11q13 Region Research Articles

Molecular analysis ofPTEN andMXI1 in primary bladder carcinoma

Loss of heterozygosity (LOH) on 10q is associated with late-stage events in urothelial neoplastic progression. The tumor suppressor gene PTEN, which is mutated or homozygously deleted in numerous cancers, maps to a region of 10q within the reported region of minimal loss in bladder tumors. In two recent studies alterations in the PTEN gene occur at a low frequency in bladder tumors displaying 10q LOH. We have screened 35 late-stage bladder tumors for mutations in PTEN and MXI1, both genes mapping to chromosome 10q. Using single-strand conformation polymorphism analysis, we identified 6 tumors harboring mutations in PTEN and 2 additional tumors displaying homozygous deletion at this locus. No MXI1 mutations were identified within the same tumor panel. Of 16 bladder tumor cell lines analyzed, 2 showed homozygous deletion of PTEN and 3 harbored point mutations resulting in an amino acid change. Two cell lines harbored missense mutations in MXI1. We report a significantly higher frequency of PTEN alterations in bladder carcinoma (23%) than was previously recorded, with no accompanying mutations in the MXI1 gene.

Delineation of the Critical Interval of Bardet-Biedl Syndrome 1 ( BBS1) to a Small Region of 11q13, through Linkage and Haplotype Analysis of 91 Pedigrees

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous recessive disease characterized primarily by atypical retinitis pigmentosa, obesity, polydactyly, hypogenitalism, and mental retardation. Despite the presence of at least five loci in the human genome, on chromosomes 2q, 3p, 11q, 15q and 16q, as many as 50% of the mutations appear to map to the BBS1 locus on 11q13. The recessive mode of inheritance and the genetic heterogeneity of the syndrome, as well as the inability to distinguish between different genetic loci by phenotypic analyses, have hindered efforts to delineate the 11q13 region as a first step toward cloning the mutated gene. To circumvent these difficulties, we collected a large number of BBS pedigrees of primarily North American and European origin and performed genetic analysis, using microsatellites from all known BBS genomic regions. Heterogeneity analysis established a 40.5% contribution of the 11q13 locus to BBS, and haplotype construction on 11q-linked pedigrees revealed several informative recombinants, defining the BBS1 critical interval between D11S4205 and D11S913, a genetic distance of 2.9 cM, equivalent to approximately 2.6 Mb. Loss of identity by descent in two consanguineous pedigrees was also observed in the region, potentially refining the region to 1.8 Mb between D11S1883 and D11S4944. The identification of multiple recombinants at the same position forms the basis for physical mapping efforts, coupled with mutation analysis of candidate genes, to identify the gene for BBS1.

Monosomy 13 Is Associated With the Transition of Monoclonal Gammopathy of Undetermined Significance to Multiple Myeloma

Chromosomal abnormalities are present in most (if not all) patients with multiple myeloma (MM) and primary plasma cell leukemia (PCL). Furthermore, recent data have shown that numerical chromosomal changes are present in most individuals with monoclonal gammopathy of undetermined significance (MGUS). Epidemiological studies have shown that up to one third of MM may emerge from pre-existing MGUS. To clarify further possible stepwise chromosomal aberrations on a pathway between MGUS and MM, we have analyzed 158 patients with either MM or primary PCL and 19 individuals with MGUS using fluorescence in situ hybridization (FISH). Our FISH analyses were designed to detect illegitimate IGH rearrangements at 14q32 or monosomy 13. Whereas translocations involving the 14q32 region were observed with a similar incidence (60%) in both conditions, a significant difference was found in the incidence of monosomy 13 in MGUS versus MM or primary PCL. It was present in 40% of MM/PCL patients, but in only 4 of 19 MGUS individuals. Moreover, whereas monosomy 13 was found in the majority of plasma cells in MM, it was observed only in cell subpopulations in MGUS. It is noteworthy that, in a group of 20 patients with MM and a previous MGUS history, incidence of monosomy 13 was 70% versus 31% in MM patients without a known history of MGUS (P =.002). Thus, this study highlights monosomy 13 as correlated with the transformation of MGUS to overt MM and may define 2 groups of MM with possible different natural history and outcome, ie, post-MGUS MM with a very high incidence of monosomy 13 and de novo MM in which other genetic events might be involved. Serial analyses of individuals with MGUS will be needed to validate this model.

Allelic loss of the PTEN region (10q23) in breast carcinomas of poor pathophenotype.

