delta-Protocadherins: a gene family expressed differentially in the mouse brain.

Phylogenetic analysis of protocadherin genes identified a new gene subfamily, the delta-protocadherins, containing several conserved motifs in their cytoplasmic domains. This subfamily can be further subdivided into two subgroups, named delta1-protocadherins (comprising protocadherin-1, -7, -9, and -11 or X/Y) and delta2-protocadherins (comprising protocadherin-8, -10, -17, -18, and -19). The members of the delta1-protocadherin subgroup were analyzed in greater detail here. They share a similar gene structure that results in the expression of multiple alternative transcripts. All members of this subgroup have at least one transcript that contains a binding site for protein phosphatase-1alpha. Like most classic cadherins, each of three delta1-protocadherins analyzed in this study by in situ hybridization showed a unique expression pattern that differed from the patterns of the other delta1-protocadherins. Together, these results suggest that the members of the delta1-protocadherin subgroup exercise tightly regulated functions in the development, regionalization, and functional differentiation of the mouse brain.

The human and murine protocadherin-beta one-exon gene families show high evolutionary conservation, despite the difference in gene number.

Extensive cDNA analysis demonstrated that all human and mouse protocadherin-beta genes are one-exon genes. The protein sequences of these genes are highly conserved, especially the three most membrane-proximal extracellular domains. Phylogenetic analysis suggested that this unique gene family evolved by duplication of one single protocadherin-beta gene to 15 copies. The final difference in the number of protocadherin-beta genes in man (#19) and mouse (#22) is probably caused by duplications later in evolution. The complex relationship between human and mouse genes and the lack of pseudogenes in the mouse protocadherin-beta gene cluster suggest a species-specific evolutionary pressure for maintenance of numerous protocadherin-beta genes.

Characterization of three novel human cadherin genes (CDH7, CDH19, and CDH20) clustered on chromosome 18q22-q23 and with high homology to chicken cadherin-7.

Full-length coding sequences of two novel human cadherin cDNAs were obtained by sequence analysis of several EST clones and 5' and 3' rapid amplification of cDNA ends (RACE) products. Exons for a third cDNA sequence were identified in a public-domain human genomic sequence, and the coding sequence was completed by 3' RACE. One of the sequences (CDH7L1, HGMW-approved gene symbol CDH7) is so similar to chicken cadherin-7 gene that we consider it to be the human orthologue. In contrast, the published partial sequence of human cadherin-7 is identical to our second cadherin sequence (CDH7L2), for which we propose CDH19 as the new name. The third sequence (CDH7L3, HGMW-approved gene symbol CDH20) is almost identical to the mouse "cadherin-7" cDNA. According to phylogenetic analysis, this mouse cadherin-7 and its here presented human homologue are most likely the orthologues of Xenopus F-cadherin. These novel human genes, CDH7, CDH19, and CDH20, are localized on chromosome 18q22-q23, distal of both the gene CDH2 (18q11) encoding N-cadherin and the locus of the six desmosomal cadherin genes (18q12). Based on genetic linkage maps, this genomic region is close to the region to which Paget's disease was linked. Interestingly, the expression patterns of these three closely related cadherins are strikingly different.

Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members.

Cadherins play an important role in specific cell-cell adhesion events. Their expression appears to be tightly regulated during development and each tissue or cell type shows a characteristic pattern of cadherin molecules. Inappropriate regulation of their expression levels or functionality has been observed in human malignancies, in many cases leading to aggravated cancer cell invasion and metastasis. The cadherins form a superfamily with at least six subfamilies, which can be distinguished on the basis of protein domain composition, genomic structure, and phylogenetic analysis of the protein sequences. These subfamilies comprise classical or type-I cadherins, atypical or type-II cadherins, desmocollins, desmogleins, protocadherins and Flamingo cadherins. In addition, several cadherins clearly occupy isolated positions in the cadherin superfamily (cadherin-13, -15, -16, -17, Dachsous, RET, FAT, MEGF1 and most invertebrate cadherins). We suggest a different evolutionary origin of the protocadherin and Flamingo cadherin genes versus the genes encoding desmogleins, desmocollins, classical cadherins, and atypical cadherins. The present phylogenetic analysis may accelerate the functional investigation of the whole cadherin superfamily by allowing focused research of prototype cadherins within each subfamily.

