The human genome project and the discovery of genetic determinants of cancer susceptibility.

The Human Genome Project has recently provided a great deal of information on the sequence that comprises the human genome. We are now in the process of structuring and deciphering the 3 x 10(9) base sequence in order to gain insights into its functional role. Several efforts are focusing on the search for DNA sequence variations underlying common/complex diseases that constitute a real burden in terms of public health measures. As expected, the genetic architecture of these complex traits, shows tremendous complexity, and the discovery and characterisation of susceptibility alleles constitute a real challenge for the geneticist. Conceptual and experimental genetic approaches aimed at dissecting the molecular features of susceptibility genes, in particular those predisposing to cancer, are outlined and discussed in this review.

Chromosome 6 abnormalities in ovarian surface epithelial tumors of borderline malignancy suggest a genetic continuum in the progression model of ovarian neoplasms.

PURPOSE: We used conventional cytogenetics, molecular cytogenetics, and molecular genetic analyses to study the pattern of allelic loss on chromosome 6q in a cohort of borderline epithelial ovarian tumors. EXPERIMENTAL DESIGN: Fifteen tumor samples were collected from patients undergoing surgery for ovarian tumors. The tumors of borderline malignancy, classified according to the standard criteria, included 4 mucinous and 11 serous tumors. Cytogenetic and molecular cytogenetic (with yeast artificial chromosome clones from 6q26-27) studies were performed on tumor areas contiguous to those used for histological examination ensuring the appropriate sampling. Moreover loss of heterozygosity analysis was performed using PCR amplification of eight microsatellite markers mapping on 6q27 (D6S193, D6S297), 6q26 (D6S305, D6S415, D6S441), 6q21 (D6S287), 6q16 (D6S311), and 6q14 (D6S300). RESULTS: Deletions of this chromosome arm, in particular of 6q24-27, were the most frequent lesions found in this set of tumors. In a tumor with a normal karyotype the only detectable alteration was a deletion of approximately 300 kb within the D6S149-D6S193 interval at band 6q27. This is, to date, the smallest deletion described for borderline tumors. CONCLUSION: Alterations in the above-mentioned interval are a common finding in advanced ovarian carcinomas but also in benign ovarian cysts, implying that some tumors of borderline malignancy may arise from benign tumors and that malignant ones may evolve from tumors of borderline malignancy. Genes located in 6q27 seem to be crucial for this mechanism of early events in ovarian tumorigenesis.

Cloning and characterization of a senescence inducing and class II tumor suppressor gene in ovarian carcinoma at chromosome region 6q27.

Cytogenetic, molecular and functional analysis has shown that chromosome region 6q27 harbors a senescence inducing gene and a tumor suppressor gene involved in several solid and hematologic malignancies. We have cloned at 6q27 and characterized the RNASE6PL gene which belongs to a family of cytoplasmic RNases highly conserved from plants, to man. Analysis of 55 primary ovarian tumors and several ovarian tumor cell lines indicated that the RNASE6PL gene is not mutated in tumor tissues, but its expression is significantly reduced in 30% of primary ovarian tumors and in 75% of ovarian tumor cell lines. The promoter region of the gene was unaffected in tumors cell lines. Transfection of RNASE6PL cDNA into HEY4 and SG10G ovarian tumor cell lines suppressed tumorigenicity in nude mice. When tumors were induced by RNASE6PL-transfected cells, they completely lacked expression of RNASE6PL cDNA. Tumorigenicity was suppressed also in RNASE6PL-transfected pRPcT1/H6cl2T cells, derived from a human/mouse monochromosomic hybrid carrying a human chromosome 6 deleted at 6q27. Moreover, 63.6% of HEY4 clones and 42.8% of the clones of XP12ROSV, a Xeroderma pigmentosum SV40-immortalized cell line, transfected with RNASE6PL cDNA, developed a marked senescence process during in vitro growth. We therefore propose that RNASE6PL may be a candidate for the 6q27 senescence inducing and class II tumor suppressor gene in ovarian cancer.

Human allantoicase gene: cDNA cloning, genomic organization and chromosome localization.

