Characterization of t(11;19)(q23;p13.3) by fluorescence in situ hybridization analysis in a pediatric patient with therapy-related acute myelogenous leukemia.

This case presents a Caucasian girl diagnosed with early pre-B cell acute lymphoblastic leukemia at age 2 years. The only chromosomal anomaly detected in her bone marrow cells at this time was an add(12p). By age 4 years, she had a bone marrow and central nervous system (CNS) relapse of ALL and was treated with chemotherapy that included etoposide. She was in complete remission for 2 years following chemotherapy with etoposide, but later developed therapy-related acute myeloid leukemia (t-AML). At this time, a t(11;19)(q23;p13.3) rearrangement was detected in her bone marrow cells. The AML relapsed again 1 year after allogeneic bone marrow transplant (BMT). The presence of a chromosome 11 abnormality involving band 11q23 in this patient suggests that the transformation from ALL to t-AML was a consequence of etoposide included in her chemotherapy. Studies have shown that the 11q23 breakpoint in the t(11;19) rearrangement is consistent, and involves the MLL gene in t-AML patients. However, the breakpoint in 19p is variable in that it could be located either at 19p13.1 or 19p13.3 and thus could involve either of two genes: ELL (11-19 lysine-rich leukemia gene) on 19p13.1 or ENL (11-19 leukemia gene) on 19p13.3. In this study, the t(11;19)(q23;p13.3) was further characterized and the breakpoint regions were defined by fluorescence in situ hybridization (FISH) analysis.

Atypical endometrial hyperplasia shares genomic abnormalities with endometrioid carcinoma by comparative genomic hybridization.

Endometrial hyperplasia is a common disorder that is now observed with increasing frequency in women treated with hormonal replacement therapy or with tamoxifen. This study was undertaken to determine whether genomic features of various forms of endometrial hyperplasias would allow their classification as a benign, premalignant, or malignant abnormality. Comparative genomic hybridization (CGH) was performed on endometrial glands microdissected by laser capture microscope from 19 archival endometrial samples, comprising 5 normal endometria, 1 polyp, 2 simple hyperplasias, 5 hyperplasias with nuclear abnormalities (atypical hyperplasias), and 4 low-grade and 2 high-grade endometrioid carcinomas, 1 with squamous component (adenoacanthoma). Genomic DNA, extracted from the glands and the squamous component in 1 case, was amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and compared with sex-matched DNA by CGH. No genomic imbalances were observed in the normal samples, the polyp, or the simple hyperplasias. However, in atypical hyperplasia, regardless of the level of cytologic atypia, genomic abnormalities were observed that also occurred in endometrioid carcinomas. Chromosomes 1, 8, and 10 were most often affected. The results are compared with molecular genetic abnormalities recently reported in these lesions. This study strongly suggests that atypical endometrial hyperplasias are closely related to endometrioid carcinoma and should be considered precancerous lesions, contrary to simple hyperplasia, which is a benign disorder. The squamous component of one of the high-grade carcinomas showed genetic abnormalities similar to those of endometrioid carcinoma and therefore does not represent squamous metaplasia but is an integral part of the malignant process.

Identical clonal origin of synchronous and metachronous low-grade, noninvasive papillary transitional cell carcinomas of the urinary tract.

One of the most important biological features of papillary transitional cell carcinoma (pTCC) of the urinary tract is its multicentricity and its tendency for recurrences. Two possible mechanisms, field effect and intramucosal seeding/spreading, have been proposed. The former theory hypothesizes that carcinogenic agents cause synchronous or metachronous malignant transformation of multiple urothelial cells (independent clonal origin), and the latter speculates that synchronous and metachronous tumors are derived from implantation or direct spreading of tumor cells (identical clonal origin). We tested these hypotheses by analyzing the methylation patterns of the androgen receptor gene (HUMARA) located at the X-chromosome. Thirty-five metachronous and synchronous, low-grade (grade 1 or 2), noninvasive pTCCs of the urinary tract from 10 heterozygous female patients were successfully analyzed using formalin-fixed, paraffin-embedded tissue. These included 16 recurrent bladder tumors from 4 patients, 10 metachronous bladder and ureter/renal pelvis tumors from 4 patients, and 9 multifocal tumors from 2 patients. All tumors are monoclonal as indicated by unbalanced methylation of HUMARA. Furthermore, same methylated allele was detected in multiple recurrent or multifocal tumors from any given patient, indicating their identical clonal origin. We conclude that low-grade, noninvasive pTCCs are monoclonal in nature. Synchronous or metachronous pTCCs have an identical clonal origin, strongly supporting the intramucosal seeding/spreading hypothesis.