The human cadherin-10 gene: complete coding sequence, predominant expression in the brain, and mapping on chromosome 5p13-14.

In a quest for novel cadherin gene family members in the human dbEST database, an interesting EST clone was identified and chosen for subsequent analysis. Using the technique of 5' rapid amplification of cDNA ends, we isolated the complete coding sequence and a large part of the UTRs of a novel gene. The sequence appeared to correspond to the human cadherin-10 gene, whose sequence was only partially known before. The expression pattern of this cadherin was found to be largely brain-specific, with additional expression in both adult and fetal kidney, and with minor expression in prostate and fetal lung. By FISH analysis the genomic location was determined at human chromosome 5p13-14, which is nearby the reported positions of the human cadherin-6, -12, and cadherin-14 (CDH18) genes. Cadherin-10 shows high relationship to the human cadherin-6 gene.

Plakophilin-3, a novel armadillo-like protein present in nuclei and desmosomes of epithelial cells.

We report on a novel Armadillo-like protein, termed plakophilin-3. The human protein, which is encoded by a 2.8 kb messenger RNA, has a predicted molecular mass of 87 kDa. The protein comprises 10 Armadillo-like repeats, preceded by an amino-terminal region of 293 amino acid residues and followed by a short carboxy-terminal region of 27 amino acid residues. Plakophilin-3 is classified as a member of the p120(ctn)/plakophilin subfamily of Armadillo proteins based on the number and organization of the Armadillo repeats and its high sequence similarity to other members of this family. CLUSTAL W alignment of p120(ctn)/plakophilin subfamily members showed the plakophilin-3 protein to be most similar to plakophilin-1 and -2. Western blot analysis of plakophilin-3 revealed expression in all epithelial cell lines tested but not in foreskin fibroblasts and various sarcoma-derived cell lines. This is unlike most other members of the p120(ctn)/plakophilin subfamily, which are widely expressed. By immunofluorescence, the plakophilin-3 protein was colocalized with desmoglein in desmosomes of epithelial cells. In addition, an intriguing speckle-like nuclear staining was observed. Hence, like plakophilin-1 and -2, plakophilin-3 displays a dual intracellular location, i.e. in the desmosomal plaque and in the nucleus. These results suggest the involvement of plakophilin-3 in both desmosome-dependent adhesion and signaling pathways. Furthermore, the human plakophilin-3 gene was mapped on the chromosomal locus 11p15 by fluorescent in situ hybridization.

HMGIC, the gene for an architectural transcription factor, is amplified and rearranged in a subset of human sarcomas.

Amplified segments of the long arm of chromosome 12 are frequently observed in human sarcomas. In most cases there are separate amplified regions around the MDM2 and CDK4 genes. Here we show recurrent amplification of a third region encompassing HMGIC, a human architectural transcription factor gene. Reduced amplification frequency of sequences flanking the gene was observed, indicating that inclusion of this third region in the amplicons is also selected for. In three samples only the 5' part of HMGIC was amplified, suggesting preferential loss of the 3' part of the gene preceding or during amplification. In several other samples rearrangement of the gene was observed. Expression analysis showed transcripts of aberrant sizes, lacking 3' sequences, and 3' RACE of one sample revealed replacement of exons 4 and 5 with ectopic sequences. This truncation of HMGIC resembles that reported for translocations of HMGIC in benign tumors, including lipomas, and it is striking that the gene was frequently amplified or rearranged in well differentiated liposarcomas, the malignant counterpart of lipomas. It seems conceivable that high levels of either full length or truncated hmgic could be relevant for the etiology of these tumors.

Amplification of a rearranged form of the high-mobility group protein gene HMGIC in OsA-CI osteosarcoma cells.

