Intrachromosomal genomic instability in human sporadic colorectal cancer measured by genome-wide allelotyping and inter-(simple sequence repeat) PCR.

We have used genome-wide allelotyping with 348 polymorphic autosomal markers spaced, on average, 10 cM apart to quantitate the extent of intrachromosomal instability in 59 human sporadic colorectal carcinomas. We have compared instability measured by this method with that measured by inter-(simple sequence repeat) PCR and microsatellite instability assays. Instability quantitated by fractional allelic loss rates was found to be independent of that detected by microsatellite instability analyses but was weakly associated with that measured by inter-(simple sequence repeat) PCR. A set of seven loci were identified that were most strongly associated with elevated rates of fractional allelic loss and/or inter-(simple sequence repeat) PCR instability; these seven loci were on chromosomes 3, 8, 11, 13, 14, 18, and 20. A lesser association was seen with two loci flanking p53 on chromosome 17. Coordinate loss patterns for these loci suggest that at least two separate sets of cooperating loci exist for intrachromosomal genomic instability in human colorectal cancer.

Genome-wide allelotyping indicates increased loss of heterozygosity on 9p and 14q in early age of onset colorectal cancer.

Colorectal cancer remains a significant public health challenge, despite our increased understanding of the genetic mechanisms involved in the initiation and progression of this disorder. It has become clear that multiple mechanisms lead to the tumorigenic phenotype, with familial predisposition syndromes accounting for less than 15% of all colorectal cancers. A genome-wide scan for loss of heterozygosity (LOH) was carried out with 150 highly polymorphic markers in an effort to identify additional loci involved in colorectal tumorigenesis in DNA samples from 42 colorectal cancer patients. The results confirm earlier observations that tumor DNAs from patients with hereditary nonpolyposis colon cancer (HNPCC) either maintain heterozygosity or exhibit altered or additional alleles. DNAs from patients with early onset colorectal carcinomas (diagnosed prior to age 50) revealed a higher overall degree of LOH than DNAs from patients with sporadic colorectal cancers diagnosed later in life (after age 50). While regions on 1p, 10q and 14q are suggestive, statistical analysis of LOH at these regions failed to reach significance. However, LOH at 9p did reveal a statistically significant increase in the early onset patient group, compared to the greater than age 50 group. LOH on 9p may involve inactivation of p16/CDKN2 through aberrant DNA methylation on the remaining chromosome, resulting in a situation analogous to a homozygous deletion of p16 and providing a selective growth advantage to these cells. This marker may prove to be a useful prognostic indicator for patient stratification in the design of therapy for early onset colorectal cancer patients.

A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome.

Loss of imprinting at IGF2, generally through an H19-independent mechanism, is associated with a large percentage of patients with the overgrowth and cancer predisposition condition Beckwith-Wiedemann syndrome (BWS). Imprinting control elements are proposed to exist within the KvLQT1 locus, because multiple BWS-associated chromosome rearrangements disrupt this gene. We have identified an evolutionarily conserved, maternally methylated CpG island (KvDMR1) in an intron of the KvLQT1 gene. Among 12 cases of BWS with normal H19 methylation, 5 showed demethylation of KvDMR1 in fibroblast or lymphocyte DNA; whereas, in 4 cases of BWS with H19 hypermethylation, methylation at KvDMRl was normal. Thus, inactivation of H19 and hypomethylation at KvDMR1 (or an associated phenomenon) represent distinct epigenetic anomalies associated with biallelic expression of IGF2. Reverse transcription-PCR analysis of the human and syntenic mouse loci identified the presence of a KvDMR1-associated RNA transcribed exclusively from the paternal allele and in the opposite orientation with respect to the maternally expressed KvLQT1 gene. We propose that KvDMR1 and/or its associated antisense RNA (KvLQT1-AS) represents an additional imprinting control element or center in the human 11p15.5 and mouse distal 7 imprinted domains.

Structure and chromosomal localization of the human and murine genes for the macrophage MARCO receptor.

