Detailed deletion mapping of the long arm of chromosome 6 in adult T-cell leukemia

Previously, we have found that the loss of heterozygosity (LOH) was frequently observed on chromosome 6q in acute/lymphoma-type adult T-cell leukemia (ATL), suggesting a putative tumor-suppressor gene for ATL may be present on chromosome 6q. To further define a region containing this gene, we performed fine-scale deletional mapping of chromosome 6q in 22 acute/lymphomatous ATL samples using 24 highly informative microsatellite markers. LOH was found in 9 samples (40. 9%) at 1 or more of the loci examined. Of the 9 samples, 8 shared the same smallest commonly deleted region flanked by D6S1652 and D6S1644 (6q15-21). The genetic distance between these two loci is approximately 4 cM. These results suggest that a putative tumor-suppressor gene on chromosome 6q15-21 probably plays a very important role in the evolution of acute/lymphomatous ATL. Our map provides key information toward cloning the gene.     

Loss of heterozygosity in sporadic oesophageal tumors in the tylosis oesophageal cancer (TOC) gene region of chromosome 17q

From the genotyping of UK and US tylotic families with a high risk of oesophageal cancer we have previously localized the tylosis-associated cancer susceptibility gene (TOC gene, tylosis oesophageal cancer gene) to a 1 cM region on the long arm of chromosome 17 (Kelsell et al., 1996). In the present study we investigated loss of heterozygosity (LOH) patterns of 35 sporadic squamous cell carcinomas of the oesophagus using six polymorphic microsatellite markers encompassing this locus. Twenty-four of the 35 cases (69%) revealed LOH at one or more loci. Deletion was most frequently observed with the marker D17S801 (64% LOH, informative cases), which shows significant linkage to the TOC locus. The LOH analysis in sporadic oesophageal cancer we report here is thus consistent with the hypothesis that the tylosis oesophageal cancer susceptibility gene is also involved in the pathogenesis of a proportion of sporadic squamous cell carcinomas of the oesophagus.     

Microsatellite analysis of plasma DNA from patients with clear cell renal carcinoma

Deletions of DNA sequences on chromosome 3p [loss of heterozygosity (LOH)] are characteristic of clear cell renal carcinoma, which accounts for about 80% of all renal malignancies. Comparing tumor DNA to DNA from normal cells, LOH analysis of microsatellite sequences has aided in molecular diagnosis of renal carcinoma. Because clinically useful tumor markers do not exist for this cancer entity, the aim of the present study was to detect chromosome 3p microsatellite alterations (LOH and microsatellite instability) in plasma DNA from patients with clear cell renal carcinoma. Four chromosome 3p microsatellites (D3S1307, D3S1560, D3S1289, and D3S1300) were amplified by fluorescent PCR using DNA isolated from normal blood cells and plasma of 40 patients. Corresponding tumor DNA was available from 21 patients. Analyzing PCR products on an automated DNA sequencer, we found LOH in at least one locus in 25 plasma samples (63%), and 14 plasma samples (35%) exhibited LOH at more than one locus. Microsatellite instability of plasma DNA was detectable in one patient (3%). No significant association of advanced (>T2N0M0) tumor stages with LOH in plasma DNA could be demonstrated. If present, modifications of plasma DNA and tumor DNA were identical. No alterations of plasma DNA were found in healthy controls. Analysis of plasma DNA from patients with clear cell renal carcinoma reveals tumor-specific microsatellite alterations and may therefore have diagnostic potential as a molecular tumor marker.     

Chromosome band 1p36 contains a putative tumor suppressor gene important in the evolution of chronic myelocytic leukemia

Chronic myelocytic leukemia (CML) is a common neoplasm of hematopoietic pluripotent stem cells. Although the evolution from chronic phase to blast crisis (BC) in CML patients is an inevitable clinical feature, little is understood about the mechanisms responsible for the transformation. We have previously performed allelotype analysis in CML BC and have detected frequent loss of heterozygosity (LOH) on the short arm of chromosome 1. To know the common region of LOH where a putative tumor suppressor gene may reside, deletional mapping was performed using 33 microsatellite markers spanning chromosome 1 in 30 patients with CML BC (21 myeloid and 9 lymphoid). DNA was extracted from slides of bone marrow smears or from bone marrow mononuclear cells. In each patient, DNA from chronic phase was analyzed alongside DNA from either their BC or accelerated phase. Allelic loss on 1p was observed in 14 of the 30 individuals (47%): 10 of the 21 myeloid and 4 of the 9 lymphoid BC cases. Serial cytogenetic information was available in 10 cases with LOH on 1p; interestingly, deletions in this region were not detected. Two samples showed LOH at all informative loci on 1p, whereas the other 12 samples showed LOH on at least one but not all loci on 1p. The common region of LOH resided proximal to D1S508 and distal to D1S507 (1p36). Our results suggest that a tumor suppressor gene that frequently plays an important role in the evolution to BC resides on 1p36 in CML.     

