Common Region Of Loss Of Heterozygosity Research Articles

Identification of Novel Somatic Mutations, Regions of Recurrent Loss of Heterozygosity (LOH) and Significant Clonal Evolution From Diagnosis to Relapse in Childhood AML Determined by Exome Capture Sequencing – an NCI/COG Target AML Study

AbstractAbstract 123▪▪This icon denotes a clinically relevant abstractComprehensive cataloguing of somatic changes in acute myeloid leukemia (AML) has revealed novel molecular pathways that contribute to the development and progression of the disease, however many lack prognostic significance. The challenge that lies ahead is to understand how these molecular changes relate to transcription and protein deregulation to influence treatment responses and outcomes. Also, some mutations found in adult AML are detected at lower frequency or not at all in pediatric AML.To better define the genomic profile of somatic mutations in childhood AML and evaluate the extent of clonal evolution from diagnosis to relapse, we performed whole exome sequencing in matched trios of specimens (diagnostic, germline and relapse) from 20 children with AML who lacked known karyotypic high-risk features. Candidate variants were verified by 454 sequencing. In total, 364 somatic variants were successfully verified, of which 263 were non-silent mutations (127 at diagnosis and 136 at relapse). Of the 195 unique nonsynonymous mutations present in 180 genes, mutations in 6 genes were detected in more than one patient, including ETV6, KIT, KRAS, NRAS in 2 patients and TET2 and WT1 in 3 patients. A polyphen prediction estimated 72% of the missense and nonsense mutations as possibly and/or probably damaging. Mutations identified in single patients included known genes implicated in leukemias (e.g., GATA2, IKZF1), as well novel mutations in genes not previously implicated in AML.Prevalence and clinical significance of ETV6 mutations were assessed in a large cohort of pediatric AML. The entire coding sequence of ETV6 gene was sequenced in 180 cases of pediatric AML. ETV6 mutations, including missense (N=5), Indels (N=4) or splice site mutations (N=1) were detected in 6% of the patients without karyotypic or molecular risk factors. Those with and without ETV6 mutations had an overall survival of 33% and 71%, respectively (p=0.006) with a corresponding relapse rate of 80% and 29% (p=0.004).We examined the clonal evolution of mutations from diagnosis to relapse by comparing the mutation profile at diagnosis to that in relapse. In the discovery phase, at diagnosis, 234 somatic mutations in 202 genes were detected, of which 174 were non-silent events, including 36 (21%) indels, 126 (72%) missense, 11 (6%) nonsense mutations and 1 (1%) splice site mutation. At relapse, 350 mutations in 309 genes were detected, of which only 112 mutations were detectable at diagnosis (29%). In verification phase, 71 mutations in 70 genes were identified only in diagnostic specimens, and 105 mutations in 104 genes were detected in relapse only, with an overlap of 192 variants, suggesting significant molecular evolution and loss or gain of genomic events from diagnosis to relapse. With secondary targeted deep sequencing the overlap of mutations present at diagnosis and relapse increased to 52%. Comparison of allele fractions between diagnosis and relapse revealed a significant enrichment of the variant from diagnosis to relapse with evolution of a minor clone at diagnosis to a more dominant presence at relapse. It also highlights that in a substantial subset of patients, novel genomic events may emerge, leading to disease relapse.In addition, analysis of the allele fractions of SNPs in the normal and tumors of these patients allowed us to detect regions of recurrent loss of heterozygosity (LOH). Six of 20 patients exhibited LOH in one or more regions. The most common region of LOH, found in 4 patients (20%) was the q-arm of chromosome 13 (3 with FLT3/ITD), including 10 genes with germ line variation across 4 patients, and in two patients the q-arm of chromosome 16 was involved. Two of the 4 patients had LOH only in the relapse specimen.This study highlights the potential impact of applying novel sequencing approaches to pediatric AML and not only identifies clinically significant somatic mutations, but defines genomic complexity in childhood AML by demonstrating significant clonal evolution from diagnosis to relapse. Mutations emerging at relapse may represent initially rare, chemotherapy-resistant events that cooperate with other mutations and lead to adverse outcome. Identifying mutations that may be therapeutically targeted early in the course of therapy may provide improved outcomes for patients. Disclosures:No relevant conflicts of interest to declare.

