Physical localisation of the breakpoints of a constitutional translocation t(5;6)(q21;q21) in a child with bilateral Wilms'' tumour.

A 6 month old boy presented with bilateral Wilms' tumour. Cytogenetic analysis of the lymphocytes from the patient showed a de novo balanced translocation t(5;6)(q21;q21), which was also present in the tumour material as the sole cytogenetic abnormality. To facilitate the identification of the translocation breakpoints, we have established a lymphoblastoid cell line (MA214L) from the patient which maintains the translocation in culture. We have used Genethon microsatellite markers as sequence tagged sites (STSs) to isolate yeast artificial chromosome (YAC) clones to 5q and 6q from human genomic libraries. Using fluorescence in situ hybridisation (FISH) on metaphase preparations of MA214L, we have physically defined the translocation breakpoints between YAC clones on each chromosome arm. The genetic distance separating the flanking YACs on 6q21 is 3 cM, while that on 5q21 is 4 cM. To date this is the first report of these chromosomal regions being implicated in Wilms' tumourigenesis.     

Chromosome 18 breakpoint in t(11;18)(q21;q21) translocation associated with MALT lymphoma is proximal to BCL2 and distal to DCC

The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnormality in a subset of extranodal marginal zone B‐cell lymphoma, a low‐grade lymphoma of mucosa‐associated lymphoid tissue (MALT). Neither the 11q21 nor the 18q21 breakpoints have been characterized by molecular genetic analysis. As a prelude to isolation of the gene(s) involved in this translocation, we have mapped the 18q21 breakpoint region by fluorescence in situ hybridization (FISH) of YAC and PAC clones. We mapped 37 YACs assigned to a 29‐cM region within the chromosomal band 18q21. Using nine of these YACs in single‐ and/or dual‐color FISH to analyze three cases of MALT lymphomas with the t(11;18)(q21;q21) translocation, we localized the breakpoints within a 1.6‐Mb nonchimeric YAC (938E1). This YAC is useful for the detection of the translocation in metaphase and in interphase cells. A nonchimeric YAC contig of an 8‐cM region around the breakpoint comprising nine YACs and a PAC contig of YAC 938E1 were constructed, which enabled the refinement of the breakpoint region in the proximal region of the YAC within a <820‐kb segment. This breakpoint is proximal to the BCL2 locus and distal to DCC and DPC4 loci in chromosomal band 18q21. Genes Chromosomes Cancer 24:156–159, 1999. © 1999 Wiley‐Liss, Inc.     

Molecular cytogenetics localizes two new breakpoints on 11q23.3 and 21q11.2 in myelodysplastic syndrome with t(11;21) translocation.

Translocation t(11;21)(q24;q11.2) is a rare but recurrent chromosomal abnormality associated with myelodysplastic syndrome (MDS) that until now has not been characterized at the molecular level. We report here results of a molecular cytogenetic analysis of this translocation in a patient with refractory anemia. Using FISH with a panel of 11q and 21q cosmid/YAC probes, we localized the chromosome 11 breakpoint at q23.3 in a region flanked by CP-921G9 and CP-939H3 YACs, distal to the HRX/MLL locus frequently involved in acute leukemias. The chromosome 21 breakpoint was mapped in a 800-kb fragment inserted into the CP-145E3 YAC at 21q11.2, proximal to the AML1 gene. It is noteworthy that in all four cases with a t(11;21) reported until now, a second der(11)t(11;21) and loss of normal chromosome 11 could be observed either at diagnosis or during the course of the disease. Since in our case heteromorphism was detected by FISH on the centromeric region of the two der(11), the second der(11) chromosome could be the result of a mitotic recombination that had occurred on the long arm of chromosome 11, rather than of duplication of the original der(11). Constancy of secondary karyotypic changes resulting in an extra copy of the putative chimeric gene at der(11), loss of 11 qter sequences, and partial trisomy 21 suggest that neoplastic progression of MDS cases with a t(11;21) may be driven by the same mechanism(s).     

Molecular characterization of the breakpoint region associated with a constitutional t(2;15)(q34;q26) in a patient with multiple myeloma

The molecular cloning of the translocation breakpoints from constitutional chromosome rearrangements in patients with a variety of human diseases has consistently led to the isolation of genes important in the development of the phenotype. We used fluorescence in situ hybridization (FISH) to analyze the breakpoint region of a constitutional chromosome translocation involving regions 2q34 and 15q26 observed in a patient with multiple myeloma (MM), a malignant disorder of plasma cells secreting monoclonal immunoglobulin. FISH analysis of this rearrangement showed that the chromosome 2-specific yeast artificial chromosome (YAC) 914E7 and the chromosome 15-specific YAC 757H6 span the translocation breakpoints, respectively. In order to characterize the location of the breakpoints further, somatic cell hybrids were constructed between mouse NIH3T3 cells and t(2;15)-bearing lymphoblastoid cells. Using these somatic cell hybrids, we have shown that the breakpoint on chromosome 2 lies between D2S3007 and D2S3004 and the chromosome 15 breakpoint lies between D15S107 and WI5967 (D15S836). YAC fragmentation has been used to define a 350 kb region containing the 15q26 breakpoint.     

