Abstracts
Cloning genes on chromosome 1 that are altered in cancer cell lines using micro-arrays of genomic clones.
K. Aubin, J. MacDonald and R. Wooster
Institute of Cancer Research, 15 Cotswold Rd, Sutton, Surrey, SM2 5NG.
A number of regions of chromosome 1 have been implicated in the development and progression of cancer by the occurrence of somatic deletions and amplifications in tumours. Previous techniques for defining these intervals, for example Southern blotting and comparative genomic hybridisation, can be time consuming, some have poor resolution and few have taken advantage of the information being produced as part of the human genome mapping project. We have produce a high density array of genomic clones, P1 artificial chromosome (PACs) that span chromosome 1. The location of the PACs is determined by their relationship to STSs placed on radiation hybrid maps and from the PAC contigs that are being built to cover the chromosome. Genomic DNA from the cell lines and normal DNA will be differentially labeled with fluorescent dyes and hybridized to the micro-arrays of PAC DNA. Specifically bound fluorescent DNA will be detected with an automated scanner which will determine the ratio of normal to cell line signals. Regions that are consistently lost or gained will be refined by producing higher density arrays of PACs towards the aim of identifying the genes that are the targets of these aberrations.
Construction of a cosmid pocket map of a 10 Mb region in 1p31.1 including PAC coverage of a locus with an elevated LOH in human breast cancer.
P.R. Baptista, 1 R.H. Flomen, 1 C.H. James, 1 J. M. Varley 2 and D. Nizetic 1
1 Centre for Applied Molecular Biology, School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK. 2 CRC - Section of Molecular Genetics, Paterson Institute for Cancer Research, Manchester, UK.
There is now increasing evidence that one or more tumour suppressor genes map to the short arm of chromosome 1, and recent mapping efforts of those regions in 1p has been reported in both invasive and pre-invasive breast tumours. High resolution mapping, both at the genomic level and using YACs with cloned DNA from the region has been carried out and this has enabled the identification of a common target for LOH between the loci D1S430 and D1S207 (Hoggard et al., 1995).
In order to refine the physical map in this region a high resolution cosmid pocket map was constructed spanning over 10 Mb in 1p31.1 (from D1S216 to D1S488). YACs from public databases have been confirmed by their STS content and used as probes for screening a six chromosome equivalent cosmid library constructed from flow sorted chromosome 1 material. This map was further enriched with PAC clones spanning over the minimal region of LOH in human breast cancer mapped to 1p31.1. These cosmid and PAC clones can be used to anchor and improve the integrity of any future sequence ready PAC/BAC maps constructed in this region. These clone collections may also help the search for the putative gene(s) involved in the pathogenesis of breast cancer in this region.
|
YAC |
Size ( Kb) |
STS content |
Cosmid hits |
|
9ag4 |
460 |
nt |
27 |
|
922a7 |
1600 |
D1S216, D1S499 |
223 |
|
27bd3 |
400 |
nt |
34 |
|
811h5 |
1350 |
AFM056XC9 |
85 |
|
816a10 |
1500 |
AFM056XC9, WI71338 |
113 |
|
904c1 |
1200 |
WI71338, D1S500 |
124 |
|
949b7 |
1500 |
D1S500, D1S3336 |
198 |
|
979a6 |
1500 |
D1S3336, D1S465, D1S430 |
90 |
|
20df1 |
500 |
nt |
67 |
|
26gb2 |
370 |
D1S500 |
31 |
|
817a12 |
680 |
D1S430 |
117 |
|
820e4 |
1300 |
D1S465, D1S430 |
169 |
|
13fc10 |
200 |
D1S430 |
16 |
|
883e11 |
1500 |
D1S430, D1S2856, D1S207 |
120 |
|
890b3 |
600 |
nt |
76 |
|
885c5 |
1600 |
D1S2856, D1S207, D1S208, D1S488 |
nt |
|
965f9 |
1020 |
D1S207, D1S208, D1S488 |
nt |
|
6fg7 |
400 |
nt |
16 |
Refined genetic mapping of the Wld candidate region.
M.P. Coleman, 1 L. Conforti, 1 E.A. Buckmaster, 1 A. Tarlton, 2 M.C. Brown, 2 M.F. Lyon 3 and V.H. Perry 1
Departments of 1 Pharmacology, and 2 Physiology, University of Oxford, UK, 3 MRC Mammalian Genetics Unit, Harwell, Oxford, U.K.
The separation of a part of an axon from the neuronal cell body results in the degeneration of the distal axonal segment. This process, that occurs both in the Peripheral Nervous System (PNS) and in the Central Nervous System (CNS), is known as Wallerian degeneration. A natural mutant strain of mice (C57BL/Wlds) which shows abnormally slow Wallerian degeneration has been discovered (Lunn et al.). The putative gene which controls this phenotype has been named Wld (Wallerian degeneration) and the mutant allele Wlds (Wallerian degeneration-slow).Wlds arose on a C57BL background. C57BL/6J and C57BL/Wlds appear to be otherwise identical. The slow degeneration phenotype can be observed in vivo, both morphologically and electrophysiologically, in the CNS and PNS, and in vitro, in neurites grown from explants of superior cervical ganglion, dorsal root ganglion and cerebellum from neonatal mice.
Backcrosses of (C57BL/Wlds x Balb/c) with each parent strain indicated that the slow degeneration phenotype is inherited in an autosomal dominant pattern (Perry et al.).
Wld has previously been mapped to distal mouse chromosome 4 (Lyon et al.)) using a 282 animal cross of (C57BL/Wlds x M. spretus) x C57BL/6J. We have extended this cross to 1262 animals and made a second 227 animal cross in which M. spretus was replaced by DBA, bringing the total to 1489. Both crosses indicated the same location for Wld between Nppa and D4Mit33 and, in addition to this, the DBA cross placed Wld proximal to D4Mit127. The composite locus order is centromere - (Tnfr2, Nppa) - (0.40 ± 0.16) - (Wld, D4Mit49, D4Mit225, D4Mit310) - (0.07 ± 0.07) - D4Mit127 - (0.07 ± 0.07) - D4Mit33 - (0.64 ± 0.28) - 4-1bb - (0.5 ± 0.3) - Kcnb3 - telomere.
