Genetic Mutations

Research into NF1 has tended to follow trends in molecular and biochemical techniques. With advancements in technology, mechanisms at the genetic, molecular, and cellular levels that NF1 utilizes for its pathogenesis have begun to become elucidated, providing greater insight into how the disease functions. Through all levels of our discussion of NF1, the trend of extreme variability will be present. This variability in onset of disease makes it difficult for biochemical characterization, and even treatment, of the disease.

With the advent of recombinant DNA techniques in the 1970s, research into the genetic basis of NF1 quickly followed suit. Understanding the genetic mutations conferring NF1 will provide a good foundation for understanding the downstream effects of the disease. The story of the location of the genetic mutation leading to NF1 does not start with NF1, but neurofibromatosis type 2 (NF2). NF2 is more rare than NF1, and is characterized by tumors on the eighth cranial nerve.¹ Extensive genetic studies were conducted by the Seizinger group on NF2 using polymorphic DNA markers to center its genetic mutation to chromosome 22.^2,3 Using the same technique, Seizinger extended this work to NF1, and found that the NF1 mutation generally lies on the locus encoding the receptor for nerve growth factor on chromosome 17.^3,4

Allelic location of the NF1 mutation set the stage for deeper genotype work on the disease. Mutations have been found to occur near translocation breakpoints on chromosome 17.^3,4 Specifically, a t(1;17) translocation or a t(17;22) translocation in the NF1 gene causes NF1 disease.^3,4 This gene mutation is dominant and can be inherited from a parent or occur spontaneously (the number of cases inherited and acquired due to spontaneous mutation are roughly equal).⁵  As genotyping techniques improved, an increased number of genetic mutations leading to NF1 have been found. The genetic mutations are evenly distributed throughout the NF1 gene, and the number of them seems arbitrary to a certain extent (there are at least 33 publications on novel mutations leading to NF1). The mutations causing NF1 include ones leading to errors in mRNA splicing of the NF1 gene product, leading to prematurely truncated proteins being translated and mutations directly affecting the fragile structure of the protein, both leading to decreased functionality of the gene product.⁶ With the discovery of the location of mutations causing NF1, Xu et al. were able to utilize cDNA technology to find that the product of the mutated gene has significant homology to the catalytic domain of the yeast IRA1 product, which was known at the time to be very similar to the mammalian GTPase-activating protein (GAP).³ The GTPase activity of the NF1 gene product, neurofibromin, was confirmed in the same year as it was discovered that it was able to change the phosphorylation state of ras p21.⁷

Protein Mutations

Concurrently with the discovery of the translocation causing NF1, it was found that neurofibromin has significant homology to the yeast IRA1 product, which is very similar to the mammalian GTPase-activating protein (GAP).³ The importance of the mammalian GAP to cell-signaling pathways led to hypotheses heralding the role of neurofibromin to be that of regulating cell-signaling pathways. Later studies found that neurofibromin stimulates GTPase activity of ras p21, with a much higher affinity than mammalian GAP, confirming the GAP activity of neurofibromin.⁷ Although the mutations leading to NF1 are numerous and may vary significantly from one another, general trends can be understood at the level of the neurofibromin protein. The neurofibromin crystal structure was only solved in 2021, but this breakthrough allowed for illuminating insight into the molecular scaffolding governing NF1 disease. Neurofibromin must assemble into a homodimer to be functional. The quaternary structure of the protein takes on a “figure-eight”-like shape. The “core” of the protein, required for assembly of the homodimer, is formed by the N- and C-HEAT domains of each protein comprising the dimer coming together, in a “helical packing” interface.⁸ Mutations have been found affecting both the N-HEAT (L844F) and C-HEAT (L1834R, N1840K, L2104R) domains, interfering with helical packing of the homodimer, leading to neurofibromin being ineffective and causing NF1.⁸ Other mutations have been associated with destabilizing the neurofibromin dimer interface.⁸ The variety and seemingly uniform spread of these mutations suggest that the complexity of the folds of the neurofibromin protein make it so that any mutations are likely to lead to disease, regardless of location.¹⁸ This suggestion is strengthened when the primary sequence of human neurofibromin is compared across different species. Neurofibromin has been found to be highly conserved in mammals, with the human neurofibromin primary sequence being 98.5% to that found in rats, and 99.4% to that found in dogs, implying strong selective pressures on the structure and function of neurofibromin.⁷ Nature tends to conserve the structure of key proteins and enzymes. This significant conservation in sequence implies the importance of the specific amino acids that comprise neurofibromin, elucidating the adverse effects that point mutations can have.

