Mutational analysis of the tumour suppressor gene MMAC1/PTEN in malignant myeloid disorders

The candidate tumour suppressor gene MMAC1/PTEN located at chromosome 10q23.3 has been reported to be frequently mutated in a number of solid tumours. Less is known about its status in leukaemia. In the present study we first analysed 13 leukaemia cell lines for mutations and homozygous deletions in MMAC1/PTEN using PCR and denaturing gradient gel electrophoresis (DGGE). We identified an intragenic deletion including MMAC1/PTEN exons 2-5 in an acute myelocytic leukaemia cell line, HL-60 blast, and an insertion of four nucleotides in exon 5 in an acute monocytic leukaemia cell line, U937. Analysis of 59 patients with acute myeloid leukaemia (AML), 26 patients with myelodysplastic syndromes (MDS) and 10 patients with chronic myeloid leukaemia (CML) only revealed a polymorphic base substitution in codon 44 in one AML patient, suggesting that mutations in the MMAC1/PTEN gene are infrequent genetic aberrations in myeloid leukaemia.     

Polymerase chain reaction-based diagnosis of del (5q) in acute myeloid leukemia and myelodysplastic syndrome identifies a minimal deletion interval

Loss of all or part of the long arm of human chromosome 5 is a recurrent abnormality in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), especially after chemotherapy for a prior malignancy. It is one of the worst prognostic indicators in AML, associated with chemotherapy resistance and short survival. These deletions center at band 5q31, which has thus been proposed as the location of a tumor suppressor gene; this site is to be distinguished from that observed in 5q- syndrome, centering at 5q32. To define the molecular extent and the clinical prevalence of 5q31 deletions, we collected a series of AML and MDS cases of mixed karyotype, taking care to exclude MDS cases with 5q- syndrome. The samples were analyzed for loss of heterozygosity (LOH) using a panel polymerase chain reaction (PCR)-based microsatellite markers from 5q, comparing malignant cells with normal tissue derived from lymphoblastoid cell lines or buccal mucosa scrapings. Losses were detected in seven of 29 matched samples, including four of 17 with MDS, and three of 12 with AML; six of these seven also had a cytogenetically-visible del(5q) or -5. The one case without a cytogenetic deletion showed molecular loss of three contiguous markers, with retention of flanking markers interleukin-9 (IL-9) and D5S414, and thus contained a small deletion that is below cytogenetic resolution. PCR failed to detect 5q loss in two cases with large cytogenetic deletions, but both had been treated and contained low percentages of malignant cells in the samples. This study thus led to the identification of a case with a minimal deletion for the 5q31 tumor suppressor gene, specified by IL-9-D5S414, that is approximately 1 Mb (2 cM) in extent. Additionally, we demonstrate that PCR-based allelotyping is a reliable method for the detection of chromosomal deletion in myeloid malignancy, providing the specimens contain a high proportion of malignant cells. These studies will help to identify the tumor-suppressor gene at 5q31, and will help to develop molecular methods for diagnosis and monitoring of these disorders.     

Genomic structure of the PIK3CG gene on chromosome band 7q22 and evaluation as a candidate myeloid tumor suppressor.

PIK3CG, which encodes the catalytic subunit p110 gamma of phosphoinositide 3-OH-kinase-gamma (PI3K gamma), has been assigned to chromosome band 7q22, a region that is frequently deleted in myeloid malignancies. PI3K gamma-mutant mice have hematologic defects and are predisposed to colon cancer. On the basis of these data, PIK3CG was evaluated as a candidate myeloid tumor suppressor gene (TSG). PIK3CG was mapped by fluorescence in situ hybridization adjacent and telomeric to a commonly deleted segment defined previously in myeloid leukemias with breakpoints within 7q22. PIK3CG contains 10 exons and spans approximately 37 kilobases of genomic DNA. Forty leukemias with monosomy 7 or a del(7q) were screened for PIK3CG mutations. Two patients had missense variations affecting residue 859 in the N-terminal catalytic domain of the protein. This allele was also detected in unaffected parents and in 1 of 60 control alleles; it probably represents a polymorphism. PIK3CG is unlikely to act as a recessive TSG in myeloid leukemias with monosomy 7.     