The identification of the Wld gene will be important not only for understanding the early events in Wallerian degeneration but also mechanisms of axonal degeneration in general. Interestingly, the axonal form of Charcot-Marie Tooth (Type 2A) has been mapped to human 1p36, which is syntenic with distal mouse chromosome 4. It may be possible to use the Wld mouse as a model to investigate the critical steps of other axonal pathologies, of which Wallerian degeneration is the common endpoint.
(2) Perry, V.H. et al. (1990). Eur. J. Neurosci. 2: 408-413.
Construction of a YAC/PAC physical map of a gene rich region in 1p13.3.
R.H. Flomen, P. Baptista, C. James and D. Nizetic.
Centre for Applied Molecular Biology, School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX.
A gene rich region spanning 2 cM of 1p13.3 around D1S2085 and D1S2809 shows a clustering of several genes, which include one or more members of a number of multigene families. The proximal boundary of this area is marked by the YKL39 precursor gene (Hu et al. 1996), adjacent to which lie a cGMP activated potassium family gene (KCNA3)(Attali et al. 1992), four members of the glutathione-S-transferase family (GSTM1-2 and 4-5) (Pearson et al. 1993) and a guanine nucleotide binding protein family member (GNAT2)(Morris and Fong 1993). Distally located is macrophage colony stimulating factor (MCSF) (Kawasaki et al. 1981), and a number of mapped ESTs (Whitehead Institute).
Work is in progress to construct PAC contigs in this region using YAC clones and STSs/ESTs to isolate PACs in the first instance. PAC contigs are extended using PAC inserts, and PAC/YAC ends as probes both on the 1p13.3 sublibrary and on the whole PAC library. Completed contigs are verified by STS/EST content and by EcoRI fingerprinting. A conservation of gene grouping between humans and rodents has been previously described in this region (Baron et al. 1996), and a high resolution physical map in 1p13.3 will enable a full cross species comparison to be carried out. This may lead to the identification of additional gene family members. The bacterial contigs will also allow the detailed mapping of other genes in the area and will form the basis of future efforts to isolate novel transcripts.
|
Major locus |
Map location |
Clone |
Size (Kb) |
Markers |
|
MCSF-KCNA3 |
1p13.3 |
YAC 944H4 |
850 |
D1S2085 |
|
YAC 14HD10 |
360 |
MCSF |
||
|
YAC 14GB2 |
500 |
[53H1R] |
||
|
YAC62H10 |
540 |
KCNA3 |
||
|
YAC788B3 |
1300 |
D1S2809 |
[x] = YAC end probe
MCSF = WI9116 (macrophage colony stimulating factor)
GNAT2 = WI9136 (guanine nucleotide binding protein)
GST = WI7167 (glutathione S transferase)
KCNA3 = UTR9869 (potassium channel gene)
YKL39 = IMAGE 284640 (YKL 39 precursor)
Characterisation of supernumerary rings and giant rod markers in adipose tissue sarcomas reveals frequent involvement of 1q21- q22 and 1q24.
A. Forus, 1 F. Pedeutour, 2 J-M. Coindre, 3 J-M. Berner, 1 O. Myklebost and C. Turc-Carel 2
1 Department of Tumor Biology, The Norwegian Radium Hospital, Oslo, Norway, 2 Laboratoire de Génétique Chromosomique des Tumeurs, Faculté de Médecine, Université de Nice- Sophia Antipolis, France, 3 Laboratoire d´Anatomie Pathologique, Fondation Bergonié, Bordeaux, France.
Supernumerary ring or giant rod marker chromosomes are a characteristic of well-differentiated liposarcomas (WDLPS) and atypical lipomas (ALP) (Orndal, et al., 1992) but has also been observed in other sarcoma subtypes (Szymanska, et al., 1996) . These marker chromosomes represent a new kind of amplification structures, midway between dmin and HSRs, but the mechanisms accounting for the formation of these structures remain obscure (Pedeutour, et al., 1994) . Using a combination of different methods, we have extensively investigated the structure and composition of rings and giant rods in a series of 14 WDLPS-ALP and three intro or inter muscular lipomas (IMLP), and revealed a unique combination of particular features strikingly related to these tumours. Although the rings and rods displayed in vitro and in vivo stability, neither the presence of alpha-satellites nor positive C-banding could be detected on any of these supernumerary structures. Comparative Genomic Hybridization analysis (CGH) in combination with fluorescent in situ hybridization (FISH) has identified the chromosomal regions contributing to the formation of these markers: All markers carried amplifications of 12q14-15, in WDLPS always involving amplification of the MDM2 gene. Variable non-syntenic regions from other chromosomes were amplified together with the chromosome 12 region, and sequences from chromosome 1, and more particularly, 1q21-q24, was frequently included. This has also been observed by other investigators (Szymanska, et al., 1997) . Using YACs from the 1q21-q22 region in combination with chromosome 1 painting probes, we are studying the composition of this marker-associated amplicon in more detail.
Structural analysis of the SCL gene locus in human, mouse, chicken and fugu.
B. Gottgens, 1 L.M. Barton, 1 J.G.R. Gilbert, 1 A.J. Bench, 1 M-J. Sanchez, 1 S. Mistry, 1 A. M McMurray, 2 J. Wilikson, 2 D. Grafham, 2 M. Berks, 2 D. Bentley, 2 M. Vaudin 2 and A.R. Green1
1 University of Cambridge, Department of Haematology, Cambridge, U.K., 2 The Sanger Centre, Wellcome Trust Genome Campus, Hinxton Cambridge
The advent of large scale sequencing projects has heightened expectations that exhaustive analysis of sequence data will be a major contributor to a better understanding of key biological processes. One area of expected progress within this emerging field of functional genomics is the study of gene hierarchies during development. This hierarchy is imposed through the transcriptional control of developmental master-regulators and is crucial for ontogenesis to proceed normally. We explored large scale sequencing of the SCL/TAL-1 gene, a master regulator of haemopoiesis, as a means to further our understanding of its transcriptional regulation. We cloned and fully sequenced the SCL locus from human, mouse, chicken and Fugu genomic DNA. We detected a large number of conserved stretches of non-coding DNA between the human and mouse loci, and showed that known areas of open chromatin precisely coincided with blocks of homology. Only a limited number of these homologous regions could be readily when the chicken or fugu sequence were included in the alignments. One of these was the promoter of the SCL gene, for which we show the alignment across all four species allowed us to zoom in on potential key transcriptional binding sites. Finally we show that a fragment of the chicken SCL genomic locus can direct expression of a linked lacZ reporter gene to a subset of the SCL expression domain in transgenic mice.
