Deletion mapping and linkage analysis provide strong indication for the involvement of the human chromosome region 8p12-p22 in breast carcinogenesis

We have identified a high frequency of loss of heterozygosity (LOH) on the human chromosome region 8p12-p22 in a panel of microdissected familial (86% LOH) and sporadic (74% LOH) breast tumours. The two most frequently deleted regions were defined around marker D8S133 and in a broader centromeric region bounded by markers D8S137 and D8S339. We cannot unequivocally characterize the 8p12-p22 loss as an early or a late event in breast carcinogenesis. In parallel, we have performed linkage analysis in four German breast cancer families. A location score greater than 13.67 corresponding to a LOD score of 2.97 at the marker D8S137 has been obtained. Our results considerably strengthen the evidence for a breast cancer susceptibility gene(s) located on the short arm of the chromosome region at 8p12-p22.     

The relative bioavailability and in vivo-in vitro correlations for four marketed carbamazepine tablets

Neuroblastoma is a heterogeneous childhood tumour of the sympathetic nervous system, in which deletions of chromosomal region 1p and amplification of the MYCN oncogene correlate with aggressive tumour behaviour. However, the majority of neuroblastoma tumours show neither of these aberrations, indicating that other chromosomal regions may be involved in tumorigenesis. Here, we report findings of loss of heterozygosity (LOH) on chromosome 3. In our neuroblastoma material, nine of 59 (15.3%) tested tumours showed allelic loss of chromosome 3p markers. We found significant clinical and biological differences between tumours with the loss of one entire chromosome 3 vs tumours with partial loss in chromosome region 3p. All children with tumours with whole chromosome 3 loss are long-term survivors, whereas all children with tumours showing partial 3p LOH have died from tumour progression. A consensus region found to be deleted in all the tumours with 3p deletions was defined by markers D3S1286 and D3S1295, i.e. 3p25.3-p14.3, distal to the FHIT gene.     

A 1.5-Mb deletion in 17p11.2-p12 is frequently observed in Italian families with hereditary neuropathy with liability to pressure palsies

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder characterized by recurrent mononeuropathies. A 1.5-Mb deletion in chromosome 17p11.2-p12 has been associated with HNPP. Duplication of the same 1.5-Mb region is known to be associated with Charcot-Marie-Tooth disease type 1 (CMT1A), a more severe peripheral neuropathy characterized by symmetrically slowed nerve conduction velocity (NCV). The CMT1A duplication and HNPP deletion appear to be the reciprocal products of a recombination event involving a repeat element (CMT1A-REP) that flanks the 1.5-Mb region involved in the duplication/deletion. Patients from nine unrelated Italian families who were diagnosed with HNPP on the basis of clinical, electrophysiological, and histological evaluations were analyzed by molecular methods for DNA deletion on chromosome 17p. In all nine families, Southern analysis using a CMT1A-REP probe detected a reduced hybridization signal of a 6.0-kb EcoRI fragment mapping within the distal CMT1A-REP, indicating deletion of one copy of CMT1A-REP in these HNPP patients. Families were also typed with a polymorphic (CA)n repeat and with RFLPs corresponding to loci D17S122, D17S125, and D17S61, which all map within the deleted region. Lack of allelic transmission from affected parent to affected offspring was observed in four informative families, providing an independent indication for deletion. Furthermore, pulsed-field gel electrophoresis analysis of SacII-digested genomic DNA detected junction fragments specific to the 1.5-Mb HNPP deletion in seven of nine Italian families included in this study. These findings suggest that a 1.5-Mb deletion on 17p11.2-p12 is the most common mutation associated with HNPP.     