Uric-acid-degrading enzymes (uricase, allantoinase, allantoicase, ureidoglycolate lyase and urease) were lost during vertebrate evolution and the causes for this loss are still unclear. We have recently cloned the first vertebrate allantoicase cDNA from the amphibian Xenopus laevis. Surprisingly, we have found some mammalian expressed sequence tags (ESTs) that show high similarity with Xenopus allantoicase cDNA. From a human fetal spleen cDNA library and adult kidney EST clone, we have obtained a 1790 nucleotide long cDNA. The 3' end of this sequence reveals a substantial high identity with the corresponding portion of Xenopus allantoicase cDNA. In contrast, at the 5' end the human sequence diverges from that of Xenopus; since no continuous open reading frame can be found in this region, the hypothetical human protein appears truncated at its N-terminus. We proposed that such a transcript could be due to an incorrect splicing mechanism that introduces an intron portion at the 5' end of human cDNA. Allantoicase cDNA is expressed in adult testis, prostate, kidney and fetal spleen. By comparison with available genomic sequences deposited in database, we have determined that the human allantoicase gene consists of five exons and spans 8kb. We have also mapped the gene in chromosome 2.

Xenopus allantoicase: molecular cloning, enzymatic activity and developmental expression.

Allantoicase is one of the enzymes of the purine degradation pathway and, interestingly, it appears to be lost, together with uricase and allantoinase, during mammalian evolution. Only allantoicases from the ascomycetes S. pombe, S. cerevisiae, and N. crassa have already been cloned, although the activity has been reported also in fishes and amphibians. By screening a cDNA expression library of Xenopus liver, we have cloned a 1491-bp-length cDNA coding for a 389 amino acid protein that shows an high similarity with the enzyme allantoicase. We have found that allantoicase mRNA is abundantly expressed in kidney and liver, but at much lower level is also present in brain, testis, intestine, and lung. We have detected enzymatic activity in crude extract from kidney, liver, and lung; we have also determined kinetic parameters (K(m) = 8.44 mM, V(max) = 6. 94 micromol min(-1) per mg protein) in kidney. During embryo development, we have detected allantoicase transcript and activity starting from 1 and 5 days after fertilization, respectively.

A large 6q deletion is a common cytogenetic alteration in fibroadenomas, pre-malignant lesions, and carcinomas of the breast.

To assess whether early breast lesions are the precursors of invasive carcinomas, three classes of breast lesions, namely benign tumors (including fibroadenomas), putative premalignant lesions (including cases of atypical hyperplasia), and invasive carcinomas, were compared at the cytogenetic and molecular cytogenetic levels. Genetic relatedness was clearly demonstrated by the sharing of several anomalies, among which 6q deletions outnumbered all of the other alterations detected. Indeed, deletions of the long arm of chromosome 6, most likely occurring in epithelial cells, were present in 83.9% of benign breast tumors, 64% of putative premalignant lesions, and 77.4% of analyzable carcinomas. Furthermore, the interval between 6q24 and qter appeared to be the common region of deletion in all three classes of breast lesions, whereas the minimal common region of deletion was 6q27-qter. Interestingly, the latter region was reported previously to be deleted in benign ovarian tumors and recently found to harbor a gene (SEN6) that is important for SV40-mediated immortalization of human cells.

The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the down critical region.

We applied cDNA selection methods to a genomic clone (YAC 761B5) from chromosome 21 located in the so-called 'Down critical region' in 21q22.3. Starting from human fetal heart and brain mRNAs we obtained and sequenced several cDNA clones. One of these clones (Down region aspartic protease (DRAP), named also BACE2 according to the gene nomenclature) revealed a striking nucleotide and amino acid sequence identity with several motifs present in members of the aspartic protease family. In particular the amino acid sequences comprising the two catalytic sites found in all mammalian aspartic proteases are perfectly conserved. Interestingly, the predicted protein shows a typical membrane spanning region; this is at variance with most other known aspartic proteases, which are soluble molecules. We present preliminary evidence, on the basis of in vitro translation studies and cell transfection, that this gene encodes a glycosylated protein which localizes mainly intracellularly but to some extent also to the plasma membrane. Furthermore DRAP/BACE2 shares a high homology with a newly described beta-secretase enzyme (BACE-1) which is a transmembrane aspartic protease. The implications of this finding for Down syndrome are discussed.

Exon structure and promoter identification of STIM1 (alias GOK), a human gene causing growth arrest of the human tumor cell lines G401 and RD.

