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.     

Lack of ETV6 (TEL) gene rearrangements or p16INK4A/p15INK4B homozygous gene deletions in infant acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) occurring in infants less than 1 year of age differs clinically and biologically from that observed in older children. Cytogenetically, 11q23 translocations are detected in approximately 50% of infant ALLs and fuse the 11q23 gene HRX with a variety of partner chromosomal loci. Overall, HRX rearrangements are detected molecularly in 70-80% of infant ALLs as compared to 5-7% of ALLs arising in older children. Two recently described molecular abnormalities in childhood ALL are ETV6 gene rearrangements and homozygous deletions of p16(INK4A) and/or p15(INK4B). Each of these abnormalities occurs in 15-20% of all childhood ALLs, and neither can be accurately identified by routine cytogenetic analyses. The incidence of these genetic abnormalities and their potential relationship to HRX gene status in infant ALL is unknown. Using Southern blot analyses, we determined ETV6 and p16(INK4A)/p15(INK4B) gene status in a cohort of infant ALLs. No ETV6 rearrangements or homozygous deletions (n=69) or homozygous p16(INK4A) and/or p15(INK4B) gene deletions (n=54) were detected in any of the infant ALLs. Therefore, ETV6 and p16(INK4A)/p15(INK4B) do not play a significant role in the pathogenesis of infant ALL, further emphasizing the distinctive biology of this subset of leukemias.     

Frequent loss of heterozygosity and three critical regions on the short arm of chromosome 8 in ovarian adenocarcinomas

BACKGROUND: Familial arteriolar tortuosity is an autosomal dominant disorder affecting the retinal arterioles. OBJECTIVES: To report a pedigree with this disorder and describe a systemic workup to determine whether this vascular abnormality is limited to the eye. RESULTS: A 58-year-old woman referred for retinal hemorrhages was found to have retinal arteriolar tortuosity of both eyes, especially in the macular area. Her 63-year-old brother had a history of retinal hemmorhages beginning at age 18 years and had similar fundoscopic examination findings. The proband had an extensive systemic workup, including magnetic resonance imaging, and cardiac and renal angiography, that failed to demonstrate any other sequelae of this inherited ocular syndrome. However, each member of the family expressing this phenotype did have hypertension. CONCLUSION: Inherited retinal arteriolar tortuosity is an autosomal dominant disorder limited to the eye, at least in this pedigree, within the sensitivity of the systemic workup we used.     

Exon 5 mutations in the p53 gene in relapsed childhood acute lymphoblastic leukemia

Thirty seven children with relapsed acute lymphoblastic leukemia (ALL), 25 B-lineage and 12 T-lineage, were analyzed for p53 alterations at different stages of the disease. Loss of heterozygosity (LOH) was detected in the relapse phase in three patients. p53 mutations were identified by single strand conformation polymorphism (SSCP) and sequencing analyzes in seven of the 37 ALL patients (19%); three B-lineage (12%) and four T-lineage (33%). Most of the mutations were identified in the relapse phase. In two exceptional cases, one of the mutations was indicated as a germ line and the other was already present at diagnosis. No p53 mutation was identified in any of the other 20 available bone marrow samples obtained at diagnosis. No correlation between the p53 status and clinical outcome could be determined. The majority of the mutations (four out of seven, 57%) were clustered at exon 5. Our data implicate that p53 exon 5 is a frequent site of mutations in relapsed childhood ALL.     

Sensitive non-radioactive PCR detection of clonal T-cell receptor-gamma gene rearrangements in childhood acute lymphoblastic leukaemia

The aim of the present study was to investigate some putative neurotransmitters involved in nociception and pain in parturients during active labour experiencing intense visceral pain. The concentration of the excitatory amino acid aspartate was significantly increased, and there was a tendency for an increase in glutamate, in lumbar cerebrospinal fluid (CSF) of parturients in active vaginal labour compared with control patients without pain subjected to elective caesarean section. The CSF concentration of the nitric oxide breakdown product nitrate was significantly decreased in parturients compared with control patients and healthy volunteers. No significant differences in the concentrations of substance P, substance P-endopeptidase or met-enkephalin were detected between parturients and controls. Our data suggest a paradoxical negative relationship between CSF concentrations of excitatory amino acids and nitric oxide in labour pain. The mechanisms behind this finding is unclear at present.     

Hypermethylation of p15/ink4b/MTS2 gene is differentially implicated among non-Hodgkin''s lymphomas

Germinated barley foodstuff (GBF), derived from the aleurone layer, scutellum and germ of germinated barley, contains a large quantity of fermentable dietary fibers, especially hemicellulose. Ten grams of GBF were given to 10 healthy volunteers 3 times a day (30 g/day/person) for 28 consecutive days. Fecal weight, water contents and short chain fatty acid content were measured before GBF administration and from days 25 to 28 after initiation of GBF administration. GBF intake significantly increased fecal butyrate content as well as fecal weight and water content. No significant change in body weight resulted from consumption of GBF for 28 days. No major laboratory abnormalities were found in hematologic and urinary analysis. These findings indicate that GBF promotes defecation, produces bacterial short chain fatty acids, especially butyrate, without adverse effects, and is a safe foodstuff for humans.     

