Allelic loss of chromosome 1p36 in neuroblastoma is of preferential maternal origin and correlates with N-myc amplification.

Neuroblastomas frequently have deletions of chromosome 1p and amplification of the N-myc oncogene. We analysed 53 neuroblastomas for the N-myc copy number, loss of heterozygosity (LOH) of chromosome 1p36 and the parental origin of the lost alleles. Allelic loss of 1p36 was found in 15 tumours. All N-myc amplified tumours belonged to this subset. In 13/15 tumours with LOH of 1p36 the lost allele was of maternal origin. This non-random distribution implies that the two alleles of the putative neuroblastoma suppressor gene on chromosome 1p36 are functionally different, depending on their parental origin. This is the first evidence as far as we know for genomic imprinting on chromosome 1p.

Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12).

Retinitis pigmentosa (RP) comprises a clinically and genetically heterogeneous group of diseases that afflicts approximately 1.5 million people worldwide. Affected individuals suffer from a progressive degeneration of the photoreceptors, eventually resulting in severe visual impairment. To isolate candidate genes for chorioretinal diseases, we cloned cDNAs specifically or preferentially expressed in the human retina and the retinal pigment epithelium (RPE) through a novel suppression subtractive hybridization (SSH) method. One of these cDNAs (RET3C11) mapped to chromosome 1q31-q32.1, a region harbouring a gene involved in a severe form of autosomal recessive RP characterized by a typical preservation of the para-arteriolar RPE (RP12; ref. 3). The full-length cDNA encodes an extracellular protein with 19 EGF-like domains, 3 laminin A G-like domains and a C-type lectin domain. This protein is homologous to the Drosophila melanogaster protein crumbs (CRB), and denoted CRB1 (crumbs homologue 1). In ten unrelated RP patients with preserved para-arteriolar RPE, we identified a homozygous AluY insertion disrupting the ORF, five homozygous missense mutations and four compound heterozygous mutations in CRB1. The similarity to CRB suggests a role for CRB1 in cell-cell interaction and possibly in the maintenance of cell polarity in the retina. The distinct RPE abnormalities observed in RP12 patients suggest that CRB1 mutations trigger a novel mechanism of photoreceptor degeneration.

Genetic heterogeneity in the cerebrohepatorenal (Zellweger) syndrome and other inherited disorders with a generalized impairment of peroxisomal functions. A study using complementation analysis.

We have used complementation analysis after somatic cell fusion to investigate the genetic relationships among various genetic diseases in humans in which there is a simultaneous impairment of several peroxisomal functions. The activity of acyl-coenzyme A:dihydroxyacetonephosphate acyltransferase, which is deficient in these diseases, was used as an index of complementation. In some of these diseases peroxisomes are deficient and catalase is present in the cytosol, so that the appearance of particle-bound catalase could be used as an index of complementation. The cell lines studied can be divided into at least five complementation groups. Group 1 is represented by a cell line from a patient with the rhizomelic form of chondrodysplasia punctata. Group 2 consists of cell lines from four patients with the Zellweger syndrome, a patient with the infantile form of Refsum disease and a patient with hyperpipecolic acidemia. Group 3 comprises one cel line from a patient with the Zellweger syndrome, group 4 one cell line from a patient with the neonatal form of adrenoleukodystrophy, and group 5 one cell line from a patient with the Zellweger syndrome. We conclude that at least five genes are required for the assembly of a functional peroxisome.

UV stimulation of DNA-mediated transformation of human cells.

Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells (complementation groups A and F), which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. No stimulation was found after damaging vector DNA by treatment with DNase or gamma rays. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome.

Molecular cloning of the human DNA excision repair gene ERCC-6.

The UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5. It harbors a deficiency in the repair of UV-induced cyclobutane pyrimidine dimers but permits apparently normal repair of (6-4) photoproducts. Genomic (HeLa) DNA transfections of UV61 resulted, with a very low efficiency, in six primary and four secondary UV-resistant transformants having regained wild-type UV survival. Southern blot analysis revealed that five primary and only one secondary transformant retained human sequences. The latter line was used to clone the entire 115-kb human insert. Coinheritance analysis demonstrated that five of the other transformants harbored a 100-kb segment of the cloned human insert. Since it is extremely unlikely that six transformants all retain the same stretch of human DNA by coincidence, we conclude that the ERCC-6 gene resides within this region and probably covers most of it. The large size of the gene explains the extremely low transfection frequency and makes the gene one of the largest cloned by genomic DNA transfection. Four transformants did not retain the correcting ERCC-6 gene and presumably have reverted to the UV-resistant phenotype. One of these appeared to have amplified an endogenous, mutated CHO ERCC-6 allele, indicating that the UV61 mutation is leaky and can be overcome by gene amplification.

