Stephan M. Tanner

Downregulation of Death-Associated Protein Kinase 1 (DAPK1) in Chronic Lymphocytic Leukemia

Summary The heritability of B cell chronic lymphocytic leukemia (CLL) is relatively high; however, no predisposing mutation has been convincingly identified. We show that loss or reduced expression of death-associated protein kinase 1 (DAPK1) underlies cases of heritable predisposition to CLL and the majority of sporadic CLL. Epigenetic silencing of DAPK1 by promoter methylation occurs in almost all sporadic CLL cases. Furthermore, we defined a disease haplotype, which segregates with the CLL phenotype in a large family. DAPK1 expression of the CLL allele is downregulated by 75% in germline cells due to increased HOXB7 binding. In the blood cells from affected family members, promoter methylation results in additional loss of DAPK1 expression. Thus, reduced expression of DAPK1 can result from germline predisposition, as well as epigenetic or somatic events causing or contributing to the CLL phenotype.

MTM1 mutations in X-linked myotubular myopathy

X‐linked myotubular myopathy (XLMTM; MIM# 310400) is a severe congenital muscle disorder caused by mutations in the MTM1 gene. This gene encodes a dual‐specificity phosphatase named myotubularin, defining a large gene family highly conserved through evolution (which includes the putative anti‐phosphatase Sbf1/hMTMR5). We report 29 mutations in novel cases, including 16 mutations not described before. To date, 198 mutations have been identified in unrelated families, accounting for 133 different disease‐associated mutations which are widespread throughout the gene. Most point mutations are truncating, but 26% (35/133) are missense mutations affecting residues conserved in the Drosophila ortholog and in the homologous MTMR1 gene. Three recurrent mutations affect 17% of the patients, and a total of 21 different mutations were found in several independent families. The frequency of female carriers appears higher than expected (only 17% are de novo mutations). While most truncating mutations cause the severe and early lethal phenotype, some missense mutations are associated with milder forms and prolonged survival (up to 54 years). Hum Mutat 15:393–409, 2000.

BAALC, the human member of a novel mammalian neuroectoderm gene lineage, is implicated in hematopoiesis and acute leukemia

The molecular basis of human leukemia is heterogeneous. Cytogenetic findings are increasingly associated with molecular abnormalities, some of which are being understood at the functional level. Specific therapies can be developed based on such knowledge. To search for new genes in the acute leukemias, we performed a representational difference analysis. We describe a human gene in chromosome 8q22.3, BAALC (brain and acute leukemia, cytoplasmic), that is highly conserved among mammals but evidently absent from lower organisms. We characterized BAALC on the genomic level and investigated its expression pattern in human and mouse, as well as its complex splicing behavior. In vitro studies of the protein showing its subcellular localization suggest a function in the cytoskeleton network. Two isoforms are specifically expressed in neuroectoderm-derived tissues, but not in tumors or cancer cell lines of nonneural tissue origin. We show that blasts from a subset of patients with acute leukemia greatly overexpress eight different BAALC transcripts, resulting in five protein isoforms. Among patients with acute myeloid leukemia, those overexpressing BAALC show distinctly poor prognosis, pointing to a key role of the BAALC products in leukemia. Our data suggest that BAALC is a gene implicated in both neuroectodermal and hematopoietic cell functions.

Amnionless, essential for mouse gastrulation, is mutated in recessive hereditary megaloblastic anemia

The amnionless gene, Amn, on mouse chromosome 12 encodes a type I transmembrane protein that is expressed in the extraembryonic visceral layer during gastrulation. Mice homozygous with respect to the amn mutation generated by a transgene insertion have no amnion. The embryos are severely compromised, surviving to the tenth day of gestation but seem to lack the mesodermal layers that normally produce the trunk. The Amn protein has one transmembrane domain separating a larger, N-terminal extracellular region and a smaller, C-terminal cytoplasmic region. The extracellular region harbors a cysteine-rich domain resembling those occurring in Chordin, found in Xenopus laevis embryos, and Sog, found in Drosophila melanogaster. As these cysteine-rich domains bind bone morphogenetic proteins (Bmps), it has been speculated that the cysteine-rich domain in Amn also binds Bmps. We show that homozygous mutations affecting exons 1–4 of human AMN lead to selective malabsorption of vitamin B12 (a phenotype associated with megaloblastic anemia 1, MGA1; OMIM 261100; refs. 5,6) in otherwise normal individuals, suggesting that the 5′ end of AMN is dispensable for embryonic development but necessary for absorption of vitamin B12. When the 5′ end of AMN is truncated by mutations, translation is initiated from alternative downstream start codons.

Germline Allele-specific Expression of TGFBR1 Confers an Increased Risk of Colorectal Cancer

Much of the genetic predisposition to colorectal cancer (CRC) in humans is unexplained. Studying a Caucasian-dominated population in the United States, we showed that germline allele-specific expression (ASE) of the gene encoding transforming growth factor–β (TGF-β) type I receptor, TGFBR1, is a quantitative trait that occurs in 10 to 20% of CRC patients and 1 to 3% of controls. ASE results in reduced expression of the gene, is dominantly inherited, segregates in families, and occurs in sporadic CRC cases. Although subtle, the reduction in constitutive TGFBR1 expression alters SMAD-mediated TGF-β signaling. Two major TGFBR1 haplotypes are predominant among ASE cases, which suggests ancestral mutations, but causative germline changes have not been identified. Conservative estimates suggest that ASE confers a substantially increased risk of CRC (odds ratio, 8.7; 95% confidence interval, 2.6 to 29.1), but these estimates require confirmation and will probably show ethnic differences.

