Disease evolution and outcomes in familial AML with germline CEBPA mutations

In-depth molecular investigation of familial leukemia has been limited by the rarity of recognized cases. This study examines the genetic events initiating leukemia and details the clinical progression of disease across multiple families harboring germ-line CEBPA mutations. Clinical data were collected from 10 CEBPA-mutated families, representing 24 members with acute myeloid leukemia (AML). Whole-exome (WES) and deep sequencing were performed to genetically profile tumors and define patterns of clonal evolution. Germline CEBPA mutations clustered within the N-terminal and were highly penetrant, with AML presenting at a median age of 24.5 years (range, 1.75-46 years). In all diagnostic tumors tested (n = 18), double CEBPA mutations (CEBPAdm) were detected, with acquired (somatic) mutations preferentially targeting the C-terminal. Somatic CEBPA mutations were unstable throughout the disease course, with different mutations identified at recurrence. Deep sequencing of diagnostic and relapse paired samples confirmed that relapse-associated CEBPA mutations were absent at diagnosis, suggesting recurrence was triggered by novel, independent clones. Integrated WES and deep sequencing subsequently revealed an entirely new complement of mutations at relapse, verifying the presentation of a de novo leukemic episode. The cumulative incidence of relapse in familial AML was 56% at 10 years (n = 11), and 3 patients experienced ≥3 disease episodes over a period of 17 to 20 years. Durable responses to secondary therapies were observed, with prolonged median survival after relapse (8 years) and long-term overall survival (10-year overall survival, 67%). Our data reveal that familial CEBPA-mutated AML exhibits a unique model of disease progression, associated with favorable long-term outcomes.

Superorganism resilience: eusociality and susceptibility of ecosystem service providing insects to stressors

Insects provide crucial ecosystem services for human food security and maintenance of biodiversity. Therefore, major declines in wild insects combined with losses of managed bees have raised great concern. Recent data suggest that honey bees appear to be less susceptible to stressors compared to other species. Here, we argue that eusociality plays a key role for the susceptibility of insects to environmental stressors due to superorganism resilience, which can be defined as the ability to tolerate the loss of somatic cells (= workers) as long as the germ line (= reproduction) is maintained. Life history and colony size appear critical for such resilience. Future conservation efforts should take superorganism resilience into account to safeguard ecosystem services by insects.

Developmental programming of tumor suppressor gene penetrance

Epidemiological studies have led to the hypothesis that major risk factors for developing diseases such as hypertension, cardiovascular disease and adult-onset diabetes are established during development. This developmental programming hypothesis proposes that exposure to an adverse stimulus or insult at critical, sensitive periods of development can induce permanent alterations in normal physiological processes that lead to increased disease risk later in life. For cancer, inheritance of a tumor suppressor gene defect confers a high relative risk for disease development. However, these defects are rarely 100% penetrant. Traditionally, gene-environment interactions are thought to contribute to the penetrance of tumor suppressor gene defects by facilitating or inhibiting the acquisition of additional somatic mutations required for tumorigenesis. The studies presented herein identify developmental programming as a distinctive type of gene-environment interaction that can enhance the penetrance of a tumor suppressor gene defect in adult life. Using rats predisposed to uterine leiomyoma due to a germ-line defect in one allele of the tuberous sclerosis complex 2 (Tsc-2) tumor suppressor gene, these studies show that early-life exposure to the xenoestrogen, diethylstilbestrol (DES), during development of the uterus increased tumor incidence, multiplicity and size in genetically predisposed animals, but failed to induce tumors in wild-type rats. Uterine leiomyomas are ovarian-hormone dependent tumors that develop from the uterine myometrium. DES exposure was shown to developmentally program the myometrium, causing increased expression of estrogen-responsive genes prior to the onset of tumors. Loss of function of the normal Tsc-2 allele remained the rate-limiting event for tumorigenesis; however, tumors that developed in exposed animals displayed an enhanced proliferative response to ovarian steroid hormones relative to tumors that developed in unexposed animals. Furthermore, the studies presented herein identify developmental periods during which target tissues are maximally susceptible to developmental programming. These data suggest that exposure to environmental factors during critical periods of development can permanently alter normal physiological tissue responses and thus lead to increased disease risk in genetically susceptible individuals. ^

