Isolation and characterization of the mouse homolog of SYT, a gene implicated in the development of human synovial sarcomas.

In a previous study we reported the isolation of the human synovial sarcoma-associated t(X;18) breakpoint. As a result of this translocation, the SYT gene on chromosome 18 fuses to either the SSX1 or the SSX2 gene on the X chromosome, depending on the exact location of the breakpoint within band Xp11.2. As yet, little is known about the modes of action of the SYT and SSX genes and their respective (fusion) products. Here we report the isolation of the mouse homolog of SYT, its full length cDNA sequence, its chromosomal localization, and its spatio-temporal expression patterns in adult and embryonic tissues. The SYT gene was found to be well conserved during evolution and is part of a region of synteny between the human and mouse chromosomes 18. In early embryogenesis, Syt is ubiquitously expressed. In later stages, the expression becomes confined to cartilage tissues, specific neuronal cells and some epithelial derived tissues. In mature testis, expression was specifically observed in primary spermatocytes.

Towards the isolation of a human malignant extragonadal germ cell tumour-associated breakpoint in chromosome 11q13.

In a previous study we have defined a subgroup of human malignant extragonadal germ cell tumours that is characterized by complex translocations involving chromosomes 6 and 11 (Echten et al. 1995). Here we report (i) the use of fluorescent in situ hybridization, pulsed field gel electrophoresis and direct visual hybridization techniques to localize the tumour-associated breakpoint within band 11q13, and (ii) the construction of a phage library enriched for this region to facilitate genomic walks towards the breakpoint. Extensive breakpoint-flanking contigs were generated and within these contigs six candidate genes could be identified.

Increasing levels of MYC and MET co-amplification during tumor progression of a case of gastric cancer.

The cytogenetic study of a nodal metastasis from a gastric carcinoma, after two passages in nude mice, revealed a large number of double minutes. Comparative genomic in situ hybridization (CGH) analysis using DNA extracted from this xenograft revealed the existence of three clear amplification units that originated from the chromosomal subregions 6q24-25, 7q31-32, and 8q24 in the xenograft DNA. Similar, though less prominent, CGH results were found with DNAs extracted from the primary tumor and its metastasis, implying that the same amplicons were also present, albeit less abundantly, in the DNAs of these neoplastic tissues. Southern analysis of the second-passage xenograft detected 18- and 10-fold amplification of MET (located at 7q31) and MYC (located at 8q24), respectively. The retrospective study of the first passage of the xenograft, as well as of the metastatic and primary tumors before xenografting, showed amplification levels of MET of, respectively, 12-, 9-, and 5-fold and MYC of, respectively, 8-, 7-, and 5-fold. Our results suggest that increased levels of co-amplification of MYC and MET correlate with enhanced growth potential in this case of gastric carcinoma.

Fluorescence in situ hybridization-based approaches for detection of 12p overrepresentation, in particular i(12p), in cell lines of human testicular germ cell tumors of adults.

Overrepresentation of the short arm of chromosome 12 is frequently detected in human testicular germ cell tumors of adolescents and adults (TGCT). This overrepresentation mostly results from the formation of an isochromosome: i(12p). Whether the overrepresentation consistently involves the complete 12p arm including the centromere is still unclear. We studied five TGCT-derived cell lines (NT2, 2102Ep, H12.1, NCCIT, and S2), combining conventional chromosome banding, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) to investigate the suitability of each of these techniques to detect aberrations involving chromosome 12. Karyotyping showed one or more i(12p)s in NT2, 2102Ep, H12.1, and S2. However, FISH with a centromere-specific probe (p alpha 12H8), a 12p "paint" and a 12p11.2--p12.1 region-specific probe yeast artificial chromosome (YAC)#5 and CGH could not confirm the presence of an i(12p) in S2. Additional randomly distributed 12p sequences were detected by FISH in H12.1, NCCIT, and S2. In most of these cases, (a part of) the centromere was included. All overrepresented 12p regions, except for those in S2, showed hybridization with YAC#5. CGH showed increased copy numbers of the complete 12p arm in the cell lines with one or more i(12p)s but no overrepresentation was noted in the cell lines without i(12p). In metaphase spreads, the centromeric block of the i(12p)s differed in size as compared with those of normal chromosomes 12. This was rarely noted in interphase nuclei. A decrease in size of the centromeric block in 2102Ep and H12.1 caused a weak FISH signal, which was difficult to detect, especially in interphase nuclei. The ratio between p alpha 12H8- and YAC#5-derived signals reflected the presence or absence of one or more i(12p)s. Our results indicate that double FISH with a centromere- and a 12p-specific probe can be used to detect 12p overrepresentation [including i(12p)] in TGCT both in metaphase spreads and interphase nuclei. CGH confirmed the relative overrepresentation of 12p sequences as detected by FISH and showed that in these cell lines the complete 12p was involved.

