A relationship between methylenetetrahydrofolate reductase variants and the development of invasive cervical cancer.

OBJECTIVE: Low red blood cell folate levels have been associated with hypomethylation of DNA in dysplastic tissue and an increased risk for cervical intraepithelial neoplasia in human papillomavirus (HPV)-infected women. Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme regulating the metabolism of folate and methionine, the important components of DNA synthesis and methylation. Two common genetic polymorphisms, causing reduced MTHFR activity, have been identified. Therefore, the goal of this study was to evaluate these MTHFR variations as risk factors for invasive cervical cancer. METHODS: To overcome the failure to properly match cases and controls that can cause false-positive inferences due to population stratification and unrecognized variables in a traditional case-control study, a family-based transmission/disequilibrium test (TDT) was used. We obtained samples from nuclear families of 102 women with invasive cervical cancer (ICC). One polymorphism was typed by a PCR-RFLP method, while a template-directed dye-terminator assay was developed for the other. RESULTS AND CONCLUSIONS: We were unable to confirm a strong association of MTHFR polymorphisms and ICC using family-based controls and a transmission/disequilibrium test. The overall results of the TDT showed chi(2) (1 df) of 0.28 (P = 0.60) for exon 4, chi(2) (1 df) of 0.81(P = 0.37) for exon 7, and chi(2) (3 df) of 2.56 (P = 0.46) for the haplotype, meaning that there was no transmission of those alleles significantly in excess of Mendelian expectations to affected women. In addition, there was no effect of these variants with increased parity or infection with high-risk-type human papillomavirus.

Polymorphisms in the prostate cancer susceptibility gene HPC2/ELAC2 in multiplex families and healthy controls.

Two polymorphisms in the newly cloned prostate cancer susceptibility gene, HPC2/ELAC2, are suspected to be associated with an increased risk of developing the disease. These missense variants result in a serine (S) to leucine (L) substitution at amino acid residue 217 and an alanine (A) to threonine (T) substitution at residue 541. We genotyped these polymorphisms in 257 multiplex prostate cancer sibships and in 355 race-matched healthy unrelated controls. A significant increase in the frequency of the T allele is seen in the prostate cancer subjects compared with controls. There is, however, little evidence for excess clustering of the T allele within the multiplex families known to be segregating this allele, and there is no evidence for linkage of prostate cancer to the HPC2/ELAC2 region of chromosome 17p11.2 in these families. The T allele shows no association with either Gleason score or age-of-onset in segregating families.

Loss of heterozygosity at 11q23.3 in vasculoinvasive and metastatic squamous cell carcinoma of the cervix.

We have previously demonstrated a strong relationship between loss of heterozygosity (LOH) at chromosome 11q23.3 and the presence of extensive tumor plugs in lymphvascular spaces (LVS) in stage 1B cervical carcinoma, suggesting that genes at this locus may regulate vasculoinvasion. This study examined LOH at 11q23.3 in microdissected tumor plugs within LVS and in metastatic foci in lymph nodes (MFLN), as well as corresponding invasive tumor and adjacent cervical intraepithelial neoplasia (CIN) 3 in stage 1B squamous cell carcinoma. Of 49 invasive carcinomas, 38.8% had LOH at 11q23.3. Of 36 tumor plugs in LVS, 39% had LOH at 11q23.3. Twenty percent of 15 MFLN demonstrated LOH at 11q23.3. Patients with LOH at 11q23.3 are significantly more likely to have disease recurrence than patients without LOH at 11q23.3 (P =.02). Of 10 foci of CIN 3, none showed LOH at 11q23.3. Although unlikely to have an impact early in carcinogenesis, tumor-suppressor genes located in the region of 11q23.3 appear to be important in tumor progression, facilitating lymphvascular space invasion and, by inference, spread to lymph nodes in squamous cell carcinoma of the cervix.

The pericentromeric region of human chromosome 11: evidence for a chromosome-specific duplication.

