Chromosome 9p21 Region Research Articles

Positive tetracycline control of expression of p15INK4B from an Epstein–Barr autonomous plasmid in a human melanoma cell line

Homozygous deletions in the region of chromosome 9p21 are frequent in human melanoma. Mutations in the p16INK4A cyclin-dependent kinase inhibitor (CDI) gene at this locus have implicated the product of this gene as a tumor suppressor. Less attention has been focused on the homologous, closely linked p15INK4B gene. To facilitate study of the phenotypic effects of restoring expression of the latter in aggressive melanoma cells lacking INK4 expression, we inserted the cDNA encoding p15INK4B into an autonomously maintained plasmid under positive tetracycline control (‘TET ON’ system). Similarly regulated luciferase and herpes thymidine kinase sequences were used as controls. We demonstrate that this system enabled efficient, and reasonably uniform, induction of p15INK4B expression in a human melanoma cell line exposed to the tetracycline derivative, doxycycline. Flow cytometry showed that this induction resulted in substantial accumulation of cells in the G0/G1 phase of the cell cycle. This system will facilitate detailed analysis of the cell cycle inhibitory mechanisms of this CDI in human melanoma cells.

PMM2 mutation spectrum, including 10 novel mutations, in a large CDG type 1A family material with a focus on Scandinavian families.

Carbohydrate-deficient glycoprotein syndrome type IA (CDG IA) is an autosomal recessive disease characterized clinically by severe involvement of the central and peripheral nervous system, and biochemically by complex defects in carbohydrate residues in a number of serum glycoproteins. CDG IA is caused by mutations in the PMM2 gene located in chromosome region 16p13. In this study, 61 CDG type IA patients (122 chromosomes) were screened for mutations in the PMM2 gene using a combination of SSCP and sequence analysis. More than 95% of the mutations could be detected. All of them were missense mutations. Mutations 422G>A and 357C>A were strikingly more common in the material and comprised 58% of mutations detected. Of the 20 mutations found, 10 were not reported previously. Seven mutations, e.g. 26G>A (five alleles) and 548T>C (seven alleles), were found only in Scandinavian families. The most common genotype was 357C>A/422G>A (36%). Three patients were homozygous, 357C>A/357C>A (two cases), and 548T>C/548T>C (one case). No patients homozygous for the most common mutation 422G>A were detected. The different mutations were clustered e.g., in that most were located in exon 5 (five) and exon 8 (six), while no mutation was detected in exon 2. When the frequencies of each mutation were included, exon 5 comprised 61% (65 chromosomes) of the mutations; in Scandinavian patients the frequency of these mutations was 72%. Thus, analysis of exon five in these patients enables both reliable and time-saving first screening in prenatal diagnostic cases. This could be followed by a second step of additional strategies for the detection of other mutations.

Analysis of p73 and p53 gene deletions in multiple myeloma.

Recently, p73, a protein with structural and functional similarities to p53, an extensively studied tumor suppressor gene, has been cloned. After being mapped to the chromosomal region 1p35-1p36, it has been postulated to act as a tumor suppressor gene, too, as this region is altered in several human malignancies. Deletions of the short arm of chromosome 1 have frequently been described in multiple myeloma (MM) whereas structural abnormalities of the 17p13 region including p53 are rare events in this disease. Since it has been proposed that especially neoplasias lacking p53 alterations might show a loss of heterozygosity at 1p35-1p36, we studied the frequency of p53 and p73 deletions in bone marrow mononuclear cells of 68 patients with MM, two patients with monoclonal gammopathy of undetermined significance and four patients with plasma cell leukemia. Dual-color fluorescence in situ hybridization (FISH) for p53 and p73 was performed using commercially available DNA probes for 17p13.3 and the microsatellite marker D1Z2, respectively. Centromeric DNA probes served to distinguish gene deletions from whole chromosome losses. In contrast to recently published FISH results, we only detected heterozygous p53 deletions in eight out of the 74 patients, three of them showing a monosomy 17. Heterozygous deletions of the D1Z2 region at 1p36 were found in six cases with one patient having a monosomy 1. Neither homozygous deletions of either chromosomal region nor nullisomies 1 or 17 could be detected. These results argue against a major role of p73 deletions in MM. As MM patients with 1p structural abnormalities have a significantly poorer survival rate than those with normal karyotypes, the role of other putative tumor suppressor genes located at the chromosomal region 1p36 in the pathogenesis of MM has to be determined.