Fish mapping of a translocation breakpoint at 6q21 (or q22) in a patient with heterotaxia

Summary Heterotaxia is a congenital lateralization defect of visceral organs. As several single-genes that act on the formation of left-right asymmetry during embryogenesis have been identified in animals, a defect in the similar system may play a role in heterotaxia in man. We previously reported a Japanese girl with heterotaxia associated with ade novo balanced translocation (6;18)(q21 or q22;q21.3 or q22). In the present study, based on a hypothesis that one of the putative situs-determining genes is disrupted at a breakpoint of the translocation, we first isolated a yeast artificial chromosome (YAC) clone covering a breakpoint, 6q21 (or q22) of the translocation. Then, using STSs mapped on the YAC, we isolated bacterial artificial chromosome (BAC) clones spanning the breakpoint. FISH analysis using the BAC clones as probes revealed that the breakpoint is confined to a segment between two STS loci, WI-4066 and the CHLC.GATA6B06.192, within a genetic distance of 1.4 cM. The human connexin43 gene was not disrupted in our patient, although mutations of this gene have been reported in patients with complex heart disease and heterotaxia. The molecular localization of the translocation breakpoint in our patient may contribute to the positional cloning of a putative heterotaxia gene.     

Cryptic subtelomeric translocation t(2;16)(q37;q24) segregating in a family with unexplained stillbirths and a dysmorphic, slightly retarded child

We here describe a submicroscopic translocation affecting the subtelomeric regions of chromosomes 2q and 16q, and segregating in a family with stillbirths, early pregnancy losses, and two dysmorphic and slightly retarded babies. FISH analysis showed a 46,XY,der(2)t(2;16)(q37.3;q24.3) in the propositus, and a balanced t(2;16) in his mother, her conceptus and maternal grandfather. FISH with YACs and BACs made it possible to map the 2q37 breakpoint precisely between the regions covered by y952E1 and y746H1, and the 16q breakpoint between the regions encompassed by bA 309g16 and bA 533d19. The contribution of 2q37.3 monosomy and 16q24.3 trisomy to the proband's phenotype is compared with that in reported patients with similar imbalances of either chromosome.     

t(15;21)(q15;q22.1)pat resulting in partial trisomy and partial monosomy of chromosomes 15 and 21 in two offspring

Two sibs, carriers of unbalanced products of the translocation t(15;21)(q15;q22.1)pat, are described. The sister had Prader-Willi syndrome due to deletion 15 (pter > q15) and partial trisomy 21 (pter > q22.1); her brother had partial trisomy 15 (pter > q15) and partial monosomy 21 (pter > q22.1). The translocation breakpoint on chromosome 21 was located proximal to the SOD1 gene, within a region of 4.0 cM (2.3 Mb) between the loci D21S217 and D21S213. The correlations between the clinical presentation and the molecular findings of the two sibs are discussed in relation to other patients with partial trisomy and monosomy 21. © 1996 Wiley-Liss, Inc.     

DNA sequences of chromosome 21-specific YAC detect the t(8;21) breakpoint of acute myelogenous leukemia.

The t(8;21)(q22;q22) is a nonrandom translocation specifically marking blasts of acute myelogenous leukemia (AML) with undifferentiated phenotype. The breakpoint on chromosome 21 involved by this rearrangement has been precisely localized relative to cloned DNA markers by physical and genetic linkage analysis enabling the use of positional cloning for its isolation. Yeast artificial chromosome (YAC) clones for loci proximal (D21S65) and distal (ERG) to the (21q22) breakpoint have been developed and their chromosome 21 origin and location relative to the breakpoint has been established. By using in situ hybridization analysis, a 240 kb YAC clone for the D21S65 locus clearly identified both derivative chromosomes of the (8;21) translocation in metaphase spreads of leukemia blasts with the rearrangement. The characterization of the DNA sequences contained in this 240 kb YAC can reveal the functional consequences of their derangement in leukemia with abnormalities of the (21q22) region.     

Double partial trisomy 9q34.1-->qter and 21pter-->q22.11: FISH and clinical findings.