Essential role of STAT3 for embryonic stem cell pluripotency.

Propagation of mouse embryonic stem (ES) cells in vitro requires exogenous leukemia inhibitory factor (LIF) or related cytokines. Potential downstream effectors of the LIF signal in ES cells include kinases of the Src, Jak, and mitogen-activated protein families and the signal transducer and transcriptional activator STAT3. Activation of nuclear STAT3 and the ability of ES cells to grow as undifferentiated clones were monitored during LIF withdrawal. A correlation was found between levels of STAT3 activity and maintenance of an undifferentiated phenotype at clonal density. In contrast, variation in STAT3 activity did not affect cell proliferation. The requirement for STAT3 was analyzed by targeted mutagenesis in ES cell lines exhibiting different degrees of LIF dependency. An insertional mutation was devised that abrogated Stat3 gene expression but could be reversed by Cre recombination-mediated excision. ES cells heterozygous for the Stat3 mutation could be isolated only from E14 cells, the line least dependent on LIF for self-renewal. Targeted clones isolated from other ES cell lines were invariably trisomic for chromosome 11, which carries the Stat3 locus, and retained normal levels of activated STAT3. Cre-regulated reduction of Stat3 gene copy number in targeted, euploid E14 clones resulted in dose-dependent losses of STAT3 activity and the efficiency of self-renewal without commensurate changes in cell cycle progression. These results demonstrate an essential role for a critical amount of STAT3 in the maintenance of an undifferentiated ES cell phenotype.

FHIT gene transcript alterations occur frequently in myeloproliferative and myelodysplastic diseases.

Twenty-five primary biopsy samples, obtained from patients diagnosed with chronic/acute myeloproliferative disorders, myelodysplastic disorders, in addition to seven cell lines established from patients with leukemias arrested at different stages of myeloid differentiation, were examined for alterations in an alternatively spliced form of the FHIT gene. Transcript alterations of this gene were detected in 80% of the primary biopsies and in two of the cell lines. Reverse transcription PCR (RT-PCR) detected deletions of one or more specific exons in the translated or untranslated portion of the FHIT gene. Point mutations in exons 3, 4, and 5 of the FHIT gene were also detected in 4 patients by single stranded conformational PCR analysis. Transcript alterations were detected in all primary hematopoietic samples which contained chromosome abnormalities, as well as, in hematopoietic disorders which did not show alterations of the 3p14 region. These findings suggest FHIT gene involvement in the transformation of hematopoietic stem cells to leukemia.

Clonal chromosome aberrations with monosomy of chromosome 8 in a case of grade III chondrosarcoma.

We report cytogenetic findings in a case of grade III chondrosarcoma. Complex clonal chromosome aberrations including monosomy of chromosomes 4, 8, 13, and a consistent t(5;14)(q23;p12) were observed in all cells. There were no structural or numerical anomalies involving chromosome 12. The complexity of the chromosome aberrations reflect the advanced stage of this chondrosarcoma; we suggest a possible involvement of the EXT1 gene located on chromosome 8.

The FHIT gene is alternatively spliced in normal kidney and renal cell carcinoma.

FHIT (Fragile Histidine Triad), a putative tumor suppressor gene, was cloned from fetal brain and colon cDNA libraries. Portions of this gene are deleted in esophageal, colon, lung and breast tumors, but this gene has not been found altered in sporadic renal cell carcinomas. We report here an alternatively spliced form of this gene cloned from a kidney cDNA library. This cDNA is 1189 bp in length, and contains an additional 94 bp exon, designated exon 2a (E2a). This novel sequence is located between exon 2 and exon 3 of the FHIT gene's untranslated region and exon 2a is present in all normal kidney tissues and cell lines. Analyses performed on sporadic renal cell carcinoma (RCC) tissues and cell lines, show consistent loss of exon 8 of the FHIT cDNA in almost 60% of the cases. Interestingly, in a familial, as well as, in a metastatic RCC, derived from a patient with the sporadic form, exon 2a and exon 3 are also deleted. Northern analyses with the exon 2a of the familial and the metastatic RCC demonstrates concurrent loss of expression of a 4.4 kb transcript with the loss of the E2a sequence, suggesting that exon 2a of the FHIT gene may play an important role in the oncogenesis of renal cell carcinoma.