Searching for a tumour suppressor gene on human chromosome 1p31.1.
Y. Hey, B. Brintnell, L. James, J. Varley
CRC Section of Molecular Genetics, Paterson Institute for Cancer Research, Wilmslow Road, Manchester M20 9BX
We have previously reported a region showing high loss of heterozygosity (LOH) in human breast cancer on human chromosome band 1p31.1. We have constructed a 7 Mb contig comprising YACs from the CEPH and Zeneca libraries, and BACs (Research Genetics). The minimal region of loss is encompassed by two overlapping BACs, and is approximately 200 Kb. We have subcloned the BACs into plasmid vectors, and generated sequence from both BACs which we are currently assembling. The sequencing is near completion, and we will be presenting our analysis of this region at the workshop. So far we have excluded all genes and ETSs known to map to the region as candidates, including one EST which we have characterized as a pseudogene. In a parallel approach we are carrying out direct selection of cDNAs using both BACs.
Physical mapping of the candidate region for Paroxysmal Choreoathetosis/Spasticity on chromosome 1p33.
K. Hofele and G. Auburger
Dept. Neurology, University Hospital Düsseldorf.
Paroxysmal choreoathetosis is a heterogeneous neurological syndrome usually with autosomal dominant inheritance. We have mapped one form of this syndrome, paroxysmal choreoathetosis/spasticity (CSE) to a region of probably 2.8 cM within markers D1S443 and D1S2802 on chromosome 1p33. In order to find the corresponding gene we are constructing a YAC-contig comprising approximately 8 cM between D1S509 and D1S417 containing this region. Currently, the contig comprises more than 60 YACs from the CEPH, ICI and ICRF libraries that have been obtained by screening for STSs from the region, both for previously identified loci and for 15 newly isolated STSs. The currently existing contig still shows two gaps, one between markers D1S443 and D1S2733 and the other between D1S2391 and D1S2874. In order to close these gaps and to get a full contig we are screening the available YAC-libraries as well as a PAC library with new STSs generated from YAC ends. Our physical mapping studies also include mapping of ESTs and possible candidate genes that have been mapped to the region previously. So far, 12 ESTs have been mapped on the current contig with 9 of these ESTs being generated from human brain cDNA libraries. The only known gene that maps to our candidate region is the gene for the Glycine-transporter GlyT1. Glycine is one of the major inhibitory transmitters in the central nervous system and it acts as activator of a chloride channel. The reuptake-transport of neurotransmitters from the Synaptic cleft into presynaptic nerve endings or glial cells is a major determinant of signal duration. A point-mutation in the gene for Glyt1 could interfere with transporter function and thus affect control of motor functions.
Gene organization of human chromosome 1q22-q23 which is homologous to the HLA region on chromosome 6p21.3.
H. Inoko, 1 A. Shigenari, 1 Y. Suto, 2 H. Kawata, 1 T. Shiina, 1 E. Soeda, 3 K. Sugaya, 4 K. Tokunaga, 5 T. Ikemura 6 and A. Ando 1
1 Dept. Molecular Life Science, Tokai University School of Medicine., 2 Dept. Research, The Japanese Red Cross Central Blood Center, 3 RIKEN Gene Bank, The Institute of Physical and Chemical Research, 4 Genome Research, National Institute Radiological Science, 5 Dept. Human Genetics, Tokyo University, 6 Dept. Developmental Genetics, National Institute of Genetics.
The human leukocyte antigen (HLA) gene complex contains plenty of genes playing a central role in immune response and spans about a 4 Mb segment of the short arm of chromosome 6 (6p21.3). Through the analyses of the gene organization of the HLA region, we have found that more than 13 genes including PBX2, RXRB, TAP, LMP, and HSP70 in the HLA region have homologous genes or sequences in the 1q21-q25, 9q33-q34, and 19p13.1-p13.3 indicating segmental chromosome duplication during the course of evolution.
In order to clarify the gene organization of these four paralogous regions we have first analyzed the genomic structure of the 1q22-q23 region around the CD1 (non-classical MHC antigen) genes using YAC and PAC clones. An approximately 1.4 megabase YAC and PAC contig containing the five CD1 genes were constructed by PCR using locus-specific and STS primer pairs and sequencing of the end regions of PAC clones. Nine genes were located in this region, with the order of CD1D - CD1A - CD1C - CD1B - CD1E - SPTA1 - FY - IFI-16 - FCERIA from centromere to telomere. +P5 (D1S3309E), a target binding site for the Wilms' tumor suppressor 1 (WT1) was also localized at the 200- Kb centromeric side of the CD1D genes. Ordering of these PAC clones in a contig were confirmed using the fiber-FISH method. Further, from the PCR analyses of the YAC clones which were located in the telomeric side of FCERIA, nine genes were mapped, FCERIA - PBX1 - ALDH9 - RXRG - POU2F1 - CD3Z -ATAC - FV -FMO1 - FASL between D1S194 and WI-6210.
Thus, it was revealed that the 1q22-q23 region around the CD1 genes contained a cluster of genes which had immunological importance, such as SPTA1, FY, IFI-16, FCERIA, and CD3Z .
Frequent interstitial deletions define multiple smallest regions of overlap at chromosome 1p34-pter in neuroblastomas found in Japan.
H. Kageyama, 1 Y. Nimura, 1 I. Ashraful, 1 N. Y. Nakamura, 1 M. Hirose, 1, T. Iijima1, 1 S. Sakiyama1, 1Y Kaneko, 2 and A. Nakagawara 1
1 Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba, Japan, and 2 Saitama Cancer Center Hospital, Saitama, Japan
The distal region of the short arm of chromosome 1 is supposed to be loci for multiple tumor suppressor genes of neuroblastoma (NB) and other cancers. However, the identification of the smallest regions of overlap (SROs) has long been difficult because most of allelic losses are large terminal deletions. To determine the SROs in the region of chromosome 1p34-pter, we here used a large number of NB tissues found by the mass screening (MS) program in Japan. The 212 paired NB tissue and blood samples which include 126 NBs found by MS were subjected for the loss of heterozygosity (LOH) study. The markers used were 16, in which D1Z2 and MYCL were the most distal and proximal markers, respectively, and 14 were markers at 1p36. The LOH at the region of 1p34-pter was observed in 87 (41%) NBs. Unexpectedly, we found frequent interstitial or small terminal deletions in 59% of the tumors with LOH. Those small deletions were more frequent in MS tumors (78%) than in sporadic tumors (47%) (p<.01). As reported previously, large terminal deletions including 1p35-pter were commonly observed in NBs with N-myc amplification. Based on these data, we defined 5 possible SROs, two of which were within the most commonly deleted region between D1S80 and TNFR2 so far reported. The estimated intervals of those SROs were between less than 1 and 3 cM. We are currently constructing contigs of each region by using PAC, P1 and BAC.
