“This is the House that Jack Built” is a popular and beloved children’s rhyme. But if children’s rhymes were written by scientists instead, it might look something like this: There’s an oxidation reaction on a methyl group, on a carbon in a cytosine, in a region of a gene, in the genome of a neuron, in the brain of an Alzheimer’s patient. There are many genes linked to Alzheimer’s disease (AD) progression and intriguingly aberrant DNA methylation has also been implicated.
Noting the importance of 5-methyl-cytosine (5mC) and its oxidized derivatives 5-hydroxymethyl-cytosine (5hmC), 5-formyl-cytosine (5fC) and 5-carboxy-cytosine (5caC) in neuronal development, the labs of Yujiang Shi (Harvard Medical School, Boston, USA) and Feizhen Wu (Fudan University, Shanghai, China) set out to compare the methylomes of healthy and AD-affected patients. A previous barrier to this kind of deep dive was the inability to distinguish between 5mC and its oxidized products at the single-base level. To overcome this, the team cunningly combined oxidative bisulfite deep sequencing (OXBS-seq) and methylase-assisted bisulfite deep sequencing (MAB-seq) to generate genome-wide maps of the distribution and levels of 5mC, 5hmC, and combined 5fC/5caC.
This fusion sequencing technique was then applied to cell line models: iPSCs were derived from healthy patients, familial early-onset Alzheimer’s disease (EOAD) patients, and sporadic late-onset Alzheimer’s disease (LOAD) patients. The iPSCs were first differentiated into neural precursor cells (NPCs) and then into neurons. The levels and distribution of 5mC and its derivatives were measured for each of the models and differentiation states.
Here’s what they found:
- In disease-free patients, 5fC/5caC levels are low in iPSCs, but then they peak in NPCs (specifically near transcription start sites), and drop again in neurons
- In both EOAD and LOAD models, there is no peak in 5fC/5caC levels at the NPC stage
- This indicates a disease-specific change in the methylation signature
- When comparing all AD models versus healthy models, there are specific regions and CpG sites that contain differential levels of 5mC, 5hmC and 5fC/5caC during differentiation
- They identified 27 region-specific and 39 CpG site-specific differences
- These regions and sites were directly related to genes involved in neuron function or had been previously implicated in AD
- These AD-specific signatures were validated against an independent patient cohort (ROS/MAP cohort) and tested for predictive power
- The best of several tested methods had a predictive capacity of 0.91, 98% specificity and 48% sensitivity, showcasing its potential value as a diagnostic tool
These newly identified AD-specific markers could allow for earlier detection of Alzheimer’s disease and conceivably be used to mark disease progression. What’s more, this novel 5mC mapping technique will certainly be of use for neurodevelopmental research and for studying other DNA methylation disorders.