Chromosome microdissection: a brief overview.

Chromosome microdissection arose as a means of facilitating long range physical mapping of chromosome regions involved in either a genetic or malignant disorder. However, with the rapid development of improved techniques for mapping and sequencing the human genome, microdissection is considered by many investigators to be a cumbersome and time consuming procedure. Nonetheless, based on the impressive number of informative diagnostic DNA markers that are now available as a result of this technology, microdissection still must be considered one of the most rapid and direct methods available for generating new DNA markers from any chromosome region, irrespective of its sequence composition. In addition, it remains an important means to dissect DNA markers from any organism, eukaryotic and prokaryotic, and has resulted in generating disease associated DNA sequences from both human and animal genomes. Recently, microdissection of single cells has emerged as a viable alternative for isolating pure populations of specific cell types, especially tumor cells, which can then be studied without background contamination from any other cellular constituents. This overview will provide a glimpse into the present applications of the microdissection technology, as well as the importance this technology will have for future exploration into the human genome.

Mapping of 29 YAC clones and identification of 3 YACs spanning the translocation t(3;8)(p14.2;q24.1) breakpoint at 8q24.1 in hereditary renal cell carcinoma.

The constitutional balanced translocation (3;8)(p14.2:q24.1) has been described in a family in which a arge number of individuals developed renal cell carcinoma RCC) at an early age. The translocation event in which genes from the 3p14.2 and the 8q24.1 sites are brought in close proximity is considered a critical, initial step for the development and progression of RCC. Even though the 3p14.2 breakpoint region has been cloned, a gene has not yet been identified, which may be responsible for either the initiation or progression of hereditary RCC. As a crucial step toward cloning the 3;8 breakpoint at the 8q24.1 site, we have mapped a series of YACs which surround this region by fluorescence in situ hybridization (FISH). Three YACs have been identified that span the 8q24.1 breakpoint region. One of these YACs is approximately 180 kb in length, and has been used to initiate construction of a high resolution cosmid contig. Several cosmids have been isolated which have been positioned in relation to the 8q24.1 breakpoint region. In addition, we have positioned 26 other YACs in relation to the 3;8 translocation breakpoint. These results provide a basis for the isolation of genes surrounding 3;8 RCC translocation breakpoint region at 8q24.1.

Chromosome localization of human genes for clathrin adaptor polypeptides AP2 beta and AP50 and the clathrin-binding protein, VCP.

Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed "assembly proteins," or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2 alpha and AP2 beta, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2 beta (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with APs. Chromosomal in situ hybridization of a human genomic clone demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2 beta fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34.

Chromosomal mapping of the genes GPRK5 and GPRK6 encoding G protein-coupled receptor kinases GRK5 and GRK6.

G protein-coupled receptor kinases (GRKs) play an important role in phosphorylating and regulating the activity of a variety of G protein-coupled receptors. Chromosomal mapping of the human genes for the two most recently identified members of the GRK family, GRK5 (GPRK5) and GRK6 (GPRK6), was accomplished by correlation of the presence of the GPRK5 and GPRK6 loci with specific chromosome regions in a rodent-human hybrid panel. These analyses revealed that GPRK5 maps to chromosome region 10q24-->qter while GPRK6 maps to 5q35. A GPRK6 related locus maps to 13pter-->q21.

High-resolution mapping of 10 unique DNA sequences to human chromosome 3 subregions by in situ hybridization.

Ten single-copy DNA probes derived from a human chromosome 3-specific genomic library were mapped by in situ hybridization to subregions of this chromosome. Seven sequences were assigned to subregions of 3q and two sequences were assigned to subregions of 3p. One single-copy DNA probe was assigned to the centromeric region of chromosome 3 by Southern blot analysis of DNA isolated from a somatic cell hybrid containing centromeric sequences of this chromosome. These DNA clones mapped by in situ hybridization can provide useful landmarks for mapping various disease loci on chromosome 3. They may also be useful for the generation of physical and genetic maps.