The stromal interaction molecular 1 gene (STIM1) encodes a type I trans-membrane protein of unknown function, which induces growth arrest and degeneration of the human tumor cell lines G401 and RD but not HBL100 and CaLu-6, suggesting a role in the pathogenesis of rhabdomyosarcomas and rhabdoid tumors. Here, we describe the STIM1 genomic organization including the identification of the promoter region. The gene consists of 12 exons that span a region larger than 250 kb between the genes RRM1 and NUP98. Nucleotide sequences of all exon-intron boundaries were determined and oligonucleotide primers for the amplification of individual exons were designed. The promoter region was identified within a 1.8-kb SacI fragment at the 5' end of the gene. In vitro CpG methylation of the promoter region indicated that transcription can be downregulated by this mechanism. The genetic tools developed in the present work will help to determine whether pathogenetic mechanisms that associate STIM1 with tumorigenesis involve mutations in coding sequences and/or promoter, and whether methylation could determine STIM1 transcriptional down-regulation in tumor samples.

Transgenic mice expressing a human apolipoprotein[a] allele.

The most important determinant of plasma levels of Lp[a] are sequence differences at the highly polymorphic apolipoprotein[a] (apo[a]) locus. To define the sequences that mediate the regulation of apo[a] expression, we cloned a 370 kb DNA fragment that included a 130 kb apo[a] gene, and 40 kb 5'- and 200 kb 3'-flanking region from an individual with high plasma levels of Lp[a] using a YAC vector. This genomic clone was used to generate transgenic mice. In the YAC-apo[a] transgenic mouse, apo[a] was only expressed in the liver, as it is in humans. The mean serum level of apo[a] in 4-week-old YAC-apo[a] transgenic mice was 20 mg/dl. In the female mice the levels of apo[a] varied over a 1.5-fold range during the 4-day estrus cycle and the levels correlated directly with serum progesterone levels. The serum levels of apo[a] decreased to almost undetectable level in male mice after puberty and this decrease was reversed by castration. Ingestion of a high-fat diet resulted in a approximately 100-fold fall in hepatic apo[a] mRNA levels and >60-fold decrease in serum apo[a] levels. To delimit the control elements that mediate tissue-specific and sex hormone-responsive apo[a] transcription, we derived a reporter YAC in which 40 kb of 5' flanking sequences from the cloned apo[a] allele were linked to a luciferase reporter gene. Analysis of four independent transgenic lines revealed no hepatic luciferase expression, suggesting that important cis -acting elements located outside the apo[a] 5'-flanking region are necessary for in vivo expression of apo[a].

Microsatellite instability in endometrial cancer: relation to histological subtypes.

Fifty-one endometrial cancers were analyzed with regard to whether or how microsatellite instability (MI) was associated with the development of different types of endometrial malignant neoplasms. We investigated 6 loci previously reported as informative for colorectal cancer and a group of 8 loci located on 6q. Replication error (RER+) phenotype was detected in 10 of 51 (19.6%) endometrial cancers (ECs), all but one of which showed endometrioid differentiation. On the contrary, the RER+ phenotype was not detected in serous carcinomas and malignant mixed Müllerian tumors. MI was present in both early and advanced stage ECs. No correlation was found between age, grade, stage, familial pattern, mitotic index, and the RER+ phenotype of ECs. Only 1 of 8 endometrial carcinomas showing MI was associated with mutant p53 expression, while the majority of RER+ tumors were positive for estrogen and progesterone receptors. Our findings suggest that MI plays an early role in endometrial tumorigenesis and is significantly correlated with adenocarcinomas showing endometrioid features (EAs). The frequent involvement of the telomeric region of chromosome 6 in the MI of EA is an indication that this region may be crucial in the process of EA tumorigenesis.

Refinement of the LOH region 1 at 11q23.1 deleted in human breast carcinomas and sublocalization of 11 expressed sequence tags within the refined region.

Loss of constitutive heterozygosity at 11q23 has been detected in various human solid tumors. Here, we described the analysis of a series of normal and tumor pairs from 110 breast carcinomas for the presence of loss of heterozygosity at 11q23 loci. The overall frequency of LOH was 48%, confirming the importance of deletions at 11q23 in breast tumorigenesis. Previously, we have identified two independent regions of LOH at 11q23, the LOH region 1 at 11q23.1 and the LOH region 2 at 11q23.3. The most telomeric region was recently refined between loci D11S1345 and D11S1316, a region of about 1 Mb. However, the LOH region 1, most centromeric, was still not finely refined: the boundaries were defined by loci D11S2000 and D11S897, separated by about 8 Mb. Here, we refined its boundaries between loci D11S1347 and D11S927, a region of about 2 Mb. We have mapped 11 expressed sequence tags (ESTs) within this region and excluded another 20. This study represents a further step toward the identification of the putative tumor suppressor gene found within the LOH region 1 at 11q23.1.