Aberrant promoter methylation of previously unidentified target genes is a common abnormality in medulloblastomas–Implications for tumor biology and potential clinical utility

Medulloblastomas exhibit an array of diverse cytogenetic abnormalities. To evaluate the significance of epigenetic rather than genetic lesions in medulloblastomas and other primitive neuroectodermal tumors (PNETs) of the childhood CNS we performed a systematic analysis of gene specific and global methylation. Methylation-specific PCR detected no methylation for p15INK4B, von Hippel Lindau and TP53 and only limited methylation for E-Cadherin and p16INK4A in tumors. The cell lines Daoy and MHH-PNET-5 in which the p16INK4A promoter was methylated did not express the gene, but demonstrated abnormalities by SSCP. Immunohistochemistry for p16 was negative in all examined normal cerebella and medulloblastomas. Using the technique of Restriction Landmark Genomic Scanning we detected methylation affecting up to 1% of all CpG islands in primary MB/PNETs and 6% in MB cell lines. Methylation patterns differed between medulloblastomas and PNETs. Examination of several methylated sequences revealed homologies to known genes and expressed sequences. Analysis of survival data identified seven of 30 hypermethylated sequences significantly correlating with poor prognosis. We suggest that DNA hypermethylation has an outstanding potential for the identification of novel tumor suppressors as well as diagnostic and therapeutic targets in MBs and other PNETs of the CNS.

RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair

Nuclear factor (NF)‐κB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF‐κB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65−/− murine fibroblasts immortalize at considerably faster rates than RelA/p65+/+ cells. The ability of RelA/p65−/− fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65−/− fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF‐κB. Altogether, our findings present a fresh perspective on the role of NF‐κB as a tumour suppressor, which acts in pre‐neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability.

miR-3151 interplays with its host gene BAALC and independently affects outcome of patients with cytogenetically normal acute myeloid leukemia

High BAALC expression levels are associated with poor outcome in cytogenetically normal acute myeloid leukemia (CN-AML) patients. Recently, miR-3151 was discovered in intron 1 of BAALC. To evaluate the prognostic significance of miR-3151 expression levels and to gain insight into the biologic and prognostic interplay between miR-3151 and its host, miR-3151 and BAALC expression were measured in pretreatment blood of 179 CN-AML patients. Gene-expression profiling and miRNA-expression profiling were performed using microarrays. High miR-3151 expression was associated with shorter disease-free and overall survival, whereas high BAALC expression predicted failure of complete remission and shorter overall survival. Patients exhibiting high expression of both miR-3151 and BAALC had worse outcome than patients expressing low levels of either gene or both genes. In gene-expression profiling, high miR-3151 expressers showed down-regulation of genes involved in transcriptional regulation, posttranslational modification, and cancer pathways. Two genes, FBXL20 and USP40, were validated as direct miR-3151 targets. The results of the present study show that high expression of miR-3151 is an independent prognosticator for poor outcome in CN-AML and affects different outcome end points than its host gene, BAALC. The combination of both markers identified a patient subset with the poorest outcome. This interplay between an intronic miR and its host may have important biologic implications.

Confirmation of prenatal diagnosis results of X-linked recessive myotubular myopathy by mutational screening, and description of three new mutations in the MTM1 gene

X‐linked recessive myotubular myopathy (XLMTM; MTM1) is a severe neonatal disorder often causing perinatal death of the affected males. The responsible gene, designated MTM1, was localized to proximal Xq28 and recently isolated. The characterization of MTM1 allowed us to screen for causing mutations in three families, previously investigated by linkage analysis. Using exon amplification, single strand conformation polymorphism, and subsequent sequencing analysis, three new mutations and their mutational origin were characterized by analyzing 10 exons. An acceptor splice site and a frameshift mutation were correlated with the concurrent appearance of XLMTM in two families. A third intronic mutation was also analyzed by reverse transcription PCR and revealed a cryptic splice site mutation cosegregating with the presumed XLMTM haplotype in the third family. These results further support the implication of the MTM1 gene in XLMTM and allow efficient and reliable carrier and prenatal diagnosis in these families. Direct mutational diagnosis of families at risk in combination with haplotype analysis avoid the drawbacks using only linkage analysis, make genetic counselling far more reliable, and early clinical management of this disease more appropriate. Moreover, pedigree analyses provide first information on de novo mutation frequency in this newly identified human disease gene. Hum Mutat 11:62–68, 1998.

Molecular profiling of chronic lymphocytic leukaemia : genetics meets epigenetics to identify predisposing genes

Molecular profiling may lead to a better understanding of a disease. This knowledge is especially important in malignancies, where multiple alterations are required during the progression from premalignant to malignant stages. Such information can be useful for the development of novel biomarkers that allow the prediction of a clinical course, response to treatment or early detection. Molecular data is also utilized to develop targeted therapies. Moreover, gene defects identified in profiling studies will help to understand the molecular pathways disrupted in the disease. This review provides an overview of molecular profiling approaches in chronic lymphocytic leukaemia (CLL). We will describe our current understanding of genetic alterations in CLL, the use of familial CLL for the identification of predisposing mutations, and the search for epigenetic alterations in CLL.