Association study of cannabinoid receptor gene (CNR1) alleles and anorexia nervosa: differences between restricting and binging/purging subtypes.

Anorexia nervosa (AN) is a severe and disabling psychiatric disorder, characterized by profound weight loss and body image disturbance. Family and twin studies indicate a significant genetic contribution to this disorder although no genetic mutation has yet been identified. The endocannabinoid system has recently been implicated in many physiological functions including appetite regulation. We, therefore, undertook a family based study to test the hypothesis whether a polymorphism of the CNR1 gene, which encodes human CB1 receptor, a subclass of the central cannabinoid receptor, contributes to the susceptibility to AN. Fifty two families (parents with one or two affected siblings) were genotyped for the (AAT) trinucleotide repeat of CNR1 gene. Using the haplotype relative risk (HRR) method, the distribution of alleles transmitted to the patients was not found to be significantly different from the non-transmitted parental alleles. However, upon dividing the samples to restricting and binging/purging subtypes of AN, the extended transmission disequilibrium test (ETDT) revealed that there is preferential transmission of different alleles in each of the subtypes. The 14 repeat allele was preferentially transmitted in the binging/purging AN group (P = 0.05) but not in the restricting AN group, whereas the 13 repeat allele was preferentially transmitted in the restricting AN group (almost significant, P = 0.07) but not in the binging/purging AN group. Our study suggests that restricting AN and binging/purging AN may be associated with different alleles of the CNR1 gene.

A meta-analysis of the association between DRD4 polymorphism and novelty seeking.

A meta-analytical review of 20 studies (n = 3907) of the association between DRD4 polymorphism and novelty seeking suggests the following conclusions: (a) on average, there is no association between DRD4 polymorphism and novelty seeking (average d = 0.06 with 95% CI of +/- 0.09), where 13 reports suggest that the presence of longer alleles is associated with higher novelty seeking scores and seven reports suggest the opposite; (b) there is a true heterogeneity among the studies (ie, unknown moderators do exist) but the strength of the association between DRD4 polymorphism and novelty seeking in the presence of any (unknown) moderator is likely to be weak; (c) search for moderators has not yielded any reliable explanation for the variability among studies. We propose that to find such moderators, theory-driven research for potential interaction, coupled with larger sample sizes should be employed. The growing availability of powerful statistical techniques, high-throughput genotyping and large numbers of polymorphic markers such as single nucleotide polymorphisms makes such proposed studies increasingly feasible.

ABL1 methylation in Ph-positive ALL is exclusively associated with the P210 form of BCR-ABL.

In human Ph-positive leukemia there is a clear association of different forms of the BCR-ABL oncogene with distinct types of leukemia. The P190 form of BCR-ABL is rarely observed in chronic myeloid leukemia (CML) but is present in 50% of Ph-positive acute lymphoblastic leukemia (ALL). In contrast, the P210 form is observed both in CML and 50% of Ph-positive ALL. Methylation of the proximal promoter of the ABL1 gene has been shown to be a nearly universal event associated with clinical progression of CML. This raises the question of whether methylation of the ABL1 promoter is an epigenetic modification also associated with Ph-positive ALL. To study this issue, we used methylation-specific PCR and bisulfite sequencing to determine the methylation status of the ABL1 promoter in 18 Ph-positive ALL samples. We report here that gene-specific ABL1 promoter methylation is associated mainly with the P210 form of BCR-ABL and not the P190 form. While six out of the seven P210-positive ALL samples had ABL1 promoter methylation, none of the 11 P190-positive ALL samples demonstrated ABL1 promoter methylation. In addition, we estimated the extent and relative abundance of ABL1 promoter methylation in several Ph-positive ALL samples and compared it to the methylation pattern in chronic, accelerated and blastic crisis phases of CML. We put forth a model that correlates the different types of leukemias with the different levels of ABL1 promoter methylation.

DNA methylation represses transcription in vivo.

DNA in somatic tissue is characterized by a bimodal pattern of methylation, which is established in the animal through a series of developmental events. In the mouse blastula, most DNA is unmethylated, but after implantation a wave of de novo methylation modifies most of the genome, excluding the majority of CpG islands, which are mainly associated with housekeeping genes. This genomic methylation pattern is broadly maintained during the life of the organism by maintenance methylation, and generally correlates with gene expression. Experiments both in vitro and in vivo indicate that methylation inhibits transcription. It has not yet been possible, however, to determine the role of DNA methylation on specific sequences during normal development. Cis-acting regulatory elements and trans-acting factors appear to be involved in both stage- and tissue-specific demethylation processes. Sp1-like elements have a key role in protecting the CpG island of Aprt (encoding adenine phosphoribosyltransferase) from de novo methylation, and when these elements are specifically mutated, the Aprt CpG island becomes methylated in transgenic mice. We have now characterized an embryo-specific element from the CpG island sequence upstream of Aprt that can protect itself from de novo methylation in transgenic mice as well as reduce methylation of flanking sequences. We placed this element on a removable cassette adjacent to a human HBB (encoding beta-globin) reporter and generated a transgene whose methylation pattern can be switched in vivo. Analysis of globin transcription in this system showed that methylation in cis inhibits gene expression in a variety of tissues, indicating that DNA modification may serve as a global genomic repressor.