We have identified a chromosome duplication in the pericentromeric region of human chromosome 11 located in 11p11 and 11q14. A detailed physical map of each duplicated region was generated to describe the nature of the duplication, the involvement at the centromere and to resolve the correct maps. All clones were evaluated to ensure they were representative of their genetic origin. The order of clones, based on their marker content, as well as the distance covered was determined by SEGMAP. Each duplication encompasses more than 1 Mb of DNA and appears to be chromosome 11 specific. Ten STS markers were mapped within each duplication. Comparative sequence analysis along the duplication identified 35 nucleotide changes in 2,036 bp between the two copies, suggesting the duplication occurred over 14 million years ago. A suggested organization of the pericentromeric region, including the duplications and alpha-related repetitive sequences, is presented.

Cervical cancer suppressor gene is within 1 cM on 6p23.

We previously showed loss of heterozygosity at 6p to be a common genetic alteration in cervical cancer and cervical intraepithelial neoplasia. To characterize this critical area of deletion in chromosome 6, we evaluated 107 invasive cervical cancers, using 23 polymorphic markers. Genomic DNA from microdissected frozen or paraffin-embedded cervical tumors and corresponding normal tissue was analyzed. Fifty-three percent (57/107) of the cervical tumors showed loss in 6p. Deletions were found in all stages and histologic types. Ninety-one percent (52/57) of these tumors had a loss at 6p23. One tumor defined the distal area of deletion at marker D6S429. Two tumors defined the proximal area of deletion at marker D6S1578. Genotyping of parental DNA was done on 16 cases to evaluate the origin of chromosomal loss. The deletion occurred in the paternal chromosome in 10 tumors and in the maternal in six. Within each tumor, the same parental chromosome was lost at all tested heterozygous 6p markers. The order of the polymorphic markers and estimate of distances in the critical region were confirmed by generation of a yeast artificial chromosome (YAC) contig and pulse-field gel electrophoresis. Our data strongly suggest that a gene important in cervical cancer tumorigenesis is located within a 1-cM region of 6p23, and it is not imprinted.

Loss of heterozygosity in clinical stage IB cervical carcinoma: relationship with clinical and histopathologic features.

Loss of heterozygosity (LOH) has been shown to be an important prognostic factor in a variety of malignant neoplasms. The relationship between LOH and established histopathological prognostic factors in cervical carcinoma has not been examined. We studied LOH in 58 FIGO stage IB cervical cancers treated by radical hysterectomy. In a randomly selected subset of 37 of these cases, LOH was examined using markers for all 41 chromosomal arms. Seventy-six percent of the 58 cases and 95% of the extensively studied cases showed LOH at one or more loci. The three most common sites of LOH were 3p21, 6p24-p23, and 11q23.3. In the extensively studied group, LOH on 11q was associated with extensive lymphvascular space invasion (P = .009) and less deeply invasive tumor (P = .042). There was a trend for tumors with LOH on 11q to recur, but this was not statistically significant. No correlation between the presence of LOH on 3p or 6p and lymphvascular space invasion or tumor depth was present. There was no correlation between the number of sites of LOH or between the presence of LOH on 3p, 6p, and 11q and the presence of metastatic tumor in regional lymph nodes, histologic type (squamous v nonsquamous), tumor differentiation, maximum tumor size, degree of inflammation, pattern of invasion, mitotic rate, or clinical recurrence. In summary, tumors with 11q LOH may behave in a more aggressive fashion. Future studies of LOH in cervical carcinoma should include histopathological prognostic information so that the relationship between LOH and these factors can be determined on larger numbers of patients.

Cervical intraepithelial neoplasia III shows frequent allelic loss in 3p and 6p.

We have shown previously that a significant number of invasive cervical cancers (ICC) have nonrandom chromosomal losses in 3p, 6p, 11q, 2q, 6q, and 19q, thereby suggesting that genes involved in the suppression of tumor development or progression are located in these regions. Cervical intraepithelial neoplasia (CIN) III is considered the precursor lesion for ICC of squamous type and occurs frequently with ICC of glandular type. In an effort to define which chromosomal losses are present in the precursor lesions, we identified CIN III lesions from 24 ICC treated by radical hysterectomy. Thirty-three CIN III associated with 22 squamous carcinomas and 2 adenocarcinomas were carefully microdissected from the paraffin-embedded sections. The whole genomic DNA from CIN III was amplified with short random primers. DNA from ICC, CIN III, and normal tissue was analyzed at the six chromosomal regions with polymorphic markers. Thirty-eight percent of hysterectomy specimens had loss of heterozygosity (LOH) in at least one of the CIN III lesions from each case. Loss occurred in 30% of cases in 3p14.1-12 (37% for associated ICC), 21% in 6p23 (33%), 14% in 2q33-37 (27%), 0 in 11q23.3 (33%), 4% in 19q13.4 (13%), and 0 in 6q21-23.3 (18%). These results suggest that mutations in 3p and 6p are important early in tumorigenesis, whereas 11q and 6q contain genes important later in tumor progression. Invasive and preinvasive cervical lesions appear to develop from multifocal genetic events since consistent losses do not occur within all precursor lesions in the same patient.