Loss of heterozygosity (LOH) in loci of the 10q23 region that harbor the PTEN gene and mutations in the sequence of this gene have been found in several primary human tumors including breast carcinomas, suggesting that this gene could be implicated in their pathogenesis. We investigated allelic losses in microsatellites of the 10q23 region, and their correlations with nine pathologic parameters in 105 breast carcinomas. The LOH analysis was performed by amplifying DNA by PCR, using five markers of the 10q23 region (D10S1687, D10S541, D10S2491, D10S583 and D10S571). LOH in at least one marker of the PTEN region was found in 29.5% of tumors. The statistical comparison between carcinomas with and without LOH in terms of the pathologic parameters showed significant differences in age (p = 0.03), lymph node metastases (p = 0.02), and higher histological grade (p = 0.02); a trend toward significance was found for progesterone receptors (p = 0.05). LOH in an individual marker and statistically significant relationships to tumor characteristics were observed at locus D10S541 for lymph node metastases (p = 0.04), at D10S2491 (intragenic to the PTEN gene) for lymph node metastases (p = 0.02), and at D10S583 for progesterone receptors (p = 0.01) and for high grade (p = 0.03). These results suggest the PTEN gene, or other genes of the 10q23 region, could be functionally related to breast cancer, probably influencing the development of histological features associated with poor prognosis.

The HNF-4/HNF-1alpha transactivation cascade regulates gene activity and chromatin structure of the human serine protease inhibitor gene cluster at 14q32.1.

Hepatocyte-specific expression of the alpha1-antitrypsin (alpha1AT) gene requires the activities of two liver-enriched transactivators, hepatocyte nuclear factors 1alpha and 4 (HNF-1alpha and HNF-4). The alpha1AT gene maps to a region of human chromosome 14q32.1 that includes a related serine protease inhibitor (serpin) gene encoding corticosteroid-binding globulin (CBG), and the chromatin organization of this approximately 130-kb region, as defined by DNase I-hypersensitive sites, has been described. Microcell transfer of human chromosome 14 from fibroblasts to rat hepatoma cells results in activation of alpha1AT and CBG transcription and chromatin reorganization of the entire locus. To assess the roles of HNF-1alpha and HNF-4 in gene activation and chromatin remodeling, we transferred human chromosome 14 from fibroblasts to rat hepatoma cell variants that are deficient in expression of HNF-1alpha and HNF-4. The variant cells failed to activate either alpha1AT or CBG transcription, and chromatin remodeling failed to occur. However, alpha1AT and CBG transcription could be rescued by transfecting the cells with expression plasmids encoding HNF-1alpha or HNF-4. In these transfectants, the chromatin structure of the entire alpha1AT/CBG locus was reorganized to an expressing cell-typical state. Thus, HNF-1alpha and HNF-4 control both chromatin structure and gene activity of two cell-specific genes within the serpin gene cluster at 14q32.1.

Analysis of the MEN1 gene in sporadic pituitary adenomas from Japanese patients

An autosomal-dominant syndrome known as multiple endocrine neoplasia type 1 (MEN1) is characterized by tumors in parathyroid glands, pancreatic endocrine tissues and the anterior pituitary gland. The predisposing gene was identified at 11q13 when germline mutations in the MEN1 gene were detected in affected pedigrees. To investigate a possible role of this gene in tumorigenesis of non-familial pituitary adenomas, we examined 24 sporadic tumors from Japanese patients for loss of heterozygosity (LOH) at the 11q13 region and for somatic mutations in the entire coding region and exon-intron boundaries of MEN1. Although three common sequence variants were detected, none of the tumors exhibited either LOH or somatic mutations of this gene. Our results indicate that inherited and sporadic forms of pituitary adenomas are different in terms of the genetic events that contribute to their development, and that other loci associated with pituitary neoplasia must still be sought.

11q13 Allelic Imbalance Discriminates Pulmonary Carcinoids from Tumorlets: A Microdissection-Based Genotyping Approach Useful in Clinical Practice