The Molecular Basis of Congential Artrichia in Humans and Mice: Mutations in the Hairless Gene

Congenital atrichia is a form of total alopecia inherited in an autosomal recessive pattern. In individuals affected with this form of hair loss, hairs are typically absent from the scalp, and patients are nearly completely devoid of eyebrows, eyelashes, axillary and pubic hair, following shedding of the natural hair shortly after birth. We have recently linked this disorder to the chromosomal region 8p12, and cloned the human hairless gene, which resides within this interval. We have identified several mutations in the hairless gene in atrichia families from around the world. In hairless mice, the hair matrix cells appear to undergo a premature and massive apoptosis, together with a concomitant decline in Bcl-2 expression, a loss of NCAM positivity, and a disconnection with the overlying epithelial sheath essential for the movement of the dermal papilla. As a consequence, the hair bulb and dermal papilla remain stranded in the dermis, and indispensible messages between the dermal papilla and stem cells in the bulge are not transmitted, so no further hair growth occurs. These findings suggest that the hairless gene product may play a crucial role in maintaining the delicate balance between cell proliferation, differentiation and apoptosis in the hair follicle, as well as in the interfollicular epidermis.

Multiple MSP Pseudogenes in a Local Repeat Cluster on 1p36.2: An Expanding Genomic Graveyard?

Chromosomal region 1p36.2 harbors an intriguing gene cluster of about 1 Mb. In addition to normal high-copy-number repeats, this cluster consists entirely of locally repeated sequences among which there are tRNA and small nuclear RNA (snRNA) genes. In 23 PACs and YACs from the 1p36.2 cluster, we identified eight different copies of a sequence with about 97% homology to the macrophage stimulating protein (MSP) gene located on chromosomal band 3p21. These MSP-like (MSPL) sequences on 1p36.2 are scattered over the repeat region. Nucleotide substitutions and single nucleotide deletions in exons of all identified MSPL genes on 1p36.2 mark them as pseudogenes. We constructed a phylogenetic tree of these sequences with their most likely order of origin in evolution. MSP from 3p21 could be identified as the ancestral sequence, a copy of which was captured into the cluster of tRNA and snRNA genes on 1p36.2 about 6 million years (MY) ago. MSP subsequently coamplified with the other sequences in the cluster. Analysis of the DNA of 18 individuals shows that the MSPL copy number is polymorphic, with a range of four to seven or more copies per haploid genome. Analysis of corresponding clusters in macaque chromosomes indicated an age for the tRNA/snRNA cluster of at least 30 MY. The MSPL sequence thus functions as a probe for the more recent primate evolution of this cluster and suggests a continuation of its unusual activity over the last 6 MY.

Isolation and characterization of glioblastoma-associated homozygously deleted DNA fragments from chromosomal region 9p21 suggests involvement of multiple tumour suppressor genes.

Representational difference analysis (RDA) of a human glioblastoma xenograft resulted in the isolation of five tumour-associated homozygously deleted DNA fragments, all originating from chromosome 9, region p21. Subsequent analysis of a series of ten glioblastomas using the newly isolated RDA fragments in conjunction with a series of known 9p21 DNA markers revealed homozygous deletions in nine of the ten (90 per cent) tumours. These deletions encompass the p15/p16 complex and two additional putative tumour suppressor loci. The RDA fragments correspond to the latter two loci. Taken together, these results suggest the involvement of multiple tumour suppressor genes from the 9p21 region in glioblastoma tumourigenesis. The novel RDA fragments will be instrumental in the isolation of the relevant genes.

Human chromosomes 11p15 and 4p12 and alcohol dependence: possible association with the GABRB1 gene.