Fine mapping of the human renal oncocytoma-associated translocation (5;11)(q35;q13) breakpoint.

Recent cytogenetic analysis of a series of human renal oncocytomas revealed the presence of a recurring chromosomal translocation (5;11)(q35;q13) as sole anomaly in a subset of the tumors. The molecular characterization of this translocation was initiated using two primary t(5;11)-positive renal oncocytomas and a panel of somatic cell hybrids derived from one of these tumors, in conjunction with fluorescence in situ hybridization (FISH) and Southern blot analysis. The breakpoint in chromosome band 11q13 could be located within a genomic interval of at maximum 400 Kb immediately centromeric to the BCL1 locus.

Comparative genomic hybridization of germ cell tumors of the adult testis: confirmation of karyotypic findings and identification of a 12p-amplicon.

Comparative genomic hybridization (CGH) was carried out on 15 primary testicular germ cell tumors (TGCT) of adolescents and adults and two metastatic residual tumors after chemotherapeutic treatment. The results were compared with karyotypic data obtained form the same tumor specimens after direct harvesting of metaphases or short-term in vitro culture. Both techniques revealed that the most consistent abnormality in primary TGCT is gain of 12p-sequences. Although in most cases over-representation of the complete short arm was observed, CGH revealed a specific amplification of 12p11.1-p12.1 region in two independent primary tumors. In addition, loss of (parts of) chromosome 13 (always involving q31-qter), and gain of (parts of) chromosome 7 (mostly involving q11), (parts of) chromosome 8, and the X chromosome were detected in more than 25% of the tumors by this latter technique. Loss of 6q15-q21 in both residual tumors analyzed may suggest a role for this anomaly in acquired resistance to chemotherapeutic treatment. Overall, the CGH analyses confirmed gains and losses of certain chromosomal regions in TGCT as observed by karyotyping, and thus support their role in the development of these neoplasms. The amplification of a restricted region of 12p in primary TGCT confirms and extends our previous observations and, as such, represents an important step forward in the identification of gene(s) on 12p relevant for the pathogenesis of these tumors.

Distinct Xp11.2 breakpoint regions in synovial sarcoma revealed by metaphase and interphase FISH: relationship to histologic subtypes.

Fluorescence in situ hybridization (FISH) and molecular analyses of synovial sarcomas with cytogenetically similar (X;18)(p11.2;q11.2) translocations have revealed two alternative breakpoint regions in Xp11.2, one residing in the ornithine aminotransferase-like 1 (OATL1) region and the other one in the related but distinct OATL2 region. As these results were obtained by different groups, we set out to evaluate an extended series of tumors with special emphasis on the two possible X-related breakpoint regions. Together, seven synovial sarcomas were identified with a break in the OATL1 region and six with a break near OATL2, thereby confirming the actual existence of the two alternative Xp breakpoint regions. We speculate that there seems to be a relationship between the occurrence of these breakpoint regions and the histologic phenotype of the tumors, with a predominance of OATL1-related breakpoints in the classical biphasic tumors and of OATL2-related breakpoints in the monophasic fibrous tumors.

An 8.35 Mb overlapping interstitial deletion of 8q24 in two patients with coloboma, congenital heart defect, limb abnormalities, psychomotor retardation and convulsions.

Chromosome analysis in two young patients with multiple congenital anomalies revealed a de novo interstitial deletion of 8q that has not been reported before. The deletions were overlapping by 8.35 Mb (8q24.21q24.23). The clinical features shared by our patients were coloboma, VSD, digital abnormalities, congenital dislocation of a hip, feeding problems, psychomotor delay and convulsions. The deletion included the region for Langer-Giedion syndrome (TRPS1 and EXT1) in the girl only. However, she is too young to present features of this syndrome, apart from dysmorphic features like a bulbous nose and notched alae nasi. Several genes are present in the commonly deleted region, including genes with unknown function, and genes for which haploinsufficiency is known to have no phenotypic effect in mice (Wnt1). A gene that might play a role in the convulsions of our patients is KCNQ3.