L3MBTL1 polycomb protein, a candidate tumor suppressor in del(20q12) myeloid disorders, is essential for genome stability

The l3mbtl1 gene is located on the long arm of chromosome 20 (q12), within a region commonly deleted in several myeloid malignancies. L3MBTL1 is a human homolog of the Drosophila polycomb L(3)MBT tumor suppressor protein and thus a candidate tumor suppressor in del(20q12) myeloid disorders. We used the loss-of-function approach to explore the possible tumor suppressive mechanism of L3MBTL1 and found that depletion of L3MBTL1 from human cells causes replicative stress, DNA breaks, activation of the DNA damage response, and genomic instability. L3MBTL1 interacts with Cdc45, MCM2-7 and PCNA, components of the DNA replication machinery, and is required for normal replication fork progression, suggesting that L3MBTL1 causes DNA damage, at least in part, by perturbing DNA replication. An activated DNA damage response and genomic instability are common features in tumorigenesis and a consequence of overexpression of many oncogenes. We propose that the loss of L3MBTL1 contributes to the development of 20q(-) hematopoietic malignancies by inducing replicative stress, DNA damage, and genomic instability.     

Chromosomal gains and losses indicate oncogene and tumor suppressor gene candidates in salivary gland tumors

The incidence of salivary gland tumor in Poland is growing in the last two decades. Simultaneously a progress in understanding the genetic mechanisms of formation of this tumor was achieved by detecting several genes like PLAG1 involved in its pathogenesis. In this study we perform a whole genome, CGH analysis with the aim to identify recurrent, chromosomal copy number changes possibly indicating novel tumor suppressor gene or oncogene loci. 29 salivary tumor samples: Cystadenolymphoma-warthin (15) and adenoma polymorphum (14) located in the parotid (27) and submandibular gland (2) were collected and CGH was performed. The established copy number profiles were compared in order to asses the smallest common region of gains and losses. The delineated regions were further analyzed with the UCSC Genome Browser on Human Mar. 2006 Assembly to asses their gene content. Altogether, salivary gland tumors presented a different aberration pattern than these reported for head and neck squamous cell carcinoma (HNSCC) but no significant differences were observed between Warthin and adenoma polymorphum tumors. Moreover, several potential tumor suppressor genes and oncogenes were identified in the smallest, common altered regions. We show a frequent deletion of the harakiri gene (12q24.2) in 12/29 tumors and TP53 gene (17p13.1) in 11/29 tumors as potential tumor suppressors in salivary gland cancers. Besides, we detected a frequent amplification of the 13q22.1-22.2 region in 13/29 cases harboring the KLF5 and KLF12 genes. KLF5 regulates the expression of survivin, an oncogene widely expressed in the majority of human cancers. The observed alterations may indicate important genetic events in the formation of salivary gland tumors. Especially the amplification in 13q may be a mechanism contributing to the expression of survivin and tumor progression.     

The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q

We have isolated the human GRAF gene (for GTPase regulator associated with the focal adhesion kinase pp125(FAK)). This gene was fused with MLL in a unique t(5;11)(q31;q23) that occurred in an infant with juvenile myelomonocytic leukemia. GRAF encodes a member of the Rho family of the GTPase-activating protein (GAP) family. On the protein level, it is 90% homologous to the recently described chicken GRAF gene that functions as a GAP of RhoA in vivo and is thus a critical component of the integrin signaling transduction pathway. The particular position of the human GRAF gene at 5q31 and the proposed antiproliferative and tumor suppressor properties of its avian homologue suggest that it also might be pathogenetically relevant for hematologic malignancies with deletions of 5q. To investigate this possibility, we sequenced 4-5 individual cDNA clones from 13 cases in which one allele of GRAF was deleted. We found point mutations within the GAP domain of the second GRAF allele in one patient. In two additional patients we found an insertion of 52 or 74 bp within the GRAF cDNA that generates a reading frame shift followed by a premature stop codon. GRAF maps outside the previously defined commonly deleted 5q31 region. Nevertheless, inactivation of both alleles in at least some cases suggests that deletions and mutations of the GRAF gene may be instrumental in the development and progression of hematopoeitic disorders with a del(5q).     

The gene for an inherited form of deafness maps to chromosome 5q31.

Primary--i.e., nonsyndromal-postlingual deafness is inherited as an autosomal dominant phenotype in a large kindred in Costa Rica. Genetically susceptible individuals begin to lose hearing at low frequencies at about age 10 years, after language and speaking are learned. Deafness inevitably progresses by age 30 years to bilateral hearing loss of all frequencies. Intelligence, fertility, and life expectancy are normal. The family traces its ancestry to an affected founder born in Costa Rica in 1754. We have mapped the gene for deafness in this kindred to chromosome 5q31, between the markers IL9 and GRL, by linkage analysis involving 99 informative relatives.     