Narrowing of the regions of allelic losses of chromosome 1p36 in meningioma tissues by an improved SSCP analysis

Mapping loss of heterozygosity (LOH) regions in the genomes of tumor tissues is a practical approach for identifying genes whose loss is related to tumorigenesis. Conventional LOH analyses using microsatellite or single nucleotide polymorphism (SNP) markers require the simultaneous examination of tumor- and matched normal-DNA. Here, we improved the previously developed SNP-based LOH assay using single strand conformation polymorphism (SSCP) analysis, so that LOH in tumor samples heavily contaminated with normal DNA can now be precisely estimated, even when matched normal DNA is not available. We demonstrate the reliability of the improved SSCP-based LOH detection method, called the LOH estimation by quantitative SSCP analysis using averaged control (LOQUS-AC), by comparing the results with those of the previous "LOH estimated by quantitative SSCP assay" (LOQUS) method. Using the LOQUS-AC assay, LOH was detected at a high consistency (98.1%) with the previous LOQUS method. We then applied this new method to characterize LOH profiles in 130 meningiomas, using 68 SNPs (i.e., a mean inter-SNP interval of 441 kbp) that are evenly distributed throughout chromosome 1p36. Benign, atypical and anaplastic meningiomas exhibited 1p36 LOH at frequencies of 48.39, 84.62 and 100.00%, respectively, using LOQUS-AC. Subsequently, we detected a candidate common LOH region on 1p36.11 that might harbor tumor suppressor genes related to malignant progression of meningioma.

Loss of heterozygosity on chromosome 3p in breast cancer and precancerous lesion

To study the loss of heterozygosity (LOH) on chromosome 3p in breast cancers and precancerous lesion. LOH at 11 microsatellite loci was detected in 41 cases of breast cancers and 12 cases of precancerous lesion by polymerase chain reaction and silver stain. The expressions of ER, PR, FHIT and hMLH1 were detected in breast cancer by immunohistochemistry. LOH on 3p was detected in 97% of breast cancers. D3S1295, D3S1029 and D3S1038 located at 3p14, 3p21-p22 and 3p25 were identified as the loci with most frequent LOH (53.1%, 43.6% and 52.5%). LOH of D3S1038 and expression of hMLH1 protein correlated with several clinicopathological features. LOH of D3S1295 had significant negative correlation with the expression of FHIT. In breast precancerous lesions, LOH on 3p was detected in 41.7% lesions. D3S1295 and D3S1029 were also identified as the most frequent LOH locus (27.3% and 16.7%). The smallest common LOH region seems likely lie between 3p14 and 3p25. The smallest LOH region indicates the existence of breast tumor related genes and some of them affect gene expression.

Identification of RUNX1/AML1 as a classical tumor suppressor gene.

Based on our previous results indicating the presence of a tumor suppressor gene (TSG), chromosome 21 was analysed for loss of heterozygosity (LOH) in 18 patients with acute myeloid leukemia (17, AML-M0; one, AML-M1). Allelotyping at polymorphic loci was performed on purified material, allowing unequivocal detection of allelic loss and homozygous deletions. Six AML-M0 patients shared a common region of LOH harboring a single gene: RUNX1 (AML1), the most frequent site of translocations in acute leukemia and a well-known fusion oncogene. Fluorescence in situ hybridization allowed the identification of deletions with breakpoints within RUNX1 in two patients as the cause of LOH. In the four others the LOH pattern and the presence of two karyotypically normal chromosomes 21 were in line with mitotic recombination. Further molecular and cytogenetic analyses showed that this caused homozygosity of primary RUNX1 mutations: two point mutations, a partial deletion and, most significantly, a complete deletion of RUNX1. These findings identify RUNX1 as a classical TSG: both alleles are mutated or absent in cancer cells from four of the 17 AML-M0 patients examined. In contrast to AML-M0, the AML-M1 patient was trisomic for chromosome 21 and has two mutated and one normal RUNX1 allele, suggesting that the order of mutagenic events leading to leukemia may influence the predominant tumor type.

Human pituitary adenomas infrequently contain inactivation of retinoblastoma 1 gene and activation of cyclin dependent kinase 4 gene.