Assignment of the human gene encoding the epidermal serine proteinase inhibitor SKALP (PI3) to chromosome region 20q12-->q13.

Recently we isolated a cDNA clone corresponding to an epidermal serine proteinase inhibitor named SKALP (skin-derived antileukoproteinase). Here we report the chromosomal localization of the human gene encoding SKALP by Southern blot analysis of a panel of human x hamster hybrid cell lines and by nonradioactive in situ hybridization on R-banded human lymphocyte chromosome spreads. The SKALP-encoding gene has been assigned the approved symbol PI3 by the Human Gene Mapping nomenclature committee.

Refined localization of the alpha 1-subunit of the skeletal muscle L-type voltage-dependent calcium channel (CACNL1A3) to human chromosome 1q32 by in situ hybridization.

We isolated and partially sequenced a cosmid clone containing the human skeletal muscle L-type voltage-dependent calcium channel gene (CACNL1A3). The cosmid clone, which was also found to contain a novel dinucleotide repeat marker for the CACNL1A3 gene, was used for the chromosomal localization of CACNL1A3 by in situ hybridization. Our results refine the localization of CACNL1A3 on the long arm of human chromosome 1 to band q32.

Human short-chain L-3-hydroxyacyl-CoA dehydrogenase: cloning and characterization of the coding sequence.

The cDNA encompassing the complete coding sequence of human liver short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) was isolated and characterized. Screening of a cDNA library combined with rapid amplification of 5' cDNA ends resulted in a SCHAD cDNA sequence of 1877 bp. It encodes a protein of 314 amino acids with a calculated molecular weight of 34.3 kDA containing a mitochondrial import signal peptide of 12 amino acids and 302 amino acids of mature SCHAD protein. The deduced amino acid sequence of the mature protein shows a 92 percent identity with SCHAD from pig heart. Northern blot analysis reveals SCHAD mRNA to be expressed in liver, kidney, pancreas, heart and skeletal muscle. The human SCHAD gene was mapped by fluorescence in situ hybridization to chromosome 4q22-26.

Assignment of the human gene for receptor-type protein tyrosine phosphatase IA-2 (PTPRN) to chromosome region 2q35 --> q36.1 and identification of an intragenic genetic marker.

Using a mouse protein tyrosine phosphatase cDNA fragment as a probe, cosmid clones containing segments of the human IA-2 PTPase gene (PTPRN) were isolated. The gene was assigned to chromosome region 2q35 --> q36.1 by fluorescence in situ hybridization. In an intronic region of the IA-2 gene a polymorphic microsatellite sequence was found, which will be useful as a genetic marker for the 2q35 --> q36 region.

The gene (PTPN13) encoding the protein tyrosine phosphatase PTP-BL/PTP-BAS is located in mouse chromosome region 5E/F and human chromosome region 4q21.

Both mouse and human genomic clones were isolated for protein tyrosine phosphatase PTP-BL/PTP-BAS (HGM approved gene symbols Ptpn13 and PTPN13, respectively). Using these clones as a probe, PTPN13 was assigned to human chromosome region 4q21 and mouse chromosome region 5E/F by fluorescence in situ hybridization (FISH).

Localization of the human gene for aquaporin 3 (AQP3) to chromosome 9, region p21-->p12, using fluorescent in situ hybridization.

The chromosome location of the gene encoding aquaporin 3 (AQP3), which functions as a channel for water and small polar solutes in the basolateral membrane of the collecting duct of the kidney, was determined. In situ hybridization on metaphase chromosomes allowed the assignment of human AQP3 to chromosome 9p21-->p12.

Refined mapping of the human Ets-related gene Elk-1 to Xp11.2-p11.4, distal to the OATL1 region.

The gene for human Elk-1, an Ets-related transcription factor, has previously been localized to a region that lies on the short arm of chromosome X and that is involved in specific chromosomal translocations associated with synovial sarcoma and renal adenocarcinomas. We have used fluorescence in situ hybridization and a panel of tumor-derived somatic cell hybrids to refine the localization of Elk-1, in particular with regard to the rearrangements in these tumors. Elk-1 has been assigned to Xp11.2-p11.4, distal to the OATL1 region.