Parkin,a gene implicated in autosomal recessive juvenile parkinsonism,is a candidate tumor suppressor gene on chromosome 6q25-q27

In an effort to identify tumor suppressor gene(s) associated with the frequent loss of heterozygosity observed on chromosome 6q25-q27, we constructed a contig derived from the sequences of bacterial artificial chromosomeP1 bacteriophage artificial chromosome clones defined by the genetic interval D6S1581-D6S1579-D6S305-D6S1599-D6S1008. Sequence analysis of this contig found it to contain eight known genes, including the complete genomic structure of the Parkin gene. Loss of heterozygosity (LOH) analysis of 40 malignant breast and ovarian tumors identified a common minimal region of loss, including the markers D6S305 (50%) and D6S1599 (32%). Both loci exhibited the highest frequencies of LOH in this study and are each located within the Parkin genomic structure. Whereas mutation analysis revealed no missense substitutions, expression of the Parkin gene appeared to be down-regulated or absent in the tumor biopsies and tumor cell lines examined. In addition, the identification of two truncating deletions in 3 of 20 ovarian tumor samples, as well as homozygous deletion of exon 2 in the lung adenocarcinoma cell lines Calu-3 and H-1573, supports the hypothesis that hemizygous or homozygous deletions are responsible for the abnormal expression of Parkin in these samples. These data suggest that the LOH observed at chromosome 6q25-q26 may contribute to the initiation andor progression of cancer by inactivating or reducing the expression of the Parkin gene. Because Parkin maps to FRA6E, one of the most active common fragile sites in the human genome, it represents another example of a large tumor suppressor gene, like FHIT and WWOX, located at a common fragile site.     

The interleukin 3 gene is located on human chromosome 5 and is deleted in myeloid leukemias with a deletion of 5q.

The gene IL-3 encodes interleukin 3, a hematopoietic colony-stimulating factor (CSF) that is capable of supporting the proliferation of a broad range of hematopoietic cell types. By using somatic cell hybrids and in situ chromosomal hybridization, we localized this gene to human chromosome 5 at bands q23-31, a chromosomal region that is frequently deleted [del(5q)] in patients with myeloid disorders. By in situ hybridization, IL-3 was found to be deleted in the 5q-chromosome of one patient with refractory anemia who had a del(5)(q15q33.3), of three patients with refractory anemia (two patients) or acute nonlymphocytic leukemia (ANLL) de novo who had a similar distal breakpoint [del(5)(q13q33.3)], and of a fifth patient, with therapy-related ANLL, who had a similar distal breakpoint in band q33 [del(5)(q14q33.3)]. Southern blot analysis of somatic cell hybrids retaining the normal or the deleted chromosome 5 from two patients with the refractory anemia 5q- syndrome indicated that IL-3 sequences were absent form the hybrids retaining the deleted chromosome 5 but not from hybrids that had a cytologically normal chromosome 5. Thus, a small segment of chromosome 5 contains IL-3, GM-CSF (the gene encoding granulocyte-macrophage-CSF), CSF-1 (the gene encoding macrophage-CSF), and FMS (the human c-fms protooncogene, which encodes the CSF-1 receptor). Our findings and earlier results indicating that GM-CSF, CSF-1, and FMS were deleted in the 5q-chromosome, suggest that loss of IL-3 or of other CSF genes may play an important role in the pathogenesis of hematologic disorders associated with a del(5q).     

4q loss is potentially an important genetic event in MM tumorigenesis: identification of a tumor suppressor gene regulated by promoter methylation at 4q13.3, platelet factor 4

In this study, we have elucidated the chromosomal imbalances in the multistep pathogenesis and delineated several critical tumor suppressor gene (TSG) loci in multiple myeloma (MM). By using comparative genomic hybridization, allelotyping, and multicolor interphase fluorescence in situ hybridization, 5 MM cell lines and bone marrow CD138+ plasma cells from 88 Chinese patients with monoclonal gammopathy of undetermined significance (MGUS) and early and advanced stages of MM were investigated. In all MGUS and MM samples, chromosome copy number abnormalities were detected. A higher number of chromosomal imbalances and specific genetic alterations are involved in MGUS to MM transition (-6q, +3p, and +1p) and MM progression (+2p and +9q). In addition to -13q, we first found high frequencies (42% to 46%) of -4q involving high percentages (70% to 74%) of clonal plasma cells in both MGUS and MM, suggesting that inactivation of TSG in this region is also a potentially critical genetic event in MM tumorigenesis. By high-resolution allelotyping, we defined a common deletion region on 4q13.3 and found that a candidate TSG, platelet factor 4, was frequently silenced by promoter hypermethylation in MM (15 of 28) and MM cell lines (5 of 5). These data have opened up a new approach in the molecular targeting therapy and provide novel insights into MM tumorigenesis.     