The structures of the human and mouse genes for the macrophage receptor with collagenous structure were determined. Both genes have 17 exons, of which exons 4-15 encode the collagenous domain. The transcription initiation sites in the mouse gene were identified using primer extension, SI nuclease mapping, and 5' capturing rapid amplification of cDNA ends assays. All three methods revealed two major initiation sites, one starting 27 bp downstream of a TATA box and another at positions -63 and -66 downstream of an AT-rich region. Several potential binding sites for transcription factors were identified in the promoter region, neither gene has a CAAT box or GC boxes. The human and mouse genes were localized to syntenic regions on chromosomes 2 and 1, respectively, using fluorescence in situ hybridization.

Mapping, genomic organization and promoter analysis of the human prostate-specific membrane antigen gene.

Prostate-specific membrane antigen (PSMA) is a 100 kDa type II transmembrane protein with folate hydrolase and NAALAdase activity. PSMA is highly expressed in prostate cancer and the vasculature of most solid tumors, and is currently the target of a number of diagnostic and therapeutic strategies. PSMA is also expressed in the brain, and is involved in conversion of the major neurotransmitter NAAG (N-acetyl-aspartyl glutamate) to NAA and free glutamate, the levels of which are disrupted in several neurological disorders including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease and schizophrenia. To facilitate analysis of the role of PSMA in carcinoma we have determined the structural organization of the gene. The gene consists of 19 exons spanning approximately 60 kb of genomic DNA. A 1244 nt portion of the 5' region of the PSMA gene was able to drive the firefly luciferase reporter gene in prostate but not breast-derived cell lines. We have mapped the gene encoding PSMA to 11p11-p12, however a gene homologous, but not identical, to PSMA exists on chromosome 11q14. Analysis of sequence differences between non-coding regions of the two genes suggests duplication and divergence occurred 22 million years ago.

NUP98-HOXD13 gene fusion in therapy-related acute myelogenous leukemia.

A novel chromosomal translocation, t(2;11)(q31;p15), was identified in a patient with therapy-related acute myelogenous leukemia (t-AML). Fluorescence in situ hybridization experiments mapped the breakpoint near NUP98; Southern blot analysis demonstrated that the nucleoporin gene NUP98 was disrupted by this translocation. We used rapid amplification of cDNA ends to identify a chimeric mRNA. An in-frame, chimeric mRNA that fused NUP98 sequences to the homeobox gene HOXD13 was cloned; the predicted fusion protein contains both the GLFG repeats from NUP98 as well as the homeodomain from HOXD13. The NUP98-HOXD13 fusion is structurally similar to the NUP98-HOXA9 fusion previously identified in patients with AML, leading to the speculation that NUP98-homeobox gene fusions may be oncogenic. Moreover, this report, along with a recent study that demonstrated NUP98-DDX10 fusions in patients with t-AML, raises the possibility that NUP98 may be a previously unsuspected target for chromosomal translocations in patients with t-AML.

Promoter sequence, expression, and fine chromosomal mapping of the human gene (MLP) encoding the MARCKS-like protein: identification of neighboring and linked polymorphic loci for MLP and MACS and use in the evaluation of human neural tube defects.