Loss of heterozygosity on the long arm of human chromosome 7 in sporadic renal cell carcinomas

Cytogenetic and molecular analysis of DNA sequences with highly polymorphic microsatellite markers have implicated allele loss in several chromosomal regions including 3p, 6p, 6q, 8p, 9p, 9q, 11p and 14q in the pathogenesis of sporadic renal cell carcinomas (RCCs). Deletions involving the long arm of chromosome 7 have not been described in RCCs although they have been seen in several other tumor types. However, there have been no detailed analysis of loss of heterozygosity (LOH) of 7q sequences in sporadic RCCs. We therefore studied LOH for DNA sequences on 7q with 10 highly polymorphic markers in 92 matched normal/tumor samples representing sporadic RCCs including papillary, nonpapillary, and oncocytomas in order to determine whether allelic loss could be detected in a tumor type with no visible 7q rearrangements at the cytogenetic level. We found chromosome 7q allele loss in 59 of 92 cases (64%) involving one, two, or more microsatellite markers. The most common allele loss included loci D7S522 (24%) and D7S649 (30%) at 7q31.1-31.2, a region that contains one of the common fragile sites, FRA7G. By comparative multiplex PCR analysis, we detected a homozygous deletion of one marker in the 7q 31.1-31.2 region in one tumor, RC21. These results support the idea that a tumor suppressor gene in 7q31 is involved in the pathogenesis of sporadic renal cell carcinomas.     

Frequent loss of heterozygosity on the long arm of chromosome 6: identification of two distinct regions of deletion in childhood acute lymphoblastic leukemia

Cytogenetic analysis of childhood acute lymphoblastic leukemia (ALL) identified nonrandom chromosomal abnormalities of the long arm of chromosome 6. Most of the alterations are deletions that are thought to be indicative of the presence of a tumor suppressor gene that is mutated on the remaining allele. These observations led us to consider whether 6q loss may contribute to the pathogenesis of childhood ALL. To define further a region containing this gene, we analyzed the loss of heterozygosity (LOH) of chromosome 6 in 113 primary ALL samples with matched normal DNA using 34 highly informative microsatellite markers. LOH was found in 17 (15%) samples at one or more of the loci, and partial or interstitial deletions of 6q were detected in 11 of these tumors. On the basis of these results, we performed a detailed deletional map and identified two distinct regions of deletion. The first region is flanked by D6S283 and D6S302 loci at 6q21-22. The second region is flanked by D6S275 and D6S283 loci at 6q21. Clinical analysis determined that LOH of 6q was demonstrated both in precursor-B cell ALLs (15 of 93; 16%) and in T cell ALLs (2 of 19; 11%). In addition, 19 patients have been studied at diagnosis and relapse; 18 showed the same 6q21-22 structural abnormality at relapse (normal, 16 patients; LOH, 2 patients) as their initial presentation, suggesting, albeit with a small patient sample size, that 6q21-22 deletions may be an initial event in leukemogenesis and may occur less frequently during the progression of childhood ALL. These data suggest the presence of putative tumor suppressor genes on chromosome arm 6q that are important in the development of both T and precursor-B childhood ALLs. Our map provides important information toward cloning putative ALL tumor suppressor genes.     

Allelic loss of 16q23.2-24.2 is an independent marker of good prognosis in primary breast cancer

Allelic loss, detected as a loss of heterozygosity (LOH) on the long arm of chromosome 16, is an early and frequent event in breast cancer. Despite this, the clinical significance of LOH on 16q has been very poorly studied. In this study, corresponding blood and tumor samples from 199 clinically well-characterized primary breast cancer patients were analyzed for LOH with the highly polymorphic microsatellite marker D16S511, located at 16q23.2-24.2. 61% of 168 informative tumors showed LOH. Univariate and multivariate analysis found a highly significant association between LOH at 16q23.2-24.2 and freedom from distant metastases, disease-free survival, and overall survival, respectively. No association was found with other clinical parameters such as menopausal status, tumor size, lymph node status, histopathology, and lymph node capsule invasion. This makes allelic loss of 16q23.2-24.2 an independent marker of good prognosis for primary breast cancer.     

