DALLAS—Genetic testing is an integral part of diagnosing neuromuscular diseases such as Duchenne muscular dystrophy, and the field continues to evolve with new technologies. At the 2025 Muscular Dystrophy Association Clinical & Scientific Conference, Jordan Bontrager, M.S., from the University of Rochester Medical Center, discussed the latest advancements.
Genetic testing is a test that finds abnormal changes occurring in genes, chromosomes, or proteins. It can be used to confirm a diagnosis, cross out a suspicion of a genetic condition, or determine the chance of developing a disorder or passing it onto a child.
Emerging technologies under investigation include optical genome mapping (OGM), long-read sequencing (LRS), and RNA sequencing. Below is an overview of genetic testing technologies, from the oldest methods to the newest.
Karyotyping and FISH
Chromosomal analysis and karyotyping were introduced in the 1950s and 1960s, while fluorescence in situ hybridization (FISH) was developed in 1982. They are primarily used to detect large chromosomal abnormalities, such as whole, extra, or missing chromosomes, Bontrager explained.
Southern blot
Developed in 1973, Southern blot is often used to diagnose repeat-expansion disorders —genetic conditions caused by the abnormal repetition of DNA sequences — including myotonic dystrophy, Huntington disease, and Friedreich ataxia. It uses a special protein called a restriction enzyme to cut DNA into pieces, which are then sorted by size using a technique called electrophoresis (a method that moves DNA fragments through a gel using electricity). This process requires a large amount of high-quality DNA, meaning the fragments must be intact and free from interfering substances. Due to these limitations, “many labs are moving away from Southern blots,” Bontrager noted.
Sanger sequencing
Sanger sequencing, developed in 1977, assesses small, specific changes in a person’s genes by determining the exact order of DNA building blocks. Once the standard for genetic testing, it is now used less often. “It is becoming much less used as routine care because of A) the cost, B) the limitations of the technology, and C) we have easier methods with next generation sequencing (NGS) approaches,” Bontrager told attendees.
Microarray
Microarray (array CGH or aCGH) is a common genetic test for individuals with developmental delays, intellectual disabilities, birth defects, or certain neurological conditions. It can detect missing or extra pieces of DNA, offering more detailed results than older methods like karyotyping. First used in the early 2000s, it helps identify genetic changes where the number of DNA copies differs from what is typically expected.
Next generation sequencing
Developed in the mid-2000s, NGS allows doctors to quickly examine many genes at once or even entire genomes. “Almost all genetic testing . . . is done via next generation sequencing technologies,” Bontrager noted.
Optical genome mapping
Originally used mainly for research, OGM is now being considered for clinical diagnoses. It works by labeling sections of DNA with a unique “barcode” to create a map, which can be compared to a reference genome. This helps doctors identify large-scale changes in DNA, similar to older tests like karyotyping or microarrays.
Compared to older testing methods, OGM offers several advantages, including better detection of certain genetic changes, greater detail than microarrays, and the ability to identify changes in conditions related to repeat expansions or mixed genetic backgrounds.
However, OGM has some challenges. It requires a very high-quality DNA sample, which can be difficult to obtain, especially from young patients who might not have enough DNA in their blood samples. Additionally, it has limitations in detecting more complex genetic mutations.
Long-read sequencing
LRS allows doctors to examine longer strands of DNA or RNA, ranging from 1,000 to more than 20,000 bases, or DNA building blocks. It can help identify important alterations in the DNA, such as structural changes, certain genetic variations, repeat expansions, and patterns linked to rare disorders. LRS helps eliminate uncertainty in assembling the complete genome by reducing the risk of mapping errors or missing information.
However, LRS is more expensive than the more common short-read sequencing method and is often not covered by insurance.
“Right now, short-read and long-read are being friends,” Bontrager said. “Long-read is most useful at this point if you have a . . . high suspicion for a specific disorder, you’ve really done everything under the sun from typical testing methodologies, and are very suspicious that there is some complex variant present that we haven’t been able to detect yet.”
Limitations in reliability
Despite advances in genetic testing, it is not always fully accurate. At the same conference session, Natalie Katz, M.D., Ph.D., from Duke University, presented a case highlighting these limitations.
A young girl was first seen at age 13 for muscle weakness she had been experiencing for two years. Despite showing all the signs of a condition called facioscapulohumeral muscular dystrophy (FSHD), her genetic test and muscle biopsy did not show any problems. When the test was repeated at another time, the results were still normal.
At age 33, a different test suggested FSHD, and further testing finally confirmed the diagnosis. The patient had lived with an undiagnosed condition for 33 years because of repeated negative test results. Dr. Katz explained that the misdiagnosis might have been caused by a small change in the DNA that tests at the time couldn’t detect, though the exact reason remains unclear.
Dr. Katz emphasized that while FSHD might be obvious based on symptoms, it can be challenging to confirm through genetic testing alone.