The MARCKS-like protein (MLP), also known as F52, MacMARCKS, or MARCKS-related protein, is a widely distributed substrate for protein kinase C (PKC). Recent studies using gene disruption in vivo have demonstrated the importance of both MARCKS and MLP to the development of the central nervous system; specifically, mice lacking either protein exhibit a high frequency of neural tube defects. We isolated a genomic clone for human MLP and discovered a directly linked polymorphism (MLP1) useful for genetic linkage analysis. The MLP promoter was 71% identical over 433 bp to that of the corresponding mouse gene, Mlp, with conservation of many putative transcription factor-binding sites; it was only 36% identical over 433 bp to the promoter of the human gene, MACS, which encodes the MLP homologue MARCKS. This 433-bp fragment drove expression of an MLP-beta-galactosidase transgene in a tissue-specific and developmental expression pattern that was similar to that observed for the endogenous gene, as shown by in situ hybridization histochemistry. In contrast to MACS, the MLP and Mlp promoters contain a TATA box approximately 40 bp 5' of the presumed transcription initiation site. MLP was localized to chromosome 1p34-->1pter by analysis of human-mouse somatic cell hybrid DNA and to 1p34 by fluorescence in situ hybridization. Radiation hybrid mapping of MLP placed it between genetic markers D1S511 (LOD > 3.0) and WI9232. MACS was localized to 6q21 between D6S266 (LOD > 3.0) and AFM268uh5 by the same technique. We tested the novel MLP1 polymorphism and the MACS flanking markers in a series of 43 Caucasian simplex families in which the affected child had a lumbosacral myelomeningocele. We found no evidence of linkage disequilibrium, suggesting that these loci were not major genes for spina bifida in these families. Nonetheless, the identification of linked and neighboring polymorphisms for MACS and MLP should permit similar genetic studies in other groups of patients with neural tube defects and other neurodevelopmental abnormalities.

Divergently transcribed overlapping genes expressed in liver and kidney and located in the 11p15.5 imprinted domain.

Human chromosomal band 11p15.5 has been shown to contain genes involved in the development of several pediatric and adult tumors and in Beckwith-Wiedemann syndrome (BWS). Overlapping P1 artificial chromosome clones from this region have been used as templates for genomic sequencing in an effort to identify candidate genes for these disorders. PowerBLAST identified several matches with expressed sequence tags (ESTs) from fetal brain and liver cDNA libraries. Northern blot analysis indicated that two of the genes identified by these ESTs encode transcripts of 1-1.5 kb with predominant expression in fetal and adult liver and kidney. With RT-PCR and RACE, full-length transcripts were isolated for these two genes, with the largest open reading frames encoding putative proteins of 253 and 424 amino acids. Database comparison of the predicted amino acid sequence of the larger transcript indicated homology to integral membrane organic cation transporters; hence, we designate this gene ORCTL2 (organic cation transporter-like 2). An expressed sequence polymorphism provided evidence that the ORCTL2 gene exhibits "leaky" imprinting in both human fetal kidney and human fetal liver. The mouse orthologue (Orctl2) was identified, and a similar polymorphism was used to demonstrate maternal-specific expression of this gene in fetal liver from interspecific F1 mice. The predicted protein of the smaller gene showed no significant similarity in the database. Northern and RACE analyses suggest that this gene may have multiple transcription start sites. Determination of the genomic structure in humans indicated that the 5'-end of this transcript overlaps in divergent orientation with the first two exons of ORCTL2, suggesting a possible role for antisense regulation of one gene by the other. We, therefore, provisionally name this second transcript ORCTL2S (ORCTL2-antisense). The expression patterns of these genes and the imprinted expression of ORCTL2 are suggestive of a possible role in the development of Wilms tumor (WT) and hepatoblastoma. Although SSCP analysis of 62 WT samples and 10 BWS patients did not result in the identification of any mutations in ORCTL2 or ORCTL2S, it will be important to examine their expression pattern in tumors and BWS patients, since epigenetic alteration at these loci may play a role in the etiology of these diseases.

A 1-Mb physical map and PAC contig of the imprinted domain in 11p15.5 that contains TAPA1 and the BWSCR1/WT2 region.