Physical map of the D6S149-D6S193 region on chromosome 6Q27 and its involvement in benign surface epithelial ovarian tumours.

A detailed long range restriction map of the region defined by markers D6S149 and D6S193 on chromosome 6q27 has been constructed. This was achieved by YAC cloning and contig assembling of the same region. Seven YAC clones were found to span the almost 1000 Kb region flanked by the two markers which on the genetic map resulted to be 1.9 cM apart. With some of the characterized YAC clones we undertook a molecular cytogenetic analysis of 20 benign ovarian tumors. The rationale for this was the recent mapping to a region of chromosome 6q27, flanked by markers D6281 and D6S133, of a locus for the SV40-mediated immortalization of human cells (SEN6 gene). Noteworthy we found that the the D6S149-D6S193 region (comprised in the larger D6S281-D6S133 physical interval) was altered in all samples analysed adding support to the occurrence of a immortalization step in this type of tumors.

Human NRD convertase: a highly conserved metalloendopeptidase expressed at specific sites during development and in adult tissues.

We report the cloning of the human homologue of the rat metalloprotease N-arginine dibasic convertase (NRD convertase). This endopeptidase is responsible for the processing, at the Arg-Lys dibasic site on the N-terminal side of the arginine residue, of propeptides and proproteins. Comparisons of the human and rat full-length cDNAs show similarity and identity of 94 and 91%, respectively. In humans NRD convertase is predominantly expressed in heart, skeletal muscle, and testis. We have also studied the expression of this gene in mouse at various developmental stages and found that the neural tissue is the almost exclusive site of expression in early development (between E 10.5 and E 16.5). To gain information about the possibility that defects in this gene are linked to inherited neuromuscular disorders, we determined the chromosomal location of the human NRD convertase gene by FISH analysis, showing that the gene resides at 1p32.2.

Characterization of multiple enhancer regions upstream of the apolipoprotein(a) gene.

Plasma concentrations of the atherogenic lipoprotein(a) (Lp(a)) are predominantly determined by inherited sequences within or closely linked to the apolipoprotein(a) gene locus. Much of the interindividual variability in Lp(a) levels is likely to originate at the level of apo(a) gene transcription. However, the liver-specific apo(a) basal promoter is extremely weak and does not exhibit common functional variations that affect plasma Lp(a) concentrations. In a search for additional apo(a) gene control elements, we have identified two fragments with enhancer activity within the 40-kilobase pair apo(a)-plasminogen intergenic region that coincide with DNase I-hypersensitive sites (DHII and DHIII) observed in liver chromatin of mice expressing a human apo(a) transgene. Neither enhancer exhibits tissue specificity. DHIII activity was mapped to a 600-base pair fragment containing nine DNase I-protected elements (footprints) that stimulates luciferase expression from the apo(a) promoter 10-15-fold in HepG2 cells. Binding of the ubiquitous transcription factor Sp1 plays a major role in the function of this enhancer, but no single site was indispensable for activity. DHIII comprises part of the regulatory region of an inactive long interspersed nucleotide element 1 retrotransposon, raising the possibility that retrotransposon insertion can influence the regulation of adjacent genes. DHII enhancer activity was localized to a 180-base pair fragment that stimulates transcription from the apo(a) promoter 4-8-fold in HepG2 cells. Mutations within an Sp1 site or either of two elements composed of direct repeats of the nuclear hormone receptor half-site AGGTCA in this sequence completely abolished enhancer function. Both nuclear hormone receptor elements were shown to bind peroxisome proliferator-activated receptors and other members of the nuclear receptor family, suggesting that this enhancer may mediate drug and hormone responsiveness.

Genomic structure and organization of kringles type 3 to 10 of the apolipoprotein(a) gene in 6q26-27.