Recently the high-mobility group protein gene HMGIC has been found to be rearranged in a variety of benign mesenchymal tumors with 12q13-q15 aberrations, such as angiomyxoma, fibroadenomas of the breast, lipomas, pleomorphic salivary gland adenomas, polyps of the endometrium, pulmonary chondroid hamartomas, and uterine leiomyomas. Here we report on HMGIC aberrations in the osteosarcoma cell line OsA-CI. In Northern blot studies, aberrant HMGIC transcripts were detected. Analysis of cDNA sequence data obtained after 3' rapid amplification of cDNA ends, indicated these to consist of 5' HMGIC sequences encoding the three DNA binding domains fused to ectopic sequences apparently derived from part of the human lumican (keratan sulphate proteoglycan) gene (LUM), which we mapped by fluorescence in situ hybridization (FISH) to chromosome 12q22-q23. Moreover, Southern blot analysis revealed amplification of this fusion gene but not of the 3'HMGIC sequences. This observation was independently confirmed by FISH analysis using yeast artificial chromosome (YAC) and cosmid clones, which furthermore indicated that the amplified 5'HMGIC sequences were contained within an amplicon of about 200 kb. Our results indicate that aberrations in HMGIC might not be restricted to benign mesenchymal tumors.

cDNA cloning, expression and chromosomal localization of the human sarco/endoplasmic reticulum Ca(2+)-ATPase 3 gene.

cDNA and genomic clones encoding human sarco/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3) were isolated. The composite nucleotide sequence of the 4.6 kb cDNA, as well as the partial structure of 25 kb of genomic DNA encoding all but the 5' region of the gene, was determined. The nucleotide sequence coding for the last six amino acids of the pump and the 3'-untranslated region were identified within the sequence of the last exon. Northern blot hybridization analysis using cDNA probes derived from this exon detected a 4.8 kb transcript in several human tissues. Using a cDNA probe derived from the 5'-coding region an unexpected mRNA distribution pattern, consisting of two mRNA species of 4.8 and 4.0 kb, was detected in thyroid gland and bone marrow only. This is the first indication of an alternative splicing mechanism operating on the SERCA3 gene transcript, which most likely generates SERCA3 isoforms with altered C-termini. Human SERCA3 expressed in platelets and in COS cells transfected with the corresponding cDNA was detected with the previously described antibody N89 (directed against the N-terminal region of rat SERCA3) and with a new SERCA3-specific antiserum C91, directed against the extreme C-terminus of the human isoform. A monoclonal antibody PL/IM430, previously assumed to recognize SERCA3 in human platelets, does not react with the 97 kDa human SERCA3 transiently expressed in COS cells. Therefore the 97 kDa isoform detected by PL/IM430 more likely represents a novel SERCA pump, as recently suggested [Kovács, Corvazier, Papp, Magnier, Bredoux, Enyedi, Sarkadi and Enouf (1994) J. Biol. Chem. 269, 6177-6184]. Finally, by fluorescence in situ hybridization and chromosome G-banding analyses, the SERCA3 gene was assigned to human chromosome 17p13.3.

Mapping of the translocation breakpoints of primary pleomorphic adenomas and lipomas within a common region of chromosome 12.

Recent molecular cytogenetic analysis of uterine leiomyoma cell lines with chromosomal aberrations of 12q14-q15 have indicated that the chromosome 12 breakpoints cluster in a 445-kb region designated ULCR12 (uterine leiomyoma cluster region of the chromosome 12 breakpoints). Here we report the results of FISH studies of five primary pleomorphic adenomas and six primary lipomas and established cell lines of these tumor types characterized by translocations involving the chromosomal segment 12q13-q15. The results reveal that for nearly all tumors and cell lines analyzed, the chromosome 12 breakpoints map within a 350-kb region included in ULCR12, despite the previous cytogenetic assignment of the breakpoints to different bands of that region. In some cases the primary material and additionally analyzed cell lines allowed an even more precise localization of the breakpoints to less than 100 kb. Furthermore, a previously hidden translocation of ULCR12 in one primary tumor could be detected by FISH.

