New genetic test for difficult neurological diseases


WE have understood the hereditary nature of diseases hemophilia For thousands of years. However, it was only in the last century that scientists discovered this underpinning mechanism.

DNA is a long sequence of basic molecules, A, T, C, and G, that provide the genetic blueprint for our development and physiological functions. The complete DNA sequence of an individual is called the genome.

The first draft of a human genome was Completed in 2001 at a cost of US$3 billion. However, this draft of the genome was incomplete. Many large repetitive regions of DNA were missing. Next-generation sequencing platforms have since fueled a genetic revolution in medicine, with the current price of a whole genome under $1000, but these technologies are now reaching their technical limits. Current genomic tests still struggle to resolve large repetitive DNA sequences known as short tandem repeat (STR) expansion.

What is a short tandem rep?

An STR is a short DNA motif that is continuously repeated at a position within the genome. There are thousands of STR regions in the genome, which make up about 7% of the total human genome sequence. They are described by the number of repetitions of a specific motif in a specific location. For example, an STR region “CAG CAG CAG CAG CAG” would consist of five repeats of the motif “CAG”. The number of repetitions usually varies between different individuals. Because of this variation, we can identify individuals based on their repeat copy numbers in several known STR regions. The variation between STR regions is so unique that we often use this method for forensic DNA profiling.

In some cases, STR repeats can undergo large expansion, resulting in hundreds or even thousands of repeat copies at a given location. This causes gene instability, silencing and/or loss of function and can manifest itself in over 50 different degenerative neurological and neuromuscular diseases.

STR expansion disorders represent a large disease burden

In 1991, the first neurological disorders were associated with extension of an STR—that was Fragile X Syndrome and Kennedy disease (spinal and bulbar muscular atrophy). Since then there is about 50 disorders related to STR expansion, with clinical presentations including amyotrophic lateral sclerosis, frontotemporal dementia, myotonic dystrophy, Fredrich’s ataxia, hereditary cerebellar ataxia, and more. While each disorder is individually fairly rare, collectively they represent a major burden of degenerative disease in our community.

Because of the wide variety of disorders and presentations, these disorders are often difficult to diagnose clinically. For example, they can manifest as balance problems, cognitive decline, muscle degeneration, chronic cough, loss of sensation, uncontrollable movements, psychiatric disorders, seizures, or intellectual disability. These symptoms often develop insidiously, vary in severity between patients and are therefore difficult to diagnose.

Limitations in current clinical tests

Modern next-generation sequencing methods struggle to determine the number of iterations in larger expansions. Therefore, an old-fashioned DNA fragment analysis method known as Southern blotting is often still used as a reference for detecting large expansions and has remained virtually unchanged since then It was first used in the 20th century to diagnose fragile X syndrome. Southern blotting is slow, labor intensive, imprecise and requires a separate assay for each different STR on the list of possible gene candidates.

A newer, more efficient method known as repeat-primed polymerase chain reaction (RP-PCR) is widely used; but, these assays cannot accurately count large repeat expansions and are imprecise within certain complex regions. Likewise, a separate assay must be created for each different STR.

Since 2017, at least 13 new STR genes have been linked to hereditary diseases. Creating a new assay for each STR region is difficult and slow as there is still no robust clinical test available to clinicians for many of these disorders.

The diagnostic odyssey for recurrent disorders

Current genetic tests are 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 might be. These patients often go through a long diagnostic pathway, often lasting years, as the clinician must test every suspicious gene. In addition, tests for recently discovered genes/diseases are often unavailable in Australia.

Six patients in our study, Published in scientific advances, had a debilitating neurological syndrome called CANVAS (cerebellar ataxia, neuropathy, and vestibular areflexia syndrome) that went undiagnosed for years due to a lack of clinical testing. Currently, to our knowledge, there is only one accredited test for this disorder offered by a clinical laboratory abroad. This test is expensive, slow and often not offered to patients. For other disorders, such as oculopharyngodistal myopathy (OPDM), there are no commercially available tests for newly described STR enhancements.

This can be very distressing for patients and their families. Although there are no curative treatments for these conditions, a definite genetic diagnosis can be very beneficial. It can help patients manage their symptoms and overcome the 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 monitoring of other complications associated with each disorder, such as: Cardiac complications associated with Friedreich’s ataxia. A genetic diagnosis can be used for family planning, testing other family members, and performing prenatal or preimplantation genetic testing.

In addition, a genetic diagnosis can facilitate the enrollment of patients into clinical drug trials.

A new simple test for all 50 STR disorders at once

In our studywe have developed a single diagnostic test that is faster, more efficient, and more accurate than existing methods for genetically diagnosing disorders caused by STR expansion.

We used a sequencing technology known as nanopore sequencing, which allowed us to target any region within the genome using programmable coordinates. The sequencer can be programmed to recognize and reject specific DNA sequence fragments during a sequencing experiment. Eliminating laboratory-based molecular targeting allows us to choose new diagnostic targets at will, such as: B. newly discovered STR extensions.

This means we can target all 50 conditions in a single test, plus any additional targets needed by the clinician. Notably, we have already added certain pharmacogenetic genes that can predict adverse drug side effects in predisposed individuals.

For example, our custom test can diagnose a person with HD, predict the likelihood of passing the mutation on to their children, and create an individual drug metabolism profile for commonly prescribed antidepressants in HD 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 $1,000 to purchase compared to the hundreds of thousands needed for mainstream sequencing technology. We anticipate that these factors will provide incentives for enrollment in pathology labs within the next 2-5 years. We anticipate that state-of-the-art nanopore devices will readily replace older molecular methods such as Southern blotting.

Due to the flexible 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 rare disease patients receive an accurate genetic diagnosis, even with extensive whole-genome testing. It is likely that many of the unresolved patients have more difficult genetic alterations in difficult-to-sequence regions such as STR regions. With more robust targeted genome sequencing, potentially pathogenic regions are likely to be implicated in many diseases of unknown cause.

Sanjog Chintalaphani is a medical student at UNSW Sydney School of Medicine and St Vincent Clinical School. He collaborates with the Kinghorn Center 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 constitute the official policy of the AMA, the MJA or InSight+ unless otherwise stated.


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