Components of cyclinD1/cyclin-dependent kinase 4 (CDK4)/p16INK4a/pRb pathway are the frequent target of many tumor types. We examined the role of retinoblastoma susceptibility gene (RB1) and the CDK4 gene in human pituitary tumorigenesis. For the RB1 gene, pRb expression and loss of heterozygosity (LOH) on 13q in pituitary adenomas were analysed. Immunostaining of pRb revealed lack of expression in 1 of 29 pituitary adenomas. In 4 of 31 pituitary adenomas, allelic imbalances including LOH of RB1 on 13q14 were detected. Three of 4 pituitary adenomas, in which one adenoma lacked pRb expression, had a common LOH region at least from D13S219 on 13q12.3-q13 to D13S265 on 13q31-32. Interphase fluorescence in situ hybridization with a probe of RB1 showed 2 copies of RB1 gene suggesting that mitotic recombination events, not deletion or chromosome loss, led to LOH in the 3 pituitary adenomas analyzed. All 27 exons, intron-exon boundaries, and essential promoter region of RB1 gene were then sequenced in genomic DNA from 4 pituitary adenomas with allelic imbalance on 13q14 including one adenoma without pRb expression and 3 adenomas with pRb expression. Any somatic mutations, insertions, or microdeletions in the RB1 gene were not detected in 4 pituitary adenomas. Methylation sensitive (MS)-polymerase chain reaction (PCR) and bisulfite sequencing analysis revealed hypomethylated status of CpG islands in the promoter region of the RB1 genes of 4 pituitary adenomas. In addition, activating mutations of CDK4 gene, which is a component of cyclinD1/CDK4/p16INK4a/pRb pathway, were not detected in 31 pituitary adenomas. Based on these results, it is concluded that somatic mutations of the RB1 gene or CDK4 gene do not appear to play a major role in pituitary tumorigenesis. This supports the presence of potential tumor suppressor gene(s) on 13q12.3-q13 to 13q31-32 in pituitary adenomas.

Molecular characterization of a 7p15–21 homozygous deletion in a Wilms tumor

Recent molecular studies have shown a relatively high rate of loss of heterozygosity (LOH) at band 7p15-21 in Wilms tumor. We previously reported that the minimal common region of LOH was located between markers D7S517 and D7S503 in bands 7p15-21. We also reported the identification of one Wilms tumor (GOS44) bearing a homozygous, interstitial deletion at a locus within this region. Homogeneous primary cell cultures have been derived from this tumor and have been used for all the subsequent analyses. Using PCR and a panel of STS markers mapping between D7S517 and D7S503, the physical boundaries of the homozygous deletion were determined to be between D7S638 and D7S644. The deleted region spans approximately 3 Mbp of genomic sequence and includes seven known genes (KIAA0744, KIAA0713, AHR, AGR2, NET6, HSPC028, and DGKB.) as well as five predicted genes with similarities to genes of known function (LOC-91802, -116364, -96009, -92511, and -92512). The proximal breakpoint was found to lie between exon 6 and exon 7 of KIAA0744, and the distal breakpoint lay between exon 17 and exon 18 of DGKB. It is unlikely that a functional fusion gene product was generated as a consequence of the fusion between these two genes, because they are oriented in opposite directions on the chromosome. This is the only reported homozygous deletion recorded so far in Wilms tumor, and it provides the means to identify the tumor-suppressor gene located in this deletion.

Deletion at 3p25.3-p23 is frequently encountered in endocrine pancreatic tumours and is associated with metastatic progression.

For several reasons, chromosome 3p is thought to be involved in the pathogenesis of sporadic endocrine pancreatic tumours (EPTs): von Hippel-Lindau's disease (VHL gene at 3p25.5) is associated with EPTs; 3p is frequently involved in solid human tumours; and comparative genomic hybridization has identified frequent losses at 3p in EPTs. This study investigated 99 benign and malignant tumours, including 20 metastases, from 82 patients, by microsatellite loss of heterozygosity (LOH) analysis and fluorescence in situ hybridization (FISH) in order to evaluate the importance of chromosome 3p deletions in the molecular pathogenesis and biological behaviour of EPTs, to elaborate a common region of deletion, and to narrow down putative tumour suppressor gene loci. Allelic losses of 3p were found in 58/99 (58.6%) of tumours in 45/82 (54.9%) patients; analysis of seven microsatellite markers (3p26-p21) revealed a common region of LOH at 3p25.3-p23. The LOH frequency was significantly higher in malignant than in benign neoplasms (70.2% versus 28.0%; p=0.001). In addition, a strong correlation was found between the loss of alleles on chromosome 3p and clinically metastatic disease (LOH of 73.7% in metastasizing versus 41.5% in non-metastasizing tumours; p=0.008). EPTs from these patients showed a tendency towards losing large parts or the entire short arm of chromosome 3 with tumour progression. Furthermore, FISH analysis revealed complete loss of chromosome 3 in ten out of 37 EPTs (27%). These results indicate that a putative tumour suppressor gene at 3p25.3-p23 may play a role in the oncogenesis of sporadic EPTs and that losses of larger centromeric regions are associated with metastatic progression.