To determine the role of genes in the chromosomal regions 11p15 and 4p12 in the development of alcohol dependence, a sample of alcoholics (n = 133) and normal controls (n = 89) were screened using polymorphisms in the dopamine D4 receptor (DRD4), tyrosine hydroxylase (TH), and GABA receptor beta1 (GABRbeta1) genes. Comparison of total alcoholics with normal controls for GABRbeta1 gene was highly significant (p = 0.004). The difference between type II alcoholics and normal controls for the same allele frequencies was also significant (p = 0.029). The allele distributions of the polymorphisms in the DRD4 and TH genes in alcoholics and normal controls were similar and their differences were not significant. Our association studies indicate that the GABRbeta1 gene may play a role in the development of alcoholism. Therefore, it is important to screen a sample of well-characterized alcoholics with functional polymorphisms in all of the GABAalpha receptor subunit genes and determine their relationship with alcoholism phenotypes. Results with TH and DRD4 genes indicate that these two genes may not play major roles in the development of alcoholism.

Disruption of Imprinted Genes at Chromosome Region 11p15.5 in Paediatric Rhabdomyosarcoma

Rhabdomyosarcomas are characterized by loss of heterozygosity (LOH) at chromosome region 11pl5.5, a region known to contain several imprinted genes including insulin-like growth factor 2 (IGF2), H19, p57KIP2. We analyzed 48 primary tumour samples and found distinct genetic changes at 11p15.5 in alveolar and embryonal histological subtypes. LOH was a feature of embryonal tumours, but at a lower frequency than previous studies. Loss of imprinting (LOI) of the IGF2 gene was detected in 6 of 13 informative cases, all harbouring PAX3—FKHR or PAX7—FKHR fusion genes characteristic of alveolar histology. In contrast, H19 imprinting was maintained in 14 of 15 informative cases and the case with H19 LOI had maintenance of the IGF2 imprint indicating separate mechanisms controlling imprinting of IGF2 and H19. The adult promoter of IGF2, P1, was used in 5 of 14 tumours and its expression was unrelated to IGF2 imprinting status implying a further mechanism of altered IGF2 regulation. The putative tumour suppressor gene p57KIP2 was expressed in 15 of 29 tumours and expression was unrelated to allele status. Moreover, in tumours with p57KIP2 expression, there was no evidence for inactivating mutations, suggesting that p57KIP2 is not a tumour suppressor in rhabdomyosarcoma.

Cloning of the human phospholipase A2 activating protein (hPLAP) gene on the chromosome 9p21 melanoma deleted region.

Cutaneous malignant melanoma (CMM) is a common skin cancer. About 50% of CMM sporadic tumours have lost one copy of the chromosome 9p21 region. To identify genes involved in the initiation and/or progression of CMM we have characterised the 9p21 melanoma deleted region and screened the human expressed sequence tag (EST) databases (dbEST) to search for expressed genes. We have identified the gene that encodes the human orthologue of the rat phospholipase A2 activating protein (PLAP). PLAP was considered a potential candidate to be involved in malignant melanoma because it maps to the critical region for CMM and because the PLA2 gene has been identified as a modifier of the APC gene, responsible for the adenomatous polyposis phenotype in the mouse. PLAP encodes a protein of 738 amino acids and has a high DNA (90%) and protein (97%) sequence similarity with the rat and mouse PLAP protein. PLAP has a region of WD40 repeats in the amino-terminus, which allows us to include this protein in the superfamily of beta-transducin proteins. Northern blot hybridisation gave a fragment of 4.5 kb, with higher expression in heart compared to other tissues. PLAP was localised at chromosome 9p21, between marker AFM218xg11 and TEK. SSCP analysis of the coding region of PLAP revealed no variants in the studied samples, but one of six CMM samples analysed by RT-PCR showed specific inactivation of PLAP. Despite PLAP's important role in mediating several cellular responses and its localisation to the chromosome 9p21 region deleted in CMM, it is unlikely that point mutations or deletions in the coding region of PLAP are responsible for the initiation or progression of CMM. Further studies on PLAP inactivation should be performed to clarify its potential involvement in CMM.