A method of dynamic foot-pressure measurement for the evaluation of pediatric orthopaedic foot deformities

We analysed 42 differentiated thyroid tumors including 15 follicular adenomas (FA), 13 papillary thyroid cancers (PTC) and 14 follicular thyroid carcinomas (FTC) with 13 microsatellite markers specific for the long arm of human chromosome 7 within 7q31; this region is deleted frequently in several other tumor types. Overall, 20 of the 42 samples analysed (48%) displayed LOH with one or more of the markers tested. LOH was detected most frequently (78%) in FTC, the most malignant of the thyroid tumors. A smallest common deleted region (SCDR) was defined in this tumor type flanked by markers D7S480 and D7S490. This SCDR is distinct from D7S522, the most commonly deleted locus in many other tumors, which was deleted in only one FTC. D7S522 did show LOH in two of six informative PTCs. None of the PTC and only two of the FAs showed LOH in the FTC SCDR. Since FA is considered a premalignant stage of FTC, our results suggest that inactivation of a putative tumor suppressor at 7q31.2 may be acquired during adenoma to carcinoma progression. The absence of LOH at this locus amongst PTC suggests that inactivation of this tumor suppressor is specific for FTC. In conclusion, LOH at 7q31 is a frequent event in differentiated thyroid cancer, and we have defined a 2 cM SCDR specific for FTC.     

A comparison between microsatellite and quantitative PCR analyses to detect frequent p16 copy number changes in primary bladder tumors

We tested 70 primary bladder tumors for altered copy number of p16 (D9S1752) by microsatellite analysis and by a quantitative PCR (QPCR) assay. These two approaches were fully concordant for 53 tumors, including all 39 tumors in which microsatellite analysis detected loss. In addition, the QPCR method detected useful anomalies in 17 additional cases, including those in which D9S1752 was uninformative. QPCR was abnormal in 56 of 70 (80%) cases, whereas microsatellite analysis was abnormal in 39 of 70 (56%) cases. Although QPCR uses more DNA than microsatellite analysis, it represents a rapid, informative technique that can readily detect both chromosome 9p21 deletions and amplifications in primary bladder tumors without the need for electrophoretic separation.     

Allelic loss on chromosome 7q in ovarian adenocarcinomas: two critical regions and a rearrangement of the PLANH1 locus

The presence of a tumour suppressor gene on chromosome 7q is indicated by cytogenetic, loss of heterozygosity (LOH) and chromosome transfer studies. One candidate gene in this region is Plasminogen Activator Inhibitor-1 (PAI-1). The PAI-1 gene product is involved in proteolysis and may therefore influence tumour spread and invasion. We have analysed a series of 139 ovarian epithelial tumours at four loci in the region 7q21-q31 which includes the PAI-1 gene. The highest rates of loss were found in malignant tumours (FIGO stages I-IV) at markers D7S471 (38%, 20/52 informative cases) and D7S522 (34%, 15/44). No loss was seen in benign tumours and only one out of 27 (4%) informative LMP tumours demonstrated LOH. The smallest region of overlap (SRO) lies between D7S471 and PAI-1. We also identified a rearrangement in one tumour in the PAI-1 gene, suggesting that this may be the inactivated gene in this region. In addition LOH at the more distal marker, D7S522, which lies outside the SRO, shows significant association with stage (P=0.0343) and with LOH on chromosome 13 (P=0.0024). This is in contrast to all other markers examined. These data suggest the presence of two critical regions on 7q which may be important in subsets of epithelial ovarian tumours.     

