Copy Number Variations Helpful in Spotting Potential CMT Mutations

Testing for large deletions or duplications in certain DNA regions, a form of mutation called copy number variations, is important in identifying new mutations that cause Charcot-Marie-Tooth disease (CMT), a study reports.

The study, “Comprehensive genetic sequence and copy number analysis for Charcot-Marie-Tooth disease in a Canadian cohort of 2517 patients,” was published in the Journal of Medical Genetics.

Mutations in over 70 different genes have been reported to cause CMT, though certain genes are more frequently implicated than others. The most common genetic cause of CMT is duplication of a gene called PMP22, leading to an extra copy of the gene — and, therefore, too much of the protein it encodes.

Understanding the spectrum of CMT mutations is important to understanding the underlying disease, and it provides a theoretical basis for developing targeted therapies.

This study details results of 2,517 genetic tests given people suspected of having CMT. They were carried out between 2015 and 2019 at the Molecular Genetics Laboratory at the London Health Sciences Centre (LHSC), which is the main center for CMT genetic testing in Ontario, Canada. The average age of individuals tested was 51.1, and 60% were males.

The researchers noted that males are more likely to develop conditions that resemble CMT, such as peripheral neuropathy associated with diabetes. This would be expected to lead to more referrals, but fewer positive tests.

The LHSC pipeline examines 34 genes previously linked to CMT. These genes are analyzed with two methods: one is genetic sequencing, which, as its name suggests, involves determining the exact sequence of a gene to identify small mutations. The other is an analysis of copy number variations (CNVs).

CNV refers to any genetic change where a gene, or a substantial part of a gene, is duplicated or deleted, meaning there are an abnormal number of copies of that gene or gene segment. The duplication of PMP22 is an example of a copy number variation.

Testing for CNVs in addition to genetic sequencing is important because sequencing alone cannot always detect copy number variations. For instance, in the case of PMP22 duplication, there wouldn’t be any sequence differences detected, because the sequence of the gene itself has not changed.

Even in instances where the genetic sequence is changed — for instance, if a large part of a gene is deleted — sequencing isn’t always able to detect these changes because of the particular molecular biology and computer analysis methods that are involved in sequencing.

Of the 2,517 tests performed at LHSC, 17.5% detected a genetic change that was definitely or likely a cause of CMT, leading to a positive diagnosis. As expected, there were more positive results in females than in males (21.5% vs. 15%).

Copy number variations in PMP22 accounted for over half (54%) of all positive results. Other commonly implicated genes included GJB1 (10%), MPZ (4.8%), FIG4 (4.1%), SH3TC2 (3.9%) and MFN2 (2.5%). Collectively, these six genes accounted for nearly four in every five positive tests.

A number of variants of uncertain significance (VUS) were also detected. As the name implies, a variant of uncertain significance is a mutation in a CMT-associated gene that is not known to actually cause disease, or be a benign happenstance. In the gene PRX, seven disease-causing mutations were detected, as were 117 VUS — a number the researchers described as “staggering.”

“Compiling and sharing this data may shed more light on pathogenicity mechanisms of genes, and may assist other clinical genomics professionals in the interpretation and classification of these less certain genetic findings,” they wrote.

The researchers also highlighted a copy number variation in the gene MARS, specifically a deletion of a portion of the gene. According to the researchers, this is “the first MARS CNV [reported] in a clinical testing setting,” emphasizing the importance of testing for copy number variations.

Mutations in MARS have been implicated in both CMT type 2U (CMT2U) and interstitial lung and liver disease (ILLD), but little is known about how different mutations in this gene contribute to either of these diseases.

“Thus, reporting of this deletion in a patient undergoing screening for CMT is important for enhancing our knowledge base of MARS’ mutational spectrum,” the researchers wrote.

“Taken together,” the researchers concluded, “we significantly expand the mutational spectrum of CMT genes and share our experience and variant interpretations for the benefit of genetics professionals engaged in hereditary neuropathy testing and patient counselling.”