Pulmonary tumorlets are minute neuroendocrine cell proliferations believed to be precursor lesions to pulmonary carcinoids. Little is known of their molecular pathogenesis because of their small size. Using tissue microdissection, we evaluated 11q13 region allelic imbalance in the pathogenesis of pulmonary tumorlet/carcinoid lesions. The int-2 gene was selected because of its chromosomal location at 11q13 in close proximity to MEN1, a tumor suppressor gene frequently mutated in familial forms of neuroendocrine cancer. Three cohorts of patients were studied: subjects with typical carcinoid tumors and coexisting tumorlets (n = 5), typical carcinoids without tumorlets (n = 6), and tumorlets alone without carcinoid lesions (n = 5). A total of 11 carcinoids and 11 tumorlets were microdissected from 4-micrometer-thick histological sections. Genotyping was designed to detect allelic imbalance of the int-2 gene and involved DNA sequencing of two closely spaced deoxynucleotide polymorphisms. Subjects shown to be informative were evaluated for allelic imbalance in tumorlet/carcinoid tissue. Eight of 11 (73%) carcinoids manifested allelic, in contrast to only one of 11 (9%) of tumorlets. Int-2 allelic imbalance was significantly associated with carcinoid tumor formation (P < 0.01). In patients having both carcinoid tumors and tumorlets, the latter showed allelic balance and were thus discordant in genotype with coexisting carcinoid excluding pathogenesis of tumorlets from intramucosal spread from carcinoid tumors. Int-2 allelic imbalance was shown to be an early event in carcinoid tumor formation by virtue of the absence of allelic imbalance for other common cancer-related gene disturbances involving 11p13 (Wilms' tumor), 3p25 (von-Hippel-Lindau), and 17p13 (p53). Demonstration of 11q13 allelic imbalance by microdissection/genotyping may be a useful discriminatory marker for pulmonary neuroendocrine neoplasia.

Is a Duplication of 14q32 a New Recurrent Chromosomal Alteration in B-Cell Non-Hodgkin Lymphoma?

Identification of clonal chromosomal abnormalities involving 14q32 and its association with specific histological subtypes of non-Hodgkin lymphoma (NHL) has provided substantial insight to the genetic events leading to the disease. However, in some cases with inferior morphology of tumor cell chromosomes, the additional segment on chromosome 14 remains unidentified by cytogenetic banding techniques alone. To elucidate the origin of the additional chromosomal segment and to correlate the newly determined alterations with histology, metaphases from 15 NHL patients with add(14)(q32) were examined using fluorescence in situ hybridization (FISH) techniques after cytogenetic analysis had been performed. We found the duplication of 14q involving the q32 region in 6 cases with a dup(14)(q32) in 4 cases and a dup(14)(q24q32) in 2 cases. In 8 cases, FISH unveiled known NHL associated translocations; a t(14;18)(q32;q21) in 4 cases, a t(11;14)(q13;q32) in 2 cases, a t(8;14)(q24;q32) and a t(9;14)(p13;q32) in 1 case each. We also noted a t(14;17)(q32;q21) in 1 case. The use of FISH was a valuable asset in determining the origin of the additional material on chromosome 14q32, and helped resolve a group of B-cell NHLs with involvement of a duplicated 14q32 region.

Assignment of the human poly(A) polymerase (PAP) gene to chromosome 14q32.1-q32.2 and isolation of a polymorphic CA repeat sequence.

We report the chromosomal localization of the gene for human poly(A) polymerase (PAP) and the characterization of a newly isolated CA repeat near the PAP locus. By fluorescence in situ hybridization and polymerase chain reaction (PCR)-based analysis with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel, this gene was mapped on the q32.1-q32.2 region of chromosome 14. From a genomic clone containing the human PAP locus, we have isolated a polymorphic dinucleotide (CA) sequence. High heterozygosity (0.81) makes this polymorphism a useful marker in the genetic study of disorders localized at the 14q32 region, such as autosomal recessive congenital microphthalmia (CMIC).

Genetic aberrations detected by comparative genomic hybridization in hepatocellular carcinomas: their relationship to clinicopathological features.

To elucidate cytogenetic alterations underlying human hepatocellular carcinomas (HCCs), we used a comparative genomic hybridization (CGH) method to analyze 41 cases of hepatocellular carcinoma (HCC) including 15 well differentiated HCCs, 14 moderately differentiated HCCs, and 12 poorly differentiated HCCs. Of these, 27 patients were chronically infected with hepatitis C virus (HCV), and the remaining patients were positive for hepatitis B virus (HBV). The most common sites of increase in DNA copy number were 1q (78% of the cases) and 8q (66%) with minimal overlapping regions at 1q24-25 and 8q24, respectively. Frequent decreases in copy number were observed at 17p (51%), 16q (46%), 13q13-14 (37%), 4q13-22 (32%), 8p (29%), and 10q (17%). In 6 cases (15%), an amplification was found in the region of 11q13. A gain of 8q24 was significantly associated with well-differentiated HCCs (P<.05), whereas a loss of 13q13-14 and amplification of 11q13 were linked to moderately and poorly differentiated HCCs (P<.01). These observations suggest that a gain of 8q24 is an early event and that a loss of 13q13-14 and amplification of 11q13 are a late event in the course of liver carcinogenesis. A gain of 10q (7/41) was detected exclusively in cases with HCV infection. In contrast, an amplification of 11q13 was preferentially found in HBV-positive HCCs. These findings raise the hypothesis that, although many genetic alterations are basically common to both HCV-positive and HBV-positive tumors, the process of carcinogenesis may be to some extent different between these two types of tumors.