Identification of two distinct deletion targets at 11q23 in cutaneous malignant melanoma

Karyotypic and molecular data indicate that genetic alterations of the long arm of chromosome 11 (11q) are involved in the pathogenesis of malignant melanoma as well as of other malignancies. We have shown previously, by analysis of loss of heterozygosity (LOH), that a tumor-suppressor gene playing an important role in malignant melanoma is likely to be located within a 51-cM region at 11q23. Its loss appeared to be a late event in tumor progression and an indicator of a less favorable clinical outcome. To further test this hypothesis on a larger set of tumors and to refine the region(s) of common allelic loss, we analyzed 21 polymorphic microsatellite repeats on 11q. A PCR-based assay for LOH was used to study normal and tumor tissues from 53 individuals with primary cutaneous malignant melanoma or metastatic disease. Our findings indicate that in cutaneous malignant melanoma there are at least 2 distinct regions of common allelic loss on 11q, one of them centered around marker APOC3 at 11q23.1-q23.2 delineated by markers D11S1347 and D11S4142 and spanning approximately 5 Mb and a second 3-Mb region around marker D11S925 at 11q23.3 delineated by markers D11S528 and D11S1345. Both regions have been described as deletion targets or as being included within larger allelic deletions detected in several other common tumor types. Thus, these 2 putative melanoma-suppressor loci are likely to harbor tumor-suppressor genes relevant to tumorigenesis of melanoma and a number of other common human malignancies.     

Detection of loss of heterozygosityat microsatellite loci in esophageal squamous-cell carcinoma

Microsatellite alterations at 3 genetic loci (chromosomes 2p, 3p and 17p) were analyzed in 25 tumors (20 primary tumors and 5 metastatic lymph nodes) from 20 patients after surgical treatment for esophageal cancer. DNA samples from tumors were compared with control DNA from lymphocytes obtained from the peripheral blood of the individual patients. Microsatellite alterations [microsatellite instability (MSI) and loss of heterozygosity (LOH)] were detected in 15% of 20 primary tumors with marker D2S123 (chromosome 2p), 55% with marker D3S1067 (chromosome 3p) and 50% with marker TP53 (chromosome 17p). The 3-year disease-free survival rate of the 10 patients who had tumors without alterations or with an alteration at only 1 of 3 microsatellite loci was 75% and it was better than that of the 10 patients who had tumors with alterations at 2 or 3 microsatellite loci (48%, p = 0.049). This finding suggests that esophageal cancer with alterations at multiple microsatellite loci might have strong malignant potential. However, MSI was only detected in one of 20 patients, which suggests that MSI might not play an important role in the development of this cancer. Three of 5 metastatic lymph nodes showed no LOH even though primary tumors of these patients exhibited LOH with 1 or 2 markers, and 1 metastatic lymph node had LOH that was detected with D3S1067 even though the primary tumor of this patient had no LOH with all markers. Thus, clonal heterogeneity might exist in esophageal squamous-cell carcinomas.     

Allelic imbalance study of 16q in human primary breast carcinomas using microsatellite markers

The high incidence of allelic imbalance on the long arm of chromosome 16 in breast cancer suggests its involvement in the development and progression of the tumor. Several loss of heterozygosity (LOH) studies have led to the assignment of commonly deleted regions on 16q where tumor suppressor genes may be located. The most recurrent LOH regions have been 16q22.1 and 16q22.4-qter. The aim of this study was to gain further insight into the occurrence of one or multiple "smallest regions of overlap" on 16q in a new series of breast carcinomas. Hence, a detailed allelic imbalance map was constructed for 46 sporadic breast carcinomas, using 11 polymorphic microsatellite markers located on chromosome 16. Allelic imbalance of one or more markers on 16q was shown by 30 of the 46 tumors (65%). Among these 30 carcinomas, LOH on the long arm of chromosome 16 was detected at all informative loci in 19 (41%); 13 of them showed allelic imbalance on the long but not on the short arm, with the occurrence of variable "breakpoints" in the pericentromeric region. The partial allelic imbalance in 11 tumors involved either the 16q22.1-qter LOH region or interstitial LOH regions. A commonly deleted region was found between D16S421 and D16S289 on 16q22.1 in 29 of the 30 tumors. The present data argue in favor of an important involvement of a tumor suppressor gene mapping to 16q22.1 in the genesis or progression of breast cancer.     