Molecular cloning and biological characterization of the human excision repair gene ERCC-3.

In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion (L. H. Thompson, A. V. Carrano, K. Sato, E. P. Salazar, B. F. White, S. A. Stewart, J. L. Minkler, and M. J. Siciliano, Somat. Cell. Mol. Genet. 13:539-551, 1987).

Molecular characterization of areas with low grade tumor or satellitosis in human malignant astrocytomas.

Malignant astrocytomas often display histopathological heterogeneity. In the present study, we have molecularly characterized different areas within 4 such tumors to determine whether the tissue heterogeneity can be explained by differences in DNA constitution. Two tumors contained low grade areas, and the other 2 had areas with satellitosis. The tumors were examined for loss of heterozygosity with markers from chromosomes 9p, 10, and 17p and for amplification of the epidermal growth factor receptor gene. In each case, the high grade portion of the tumor displayed at least one of these structural alterations. However, identical alterations were found in the associated low grade or satellitosis areas of each tumor. Our data suggest that: (a) genetic alterations associated with tumor progression already occur in histopathologically low grade areas of high grade astrocytoma; (b) satellitosis associated with a high grade astrocytoma has to be considered as part of that tumor; and (c) tissue heterogeneity within a high grade astrocytoma is not a consequence of differences in DNA constitution at the loci that were examined.

Loss of heterozygosity in Wilms' tumors, studied for six putative tumor suppressor regions, is limited to chromosome 11.

Studies on the loss of heterozygosity (LOH) in human malignancies have shown that a number of different chromosomal regions associated with putative tumor suppressor genes may be involved in any one given tumor. We have carried out a similar study on Wilms' tumor using a range of DNA markers for a number of tumor suppressor regions. We tested a total of 44 Wilms' tumors including material from bilateral cases and from patients with Beckwith-Wiedemann syndrome, Drash syndrome, Perlman syndrome, and hemihypertrophy. In 11 of 36 informative tumors we found LOH for markers for the short arm of chromosome 11; only one of these tumors had additional LOH for regions 5q and 17p. No LOH was found for regions 3p, 13q, and 22q. Thus our findings support a major role for chromosome 11p in Wilms' tumor development and apparent noninvolvement of other tumor suppressor genes. No correlation was found between allelic losses and the International Society of Paediatric Oncology tumor stage or histology.

Deletion mapping in neuroblastoma cell lines suggests two distinct tumor suppressor genes in the 1p35-36 region, only one of which is associated with N-myc amplification.

Neuroblastoma is characterized by deletions of the short arm of chromosome 1 (1p) and amplification of the N-myc oncogene. We have made somatic cell hybrids of two human neuroblastoma cell lines, one with and one without N-myc expression and amplification. The expression of the amplified N-myc gene is completely switched off in the hybrids. This suggests that N-myc expression results from loss of a repressor function. As N-myc amplification is associated with loss of heterozygosity (LOH) of 1p36, we analysed 1p deletions in 16 neuroblastoma cell lines. The seven cell lines without N-myc amplification have no deletions or relatively small deletions, with an SRO on 1p36.23-33. This suggests that a tumor suppressor gene maps in this region. All nine cell lines with N-myc amplification have larger deletions, with an SRO from 1p35-36.1 to the telomere. This suggests that a second tumor suppressor gene which is associated with N-myc amplification maps more proximally. Fine mapping of 1p36 deletions in the two cell lines of the fusion experiment suggests that the distal locus is not a repressor of N-myc expression, but the more proximal locus could be a candidate for this function.

Lack of class I HLA expression in neuroblastoma is associated with high N-myc expression and hypomethylation due to loss of the MEMO-1 locus.