Positional identification of an asthma susceptibility gene on human chromosome 5q33

RATIONALE: Asthma is a common respiratory disease with complex genetic components. We previously reported strong evidence for linkage between mite-sensitive asthma and markers on chromosome 5q33. This area of linkage includes a region homologous to a mouse area that contains a locus involved in regulation of airway hyperreactivity. OBJECTIVE: The aim of the present study is to identify asthma susceptibility genes on chromosome 5q33. METHODS AND RESULTS: We performed mutation screening and association analyses of genes in the 9.4-Mb human linkage region. Transmission disequilibrium test analysis of 105 polymorphisms in 155 families with asthma revealed that six polymorphisms in cytoplasmic fragile X mental retardation protein (FMRP)-interacting protein 2 gene were associated significantly with the development of asthma (p = 0.000075; odds ratio, 5.9). These six polymorphisms were in complete linkage disequilibrium. In real-time quantitative polymerase chain reaction analysis, subjects homozygous for the haplotype overtransmitted to asthma-affected offspring showed significantly increased level of cytoplasmic FMRP interacting protein 2 gene expression in lymphocytes compared with ones heterozygous for the haplotype (p = 0.038). CONCLUSIONS: Our data suggest that cytoplasmic FMRP interacting protein 2 are associated with the development of atopic asthma in humans, and that targeting cytoplasmic FMRP interacting protein 2 could be a novel strategy for treating atopic asthma.     

Loss of heterozygosity on 10q23.3 and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions

AIM: To investigate the loss of heterozygosity (LOH) and mutation of tumor suppressor gene PTEN in gastric cancer and precancerous lesions. METHODS: Thirty cases of normal gastric mucosa, advanced and early stage gastric cancer, intestinal metaplasia, atrophic gastritis, and atypical hyperplasia were analyzed for PTEN LOH and mutations within the entire coding region of PTEN gene by PCR-SSCP denaturing PAGE gel electrophoresis, and PTEN mutation was detected by PCR-SSCP sequencing followed by silver staining. RESULTS: LOH rate found in respectively atrophic gastritis was 10% (3/30), intestinal metaplasia 10% (3/30), atypical hyperplasia 13.3% (4/30), early stage gastric cancer 20% (6/30), and advanced stage gastric cancer 33.3% (9/30), None of the precancerous lesions and early stage gastric cancer showed PTEN mutations, but 10% (3/30) of the advanced stage gastric cancers, which were all positive for LOH, showed PTEN mutation. CONCLUSION: LOH of PTEN gene appears in precancerous lesions, and PTEN mutations are restricted to advanced gastric cancer, LOH and mutation of PTEN gene are closely related to the infiltration and metastasis of gastric cancer.     

Identification of GLIPR1 tumor suppressor as methylation-silenced gene in acute myeloid leukemia by microarray analysis

Purpose

To identify methylation-silenced genes in acute myeloid leukemia (AML).

Methods

Microarray analyses were performed in AML cell line HL-60 cells exposed to the demethylating agent 5-aza-2dC. The methylation status and expression of glioma pathogenesis-related protein 1 (GLIPR1), one of highly induced genes by demethylation, were further detected in six hematopoietic malignancy cell lines and 260 bone marrow samples from leukemia patients and nonmalignant diseases as control, as well as pre-treated and post-treated bone marrow samples from 24 complete remission AML patients received chemotherapy using MS-PCR, bisulfite DNA sequencing, RT-PCR, and Western blotting.

Results

One hundred and nine genes were significantly induced by demethylation in HL-60 cells, 12 genes of which were confirmed by RT-PCR. GLIPR1, a tumor suppressor gene, was frequently methylation-silenced in AML cell lines and AML patients, but not in the other hematopoietic malignancy cell lines and patients. The frequencies of methylation-silenced GLIPR1 in the pre-treatment were significantly higher than those in the post-treatment in complete remission AML patients.

Conclusion

We identify 109 genes induced by demethylation in HL-60 cells, and demonstrate that GLIPR1 is a methylation-silenced gene in the AML patients, and may serve as a marker for monitoring disease activity during therapy in the AML patients. The data provide the important information for studying the pathogenesis of AML and discovering the target genes of methylating agents.