We describe a patient with double trisomy 9q34.1-->qter and 21pter-->q22.1 resulting from 3:1 segregation of a maternal balanced translocation. The patient shows a clinical syndrome similar to that observed in patients with duplication of the chromosome 9q distal region, while no signs of trisomy 21 were observed. The use of high resolution banding and FISH were of fundamental importance for the cytogenetic diagnosis and for definition of the breakpoints on both chromosomes 9 and 21.     

Analysis of a familial three way translocation involving chromosomes 3q, 6q, and 15q by high resolution banding and fluorescent in situ hybridisation (FISH) shows two different unbalanced karyotypes in sibs.

We report on a familial three way translocation involving chromosomes 3, 6, and 15 identified by prometaphase banding and fluorescence in situ hybridisation (FISH). Two mentally retarded sibs with different phenotypic abnormalities, their phenotypically normal sister and mother, and two fetuses of the phenotypically normal sister were analysed. The terminal regions of chromosomes 3q, 6q, and 15q were involved in a reciprocal translocation, in addition to a paracentric inversion of the derivative chromosome 15. Conventional cytogenetic studies with high resolution GTG banding did not resolve this rearrangement. FISH using whole chromosome paints (WCPs) identified the chromosomal regions involved, except the aberrant region of 3q, which was undetectable with these probes. Investigation of this region with the subtelomeric FISH probe D3S1445/D3S1446 showed a balanced karyotype, 46,XX,t(3;15;6) (q29;q26.1;q26), inv der(15) (q15.1q26.1) in two adult females and one fetus. It was unbalanced in two sibs, showing two different types of unbalanced translocation resulting in partial trisomy 3q in combination with partial monosomy 6q in one patient and partial trisomy 15q with partial monosomy 6q in the other patient and one fetus. These represent apparently new chromosomal phenotypes.     

Physical mapping of the chromosome 7 breakpoint region in an SLOS patient with t(7;20) (q32.1;q13.2)

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder characterized by multiple congenital anomalies and mental retardation. SLOS has an associated defect in cholesterol biosynthesis, but the molecular genetic basis of this condition has not yet been elucidated. Previously our group reported a patient with a de novo balanced translocation [t(7;20)(q32.1;q13.2)] fitting the clinical and biochemical profile of SLOS. Employing fluorescence in situ hybridization (FISH), a 1.8 Mb chromosome 7-specific yeast artificial chromosome (YAC) was identified which spanned the translocation breakpoint in the reported patient. The following is an update of the on-going pursuit to physically and genetically map the region further, as well as the establishment of candidate genes in the 7q32.1 breakpoint region. Am. J. Med Genet. 68:279–281, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of a complex chromosome rearrangement involving 6q in a melanoma cell line by chromosome microdissection

Deletion of 6q is one of the most frequent chromosomal alterations in human malignant melanoma. Recently, we used chromosome painting probes of 6p and 6q to study 21 melanoma cell lines. A reciprocal translocation between chromosomes 6q and 17p was detected in one cell line (UACC-930). Upon further characterization of the translocation marker using the micro fluorescence in situ hybridization (FISH) technique, a complex rearrangement including an inversion of 6q and a translocation between the inverted 6q and 17p, [der(6)inv(6)(q16q27)t(6;17)(q26;p13)], was detected. A yeast artificial chromosome (YAC) clone spanning the breakpoint at 6q16 was isolated by the FISH screen. Loss of one or more copies of the YAC clone was also detected in 10 of 12 melanoma cell lines. This result implies that the YAC clone may contain a putative tumor suppressor gene related to the pathogenesis of malignant melanoma. Further characterizations of the breakpoint at 6q16 and molecular cloning breakpoints at 6q27 and 17p13 are in progress.     

Hematologic malignancies with the t(10;11)(p13;q21) have the same molecular event and a variety of morphologic or immunologic phenotypes

Previous studies described the t(10;11)(p13-14;q14-21) as a recurring translocation associated with T-cell acute lymphoblastic leukemia (ALL). This translocation has also been reported in monocytic leukemia or ALL with a very early pre-B phenotype. However, whether these cytogenetically similar translocations involve the same molecular breakpoint is unknown. Using fluorescence in situ hybridization (FISH) with a series of probes on 11q, we mapped the 11q breakpoint of the U937 cell line, which was derived from a patient with diffuse histiocytic lymphoma and was shown by FISH to have the t(10;11)(p13-14;q14-21). Subsequently, we identified a yeast artificial chromosome (YAC) clone, y960g8, that included the breakpoint on 11q. From this YAC, we isolated a P1 clone, P91B1, that was split by the 10;11 translocation. We studied four patients with a t(10;11), one of whom had acute monocytic leukemia (AMoL), one had acute lymphoblastic leukemia (ALL), one had lymphoblastic lymphoma (LBL), and one had granulocytic sarcoma, by using FISH with y960g8 and P91B1. Y960g8 and P91B1 were split by the translocation in each patient. We showed that P91B1 included a recently identified gene, CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene), and that AF10 was also rearranged in each patient by FISH when we used y807b3, which contains the AF10 gene. These findings indicate that hematologic malignant diseases with fusion of AF10 and CALM show various morphologic and immunologic phenotypes, suggesting that this fusion occurs in multipotential or very early precursor cells. Genes Chromosomes Cancer 20:253–259, 1997. © 1997 Wiley-Liss, Inc.     