Frequent allelic imbalance of tumor suppressor gene loci in cervical dysplasia.

In addition to human papillomavirus (HPV) infection, loss of heterozygosity (LOH) at tumor suppressor gene loci has been frequently observed in cervical cancer. Thus, it may be assumed that detection and characterization of specific LOH profiles in preneoplastic lesions, in addition to HPV typing, might facilitate assessment of progression risk of cervical dysplasia. In this study, the type and frequency of allelic imbalance (allelic loss or allelic reduction) were analyzed in 24 unrelated cervical lesions using 14 polymorphic microsatellite markers at different tumor suppressor gene loci. No allelic loss was observed in four condylomatous lesions, whereas 2 of 13 (15%) CIN I lesions displayed allelic loss at 3p25 and 5q11-13. In high-grade lesions, however, allelic loss occurred in four of six (66%) cases at multiple chromosomal regions (3p14-25, 5p15, 5q11, 5q21, 11p15, and 17q21). Allelic reduction was observed in 4 of 13 (30%) low-grade lesions and 3 of 6 (50%) high-grade lesions. LOH was confined to lesions infected by high-risk HPV types. These data suggest that chromosomal instability is an early event in cervical carcinogenesis. The detection of LOH on multiple chromosome 3p loci in 50% of high-grade lesions suggests that a specific marker panel encompassing this region might enable better assessment of which lesions are likely to regress, persist, or progress.

Candidate gene involved in genital ontogeny in chromosome region 16p13.3

Candidate gene involved in genital ontogeny in chromosome region 16p13.3

Candidate gene involved in genital ontogeny in chromosome region 16p13.3

Candidate gene involved in genital ontogeny in chromosome region 16p13.3

Candidate gene involved in genital ontogeny in chromosome region 16p13.3

Candidate gene involved in genital ontogeny in chromosome region 16p13.3

Novel tumor suppressor locus in human chromosome region 3p14.2.

Alterations of chromosome region 3p14 are observed in numerous human malignancies. Because the pattern of allelic losses suggests the existence of at least one tumor suppressor gene within this region, we established a library of yeast artificial chromosomes (YACs) containing contiguous human 3p14 sequences to permit a search for tumor suppressor loci within the 3p14 region by use of functional complementation. YACs specific for human chromosome region 3p14 were transduced by spheroplast fusion into cells of the human nonpapillary renal carcinoma cell line RCC-1, which shows a cytogenetically detectable 3p deletion and is tumorigenic in nude mice. We identified a 3p14.2-specific YAC clone, located in the vicinity of the fragile histidine triad (FHIT) gene (but toward the telomere), that is capable of inducing sustained suppression of tumorigenicity in nude mice and of activating cellular senescence in vitro. Among 23 mice given injections of RCC-1 cells containing this YAC, 16 (70%) remained tumor free for at least 6 months, whereas tumor formation occurred after a median of 6 weeks in control mice given injections of either RCC-1 parental cells or a revertant cell line (in which the YAC had lost all human sequences) or RCC-1 parental cells containing other, unrelated YACs. Similar results were obtained following microcell-mediated transfer of the entire human chromosome 3. These data provide strong evidence for the existence of a novel tumor suppressor locus adjacent to the previously identified candidate tumor suppressor gene, FHIT, in 3p14.2. Positional cloning of the novel suppressor element within the 3p14.2-specific YAC and the sequence's molecular and functional characterization should add to the understanding of the pathogenesis of renal cell carcinoma and other human tumors that exhibit 3p14 aberrations.

HLA class I expression and chromosomal deletions at 6p and 15q in head and neck squamous cell carcinomas.