High resolution YAC fragmentation map of 1p21.
M Lioumi,1,3 GM Olavesen,1 D Nizetic, 2 J Ragoussis 1
1 Division of Medical and Molecular Genetics, United Medical and Dental School of Guy's and St. Thomas's Hospital London, 2 Centre of Applied
Molecular Biology, University of London, School of Pharmacy, London, 3 Paediatric Research Unit, Guy's Hospital, London.
Chromosomal band 1q21 contains a number of genes, consisting the Epidermal Differentiation Complex (EDC), most of which are involved in the process of terminal differentiation of the human epidermis and implicated in several disorders of keratinization and cancer. The physical map of 1q21 has been refined by generating 400 YAC derivatives. These products have allowed us to precisely localise EDC genes and additional ESTs. The transcriptional map of the region has been extended by positioning 20 ESTs reported to map between D1S442 and D1S305. The ESTs are localized in two distinct clusters, confirmed by isolating PACs and chromosome 1 specific cosmids. Two of the ESTs correspond to the genes for YL1 and selenium binding protein, both of which have potential tumor suppressor activity. Through the use of fragmented YACs and bacterial clones the order of markers and ESTs in the region has been established as follows: CEN -A002O32-Bda44g03-Cda10d12- Bdab5d06, H60056, A005K39-D1S442-WI5663-WI7969-Cx40-Cda0ge12-Cda0kh05-A002D26-A008S07-Cda0ff08-D1S498-S100A10-WI7815(THH)-WI7217(FLG)-D1S1664-INV-SPRR2A-LOR-A001X21-D1S305-TEL (to be published in Genomics).
We are currently characterizing two of the above transcripts, Bdab5d06 and A001X21. While the former is linked to an EST contig part of which is probably coding based on the similarities to several mouse ESTs, the latter identifies several cDNA clones that show significant homology with the growth arrest inducible gene product
A yeast artificial chromosome contig across an interval on 1q25-31 containing the Camptodactyly-Arthropathy-Coxa Vara-Pericarditis syndrome locus.
J. Marcelino, W Suwairi, O. Gutierrez, S. Schwartz, S. Bahabri and M Warman.
Departments of Genetics and Pediatrics, Case Western reserve University School of Medicine, Cleveland, OH; Centre for Human Genetics, University Hospitals of Cleveland, Cleveland, OH; King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
We report the construction and characterisation of a yeast artificial chromosome (YAC) contig across and interval on human chromosome 1q25-31 which contains the locus responsible for causing the Camptodactyly-Arthropathy-Coxa Vara-Pericarditis Syndrome (CACP). CACP is an autosomal recessive disorder whose principal features include congenital flexion contractures of the fingers and toes (camptodactyly) and childhood-onset joint pain, swelling, and/or restricted range of motion (arthropathy) Less frequently occurring component features include progressive deformity of the femoral neck (coxa vara) and non-inflamatory pericarditis. Performing homozygosity mapping with DNA from 4 consanguineous kindreds affected with CACP permitted us to map the CACP locus to a 1.9 cM genetic interval (Bahabri et al., Arthritis & Rheumatism, 41:730-735, 1998); the simple sequence repeat polymorphic (SSRP) marker D1S2701 defined the centromeric boundary of this interval and the SSRP marker D1S202 defined the telomeric boundary. YACs containing genomic DNA from this interval were identified from the Human Physical Mapping Project database at the Whitehead Institute. Four YACs from the CEPH mega-YAC library (940-F12, 910-F9, 956-B9, and 815-G3) were chosen for detailed analysis. At least one of these four YAVS contains the boundary SSRP (D1S2701 or D1S202) or a completely linked SSRP (e.g. D1S191, D1S444). Fluorescence in situ hybridisation analyses using the YAC DNAs on human metaphase chromosome spreads indicated that they were non-chimeric and contained no sizable internal deletions. That the YACs formed an overlapping contig was established by both PCR and Southern blot analysis using SSRP and sequence tagged sites (STS) as probes. Unique sequences derived from YAC end clones were used to generate novel STS probes to confirm this overlap. Pulsed filed gel electrophoresis of uncut and NotI digested YAC DNA suggests that the physical size of the contig is approximately 4 megabases.
We have placed and roughly ordered two known genes and twelve expressed sequence tags (ESTs) within the contig. Several bacterial artificial chromosomes (BACs) containing inserts from the CACP candidate region were identified by screening the California Institute of Technology BAC library
These BACs are being used to generate new SSRPs in order to narrow the candidate interval; once narrowed a complete BAC contig will be constructed.
Fine mapping of region 1p36.2-3, commonly deleted in neuroblastoma and germ cell tumors.
T. Martinsson, 1 K. Ejeskar, 1 R. M. Sjoberg, 1 and P.F. Ambros 2
1 Dept. Clin. Genet., Sahlgrenska University, Hospital /East, S-416 85, Gothenburg, Sweden and 2 Children's Cancer Res. Inst., St. Anna, Kinderspital, Kinderspitalg. 6, A-1090 Vienna, Austria
A common genetic feature of neuroblastomas (NB), also being an important prognostic factor, are deletions of chromosome region 1p. The deletion of 1p often involves a deletion of varying size, with a consensus region within the most distal bands 1p36.2-3. The NB SRO (shortest region of overlap of deletions) presented earlier by our group was defined distally by the cluster of loci D1S80/D1Z2/CDC2L1 and proximally by loci D1S244 i.e. app. 25 cM. The 1p deletions are, however, not restricted to NB tumors. In fact a large spectrum of tumor types display deletions to varying degree of 1p. We have exploited the possibility of using deletions of other tumor types, preferentially that of germ cell tumors (GCT), and combining it with that of the NB SRO. Also in GCT distal 1p-deletion have been shown to have prognostic significance. We found in our GCTs a SRO ranging from D1S508 to D1S200. Interestingly this region only partially overlapped with our NB SRO in region D1S508 to D1S244, which constitute app 5 cM. We have thus focused on analysis of this smaller region in search for genes involved in the genesis of different cancer. We have performed radiation hybrid mapping of a large number of markers, STSs, ESTs and others known to reside in 1p. We have also initiated the development of a BAC contig of the region. FISH Mapping of BACs were also performed. We have also analyzed a number of transcribed genes in the region for mutations and methylation pattern. The data presented here constitute an ongoing work with the aim to identify and clone gene(s) important for development of GCTs, NBs and possibly other tumors.