We have constructed a 1-Mb contig in human chromosomal band 11p15.5, a region implicated in the etiology of several embryonal tumors, including Wilms tumor, and in Beckwith-Wiedemann syndrome. Cosmid, P1, PAC, and BAC clones were characterized by NotI/SalI digestion and hybridized to a variety of probes to generate a detailed physical map that extends from D11S517 to L23MRP. Included in the map are the CARS, NAP2, p57/KIP2, KVLQT1, ASCL2, TH, INS, IGF2, H19, and L23MRP genes as well as end probes isolated from PACs. The TAPA1 gene, whose protein product can transmit an antiproliferative signal, was also localized in the contig. However, Northern blot analysis demonstrated that its expression did not correlate with tumorigenicity in G401 Wilms tumor hybrids, suggesting that TAPA1 is not responsible for the tumor suppression associated with 11p15.5. Genomic clones were used as probes in FISH analysis to map the breakpoints from three Beckwith-Wiedemann syndrome patients and a rhabdoid tumor. Interestingly, each of the breakpoints disrupts the KVLQT1 gene, which is spread over a 400-kb region of the contig. Since 11p15.5 contains several genes with imprinted expression and one or more tumor suppressor genes, our contig and map provide a framework for characterizing this intriguing genetic environment.

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.

Human fertilin beta: identification, characterization, and chromosomal mapping of an ADAM gene family member.

Fertilin alpha/beta (PH30 alpha/beta) is a heterodimeric sperm surface protein containing binding and fusion domains with potential for interaction with integrin receptors on the oocyte. We report the cDNA cloning, deduced amino acid sequence, tissue specificity, and chromosomal mapping of human fertilin beta. Encoded by a 2205 nucleotide open reading frame, the deduced amino acid sequence of human fertilin beta contains pro-, metalloprotease-like, disintegrin-like, cysteine-rich, epidermal growth factor-like (EGF) repeat, transmembrane, and cytoplasmic domains. Due to this domain organization, human fertilin beta has been identified as a member of the ADAM family, which is composed of membrane-anchored proteins having A Disintegrin And Metalloprotease domain. The amino acid sequence of human fertilin beta shares 90%, 56%, and 55% identity, respectively, to monkey, guinea pig, and mouse fertilin beta homologs. A phenylalanine-glutamate-glutamate (FEE) binding tripeptide within the disintegrin-like domain of human fertilin beta, homologous to other fertilin beta RGD-like (arginine-glycine-aspartic acid) tripeptides, could compete for recognition by integrins and other receptors. Northern analysis from 16 human tissues revealed human fertilin beta's 2.9 kb message only in testis, which raises interest in possible clinical applications of this molecule as a contraceptive vaccinogen. Human fertilin beta maps to chromosome 8, band p11.2, by fluorescence in situ hybridization and mouse/human somatic cell hybrid Southern hybridization.

Structure and chromosomal location of the human CD6 gene: detection of five human CD6 isoforms.

The CD6 protein has been shown to play important roles in T cell costimulation and adhesion. Recently, variably spliced isoforms of CD6 mRNA have been identified in both human and murine T cells. Here we report on the genomic organization of the human CD6 gene, its chromosomal localization, and the characterization of novel isoforms. Human CD6 is encoded by at least 13 exons. The amino terminal signal sequence, extracellular region, and transmembrane domain are encoded by seven exons, while the cytoplasmic domain of CD6 is encoded by six exons. Each of the three extracellular scavenger receptor cysteine-rich domains is encoded by a separate exon. Fluorescence in situ hybridization studies and screening of a chromosome-specific YAC (yeast artificial chromosome) library revealed that the gene encoding CD6 is located on chromosome 11 at 11q13 in close proximity to the gene encoding the related molecule CD5 and within 600 kb of CD20. Analysis of mRNA transcripts encoding CD6 isolated from mitogen-activated PBMC and from B cells obtained from patients with chronic lymphocytic leukemia revealed the presence of at least five different CD6 transcripts. These transcripts arise via variable splicing of exons encoding the cytoplasmic domain of CD6. The existence of these isoforms suggests that signaling through CD6 could be regulated via alternative splicing of cytoplasmic encoding exons.

Framework YAC contig anchored into a 3.2-Mb high-resolution physical map in proximal 11q13.

