Genetic sequencing may aid accurate tally of FA-causing GAA repeats
Aim is to better understand correlation between genotype, clinical status
Researchers have identified a more high-throughput genetic sequencing method to quantify the GAA repeats in the FXNÂ gene that cause Friedreich’s ataxia.
Since the number of repeats has been linked to clinical disease presentation, a better way to show their quantity will aid a more accurate understanding of how a person’s genetic status (genotype) correlates with their clinical status (phenotype), according to the authors of “Sequencing through hyperexpanded Friedreich’s ataxia-GAA repeats by nanopore technology: implications in genotype–phenotype correlation,” which was published in Brain Communications.Â
The researchers believe it’s “imperative to deploy this technology,” not only for FA, but for other diseases associated with a similar type of mutation, such as fragile X syndrome or Huntington’s disease.
FA is caused by mutations in FXN, leading to the disrupted production of the frataxin protein that’s important for cellular energy production. These mutations consist of the excessive repetition of a trio of nucleotides — DNA building blocks — one guanine (G) and two adenines (A).
Normally, this trio is repeated in the general population from 5 to 33 times, but people with FA have significantly more. At least 66 repeats are generally considered disease-causing, but there is usually between 600 to 1,200 with FA.
The number of repeats has been found to correlate with clinical disease characteristics, with more linked to an earlier disease onset, more severe symptoms, and faster progression.
That could in part inform the large variability in FA symptoms and presentation, according to researchers. It’s thus important to accurately quantify these repeats to understand their relationship to the disease’s clinical course.
Current options have technical limitations or aren’t practical for diagnostic use. The gold standard method is called southern blotting, which detects the GAA sequence, but this technique is low-throughput and time-consuming.
To overcome some of these limitations, researchers in India developed a new protocol for determining GAA repeat length. It relies in part on genetic sequencing using Oxford Nanopore technology, which allows for a real-time analysis of specific DNA sequences.
Quantifying GAA repeats
The scientists tested their approach on 11 patients with FA, who had varying ages of disease onset and clinical presentation.
Two of them, a 69-year-old man and a 55-year-old woman, were from the same family and had difficult to diagnose symptoms of cerebellar ataxia, a loss of muscle coordination that’s a symptom of FA, and other neuromuscular problems.
Standard genetic testing revealed no known disease-causing mutations for their symptoms. After several failed diagnoses, they were tested with the researchers’ pipeline.
GAA repeats were identified in both copies of the FXNÂ gene for both patients, with an estimated repeat number of more than 200 in one gene copy and more than 1,000 in the other. Other unaffected family members carried GAA repeats in one copy of their FXNÂ gene, with large variability in the length of repeats.
These two cases and the nine others were used to validate the approach for more accurately estimating GAA repeat length. The scientists ultimately found their technique was able to accurately quantify GAA repeats in a range from about 120 to 1,100.
It was also sensitive enough to identify “interruptions” to the GAA repeat sequence, places where nucleotides other than GAA are inserted. These interruptions have been found to potentially influence disease phenotypes.
As has been previously found, GAA repeat lengths on a person’s shorter copy of the FXN gene were found to correlate more accurately with clinical presentation, namely a person’s age at disease onset.
The finding shows the approach can establish genotype-phenotype relationships and potentially guide clinical management, according to the researchers.
Along with its accuracy, the researchers noted advantages to the high-throughput nature of the sequencing approach, which can cost-effectively screen up to 96 samples in under 24 hours. Taken together, the data indicate the method is “clinically scalable and deployable for day-to-day diagnostics,” the team wrote.
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