A gene map of the Best's vitelliform macular dystrophy region in chromosome 11q12-q13.1.

Best's vitelliform macular dystrophy is an autosomal dominant disorder of unknown causes. To identify the underlying gene defect the disease locus has been mapped to an approximately 1.4-Mb region on chromosome 11q12-q13.1. As a prerequisite for its positional cloning we have assembled a high coverage PAC contig of the candidate region. Here, we report the construction of a primary transcript map that places a total of 19 genes within the Best's disease region. This includes 14 transcripts of as yet unknown function obtained by EST mapping and/or cDNA selection and five genes mapped previously to the interval (CD5, PGA, DDB1, FEN1, and FTH1). Northern blot analyses were performed to determine the expression profiles in various human tissues. At least three genes appear to be good candidates for Best's disease based on their abundant expression in retina or retinal pigment epithelium. Additional information on the functional properties of these genes, as well as mutation analyses in Best's disease patients, have to await their further characterization. [The GenBank/EMBL accession numbers and details of the isolation, localization, and characterization of ESTs and selected cDNAs are available as online supplements in Online Tables 1-3 at http://www.genome.org.]

A transcript map of an 800-kb region on human chromosome 11q13, part of the candidate region for SCA5 and BBS1.

The exon-amplification method was used to identify putative transcribed sequences from an 800-kb region that includes the genes for phospholipase Cbeta3 and PYGM on human chromosome 11q13. The clone contig consisted of ten cosmids, three bacterial artificial chromosomes, and one P1 artificial chromosome. A total of 83 exons were generated of which 23 were derived from known genes and expressed sequence tags (ESTs). Five different EST cDNA clones were identified and mapped on the contig. One is a homolog of the human p70S6 kinase (p70s6 k) gene whose function involves the translational regulation of ribosomal protein synthesis and thereby impacts on ribosomal biogenesis. The gene for p70s6 k is expressed universally, including within adipose cells and retina, and it could play a role in Bardet-Biedl syndrome type 1, which has been mapped to 11q13.

A 3-Mb contig from D11S987 to MLK3, a gene-rich region in 11q13.

We have combined genetic, radiation-reduced somatic cell hybrid (RRH), fluorescent in situ hybridization (FISH), and physical mapping methods to generate a contig of overlapping YAC, PAC, and cosmid clones corresponding to > 3 continuous Mb in 11q13. A total of 15 STSs [7 genes (GSTP1, ACTN, PC, MLK3, FRA1, SEA, HNP36), 4 polymorphic loci (D11S807, D11S987, GSTP1, D11S913), 3 ESTs (D11S1956E, D11S951E, and W1-12191), and 1 anonymous STS (D11S703)], mapping to three independent RRH segregation groups, identified 26 YAC, 7 PAC, and 16 cosmid clones from the CGM, Roswell Park, CEPH Mark I, and CEPH MegaYAC YAC libraries, a 5 genome equivalent PAC library, and a chromosome II-specific cosmid library. Thirty-six Alu-PCR products derived from 10 anonymous bacteriophage lambda clones, a cosmid containing the polymorphic marker D11S460, or STS-positive YAC or cosmid clones were identified and used to screen selected libraries by hybridization, resulting in the identification of 19 additional clones. The integrity and relative position of a subset of clones was confirmed by FISH and were found to be consistent with the physical and RRH mapping results. The combination of STS and Alu-PCR-based approaches has proven to be successful in attaining contiguous cloned coverage in this very GC-rich region, thereby establishing for the first time the absolute order and distance between the markers: CEN-MLK3-(D11S1956E/D11S951E/W1-12191)-FRA1-D 11S460-SEA-HNP36/ D11S913-ACTN-PC-D11S703-GSTP1-D11S987-TEL.

