In the pediatric group, 7 CpG island loci (CDH1, DAPK1
, CRABP1, GR1N2B, T1MP3, CALCA, and TW1ST1) showed significantly higher methylation levels in results from H pylori-positive samples than those from H pylori-negative samples (all values, P < .
For a preliminary test of the reliability and performance of the MS-LAMP assay without the variation introduced by bisulfite treatment, we performed experiments to assess specificity, the detection limit, and selectivity with plasmid targets containing the DAPK1 promoter sequence in either a methylated state or a unmethylated state after bisulfite treatment.
We used 1 or 2 [micro]L (approximately 25-50 ng) of DNA as the target for investigating the hypermethylation status of the CDKN2A, DAPK1, and GATA5 promoters.
MS-LAMP primers (F3, B3, FIP, BIP, LF, LB), which specifically amplify only the methylated sequences of the CDKN2A, DAPK1, and GATA5 promoters after bisulfite treatment, were designed with the aid of PrimerExplorer V4 (Eiken).
For the multiplex LAMP reactions assessed by fluorescence, the LF primers of the DAPK1 and CDKN2A assays were substituted by oligonucleotides that had the same DNA sequence but were labeled at the 5' end with BODIPY FL (dipyrromethene boron difluoride FL) and 6-carboxytetramethylrhodamine (TAMRA) dyes, respectively.
The thermocycling protocols for the PCR reactions were as follows: CDKN2A, initial denaturation at 94 [degrees]C for 2 min and 50 cycles of 94 [degrees]C for 30 s, 65 [degrees]C for 30 s, and 72 [degrees]C for 30 s; DAPK1, initial denaturation at 94 [degrees]C for 2 min and 45 cycles of 94 [degrees]C for 30 s, 68 [degrees]C for 30 s, and 72 [degrees]C for 30 s; GATA5, initial denaturation at 94 [degrees]C for 2 min and 50 cycles of 94 [degrees]C for 30 s, 66 [degrees]C for 30 s, and 72 [degrees]C for 30 s.
The DAPK1 gene has been a target for drug discovery in other diseases, and a variety of drug compounds can now be tested for their effect on learning and memory in animal models of Alzheimer's disease.
DAPK1 is an enzyme involved in the programmed cell death cascade and evidence suggests that one of its functions is to control the death of nerve cells, and it is predominantly expressed in regions of the brain, such as the hippocampus and cortex, that are most severely affected by Alzheimer's disease.
The previously reported associated markers and this marker in DAPK1 each consistently show an involvement in apoptotic cell death shedding light on the fundamental underlying mechanism of disease, critical to new therapeutic options.
These research findings also hold promise for enabling the development of diagnostic tests to identify people who may be at risk for Alzheimer's and would potentially benefit from treatment with DAPK1 inhibitors," said Thomas White, Ph.
The DAPK1 SNP survived replication in multiple data sets and was then further tested in three additional validation sets.