Balanced X;15 translocation 46,X,t(X;15)(q21;q23) associated with primary amenorrhea

Cytogenetic studies on a woman with primary amenorrhea showed an X;15 translocation, karyotype 46,X,t(X;15)(q21;q23). Fifteen percent of the buccal cells showed a normal-sized sex chromatin body. The normal X chromosome was uniformly inactivated. Many balanced X;15 translocations have been reported; however, breakpoints in our patient differ from those reported previously. This case also supports earlier evidence that ovarian development fails when the breakpoint of the X chromosome is in the region X q13-q25 or q13-q27.     

FISH characterization of the Xq21 breakpoint in a translocation carrier with premature ovarian failure

A panel of ordered YAC clones, isolated using STSs in the Xq13-Xq23 region, was used to characterize by Fluorescent In Situ Hybridization (FISH) the Xq21 breakpoint in a t(X;1)(q21;p34) translocation female with premature ovarian failure. The YAC 949E11 was found to span the breakpoint, but also to join the two non-overlapping YACs 36CB1 and 40AB3, proximal and distal, respectively, to the patient's Xq21 breakpoint.     

Sorting nexin 3 (SNX3) is disrupted in a patient with a translocation t(6;13)(q21;q12) and microcephaly,microphthalmia, ectrodactyly,prognathism (MMEP) phenotype

A patient with microcephaly, microphthalmia, ectrodactyly, and prognathism (MMEP) and mental retardation was previously reported to carry a de novo reciprocal t(6;13)(q21;q12) translocation. In an attempt to identify the presumed causative gene, we mapped the translocation breakpoints using fluorescence in situ hybridisation (FISH). Two overlapping genomic clones crossed the breakpoint on the der(6) chromosome, locating the breakpoint region between D6S1594 and D6S1250. Southern blot analysis allowed us to determine that the sorting nexin 3 gene (SNX3) was disrupted. Using Inverse PCR, we were able to amplify and sequence the der(6) breakpoint region, which exhibited homology to a BAC clone that contained marker D13S250. This clone allowed us to amplify and sequence the der(13) breakpoint region and to determine that no additional rearrangement was present at either breakpoint, nor was another gene disrupted on chromosome 13. Therefore, the translocation was balanced and SNX3 is probably the candidate gene for MMEP in the patient. However, mutation screening by dHPLC and Southern blot analysis of another sporadic case with MMEP failed to detect any point mutations or deletions in the SNX3 coding sequence. Considering the possibility of positional effect, another candidate gene in the vicinity of the der(6) chromosome breakpoint may be responsible for MMEP in the original patient or, just as likely, the MMEP phenotype in the two patients results from genetic heterogeneity.     

Systematic characterisation of disease associated balanced chromosome rearrangements by FISH: cytogenetically and genetically anchored YACs identify microdeletions and candidate regions for mental retardation genes

Disease associated balanced chromosome rearrangements (DBCRs) have been instrumental in the isolation of many disease genes. To facilitate the molecular cytogenetic characterisation of DBCRs, we have generated a set of >1200 non-chimeric, cytogenetically and genetically anchored CEPH YACs, on average one per 3 cM, spaced over the entire human genome. By fluorescence in situ hybridisation (FISH), we have performed a systematic search for YACs spanning translocation breakpoints. Patients with DBCRs and either syndromic or non-syndromic mental retardation (MR) were ascertained through the Mendelian Cytogenetics Network (MCN), a collaborative effort of, at present, 270 cytogenetic laboratories throughout the world. In this pilot study, we have characterised 10 different MR associated chromosome regions delineating candidate regions for MR. Five of these regions are narrowed to breakpoint spanning YACs, three of which are located on chromosomes 13q21, 13q22, and 13q32, respectively, one on chromosome 4p14, and one on 6q25. In two out of six DBCRs, we found cytogenetically cryptic deletions of 3-5 Mb on one or both translocation chromosomes. Thus, cryptic deletions may be an important cause of disease in seemingly balanced chromosome rearrangements that are associated with a disease phenotype. Our region specific FISH probes, which are available to MCN members, can be a powerful tool in clinical cytogenetics and positional cloning.