A novel tumor suppressor locus on chromosome 18q involved in the development of human lung cancer

The high incidence of loss of heterozygosity (LOH) on chromosome 18q in advanced non-small cell lung carcinomas indicates the presence of tumor suppressor gene(s) on this chromosome arm, which plays an important role in the acquisition of malignant phenotypes in lung cancers. In the present study, we examined 62 lung cancer specimens and 54 lung cancer cell lines for allelic imbalance at 11 microsatellite loci to define common regions of 18q deletions. Allelic imbalance of 18q was detected in 24 (55.8%) non-small cell lung carcinoma specimens and in 6 (31.6%) small cell lung carcinoma specimens, whereas a similar frequency of LOH was statistically inferred to occur in cell lines by analyzing marker homozygosity as an indirect measure of LOH. Five specimens and 11 cell lines showed partial or interstitial deletions of chromosome 18q, and 2 of them had homozygous deletions at the 18q21.1 region. A commonly deleted region was assigned between the D18S46 and y953G12R loci. The size of this region is less than 1 Mb, and the coding exons of three candidate tumor suppressor genes, Smad2, Smad4, and DCC, were mapped outside the region. This result suggests that the common region harbors a novel tumor suppressor gene involved in the progression of lung cancer.     

The retinopetal visual system in the chameleon (Chameleo chameleon)

Wolf-Hirschhorn syndrome (WHS) caused by 4p16.3 deletions comprises growth and mental retardation, distinct facial appearance and seizures. This study characterized a subtle interstitial deletion of 4p16.3 in a girl with mild retardation and possessing facial traits characteristic of WHS. The patient had generalized seizures in conjunction with fever at 3 and 5 years of age. Fluorescence in situ hybridization (FISH) with a series of markers in the 4p16.3 region showed that the interstitial deletion in this patient was between the probes D4S96 and D4S182, enabling the size of the deletion to be estimated as less than 1.9 Mb. This is the smallest interstitial deletion of 4p16.3 which has been reported. The patient contributes to a refinement of the phenotypic map of the WHS region in 4p16.3. The critical region for the characteristic facial changes of WHS, failure to thrive and developmental delay is now localized to a region of less than 700 kb. The mental retardation of this patient was mild suggesting that small interstitial deletion may have less severe phenotypic consequences.     

Allelic deletion on chromosome 17p13.3 in early ovarian cancer

Multiple chromosome 17 loci may be involved in ovarian carcinogenesis. Fifty-seven sporadic ovarian epithelial tumors were examined for loss of heterozygosity at 15 loci on chromosomes 17p. Eighty % (39 of 49) of informative tumors had allelic loss in 17p13.3 at D17S30, D17S28, or both loci within this region, including 3 of 7 tumors of low malignant potential and 4 of 5 nonmetastatic carcinomas. The smallest region of overlapping deletions extends from D17S28 to D17S30, a distance of 15 kb. Furthermore, several tumors have breakpoints within the region detected by the D17S30 probe. Chromosome 17p13.3 genes with potential tumor suppressor function include HIC-1, DPH2L (N. J. Phillips et al. Isolation of a human diphthamide biosynthesis gene on chromosome 17p13.3, submitted for publication)/OVCA1, PEDF, and CRK. The HIC-1 coding sequence lies i kb centromeric to the D17S28-S17S30 region of deletion (M. Makos Wales et al., Nat. Med., 1:570-577, 1995) but remains a candidate because 5'-regulatory elements may lie within the critical region. Portions of the DPH2L/OVCA1 coding sequence lie within the D17S28-D17S30 interval. Somatic cell hybrid analysis places PEDF in an interval including D17S28, D17S30, and D17S54, whereas CRK is excluded from this interval. Chromosome 17p13.3 loss precedes TP53 and BRCA1 region deletions because the latter changes are see only in high-stage carcinomas. Microsatellite instability plays only a minor role in sporadic ovarian carcinogenesis because only 1 of 57 tumors showed this finding.     