Despite the presence on band q13 of chromosome 11 of a number of genes predisposing individuals to various human diseases, most of this genomic region remains loosely mapped. Moreover, there is a relative dearth of yeast artificial chromosome (YAC) contigs from genome-wide studies: YACs are irregularly distributed over this chromosomal region and have not been arranged into contigs. We have thus undertaken fine-scale mapping of a 3.2-Mb region flanked by ACTN3 and FGF3. Since this region has demonstrated a high degree of YAC instability, we have established a framework contig by anchoring YACs and cosmids into a high-resolution physical map based on fluorescence in situ hybridization and long-range restriction mapping. The 3.2-Mb area studied includes the boundaries of regions thought to contain genes predisposing individuals to osteoporosis-pseudoglioma syndrome and insulin-dependent diabetes mellitus, as well as genes driving amplification events in human carcinomas. Another feature of this genomic area is that it cross-hybridizes to nonsyntenic regions of the genome. In addition, it spans the region where syntenic conservation with mouse chromosome 19 ends, making clones that we have anchored there valuable tools in understanding genome evolution.

A 5.5-Mb high-resolution integrated map of distal 11q13.

The distal part of 11q13, which contains several genes relevant to human diseases, has been poorly mapped as part of genome-wide mapping efforts. In the prospect of drawing a fine-scale integrated map of the area containing KRN1 and OMP, we have established a framework of markers by hybridization to DNA of somatic cell hybrids and by fluorescence in situ hybridization (FISH) on metaphase chromosomes. The probes studied were used to isolate 27 YACs and 16 cosmids that could be organized in three contigs covering approximately 6 Mb. These contigs were separated by two gaps that are likely to contain sequences underrepresented in YAC libraries. They were then integrated based on long-range restriction mapping and DNA-fiber FISH into a high-resolution physical map, which covers a 5.5-Mb region and includes 36 anonymous markers and 10 genes. This map will be used to search for genes within the 2/3 of this region where none have been localized as yet. It will also lay the ground for the characterization of an amplicon surrounding GARP in breast cancer and for the search of disease genes within this region.

Novel transcribed sequences within the BWS/WT2 region in 11p15.5: tissue-specific expression correlates with cancer type.

Chromosome band 11p15.5 has proven to be an intriguing area of the human genome. Various studies have linked alterations in this region to growth-related disorders such as Beckwith-Wiedemann syndrome and a variety of human cancers. Furthermore, functional assays in G401 Wilms tumor cells and RD rhabdomyosarcoma cells support the existence of a tumor suppressor gene on 11p15.5, sometimes called WT2. In addition, several genes mapping to this region show imprinted expression, suggesting that 11p15.5 contains an imprinted domain. We have employed solution hybrid capture in combination with sequence analysis to identify 16 genes within the approximately 700-kb critical region of 11p15.5 between D11S601 and D11S1318. Two of these genes, NAP1L4 and KCNA9, had been previously reported. Ten novel transcripts were identified with partial cDNA sequences selected by solution hybrid capture. Sequence homology to known ESTs was used to identify the remaining gene transcripts. Interestingly, the tissue-specific mRNA expression of these genes correlates with the tumor types linked to this region. This work can be compiled into a transcript map, important in the elucidation of tumor suppressor activity on chromosome 11p15.5.

A 1.5-megabase physical map encompassing the multiple endocrine neoplasia type-1 (MEN1) locus on chromosome 11q13.

Linkage analysis and loss of heterozygosity studies have shown that the gene responsible for the multiple endocrine neoplasia type-1 (MEN1) syndrome localizes to a small interval between D11S427 and D11S460 on chromosome 11q13. As an initial step to clone this tumor suppressor gene, our group is the first to map the MEN1 region physically using yeast artificial chromosome, bacterial artificial chromosome (BAC), and cosmid contigs. The 1.5-Mb high-resolution, contiguous map extends from PYGM to 300 kb telomeric of D11S460. Of this, the 1.2-Mb interval between PYGM and D11S460 is isolated in cosmids and BACs and will be useful for the development of genomic sequences and transcription maps of this important region. Nine new sequence-tagged sites (STS) are also characterized from this region. The physical map and the STSs will be valuable tools for the cloning of the MEN1 gene.