Loss at the chromosomal region 6p21.3 is a frequent event in head and neck squamous cell carcinomas (HNSCC). Since the human leukocyte antigen (HLA) complex is located at 6p21.3, loss of heterozygosity (LOH) of this region may provide tumour cells with an immune-escape tumour phenotype. In the present study, we have studied the correlation of HLA class I, TAP1 and TAP2 expression and LOH at 6p21.3. HLA class I and TAP1 and TAP2 protein expression was analysed by immunohistochemical procedures. A panel of 41 HNSCC with downregulated HLA class I expression was selected for LOH studies using 5 microsatellite markers located at 6p21.3 (D6S105, D6S265, D6S276, D6S273, D6S291) and 2 markers located at the chromosome 6 centromere (D6S473) and the 6p telomere (D6S277). In addition, LOH of the beta-2-nmicroglobulin (beta2m) gene was studied using 2 microsatellite markers flanking the beta2m gene (D15S126 and D15S153) and was correlated with beta2m and HLA class I expression. In 20/41 (49%) of the HNSCC, allelic loss for at least one locus at 6p21.3 was found. Loss at 15q was found in 4/10 (40%) HNSCC with downregulated beta2m expression and in 12/41 (29%) HNSCC with downregulated HLA class I expression. Our data show that downregulation of HLA class I expression is correlated with loss of chromosomal regions at 6p21.3 in HNSCC. In addition, LOH at 6p21.3 and 15q in 10 paired samples of DNA derived from the primary HNSCC, the lymph node metastases and from peripheral blood lymphocytes (PBLs) was studied. Five (5/10) primary tumours contained the same deletion as the corresponding lymph node metastases. The other cases contained deletions either in the primary tumour (3 cases) or in the lymph node metastases (1 case) or no deletions at all (1 case).

A Putative Susceptibility Locus on Chromosome 18 Is Not a Major Contributor to Human Selective IgA Deficiency: Evidence from Meiotic Mapping of 83 Multiple-Case Families

Previous reports of an association between constitutional chromosome 18 abnormalities and low levels of IgA suggested that this chromosome contains a susceptibility locus for selective IgA deficiency (IgAD), the most frequent Ig deficiency in humans. IgAD is genetically related to common variable immunodeficiency (CVID), characterized by a lack of additional isotypes. Our previous linkage analysis of 83 multiple-case IgAD/CVID families containing 449 informative pedigree members showed a significantly increased allele sharing in the chromosome region 6p21 consistent with allelic associations in family-based and case-control studies and provided the evidence for a predisposing locus, termed IGAD1, in the proximal part of the MHC. We have typed the same family material at 17 chromosome 18 marker loci with the average intermarker distance of 7 cM. A total of 7633 genotypes were analyzed in a nonparametric linkage analysis, but none of the marker loci exhibited a significantly increased allele sharing in affected family members. In addition, reverse painting and deletion mapping of a panel of constitutional chromosome 18 deletions/translocations showed the presence of IgA-deficient and IgA-proficient patients with the same abnormality and did not reveal a region commonly deleted. The linkage analysis of chromosome 8 and 21 regions involved in reciprocal translocations t(8;18) and t(18;21), which were identified in two patients lacking IgA, did not disclose a significant allele sharing. Although these results do not exclude the presence of a minor predisposing locus on this chromosome, such a putative locus would confer a population risk of developing IgAD/CVID much lower than IGAD1.

Inactivation patterns of the p16 (INK4a) gene in oral squamous cell carcinoma cell lines.

To determine whether inactivation of the p16 gene mapped to the chromosome 9p21 region is associated with the development of oral squamous cell carcinoma (SCC), we investigated the mutational states of two forms of alternative transcripts (alpha and beta) from the p16 gene in 14 oral SCC cell lines by means of RT-PCR, PCR, direct sequencing and methylation analyses. Alterations of the alpha transcript were detected in all of the cell lines examined: homozygous deletions in three lines; subtle mutations in exons 1 alpha or 2 in four lines; skipping of exon 2 in two lines; hypermethylation of the 5' CpG island of the p16 gene in four lines; and an unknown mechanism in one line. On the other hand, abnormalities of the beta transcript were observed in seven of the 14 cell lines. Nonetheless, the mutations that essentially affect the function of the encoded protein were found only in five cell lines, including three lines with homozygous deletion. There was no cell line having only beta transcript alterations. Thus, alteration of the alpha transcript of the p16 gene was a highly frequent event in oral SCC. Since this type of alteration resulted in gene inactivation through multiple pathways, it may play a major role in the process of oral SCC development.