Analysis of loss of heterozygosity on chromosome 11 and infrequent inactivation of the MEN1 gene in sporadic pituitary adenomas

To investigate the role of tumor suppressor genes in sporadic pituitary adenomas, we first analyzed loss of heterozygosity on 11q13 with microsatellite analysis in 31 tumors. Loss of heterozygosity on 11q13 was detected in 1 mixed GH/PRL adenoma, and the somatic 22-bp deletion of the multiple endocrine neoplasia type 1 (MEN1) gene encoding menin was detected in this tumor. Trisomy 11 suggested by the decreased mean allelic ratios of 66% or 65% for 16 or 13 microsatellite markers, respectively, in 2 of 31 pituitary adenomas was confirmed by interphase fluorescence in situ hybridization. Screening for mutations of the MEN1 gene did not find mutations with PCR-single strand conformation polymorphism analysis in other pituitary adenomas retaining heterozygosity on 11q13. Based on these, it is concluded that inactivation of the MEN1 gene comprises a rare etiology for tumorigenesis of the pituitary gland, and that trisomy 11 or another gene(s) may contribute to the pathogenesis of sporadic pituitary adenomas.     

Inactivation of the p15INK4B and p16INK4 genes in hematologic malignancies

The recently discovered p15INK4B and p16INK4 genes encoding cell cycle regulating proteins, map to a region on chromosome 9p21 that is commonly deleted in a variety of malignant diseases. The p16INK4 gene has now been shown to be a tumor suppressor gene. It is frequently inactivated in cancer and is possibly the second most often mutated gene in human malignant disease after p53. The role of the p15INK4B and p16INK4 genes in hematologic malignancies has been the subject of intense investigation since their discovery. In this review we address the function and possible role in tumorigenesis of the p15INK4B and p16INK4 genes and discuss their significance as prognostic markers in hematologic malignancies.     

Alterations of p16 and p15 genes in acute leukemia with MLL gene rearrangements and their correlation with clinical features

p16 and p15 genes are putative tumor suppressor genes located on chromosome 9p21. In acute leukemias, alterations of p16 and p15 genes have been reported to occur exclusively in lymphoid lineage. We analyzed alterations of p16 and p15 genes in 46 acute leukemias with MLL gene rearrangements by Southern blot analysis, and investigated the association with clinical characteristics. We identified homozygous deletion of p16 and p15 genes in five (19%) of 27 acute lymphoblastic leukemias (ALLs) and in two (11%) of 19 acute myeloid leukemias (AMLs). Patients with homozygous deletion of p16 and p15 genes showed higher average leukocyte counts (343 x 10(9)/l vs 271 x 10(9)/l) and lower estimated 2-year survival rates than those with normal p16 and p15 genes (14.3 vs 30.7%), although the differences were not statistically significant. In addition, we investigated mutation of p16 gene by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) in 31 patients, but no mutation was found in the patients tested. Our results suggest that alterations of p16 and p15 genes are involved in a subset of acute leukemias with MLL gene rearrangement not only of lymphoid but also of myeloid phenotype. Homozygous deletion of p16 and p15 genes may be a possible adverse prognostic factor, although further analysis would be needed to confirm it.     

p16/INK4a and p15/INK4b gene methylation and absence of p16/INK4a mRNA and protein expression in Burkitt''s lymphoma

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the Aquifex-Hydrogenobacter complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the Aquifex-Hydrogenobacter complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.     

Deletions of the short arm of chromosome 9, including the interferon-alpha/-beta genes, in acute lymphocytic leukemia. Studies on loss of heterozygosity, parental origin of deleted genes and prognosis

A restriction fragment length polymorphism (RFLP) analysis of the alpha- and beta-interferon (IFN) genes was performed in malignant cells from 52 patients with acute lymphocytic leukemia (ALL). Normal cell DNA was available for comparison in 23 of the patients. Ten patients were found to have gross alterations of their alpha- and beta-IFN genes. Leukemic cells from 2 ALL patients showed a complete loss of alpha- and beta-IFN genes. Seven patients had a hemizygous loss of one of the alpha- and beta-IFN alleles, as shown by RFLP analysis and/or loss of signal intensity. In one other patient the malignant clone was reduced to homozygosity with regard to the alpha- and beta-IFN genes, without loss of signal intensity. In patients without hemizygous deletions, the overall incidence of complete homozygosity for the alpha- and beta polymorphisms was higher than expected. Analysis of the data indicates that the total frequency of ALL clones with gross alterations of the IFN-loci is around 30%. A 9p24 probe detected hemizygous deletions in 2 cases of IFN gene deletions. In the other tested cases the deletions were interstitial. No deletions of 9p24 were detected in patients without allelic losses of IFN genes. In 5 cases of allelic IFN gene deletions, DNA from parents was available for comparison. In 4 cases the deleted allele was derived from the mother, whereas in the fifth it originated from the father. Pediatric ALL patients with IFN-gene deletions or homozygosity for all polymorphisms in the IFN-loci had a significantly worse prognosis than heterozygotes. We conclude that deletion of alpha- and beta-IFN genes is a relatively common event in ALL and that RFLP analysis of the IFN genes may provide additional prognostic information in childhood ALL. Whether or not the IFNs act as tumor-suppressor genes in this disease is not yet known.     