DNA Fragmentation Factor 45 (DFF45) Gene at 1p36.2 Is Homozygously Deleted and Encodes Variant Transcripts in Neuroblastoma Cell Line

Recently, loss of heterozygosity (LOH) studies suggest that more than two tumor suppressor genes lie on the short arm of chromosome 1 (1p) in neuroblastoma (NB). To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in 20 NB cell lines using a high-density STS map spanning chromosome 1 p36, a common LOH region in NB. We found that the 45-kDa subunit of the DNA fragmentation factor (DFF45) gene was homozygously deleted in an NB cell line, NB-1. DFF45 is the chaperon of DFF40, and both molecules are necessary for caspase 3 to induce apoptosis. DFF35, a splicing variant of DFF45, is an inhibitor of DFF40. We examined 20 NB cell lines for expression and mutation of DFF45 gene by reverse transcription (RT)-polymerase chain reaction (PCR) and RT-PCR-single-strand conformation polymorphism. Some novel variant transcripts of the DFF45 gene were found in NB cell lines, but not in normal adrenal gland and peripheral blood. These variants may not serve as chaperons of DFF40, but as inhibitors like DFF35, thus disrupting the balance between DFF45 and DFF40. No mutations of the DFF45 gene were found in any NB cell line, suggesting that the DFF45 is not a tumor suppressor gene for NB. However, homozygous deletion of the DFF45 gene in the NB-1 cell line may imply the presence of unknown tumor suppressor genes in this region.

Allelic deletion at chromosome bands 11q14-23 is common in neuroblastoma.

Neuroblastoma tumorigenesis may involve the differential inactivation of multiple tumor suppressor genes. Recent data have suggested that a neuroblastoma suppressor gene may be located on the long arm of chromosome 11 (11q). We therefore analyzed 295 primary neuroblastomas from a representative group of patients for loss of heterozygosity (LOH) at 25 polymorphic markers spanning 11q. LOH was observed in 129 primary neuroblastomas (44%), and a common region of LOH mapped to 11q14-23. No correlation was found between 11q LOH and adverse prognostic variables, but a strong inverse relationship between 11q LOH and MYCN amplification (P < 0.001) was observed. There was no difference in overall survival when patients were stratified by 11q LOH status. However, 11q LOH was associated with a decreased overall survival probability when patients whose tumors had a single copy of MYCN were analyzed separately (P = 0.008). These data support the hypothesis that a tumor suppressor gene mapping within 11q14-23 is frequently inactivated during the malignant evolution of neuroblastoma.

Involvement of chromosomes 9 and 11 in familial and sporadic polycythemia vera

Polycythemia vera (PV) is an acquired clonal myeloproliferative disorder. Although the vast majority of PV cases are sporadic, few familial cases have been reported. We identified six families with multiple members with PV all having clonal myelopoiesis. The predisposition to PV follows an autosomal dominant trait with incomplete penetrance. Based on the clonality and genetic studies of these PV families, we propose a model of the PV pathogenesis according to which loss of heterozygosity (LOH) in the PV locus is the disease-initiating event. We excluded the linkage between the PV phenotype and markers on 20q (del20q is the most common cytogenetic lesion found in ∼8% of PV cases), the SHP-1 gene locus, and the c-mpl gene. Since none of the familial PV subjects had cytogenetic abnormalities that would identify regions with possible LOH, we performed a genome-wide screening for LOH by microsatellite PCR at 10 cM resolution in one of the familial PV subjects. We identified two loci with LOH one on chromosome 9p and the other on chromosome 11q. We examined 10 sporadic PV subjects for the presence of both of these defects. The 9p LOH was present in 3/10 (33%) of sporadic and 2/15 (13%) familial PV cases. We mapped the common LOH region on 9p to be located between D9S1813-D9S1833 that spans 37 cM. In all examined 9pLOH+ PV subjects, the 9pLOH was present in 100% of the EPO-independent colonies suggesting the 9pLOH was present in 100% of the EPO-independent colonies suggesting the 9pLOH might be the primary PV lesion or a secondary lesion providing clonal advantage to the PV clone. Mitotic recombination is the most frequent cause of LOH in both loci (LOH remains undetectable by cytogenetic analysis). The 11q LOH was present in 2/15 (13%) familial PV cases. The 11q LOH spans a 70 cM region between markers D11S2002-11qTER. The linkage analysis of both regions (that would eliminate secondary loci and further map the primary PV locus) is in progress.