Cytogenetic findings in primary uveal melanoma.

We analyzed cytogenetic abnormalities in 10 cases of primary uveal melanoma. Clonal chromosomal abnormalities were present in nine cases. Chromosome 6 was most commonly affected (seven cases) and included gain of material from 6 and/or loss of material from 6q. Trisomy of chromosome 8 or gain in material from 8q, mostly in the form of an i(8q) resulting in three to five copies of the 8q segment was seen in six cases. Monosomy of chromosome 3 and rearrangements of chromosome 9 were less frequent and were altered in three cases each. Clinical, histopathologic, and cytogenetic abnormalities are correlated.

Regional mapping of the human galactocerebrosidase gene (GALC) to 14q31 by in situ hybridization.

The cDNA for human galactocerebrosidase (GALC) has recently been cloned and expressed. A portion of this cDNA was used for in situ hybridization, and the region of strongest signal corresponded to human chromosome region 14q31. This agrees with recent linkage studies that localized Krabbe disease (globoid cell leukodystrophy) to the same region. This information will be useful in future studies for mapping this gene in animal models of GALC deficiency.

Molecular cloning of the human proto-oncogene Wnt-5A and mapping of the gene (WNT5A) to chromosome 3p14-p21.

The highly conserved Wnt genes belong to a widely distributed family of presumptive signaling molecules that have been implicated not only in the regulation of normal pattern formation during embryogenesis and differentiation of cell lineages, but also in oncogenic events. All of the known vertebrate Wnt genes encode for 38- to 43-kDa cysteine-rich putative glycoproteins, which have features typical of secreted growth factors: a hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence, and 22 conserved cysteine residues whose relative spacing is maintained. In this study, we report the cloning and sequencing of several overlapping cDNAs encoding approximately 4.1 kb of the human homologue of Wnt-5A. The mature protein contained 343 residues (M(r) approximately 38,000 excluding any post-translational modifications) with a > 93% homology to the reported sequences of other Wnt-5A proteins (> 99% homologous to mouse Wnt-5A). This protein maintained certain features--a hydrophobic signal sequence, the Wnt-1 family "signature sequence" (CKCHGvSGSC), and a number of other conserved amino acid residues: 24 cysteine residues, 4 asparagine-linked oligosaccharide consensus sequences, and a tyrosine sulfation site--that have been found in all other Wnt-5A proteins. Reverse transcriptase PCR analysis of RNA from a variety of human embryonic, neonatal, and adult cells and/or tissues showed that human Wnt-5A expression was detected only in neonatal heart and lung. It may be relevant, however, that the 3'-untranslated region contained numerous AT-rich motifs that could be involved in the rapid degradation of mRNA. Finally, using a combination of Southern blotting, PCR amplification, and in situ hybridization, the human Wnt-5A (WNT5A) gene was mapped to chromosome 3p14-p21.

Detailed genetic and physical map of the 3p chromosome region surrounding the familial renal cell carcinoma chromosome translocation, t(3;8)(p14.2;q24.1).

Extensive studies of loss of heterozygosity of 3p markers in renal cell carcinomas (RCCs) have established that there are at least three regions critical in kidney tumorigenesis, one most likely coincident with the von Hippel-Lindau gene at 3p25.3, one in 3p21 which may also be critical in small cell lung carcinomas, and one in 3p13-p14.2, a region which includes the 3p chromosome translocation break of familial RCC with the t(3;8)(p14.2;q24.1) translocation. A panel of rodent-human hybrids carrying portions of 3p, including a hybrid carrying the derivative 8 (der(8)(8pter-->8q24.1::3p14.2-->3pter)) from the RCC family, have been characterized using 3p anchor probes and cytogenetic methods. This 3p panel was then used to map a large number of genetically mapped probes into seven physical intervals between 3p12 and 3pter defined by the hybrid panel. Markers have been physically, and some genetically, placed relative to the t(3;8) break, such that positional cloning of the break is feasible.

Human zinc finger gene ZNF23 (Kox16) maps to a zinc finger gene cluster on chromosome 16q22, and ZNF32 (Kox30) to chromosome region 10q23-q24.

