Faculty Bibliography

Cellular, cytogenetic, and molecular evidence indicates that chromosome band 1p36 is often deleted in neuroblastoma cell lines and tumours, suggesting the presence of one or more tumour suppressor genes in this region. We used a multifaceted approach to analyse the commonly deleted region, 28 distal 1p-specific polymorphic loci were used to detect loss of heterozygosity (LOH) in a panel of primary neuroblastoma tumours. Thirty-two of 122 tumours (26%) demonstrated LOH at three or more loci. In addition, a patient with a constitutional deletion of 1p36.2-.3 and two neuroblastoma cell lines with 1p36 abnormalities were characterised by FISH. When combined with the LOH data, a single consensus region of deletion was defined proximally by PLOD and distally by D1S80, a region spanning approximately five megabases. Several proposed candidate tumour suppressor genes, including ID3, CDC2L1, DAN, PAX7, E2F2, TNFR2 and TCEB3, map outside of this region; however, the transcription factor HKR3 cannot be excluded. LOH for 1p is correlated with adverse clinical and biological features and a poor prognosis, but 1p LOH is not an independent predictor of overall survival. To identify additional candidate genes, an integrated physical map of 1p35-36 is being constructed. The current map includes 445 polymerase chain reaction (PCR)-formatted markers and 608 YACs. This map will help identify region-specific transcripts by direct selection and sequencing.

Human Krüppel-related 3 (HKR3) is a zinc finger gene that maps within chromosome subbands 1p36.2-.3, a region postulated to contain a tumour suppressor gene associated with advanced neuroblastomas. Genomic clones of HKR3 were isolated from a P1 library and physically mapped to within 40 kb of D1S214 at 1p36.3. The gene is ubiquitously expressed in human tissues, but especially high levels are present in human fetal and adult nervous tissues. Hemizygous deletion of HKR3 in a lymphoblastoid cell line derived from a neuroblastoma patient with a constitutional 1p36 interstitial deletion and in the neuroblastoma cell line SK-N-AS, which also has a small interstitial 1p36 deletion, has been observed. Allelic loss at D1S214 in 15/15 informative primary neuroblastoma specimens with 1p36 deletions has also been observed. In a panel of 16 neuroblastoma cell lines, no gross genomic DNA rearrangements were noted, the gene was always expressed (albeit at variable levels) and there was no evidence for truncating mutations. Furthermore, there were no mutations detected in the zinc finger coding region in four neuroblastoma cell lines with 1p deletions analysed by direct sequence analysis. We conclude that HKR3 is a novel zinc finger gene that maps to a region of the genome commonly rearranged or deleted in neuroblastoma and other human cancers.

Neuroblastoma has several clinical and molecular genetic parallels with the other paediatric embryonal tumours, such as retinoblastoma, including a hereditary form of the disease. We hypothesised that neuroblastoma susceptibility is due to germline mutations in a tumour suppressor gene and that this predisposition gene may be involved in sporadic neuroblastoma tumorigenesis as well. We therefore aimed to localise the familial neuroblastoma predisposition gene by linkage analysis in neuroblastoma kindreds. Eighteen families segregating for neuroblastoma were ascertained for candidate locus linkage analysis. Although many of the 49 affected individuals in these families were diagnosed as infants with multifocal primary tumours, there was marked clinical heterogeneity. We originally hypothesised that familial neuroblastoma predisposition would map to the telomeric portion of chromosome band 1p36, a genomic region likely to contain a sporadic neuroblastoma suppressor gene. However, neuroblastoma predisposition did not map to any of eight polymorphic markers spanning 1p36.2-.3 in three large kindreds. In addition, there was strong evidence against linkage to two Hirschsprung disease susceptibility genes (RET and EDNRB), a condition that can cosegregate with neuroblastoma as in one of the kindreds tested here. We conclude that the neuroblastoma susceptibility gene is distinct from the 1p36 neuroblastoma suppressor and two of the currently identified Hirschsprung disease susceptibility genes.

The distal short arm of human chromosome 1 (1p) is rearranged in a variety of malignancies, and several genetic diseases also map to this region. We have constructed an integrated transcript map to precisely define the positions of genes and expressed sequence tags (ESTs) previously mapped to 1p35-p36, a region spanning approximately 40 Mb. To anchor the integrated map, a framework genetic map was constructed with 24 genetic markers and a marker order of 1000:1 odds, yielding an average resolution of 2.8 cM. An additional 106 genetic markers were localized relative to the framework genetic map. To place markers more precisely within 1p35-p36, a chromosome 1-specific, radiation-reduced hybrid (RH) panel was created. Individual DNA fragments of the RH panel were identified and ordered by PCR with the framework genetic map. A total of 250 markers, including 142 genes and ESTs, were mapped by PCR against the RH panel. The map has an observed resolution of 800 kb, and the results closely match and more precisely define previous mapping information for most markers. This map will help to identify candidate genes for genetic diseases mapping to distal 1p and is fully integrated with existing genetic and RH maps of the human genome.

Ror1 is an orphan cell surface receptor with strong homology to the tyrosine kinase domain of growth factor receptors, in particular the Trk family. Southern blot analysis of genomic DNA from somatic cell hybrids revealed that Ror1 is located on chromosome 1. We have mapped the Ror1 gene to chromosome 1p12-p32 using PCR on a somatic cell hybrid panel that subdivides chromosome 1p. We have further localized the gene to chromosome 1p31-p32 by fluorescence in situ hybridization using a PAC clone that contains the Ror1 gene.

