Abstracts

A third P-domain peptide gene (TFF3), human intestinal trefoil factor, maps to 21q22.3

Holger Schmitt, Iris Wundrack, Stefanie Beck, Peter Goett, Cornelius Welter, Hiroaki Shizuya, Melvin I. Simon and Nikolaus Blin

Small peptides displaying a cysteine-rich module (termed P-domain or trefoil motif) form a recently increasing group of peptides abundantly expressed at mucosal surfaces of specific tissues and are associated with the maintenace of surface integrity. The estrogen-inducible pS2/BCEI gene and the human homolog to the porcine spasmolytic peptide gene (hSP/SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be localized at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene (TFF3) were described recently. By using suitable oligonucleotide primers and PCR in a somatic hybrid cell panel and furtheron, isolating two large (110 kb, 210 kb) genomic recombinants cloned in the Bacterial Artificial Chromosome (BAC) system and fluorescence in situ hybridization (FISH), we have now shown that this gene sequence coding for the new member of human P-domain/trefoil peptides also maps to chromosomal region 21q22.3 suggesting a physical linkage of all three trefoil peptide genes.

Cytogenet Cell Genet 72: 299-302 [1996]

A family with a germline mutation of the RET proto-oncogene with multiple endocrine neoplasia type 2A (MEN2A)

Wundrack, I., Reichert, J., Langer, H., Leicht, E., Herrmann, M., Subke, F., Meese, E. and Blin N.

MEN2A, a dominantly inherited cancer syndrome, is defined by the presence of medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. Along with MEN2B and familial medullary thyroid carcinoma (FMTC) it is associated with germline mutations of the RET proto-oncogene localized in10q11.2. In FMTC and MEN2A, point mutations result in the substitution of one of five Cys residues in the extracellular domain of RET. In a larger pedigree from Saarland, several individuals were observed with C-cell thyroid carcinoma. We screened 16 members of this extended family by single strand conformation assay, PCR followed by restriction enzyme analysis and by sequencing the mutated regions. In 7 family members, all of whom had been earlier operated on because of MTC, a DNA transition from T to C was observed, causing an amino acid subsitution Cys ⁶³⁴ Arg. Nine members of the kindred did not carry the mutation and may be excluded from yearly biochemical testing. One of these persons seems to have been unnecessarily operated on due to a borderline pentagastrin test.

Endocrin Pathol 7: 71-76 [1996]

pS2 protein expression in gastric carcinoma. An immunohistochemical and immunoradiometric study

Jose-Carlos Machado, Fatima Carneiro, Paula Ribeiro, Nikolaus Blin, Manuel Sobrinho-Simoes

We analysed pS2 protein expression in 50 gastric carcinomas and respective adjacent mucosas by immunohistochemistry and immunoradiometric assay (IRMA). pS2 was consistently expressed in superficial and foveolar epithelium of non-neoplastic mucosa and in 66.0% of the carcinomas. pS2 immunoreactivity was significantly higher in diffuse than in intestinal carcinomas and in cases with nodal metastases than in those without. No correlation was found between pS2 immunostaining and gender, age, staging, wall penetration, venous invasion, ploidy and S-phase fraction. The mean levels of pS2 (IRMA) were significantly lower in gastric carcinomas than in non-neoplastic mucosas and were not correlated with any of the aforementioned clinicopathologic features. The survival of patients with pS2-positive tumours was not significantly different from that of patients with pS2-negative tumours. We conclude that pS2 expression, which can be used as a marker of gastric-type differentiation, is associated with gastric carcinoma of diffuse type. The lack of correlation between pS2 expression and most features of tumour aggressiveness and patients' survival precludes its use as a prognostic tool in gastric carcinoma.