Mapping of chromosome 3p deletions in human epithelial ovarian tumors

Epithelial ovarian tumors frequently display deletions on the short arm of chromosome 3 suggesting the existence of tumor suppressor genes within the deleted regions. We have recently established a primary tissue culture system as a model to investigate the genetic events associated with ovarian cancer. The frequencies of loss of heterozygosity (LOH) at 16 loci representative of chromosome 3p in 33 tumor biopsies and 47 ovarian primary cultures derived from unselected ovarian cancers were examined. This repertoire also included benign and borderline tumors as well as malignant ovarian ascites. LOH was observed in 25 (31%) samples for at least one marker: 21 of 58 malignant, two of 12 borderline and two of 10 benign specimens. Chromosome 3p loss was not restricted to ovarian tumors of high grade and stage. LOH was observed in both cultured and non cultured tumors and ascites. A spontaneously immortalized cell line derived from a malignant ovarian ascites, OV-90, displayed LOH of the majority of markers suggesting loss of one homolog of chromosome 3p. The pattern of deletion displayed by these 25 samples enabled the determination of at least two distinct regions of overlapping deletions on chromosome 3p extending from D3S1270 to D3S1597 and from D3S1293 to D3S1283. In addition, a region proximal to D3S1300 was deleted in a subset of samples. Although loss of loci overlapping these three regions (Regions I, II and III) were observed in malignant and benign tumors, in borderline tumors loss was observed of markers representative of Region III only. While RARbeta is presently included in Region II, the minimal regions of deletion exclude VHL, TGFBR2, PTPase(gamma) and FHIT as candidate tumor suppressors in ovarian tumorigenesis.     

Preliminary mapping of the deleted region of chromosome 9 in bladder cancer

Inactivation of a suppressor gene by deletion of chromosome 9 is a candidate initiating event in bladder carcinogenesis. We have used 13 polymorphic markers spanning the length of chromosome 9 in order to map the region of deletion in human bladder carcinomas. In the majority of tumors loss of heterozygosity was found at all informative sites along the chromosome, indicating deletion of the entire chromosome. Nine tumors had selective deletions of chromosome 9. Mapping of the deleted region in these tumors suggests that the target gene is located between D9S22 at 9q22 and D9S18 at 9p12-13.     

Homozygous deletions at chromosome 9p21 and mutation analysis of p16 and p15 in microdissected primary non-small cell lung cancers

Loss of heterozygosity on chromosome 9p has been detected in many primary human tumors and cell lines, suggesting that this chromosomal arm harbors one or more tumor suppressor genes. The recently cloned p16 and p15 genes, mapped to 9p21, are likely candidates for such tumor suppressors. To map the deletion at chromosome 9p21 in non-small cell lung tumors, we analyzed DNA from 25 tumors and matching normal DNAs at six microsatellite markers that flank the region occupied by the p16 and p15 genes. Loss of heterozygosity of at least one microsatellite marker on chromosome 9p21 was detected in 13 (52%) of 25 tumors, including one tumor that exhibited homozygous deletion of both human IFNalpha and D9S171. Six tumors analyzed by a comparative multiplex PCR technique showed homozygous deletions of the sequence tag site marker c5.1 (within p16). Screening for mutations in p16 and p15 revealed one tumor with a non-sense mutation in exon 2 of p16, but no mutations were detected in p15 in any of the tumors. Thus, in these analyses approximately one-half of the non-small cell lung tumors had loss of heterozygosity at chromosome 9p21, and of these tumors, one-half had homozygous deletions of the region that includes p16. This appears to confirm the importance of a locus in this region critical to growth control in lung. The apparent lack of other mutations in p16 and p15 in the tumors with loss of heterozygosity leaves open the possibility of an unidentified gene in this region that may function as a tumor suppressor.     

Memory-impairing effects of local anesthetics in an elevated plus-maze test in mice

Detailed deletion mapping of chromosome 6q has shown that the highest percentage of loss of heterozygosity (LOH) is located at 6q25-q27 and suggested that an ovarian cancer associated tumor suppressor gene may reside in this region. To further define the smallest region of common loss, we used 12 tandem repeat markers spanning a region no more than 18 cM, located between 6q25.1 and 6q26, to examine allelic loss in 54 fresh and paraffin embedded invasive ovarian epithelial tumor tissues. Loss of heterozygosity was observed more frequently at the loci defined by marker D6S473 (14 of 32 informative cases, 44%) and marker D6S448 (17 of 40 informative cases, 43%). Detailed mapping of chromosome 6q25-q26 in these tumor samples identified a 4 cM minimal region of LOH between markers D6S473 and D6S448 (6q25.1-q25.2). Loss of heterozygosity at D6S473 correlated significantly both with serous versus non-serous ovarian tumors (P=0.040) and with high grade versus low grade specimens (P=0.023). The results suggest that a 4 cM deletion unit located at 6q25.1-q25.2 may contain the putative tumor suppressor gene which may play a role in the development and progression of human invasive epithelial ovarian carcinomas (IEOC).     