Class I HLA expression is low in neuroblastoma tumours and cell lines. We have recently mapped a modifier of methylation for HLA-C (MEMO-1) to chromosomal bands 1p35-36.1, a region deleted in many neuroblastomas. Hypomethylation of HLA-C is strongly correlated with allelic loss of the MEMO-1 locus. Here, we show that loss of MEMO-1 is associated with hypomethylation of both the 5' and 3' regions of class I HLA loci. We next investigated the relationship between methylation and expression of class I HLA in 28 cell lines of neuroectodermal tumours. Cell lines with hypermethylated HLA-C and HLA-A loci have relatively high expression, while most cell lines with hypomethylated loci have no or a reduced expression. It was reported earlier that high expression of c- or N-myc can suppress class I HLA expression. Remarkably, also N-myc amplification in neuroblastomas is associated with allelic loss of 1p35-36. Therefore, we have analysed the relationships between allelic loss of the MEMO-1 locus, class I HLA methylation and expression, and N-myc amplification and expression. This study shows a tight inter-relationship between these phenomena. Our data suggest a model in which hypomethylation of class I HLA due to loss of the MEMO-1 locus and high N-myc expression could collaborate in the down-regulation of class I HLA expression.

Genome-wide linkage in a large Dutch consanguineous family maps a locus for intracranial aneurysms to chromosome 2p13.

BACKGROUND AND PURPOSE: Familial occurrence of intracranial aneurysms suggests a genetic factor in the development of these aneurysms. In this study, we present the identification of a susceptibility locus for the development of intracranial aneurysms detected by a genome-wide linkage approach in a large consanguineous pedigree. METHODS: Patients with clinical signs and symptoms of intracranial aneurysms, confirmed by radiological, surgical, or postmortem investigations, were included in the study. Magnetic resonance angiography was used to detect asymptomatic aneurysms in relatives. RESULTS: Seven out of 20 siblings had an intracranial aneurysm. Genome-wide multipoint linkage analysis showed a significant logarithm of the odds score of 3.55. CONCLUSIONS: In a large consanguineous pedigree intracranial aneurysms are linked to chromosome 2p13 in a region between markers D2S2206 and D2S2977.

Microsatellite instability and promoter methylation as possible causes of NF1 gene inactivation in neurofibromas.

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder. One of the characteristic features of this disease is the development of neurofibromas. Since the NF1 gene is supposed to be a tumour suppressor gene, these neurofibromas should develop upon inactivation of both NF1 alleles. So far, mutation and deletion have been found to be involved in NF1 gene inactivation. However, these inactivating mechanisms explain the development of only a limited fraction of analysed neurofibromas. In this study, we investigated microsatellite instability (MSI) and promoter methylation as potential contributors to NF1 gene inactivation. As site-specific methylation in the NF1 promoter inhibits binding of transcription factors Sp1 and CREB, we studied the methylation status of their binding sites in particular. We analysed 20 neurofibromas and three neurofibrosarcomas, but did not find evidence for microsatellite instability or NF1 promoter methylation in any of the tumours. Thus, our data suggest that both microsatellite instability and promoter methylation are unlikely to be the major causes of NF1 gene inactivation in these tumours.

Mechanism of spreading of the highly related neurofibromatosis type 1 (NF1) pseudogenes on chromosomes 2, 14 and 22.

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder that involves tissues derived from the embryonic neural crest. Besides the functional gene on chromosome arm 17q, NF1-related sequences (pseudogenes) are present on a number of chromosomes including 2, 12, 14, 15, 18, 21, and 22. We elucidated the complete nucleotide sequence of the NF1 pseudogene on chromosome 22. Only the middle part of the functional gene but not exons 21-27a, encoding the functionally important GAP-related domain of the NF1 protein, is presented in this pseudogene. In addition to the two known NF1 pseudogenes on chromosome 14 we identified two novel variants. A phylogenetic tree was constructed, from which we concluded that the NF1 pseudogenes on chromosomes 2, 14, and 22 are closely related to each other. Clones containing one of these pseudogenes cross-hybridised with the other pseudogenes in this subset, but did not reveal any in situ hybridisation with the functional NF1 gene or with NF1 pseudogenes on other chromosomes. This suggests that their hybridisation specificity is mainly determined by homologous sequences flanking the pseudogenes. Strong support for this concept was obtained by sequence analysis of the flanking regions, which revealed more than 95% homology. We hypothesise that during evolution this subset of NF1 pseudogenes initially arose by duplication and transposition of the middle part of the functional NF1 gene to chromosome 2. Subsequently, a much larger fragment, including flanking sequences, was duplicated and gave rise to the current NF1 pseudogene copies on chromosomes 14 and 22.

Anticipation in familial intracranial aneurysms in consecutive generations.