Epstein-Barr virus DNA in Reed-Sternberg cells of Hodgkin's disease is frequently associated with CR2 (EBV receptor) expression.

We studied 44 cases of Hodgkin's disease for the presence of Epstein-Barr virus (EBV) DNA, its localization and the expression of the EBV receptor on the tumour cells. EBV DNA was found in 52% (16/31) of the Hodgkin's lymphomas using the polymerase chain reaction. With a very sensitive non-radioactive DNA in situ hybridization technique in combination with immunohistochemistry for CD 30 or CD 15 antigens, EBV DNA was localized to Reed-Sternberg cells and its mononuclear variants. The relationship between the presence of EBV DNA and the expression of the EBV-receptor CR2 (CD 21) on Reed-Sternberg cells was studied using the same techniques and two different monoclonal anti-CD 21 antibodies. CR2 could be detected on a substantial number of the Reed-Sternberg cells in EBV DNA positive Hodgkin's lymphomas (9/12; 75%), whereas in EBV negative cases positivity with anti-CD 21 was rare (1/13; 8%). The results indicate that CR2 expression on Reed-Sternberg cells and the presence of EBV DNA sequences are frequently associated in Hodgkin's lymphomas.

Comparative genomic hybridization as a tool to define two distinct chromosome 12-derived amplification units in well-differentiated liposarcomas.

Well-differentiated liposarcomas (WDLPS) are frequently characterized by a near-diploid karyotype with supernumerary ring and/or giant rod-shaped marker chromosomes. We have shown, using fluorescence in situ hybridization (FISH) and molecular strategies, that these markers contain chromosome 12-derived sequences. Here we report the analysis of six WDLPS for the presence of amplified DNA segments by means of the recently developed comparative genomic hybridization (CGH) strategy. Two distinct chromosome 12-derived amplification units could be identified in all tumors examined, one located in the q14-q15 region as expected, the second unexpectedly mapping to q21.3-q22. Our results indicate that the concerted amplification of these two distinct regions on the long arm of chromosome 12 may be a consistent characteristic of WDLPS. These amplifications are most likely directly related to the presence of supernumerary ring and/or giant marker chromosomes in this group of soft tissue tumors.

A synovial sarcoma with a complex t(X;18;5;4) and a break in the ornithine aminotransferase (OAT)L1 cluster on Xp11.2.

The initial cytogenetic analysis of a biphasic synovial sarcoma revealed complex anomalies involving six different chromosomes: 46,Y,t(X;18;5;4)(p11;q11;p13;q12),t(2;5)(q35;q11). After fluorescence in situ hybridization (FISH) analysis, using chromosome X-specific plasmid library and YAC probes, the situation appeared to be even more complex, with an insertion of part of the X chromosome short arm into the der(5)t(5;18). In spite of these complex chromosomal rearrangements, the Xp11 breakpoint could be mapped to within the ornithine aminotransferase (OAT)L1 cluster, very similar to that reported previously for the standard t(X;18)(p11;q11) in synovial sarcomas. These findings suggest common pathogenetic pathways in these cytogenetically different but morphologically similar tumors.

Localization of the gene encoding the alpha 2/delta-subunits of the L-type voltage-dependent calcium channel to chromosome 7q and analysis of the segregation of flanking markers in malignant hyperthermia susceptible families.

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant disorder of skeletal muscle which manifests as a potentially fatal hypermetabolic crisis triggered by commonly used anaesthetic agents. The demonstration of genetic heterogeneity in MHS prompted the investigation of the roles played by calcium regulatory proteins other than the ryanodine receptor (RYR1), which is known to be linked to MHS in fewer than half of the European MHS families studied to date. Previously, we have excluded the genes encoding the skeletal muscle L-type voltage-dependent calcium channel alpha 1-, beta 1- and gamma-subunits as candidates for MHS. In this report, we describe the cloning and partial DNA sequence analysis of the gene encoding the alpha 2/delta-subunits, CACNL2A, and its localization on the proximal long arm of chromosome 7q. A new dinucleotide repeat marker close to CACNL2A was identified at the D7S849 locus and tested for linkage in six MHS families. D7S849 and flanking genetic markers were found to co-segregate with the MHS locus through 11 meioses in one, three-generation family. These results suggest that mutations in or near CACNL2A may be involved in some forms of this heterogeneous disorder.