Modifier genes and susceptibility to colorectal cancer in individuals with Lynch syndrome

Lynch syndrome, is caused by inherited germ-line mutations in the DNA mismatch repair genes resulting in cancers at an early age, predominantly colorectal (CRC) and endometrial cancers. Though the median age at onset for CRC is about 45 years, disease penetrance varies suggesting that cancer susceptibility may be modified by environmental or other low-penetrance genes. Genetic variation due to polymorphisms in genes encoding metabolic enzymes can influence carcinogenesis by alterations in the expression and activity level of the enzymes. Variation in MTHFR, an important folate metabolizing enzyme can affect DNA methylation and DNA synthesis and variation in xenobiotic-metabolizing enzymes can affect the metabolism and clearance of carcinogens, thus modifying cancer risk. ^ This study examined a retrospective cohort of 257 individuals with Lynch syndrome, for polymorphisms in genes encoding xenobiotic-metabolizing enzymes-- CYP1A1 (I462V and MspI), EPHX1 (H139R and Y113H), GSTP1 (I105V and A114V), GSTM1 and GSTT1 (deletions) and folate metabolizing enzyme--MTHFR (C677T and A1298C). In addition, a series of 786 cases of sporadic CRC were genotyped for CYP1A1 I462V and EPHX1 Y113H to assess gene-gene interaction and gene-environment interaction with smoking in a case-only analysis. ^ Prominent findings of this study were that the presence of an MTHFR C677T variant allele was associated with a 4 year later age at onset for CRC on average and a reduced age-associated risk for developing CRC (Hazard ratio: 0.55; 95% confidence interval: 0.36–0.85) compared to the absence of any variant allele in individuals with Lynch syndrome. Similarly, Lynch syndrome individuals heterozygous for CYP1A1 I462V A>G polymorphism developed CRC an average of 4 years earlier and were at a 78% increased age-associated risk (Hazard ratio for AG relative to AA: 1.78; 95% confidence interval: 1.16-2.74) than those with the homozygous wild-type genotype. Therefore these two polymorphisms may be additional susceptibility factors for CRC in Lynch syndrome. In the case-only analysis, evidence of gene-gene interaction was seen between CYP1A1 I462V and EPHX1 Y113H and between EPHX1 Y113H and smoking suggesting that genetic and environmental factors may interact to increase sporadic CRC risk. Implications of these findings are the ability to identify subsets of high-risk individuals for targeted prevention and intervention. ^

Cross-lineage expression of Ig-β (B29) in thymocytes: Positive and negative gene regulation to establish T cell identity

Developmental commitment involves activation of lineage-specific genes, stabilization of a lineage-specific gene expression program, and permanent inhibition of inappropriate characteristics. To determine how these processes are coordinated in early T cell development, the expression of T and B lineage-specific genes was assessed in staged subsets of immature thymocytes. T lineage characteristics are acquired sequentially, with germ-line T cell antigen receptor-β transcripts detected very early, followed by CD3ɛ and terminal deoxynucleotidyl transferase, then pTα, and finally RAG1. Only RAG1 expression coincides with commitment. Thus, much T lineage gene expression precedes commitment and does not depend on it. Early in the course of commitment to the T lineage, thymocytes lose the ability to develop into B cells. To understand how this occurs, we also examined expression of well defined B lineage-specific genes. Although λ5 and Ig-α are not expressed, the μ0 and Iμ transcripts from the unrearranged IgH locus are expressed early, in distinct patterns, then repressed just before RAG1 expression. By contrast, RNA encoding the B cell receptor component Ig-β was found to be transcribed in all immature thymocyte subpopulations and throughout most thymocyte differentiation. Ig-β expression is down-regulated only during positive selection of CD4+CD8– cells. Thus several key participants in the B cell developmental program are expressed in non-B lineage-committed cells, and one is maintained even through commitment to an alternative lineage, and repressed only after extensive T lineage differentiation. The results show that transcriptional activation of “lymphocyte-specific” genes can occur in uncommitted precursors, and that T lineage commitment is a composite of distinct positive and negative regulatory events.