Eur J Cancer 32A: 1585-1590 [1996]

Distribution and linkage of repetitive clusters from the heterochromatic region of human chromosome 22

Roman Muellenbach, Carsten Pusch, Karlheinz Holzmann, Ron Suijkerbuijk, Nikolaus Blin

The pericentric regions of eukaryotic chromosomes consist of several types of repetitive DNA families. In human chromosome 22, the organization of such families was studied in more detail. In addition to the known families of alpha and beta repeats an additional repeat with a 48 bp motif was previously assigned to 22pter-q11. Here, we report in more detail the distribution of these repeat families applying pulsed field gel electrophoresis, fluorescence in situ hybridization and physical linkage on cosmid recombinants. At least two clusters of 48 bp repeats are localized on chromosome 22: one on the distal p-arm and one in the region 22cen-q11, respectively. Cosmids from a chromosome 22 library containing both, 48 bp and beta repeats, link both arrays on 22p and define their maximum distances to less than 44 kb. Loss of 48 bp repeat sequences in a DiGeorge cell line carrying a deletion in 22q11 suggests the presence of a second cluster in 22q11, a distribution supported by FISH signal analysis. Since additional members of the 48 bp repeat family can be found on all acrocentric chromosomes it remains to be determined whether the distribution seen on chromosome 22 is also common in other human acrocentric chromosomes.

Chromos Res 4: 282-287 [1996]

Framework for a physical map of the human 22q13-region using Bacterial Artificial Chromosomes (BACs)

Holger Schmitt , Ung-Jin Kim, Tatiana Slepak, Nikolaus Blin, Melvin I. Simon and Hiroaki Shizuya

Detailed physical maps of entire chromosomes based on combined genetic, cytogenetic and structural information are essential components for positional cloning, and genomic sequencing. Despite the wealth of genetic information of the known diseases in the chromosome 22q13, the construction of a detailed physical map of the terminal region is difficult due to the sparsity of the genetic markers. We present here a map of BAC contigs that cover a number of genetic loci in 22q13 region. One hundred thirty-six BACs with an average insert size of 140 kb are assembled into 35 contigs defined by 64 markers in 22q13-qter. Twenty-three anonymous markers are now linked to the previously mapped genetic anchor points.

Genomics 33: 9-20 [1996]

Cloning of contiguous genomic fragments from human chromosome 21 harbouring three trefoil peptide genes

Stefanie Beck, Holger Schmitt, Hiroaki Shizuya, Nikolaus Blin, Peter Goett

A group of small peptides with a typical cysteine-rich domain (termed trefoil motif or P-domain) is abundantly expressed at mucosal surfaces of specific normal and neoplastic tissues. Their association with the maintenance of surface integrity was suggested. The first known human trefoil peptide (pS2) was isolated from breast cancer cells (MCF7). Its oestrogen-inducible gene, and the human homologue to the porcine spasmolytic peptide gene (hSP/SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be localised at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene were described recently. By using suitable oligonucleotide primers and PCR and isolating large (110-250 kb) genomic recombinants cloned in the bacterial artificial chromosome (BAC) system, we present a genomic region from chromosome band 21q22.3 cloned in contiguous sequences and encoding all three members of human P-domain/trefoil peptides proving a physical linkage of all three trefoil peptide genes. Such genomic sequences will provide useful experimental material for analysis of gene regulation, for gene modification experiments and for establishing transgenic cells or animals.

Hum Genet 98: 233-235 [1996]

Cosmid-derived transcripts and sequence tags mapped to three subregions of human chromosome 22

Carsten Pusch, Roman Mⁿllenbach, Peter Goett, Holger Schmitt, Zhili Wang, Bruce Roe, Nikolaus Blin

Fifty cosmids from the ICRF, London, and Lawrence Livermore Laboratory, California, human chromosome 22 cosmid libraries were isolated, regionally assigned and tested for their ability to detect repeats or single copy sequences. The search resulted in nine cosmids containing repetitive motifs from the pericentric region of chromosome 22. An additional 19 cosmids, that detected single copy sequences in the long arm of chromosome 22q: 7 in the region 22q11.2-q13.1 and 12 in 22q13.1-qter, were mapped more precisely by fluorescence in situ hybridization. Three out of these 19 recombinants displayed restriction fragments containing (CA)n repeats, were subcloned and sequenced. One cosmid, representing a region coding for an ubiquitous 300 bp transcript, is localized 600 kb from PDGFB, and four cosmids contained sequences surrounding the ARSA gene at 22q13.3. Presently, long range physical maps, that may be useful for analysing structural alterations of chromosome 22q13, are being constructed from these additional, regionally assigned markers from chromosome 22q13 employing both existing cosmid and new bacterial artificial chromosome (BAC) libraries.