A 6-Mb yeast artificial chromosome contig and long-range physical map encompassing the region on chromosome 12q15 frequently rearranged in a variety of benign solid tumors.

Cytogenetic analysis of a variety of benign solid tumors, among which uterine leiomyoma, lipoma, pleomorphic salivary gland adenoma, and pulmonary chondroid hamartoma, has indicated that these tumors often display chromosome breakpoints in region q13-q15 of chromosome 12. In previous studies, we have reported that these breakpoints map between locus D12S8 and the CHOP gene, the latter of which has been shown to be consistently rearranged in myxoid liposarcomas with t(12;16)(q13;p11). Here, we report directional chromosome walking studies starting from D12S8 and resulting in the construction of a YAC contig of about 6 Mb. This YAC contig, whose orientation on chromosome 12 was determined by double-color fluorescence in situ hybridization (FISH) analysis, has at least double coverage and consists of 75 overlapping YAC clones, all isolated from CEPH YAC libraries. Their insert sizes were estimated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosomal localization and chimerism of the YACs were investigated by FISH analysis. Chimerism of YAC clones was independently determined by restriction mapping. On the basis of YAC end-derived DNA markers and sequence-tagged sites (STSs), with an average spacing of approximately 70 kb, as well as restriction enzyme analysis, a long-range physical map was established for the 6-Mb DNA region of chromosome 12 covered by the YAC contig. Within the YAC contig, the relative positions of various known genes, an expressed sequence-tagged site, and a number of CEPH/Généthon polymorphic markers were determined. The latter data allow full integration of our mapping data with those obtained by CEPH/Généthon as well as those reported at the Second International Workshop on Human Chromosome 12 Mapping. Finally, this YAC contig constitutes the basis for the contstruction of a transcriptional map of this region and is likely to facilitate identification of genes involved in the formation of various benign solid tumor types.

Four cytogenetic subgroups can be identified in endometrial polyps.

We have cytogenetically investigated a total of 33 simple benign endometrial polyps, 7 of which have been reported previously. Clonal chromosome rearrangements are found in 19 of 33 lesions (57%). Three major cytogenetically abnormal subgroups can be distinguished: (a) those with rearrangements in the 6p21-p22 region; (b) those with rearrangements of the 12q13-15 region; (c) those with rearrangements of the 7q22 region. A normal karyotype is found in a fourth subgroup. Recombinations of the 6p21-22 region with 2q35 and 10q22, as well as rearrangements of 7q22, have not been described before. It can be concluded that endometrial polyps, like several other types of benign mesenchymal tumors, present several cytogenetically different subgroups despite a seemingly identical clinical and morphological appearance. It is mandatory, therefore, to look for a common denominator of these tumors at the molecular level.

Molecular characterization of MAR, a multiple aberration region on human chromosome segment 12q13-q15 implicated in various solid tumors.

Chromosome arm 12q breakpoints in seven cell lines derived from primary pleomorphic salivary gland adenomas were mapped by FISH analysis relative to nine DNA probes. These probes all reside in a 2.8 Mb genomic DNA region of chromosome segment 12q13-q15 and correspond to previously published sequence-tagged sites (STS). Their relative positions were established on the basis of YAC cloning and long range physical and STS content mapping. The 12q breakpoints of five of the cell lines were found to be mapping within three different subregions of the 445 kb DNA interval that was recently defined as the uterine leiomyoma cluster region of chromosome 12 breakpoints (ULCR12) between STS RM33 and RM98. All seven breakpoints appeared to map within the 1.7 Mb DNA region between STS RM36 and RM103. Furthermore, the chromosome 12 breakpoints of three primary pleomorphic salivary gland adenomas were also found to be mapping between RM36 and RM103. Finally, FISH analysis of two lipoma cell lines with 12q13-q15 aberrations pinpointed the breakpoints of these to relatively small and adjacent DNA segments which, as well as those of two primary lipomas, appeared to be located also between RM36 and RM103. We conclude from the observed clustering of the 12q breakpoints of the three distinct solid tumor types that the 1.7 Mb DNA region of the long arm of chromosome 12 between RM36 and RM103 is a multiple aberration region which we designate MAR.