Characterisation of a novel amplicon at 1q21-q22 frequently observed in human sarcomas by Fluorescent in situ Hybridisation (FISH).
L.A. Meza-Zepeda, 1 A. Forus, 1 L. Godager, 1 J-M. Berner, 1 D. Mischke, 2 I. Marenholz, 2 I. Ragoussis, 3 Ø. Fodstad 1 and O. Myklebost1
1 Dept. of Tumour Biology, The Norwegian Radium Hospital, Oslo, Norway, 2 Institut für Experimentelle Onkologie und Transplantationsmedizin, Virchow-Klinikum der Humbolt-Universität, Berlin, Germany, 3 Division of Medical and Molecular Genetics, Guy's Hospital, London, UK
Sarcomas show recurrent amplification of 1q21-q22 (Forus, et al., 1995a; Forus, et al., 1995b) . By Southern blot analysis we investigated the amplification status of 14 markers in this region, including CACY/S100A6 and MUC1, that were previously found to be occasionally amplified in melanoma and breast carcinoma, respectively. Thirty-six sarcoma samples were analysed, and most of the 14 markers detected only low amplification levels (Forus, et al., 1998) . Only D1S3620, located within the Epidermal Differentiation Complex (EDC) (Mischke, et al., 1996) andAPOA2 showed high level amplifications (>10-fold increases) in one sample each. We used FISH to determine the amplification patterns of YACs from the 6 Mb YAC contig covering EDC (Marenholz, et al., 1996) , as well as two that were more distally located in selected samples. For most of the cases, FISH detected high-level amplifications only in about 50% of the nuclei. There was also considerable heterogeneity between the nuclei, indicating clonal variations within the tumours. Six samples had amplification of the YACs containing D1S3620 (789f2 and 954e4), and in three also the flanking 764a1 was included. Five of these tumours showed normal copies of the more distal YACs, thus, it seems likely that an important gene may be located within 789f2/954e4, or very close. We are now analysing the region around D1S3620 in more detail, using PAC subclones and more proximally located YACs that do not contain D1S3620. - The two most distal YACs, 883h6 and 935b12, showed high copy numbers in two samples. Taken together, FISH and molecular analyses indicate complex amplification patterns in 1q21-q22 with at least two amplicons.
Assignment of additional genes expressed in human keratinocytes to the Epidermal Differentiation Complex (EDC) in chromosomal region 1q21.
D. Mischke, 1 M. Zirra, 1 D. Fischer, 2 C. Backendorf, 2 A. Ziegler, 1 I. Marenholz1 1
1 Institut für Molekulare Immunologie, Universitaetsklinikum Charité der Humboldt-Universitaet zu Berlin, Berlin, Germany, 2 Department of Molecular Genetics, Gorleaus Laboratories, Leiden, Netherlands
The Epidermal Differentiation Complex (EDC) in region q21 on human chromosome 1 represents a gene complex comprising an extraordinary number of genes that are important for the maturation of the human epidermis. So far, twenty-seven genes belonging to three families have been mapped within 2.05 Mb of 1q21. The first family encodes the structural proteins of the cornified envelope loricrin (LOR), involucrin (IVL), and the small proline rich proteins (two SPRR1, seven SPRR2 and one SPRR3 genes). The second comprises thirteen calcium-binding proteins (S100A1 to S100A13). The intermediate filament-associated proteins trichohyalin (THH) and profilaggrin (FLG) constitute the third family. We have previously assembled a contig of yeast artificial chromosomes (YACs) that covers about 6 Mb of 1q21, including the EDC and its flanking regions.
To identify novel genes likely to be involved in epidermal differentiation, we selected a YAC from the telomeric part of the contig as a hybridization probe for a gridded human keratinocyte cDNA-library. Here we report the assignment of eight additional cDNA-clones to the EDC. The respective genes were sublocalized on our genomic long range restriction map and YAC-contig. Five of these represent already known genes, whereas the other three correspond to hitherto undetected transcripts in human keratinocytes. Our results strengthen the importance of the EDC in keratinocyte biology and should aid in identifying new candidate genes for skin diseases and tumors frequently associated with this region of chromosome 1.
Identification of a new locus for Congenital Muscular Dystrophy with Rigid Spine syndrome on chromosome 1p35-36.
B. Moghadaszadeh,1 I. Desguerre, 2 H. Topaloglu, 3 F. Muntoni, 4 S. Pavek, 5 M. Mayer, 2 C. Sewry, 4 M. Fardeau, 1 F.M.S. Tomé,1 and P. Guicheney1.
1 INSERM U153, Groupe Hospitalier Pitié-Salpêtrière, Paris; 2Service de Neuropédiatrie, Hôpital Saint-Vincent-de-Paul, Paris (France); 3 Department of Paediatric Neurology, Hacettepe Children's Hospital, Ankara (Turkey); 4 Department of Paediatrics and Neonatal Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London (UK); 5 Généthon-CNRS URA Evry (France)
Classical Congenital Muscular Dystrophies (CMD) are autosomal recessive neuromuscular disorders, characterized by early onset, contractures, general atrophy of the limbs and trunk muscles and dystrophic changes in the muscle biopsy. Only one gene, LAMA2 (6q2), which encodes the laminin alpha 2 chain (or merosin) has been identified so far. Mutations in LAMA2 cause CMD with complete or partial merosin deficiency detectable by immunohistochemistry on patient muscle biopsies and account for about 50% of CMD cases. In a large consanguineous CMD family (11 siblings) without merosin deficiency, we undertook a genome-wide research by homozygosity mapping and analyzed 380 microsatellite markers. The 3 affected children were homozygous for several markers on chromosome 1p35-36. We identified two additional consanguineous families linked to this locus. A maximum cumulative LOD score of 4.48, at a recombination rate of .00 was obtained with D1S2885. A consistent feature in these families was the presence of rigidity of the spine, scoliosis and reduced vital capacity, as it is found in the rigid spine syndrome. The present study is the first description of a locus for a CMD without merosin deficiency and will help better define the nosology of the rigid spine syndrome.