DNA methylation: a molecular lock.

In mammals, the promoters of expressed genes are generally unmethylated, whereas those of genes that are not expressed are methylated. Two recent papers help to explain the mechanism by which methylation modulates gene expression.

Sp1 elements protect a CpG island from de novo methylation.

Animal somatic cell DNA is characterized by a bimodal pattern of methylation: tissue-specific genes are methylated in most cell types whereas housekeeping genes have 5' CpG islands which are constitutively unmethylated. Because methyl moieties derived from the gametes are erased in the morula and early blastula, this profile must be re-established in every generation; this is apparently accomplished by a wave of non-CpG island de novo methylation that occurs at implantation. Using transfection into embryonic stem cells and transgenic mice as a model system, we now show that Sp1 elements play a key role in protecting a CpG island in the adenine phosphoribosyltransferase (APRT) gene from de novo methylation. This recognition mechanism represents a critical step in embryogenesis, as it is responsible for setting up the correct genome methylation pattern which, in turn, is involved in regulating basal gene expression in the organism.

An ancient family of embryonically expressed mouse genes sharing a conserved protein motif with the T locus.

The T locus encodes a product with DNA binding activity that is likely to play a role in the development of all vertebrate organisms. We have identified and characterized a novel family of mouse genes that share a protein motif, the T-box, with the prototypical T locus. The T-box domain of the T locus co-localizes with its DNA binding activity. Each T-box gene is expressed in a unique temporal and spatial pattern during embryogenesis. Phylogenetic analysis suggests that at least three T-box genes were present in the common ancestor to vertebrates and invertebrates. Thus, members of the T-box family could have played a role in the evolution of all metazoan organisms.

Altered transcriptional activity of c-fos promoter plasmids in v-raf-transformed NIH 3T3 cells.

In cells transformed by v-raf, an oncogenic counterpart of the serine/threonine kinase Raf-1, regulatory elements of the c-fos promoter were active under conditions of cell growth or stimulation for which they were inactive in untransformed control cells. This suggests that v-raf transforms by deregulating transcription of early response genes.

Localization of the ribosome-releasing factor gene in the Escherichia coli chromosome.

The ribosome-releasing factor (RRF) gene was localized at a position between 2 and 6 min on the Escherichia coli chromosome by measuring the gene-dosage-dependent production of RRF in various E. coli F' merozygotes. This position was confirmed and refined by using a nucleotide probe corresponding to a 16-amino-acid sequence in RRF. It was found that the RRF gene was contained in pLC 6-32 of the Clark-Carbon Gene Bank. Restriction enzyme mapping of E. coli genomic DNA with the above probe led us to conclude that the RRF gene is situated in the 4-min region, somewhere downstream (clockwise) of the elongation factor Ts gene, tsf. A pLC 6-32-derived DNA fragment which carries the RRF gene was found to contain a partial sequence of tsf. The exact location of the translational initiation site of the RRF gene was determined to be 1.1 kilobases downstream from the translational termination site of tsf. The RRF gene is designated frr.

Transcription activation by serum, PDGF, and TPA through the c-fos DSE: cell type specific requirements for induction.

We have investigated the sequences that are necessary and sufficient for the induction of the c-fos gene by serum, TPA or PDGF in different cell types. The dyad symmetry element (DSE) is a regulatory element of the c-fos gene previously shown to be required for induction of c-fos transcription by serum. We show that the DSE is also necessary for the induction of c-fos by either TPA or PDGF in NIH3T3 cells. We also show that in NIH 3T3 cells the DSE is sufficient to confer inducibility on a heterologous promoter, the beta-globin promoter, when serum provides the stimulus. However, it is not sufficient when either TPA or PDGF is the inducer. This suggests a requirement in 3T3 cells for cooperating sequence elements for TPA or PDGF induction but not for serum. Interestingly, the need for cooperating elements for TPA induction is abolished in HeLa cells since the DSE alone is sufficient for TPA inducibility of the beta-globin promoter in these cells. Thus, the highly transformed HeLa cell line displays diminished sequence requirements for TPA induction. We discuss the possibility that mutations which diminish the stringent transcriptional control of protooncogenes such as c-fos may contribute to the transformed state.

Mutation of the c-fos gene dyad symmetry element inhibits serum inducibility of transcription in vivo and the nuclear regulatory factor binding in vitro.

In vitro mutagenesis of a 61-base-pair DNA sequence element that is necessary for induction of the c-fos proto-oncogene by growth factors revealed that a small region of dyad symmetry within the sequence element is critical for c-fos transcriptional activation. The same c-fos dyad symmetry element was found to bind a nuclear protein in vitro, causing a specific mobility shift of this c-fos regulatory sequence. An analysis of insertion and deletion mutants established a strict correlation between the ability of the dyad symmetry element to promote serum activation of c-fos transcription and in vitro nuclear protein binding. These experiments suggest that the DNA mobility shift assay detects a nuclear protein that mediates growth factor stimulation of c-fos expression. In vitro competition experiments indicate that the c-fos regulatory factor also binds to sequences within another growth factor-inducible gene, the beta-actin gene.