Chromosomal deletions occur in restricted regions of 5q in testicular germ cell cancer.

Since the biologic behavior and molecular genetic changes observed in testicular germ cell cancer differ from those seen in more common epithelial tumors, it is likely that hitherto uncharacterized genes play a role in the development of germ cell tumors. Our previous work on testicular germ cell cancer suggested that chromosome 5q might contain one or more novel tumor suppressor genes that play a role in this malignancy. In this study, we performed a high resolution loss of heterozygosity (LOH) study of testicular cancer using 37 informative markers on chromosome 5. We detected allelic losses in 20/48 (42%) specimens and identified three common sites of loss on chromosome 5q14, 5q21 and 5q34-qter, defined respectively by minimal regions of deletion of < or = 1 cM, 10 cM and approximately 20 (cM). Using an overlapping series of YACs and radiation hybrid mapping, we have constructed a physical map of the 5q14 deletion that should aid in the isolation and characterization of the putative tumor suppressor gene located therein.

Loss of Heterozygosity of 14q32 in Colorectal Carcinoma

Previous allelotyping studies on colorectal carcinoma suggest that loss of heterozygosity (LOH) on chromosome 14q may be a common genetic alteration in this tumor type. The purpose of this study was to determine a precise frequency of LOH at 14q32 region in colorectal carcinomas and to define a minimal region of LOH. LOH at 14q32 in 66 primary colorectal carcinomas were analyzed by polymerase chain reaction-based deletion mapping using six highly polymorphic microsatellites. The average rate of informative data was 68.2% in all cases of colorectal carcinomas analyzed. The average rate of LOH at 14q32 was 44.1%, with the highest frequency of 50.0% at D14S267 locus in the informative cases. The minimal region of LOH was defined by markers D14S65 and D14S250 at 14q32. The present data provide useful information for the isolation of tumor suppressor genes from this region.

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.

Frequent chromosome arm 13q deletion in aggressive non-Hodgkin's lymphoma.

To clarify the role of allelic loss on chromosome arm 13q in lymphomagenesis, we performed fluorescence in situ hybridization (FISH) analysis of a total of 43 primary lymphomas, including both indolent and aggressive non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD), using the specific probes at RB1 and D13S319 loci on the centromeric portion of chromosome arm 13q. Monosomy at either or both RB1 and D13S319 loci was detected in 15 of 43 (35%) lymphomas (14 of 43 cases at RB1 locus and seven of 43 cases at D13S319 locus); the 13q deletion was frequently detected in the aggressive NHLs (40%; 12 of 30 cases) compared with that in indolent NHL (17%; one of six cases) and a subset of HD (29%; two of seven cases). There are only six cases of 43 which have total monosomy 13q14, all aggressive NHL, 14% of total or 20% of this subgroup. In addition, we analyzed the loss of heterozygosity in 15 of the 43 primary lymphoma samples for several polymorphic microsatellite loci (D13S168, RB1 and D13S272) on the chromosome arm 13q, and confirmed the 13q deletion in four of five cases that were positive on FISH analysis. The subchromosomal region frequently altered in lymphoma on 13q14 is the region around RB1 locus and centromeric to D13S319 locus, which is an overlapped region frequently deleted in chronic lymphocytic leukemia. Together, our data indicate that the 13q alterations are present in a variety of types of lymphoma and occur in a significant proportion of aggressive NHLs, suggesting the possible presence of common candidate gene(s) on the 13q14 region, whose alteration may play an important role in the formation or development of a wide variety of mature lymphoid malignancies.

Linkage and association studies of atopy and the chromosome 11q13 region

The clinical syndrome atopy is largely determined by genetic factors. In 1989, the first linkage of markers within and flanking the chromosomal region 11q13 and atopy was reported. In the...

Somatic mutation of PTEN in bladder carcinoma.

