Expansion and Methylation Go Hand in Hand in C9ORF72

The C9ORF72 gene stymies scientists attempting to sequence or amplify it. Researchers have now managed to analyze the methylation status of the hexanucleotide repeat expansion that has been linked to neurodegenerative diseases. Once this region extends beyond 90 GGGGCC repeats, it always picks up methyl groups, first author Zhengrui Xi and colleagues at the University of Toronto report in the February 26 Acta Neuropathologica online. Methylation typically shuts off transcription, though researchers have not yet proven that occurs in this case. Xi and senior author Ekaterina Rogaeva hypothesize that the methylation quells the production of abnormal RNAs and peptides, but also the normal C9ORF72 protein.

Expanded repeats in intron 1 of C9ORF72, which can number in the hundreds or thousands, are a common cause of both amyotrophic lateral sclerosis and frontotemporal lobar dementia. Due to abnormal transcription and translation, repeat-toting cells fill up with RNA foci and aggregates of dipeptides, and scientists suspect one or both of these formations may be neurotoxic. However, repeat expansion carriers typically produce about half the normal amount of proper C9ORF72 RNA, leading researchers to hypothesize that haploinsufficiency of the protein, which has no known function, may also damage neurons.

Because methyl groups silence gene expression, scientists have wondered if such modifications explain the lack of C9ORF72 protein. Multiple studies have detected methyl groups in the promoter region of the gene, but what of the repeats themselves (see Jun 2013 news; Liu et al., 2014; Xi et al., 2014)? Co-first authors Xi and Ming Zhang and their collaborators collected 270 DNA blood samples from expansion carriers and controls to check methylation status.

However, evaluating methylation within the repeats was not a straightforward experiment. The guanine- and cytosine-rich nucleic acids fold up into hairpins that can interfere with typical polymerase chain reaction and the sequencing protocols necessary techniques to assess methylation. To overcome these problems, Xi borrowed a two-step method used to study CG-rich expansions in a gene associated with Fragile X syndrome (Zhou et al., 2006). First, they treated the DNA with sodium bisulfite. This turns any unmethylated cytosines into uracils, which are then converted to thymines in the ensuing PCR. Methylated cytosines remain unchanged. Methylation occurs specifically on the last cytosine in the GGGGCC sequence because the first guanine in the next repeat creates a CpG dinucleotide amenable to methyl attachment. Therefore, any unmethylated repeat will be converted to GGGGTT, and methylated ones to GGGGTC.

Next, the authors amplified the modified DNA with primers specific for unmethylated (now GGGGTT) and methylated sequences (now GGGGTC). The authors caution that their technique does not indicate how much of a repeat expansion is methylated versus unmethylated, or where those methyls occur. “It is a yes/no test,” Rogaeva said. Because the repeat-primed PCR mainly amplified DNA within the first 20-30 repeats of the expansion, it identified methylation in that region, she added. The authors cannot say how far the methyl groups extend.

When Xi and Zhang examined their data from those 270 blood samples, a clear pattern emerged. For DNA with up to 70 repeats, no methylation was present. For all samples with more than 90 hexanucleotides, there were always methyl groups. There were no samples with 70-90 repeats, so the researchers need to analyze more samples to determine the length at which methylation starts, Rogaeva pointed out. This pattern held true regardless of a person’s diagnosis of ALS or FTLD. Methylation seems to be a direct consequence of expansion, Xi said.

“This is an elegant technique showing that the expansion is methylated,” commented Stuart Pickering-Brown of the University of Manchester in the United Kingdom, who was not involved with the study, in an email to Alzforum. “It is interesting that there is no difference between FTLD and ALS. The reason why one expansion carrier gets FTLD while another gets ALS still remains unexplained.”

Indeed, scientists can only guess at what the methylation means for disease. “We do not understand why this region is methylated or non-methylated,” commented Edward Lee of the University of Pennsylvania in Philadelphia, who was not part of the study. Gene silencing, he said, is the “likely” but unconfirmed reason. The cell often methylates repetitive sequences, perhaps because they are unstable, Lee explained. Such silencing would shut off production of toxic RNAs and peptides—a benefit—but also lead to haploinsufficiency of the proper C9ORF72 protein. Rogaeva and Xi suggested scientists could use their two-step method to identify methylated expansions as a quick way to predict repeat length, “although vaguely,” Xi said. The standard technique to measure repeat size, by Southern blotting, requires several micrograms of DNA and that amount is often hard to obtain, Rogaeva noted. The sodium bisulfite/PCR technique works with just half a microgram of DNA. “This method discriminates very well between long and intermediate length repeats,” she said.

Reference:

Xi Z, Zhang M, Bruni AC, Maletta RG, Colao R, Fratta P, Polke JM, Sweeney MG, Mudanohwo E, Nacmias B, Sorbi S, Tartaglia MC, Rainero I, Rubino E, Pinessi L, Galimberti D, Surace EI, McGoldrick P, McKeever P, Moreno D, Sato C, Liang Y, Keith J, Zinman L, Robertson J, Rogaeva E. The C9orf72 repeat expansion itself is methylated in ALS and FTLD patients. Acta Neuropathol. 2015 Feb 26; PubMed.


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c9orf72 disease-als disease-ftd human tissue and iPSCs topic-preclinical
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