Loss of heterozygosity at 7p in Wilms' tumour development.

Chromosome 7p alterations have been implicated in the development of Wilms' tumour (WT) by previous studies of tumour cytogenetics, and by our analysis of a constitutional translocation (t(1;7)(q42;p15)) in a child with WT and radial aplasia. We therefore used polymorphic microsatellite markers on 7p for a loss of heterozygosity (LOH) study, and found LOH in seven out of 77 informative WTs (9%). The common region of LOH was 7p15–7p22, which contains the region disrupted by the t(1;7) breakpoint. Four WTs with 7p LOH had other genetic changes; a germline WT1 mutation with 11p LOH, LOH at 11p, LOH at 16q, and loss of imprinting of IGF2. Analysis of three tumour-associated lesions from 7p LOH cases revealed a cystic nephroma-like area also having 7p LOH. However, a nephrogenic rest and a contralateral WT from the two other cases showed no 7p LOH. No particular clinical phenotype was associated with the WTs which showed 7p LOH. The frequency and pattern of 7p LOH demonstrated in our studies indicate the presence of a tumour suppressor gene at 7p involved in the development of Wilms' tumour. © 2000 Cancer Research Campaign

A 2-Mb Sequence-Ready Contig Map and a Novel Immunoglobulin Superfamily Gene IGSF4 in the LOH Region of Chromosome 11q23.2

Human chromosome 11q23.2 has been proposed to contain a tumor suppressor gene(s) whose deletion has been associated with cancer of the lung and breast and with neuroblastoma. To analyze the genomic structure and to isolate a candidate tumor suppressor gene from this region, we constructed a 2-Mb sequence-ready contig map using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC). The map comprises a contig of 24 overlapping P1, BAC, and PAC clones. To isolate gene fragments from the region, we performed direct cDNA library screening, exon trapping, EST mapping, and genomic sequencing using the P1, BAC, and PAC clones. Sequence analysis of 5 clones, which spans 23% (458,738 bp) of the region, and extensive gene scanning along the entire region revealed that the region is extraordinarily scarce in genes, but we identified one ubiquitously expressed novel gene and one testis-specific gene fragment. The novel gene, which we call IGSF4 (immunoglobulin superfamily 4), is transcribed into a 1.6- or 4.4-kb RNA encoding a 442-amino-acid protein. It shares strong homology with mouse IGSF-B12 and cell adhesion molecules NCAM1 and NCAM2 within their Ig-like C2-type domains. The IGSF4 gene, a novel gene that is shown to be located in the common loss of heterozygosity region, possesses a number of interesting features and may be good candidate for a tumor suppressor gene.

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. © 1998 by The American Society of Hematology.

Chromosome Band 1p36 Contains a Putative Tumor Suppressor Gene Important in the Evolution of Chronic Myelocytic Leukemia

AbstractChronic 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.© 1998 by The American Society of Hematology.

Construction of a bacterial artificial chromosome (BAC) contig across the minimally deleted region in 13q14.3 in B-cell chronic lymphocytic leukemia.

Loss of heterozygosity (LOH) analysis in B-cell chronic lymphocytic leukemia (BCLL) has indicated that a frequent genetic event is loss of alleles from an approximately 500 kb region in 13q14.3, distal to the retinoblastoma gene. We have used DNA markers from this region to isolate and characterize a series of bacterial artificial chromosomes (BACs) which span the region between markers D13S319 and D13S25, which represents the common region of LOH. This entire region appears to be contained within only two minimally overlapping BACs, representing a maximum distance of approximately 350 kb. This BAC contig has been used to position known STS, EST and gene markers within the region. We have also used a modified differential display/RNA fingerprinting procedure designed to isolated transcribed sequences from YACs to isolate two transcribed units from the region which have also been positioned within the contig. The construction of a BAC contig with minimal redundancy provides the ideal resources from which to begin to identify candidate genes related to BCLL.

