromosomes. Amplification of 11q23 region was associated with complex karyotypes at the time of diagnosis or following treatment in secondary leukemias. Three were AML cases belonging to either AML-M5a or AML-1 subtypes and one was a myeloproliferative disorder. These cases were resistant to treatment. Conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) studies using MLL, 11 painting, or 11 centromere probes ascertained the segmental amplification. Since the patients did not respond to treatment the amplification of gene or genes that map to 11q23 may be responsible for the unfavorable prognosis. Hence, this type of amplifications could have clinical significance.