Chromosome 5 allelic losses are early events in tumours of the papilla of Vater and occur at sites similar to those of gastric cancer

During our studies of DNA fingerprinting of tumours of the pancreas and papilla (ampulla) of Vater, using arbitrarily primed polymerase chain reaction (AP-PCR), we noticed two bands showing a decreased intensity in six of ten ampullary tumours with respect to matched normal tissues. Those bands were both assigned to chromosome 5. Such a finding was somewhat in contrast with the reportedly low frequency of APC gene mutations in ampullary cancers, located at chromosome 5q21, and suggested that loci different from that of APC might be the target of chromosome 5 allelic losses (LOH) in these tumours. Therefore, we analysed chromosome 5 LOH in a panel of 27 ampullary tumours, including eight adenomas, four early- and 15 advanced-stage cancers, using 16 PCR-amplified CA microsatellite polymorphic markers spanning the entire chromosome. Nineteen cases (70%) showed LOH, and the interstitial deletions found in these tumours described two smallest common deleted regions, in which putative suppressor genes might reside. They were at 5q13.3-q14 and at 5q23-q31 respectively, which correspond to those found in gastric tumours. In addition, the presence of 5q LOH in six of eight adenomas and in three of four early-stage cancers suggests that such phenomena occur at early stages of neoplastic progression of the ampullary epithelium.     

Suppression of tumorigenicity of rat liver epithelial tumor cell lines by a putative human 11p11.2-p12 liver tumor suppressor locus

We have previously identified and mapped a locus within human chromosome 11p11.2-p12 that suppresses the tumorigenic potential of a rat liver tumor cell line (termed GN6TF) which contains well defined chromosomal aberrations involving rat chromosomes 1, 4, 7, and 10. In the present study, we investigated the potential of this human 11p11.2-p12 liver tumor suppressor locus to suppress the tumorigenic potential of two other rat liver tumor cell lines (GN3TG and GP10TA) following microcell-mediated introduction of human chromosome 11. These tumor cell lines are aneuploid and contain chromosomal abnormalities that are similar to the GN6TF tumor line. The tumorigenic potential and other phenotypic characteristics of GN3TG-11neo and GP10TA-11neo microcell hybrid (MCH) cell lines were variable, and dependent upon the status of the introduced human chromosome 11. MCH cell lines that retained the region of 11p11. 2-p12 delineated by microsatellite markers D11S1385 and D11S903 exhibited suppression of tumorigenicity in vivo (decrease in tumorigenicity and/or elongation of latency), whereas, the tumorigenic potential of one MCH line that lacked markers in this region of human 11p11.2-p12, but retained flanking markers, was not changed from that of the parental tumor cell line. The chromosomal interval between microsatellite markers D11S1385 and D11S903 encompasses the previously localized minimal liver tumor suppressor region, suggesting that a common locus is responsible for tumor suppression among the rat liver tumor cell lines examined. The results of the present study have verified the presence of a liver tumor suppressor locus within human 11p11.2-p12, and have identified a substantial number of microsatellite markers that are closely linked to this tumor suppressor region. These chromosomal markers will facilitate positional cloning of candidate genes from this region, and may prove useful for determining the involvement of this locus in the pathogenesis of human liver cancer.     

Glomerulomegaly and proteinuria in a patient with idiopathic pulmonary hypertension

In an attempt to identify genes involved in neuroblastoma, we scanned neuroblastoma tumour DNAs for homozygous deletions by representational difference analysis (RDA). The RDA produced several difference products, nine of which represented hemizygous deletions located on chromosome 1 or 3. In order to detect deletions, a genomewide loss of heterozygosity (LOH) screening with polymorphic markers was performed. Allelic losses on a number of different chromosomes were detected, mainly in favourable neuroblastomas (stage 1, 2 and 4S). The most frequently deleted region, apart from 1p, was chromosomal region 3p. A more detailed study was made in this region, which showed that 9 out of 58 (16%) tested neuroblastoma tumours showed allelic loss in the same region on chromosome 3p, i.e. 3pter-14.2. Thus, both RDA and LOH studies showed chromosome region 3p as being frequently involved in deletions and/or rearrangements in neuroblastoma tumours. Therefore, it is possible that one or more of the 3p genes implicated in the development of other cancers also play a role in neuroblastoma development and/or progression.     

DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies

This review summarizes reports of recurrent DNA sequence copy number amplifications in human neoplasms detected by comparative genomic hybridization. Some of the chromosomal areas with recurrent DNA copy number amplifications (amplicons) of 1p22-p31, 1p32-p36, 1q, 2p13-p16, 2p23-p25, 2q31-q33, 3q, 5p, 6p12-pter, 7p12-p13, 7q11.2, 7q21-q22, 8p11-p12, 8q, 11q13-q14, 12p, 12q13-q21, 13q14, 13q22-qter, 14q13-q21, 15q24-qter, 17p11.2-p12, 17q12-q21, 17q22-qter, 18q, 19p13.2-pter, 19cen-q13.3, 20p11.2-p12, 20q, Xp11.2-p21, and Xp11-q13 and genes therein are presented in more detail. The paper with more than 150 references and two tables can be accessed from our web site http://www.helsinki.fi/lglvwww/CMG.html. The data will be updated biannually until the year 2001.     

Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer

Malignant rhabdoid tumours (MRTs) are extremely aggressive cancers of early childhood. They can occur in various locations, mainly the kidney, brain and soft tissues. Cytogenetic and molecular analyses have shown that the deletion of region 11.2 of the long arm of chromosome 22 (22q11.2) is a recurrent genetic characteristic of MRTs, indicating that this locus may encode a tumour suppressor gene. Here we map the most frequently deleted part of chromosome 22q11.2 from a panel of 13 MRT cell lines. We observed six homozygous deletions that delineate the smallest region of overlap between the cell lines. This region is found in the hSNF5/INI1 gene, which encodes a member of the chromatin-remodelling SWI/SNF multiprotein complexes. We analysed the sequence of hSNF5/INI1 and found frameshift or nonsense mutations of this gene in six other cell lines. These truncating mutations of one allele were associated with the loss of the other allele. Identical alterations were observed in corresponding primary tumour DNAs but not in matched constitutional DNAs, indicating that they had been acquired somatically. The observation of bi-allelic alterations of hSNF5/INI1 in MRTs suggests that loss-of-function mutations of hSNF5/INI1 contribute to oncogenesis.     

p53 Protein accumulates in Cushings adenomas and invasive non-functional adenomas

The p53 protein, a negative regulator of cell growth, plays an important role in the pathogenesis of many human tumours following gene mutation and/or deletion. We screened a large number of sporadic pituitary tumours for p53 protein accumulation suggestive of gene mutation. Samples were divided into benign adenomas (n = 95) and invasive tumours with local or distant invasion (n = 26). All main tumour classes were represented. Putative p53 mutations were detected by immunohistochemistry on paraffin-embedded sections using polyclonal CM-1 and monoclonal DO-7 and PAb1801 antibodies. Results were compared to normal post-mortem pituitary tissue controls (n = 17). p53 protein accumulation was detected in invasive tumours (16%), but only in corticotrophinomas (2/4) and non-functional tumours (4/15). In non-invasive adenomas, protein accumulation was observed only in ACTH-secreting tumours where 50% were positive (16/32). No protein accumulation was identified in any control tissue. These results indicate that p53 protein accumulation may play a role in the development of Cushings adenomas and in the progression of non-functional tumours to the invasive state.     

