SMYD1 is the underlying gene for the AnWj-negative blood group phenotype.

BACKGROUND: AnWj is a high-incidence blood group antigen associated with three clinical disorders: lymphoid malignancies, immunologic disorders, and autoimmune hemolytic anemia. The aim of this study was to determine the genetic basis of an inherited AnWj-negative phenotype. METHODS: We identified a consanguineous family with two AnWj-negative siblings and 4 additional AnWj-negative individuals without known familial relationship to the index family. We performed exome sequencing in search for rare homozygous variants shared by the two AnWj-negative siblings of the index family and searched for these variants in the four non-related AnWj-negative individuals. RESULTS: Exome sequencing revealed seven candidate genes that showed complete segregation in the index family and for which the two AnWj-negative siblings were homozygous. However, the four additional non-related AnWj-negative subjects were homozygous for only one of these variants, rs114851602 (R320Q) in the SMYD1 gene. Considering the frequency of the minor allele, the chance of randomly finding 4 consecutive such individuals is 2.56 × 10-18 . CONCLUSION: We present genetic and statistical evidence that the R320Q substitution in SMYD1 underlies an inherited form of the AnWj-negative blood group phenotype. The mechanism by which the mutation leads to this phenotype remains to be determined.

cAMP-dependent protein kinase type I regulates ethanol-induced cAMP response element-mediated gene expression via activation of CREB-binding protein and inhibition of MAPK.

We have shown that the two types of cAMP-dependent protein kinase (PKA) in NG108-15 cells differentially mediate forskolin- and ethanol-induced cAMP response element (CRE)-binding protein (CREB) phosphorylation and CRE-mediated gene transcription. Activated type II PKA is translocated into the nucleus where it phosphorylates CREB. By contrast, activated type I PKA does not translocate to the nucleus but is required for CRE-mediated gene transcription by inducing the activation of other transcription cofactors such as CREB-binding protein (CBP). We show here that CBP is required for forskolin- and ethanol-induced CRE-mediated gene expression. Forskolin- and ethanol-induced CBP phosphorylation, demonstrable at 10 min, persists up to 24 h. CBP phosphorylation requires type I PKA but not type II PKA. In NG108-15 cells, ethanol and forskolin activation of type I PKA also inhibits several components of the MAPK pathway including B-Raf kinase, ERK1/2, and p90RSK phosphorylation. As a result, unphosphorylated p90RSK no longer binds to nor inhibits CBP. Moreover, MEK inhibition by PD98059 induces a significant increase of CRE-mediated gene activation. Taken together, our findings suggest that inhibition of the MAPK pathway enhances cAMP-dependent gene activation during exposure of NG108-15 cells to ethanol. This mechanism appears to involve type I PKA-dependent phosphorylation of CBP and inhibition of MEK-dependent phosphorylation of p90RSK. Under these conditions p90RSK is no longer bound to CBP, thereby promoting CBP-dependent CREB-mediated gene expression.

Ethanol stimulates cAMP-responsive element (CRE)-mediated transcription via CRE-binding protein and cAMP-dependent protein kinase.

Alcoholism is characterized by tolerance, dependence, and unrestrained craving for alcohol. Adaptive responses, including changes in gene expression in neurons, are thought to account for some of these complex behavioral abnormalities. We have shown in the NG108-15 neuroblastoma x glioma hybrid cell line that ethanol increases cellular cAMP levels via activation of adenosine A(2) receptors, leading to phosphorylation of the cAMP response element-binding protein (CREB). However, phosphorylation of CREB is not sufficient to activate cAMP response element (CRE)-mediated gene expression. Here we investigate whether ethanol increases CRE-mediated gene expression via endogenous CREB using a CRE-regulated luciferase reporter construct, transfected into NG108-15 cells. We find increased luciferase activity as a function of time of exposure to ethanol. Coexpression of a dominant-negative CREB construct blocked ethanol-stimulated CRE-luciferase expression, further suggesting that CREB is required for this response. We also determined whether ethanol-induced increases in gene expression are mediated by ethanol-induced increases in extracellular adenosine. We found that CRE-mediated gene expression induced by ethanol occurs in two phases: an early phase (4 h), in which adenosine receptor blockade prevents ethanol-induced gene expression, and a later phase (14 h), which is not blocked by an adenosine receptor antagonist. In both phases, inhibition of cAMP-dependent protein kinase A (PKA) activity prevented ethanol-induced CRE-mediated luciferase expression. Our data suggest that ethanol induces cAMP-dependent gene expression regulated by CREB and PKA and that this signaling pathway may mediate some of the addictive behaviors underlying alcoholism.