Positional cloning of a gene involved in hereditary multiple exostoses.

Hereditary multiple exostosis (EXT) is an autosomal dominant condition mainly characterized by the presence of multiple exostoses on the long bones. These exostoses are benign cartilaginous tumors (enchondromata). Three different EXT loci on chromosomes 8q (EXT1), 11p (EXT2) and 19p (EXT3) have been reported, and recently the EXT1 gene was identified by positional cloning. To isolate the EXT2 gene, we constructed a contig of yeast artificial chromosomes (YAC) and P1 clones covering the complete EXT2 candidate region on chromosome 11p11-p12. One of the transcribed sequences isolated from this region corresponds to a novel gene with homology to the EXT1 gene, and harbours inactivating mutations in different patients with hereditary multiple exostoses. This indicates that this gene is the EXT2 gene. EXT2 has an open reading frame encoding 718 amino acids with an overall homology of 30.9% with EXT1, suggesting that a family of related genes might be responsible for the development of EXT.

ML-1 cell line lacks a germline MLL locus.

Gene rearrangements involving MLL (also known as ALL1, HRX, or Htrx) are among the most common molecular abnormalities associated with acute leukemia. These leukemias generally have one allele involved in a rearrangement, while the remaining allele is uninvolved and demonstrates a germline MLL configuration. In this study, we describe a leukemic cell line that does not have a germline MLL allele and thus cannot produce a normal MLL gene product. We show that the ML-1 cell line, derived from a patient with acute myeloid leukemia, has one allele involved in a t(6;11)(q27;q23), while the remaining MLL allele is deleted. Cloning of the genomic breakpoints on the derivative(6) and der(11) chromosomes demonstrated a balanced translocation between MLL on chromosome band 11q23 and AF6 on chromosome band 6q27. Sequence analysis of the derivative chromosomes revealed that a 186-bp segment of MLL intron 6, downstream of the breakpoint, had been duplicated, inverted, and inserted between MLL and AF6 on the der(11) chromosome. In light of the fact that ML-1 cells can be induced to differentiate along the granulocyte and macrophage lineages, the finding that ML-1 lacks a germline MLL allele demonstrates that a normal MLL gene is not required for survival, proliferation, or differentiation of this cell line.

Complex MLL rearrangement in a patient with T-cell acute lymphoblastic leukemia.

MLL (also known as ALL-I, HTRX, or HRX) gene translocations are among the most common chromosomal abnormalities recognized in both B-lineage acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). However, MLL gene rearrangements are uncommon in T-cell ALL. We recently detected an MLL gene rearrangement in a patient with typical T-cell ALL. We recently detected an MLL gene rearrangement in a patient with typical T-cell ALL (CD2+, CD4+, CD5+, CD7+, CD8+, HLA DR-) and an apparently normal karyotype (46,XX). The rearrangement was cloned and characterized; a DNA fragment distal to the breakpoint was mapped by fluorescence in situ hybridization (FISH) to 19p13, indicating that the leukemic blasts had undergone a cytogenetically undetected rearrangement involving chromosomes 11 and 19. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay demonstrated an in-frame fusion mRNA between the amino terminus of MLL and the carboxy terminus of ENL (also known as MLLT1 or LTG19), a gene that has been mapped to 19p13. In addition, MLL sequences distal (telomeric) to the breakpoint were deleted from the genome, which precludes the formation of a reciprocal ENL/MLL fusion protein. These findings suggest that an MLL/ENL fusion protein (and not a reciprocal ENL/MLL fusion) was likely to be pathogenic in this patient, and they reinforce previous studies showing that leukemic blasts with apparently normal karyotype may harbor MLL rearrangements. Additionally, this report provides the first conclusive evidence of an MLL/ENL gene fusion characterized at a molecular level in a patient with T-cell ALL.