Intracranial aneurysms (IA) are the major cause of subarachnoid haemorrhages (SAH). A positive family history for SAH is reported in 5-10% of the patients. The mode of inheritance is not unambiguously established; both autosomal dominant and recessive modes have been reported. In sporadic as well as in familial SAH, approximately 60% of the SAH patients are female. Recently, anticipation has been described in familial SAH. Since up to 15% of the SAHs are not caused by an IA, we have analysed anticipation, sex ratio and mode of inheritance only in families with patients with a proven IA in two consecutive generations. A total of 10 families were studied in which at least two persons in consecutive generations were affected by SAH, a symptomatic IA (SIA) or a presymptomatic IA (PIA). We also analysed published data from families with a proven IA in two consecutive generations on age of SIA onset and sex ratios among affected family members (both SIA and PIA). The age of SIA onset in the parental generation (mean 55.5 years) differed significantly from the age of onset in their children (mean 32.4 years). In the parental generation 11 men and 37 women were affected (both SIA and PIA), in the consecutive generation these numbers were 28 men and 32 women. There is a significant difference in sex ratio of affected family members when the generations are compared (P<0.02). No family could be found in which three consecutive generations were affected by an IA (SIA or PIA).

Some patients with intracranial aneurysms have a reduced type III/type I collagen ratio. A case-control study.

A reduced production of type III collagen has been reported in previous studies to be associated with intracranial aneurysms. The purpose of this prospective case-control study was to assess the possible role of a reduced type III collagen production as a risk factor for having an intracranial aneurysm. The study group consisted of 41 consecutively admitted patients with intracranial aneurysms. Intracranial aneurysms were demonstrated by intraarterial digital subtraction cerebral angiography or during operation. The control group consisted of 41 healthy volunteers matched for age and sex. Fibroblasts were cultured from skin biopsies from patients and control subjects, and the type III/type I collagen ratios were determined. The type III/type I collagen ratios in the controls ranged from 5.5 to 19.8%, with a median ratio of 10%, and none had a ratio below 5.5%. The type III/type I collagen ratios in patients ranged from 1.1 to 25.1%, with a median ratio of 10.5%, and eight patients (19.5%) had a low (< 5.5%) ratio (p = 0.005, Fisher's exact test). Our findings support the hypothesis that a reduced production of type III collagen may contribute to the formation of intracranial aneurysms in some patients.

Retinitis pigmentosa: defined from a molecular point of view.

Retinitis pigmentosa (RP) denotes a group of hereditary retinal dystrophies, characterized by the early onset of night blindness followed by a progressive loss of the visual field. The primary defect underlying RP affects the function of the rod photoreceptor cell, and, subsequently, mostly unknown molecular and cellular mechanisms trigger the apoptotic degeneration of these photoreceptor cells. Retinitis pigmentosa is very heterogeneous, both phenotypically and genetically. In this review we propose a tentative classification of RP based on the functional systems affected by the mutated proteins. This classification connects the variety of phenotypes to the mutations and segregation patterns observed in RP. Current progress in the identification of the molecular defects underlying RP reveals that at least three distinct functional mechanisms may be affected: 1) the daily renewal and shedding of the photoreceptor outer segments, 2) the visual transduction cascade, and 3) the retinol (vitamin A) metabolism. The first group includes the rhodopsin and peripherin/RDS genes, and mutations in these genes often result in a dominant phenotype. The second group is predominantly associated with a recessive phenotype that results, as we argue, from continuous inactivation of the transduction pathway. Disturbances in the retinal metabolism seem to be associated with equal rod and cone involvement and the presence of deposits in the retinal pigment epithelium.

Assessment of chromosomal gains and losses in oral squamous cell carcinoma by comparative genomic hybridisation.

Cytogenetic studies have demonstrated that oral squamous cell carcinomas (OSCCs) are usually characterised by complex karyotypes with many marker chromosomes. We analysed the genetic changes of six OSCC cell cultures by comparative genomic hybridisation (CGH). The CGH technique provides information on chromosomal gains and losses of the whole tumour genome in a single experiment and can therefore identify regions that harbour putative tumour suppressor genes (in the case of loss of chromosomal material) or oncogenes (in the case of gain or amplification of chromosomal material). Recurrent losses were detected at chromosome arms Xp and 3p (four cases). Gains consistently occurred at chromosome arms 8q and 9q (four cases) and at 1q, 3q, 5p, 7p, and 9p (three cases). The same six tumour cultures have previously been analysed by classical karyotyping. An important discrepancy between the two techniques was the number of losses detected: 55 with karyotyping versus 26 with CGH. On the basis of the cytogenetic complexity of these tumours and on FISH experiments that confirmed the CGH results, we conclude that genetic changes, particularly losses, can be more reliably detected by CGH analysis.