Tumor burden and clonality in multiple intestinal neoplasia mouse/normal mouse aggregation chimeras

Aggregation chimeras were formed between C57BL/6 mice heterozygous for the Apcmin (Min) mutation and wild-type SWR mice, that differ in their Pla2g2a status, a modifier of Apcmin, and also in their resistance to intestinal polyp formation. Variation in the dolichos biflorus agglutinin-staining patterns of the intestines of these mouse strains was used to determine the chimeric composition of the intestine in individual mice and to examine the clonal composition of adenomas. Macroscopic adenoma numbers in chimeric mice were compared with the expected adenoma numbers based on the percentage of C57BL/6J-Apcmin/+ epithelium in individual mice. These results unexpectedly show that there was no apparent inhibitory effect of the SWR-derived (Pla2g2a wild-type) tissue on adenoma formation in the C57BL/6J-Apcmin/+ epithelium. This suggests that the main genetic modifiers of the Min phenotype act at a cellular or crypt-restricted level with no discernable systemic effect. All adenomas were seen to contain C57BL/6J-Apcmin/+-derived epithelium, confirming that the germ-line mutation of the mApc gene is necessary to initiate tumorigenesis in this model system, and that the mApc gene acts in a cell autonomous fashion.

The STAR protein QKI-6 is a translational repressor

The signal transduction and activation of RNA (STAR) family of RNA-binding proteins, whose members are evolutionarily conserved from yeast to humans, are important for a number of developmental decisions. For example, in the mouse, quaking proteins (QKI-5, QKI-6, and QKI-7) are essential for embryogenesis and myelination , whereas a closely related protein in Caenorhabditis elegans, germline defective-1 (GLD-1), is necessary for germ-line development. Recently, GLD-1 was found to be a translational repressor that acts through regulatory elements, called TGEs (for tra-2 and GLI elements), present in the 3′ untranslated region of the sex-determining gene tra-2. This gene promotes female development, and repression of tra-2 translation by TGEs is necessary for the male cell fates. The finding that GLD-1 inhibits tra-2 translation raises the possibility that other STAR family members act by a similar mechanism to control gene activity. Here we demonstrate, both in vitro and in vivo, that QKI-6 functions in the same manner as GLD-1 and can specifically bind to TGEs to repress translation of reporter constructs containing TGEs. In addition, expression of QKI-6 in C. elegans wild-type hermaphrodites or in hermaphrodites that are partially masculinized by a loss-of-function mutation in the sex-determining gene tra-3 results in masculinization of somatic tissues, consistent with QKI-6 repressing the activity of tra-2. These results strongly suggest that QKI-6 may control gene activity by operating through TGEs to regulate translation. In addition, our data support the hypothesis that other STAR family members may also be TGE-dependent translational regulators.

RAG2 is regulated differentially in B and T cells by elements 5′ of the promoter

To study RAG2 gene regulation in vivo, we developed a blastocyst complementation method in which RAG2-deficient embryonic stem cells were transfected with genomic clones containing RAG2 and then assessed for their ability to generate lymphocytes. A RAG2 genomic clone that contained only the RAG2 promoter sequences rescued V(D)J recombination in RAG2-deficient pro-B cell lines, but did not rescue development of RAG2-deficient lymphocytes in vivo. However, inclusion of varying lengths of sequences 5′ of the RAG2 promoter generated constructs capable of rescuing only in vivo B cell development, as well as other constructs that rescued both B and T cell development. In particular, the 2-kb 5′ region starting just upstream of the RAG2 promoter, as well as the region from 2–7 kb 5′, could independently drive B cell development, but not efficient T cell development. Deletion of the 2-kb 5′ region from the murine germ line demonstrated that this region was not required for RAG expression sufficient to generate normal B or T cell numbers, implying redundancy among 5′ elements. We conclude that RAG2 expression in vivo requires elements beyond the core promoter, that such elements contribute to differential regulation in the B vs. T lineages, and that sequences sufficient to direct B cell expression are located in the promoter-proximal 5′ region.