The human HNP36 gene is localized to chromosome 11q13 and produces alternative transcripts that are not mutated in multiple endocrine neoplasia, type 1 (MEN I) syndrome.

Multiple endocrine neoplasia, type 1 (MEN I), is an autosomal dominant syndrome of selected endocrine neoplasms whose causative gene, a suspected tumor suppressor, has been localized to chromosome 11q13, but has not been identified. Recently, the HNP36 cDNA was identified as a novel growth factor responsive gene of undetermined biological function that is expressed in the pituitary and parathyroid glands. In studies seeking the function of the HNP36 gene product, the gene was localized by fluorescence in situ hybridization within the 11q13 segment. Further analysis of radiation-reduced hybrid DNAs and chromosome 11-specific YAC clones established that the HNP36 gene is within 80 kb of D11S913, a marker tightly linked to the MEN1 gene. Consequently, the HNP36 gene was studied as a candidate for the MEN1 gene. The human HNP36 gene was cloned and determined to consist of 12 exons. Expression of the HNP36 gene from pituitary and parathyroid tissue and four patient tumors or lymphoblasts was confirmed by RT-PCR amplification of the coding sequences, and HNP36 transcripts were analyzed for mutations. All tissues expressed three HNP36 gene transcripts that result from alternative splicing and appear to encode related, but distinct, proteins. However, DNA sequence determination of the RT-PCR products from MEN I-associated tumors found no deletions and identified a single nucleotide difference that may be a polymorphism. Thus, mutations in the coding segments of the HNP36 gene are not the cause of the MEN I syndrome. Nevertheless, the assignment of the HNP36 gene to 11q13 and identification of new potential gene products provides a novel growth-regulated genetic candidate for other disorders whose genes map to this locus.

A sequence-ready high-resolution physical map of the best macular dystrophy gene region in 11q12-q13.

Best disease, an autosomal dominant inherited macular degenerative disorder, was previously localized between D11S1765 and UGB (uteroglobin) in 11q13 by genetic linkage analysis. Since this region was found to be refractory to cloning in YAC (yeast artificial chromosome)-based vectors, a P1 artificial chromosome (PAC) contig was assembled. Gridded PAC libraries representing a 16-fold genome equivalent were screened by hybridization using PCR products representing STSs derived from YAC end sequences, markers binned to 11q13, and PAC-derived insert ends. A highly marker dense approximately 1.7-Mb PAC contig that encompassed the disease gene region was constructed, allowing us to order accurately the markers throughout the region and to provide the most precise estimate of its physical size. Using this contig, thus far we have mapped seven anonymous ESTs and five known genes into this region. This high-resolution physical map will facilitate the isolation of polymorphic markers for refinement of the disease gene region, as well as the identification of candidate genes by exon trapping, cDNA selection, and gene prediction from PAC-derived genomic sequence.

Human uteroglobin gene: structure, subchromosomal localization, and polymorphism.

Human uteroglobin (hUG) or Clara cell 10-kD protein (cc10 kDa) is a steroid-dependent, immunomodulatory, cytokine-like protein. It is secreted by mucosal epithelial cells of all vertebrates studied. The cDNA encoding hUG and the 5' promoter region of the gene have been characterized previously. Here, we report that the structure of the entire hUG gene is virtually identical to those of rabbit, rat, and mouse. It is localized on human chromosome 11q12.3-13.1, a region in which several important candidate disease genes have been mapped by linkage analyses. Our data indicate that candidate genes for atopic (allergic) asthma and Best's vitelliform macular dystrophy are in closest proximity to the hUG gene. To determine whether hUG gene mutation may be involved in the pathogenesis of these diseases, we studied two isolated groups of patients, each afflicted with either atopy or Best's disease, respectively. We detected a single base-pair change in the hUG gene in Best's disease patients and normal controls but no such change was detected in atopy patients. This alteration in hUG gene-sequence in Best disease family appears to be a polymorphism. Although the results of our investigation did not uncover mutations in hUG gene that could be causally related to the pathogenesis of either of these diseases, its conservation throughout vertebrate phyla implies that this gene is of physiological importance. Moreover, the close proximity of this gene to several candidate disease genes makes it an important chromosomal marker in cloning and characterization of those genes.

