In a recent blog, I discussed the change in nomenclature for the conditions formerly known as Neurofibromatosis type 2 (NF2) and schwannomatosis. This change was based on the results of an international effort, sponsored by the Children’s Tumor Foundation (CTF), to review and update the diagnostic criteria of neurofibromatosis to include the most current research and clinical findings.
The updated diagnostic criteria for NF2 and schwannomatosis were published in 2022 in Genetics in Medicine, the official journal of the American College of Medical Genetics and Genomics (ACMG) (Updated diagnostic criteria and nomenclature for neurofibromatosis type 2 and schwannomatosis: An international consensus recommendation - ScienceDirect). In addition to changes in NF2 diagnostic criteria, the committee recommended the term “schwannomatosis” be used as an umbrella term for conditions that predispose to schwannomas, which includes the two conditions formerly known as NF2 and schwannomatosis, while further classifying each type of schwannomatosis by the gene containing the disease-causing pathogenic variant. As a result of these recommendations, the condition formerly known as NF2 is now called NF2-related schwannomatosis, while the other schwannomatosis-related conditions are known as SMARCB1-related schwannomatosis; LZTR1-related schwannomatosis; 22q-related schwannomatosis, and schwannomatosis - Not Elsewhere Classified (NEC) if no pathogenic variant was identified in blood and tumor, as well as schwannomatosis-Not Otherwise Specified (NOS) for those who have clinical features of NF2/schwannomatosis but have not had genetic testing. It is important to note that all three known schwannomatosis causal genes reside on chromosome 22q.
For the first time, the updated diagnostic criteria for schwannomatosis include genetic testing.
This month’s blog features insights from the Director of the UAB Medical Genomics Laboratory, Yulong Fu, Ph.D., FACMG, regarding the laboratory’s advanced genetic testing capabilities for schwannomatosis and the distinctive expertise the laboratory provides to clinicians and patients.
What is the current role of molecular genetic testing in establishing a diagnosis of schwannomatosis?
Genetic testing is important in establishing a diagnosis of schwannomatosis for a few important reasons. First, genetic testing has been incorporated into the updated diagnostic criteria for schwannomatosis published in 2022, whereas it was not included in the previous diagnostic criteria. Genetic testing is particularly helpful in cases where a clinical diagnosis may be inconclusive or needs to be confirmed. For example, regarding NF2-related schwannomatosis, an individual with only one vestibular schwannoma (hearing nerve tumor) may not develop a second feature for many years to meet a clinical diagnosis of the condition. However, genetic testing can help to establish a diagnosis for such individuals by identifying the pathogenic variant. Also, the new naming conventions for schwannomatosis are based on the disease-causing pathogenic variant. Molecular genetic testing is the only way to identify and distinguish the disease-causing pathogenic variant to establish a correct diagnosis.
What is the most frequently used testing approach for schwannomatosis in the Medical Genomics Laboratory?
Tumor testing is an important component of genetic testing for schwannomatosis. While blood testing using next-generation sequencing (NGS) is considered frontline genetic testing for NF1, we often are unable to determine a schwannomatosis-related variant using only blood. When no pathogenic variant is found in the three causal genes in the blood (NF2, SMARCB1, and LZTR1) using NGS, the only way to identify the variant is through tumor testing. We are the only laboratory in the U.S. to perform biopsy testing of tumor tissue, including fresh tumor and fixed tissue samples. This testing can be critical in achieving a diagnosis in patients who have mosaic schwannomatosis, in which only some of the cells in the body contain the pathogenic variant. Based on our experience, mosaicism in schwannomatosis is common in affected individuals, and tumor testing can identify the pathogenic variant in these individuals when blood testing is negative. Additionally, sometimes we identify a variant in these three genes based on blood testing, but the clinical significance of this variant could be unknown; we call this a variant of uncertain significance (VUS). Further testing on tumor tissue can help us determine the pathogenicity of this variant. For example, if an NF2 variant identified in blood is co-present with a unique pathogenic NF2 variant in two anatomically different tumors, we can upgrade this VUS to likely pathogenic. The same logic can apply to SMARCB1 and LZTR1.
How does the Medical Genomics Laboratory advise patients and physicians in the types of genetic testing that should be done and the samples needed to confirm a suspected diagnosis of schwannomatosis?
We maintain close communication with referring clinicians as well as UAB NF experts, which allows us to advise referring clinicians regarding the most appropriate testing strategies, sample types needed, and interpretation of the results.
A comprehensive schwannoma panel analysis of tumor consists of three components: 1) NGS analysis to identify small genetic lesions (single nucleotide variant and small Indels) in NF2, SMARCB1 and LZTR1; 2) copy number analysis of the three genes by multiplex-ligation dependent probe amplification (MLPA); and 3) loss of heterozygosity analysis for chromosome 22q by comparing twelve microsatellite markers between the tumor and unaffected tissue. We cannot complete this comprehensive analysis when unaffected tissue such as blood or saliva is not provided along with tumor tissues.
Ideally, we prefer to receive fresh tumor tissue, since this allows the most comprehensive testing. Alternatively, we can work with archived fixed specimens, including specimens that were surgically removed many years ago, although the quality of DNA obtained from these may be inadequate for all forms of testing.
What is the Laboratory Looking for?
When we test tumor-derived DNA, we are looking for changes in the three causal genes on chromosome 22. For NF2-related schwannomatosis, we expect to see loss of function in both copies of the NF2 gene – one will be a pathogenic variant that occurred sometime early in development and the other will have been acquired specifically in the cells that gave rise to the tumor. Often the second variant – commonly referred to as the “second hit” – is a deletion of part of chromosome 22. For SMARCB1- or LZTR1-related schwannomatosis, we expect to see a pathogenic variant in one of these two genes, a deletion of the long arm of chromosome 22, and a pathogenic NF2 variant residing on the same chromosome with the SMARCB1 or LZTR1 variant. If we have fresh/frozen tissue, we can do a comprehensive analysis to investigate if a tumor demonstrates such molecular features. However, with older archived tissue, usually due to inferior quality of DNA, the copy number analysis by MLPA could fail. In this case, loss of heterozygosity analysis can be done, but it becomes critical to have access to unaffected tissue to compare with the result in the tumor.
The goal for tumor testing is to look for a shared pathogenic variant among tumors from anatomically different locations in the body. Therefore, testing of a single tumor is usually inconclusive if mutations of both copies of a gene are found, as we cannot determine which variant is the common first hit by just looking at one tumor. We therefore strongly recommend sending at least two tumors for analysis, if possible. For some patients, if there is only one tumor available, we can offer targeted NGS, which has very high coverage for the targeted variant, to determine if one of the variants identified from the tumor is present in the blood at a low level. This targeted NGS can detect a rate of mosaicism as low as one percent. In other words, if there is one cell that carries the pathogenic variant among one hundred cells, this method is sensitive enough to detect it. It is not uncommon, however, for this targeted NGS to be negative. In this case, the results may be inconclusive unless a second tumor is eventually submitted for analysis. The targeted NGS analysis or testing on a second tumor is performed free of charge by the UAB Medical Genomics Laboratory.
For more information about the UAB Medical Genomics Laboratory and its testing capabilities for schwannomatosis and all other forms of NF, please visit: NF2 Landing - Genetics | UAB