WE have understood the hereditary nature of diseases such as haemophilia for thousands of years. However, it was only in the past century when scientists discovered the underpinning mechanism.
DNA is a long sequence of base molecules, A, T, C and G, that provides the genetic blueprint for our development and physiological functions. The full DNA sequence of an individual is known as their genome.
The first draft of a human genome was completed in 2001 at a cost of $3 billion. However, this draft genome was incomplete. It was missing many large repetitive DNA regions. Next-generation sequencing platforms have since driven a genetic revolution in medicine, with the current price of a whole genome under $1000, but these technologies are now being stretched to their technical limits. Current genomic testing still struggles to resolve large repetitive DNA sequences known as short tandem repeat (STR) expansions.
What is a short tandem repeat?
An STR is a short DNA motif that is repeated consecutively at a position within the genome. There are thousands of STR regions in the genome, comprising around 7% of the entire human genome sequence. They are described by the number of repetitions of a given motif at a given site. For example, an STR region “CAG CAG CAG CAG CAG” would be five repeats of the motif “CAG”. The number of repeats commonly varies between different individuals. Because of this variation, we can identify individuals based on their repeat copy numbers at multiple known STR regions. Variation across STR regions is so unique that we commonly use this method for forensic DNA profiling.
In some cases, STR repeats may undergo a large expansion, resulting in hundreds or even thousands of repeat copies at a given site. This causes gene instability, silencing and/or loss of function and can manifest in over 50 different degenerative neurological and neuromuscular conditions.
STR expansion disorders represent a wide burden of disease
In 1991, the first neurological disorders were linked to an expansion of an STR – these were fragile X syndrome and Kennedy’s disease (spinal and bulbar muscular atrophy). Since then, there have been roughly 50 disorders linked to STR expansions, with clinical presentations including amyotrophic lateral sclerosis, frontotemporal dementia, myotonic dystrophy, Fredrich’s ataxia, hereditary cerebellar ataxias, and more. While each disorder is individually quite rare, collectively they represent a large burden of degenerative diseases across our community.
Due to the wide variety of diseases and presentations, these disorders are often difficult to diagnose clinically. They may present as balance issues, cognitive decline, muscular degeneration, chronic cough, loss of sensation, uncontrollable movements, psychiatric disorders, seizures, or intellectual disability, for example. These symptoms often develop gradually, vary in extent between different patients, and are therefore difficult to diagnose.
Limitations in current clinical testing
Modern next-generation sequencing methods have difficulty in determining the number of repeats in larger expansions. Thus, an old-fashioned DNA fragment analysis method known as Southern blotting is often still used as a reference to detect large expansions, and is virtually unchanged since it was first used to diagnose fragile X syndrome in the 20th century. Southern blotting is slow, labour-intensive, imprecise and requires a separate assay for every different STR on the list of possible gene candidates.
A newer more efficient method, known as repeat-primed polymerase chain reaction (RP-PCR), has been widely adopted; however, these assays cannot accurately count large repeat expansions and are inaccurate within certain complex regions. Similarly, a separate assay for every different STR must be created.
Since 2017, at least 13 new STR genes have been linked to inherited disease. Creating a new assay for each STR region is difficult and slow, with many of these disorders still lacking a robust clinical test which is available to clinicians.
The diagnostic odyssey for repeat disorders
Current genetic testing is often “hit and miss”. When a patient presents with neurological symptoms it is difficult to decide whether they have an STR disorder and which of the 50 STR disorders it may be. These patients often travel down a long diagnostic path, often lasting years, as the clinician must test each gene suspected. On top of this, testing for recently discovered genes/diseases is often unavailable in Australia.
Six patients in our study, published in Science Advances, had a debilitating neurological syndrome known as CANVAS (cerebellar ataxia, neuropathy and vestibular areflexia syndrome) which was undiagnosed for years due to a lack of clinical testing. Currently, and to our knowledge, there is only one accredited test for this disorder offered by a clinical laboratory overseas. This test is expensive, slow, and often unfeasible to offer to patients. Other disorders, such as oculopharyngodistal myopathy (OPDM), have no commercially available testing for newly described STR expansions.
This can be quite stressful for patients and families. While there are no curative treatments for these disorders, giving a clear genetic diagnosis can be very beneficial. It may help patients deal with their symptoms and overcome anxiety associated with an unknown diagnosis. A clear diagnosis also helps patients avoid unnecessary tissue biopsies or risky immunosuppressive treatment. It will also guide careful surveillance of other complications associated with each disorder, for example, cardiac complications associated with Friedreich’s ataxia. A genetic diagnosis can be used for family planning, testing other family members, and provides the opportunity for prenatal or pre-implantation genetic testing.
Furthermore, a genetic diagnosis may facilitate enrolment of patients for clinical drug trials.
A new simple test for all 50 STR disorders at once
In our study, we created a single diagnostic test that is quicker, more efficient, and more accurate than existing methodologies for genetic diagnosis of disorders caused by STR expansions.
We used a sequencing technology known as nanopore sequencing which enabled us to target any region within the genome using programmable coordinates. The sequencing device can be programmed to recognise and reject specific DNA sequence fragments during a sequencing experiment. The removal of laboratory-based molecular targeting allows us to select new diagnostic targets, such as newly discovered STR expansions, at will.
This means that we can target all 50 disorders in a single test, and any additional targets required by the clinician. Of note, we have already added certain pharmacogenetics genes that can predict harmful adverse drug reactions in predisposed individuals.
As an example, our custom test can diagnose an individual with Huntington’s disease, predict the likelihood of passing on the mutation to their children, and generate an individualised drug metabolic profile for commonly prescribed antidepressants in Huntington’s disease, such as citalopram and escitalopram, thereby avoiding high risk of drug toxicity.
The future of genetic testing
The nanopore technology used in this test is smaller and cheaper than current clinical tests. The device is the size of a stapler and costs around $1000 to purchase, compared with hundreds of thousands needed for mainstream sequencing technology. We expect that these factors will incentivise uptake into pathology labs within the next 2–5 years. We anticipate that older molecular methods such as Southern blotting will be readily replaced by cutting-edge nanopore devices.
Due to the flexible targeted nature of this technology, we also anticipate that it will be an excellent tool for discovering new genetic links to suspected hereditary diseases.
It is estimated that less than 50% of patients with rare diseases receive an accurate genetic diagnosis even with extensive whole-genome testing. It is likely that many of the unsolved patients have more challenging genetic changes in difficult-to-sequence regions, such as STR regions. With more robust targeted genomic sequencing, potential pathogenic regions will likely be linked to many diseases with an unknown cause.
Sanjog Chintalaphani is a medical student at UNSW Sydney’s School of Medicine and St Vincent’s Clinical School. He works with the Kinghorn Centre for Clinical Genomics at the Garvan Institute of Medical Research.
The statements or opinions expressed in this article reflect the views of the authors and do not represent the official policy of the AMA, the MJA or InSight+ unless so stated.