A linkage map of chromosome 6 based on 2 large kindreds segregating bipolar affective disorder.

Twenty-eight markers, both simple sequence repeats (SSRs) and restriction fragment length polymorphisms (RFLPs), were genotyped on members of 2 large pedigrees (OOA, BIP167) segregating bipolar affective disorder. Using the multipoint program "build" of CRIMAP and odds of placement 1000:1, a unique sex-averaged map was generated that spans 227 cM and includes 26 markers. The two other markers were placed on the map with a lower likelihood. The female recombination map is larger than the male recombination map by about 80%. Linkage analysis between the polymorphisms and the disease in the OOA screening pedigree did not result in any significantly positive lod scores nor did a non-parametric, identity-by-descent, method generate any significant p-values. BIP167 was analyzed for allele sharing at the simple sequence repeat loci and significant associations were not found. At present we conclude, that the pedigrees under study do not have a major predisposition gene for bipolar affective disorder on chromosome 6 under the diagnostic and transmission models analyzed by the 2 different methods.

A follow-up report of a genome search for affective disorder predisposition loci in the Old Order Amish.

Progress of a full-genome scan for predisposition loci for affective disorder in the Old Order Amish is reported. LOD-score results have been previously published for 51 loci on chromosomes 1 and 11, collectively. The present report contains results for an additional 367 loci throughout the genome with extensive coverage on chromosomes 1, 2, 3, 4, 6, 7, 9, 10, 13, 14, 18, 19, and 21 (average marker density for these chromosomes = 10.7 cM). Analyses were conducted in a four-stage process: (1) two-point LOD scores were calculated for all loci under a dominant model with reduced penetrance, consistent with results of segregation analyses of these pedigrees; (2) a screen for the sharing of alleles in similarly affected individuals was used to highlight areas potentially important for further analysis; (3) the preceding areas and markers on densely covered chromosomes were analyzed using the affected-pedigree-member (APM) method; and (4) the sharing of extended haplotypes in affected individuals was examined in areas showing apparent clustering of significant allele sharing as assessed by the APM method. Of the 367 markers analyzed, no statistically significant LOD scores resulted. Some degree (P < .05) of allele sharing was found at 74 loci, and 3.8% of all markers analyzed (N = 14) passed more stringent significance criteria suggestive of linkage (P < or = .001 for at least one of the weighting functions). Multilocus APM and detailed exploration of extended haplotype sharing in areas highlighted by the APM analyses provided methods for more informative exploration of potentially suggestive results but did not identify areas clearly involved in the etiology of affective disorder in this population.

Allelotyping of all chromosomal arms in invasive cervical cancer.

The best characterized factor in the development of cervical cancer is the integration, of human papillomavirus into cervical cell chromosomes. In addition to HPV integration, the neoplastic process probably requires the activation of cellular protooncogenes and loss of tumor suppressor gene function. Loss of heterozygosity analysis in a large sample is used to identify regions which harbor putative tumor suppressor genes (TSG) since the deletion of normal alleles unmask mutated alleles. We evaluated tumor tissue from invasive cervical carcinomas, carefully microdissected to eliminate normal stroma and lymphocytes, for LOH at all 41 chromosomal arms with 50 polymorphic markers. We have evaluated tumor and normal DNA pairs from 48 invasive cervical cancers of which 85% of the tumors are confined to the cervix. The mean loss for all chromosomal arms was 12%. Three regions exhibited LOH two standard deviations above the mean: 3p14.1-12 (40%), 11q23.3 (36%), and 6p22-21.3 (32%). Three regions showed loss one standard deviation above the mean: 19q13.4 (30%), 6q21-23.33 (25%), and 2q33-37 (24%). Our results indicate that a significant number of invasive cervical cancers have lost specific chromsomal regions, thereby suggesting that genes involved in the cell cycle regulation or the suppression of tumor development are located in these regions.

