Short-read genome sequencing test may help diagnose atypical FA

Test advised for patients whose symptoms, family history are not indicative of FA

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by Andrea Lobo |

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Short-read genome sequencing (SR-GS), a test that can identify difficult-to-detect mutations, may help to correctly diagnose atypical Friedreich’s ataxia (FA) in people whose symptoms and family history are not indicative of FA, a study in Germany shows.

The study, “Short-read genome sequencing allows ‘en route’ diagnosis of patients with atypical Friedreich ataxia,” was published in the Journal of Neurology.

FA is a genetic condition in which mutations in the FXN gene result in damage to multiple bodily systems, particularly the nervous system and muscles. The disease causes a progressive loss of muscle control during voluntary movements — a condition known as ataxia.

The most common FA-causing genetic mutation is a repeat expansion in which three nucleotides (the building blocks of DNA) are repeated an excessive number of times.

In normal circumstances, the FXN gene contains a region — called a short tandem repeat (STR) — where the GAA sequence is repeated about 5 to 33 times. In most FA cases, however, the STR region is mutated and contains a much higher number of GAA repeats — ranging from 66 to more than 1,000 repeats — in both gene copies.

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Genetic sequencing may aid accurate tally of FA-causing GAA repeats

Ataxias diagnosed based on symptoms, family history, and more common tests

Usually, diagnosis of ataxias is made based on symptoms and family history, as well as genetic testing of the specific genes that could be mutated to cause the suspected ataxia. However, common sequencing techniques have moderate specificity to detect STR expansions and fail almost completely to detect these repeats in non-coding regions, as can happen in FA.

Thus, if a patient is suspected of having FA based on symptoms and clinical history, the proper genetic test will be prescribed. However, if a person has atypical symptoms, the selected sequencing method usually will not be able to detect FA-associated mutations, and patients will not receive an adequate diagnosis.

Short-read sequencing is currently the most commonly used form of next-generation sequencing, a method that allows the sequencing of many DNA strands at the same time. In SR-GS, the genome is broken into small fragments before being sequenced, allowing the identification of mutations in coding and non-coding regions of the genome, including STR extensions.

In the recent case series, researchers in Germany demonstrated that SR-GS can help overcome these issues, by detecting mutations in several ataxia-related genes, as well as the intronic STR extensions associated with FA.

The team performed SR-GS on 127 ataxia patients recruited at the ataxia outpatient clinics of the Center of Neurology in Tübingen, Germany, from 2021 to 2022. The method identified three FA patients with GAA expansions in both FXN gene copies, in whom FA had not been considered at the initial diagnostic workup.

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The first patient, a 64-year-old woman, had slowly progressive weakness and stiffness in her leg muscles and gait ataxia since she was 53, which progressed to difficulty speaking and trouble swallowing, limb ataxia, and bladder incontinence. She had no scoliosis or foot deformities, but her brother had a similar progressive disease starting at a similar age.

Initial MRI scans of the brain and spinal cord failed to reveal any abnormalities, and sequencing of genes that commonly cause ataxias did not reveal a clear genetic cause. Sequencing of the genome coding regions revealed a genetic variant in the ABCD1 gene, which could be associated with the patient’s symptoms.

SG-GS, conducted to rule out more convincing genetic causes, revealed GAA repeat expansions in both copies of the FXN gene — 98 repeats in one copy, and 1,460 in the other, indicating an FA diagnosis.

Another patient, a 59-year-old woman, developed gait disturbances with weak leg muscles, limb ataxia, and column dysfunction since age 45. She also reported bladder incontinence and had exaggerated tendon reflexes. Her father had similar gait problems.

Laboratory tests and a brain and spinal cord MRI did not provide evidence for any common secondary causes of ataxia. Also, common genetic tests did not reveal a genetic cause.

When SR-GS was performed, it found an intronic GAA expansion in both FXN gene copies, which were determined to have 144 and 650 GAA repeats, confirming an FA diagnosis.

The third case was of a 58-year-old man with slowly progressive weakness and muscle stiffness in all four limbs starting at age 49. He also had limb ataxia, difficulty speaking and swallowing, and bladder incontinence, but no obvious cause was detected on laboratory and imaging analyses.

[Short-read genome sequencing] should therefore be considered early in the genetic diagnostic work up of patients with suspected hereditary neurological disorder.

Although initial genetic testing revealed a mutation in the PLEKHG4 gene, also identified in his father who had a similar gait impairment, the gene’s link with ataxia had not been fully demonstrated. Thus, an SR-GS analysis was performed and revealed an STR expansion in both FXN gene copies, determined to have 87 and 1,140 GAA repeats, confirming his FA diagnosis.

“Our findings demonstrate that SR-GS [can] identify ataxia subjects with … GAA expansions in FXN, where FA had not been part of the initial differential diagnosis, despite the fact that they were seen by ataxia experts with [more than] 10 years of genetics ataxia experience,” the researchers said.

The difficulty in diagnosis was associated with atypical symptoms and age of symptom onset, misleading family history, and identification of other genetic variants in genes that could be associated with the identified symptoms.

“[SR-GS] should therefore be considered early in the genetic diagnostic work up of patients with suspected hereditary neurological disorder, in particular in ataxias where intronic STRs are a common mutational mechanism,” the researchers concluded.