Identification of the chromosome 12 translocation breakpoint region of a pleomorphic salivary gland adenoma with t(1;12)(p22;q15) as the sole cytogenetic abnormality.

Cell line Ad-312/SV40, which was derived from a primary pleomorphic salivary gland adenoma with t(1;12)(p22;q15), was used in fluorescence in situ hybridization (FISH) analysis to characterize its translocation breakpoint region on chromosome 12. Results of previous studies have indicated that the chromosome 12 breakpoint in Ad-312/SV40 is located proximally to locus D12S8 and distally to the CHOP gene. We here describe two partially overlapping yeast artificial chromosome (YAC) clones, Y4854 (500 kbp) and Y9091 (460 kbp), which we isolated in the context of a chromosome walking project with D12S8 and CHOP as starting points. We present a composite long-range restriction map encompassing the inserts of these two YAC clones and show by FISH analysis that both YACs span the chromosome 12 breakpoint as present in Ad-312/SV40 cells. Subsequently, we have isolated cosmid clones corresponding to various sequence-tagged sites (STSs) mapping within the inserts of these YAC clones. These included cRM51, cRM69, cRM85, cRM90, cRM91, cRM110, and cRM111. In FISH studies, cosmid clones cRM85, cRM90, and cRM111 appeared to map distally to the chromosome 12 breakpoint, whereas cosmid clones cRM51, cRM69, cRM91, and cRM110 were found to map proximally to it. These results assign the chromosome 12 breakpoint in Ad-312/SV40 to a DNA region of less than 165 kbp. FISH evaluation of the chromosome 12 breakpoints in five other pleomorphic salivary gland adenoma cell lines indicated that these are located proximally to the one in Ad-312/SV40, at a distance of more than 0.9 Mbp from STS RM91. These results, while pinpointing a potentially critical region on chromosome 12, also provide evidence for the possible involvement of 12q13-q15 sequences located elsewhere.

Identification, molecular cloning, and characterization of the chromosome 12 breakpoint cluster region of uterine leiomyomas.

Recent molecular cytogenetic analysis of uterine leiomyoma cell lines with chromosome 12 aberrations has indicated that their chromosome 12 breakpoints map between linkage locus D12S8 and the CHOP gene. Here, we report fluorescence in situ hybridization (FISH) and molecular cloning studies of the chromosome 12 breakpoints in a panel of seven such uterine leiomyoma cell lines; five with the frequently observed t(12;14)(q15;q24), one with t(12;15)(q15;q24), and one with ins(12;11)(q14;q21qter). Directional chromosome walking studies starting from D12S8 and the CHOP gene resulted in the isolation of a relatively large number of overlapping YAC clones, including Y5355 (465 kbp), Y7673 (360 kbp), and Y9899 (275 kbp). In total, the inserts of these three YAC clones span an 800 kbp long and presumably contiguous stretch of human genomic DNA. All chromosome 12 breakpoints of the uterine leiomyoma cell lines studied were found by FISH analysis to be mapping within a 445 kbp subfragment of this region and, furthermore, to be dispersed over a DNA region which is at least 150 kbp in size. The chromosome 12 breakpoint of t(12;14)(q15;q24) in uterine leiomyoma cell line LM-30.1/SV40 was tentatively mapped within the 60 kbp region between YAC clones Y9899 and Y5355. From this 60 kbp region and close to sequence-tagged site RM99, we isolated probe pRM118-A, which showed in Southern blot analysis that it detected a rearrangement in LM-30.1/SV40 DNA, and generated restriction maps of the normal and rearranged genomic DNA regions detected with this probe. Finally, we molecularly cloned part of one of those rearranged DNA fragments using a vectorette-PCR-based technique and demonstrated that it consisted of 12q13-q15 sequences fused to DNA sequences derived from 14q23-24 and most likely represented the translocation junction on der(14) in LM-30.1/SV40 cells. Our studies strongly suggest that we have identified and isolated the uterine leiomyoma cluster region of chromosome 12 breakpoints, which we designate ULCR12, and molecularly cloned and characterized the der(14) translocation junction in cells derived from a uterine leiomyoma carrying the frequently observed t(12;14)(q15;q24).