Gene , in press

Pattern of pS2 protein expression in premalignant and malignant lesions of gastric mucosa

Jose-Carlos Machado, Fatima Carneiro, Nikolaus Blin, Manuel Sobrinho-Simoes

The aim of this study was to evaluate the pattern of pS2 protein expression in premalignant and malignant lesions of gastric epithelium. We analysed, by immunohistochemistry, the pS2 expression in 6 samples of normal gastric mucosa, 18 cases of chronic atrophic gastritis with intestinal metaplasia (IM), 10 hyperplastic polyps, 11 adenomatous polyps, and 50 gastric carcinomas together with the respective samples of adjacent non-neoplastic mucosa. pS2 is expressed throughout foveolar and superficial epithelium of normal gastric mucosa and this pattern is retained in chronic atrophic gastritis, out of IM lesions. pS2 expression is confined to goblet cells in complete IM and occurs both in goblet and columnar cells in incomplete IM. Hyperplastic polyps displayed significantly higher pS2 expression than adenomatous polyps. In gastric carcinomas, pS2 expression was observed in 66.0% of the cases, being significantly higher in diffuse (88.9%) than in intestinal type carcinomas (53.6%). A subset of carcinomas of the latter group displayed pS2 immunoreactivity in a high percentage of cells with a pattern similar to that of hyperplastic polyps. Our results demonstrate there are major changes of pS2 expression, which can be used as a marker of gastric-type differentiation, along the process of gastric carcinogenesis and support the existence of at least two pathways of malignant transformation of gastric mucosa: one via intestinal metaplasia and adenomatous dysplasia, leading to glandular carcinomas with intestinal-type differentiation; the other via hyperplastic changes, or de novo, leading to diffuse carcinomas and to a subset of glandular carcinomas displaying gastric-type differentiation.

Eur J Cancer Prev 5: 169-179 [1996]

Human trefoil peptides: Genomic structure in 21q22.3 and coordinated expression

Peter Goett, Stefanie Beck, Jose Carlos Machado, Fatima Carneiro, Holger Schmitt, and Nikolaus Blin

Trefoil peptides are small secretory proteins characterized by three intrachain disulfide bonds forming the trefoil motif or P-domain. They are abundantly expressed at mucosal surfaces, especially of the gastrointestinal tract. In pathological conditions like ulcera, meta- and neoplasia their expression is upregulated. Three human trefoil peptideshave been described: the oestrogen inducible pS2 protein, the spasmolytic protein and the intestinal trefoil factor. Recently, their role in maintenance of surface integrity and ulcer healing was discussed. We already mapped the corresponding three genes (BCEI, SML1, TFF3) to the same genomic region (21q22.3). Here we show that the three genes are clustered in a tandemly oriented fashion within 50 kb on a Bacterial Artificial Chromosome (BAC) recombinant. This cluster is located adjacent to D21S19 and the locus order is cen-D21S221-TFF3-SML1-BCEI-D21S19-tel, whereas transcription of all three genes is directed towards the centromere. The gene structure of SML1 exhibits 4 exons, two of which encode the two separate trefoil motifs. TFF3 and BCEI, both containing one trefoil motif, are composed of three exons each suggesting gene duplication and exon-shuffling events during evolution. The 5'-flanking region of SML1 was compared to the corresponding region of other trefoil genes. Two motifs with identical sequence and positions are shared between SML1 and BCEI, thus presenting possible targets for stomach specific gene regulation. Two other motifs are shared within all known human and rat trefoil genes suggesting a coordinated regulation and/or a common locus controlling region. Using RT-PCR, a change in the pattern of trefoil gene expression is detected in tissue samples from normal gastric mucosa, hyperplastic polyps, gastric cancer, and gastric cancer cell lines, respectively.

Eur J Hum Genet , in press

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