The syndrome of hypoparathyroidism, dysmorphism and growth and mental retardation (hdr) maps to 1q42-43.
R. Parvari, 1 E. Hershkovitz, 1 A. Kanis, 2 R. Gorodischer, 1 V. Shefield, and R. Carmi12
1 Soroka medical Center and the Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel. 2 Department of Pediatrics Division of Medical genetics, University of Iowa, Iowa City, Iowa, USA
The syndrome of hypoparathyroidism associated with dysmorphism, growth retardation and developmental delay (HDR) is a newly described autosomal recessive congenital disorder with severe and often fatal consequences. Since the syndrome is very rare and all the families presenting it are Bedouins, a small population where consaguinity is very common, the syndrome was presumed to be caused by a single mutation with a founder effect. To map the HDR gene, a genome screen using a combination of homozygosity mapping and DNA pooling was performed using apparently unlinked kindreds. Analysis of a panel of highly polymorphic markers revealed linkage to D1S235. The lod score obtained was 4.11 at theta=0. Analysis of additional chromosome 1 markers in all the available families revealed evidence of linkage disequilibrium and placed the HDR in an approximately 1 cM interval defined by D1S1540 and D1S2678 on chromosome 1q42-43.
The small interval makes the positional cloning of the HDR gene feasible. The identification of the gene causing HDR when mutated may contribute to our understanding of the function of the parathyroid gland. Since serum ionized calcium closely regulates the production of the parathyroid hormone, insight may be gained into the regulation of calcium homeostasis as well. In addition, the particular facial features of the patients suggests that the HDR gene may have a function in the very early development of the pharyngeal pouches from which both facial structures and the parathyroid glands are derived. The growth and mental retardation observed in HDR patients, not reported in the various isolated forms of hypoparathyroidism, and apparently not caused by hypoparathyroidism per se that still enables normal growth, remain unexplained and may represent a unique effect of the affected gene on pleiotropic aspects of fetus development and postnatal growth.
The unique local availability of the HDR patients holds a promise for a significant contribution to the study of the human genome.
Evidence for genetic homogeneity, and refined mapping of the gene for Thiamine-Responsive Megaloblastic Anemia.
T. Raz,1 T. Barrett,2 R. Szargel,1 H. Mandel,3 E.J. Neufeld,4 K. Nosaka,5 M. Viana6 and N. Cohen1
1 Department of Genetics, Tamkin Human Molecular Genetics Research Facility, Technion-Israel Institute of Technology, Bruce Rappaport Faculty of Medicine, Haifa, Israel. 2 University of Birmingham, Department of Pediatrics and Child Health, Birmingham, UK. 3 Department of Pediatrics, Rambam Medical Center, Haifa, Israel. 4 Division of Hematology, Children's Hospital, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA. 5Kyoto Prefectural University of Medicine. 6 Department of Hematology, Felichio Rocho Medical Center, Belo Horizonte, Brazil
Thiamine responsive megaloblastic anemia (TRMA) is a rare autosomal recessive disorder characterized by a triad of megaloblastic anemia, diabetes mellitus, and sensorineural deafness. Patients respond, in varying degrees, to treatment with megadoses of thiamine (vitamine B1). We have recently shown genetic linkage of the TRMA gene to a 16 centimorgan (cM) region on 1q23.2-1q23.3 based on the analysis of 4 large, inbred families (1 Alaskan, 1 Italian, and 2 Israeli Arab families). Here we bring further evidence that the TRMA gene is located in this chromosomal area and we confirm the genetic homogeneity of the disease. We also narrow the TRMA interval down to 4 cM based on genetic recombination, homozygosity mapping and linkage disequilibrium (highest lod score of 11.5 at D1S2799, at a recombination fraction of 0). In this analysis, we genotyped 7 additional families of diverse ethnic origins (Pakistani, Indian, Italian, Brazilian, and Japanese), and analyzed additional markers in 2 previously reported families showing evidence of linkage disequilibrium in a large area of their haplotypes. The multi-system manifestations of TRMA suggest that thiamine has a pivotal role in a multiplicity of physiological processes. Mapping the TRMA gene and understanding the molecular basis of the disease may, thus, shed light on the role of thiamine in common disorders such as deafness, anemia, and diabetes.
A sequence-ready BAC contig across the Van der Woude Syndrome critical region in 1q32-q41 and microdeletions as a cause of VWS.
B.C. Schutte, 1 K.B. Coppage, 1 B.C. Bork, 1 A.M. Basart, 1 M.I. Malik, 1 S.J. Edwards, 2 M.J. Dixon, 2 and Jeffrey C. Murray 1
1 Department of Pediatrics, University of Iowa, Iowa City, IA, 2 Departments of Dental Medicine and Surgery, University of Manchester, Manchester M13 9PT, UK.
Van der Woude syndrome (VWS) is an autosomal dominant disorder characterized by cleft lip and/or palate and lip pits with occasional hypodontia. The VWS locus has been mapped to a 1.6 cM region of chromosome 1q32-q41 by linkage analysis and by the discovery of a microdeletion around D1S205. One YAC, yCEPH785B2, contains both flanking markers, D1S491 and D1S205, and encompasses the microdeletion. The maximal size of the critical region is approximately 850 Kb. A complete BAC and partial cosmid contig has been constructed containing 14 BAC and 48 cosmid clones. Partial sequence analysis of these clones identified 3 genes: VWS1, the human homologue of the rat calmodulin dependent protein kinase was also identified from these clones and used to screen 45 VWS families for the presence of a microdeletion. One new microdeletion was detected in a family whose affected members display the typical VWS features, but no other apparent phenotype. Affected members of the family with the previously described microdeletion displayed developmental delays along with the typical VWS features. Surprisingly, the proximal end of the new deletion extends at least 1 Mb beyond the previously described microdeletion, while the distal end maps near the first.
Sequence ready bacterial contig spanning the Human Epidermal Differentiation Complex in 1q21.
A.P. South, 1 C.H. James, 1 D. Mischke, 2 I. Ragoussis, 3 and Dean Nizetic 1
1 School Of Pharmacy, University of London, London, UK., 2Institut fur Experimentelle Onkologie und Transplantationsmedizin, Virchow-Klinikum der Humboldt-Universitat zu Berlin, Berlin, Germany, 3 St Thomas's Hospitals (UMDS), Guy's Hospital, London, UK.