Putative tumor suppressor gene region within 0.85 cM on chromosome 12 in radiation‐induced murine lymphomas

Analyses of genetic alterations in tumors from F1 hybrid mice produced by inter-subspecific crosses between genetically well-characterized inbred strains provide precise and comprehensive evidence for genetic abnormalities such as allelic loss. We performed loss of heterozygosity (LOH) analyses of 125 radiation-induced lymphomas of (BALB/cHeA × MSM/Ms)F1 hybrid mice by polymerase chain reaction (PCR) analysis of microsatellite DNA polymorphic markers. Very frequent LOH was found at a distal region on chromosome 12. To precisely define the most common region of LOH, we first determined the order of and distances between the available microsatellite loci around the region by using 586 (CXSD × MSM/Ms)F2 hybrid mice (1172 meiosis). The locus order and distances were [centromere]—D12Mit132—(0.34 cM)—D12Mit50—(2.05 cM)—[D12Mit122, D12Mit53]—(0.85 cM)—D12Mit233—(0.43 cM)—D12Mit279—(0.17 cM)—D12Mit181—[telomere]. We then investigated the features of LOH at these loci. The highest frequency of LOH (83 of 125, 66%) was found at D12Mit233. The LOH patterns of individual lymphomas indicated that the most common region of LOH was within the 0.85 cM between D12Mit53 and D12Mit233, a region homologous to human chromosome 14q32.1. These results suggest that a putative novel tumor suppressor gene exists within this region. Mol. Carcinog. 22:175–181, 1998. © 1998 Wiley-Liss, Inc.

Putative tumor suppressor gene region within 0.85 cM on chromosome 12 in radiation-induced murine lymphomas

Analyses of genetic alterations in tumors from F1 hybrid mice produced by inter-subspecific crosses between genetically well-characterized inbred strains provide precise and comprehensive evidence for genetic abnormalities such as allelic loss. We performed loss of heterozygosity (LOH) analyses of 125 radiation-induced lymphomas of (BALB/cHeA x MSM/Ms)F1 hybrid mice by polymerase chain reaction (PCR) analysis of microsatellite DNA polymorphic markers. Very frequent LOH was found at a distal region on chromosome 12. To precisely define the most common region of LOH, we first determined the order of and distances between the available microsatellite loci around the region by using 586 (CXSD x MSM/Ms)F2 hybrid mice (1172 meiosis). The locus order and distances were [centromere]-D12Mit132-(0.34 cM)-D12Mit5O-(2.05 cM)-[D12Mit122, D12Mit53]-(0.85 cM)-D12Mit233-(0.43 cM)-D12Mit279-(O.17 cM)-D12Mit181-[telomere]. We then investigated the features of LOH at these loci. The highest frequency of LOH (83 of 125, 66%) was found at D12Mit233. The LOH patterns of individual lymphomas indicated that the most common region of LOH was within the 0.85 cM between D12Mit53 and D12Mit233, a region homologous to human chromosome 14q32.1. These results suggest that a putative novel tumor suppressor gene exists within this region.

Frequent deletion of chromosome 12p12.3 in children with acute lymphoblastic leukaemia

Cytogenetic deletions of the short arm of chromosome 12 are common recurring alterations found in a wide range of haematological neoplasias, including childhood acute lymphoblastic leukaemia (ALL), the most frequent paediatric malignancy. Such a loss of genetic material suggests the presence of a tumour suppressor gene which plays an important role in growth regulation or in the differentiation of haemopoietic stem cells. To substantiate this hypothesis and to determine more precisely the chromosomal location of this putative gene, we applied a deletion mapping strategy based on the detection of loss of heterozygosity (LOH) at specific genomic loci in tumour cells. 13 polymorphic markers were used to screen DNA samples from 20 children with ALL. LOHs at 12p12.3 were observed in almost 50% of informative B-cell precursor ALL patients analysed. This is one of the most frequent genetic alterations found in this disease. A common region of LOH was delimited by the markers D12S89 (distal) and D12S358 (proximal), separated by a genetic interval of approximately 3 cM. We refined the position of the putative 12p tumour suppressor gene to a physical interval of <1.3 Mb, a crucial step towards the identification of candidate genes. A yeast artificial chromosome clone contig that spans the entire critically deleted region includes two known genes: TEL, a member of the ets family of transcription factors, and p27KIP1, a cyclin-dependent kinase inhibitor.