Dysregulation of cyclin D1 by translocation into an IgH gamma switch region in two multiple myeloma cell lines.

Translocations involving the IgH locus at chromosomal locus 14q32.3 are a common event in many B-cell malignancies. The translocations, which generally occur into JH and switch regions, are mediated by errors in the two developmentally regulated, lymphocyte-specific pathways: VDJ-and switch-mediated recombination. Dysregulation of cyclin D1 by a t(11;14)(q13;q32) translocation occurs in most cases of mantle-cell lymphoma and in approximately 30% of multiple myeloma (MM) tumors in which a 14q32 translocation can be detected. We show here that in two of three myeloma lines that overexpress cyclin D1, there is an 11;14 translocation into a gamma switch region, suggesting an error in switch recombination. By contrast, 11;14 translocations in mantlecell lymphoma are invariably into or near a JH segment, suggesting an error in VDJ recombination. This is consistent with the fact that myeloma cells have undergone lgH switch recombination, whereas mantle-cell lymphoma cells generally have not.

A high-resolution physical map of human chromosome 11.

The development of a highly reliable physical map with landmark sites spaced an average of 100 kbp apart has been a central goal of the Human Genome Project. We have approached the physical mapping of human chromosome 11 with this goal as a primary target. We have focused on strategies that would utilize yeast artificial chromosome (YAC) technology, thus permitting long-range coverage of hundreds of kilobases of genomic DNA, yet we sought to minimize the ambiguities inherent in the use of this technology, particularly the occurrence of chimeric genomic DNA clones. This was achieved through the development of a chromosome 11-specific YAC library from a human somatic cell hybrid line that has retained chromosome 11 as its sole human component. To maximize the efficiency of YAC contig assembly and extension, we have employed an Alu-PCR-based hybridization screening system. This system eliminates many of the more costly and time-consuming steps associated with sequence tagged site content mapping such as sequencing, primer production, and hierarchical screening, resulting in greater efficiency with increased throughput and reduced cost. Using these approaches, we have achieved YAC coverage for >90% of human chromosome 11, with an average intermarker distance of <100 kbp. Cytogenetic localization has been determined for each contig by fluorescent in situ hybridization and/or sequence tagged site content. The YAC contigs that we have generated should provide a robust framework to move forward to sequence-ready templates for the sequencing efforts of the Human Genome Project as well as more focused positional cloning on chromosome 11.

Localization of a tumor suppressor gene in 11p15.5 using the G401 Wilms' tumor assay.

Multiple studies have underscored the importance of loss of tumor suppressor genes in the development of human cancer. To identify these genes, we used somatic cell hybrids in a functional assay for tumor suppression in vivo. A tumor suppressor gene in 11p15.5 was detected by transferring single human chromosomes into the G401 Wilms' tumor cell line. In order to better map this gene, we created a series of radiation-reduced t(X;11) chromosomes and characterized them at 24 loci between H-RAS and beta-globin. Interestingly, three of the chromosomes were indistinguishable as determined by genomic and cytogenetic analyses. Each contains an interstitial deletion with one breakpoint in 11p14.1 and the other breakpoint between the D11S601 and D11S648 loci in 11p15.5. PFGE analysis localized the 11p15.5 breakpoints to a 175 kb MluI fragment that hybridized to D11S601 and D11S648 probes. Genomic fragments from this 175 kb region were hybridized to DNA from mouse hybrid lines containing the delta t(X;11) chromosomes. This analysis detected the identical 11p15.5 breakpoint which disrupts a 7.8 kb EcoRI fragment in all three of the delta t(X;11) chromosomes, suggesting they are subclones of the same parent colony. Upon transfer into G401 cells, one of the chromosomes suppressed tumor formation in nude mice, while the other two chromosomes lacked this ability. Thus, our mapping data indicate that the gene in 11p15.5 which suppresses tumor formation in G401 cells must lie telomeric to the D11S601 locus. Koi et al. (Science 260: 361-364, 1993) have used a similar functional assay to localize a growth suppressor gene for the RD cell line centromeric to the D11S724 locus. The combination of functional studies by our lab and theirs significantly narrows the location of the tumor suppressor gene in 11p15.5 to the approximately 500 kb region between D11S601 and D11S724.