Molecular analysis and comparison of radiation-induced large deletions of the HPRT locus in primary human skin fibroblasts

Genetic alterations in gamma-ray- and alpha-particle-induced HPRT mutants were examined by multiplex polymerase chain reaction (PCR) analysis. A total of 39-63% of gamma-ray-induced and 31-57% of alpha-particle-induced mutants had partial or total deletions of the HPRT gene. The proportion of these deletion events was dependent on radiation dose, and at the resolution limits employed there were no significant differences between the spectra induced by equitoxic doses of alpha particles (0.2-0.4 Gy) and gamma rays (3 Gy). The molecular nature of the deletions was analyzed by the use of sequence tagged site (STS) primers and PCR amplification as a "probe" for specific regions of the human X chromosome within the Xq26 region. These STSs were closely linked and spanned regions approximately 1.7 Mbp from the telomeric side and 1.7 Mbp from the centromeric side of the HPRT gene. These markers include: DXS53, 299R, DXS79, yH3L, 3/19, PR1, PR25, H2, yH3R, 1/44, 1/67, 1/1, DXS86, D8C6, DXS10 and DXS144. STS analyses indicated that the maximum size of total deletions in radiation-induced HPRT mutants can be greater than 2.7 Mbp and deletion size appears to be dependent on radiation dose. There were no apparent differences in the sizes of the deletions induced by alpha particles or gamma rays. On the other hand, deletions containing portions of the HPRT gene were observed to be 800 kbp or less, and the pattern of the partial deletion induced by alpha particles appeared to be different from that induced by gamma rays.     

A longitudinal study of markers of bone turnover in Graves'' disease and their value in predicting bone mineral density

Alterations of chromosome 8, including deletions of 8p, occur frequently in many tumors. In this study, fluorescence in situ hybridization was used to study the relationship between 8p deletions, 8q gains, and phenotype in bladder cancer. Cells from 87 tumors were examined by dual-labeling fluorescence in situ hybridization with a centromere 8 probe (pJM12) and P1 probes for 8p22, 8p12, 8q12, and 8q24. Both 8p22 deletions and 8q24 gains were strongly associated with tumor phenotype. There was a marked difference in 8p22 deletions between noninvasive (pTa) tumors (3/33) and minimally invasive (pT1) tumors (8/19; P = 0.005) whereas there was no significant difference between pT1 and muscle-invasive (pT2-4) tumors (19/35; P = 0.3926). Six tumors with 8p22 deletion were examined at 8p12. Three of these tumors showed no 8p12 deletion, narrowing down the site of a putative tumor suppressor gene distal to 8p12. In one other case, there was a marked increase in 8p12 copy number (> 40 per cell; amplification), suggesting the presence of an oncogene involved in bladder cancer at 8p12. The marked difference in 8p22 deletions between noninvasive (pTa) and minimally invasive (pT1) tumors is consistent with a role of a putative tumor suppressor gene on 8p for development of invasive tumor phenotype.     

Management of upper urinary tract calculi with ureteroscopic techniques

Frequent loss of heterozygosity on chromosome 8p in a variety of human malignancies, including head and neck cancers, has suggested the presence of a tumor suppressor gene (or genes) associated with the pathogenesis of these cancers. To test the role of genetic alterations at 8p23 in oral carcinogenesis, we studied 51 squamous cell carcinomas of the head and neck and 29 oral squamous cell carcinoma cell lines for allelic loss using 7 microsatellite markers spanning approximately 5 cM of chromosome band 8p23. Twenty-three of 51 tumors (45%) and 23 of 29 cell lines (79%) showed allelic loss at 1 or more loci. Three cell lines showed homozygous deletion of loci within a 3 cM region defined by the markers D8S1781 and D8S262. Our results suggest that a tumor suppressor gene (or genes) is located in 8p23 and is associated with the development and/or progression of oral carcinomas.     