Neurofibromin is a regulatory protein that is very susceptible to loss-of-function (LOF) mutations. Nature may have evolved single regulatory proteins controlling a multitude of pathways because of the efficiency and energy conservation this grants. However, when one protein is responsible for the operation of multiple crucial signaling pathways, there is a cause of worry for mutations. The susceptibility of neurofibromin to mutations is intriguing due to its presence in many mammals, and its importance for survival. Nevertheless, neurofibromatosis type I is a disease that starts at the genetic level, where mutations (either inherited or acquired) on chromosome 17 have effects on neurofibromin. The severity of this disease is due to the importance of neurofibromin, and its role in regulating cytosolic cell pathways.

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Works Cited

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(2) Seizinger, B. R.; Martuza, R. L.; Gusella, J. F. Loss of Genes on Chromosome 22 in Tumorigenesis of Human Acoustic Neuroma. Nature 1986, 322 (6080), 644–647. https://doi.org/10.1038/322644a0.

(3) Xu, G.; O’Connell, P.; Viskochil, D.; Cawthon, Richard M.; Roberston, M.; Culver, M.; Dunn, D.; Stevens, J.; Gesteland, R.; White, R.; Weiss, R. The Neurofibramotosis Type 1 Gene Encodes a Protein Related to GAP. Cell 1990, 62, 559–608.

(4) Cawthon, Richard M.; Weiss, R.; Xu, G.; Viskochil, D.; Culver, M.; Stevens, J.; Roberston, M.; Dunn, D.; Gesteland, R.; O’Connell, P.; White, R. A Major Segment of the Neurofibromatosis Type 1 Gene: CDNA Sequence, Genomic Structure, and Point Mutations. 1990, 62, 193–201.

(5) Gorelick, K. Inheritance and Genetics of Neurofibromatosis Type 1 (NF1) – School of Medicine – Neurofibromatosis Program | UAB https://www.uab.edu/medicine/nfprogram/learn/neurofibromatosis-type-1-nf1/inheritance-genetics (accessed 2022 -04 -24).

(6) Ars, E.; Serra, E.; García, J.; Kruyer, H.; Gaona, A.; Lázaro, C.; Estivill, X. Mutations Affecting MRNA Splicing Are the Most Common Molecular Defects in Patients with Neurofibromatosis Type 1. Hum. Mol. Genet. 2000, 9 (2), 237–247. https://doi.org/10.1093/hmg/9.2.237.

(7) Martin, G.; Viskochil, D.; Bollag, G.; McCabe, P.; Crosier, W.; Haubruck, H.; Conroy, L.; Clark, R.; O’Connell, P.; Cawthon, Richard M.; Innis, M.; McCormick, F. The GAP-Related Domain of the Neurofibromatosis Type 1 Gene Products Interacts with Ras P21. 1990, 63, 843–849.

(8) AboutKidsHealth https://www.aboutkidshealth.ca:443/article?contentid=866&language=english (accessed 2022 -04 -26).

(9) Lupton, C. J.; Bayly-Jones, C.; D’Andrea, L.; Huang, C.; Schittenhelm, R. B.; Venugopal, H.; Whisstock, J. C.; Halls, M. L.; Ellisdon, A. M. The Cryo-EM Structure of the Human Neurofibromin Dimer Reveals the Molecular Basis for Neurofibromatosis Type 1. Nat. Struct. Mol. Biol. 2021, 28 (12), 982–988. https://doi.org/10.1038/s41594-021-00687-2.

(10) Neurofibromin Structure, Functions and Regulation – PMC https://www-ncbi-nlm-nih-gov.muhlenberg.idm.oclc.org/pmc/articles/PMC7692384/ (accessed 2022 -04 -15).

(11) Fahsold, R.; Habash, T.; Trautmann, U.; Haustein, A.; Pfeiffer, R. Familial Reciprocal Translocation t(17;19) (Q11.2;Q13.2) Associated with Neurofibromatosis Type 1, Including One Patient with Non-Hodgkin Lymphoma and an Additional t(14;20) in B Lymphocytes. Hum. Genet. 2004. https://doi.org/10.1007/BF00214188.

For a complete list of works used, please access the Annotated Bibliography.