Cisplatin increases meiotic crossing-over in mice

Genetic mapping of traits and mutations in mammals is dependent upon linkage analysis. The resolution achieved by this method is related to the number of offspring that can be scored and position of crossovers near a gene. Higher precision mapping is obtained by expanding the collection of progeny from an appropriate cross, which in turn increases the number of potentially informative recombinants. A more efficient approach would be to increase the frequency of recombination, rather than the number of progeny. The anticancer drug cisplatin, which causes DNA strand breakage and is highly recombinogenic in some model organisms, was tested for its ability to induce germ-line recombination in mice. Males were exposed to cisplatin and mated at various times thereafter to monitor the number of crossovers inherited by offspring. We observed a striking increase on all three chromosomes examined and established a regimen that nearly doubled crossover frequency. The timing of the response indicated that the crossovers were induced at the early pachytene stage of meiosis I. The ability to increase recombination should facilitate genetic mapping and positional cloning in mice.

Effects of p53 mutations on apoptosis in mouse intestinal and human colonic adenomas

We have examined the effects of inactivation of the p53 tumor suppressor gene on the incidence of apoptotic cell death in two stages of the adenoma-to-carcinoma progression in the intestine: in early adenomas where p53 mutations are rare and in highly dysplastic adenomas where loss of p53 occurs frequently. Homozygosity for an inactivating germ-line mutation of p53 had no effect on the incidence or the rate of progression of ApcMin/+-induced adenomas in mice and also did not affect the frequency of apoptosis in the cells of these adenomas. To examine the effect of p53 loss on apoptosis in late-stage adenomas, we compared the incidence of apoptotic cell death before and after the appearance of highly dysplastic cells in human colonic adenomas. The appearance of highly dysplastic cells, which usually coincides during colon tumor progression with loss of heterozygosity at the p53 locus, did not correlate with a reduction in the incidence of apoptosis. These studies suggest that p53 is only one of the genes that determine the incidence of apoptotic in colon carcinomas and that wild-type p53 retards the progression of many benign colonic adenoma to malignant carcinomas by mechanism(s) other than the promotion of apoptosis.

Activating mutations for the Met tyrosine kinase receptor in human cancer

Recently, mutations in the Met tyrosine kinase receptor have been identified in both hereditary and sporadic forms of papillary renal carcinoma. We have introduced the corresponding mutations into the met cDNA and examined the effect of each mutation in biochemical and biological assays. We find that the Met mutants exhibit increased levels of tyrosine phosphorylation and enhanced kinase activity toward an exogenous substrate when compared with wild-type Met. Moreover, NIH 3T3 cells expressing mutant Met molecules form foci in vitro and are tumorigenic in nude mice. Enzymatic and biological differences were evident among the various mutants examined, and the somatic mutations were generally more active than those of germ-line origin. A strong correlation between the enzymatic and biological activity of the mutants was observed, indicating that tumorigenesis by Met is quantitatively related to its level of activation. These results demonstrate that the Met mutants originally identified in human papillary renal carcinoma are oncogenic and thus are likely to play a determinant role in this disease, and these results raise the possibility that activating Met mutations also may contribute to other human malignancies.