Cytogenetical characterisation of Chinese hamster 43-3B transferants with the amplified or non-amplified human DNA repair gene ERCC-1.

A comparative study on the biological responses to different mutagens (UV, 4NQO, MMC, MMS and EMS) was made on CHO wild-type cells (CHO-9), its UV-hypersensitive mutant 43-3B, and 2 types of its transferants, i.e., one containing a few copies of the human repair gene ERCC-1 and the other having more than 100 copies of ERCC-1 (due to gene amplification). Cell survival, chromosomal aberrations and SCEs were used as biological end-points. The spontaneous frequency of chromosomal aberrations in the transferants was less than found in 43-3B mutant cells, but still 2-3 times higher than in wild-type CHO cells. The spontaneous frequency of SCEs in the transferants was less than in 43-3B and similar to that of wild-type cells. The induction of SCEs by all tested agents in transferants was similar to that found in CHO-9 cells, while the mutant is known to respond with higher frequencies. ERCC-1 also bestowed resistance to MMS and EMS on the mutant to induction of chromosomal aberrations and cell killing to levels comparable with those of the wild-type strain. On the other hand ERCC-1 could not completely regain the repair proficiency against cell killing and induction of chromosomal aberrations by UV or MMC to the wild-type level. These results suggest that the ERCC-1 corrects the repair defect in CHO mutant cells, but it is unable to rectify fully the defect; probable reasons for this are discussed. However, amplified transferants (having more than 100 copies of the ERCC-1 gene) restored the impaired repair function in 43-3B to UV-, MMC- or 4NQO-induced DNA damage better than non-amplified transferants with a few copies of the ERCC-1. This difference may be due to the high amount of gene product involved in the excision repair process in the amplified cells.

Biological and biochemical consequences of the human ERCC-1 repair gene after transfection into a repair-deficient CHO cell line.

The consequences of the presence of the human gene ERCC1 in repair-deficient 43-3B cells were examined. The gene restores the sensitivity of this mutant not only to UV but also to 4NQO, N-Ac-AAF and alkylating agents to the normal level. Also, the frequency of mutation induction by UV at the Na+/K+-ATPase locus returns to the level of CHO wild-type cells. Additionally, the rate of cyclobutane pyrimidine dimer removal approaches that in wild-type CHO cells. The results obtained indicate that the human gene ERCC-1 restores the impaired functions in 43-3B, and that the gene is probably functionally homologous to the defective one in the 43-3B cell line. Some evidence was found for a difference between the human gene product and its rodent counterpart, as the restoration of normal sensitivity to 4NQO, ENU and N-Ac-AAF was complete whereas it was not for UV.

Phenotypic heterogeneity within the first complementation group of UV-sensitive mutants of Chinese hamster cell lines.

A DNA-repair mutant was characterized that has the extraordinary and interesting properties of extreme sensitivity to UV killing combined with a high level of nucleotide excision repair. The mutant V-H1 isolated from the V79 Chinese hamster cell line appeared very stable, with a reversion frequency of about 3.5 X 10(-7). Genetic complementation analysis indicates that V-H1 belongs to the first complementation group of UV-sensitive Chinese hamster ovary (CHO) mutants described by Thompson et al. (1981). This corresponds with data on cross-sensitivity and mutation induction after UV irradiation published by this group. Surprisingly, the mutant V-H1 shows only slightly reduced (to approximately 70%) unscheduled DNA synthesis (UDS) after UV exposure, while the other two mutants of this complementation group are deficient in UDS after UV. In agreement with the high residual UDS, in V-H1 also the amount of repair replication in response to UV treatment is relatively high (approximately 50%). It has also been shown that the incision step of the nucleotide excision pathway takes place in V-H1 (with a lower rate than observed in wild-type cells), whereas another mutant (UV5) of the same complementation group is deficient in incision. This heterogeneity within the first complementation group indicates that the repair gene of this complementation group may have more than one functionally important domain or that the gene is not involved in the incision per se but is involved in e.g. preferential repair of active genes.