FXN gene methylation may help diagnose carrier status in FA

Small chemical tags in DNA may ID carriers with no symptoms: Study

Patricia Inácio, PhD avatar

by Patricia Inácio, PhD |

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Assessing the levels of methylation — small chemical tags on specific DNA regions — in the FXN gene may help identify Friedreich’s ataxia (FA) carriers showing no disease symptoms, a study suggests.

Making such a diagnosis is often “fraught with technical difficulties,” according to researchers, who sought “a reliable way to identify” such carriers.

“We show that the methylation pattern of the [FA gene region] allows accurate identification,” the team wrote.

The study, “FXN gene methylation determines carrier status in Friedreich ataxia,” was published in the Journal of Medical Genetics.

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Gene methylation levels may be new diagnostic tool

FA is caused by mutations in the FXN gene, which contains the instructions for the protein frataxin. The most common is excessive repeats of three nucleotides, or DNA building blocks — one guanine (G) and two adenines (A), GAA — in a region of the gene.

In healthy people this sequence is repeated up to 33 times, but those with FA may have more than 1,000 repeats. The abnormal number of repeats silences FXN gene activity and is accompanied by an epigenetic mechanism called methylation, which is highly predictive of FXN activity deficiency and age of FA onset.

Most people with FA are homozygous, which means they have excessive repeats in both copies (alleles) of the FXN gene — each copy being passed down from one biological parent. In a minority of cases (around 5%), the GAA expansion affects only one copy of the gene and a different mutation is seen in the other copy. This type of genetic profile is known as compound heterozygous.

Heterozygous carriers have GAA repeat expansions in one copy of the gene and no other mutation in the other copy. Carriers of this type of mutation retain around 50% of frataxin protein levels and are asymptomatic.

One issue for clinicians is that the detection of heterozygous carriers is technically challenging. According to researchers, certain testing that is “highly effective at diagnosing homozygous [carriers], has a high false-negative rate for heterozygous” individuals.

Starting with an assumption that asymptomatic carriers have around 50% methylation in the target FXN gene region for silencing to occur, a team of scientists U.S. now sought to determine whether profiling the level of methylation could effectively detect heterozygous carriers.

To that end, blood samples from 32 asymptomatic heterozygous carriers and five compound heterozygous carriers were collected at the Children’s Hospital of Philadelphia. These samples were used to extract DNA. All participants were positive for excessive GAA repeats, which ranged from 385 to 1,350.

Methylation levels ranged from 25.4% to 70% among the 37 carriers. Alleles methylated at all target sites ranged between 2.3 to 41.3%, and significantly correlated with the length of the repeats.

Since most modern molecular diagnostic labs have next-generation sequencing capabilities, the … test used here offers a … method to accurately identify or rule out [carrier status].

The researchers then compared the methylation profile of the heterozygous carriers to individuals without FA and to homozygous FA patients. The patients were further divided into two groups according to repeat numbers — those with more than 500 repeats in both FXN copies in one group, and individuals with fewer than 500 repeats in one copy in the other.

The amount of methylation enabled clear distinction of heterozygous carriers from individuals without FA, as well as from both groups of patients with FA, the researchers noted.

Heterozygous carriers had significantly higher levels of methylation compared with individuals without FA, but significantly less compared with FA patients.

The findings suggest that FXN gene methylation is a “reliable indicator of heterozygous carrier status in [FA],” the researchers wrote.

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Testing the method in a boy with FA symptoms

The team then tested this method in a boy with typical FA symptoms, including lack of motor coordination, an enlarged heart muscle, and scoliosis, or a sideways curvature of the spine. His condition worsened, and he needed a wheelchair when he became a teenager.

He had been previously identified to carry a mutation in the paternally inherited allele of the FXN gene (known as c.2delT), but no excessive GAA repeats were found using standard molecular analysis conducted by two different labs. His mother also did not have excessive repeats according to one of the tests.

However, the boy showed a deficiency in frataxin protein levels, which was consistent with an FA diagnosis and suggested that the expansion repeat was likely being missed with standard techniques.

The boy, his parents and several maternal relatives were then analyzed for methylation in the FXN gene. Methylation levels were consistent with heterozygous carrier status of the boy’s mother, grandmother and two maternal uncles. This means the boy was a compound heterozygous with GAA repeat expansion in one gene copy and a c.2delT mutation in the other.

Overall, assessing methylation levels of the FXN gene “reliably detects the GAA-TRE [GAA-riple nucleotide expansion] in the heterozygous state and offers a robust alternative strategy to diagnose [FA] due to compound heterozygosity and to identify asymptomatic heterozygous carriers,” the study concluded.

The team said this strategy could be used among patients.

“Since most modern molecular diagnostic labs have next-generation sequencing capabilities, the … test used here offers a … method to accurately identify or rule out” carrier status, the researchers wrote.

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