Human chromosomal region 1q21 reveals a 2.2 Mb segment containing over 25 clustered genes, many of which are expressed during terminal differentiation in stratified squamous epithelia. As such this region has been termed the Epidermal Differentiation Complex (Mischke et al. 1996). It is rare to find such clustering of functionally related genes within the genome. The most well studied example to date would be the major histocompatibility complex (MHC) on chromosome 6p which yields a gene density of at least 55 genes/ Mb (Newell et al. 1996). The number of transcripts and genes being positioned to 1q21 is increasing.
We have constructed a 2.6 Mb contig consisting of 55 PAC clones, 2 BAC clones, and 28 cosmid clones with one gap. The main contig of 2.4 Mb contains no gaps and encompasses 26 of the 28 genes in the EDC, resolving these genes and STS markers to level of EcoRI restriction fragments. All clone overlaps and the integrity of the map have been confirmed by gel fingerprinting with EcoRI. A full NotI and SalI restriction map along with a partial EcoRI restriction map has been constructed.
Bacterial clones will be the starting resource for the large scale genomic sequencing of this region by the Sanger Centre. They also provide excellent material for gene discovery experiments such as exon trapping, cDNA selection and the screening of cDNA libraries. One would hypothesize that this region on chromosome 1q21 will be extremely gene rich, facilitating the elucidation of novel transcripts. An approach to study the evolution of this complex has been initiated in the mouse, chicken and the pufferfish Fugu rubripes.
Searching for the Primary Congenital Glaucoma Gene (GLC3B) on the 1p36 region.
I. Stoilov and M. Sarfarazi
Molecular Ophthalmic Genetics Laboratory, Surgical Research Center, University of Connecticut Health Center, Farmington CT (USA)
The Primary Congenital Glaucoma (PCG) is an inherited eye condition with a reported incidences that varies between 1:1,250 to 1:22,000. In almost all of the familial cases, PCG is inherited as an autosomal recessive condition. Genetic linkage studies conducted in our laboratory identified two chromosomal loci associated with the disease. The GLC3A (26 families) and GLC3B (4 families) loci were mapped to chromosomes 2p21 and 1p36 respectively. Five families were unlinked to both loci thus indicating the existence of at least one more PCG locus in the genome. Recently, we reported that mutations affecting Cytochrome P4501B1 gene are responsible for the PCG phenotype in families linked to the GLC3A locus. Based on recombination events, the GLC3B locus is confined within a 3 cM interval that is flanked by (D1S1597/D1S489/D1S228) and (D1S1176/D1S507/D1S407). This locus is resided centromeric to Neuroblastoma and CMT2A loci. SSCP screening found no mutations in nine candidate genes: PAX7, PNDB, TNFR2, ENO1, 5-HT6, HUP-1, PLOD, NPPB and OX40. The GLC3B critical region is covered by a YAC contig that contains 8 clones (900-G-3, 889-H-10, 796-H-4, 758-C-2, 807-H-7, 922-E-6, 927-C-12, and 971-E-5). We are using this contig as an anchor for establishing a contig of BAC clones. Screening of a human BAC library (Research Genetics) with markers D1S402, D1S228, and D1S407 has already identified six positive BAC clones within the region of interest. Screening with additional markers is currently underway.
Recently, the complete sequence of PAC clone (dJ308I13, 96,608 bp) was made available by the Sanger Centre. This clone contains marker D1S2834 which maps within the GLC3B candidate region. Computer assisted evaluation of this sequence for potential coding proteins was carried out with Grail 2, Genie, and FGENEH. A total of 101 exons were predicted for this PAC clone. We selected regions predicted by at least two of the programs and carried out a series of 3 -RACE experiments with cDNAs that were prepared from normal human fibroblasts, trabecular meshwork and nonpigmented ciliary epithelial cell lines. The candidate fragments generated by this experiments are currently being further analyzed.
Construction of a PAC contig in 1p36. Utilizing chromosome 1 ACEDB databases for a positional cloning project.
Y. Tao, S. Carson, A. Lindstrom, J. Barker, C. Richardson, H. Blomeier, P. Denton, C. Haynes, B. Slotterbeck and J. Vance
Duke University Medical Centre, Durham, NC, USA
Charcot-Marie-Tooth disease type 2A (CMT2A) is an inherited neuropathy, previously localized to chromosome 1p36. The region had proven to be difficult to clone, with over 95% of YACs pulled in the area chimeric by FISH. A PAC contig was subsequently initiated as part of a positional cloning effort to identify the CMT2A gene. This was overlayed over an existing YAC contig we have completed. Identification of the PACs employed two sources: Markers were chosen from genetic and radiation hybrid maps and used to screen the PAC library in our laboratory using both PCR and hybridization. HindIII digests were used to obtain fingerprint analysis. End clones were sequenced directly from PACs. This data was incorporated with ACEDB files containing ongoing contig and sequencing information in the region produced by the Sanger Centre, which allowed multiple contigs to be connected and verified. Compiled data then placed in a private ACEDB database.
A contig was constructed spanning the CMT2A minimal candidate region. Currently this included over 239 PACs with a minimum tiling path of 22 PACs. This effort provides insight into future utilization of large public databases in positional cloning efforts, as the HGI sequencing effort progresses.
PAC analysis of 5 homologous clusters of locally repetitive sequences on chromosome 1 reveal a total length of 8 Mb.
R. Versteeg, A. Chan, P. van der Drift
Dept of Human genetics, Academic Medical Center, University of Amsterdam, P.O. Box 22700, Amsterdam, The Netherlands
Chromosome band 1p36.2 harbours a cluster of at least 1.5 Mb consisting of locally repetitive sequences, among them genes for tRNA TRE and small nuclear RNA U1. YACs from this cluster cross hybridize to regions on chromosomal band 1q21, 1q42, 1p12 and 1p36.¬33. To analyze the length and character of these five homologous regions, we isolated 122 PACs from these clusters. FISH analysis shows that the 1q21 cluster is at least 6.5 Mb long and the total length of the five clusters is at least 8 Mb. Sequence information was obtained by isolation of 150 probes from the 1p36.2 cluster. All probes recognize 5 to 40 copies on chromosome 1. The distance between two copies within a cluster typically is 75©150 Kb range. 80% of the probes recognizes sequences in different clusters, indicating that they belong to the ancestral sequence from which the five clusters originated. Examples are TRE, RNU1 and other expressed sequences. FISH analyses of macaque chromosomes shows that this ancestral cluster was split in the 1p36 and 1q21 cluster over 30 million years ago. Another 20% of the sequences cloned from 1p36.2 PACs and YACs are not encoded in other clusters, but nevertheless have 5©15 copies. Examples are genes for a MSP homologue, Espin and several anonymous genes. Analysis of MSP like genes shows that they originate from another chromosomal position and are caught and co-amplified in the repeat cluster about 6 million years ago. The MSP like genes reveal that the 1p36.2 cluster has a striking length polymorphism among healthy individuals, probably caused by meiotic recombination.