Mouse eck, a member of the EPH gene family, has been mapped to mouse chromosome 4. The syntenic relationship between this chromosome and human chromosome 1 suggests that the human ECK gene maps to the distal short arm of human chromosome 1 (1p). Since this region is frequently deleted or altered in certain tumors of neuroectodermal origin, it is important to define the specific chromosomal localization of the human ECK gene. PCR screening of a rodent-human somatic cell hybrid panel by ECK-specific primers showed that ECK is indeed localized to human chromosome 1. Additional PCR screening of a regional screening panel for chromosome 1p indicated that ECK is localized to 1p36, distal to FUCA1. Furthermore, fluorescence in situ hybridization analysis with an ECK-specific P1 clone showed that ECK maps proximal to genetic marker D1S228. Taken together, the data suggest that ECK maps to 1p36.1, a region that is frequently deleted in neuroblastoma, melanoma, and other neuroectodermal tumors.

One of the most important prognostic factors in neuroblastoma is amplification of the MYCN gene, which is strongly associated with advanced stages of disease and a poor prognosis. Although the MYCN amplicon sometimes spans more than 1 Mb, no other consistently expressed sequences from the MYCN amplicon have been reported. However, DDX1, a gene encoding a DEAD box protein, was recently mapped to chromosome 2p24 and is frequently co-amplified with MYCN. Therefore, we performed genomic mapping with YACs to determine the physical relationship between DDX1 and MYCN, and whether DDX1 was contained within the core region of amplification. Based on YAC restriction mapping and content analysis, DDX1 maps 340 kb 5' of MYCN, outside the core domain of consistent amplification. Interestingly, we also determined by sequence analysis and detailed restriction mapping that G21, previously isolated as a 'neuroblastoma-specific' cDNA clone from an MYCN amplicon, is a partial cDNA of DDX1. Our data confirm that DDX1 is amplified in some but not all MYCN-amplified tumors, and that it is rearranged in other cases. This suggests that the co-amplification of DDX1 is due to its proximity to MYCN.

Familial predisposition to neuroblastoma, a common embryonal cancer of childhood, segregates as an autosomal dominant trait with high penetrance. It is therefore likely that neuroblastoma susceptibility is due to germ line mutations in a tumor suppressor gene. Cytogenetic, functional, and molecular studies have implicated chromosome band 1p36 as the most likely region to contain a suppressor gene involved in sporadic neuroblastoma tumorigenesis. We now demonstrate that neuroblastoma predisposition does not map to any of eight polymorphic markers spanning 1p36 by linkage analysis in three families. In addition, there is no loss of heterozygosity at any of these markers in tumors from affected members of these kindreds. Furthermore, there is strong evidence against linkage to two Hirschsprung disease (a condition that can cosegregate with neuroblastoma) susceptibility genes, RET and EDNRB. We conclude that the neuroblastoma susceptibility gene is distinct from the 1p36 tumor suppressor and the currently identified Hirschsprung disease susceptibility genes.

The Krüppel-type zinc finger proteins are members of a conserved family of transcription factors that are important in developmental regulation. Altered expression of several of these proteins has been implicated in human diseases, including cancer. We report the cloning, mapping, and characterization of the zinc finger gene Human Krüppel-Related 3 (HKR3). Genomic clones of HKR3 were isolated from a P1 library and localized to human chromosome subband 1p36.3 by human-rodent somatic cell hybrid mapping and fluorescence in situ hybridization. The gene was physically mapped to within 40 kb of D1S214 by YAC content and long-range restriction mapping. HKR3 spans 9.5 kb of genomic DNA and is contained in 11 exons. Sequencing defined each of the exon/intron splice site junctions and identified a CpG island in the 5' region of the gene. HKR3 is ubiquitously expressed in human tissues as at least two major transcripts, the shorter of which excludes a conserved finger-associated box and a putative acidic activation domain contained in the full-length transcript. HKR3 is a novel zinc finger gene that maps to a region of the genome commonly rearranged or deleted in human cancers.

The tumor necrosis factor receptor 2 (TNFR2) gene localizes to 1p36. 2, a genomic region characteristically deleted in neuroblastomas and other malignancies. In addition, TNFR2 is the principal mediator of the effects of TNF on cellular immunity, and it may cooperate with TNFR1 in the killing of nonlymphoid cells. Therefore, we undertook an analysis of the genomic structure and precise physical mapping of this gene. The TNFR2 gene is contained on 10 exons that span 26 kb. Most of the functional domains of TNFR2 are encoded by separate exons, and each of the repeats of the extracellular cysteine-rich domain is interrupted by an intron. The genomic structure reveals a close relationship to TNFR1, another member of the TNFR superfamily. Based on electrophoretic analysis of yeast artificial chromosomes, TNFR2 maps within 400 kb of the genetic marker D1S434. In addition, we have identified a new polymorphic dinucleotide repeat within intron 4 of TNFR2. The genetic sequence information and exon-intron boundaries we have determined will facilitate mutational analysis of this gene to determine its potential role in neuroblastoma, as well as in other cancers with characteristic deletions or rearrangements of 1p36.