Frequent loss of heterozygosity at chromosome 3p14.2-3p21 in human pancreatic islet cell tumours.

Pancreatic islet betacell tumours occur either sporadically or as part of inherited neoplastic syndromes, most commonly multiple endocrine neoplasia (MEN) type 1. Recently, a transgenic mouse model has been established in which the expression of the SV40 large T antigen was targeted to betacells by the rat insulin promoter, leading to the development of multiple pancreatic betacell tumours. In the advanced stages of tumour evolution, these tumours exhibited a high prevalence of loss of heterozygosity (LOH) on mouse chromosomes 9 and 16, at regions syntenic with regions 3q, 3p21, 6q12, 15q24 and 22q of the human genome. Loss of heterozygosity in human islet cell tumours was analysed in a PCR based approach at regions of the human genome syntenic with the mouse loci linked to pancreatic betacell tumours as well as the MEN1 gene on chromosome 11q13. These included 35 microsatellite markers in the human chromosomal regions 3q, 3p21, 6q12, 11q13, 15q24 and 22q. 21 patients diagnosed with insulinoma were analysed. Histologically, 16 tumours were benign, while 5 were malignant insulinomas. Thirteen of 21 (62%) tumours were found to have loss of genetic material on chromosome 3. The shortest region of overlap implicated a deletion at 3p14.2-3p21 region, corresponding to the marker D3S1295. We did not detect a substantial frequency of LOH in the other syntenic regions, except for the region of MEN 1 gene on 11q13 found to be deleted in 6 (29%) cases, including 3 of 4 tumours from MEN 1 families. Deletions of 3p14. 2-3p21 were observed in 8 of 15 (53%) benign tumours, and in 5 of 6 (83%) malignant neoplasms. These results indicate the high frequency of 3p14.2-3p21 deletions in human pancreatic betacell neoplasms. These finding suggest the presence of a tumour suppressor gene in this region, that may be important in the microevolution of these tumours towards malignancy.

Role of FHIT in human cancer.

Through investigation of hemizygous and homozygous deletions in common human cancers, including lung cancer, we have cloned and characterized a gene at chromosome region 3p14.2, FHIT, that is inactivated in epithelial tumors, particularly in tumors resulting from exposure to environmental carcinogens. In some tumors, particularly those associated with environmental carcinogens, alterations in the FHIT gene occur quite early in the development of cancer. In other cancers, Fhit inactivation seems to be a later event, possibly associated with progression to more aggressive neoplasias. Thus, detection of Fhit expression by immunohistochemistry in premalignant and malignant tissues may provide important diagnostic and prognostic information.

Multiple genetic alterations, 4q28, a new suppressor region, and potential gender differences in human hepatocellular carcinoma.

Primary hepatocellular carcinomas (HCCs) of different etiologies were studied to determine the rate of alteration of several genetic regions previously associated with the HCC phenotype. The focus of our study was to identify the frequency of genetic alterations within individual HCCs and their distribution among male and female cases. Genetic differences were evaluated between DNA isolated from tumor (T) and corresponding non-tumor (N) tissue using short tandem repeat (STR)-microsatellites and restriction fragment length polymorphism (RFLP) analyses. Twenty-eight HCC cases were studied with polymorphic markers from different parts of the genome. Three or more loci were identified with genetic alterations from 28 loci tested in 63% of HCC cases. The highest frequency of alteration occurred in the chromosome regions 1p36, 13q14, 17p13, and the 4q28 region identified in HCCs for the first time. High loss of heterozygosity (LOH) in 1p36, 4q28, 13q14, and 17p13 regions indicates important HCC suppressors within the regions. The study documents no association between cause and specific genetic change or their frequency in HCCs analyzed. The data document a significant gender distortion for genetic alteration in chromosome 13q14 and 17p13 regions and a concordant gender alteration rate in the 1p36 and 4q28 regions. An overall higher frequency of genetic alterations was identified in male cases. Future study with an extended number of HCC cases should substantiate the frequency of alteration between genders and identify tentative suppressors in the 1p36 and 4q28 regions.