Molecular analysis of survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes of spinal muscular atrophy patients and their parents

We have assayed deletions of two candidate genes for spinal muscular atrophy (SMA), the survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes, in 101 patients from 86 Chinese SMA families. Deletions of exons 7 and 8 of the telomeric SMN gene were detected in 100%, 78.6%, 96.6%, and 16.7%, in type I, II, III, and adult-onset SMA patients, respectively. Deletion of exon 7 only was found in eight type II and one type III patient. One type II patient did not have a deletion of either exon 7 or 8. The prevalence of deletions of exons 5 and 6 of the NAIP gene were 22.5% and 2.4% in type I and II SMA patients, respectively. We also examined four polymorphisms of SMN genes and found that there were only two, SMN-2 and CBCD541-2, in Chinese subjects. In our study, analysis of the ratio of the telomeric to centromeric portion (T/C ratio) of the SMN gene after enzyme digestion was performed to differentiate carriers, normals, and SMA patients. We found the T/C ratio of exon 7 of the SMN gene differed significantly among the three groups, and may be used for carrier analysis. An asymptomatic individual with homozygous deletion of exons 7 and 8 of the SMN gene showed no difference in microsatellite markers in the SMA-related 5q11.2-5q13.3. In conclusion, SMN deletion in clinically presumed child-onset SMA should be considered as confirmation of the diagnosis. However, adult-onset SMA, a heterogeneous disease with phenotypical similarities to child-onset SMA, may be caused by SMN or other gene(s).     

Identification of a consistent region of allelic loss on 1p32 in meningiomas: correlation with increased morbidity

Meningioma is a common tumor of the central nervous system. Deletions of the short arm of chromosome 1 (1p) are the second most commonly observed chromosomal abnormality in these tumors. Here, we analyzed tumor and normal DNAs from 157 meningioma patients using PCR-based polymorphic loci. Loss of heterozygosity (LOH) for at least one informative marker on 1p was observed in 54 cases (34%), whereas LOH on 1q occurred in only 9 cases (8%). High-resolution deletion mapping defined a consensus region of deletion flanked distally by D1S2713 and proximally by D1S2134, which spans 1.5 cM within 1p32. LOH in this region has also been observed in several other malignancies, suggesting the presence of a tumor suppressor gene or genes that are important for several types of cancer. Statistical analysis revealed that 1p LOH was associated with chromosome 22 deletions and with abnormalities of the NF2 gene in meningioma. In addition, unlike other clinical and molecular characteristics, only 1p LOH was shown to be significantly associated with recurrence-free survival.     

Infrequent mutations of p27Kip1 gene and trisomy 12 in a subset of human pituitary adenomas

To study the etiologic roles of genes on chromosome 12 for the pituitary tumorigenesis of adenomas, mutations of the p27Kip1 gene and allelic ratios of 18 microsatellite markers on the entire chromosome 12 were studied in 33 pituitary adenomas. The p27Kip1 gene on chromosome 12p12-p13 encoding an inhibitor of complexes between cyclins and cyclin-dependent kinases is supposed to function as the tumor suppressor gene. Among 31 sporadic and 2 familial pituitary adenomas, PCR-single strand conformation polymorphism analysis detected three polymorphic changes but no tumor-specific mutations of the p27Kip1 gene. Genotyping of 18 microsatellite markers on the entire chromosome 12 detected the uniformly decreased allelic ratios ranging from 54-66% in 8 of 33 pituitary adenomas (24%), although no loss of heterozygosity was detected. Fluorescence in situ hybridization confirmed trisomy 12 in all 5 available samples out of these 8 samples. Based on these, we conclude that not mutations of the p27Kip1 gene, but trisomy 12 may be etiologically important in a subgroup of pituitary adenomas.     

Sexuality in head and neck cancer patients

We describe a patient with Hirschsprung disease and autism. High-resolution karyotyping indicated that the patient has an interstitial deletion of 20p11.22-p11.23. Microsatellite analysis showed a deletion involving a 5-6 cM region from the maternally derived chromosome 20. The deleted region is proximal to, and does not overlap, the recently characterized Alagille syndrome region. This region of 20p has not yet been implicated in Hirschsprung disease or autism. However, this region contains several genes that could plausibly contribute to any phenotype that includes abnormal neural development.