A long range restriction map spanning the myxoid liposarcoma breakpoint in the q13-14 region of human chromosome 12.

We have used pulsed-field gel electrophoresis to construct a long range restriction map of the myxoid liposarcoma (MLS) breakpoint region in 12q13-14. The CHOP/GADD153 gene, consistently translocated in myxoid liposarcomas, is located less than 55 kb from the putative oncogene GLI. We have used fluorescent in situ hybridization to orient the map with respect to the chromosome, and to show that GLI (and thus A2MR) is located proximal to the MLS breakpoint.

Physical mapping of chromosome 12q breakpoints in lipoma, pleomorphic salivary gland adenoma, uterine leiomyoma, and myxoid liposarcoma.

We report here the physical mapping of recurrent chromosome 12q13-q15 breakpoints in cell lines derived from primary myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands. In fluorescence in situ hybridization (FISH) experiments, we first mapped the position of the chromosome 12 translocation breakpoint in uterine leiomyoma cell line LM-30.1/SV40 relative to loci COL2A1, D12S4, D12S17, D12S6, D12S6, D12S19, D12S8, and D12S7. It mapped between linkage probes CRI-C86 (D12S19) and p7G11 (D12S8). We then isolated YAC clones using CRI-C86- and p7G11-derived sequence-tagged sites, constructed corresponding YAC contigs of 310 and 800 kb, respectively, and established long-range physical maps of these. Cosmid clones LLNL12NCO1-98C10 and LLNL12NCO1-113D12 were isolated using STSs within the CRI-C86- and the p7G11-derived YAC contigs, respectively, and a mixture of them was used to routinely study the various tumor cell lines by FISH analysis. The chromosome 12 breakpoints of all tumor cell lines tested mapped between cosmids LLNL12NCO1-98C10 and LLNL12NCO1-113D12. None of the breakpoints appeared to map within any of the isolated YAC clones. Furthermore, FISH analysis using cosmid LLNL12-NCO1-144G3, which maps at the CHOP locus, revealed that the chromosome 12 breakpoints in all cell lines of the three benign solid tumors that were tested were located distal to the chromosome 12 translocation breakpoint with the CHOP gene in myxoid liposarcoma cells with t(12;16). In conclusion, our studies seem to indicate that the chromosome 12 breakpoints of myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands are all clustered within the 7-cM interval between D12S19 and D12S8, with those of the benign solid tumors distal to CHOP. Finally, the MYF5 gene mapped telomeric to LLNL12NCO1-113D12, and the MIP gene mapped centromeric to the chromosome 12 translocation breakpoint in myxoid liposarcoma cells.

Regional mapping of the human NSP gene to chromosome region 14q21-->q22 by fluorescence in situ hybridization analysis.

Genetic sequences of the novel NSP gene, which encodes neuroendocrine-specific proteins, were isolated from cDNA libraries constructed with mRNA isolated from human lung carcinoma cells. Hybridization analysis of a panel of human x mouse cell hybrids with an 0.8-kb NSP cDNA probe indicated that the human NSP gene is probably located on chromosome 14. Fluorescence in situ hybridization analysis of metaphase chromosomes using overlapping genomic clones of NSP as a probe localized the NSP gene to chromosome region 14q21-->q22.

Rearrangement of 12q14-15 in pulmonary chondroid hamartoma.

Cytogenetic analysis of a pulmonary chondroid hamartoma showed an inv(12)(p13q14-15) as the sole abnormality. This finding together with previously reported data is an indication that the 12q14-15 region may be nonrandomly involved in the pathogenesis of this tumor type.