The tumour suppressor gene PTEN/MMAC1, which is mutated or homozygously deleted in glioma, breast and prostate cancer, is mapped to a region of 10q which shows loss of heterozygosity (LOH) in bladder cancer. We screened 123 bladder tumours for LOH in the region of PTEN. In 53 informative muscle invasive tumours (≥ pT2), allele loss was detected in 13 (24.5%) and allelic imbalance in four tumours (overall frequency 32%). LOH was found in four of 60 (6.6%) informative, non-invasive tumours (pTa/pT1). We screened 63 muscle invasive tumours for PTEN mutations by single-strand conformation polymorphism (SSCP) analysis and for homozygous deletion by duplex quantitative polymerase chain reaction (PCR). Two homozygous deletions were identified but no mutations. Of 15 bladder tumour cell lines analysed, three showed homozygous deletion of all or part of the PTEN gene, but none had mutations detectable by SSCP analysis. Our results indicate that PTEN is involved in the development of some bladder tumours. The low frequency of mutation of the retained allele in tumours with 10q23 LOH suggests that there may be another predominant mechanism of inactivation of the second allele, for example small intragenic deletions, that hemizygosity may be sufficient for phenotypic effect, or that there is another target gene at 10q23. © 1999 Cancer Research Campaign

Frequent allelic loss at the TOC locus on 17q25.1 in primary breast cancers.

Sporadic breast cancers often show allelic losses on the long arm of chromosome 17. Since the BRCA1 gene lies at 17q21.1 and the TOC locus, associated with esophageal cancer, lies at 17q25.1, either gene could be the target of those losses. We examined both loci in 178 primary breast cancers, using microsatellite markers covering the relevant regions of 17q, and observed allelic losses in 97 tumors (55%). Losses were most frequent at markers around the TOC locus (48% at D7S1839 and 43% at D17S1603), where we identified a distinct commonly deleted region within a I -cM interval. Another larger, separate commonly deleted region including the BRCA1 gene was also identified, which exhibited 45% of allelic loss (at D17S934). Allelic loss on 17q was more frequent in tumors of the solid-tubular histologic type (P = 0.0129) and in estrogen-negative and progesterone-negative tumors (P = 0.0281 and 0.0196, respectively). The results indicated that BRCA1 and TOC are independent targets of allelic loss on 17q in primary breast cancers, and that inactivation of the TOC locus in particular may play an important role in the genesis of sporadic breast tumors.

Molecular genetic analysis of non-astrocytic gliomas.

Oligodendroglial tumours follow genetic pathways different from but overlapping with those of astrocytic tumours. The aim of this study was to examine whether major genetic events such as loss of chromosome 10 and p53 mutation found in astrocytic gliomas are also involved in the development and anaplastic transformation of non-astrocytic gliomas and to correlate the findings with histopathological subtypes of these tumours. Sixty-one formalin-fixed, paraffin-embedded oligodendroglial and ependymal tumours (16 oligodendrogliomas, 12 anaplastic oligodendrogliomas, seven oligoastrocytomas, 24 ependymomas and two anaplastic ependymomas) were examined for allelic deletions on chromosome 10q23 and 10q25-26 regions, mutations of PTEN/MMAC1 and p53, MDM2 gene amplification and apoptosis. The frequencies of allelic deletions at marker D10S2491 (which mapped within PTEN/MMAC1) and between markers D10S209 and D10S587 (where DMBT1 was located) were found to be < 30% in both types of non-astrocytic gliomas. High frequency of allelic deletions was detected at marker D10S215 (80%) at the proximal 10q23 region in both oligodendroglial and ependymal tumours and between markers D10S216 (42%) and D10S169 (67%) at distal 10q25-26 region in oligodendroglial tumours. No mutations of PTEN/MMAC1 were found. p53 mutations were detected in three oligoastrocytomas and one ependymoma; three out of five mutations were found in exon 4. MDM2 gene amplification was found in one ependymoma harbouring wild-type p53. The apoptotic index was lower in p53-mutated tumours than in tumours with wild-type p53. The telomeric end of chromosome 10q could be involved in the development and anaplastic transformation of oligodendroglial tumours. Mutations of PTEN/MMAC1 and p53, amplification of the MDM2 gene and allelic loss on chromosome 10q do not play a major part in the pathogenesis or anaplastic transformation of oligodendrogliomas and ependymal tumours.

Isolation and mapping of a polymorphic CA repeat sequence at the human VRK1 locus.

VRK1 is a novel human putative serine/threonine kinase, and is located on chromosome 14 at band q32 where an autosomal recessive congenital microphthalmia (CMIC) is mapped. We isolated a polymorphic dinucleotide CA repeat marker from a genomic clone containing the human VRK1 gene. This polymorphism will be useful in genetic studies of disorders localized at the 14q32 region, such as CMIC.

Human Caveolin-1 and Caveolin-2 Are Closely Linked Genes Colocalized with WI-5336 in a Region of 7q31 Frequently Deleted in Tumors

Human Caveolin-1 and Caveolin-2 Are Closely Linked Genes Colocalized with WI-5336 in a Region of 7q31 Frequently Deleted in Tumors