Two members of the KOX gene family, ZNF23 (KOX16) and ZNF32 (KOX30), have been mapped by in situ hybridization to chromosome regions 16q22 and 10q23-q24, respectively. The map location of ZNF23 and ZNF32 placed these zinc finger protein genes near to chromosome loci that, under certain in vitro conditions, are expressed as fragile sites (FRA16B, FRA16C) and (FRA10D, FRA10A, FRA10B and FRA10E). Human zinc finger gene ZNF32 maps to a chromosome region on 10q23-24 in which deletions have been observed associated with malignant lymphoma on 10q22-23 and with carcinoma of the prostate on 10q24. ZNF23 is located on 16q22 in a chromosomal region that has been involved in chromosome alterations characteristic of acute myeloid leukemia. A second Kox zinc finger gene (ZNF19/KOX12) was recently mapped to the same chromosome region on human chromosome 16q22. In the analogous murine position, the murine zinc finger genes Zfp-1 and Zfp-4 are found in the syntenic 16q region of mouse chromosome 8. Thus, ZNF19 and ZNF23 might be members of an evolutionarily conserved zinc finger gene cluster located on human chromosome 16q22.

The cloning of a receptor-type protein tyrosine phosphatase expressed in the central nervous system.

We have isolated cDNA clones and deduced the complete amino acid sequence of a large receptor-type protein tyrosine phosphatase containing 2307 amino acids. The human gene encoding this phosphatase, denoted RPTP beta (or PTP zeta), has been localized to chromosome 7q31-33. RPTP beta is composed of a large extracellular domain, a single transmembrane domain, and a cytoplasmic portion with two tandem catalytic domains. We have also cloned a variant of RPTP beta lacking 859 amino acids from the extracellular domain but with intact transmembrane and cytoplasmic domains. Interestingly, the amino-terminal region of the extracellular domain of RPTP beta contains a stretch of 266 amino acids with striking homology to the enzyme carbonic anhydrase. Immunoprecipitation experiments from a human neuroblastoma cell line indicate that the apparent molecular mass of the core and glycosylated forms of RPTP beta are approximately 250 and 300 kDa, respectively. Northern blot analysis shows that RPTP beta is strictly expressed in the central nervous system. In situ hybridization was used to further localize the expression to different regions of the adult brain including the Purkinje cell layer of the cerebellum, the dentate gyrus, and the subependymal layer of the anterior horn of the lateral ventricle. Hence, RPTP beta represents the first mammalian tyrosine phosphatase whose expression is restricted to the nervous system. The high level of expression of RPTP beta transcripts in the ventricular and subventricular zones of the embryonic mouse brain suggests the importance of this tyrosine phosphatase in the development of the central nervous system.

The human decorin gene: intron-exon organization, discovery of two alternatively spliced exons in the 5' untranslated region, and mapping of the gene to chromosome 12q23.

Decorin is a chondroitin/dermatan sulfate proteoglycan expressed by most vascular and avascular connective tissues and, because of its ability to interact with collagen and growth factors, has been implicated in the control of matrix assembly and cellular growth. To understand the molecular mechanisms involved in regulating its tissue expression, we have isolated a number of genomic clones encoding the complete decorin gene. The human decorin gene spans over 38 kb of continuous DNA sequence and contains eight exons and very large introns, two of which are 5.4 and > 13.2 kb. We have discovered two alternatively spliced leader exons, exons Ia and Ib, in the 5' untranslated region. These exons were identified by cloning and sequencing cDNAs obtained by polymerase chain reaction amplification of a fibroblast cDNA library. Using Northern blotting or reverse transcriptase PCR, we detected the two leader exons in a variety of mRNAs isolated from human cell lines and tissues. Interestingly, sequences highly (74-87%) homologous to exons Ia and Ib are found in the 5' untranslated region of avian and bovine decorin, respectively. This high degree of conservation among species suggests regulatory functions for these leader exons. In the 3' untranslated region there are several polyadenylation sites, and at least two of these sites could give rise to the transcripts of approximately 1.6 and approximately 1.9 kb, typically detected in a variety of tissues and cells. Using a genomic clone as the labeled probe and in situ hybridization of human metaphase chromosomes, we have mapped the decorin gene to the discrete region of human chromosome 12q23. This study provides the molecular basis for discerning the transcriptional control of the decorin gene and offers the opportunity to investigate genetic disorders linked to this important human gene.