Apolipoprotein(a) [apo(a)] is a highly polymorphic glycoprotein covalently linked to the apolipoprotein B-100 of LDL in a particle called lipoprotein(a) [Lp(a)]. High plasma levels of Lp(a) are associated with coronary as well as peripheral atherosclerosis. Plasma levels of Lp(a) show a remarkable variation ranging from 0.1 mg/dl to over 100 mg/dl. The apo(a) gene shows a size polymorphism which resides in the variable number of kringle domains which resemble plasminogen kringle IV. Ten different types of kringle IV repeats have been described, nine of which (kringle IV type 1 and type 3-10) are each supposed to be present in a single copy. The other kringles, namely kringle IV type 2 repeats, vary in number from 3 to 42 between apo(a) alleles and form the basis for the apo(a) size polymorphism. Although an inverse relationship has been observed between the number of kringle type 2 repeats and plasma levels of Lp(a), there are exceptions to this general finding. Indeed, several individuals have been described with similar apo(a) size alleles but very different plasma levels of Lp(a). Genetic studies have linked these differences to the apo(a) locus on 6q26-27, outlining the importance, besides the kringle type 2 repeats, of other regions of the apo(a) gene in contributing to the interindividual differences in the plasma concentration of Lp(a). One of the candidate regions is represented by the non-repeated type-3 to type-10 kringles which are invariably present in each apo(a) allele and whose structural integrity is playing a critical role in the correct assembly of the Lp(a) particle. Biochemical studies with recombinant wild type and mutagenized apo(a) cDNAs with several alterations of the non-repeated kringles have well documented this latter point. As a starting point to search for genetic variations in these kringles associated with different levels of Lp(a), we are presenting the genome organization of type-3 to 10 kringle along with specific PCR primers for easy analysis from genomic DNA. Restriction as well as partial sequencing analyses of the type-3 to 10 kringles region has also provided interesting clues as to the different evolutionary origin of these types of kringle with respect to the polymorphic type-2 kringles.

Mammalian Rh/T2/S-glycoprotein ribonuclease family genes: cloning of a human member located in a region of chromosome 6 (6q27) frequently deleted in human malignancies.

We describe the cloning of a novel human gene belonging to the Rh/T2/S-glycoprotein class of extracellular ribonucleases. This gene is present in a single copy in the human genome and has been mapped to 6q27, a region of the human genome prone to rearrangements associated with several human malignancies. The predicted open reading frame of the human cDNA encodes a protein of 191 amino acids, and the pattern of expression is ubiquitous. Some of the sequence features of this gene, in particular those corresponding to the bipartite RNase motif of the active site, are perfectly conserved between distant species such as human and the plant Lycopersicon esculentum. No mammalian homologues have been described so far, and this report presents for the first time both the human and the mouse sequences of the corresponding members of this class of highly conserved extracellular ribonucleases.

Cloning a new human gene from chromosome 21q22.3 encoding a glutamic acid-rich protein expressed in heart and skeletal muscle.

The identification and functional characterization of genes on chromosome 21 is a necessary step to understand the pathogenesis of the various phenotypic anomalies that affect Down syndrome patients. Using direct cDNA selection we have identified a new gene, SH3BGR, that maps to 21q22.3, proximal to HMG14, and is differentially expressed in heart and skeletal muscle. SH3BGR encodes a novel protein that is characterized by the presence of a proline-rich region containing the consensus sequence for a SH3-binding domain and by an acidic carboxyl-terminal region containing a glutamic acid-rich domain predicted to assume a coiled coil. The presence of two functional domains involved in protein-protein interactions suggests that SH3BGR could be part of a multimeric complex. Its overexpression might alter specific functions of muscular tissue and therefore take part in the pathophysiology of muscular hypotonia in Down syndrome.

Involvement of chromosome 6 in endometrial cancer.

Cytogenetic investigation was performed on direct preparations of 15 endometrial cancers showing different histotypes. Clonal abnormalities were found in 11 out of 13 analysable cases. The modal chromosome number was near diploid in all cases. The abnormal karyotypes contained relatively simple numerical or structural aberrations in the majority of tumours. In contrast, two neoplasms with serous papillary and mixed mullerian morphological features shared multiple complex changes as well as cytogenetic evidence of intratumoral heterogeneity. The most frequent chromosome abnormality in our series of endometrial neoplasms was 6q deletion, which was detected in serous papillary, endometrioid and mixed mullerian tumours. The loss of the 6q region, which is also frequently involved in ovarian carcinoma, suggests a relationship between endometrial and ovarian cancers based on a common histogenesis.