A new method can accurately quantify genetic variants called short tandem repeats (STRs) and may improve diagnosis of fragile X syndrome and other disorders, a study suggests.
The method was described in the study, “Analysis of short tandem repeat expansions and their methylation state with nanopore sequencing,” which was published in the journal Nature Biotechnology.
STRs are multiple repetitions of short genetic sequences. They can vary in length and impact a gene’s ability to function properly. For instance, in fragile X, an overabundance of CGG repeats in the FMR1 is associated with the addition of methyl groups to DNA (a process called methylation), which disrupts the production of the FMRP protein. (Of note, C stands for cytosine and G for guanine, two of the building blocks — or nucleotides — of DNA).
Despite their importance, STRs can be difficult to study. Most current sequencing technologies have been unable to decode them with high resolution, mainly because the presence of several repeats can confuse the algorithms that translate raw biochemical inputs into letters that humans can understand.
“Most algorithms fail because they do not expect the regular patterns of repetitive sequences,” Pay Giesselmann, the study’s first author, said in a press release.
First, they created a way to use CRISPR/Cas9 gene editing to specifically cut out the DNA regions with the STR of interest.
“If we had not pre-sorted the molecules in this way, their signal would have been drowned in the noise of the rest of the genome,” said Giesselmann.
Next, the researchers developed an algorithm named STRique (short tandem repeat identification, quantification, and evaluation) to better analyze sequence data generated from nanopore sequencing, a technique that identifies nucleotides based on the different electric currents they produce when passing through a small channel.
Compared to previous alternatives, the algorithm specifically looks for the number of STRs rather than trying to determine the genetic sequence itself. Then, the technology was expanded to analyze DNA methylation.
The team tested its method in a number of models. These included artificially constructed DNA sequences with STRs, and stem cells from a patient with fragile X and autism spectrum disorder, or people with frontotemporal dementia and amyotrophic lateral sclerosis (another disorder in which STRs are implicated).
Results showed that STRique was able to accurately identify both STR length and methylation status in these samples. All expanded FMR1 STRs from the person with fragile X syndrome were found to be highly methylated, as the team had expected and in line with data from other genetic tests.
“Our method enables the study of previously inaccessible genomic regions,” the researchers said.
“We developed a unique method for the analysis of single molecules and for the darkest regions of our genome — that’s what makes this so exciting for me,” said Franz-Josef Müller, the study’s senior author.
Overall, the researchers believe that this new technique will improve diagnostics and research in fragile X and other conditions characterized by STRs.
“We are very close to clinical application,” Müller said.