Comprehensive map of chromosome 1.
P.S. White, 1 E.P. Sulman, 1 T.C. Matise 2
1 Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, PA, 19104. 2 Lab of Statistical Genetics, Rutgers University, New Brunswick, NY,
Comprehensive maps integrating multiple marker types and reflecting physical distance are essential to genetic disease locus searches, gene identification, and sequencing efforts. We have developed a method for combining mapping datasets from multiple sources that allows the integration of markers relative to a common framework, which we have tested on human chromosome 1. Typing data from version 8 of the Radiation Hybrid database (RHdb) and transcript cluster information from Unigene were imported into a relational database. The subset of chromosome 1 RHdb entries typed on the Genebridge4 panel were searched for marker typing duplications by comparing primer sequences. After elimination of duplicated typings, a radiation hybrid (RH) map was constructed from this set of chromosome 1 markers with MultiMap using a set of 62 well-typed genetic markers as an initial framework. Additional markers were added to the framework in an iterative process, first at odds >10,000:1 and then at odds >1000:1, with preferential addition of genetic markers. This process resulted in a framework map of 241 markers and an average map resolution of 1.1 Mb. The 1000:1 odds-defined locations, relative to the framework map, of an additional 2111 markers were then calculated. All markers were compared with the Unigene data to identify marker sets sharing transcript sequence clusters. Markers sharing overlapping map coordinates that were also assigned to the same Unigene cluster were presented as single transcripts. This process of combining map locations with sequence homology efficiently detects highly homologous but non-identical transcripts as well as possible RH typing errors. A subset of RH framework markers was used as a skeletal map for constructing an integrated genetic map. The genetic map had 173 framework markers (2.1 cM/1.5 Mb resolution) and a total of 810 genetic markers. Using the genetic markers on the RH framework map as reference points, we also integrated YAC contigs from the Whitehead and CEPH-Généthon physical maps. The final map contains 3142 unique markers. We are currently finishing an updated comprehensive map of chromosome 1 using RHdb version 11. This map will contain approximately 5200 markers and an estimated resolution of 900 Kb. An internet site that displays the chromosome 1 map and allows for direct database querying by graphical and text-based methods is being developed for map presentation.
Construction of sequence-ready bacterial clone contigs of human chromosome 1q23 -1q25.
H. Williams, L. McDonald, D. Scott, M. Vaudin and S. Gregory
The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, U.K.
The chromosomal region of 1q23-1q25 is known to contain a number of neurologically and immunologically important genes including the neural cell adhesion molecules tenascin-R (TNR) and astrotactin (ASTN); TIGR, a gene shown to cause juvenile onset glaucoma (Stone, 1997); a family of selectin proteins (ELAM-1, LAM-1) and GMP-140), and cathepsin (CTSE).
Here we report the construction of a high resolution PAC map of 1q23-q25 using available sequence tagged site (STS) markers spanning approximately 5 Mb between markers D1S196 and D1S242. A combination of a high resolution PAC PCR and hybridisation of pooled radiolabelled STS PCR products was used to screen the whole genome PAC library (Pieter de Jong). 1866 positive clones have been identified using 233 markers and have been analysed by HindIII/Sau3AI restriction enzyme fluorescent fingerprinting. To date, 624 PAC clones have been assembled into 10 contigs containing a total of 145 STSs. The estimated total coverage in bacterial clones is 4.8 Mb. Gaps between contigs are currently being closed using vectorette PCR.
Construction of these PAC contigs has refined the localisation of seven known genes (ELAM-1, LAM-1, GMP-140, CTSE, ASTN, TIGR, and TNR) and has produced an invaluable resource for characterisation of the region at the sequence level. To this end, PAC clones from all ten contigs have been selected for shotgun sequencing.
Large scale analysis of chromosome 1 derived Not1 fragments in neuroblastoma uncovers deletion of p73 and hypermethylation of a novel homeobox gene.
X.X. Zhu, K. Wimmer, D. Thoraval, B. Lamb, S. Motyka, R. Kuick, P. Ambros, O. Delattre, H. Kovar and S. Hanash.
Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109
We have implemented a computerized strategy for the two-dimensional analysis of Not1 genomic restriction fragments which we have utilized to investigate chromosome 1 alterations in neuroblastoma. 183 chromosome 1 derived Not1 fragments which were observed in 2-D patterns of genomic digests of flow sorted chromosome 1 were identified in whole genomic 2-D patterns. The occurrence and intensity of these fragments in 2-D patterns of neuroblastomas were investigated. Ten chromosome 1 derived fragments exhibited frequent loss or reduction in intensity in 2-D patterns of neuroblastoma cell lines and tumours. To facilitate the cloning of fragments, a chromosome 1 Not1 EcoRV genomic library was constructed. Two of the ten fragments were cloned and their sequence analyzed. One was mapped to chromosome 1p13-21. This fragment encompassed a CpG island which exhibited identity with the hamster homeobox gene Alx3. Independent analysis of the methylation status of the human Alx3 CpG island using isoschizomers provided evidence for hypermethylation of Alx3 in neuroblastoma cell lines. Hypermethylation of the Alx3 NotI restriction site was associated with reduced or absent Alx3 expression in neuroblastoma cell lines. Treatment of the neuroblastoma cell line SKNSH with the methylation inhibitor 5-aza 2'deoxycytidine, induced expression of Alx3. The second fragment was identified as containing promoter sequences for the p73 based on its content of a CpG island and of transcribed sequences of p73. The decreased intensity of the p73 fragment in 2-D patterns was found to be the result of deletion. All 5 neuroblastomas analyzed that were known to contain 1p36 deletion showed loss of p73. In addition, two of four tumors that lacked evidence of 1p36 deletion by LOH analysis, exhibited loss of p73. Our data indicate a role for both methylation and deletion in silencing gene expression in neuroblastoma.