Hypermethylation of the p15INK4B gene in myelodysplastic syndromes

Previous studies have shown that the cyclin-dependent kinase inhibitor (CDKI) genes p15INK4B and p16INK4A are frequently inactivated by genetic alterations in many malignant tumors and that they are candidate tumor-suppressor genes. Although genetic alterations in these genes may be limited to lymphoid malignancies, it has been reported that their inactivation by aberrant methylation of 5' CpG islands may be involved in various hematologic malignancies. In this study, we investigated the p15INK4B and p16INK4A genes to clarify their roles in the pathogenesis of myelodysplastic syndrome (MDS). Southern blotting analysis showed no gross genetic alterations in either of these genes. However, hypermethylation of the 5' CpG island of the p15INK4B gene occurred frequently in patients with MDS (16/32 [50%]). Interestingly, the p15INK4B gene was frequently methylated in patients with high-risk MDS (refractory anemia with excess blasts [RAEB], RAEB in transformation [RAEB-t], and overt leukemia evolved from MDS; 14/18 [78%]) compared with patients with low-risk MDS (refractory anemia [RA] and refractory anemia with ring sideroblast [RARS]; 1/12 [8%]). Furthermore, methylation status of the p15INK4B gene was progressed with the development of MDS in most patients examined. In contrast, none of the MDS patients showed apparent hypermethylation of the p16INK4A gene. These results suggest that hypermethylation of the p15INK4B gene is involved in the pathogenesis of MDS and is one of the important late events during the development of MDS.     

Loss of heterozygosity on the short arm of chromosome 3 in cervical intra-epithelial neoplasia without concomitant cervical carcinoma

OBJECTIVE: To study magnetic resonance (MR) imaging pattern of normal hyaline articular cartilage in the knee joint with regard to the contribution of the "magic angle" effect to the MR signal. DESIGN: Thirty-two healthy volunteers were imaged in a standard supine position in a 1.5-T unit using spin echo and gradient echo sequences. Nine volunteers were reimaged with the knee flexed. The signal behavior of the hyaline cartilage of the femoral condyles was evaluated qualitatively and quantitatively. The extended and flexed positions of the nine volunteers were compared. RESULTS: A superficial and a deep hyperintense layer and a hypointense middle cartilage layer were observed. Segments of increased signal intensity were visible along the condyles; a magic angle effect on signal intensity was evident in the hypointense middle layer with both gradient echo and spin echo images. CONCLUSION: The MR signal behavior of hyaline cartilage is influenced by the alignment of the collagen fibers within the cartilage in relation to the magnetic field. Failure to recognize this effect may lead to inaccurate diagnosis.     

Allelic loss of the FMS gene in acute myeloid leukaemia

The FMS proto-oncogene encodes for the colony stimulating factor-1 receptor expressed on monocytes and B lymphocytes within the peripheral blood system. Allelic loss of the FMS gene occurs in patients with refractory anaemia and the 5q- syndrome associated with the myelodysplastic syndromes. To determine the frequency of FMS gene loss in patients with myeloid malignancy, 50 DNA samples from patients with acute myeloid leukaemia (AML) and 30 samples from haematologically normal samples were analysed using a quantitative Southern blotting technique. Allelic loss of one allele (hemizygous) was detected in five of 18 samples of AM-M4 and eight of 27 samples of AML M1, M2 and M3. In addition, loss of both FMS alleles (homozygous) was demonstrated in three of 18 samples of AML M4 and 0127 samples of AML M1, M2 and M3. One patient with AML M5 and one with AML M6 were assessed although no allelic loss of FMS was detected. Three samples from patients with secondary AML were also analysed and hemizygous loss was detected in one case. Homozygous or hemizygous loss of FMS was not detected in any of 30 DNA samples isolated from haematologically normal individuals. These data indicate that loss of the FMS gene is common in AML, with an increased frequency in those patients with AML subtype M4.     

Mutation analysis of CD95 (APO-1/Fas) in childhood B-lineage acute lymphoblastic leukaemia

The delta-type phospholipase C (PLC) is thought to be evolutionally the most basal form in the mammalian PLC family. One of the delta-type isoforms, PLC-delta 1, binds to both phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) with a high affinity via its pleckstrin homology (PH) domain. We report here a missense mutation in the region encoding the C-terminal PH domain of the human PLC-delta 1. This is also the first report of a mutation in the human PLC genes. A single base substitution (G to A) causes the amino acid replacement, Arg105 to His. Site-directed mutagenesis of the glutathione-S-transferase (GST)/PLC-delta 1 fusion protein changing Arg105 to His resulted in a fourfold decrease in the affinity of specific Ins(1,4,5)P3 binding and a reduction in PtdIns(4,5)P2 hydrolysing activity to about 40% of that of the wild-type enzyme. This remarkable loss of function can be interpreted in terms of a conformational change in the PH domain.