Genetic Testing for NF1
Next-generation sequencing (NGS), using blood or saliva samples, is the most frequently used genetic test to detect variants that cause the condition (referred to as “pathogenic variants”) in the NF1 gene. NGS involves sequencing of the entire genome, or, in some cases, the entire component of the genome that is encoded for protein (referred to as the “exome”). A gene is encoded in segments, called exons, which code for the amino acids of a protein, separated by introns, which are intervening sequences. Most NGS technology is capable of detecting variants in the exons and around the borders of introns and exons. However, occasionally a variant exists deep in the intron that is more difficult to detect.
RNA-Based Testing
RNA-based testing might be used when a pathogenic variant cannot be identified using NGS. RNA is the molecule that copies the genetic information for a particular protein from DNA and moves it to the part of the cell where protein is made. The UAB Medical Genomics Laboratory can perform NF1 RNA testing, which can be done in cases of a negative or inconclusive NF1 result using NGS. RNA-based testing is a highly specialized, intensive process that requires well-trained technicians with distinct expertise. It detects an abnormality in the RNA copy (mRNA) of the NF1 gene and can be used to identify a genetic variant deep within the intron. Some individuals who have received an inconclusive result with NGS can receive an NF1 diagnosis with RNA-based testing.
Mosaicism
Mosaicism is also a possibility in individuals with clinical features of NF1 who receive a negative genetic test result. In these cases, the NF1 pathogenic variant affects some cells in the body but not all due to acquisition of a mutation during development. NGS can sometimes detect a minor cell population in the blood that can confirm mosaicism, but if cells with the variant are not present in blood in sufficient numbers, they will not be detected. The next best approach is testing of cells in neurofibromas or café-au-lait spots. The UAB Medical Genomics Laboratory is one of few labs anywhere equipped to perform testing of these tissues; this technique requires obtaining biopsies of tumors or café-au-lait spots (preferably two or more) and transporting tissue that contains Schwann cells (neurofibromas) or melanocytes (café-au-lait spots) to be grown in a cell culture system.
While the UAB laboratory stopped tumor testing during the COVID-19 pandemic, they are currently performing final validation to resume tumor testing and have already resumed offering melanocyte cultures for café-au-lait spots. Because of the “two-hit” genetic mechanism that causes the development of tumors and café-au-lait spots in individuals with NF1, one would expect two NF1 pathogenic variants to be present in the tumor Schwann cells or café-au-lait spot melanocytes; one is an acquired mutation, and one is the mosaic variant. All people have two copies of the NF1 gene, one inherited from each parent. In individuals with NF1, one copy of the NF1 gene is altered (the variant) due to either inheriting the variant-containing gene from a parent, or a new mutation that occurs in the egg or sperm prior to conception, or from a mutation that occurs early in embryonic development. This represents the “first-hit” genetic variant. For a neurofibroma or café-au-lait spot to develop in someone with NF1, a random genetic mutation must occur to the other copy of the NF1 gene in the tissue that will become the neurofibroma or café-au-lait spot; this is referred to as the “second-hit” variant.
Genetic Testing for NF2 and Schwannomatosis
Blood samples are often used to perform NGS to identify a pathogenic variant in the NF2 gene that confirms a diagnosis of NF2. However, many individuals with NF2 have mosaicism, which can only be detected by testing tumor tissue. For this testing, fixed tissue, even tissue that may have been archived for years, can be used. Mosaicism is also common in people who have schwannomatosis. Here, too, tumor testing, including testing of archived fixed tissue, can be done if blood testing is negative.
When these approaches do not produce a definitive answer, it is important to note that, regarding NF2, both vestibular schwannomas and meningiomas can occur in individuals in the general population who do not have NF2. In the case of schwannomatosis, it is possible that other genes yet to be discovered may also be associated with the condition.
Individuals with Multiple Neurofibromas
It is known that the gene product of NF1, neurofibromin, functions as a regulator of a protein called RAS. RAS is a key component of the RAS/MAPK cell signaling pathway that controls cell growth and development. This pathway involves multiple proteins that are involved in transmission of signals received at the cell surface to regulate genes in the cell nucleus. It has been learned that variants in genes that encode these other proteins in the pathway also can lead to medical disorders, which are collectively called “RASopathies.”
One of these disorders, Noonan syndrome, is a distinct disorder from NF1, but in rare instances can cause individuals to develop neurofibromas around the spine, similar to NF1. Noonan syndrome is characterized by short stature, cardiac defects, characteristic facial appearance, and learning and developmental problems. Genes associated with Noonan syndrome (there are several, all encoding proteins that act in the RAS signaling pathway) can be tested in individuals with multiple spinal neurofibromas who have negative NF1 testing. It should be noted that some people with NF1 have some features that overlap with Noonan syndrome, which is not surprising given that both conditions involve changes in genes that encode proteins acting in the RAS signaling pathway. Some of these individuals are given the label “NF-Noonan syndrome.” It should be noted, though, that this is distinct from Noonan syndrome itself, due to changes in genes other than NF1; these individuals do not have two separate disorders, but rather have NF1 with some features reminiscent of Noonan syndrome.
Also, some individuals who have NF-like symptoms such as café-au-lait spots and skin fold freckles may have a pathogenic variant in the SPRED1 gene that is associated with a condition called Legius syndrome, another disorder associated with disruptions in the RAS/MAPK pathway. This condition, which is also associated with learning disabilities, can be impossible to distinguish from NF1 in young children who have only café-au-lait spots and skin fold freckles and no family history of NF1. Diagnosis of Legius syndrome is confirmed by NGS of the SPRED1 gene, which is usually tested in a panel alongside the NF1 gene.
Explanations for Negative Genetic Tests
If all genetic testing options are exhausted, there are additional possibilities. First, the clinical diagnosis of NF may be inaccurate and should be reevaluated. For example, the individual could have symptoms of another disorder, such as a RASopathy, that overlaps with NF features. Another explanation is that there are other genetic mechanisms yet to be identified that are associated with the clinical features. Newer genomic testing technologies may be able to identify these genes in the future. Still, patients with clinical features for whom genetic testing is negative should be followed clinically by an NF specialist.