Leukocyte migration in acute colonic inflammatory bowel disease: comparison of histological assessment and Tc-99m-HMPAO labeled leukocyte scan

Loss of heterozygosity (LOH) on chromosome 11 is frequently altered in various epithelial cancers. The present study was designed to investigate LOH on chromosome 11 in microdissected samples of normal prostatic epithelium and invasive carcinoma from the same patients. For this purpose, DNA was extracted from the microdissected normal and tumor cells of 38 prostate cancers, amplified by polymerase chain reaction PCR and analyzed for LOH on chromosome 11 using 9 different polymorphic DNA markers (D11S1307, D11S989, D11S1313, D11S898, D11S940, D11S1818, D11S924, D11S1336 and D11S912). LOH on chromosome 11 was identified in 30 of 38 cases (78%) with at least one marker. Four distinct regions of loss detected were: 1) at 11p15, at loci between D11S1307 and D11S989; 2) at 11p12, on locus D11S131 (11p12); 3) at 11q22, on loci D11S898, D11S940 and D11S1818; and 4) at 11q23-24, on loci between D11S1336 and D11S912. We found 25% of the tumors with LOH at 11p15; 39% had LOH at 11p12; 66% had LOH at 11q22; and 47% had LOH at 11q23-24. These deletions at 11p15, 11p12, 11q22 and 11q23-24 loci were not related to the stage or grade of the tumor.     

Molecular support for field cancerization in the head and neck

BACKGROUND: Two competing concepts, field cancerization and micrometastatic lesions, have been postulated to account for the high frequency of second primary tumors and multicentric dysplasia in patients with head and neck carcinoma. METHODS: To provide insight into this process, the authors examined histologically normal mucosa and dysplastic tissue adjacent to invasive tumor for loss of heterozygosity (LOH) at three commonly deleted loci. Tissues from 21 patients with carcinoma of the oral cavity and oropharynx were identified and verified by a pathologist to contain histologically normal mucosa, dysplasia, and adjacent invasive squamous cell carcinoma. Each specimen was analyzed for LOH at D9S171 (9p21), D3S1007 (3p21.3-22), and D3S1228 (3p14). RESULTS: Of the 21 patients, 19 had adequate DNA for analysis. Seventeen patients were heterozygous at one or both of the 3p sites and LOH occurred in 6 of 17 invasive tumor specimens, 1 of 17 dysplasia specimens, and in none of the mucosal specimens. LOH at 9p21 occurred in 11 of 13 informative specimens of invasive tumor, 8 of 13 dysplasia specimens, and 6 of 13 normal mucosa specimens. However, one case that did not have 9p deletion in the tumor demonstrated LOH in the mucosa and two cases had LOH in both the tumor and mucosa but with deletion of the opposite allele. CONCLUSIONS: These data suggest that 9p21 but not 3p14 or 3p21 deletions occur in the absence of histologic changes. In two cases preinvasive and invasive lesions that apparently were an example of histologic progression contained disparate genetic events, calling into question the use of adjacent dysplasia as a model for premalignant lesions.     

Loss of heterozygosity in tuberous sclerosis hamartomas

We have previously described in tuberous sclerosis (TSC) hamartomas the phenomenon of loss of heterozygosity (LOH) for DNA markers in the region of both the TSC2 gene on chromosome 16p13.3 and the TSC1 gene on 9q34. We now describe the spectrum of LOH in 51 TSC hamartomas from 34 cases of TSC. DNA was extracted from leucocytes or normal paraffin embedded tissue, and from frozen paraffin embedded hamartoma tissue from the same patient. The samples were analysed for 11 markers spanning the TSC1 locus and nine markers spanning the TSC2 locus. Twenty-one of 51 hamartomas showed LOH (41%). There was significantly more LOH on 16p13.3, with 16 hamartomas showing LOH around TSC2, and five in the vicinity of TSC1. No hamartoma showed LOH for markers around both loci. All the areas of LOH on chromosome 9 were large, but the smallest region of overlap lay between the markers D9S149 and D9S114, providing independent evidence for the localisation of the TSC1 gene. These data show that LOH is a common finding in a wide range of hamartomas, affecting the same TSC locus in different lesions from the same patient but not affecting both loci. These data support the hypothesis that both the TSC genes act as tumour suppressors and that the manifestations of TSC in patients with germline TSC mutations rise from "second hit" somatic mutations inactivating the remaining normal copy of the TSC gene.