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Background Methylation capture sequencing (MC-seq), which relies on next-generation sequencing technology, offers advantages over the widely used array-based approach that Illumina Inc. developed regarding both resolution and comprehensiveness for detecting DNA methylation changes across genomes. In the present study, MC-seq was employed for the first time to identify DNA methylation markers for Alzheimer’s disease (AD). Results We compared DNA methylation in the blood of 12 AD patients with brain amyloidosis and 12 cognitively normal elderly Japanese individuals without brain amyloidosis. Candidate methylation differences were validated in the two cohorts using bisulfite amplicon sequencing. Significant differentially methylated regions were identified in the ANKH , MARS , ANKFY1 , LINC00908 , and KLF2 genes and a slight methylation change in CHRNE ( p  = 0.061). Furthermore, our AD diagnostic prediction model showed that combining the methylation levels of ANKH and MARS with the APOE genotype provided diagnostic accuracy, achieving AUCs of 0.90 and 0.81 in the discovery and validation datasets, respectively. Conclusions The present results suggest the potential of combining these markers for diagnosing AD and support the validity of our approach for identifying disease-related DNA methylation markers using next-generation sequencing.

Dementia with Lewy bodies (DLB) is the second most common form of degenerative dementia in older people. The clinical feature of DLB includes cognitive impairment, visual hallucinations, parkinsonism, and fluctuating attention. Three genes, SNCA (-synuclein), APOE (apolipoprotein E), and GBA (glucosylceramidase), have been convincingly demonstrated to be associated with DLB. Our previous studies reported that a variant in MFSD3 (rs143475431, c.888T>A:p.C296*) is associated with DLB in the Japanese population through whole-genome sequencing and association studies. However, the precise mechanisms that this variant contributes to DLB development remains unclear. We introduced the MFSD3 p.C296* variant into a human neural stem cell line using the CRISPR/Cas9 system. Subsequently, the mutant cells were analyzed for cell proliferation rate using CyQUANT Cell Proliferation Assay Kit. The mutant cells were further induced to differentiate into neurons and astrocytes to assess their differentiation ability. Additionally, we generated the homologous Mfsd3 knockout (KO) mice using the CRISPR/Cas9 system. The neurogenesis was evaluated by the number of doublecortin positive cells in the hippocampus. The behavior of Mfsd3 KO and wild-type mice was analyzed using IntelliCage. We revealed that the MFSD3 p.C296* variant was associated with decreased cell proliferation and reduced neurogenesis in the human neural stem cell line analyses. This decline in neurogenesis was also observed in the hippocampal dentate gyrus of Mfsd3 KO mice, and the Mfsd3 KO mice had smaller hippocampi compared to the wild-type mice. Furthermore, Mfsd3 KO mice exhibited a reduced level of curiosity about the new environment. Our results demonstrate that loss of function of MFSD3 affects neurons and the brain, as indicated by our studies using human neural stem cell lines and Mfsd3 KO mice. Further functional verification will contribute to elucidating the mechanism of DLB pathogenesis.

Background Dementia with Lewy bodies (DLB) is the second most common form of degenerative dementia in older people. The clinical feature of DLB includes cognitive impairment, visual hallucinations, parkinsonism, and fluctuating attention. Three genes, SNCA (‐synuclein), APOE (apolipoprotein E), and GBA (glucosylceramidase), have been convincingly demonstrated to be associated with DLB. Our previous studies reported that a variant in MFSD3 (rs143475431, c.888T>A:p.C296*) is associated with DLB in the Japanese population through whole‐genome sequencing and association studies. However, the precise mechanisms that this variant contributes to DLB development remains unclear. Method We introduced the MFSD3 p.C296* variant into a human neural stem cell line using the CRISPR/Cas9 system. Subsequently, the mutant cells were analyzed for cell proliferation rate using CyQUANT Cell Proliferation Assay Kit. The mutant cells were further induced to differentiate into neurons and astrocytes to assess their differentiation ability. Additionally, we generated the homologous Mfsd3 knockout (KO) mice using the CRISPR/Cas9 system. The neurogenesis was evaluated by the number of doublecortin positive cells in the hippocampus. The behavior of Mfsd3 KO and wild‐type mice was analyzed using IntelliCage. Result We revealed that the MFSD3 p.C296* variant was associated with decreased cell proliferation and reduced neurogenesis in the human neural stem cell line analyses. This decline in neurogenesis was also observed in the hippocampal dentate gyrus of Mfsd3 KO mice, and the Mfsd3 KO mice had smaller hippocampi compared to the wild‐type mice. Furthermore, Mfsd3 KO mice exhibited a reduced level of curiosity about the new environment. Conclusion Our results demonstrate that loss of function of MFSD3 affects neurons and the brain, as indicated by our studies using human neural stem cell lines and Mfsd3 KO mice. Further functional verification will contribute to elucidating the mechanism of DLB pathogenesis.