Cell cycle-dependent colocalization of BARD1 and BRCA1 proteins in discrete nuclear domains

Germ-line mutations of the BRCA1 gene predispose women to early-onset breast and ovarian cancer by compromising the gene’s presumptive function as a tumor suppressor. Although the biochemical properties of BRCA1 polypeptides are not understood, their expression pattern and subcellular localization suggest a role in cell-cycle regulation. When resting cells are induced to proliferate, the steady-state levels of BRCA1 increase in late G1 and reach a maximum during S phase. Moreover, in S phase cells, BRCA1 polypeptides are hyperphosphorylated and accumulate into discrete subnuclear foci termed “BRCA1 nuclear dots.” BRCA1 associates in vivo with a structurally related protein termed BARD1. Here we show that the steady-state levels of BARD1, unlike those of BRCA1, remain relatively constant during cell cycle progression. However, immunostaining revealed that BARD1 resides within BRCA1 nuclear dots during S phase of the cell cycle, but not during the G1 phase. Nevertheless, BARD1 polypeptides are found exclusively in the nuclear fractions of both G1- and S-phase cells. Therefore, progression to S phase is accompanied by the aggregation of nuclear BARD1 polypeptides into BRCA1 nuclear dots. This cell cycle-dependent colocalization of BARD1 and BRCA1 indicates a role for BARD1 in BRCA1-mediated tumor suppression.

Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA

A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3′2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5′ end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo.

Spontaneous mutations recovered as mosaics in the mouse specific-locus test

The specific-locus test (SLT) detects new mutants among mice heterozygous for seven recessive visible markers. Spontaneous mutations can be manifested not only as singleton whole-body mutants in controls (for which we report new data), but as mosaics—either visible (manifesting mottled coat color) in the scored generation (G2) or masked, among the wild-type parental generation (G1). Masked G1 mosaics reveal themselves by producing clusters of whole-body mutants in G2. We provide evidence that most, if not all, mosaics detected in the SLT (both radiation and control progenies) result from a single-strand spontaneous mutation subsequent to the last premeiotic mitosis and before the first postmeiotic one of a parental genome—the “perigametic interval.” Such events in the genomes of the G1 and G0 result, respectively, in visible and masked 50:50 mosaics. Per cell cycle, the spontaneous mutation rate in the perigametic interval is much higher than that in pregamete mitotic divisions. A clearly different locus spectrum further supports the hypothesis of different origin, and casts further doubt on the validity of the doubling-dose risk-estimation method. Because mosaics cannot have arisen in mitotic germ cells, and are not induced by radiation exposure in the perigametic interval, they should not be included in calculations of radiation-induced germ-line mutation rates. For per-generation calculations, inclusion of mosaics yields a spontaneous frequency 1.7 times that calculated from singletons alone for mutations contributed by males; including both sexes, the multiple is 2.2.

Sequence variation in the Fanconi anemia gene FAA

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA crosslinking agents, and predisposition to malignancy. The gene for FA complementation group A (FAA) recently has been cloned. The cDNA is predicted to encode a polypeptide of 1,455 amino acids, with no homologies to any known protein that might suggest a function for FAA. We have used single-strand conformational polymorphism analysis to screen genomic DNA from a panel of 97 racially and ethnically diverse FA patients from the International Fanconi Anemia Registry for mutations in the FAA gene. A total of 85 variant bands were detected. Forty-five of the variants are probably benign polymorphisms, of which nine are common and can be used for various applications, including mapping studies for other genes in this region of chromosome 16q. Amplification refractory mutation system assays were developed to simplify their detection. Forty variants are likely to be pathogenic mutations. Seventeen of these are microdeletions/microinsertions associated with short direct repeats or homonucleotide tracts, a type of mutation thought to be generated by a mechanism of slipped-strand mispairing during DNA replication. A screening of 350 FA probands from the International Fanconi Anemia Registry for two of these deletions (1115–1118del and 3788–3790del) revealed that they are carried on about 2% and 5% of the FA alleles, respectively. 3788–3790del appears in a variety of ethnic groups and is found on at least two different haplotypes. We suggest that FAA is hypermutable, and that slipped-strand mispairing, a mutational mechanism recognized as important for the generation of germ-line and somatic mutations in a variety of cancer-related genes, including p53, APC, RB1, WT1, and BRCA1, may be a major mechanism for FAA mutagenesis.