|External IDs||GeneCards: |
Neurofibromin 1 (NF1) is a gene in humans that is located on chromosome 17. NF1 codes for neurofibromin, a GTPase-activating protein that negatively regulates RAS/MAPK pathway activity by accelerating the hydrolysis of Ras-bound GTP. There are currently five known neurofibromin isoforms that are expressed in different tissues and perform different functions. NF1 has a high mutation rate and mutations in NF1 can alter cellular growth control, and neural development, resulting in neurofibromatosis type 1 (NF1, also known as von Recklinghausen syndrome). Symptoms of NF1 include cutaneous neurofibromas, café au lait pigment spots, plexiform neurofibromas, skeletal defects and optic nerve gliomas.
NF1 encodes the protein neurofibromin, a GTPase-activating protein, which primarily regulates the protein Ras. NF1 is located on the long arm of chromosome 17, position q11.2 and was identified in 1990 through positional cloning. NF1 spans over 350-kb of genomic DNA and contains 62 exons. 58 of these exons are constitutive and 4 exhibit alternative splicing ( 9a, 10a-2, 23a, and 28a). The genomic sequence starts 4,951-bp upstream of the transcription start site and 5,334-bp upstream of the translation initiation codon, with the length of the 5’ UTR being 484-bp long.
There are three genes that are present within intron 27b of NF1. These genes are EVI2B, EVI2A and OMG, which are encoded on the opposite strand and are transcribed in the opposite direction of NF1. EVI2A and EVI2B are human homologs of the Evi-2A and Evi-2B genes in mice that encode proteins related to leukemia in mice. OMG is a membrane glycoprotein that is expressed in the human central nervous system during myelination of nerve cells.
Early studies of the NF1 promoter found that there is great homology between the human and mouse NF1 promoters. The major transcription start site has been confirmed, as well as two minor transcription start sites in both the human and mouse gene.
The major transcription start is 484-bp upstream of the translation initiation site. The open reading frame is 8,520-bp long and begins at the translation initiation site. NF1 exon 1 is 544-bp long, contains the 5’ UTR and encodes the first 20 amino acids of neurofibromin. The NF1 promoter lies within a CpG island that is 472-bp long, consisting of 43 CpG dinucleotides, and extends into the start of exon 1. This CpG Island begins 731-bp upstream of the promoter and no core promoter element, such as a TATA or CCATT box, has been found within it. Although no core promoter element has been found, consensus binding sequences have been identified in the 5’ UTR for several transcription factors such as Sp1 and AP2.
A methylation map of five regions of the promoter in both mouse and human was published in 1999. This map showed that three of the regions (at approximately – 1000, – 3000, and – 4000) were frequently methylated, but the cytosines near the transcription start site were unmethylated. Methylation has been shown to functionally impact Sp1 sites as well as a CREB binding site. It has been shown that the CREB site must be intact for normal promoter activity to occur and methylation at the Sp1 sites may affect promoter activity.
Proximal NF1 promoter/5’ UTR methylation has been analyzed in tissues from NF1 patients, with the idea that reduced transcription as a result of methylation could be a “second hit” mechanism equivalent to a somatic mutation. There are some sites that have been detected to be methylated at a higher frequency in tumor tissues than normal tissues. These sites are mostly within the proximal promoter; however, some are in the 5’ UTR as well and there is a lot of interindividual variability in the cytosine methylation in these regions.
A study in 1993 compared the mouse NF1 cDNA to the human transcript and found that both the untranslated regions and coding regions were highly conserved. It was verified that there are two NF1 polyadenylated transcripts that differ in size because of the length of the 3’ UTR, which is consistent with what has been found in the mouse gene.
A study conducted in 2000 examined whether the involvement of the 3’ UTR in post-transcriptional gene regulation had an effect on the variation of NF1 transcript quantity both spatially and temporally. Five regions of the 3’ UTR that appear to bind proteins were found, one of which is HuR, a tumor antigen. HuR binds to AU-rich elements which are scattered throughout the 3' UTR and are thought to be negative regulators of transcript stability. This supports the idea that post-transcriptional mechanisms may influence the levels of NF1 transcript.
NF1 has one of the highest mutation rates amongst known human genes, however mutation detection is difficult because of its large size, the presence of pseudogenes, and the variety of possible mutations. The NF1 locus has a high incidence of de novo mutations, meaning that the mutations are not inherited maternally or paternally. Although the mutation rate is high, there are no mutation “hot spot” regions. Mutations tend to be distributed within the gene, although exons 3, 5, and 27 are common sites for mutations.
The Human Gene Mutation Database contains 1,347 NF1 mutations, but none are in the “regulatory” category. There have not been any mutations conclusively identified within the promoter or untranslated regions. This may be because such mutations are rare, or they do not result in a recognizable phenotype.
There have been mutations identified that affect splicing, in fact 286 of the known mutations are identified as splicing mutations. About 78% of splicing mutations directly affect splice sites, which can cause aberrant splicing to occur. Aberrant splicing may also occur due to mutations within a splicing regulatory element. Intronic mutations that fall outside of splice sites also fall under splicing mutations, and approximately 5% of splicing mutations are of this nature. Point mutations that effect splicing are commonly seen and these are often substitutions in the regulatory sequence. Exonic mutations can lead to deletion of an entire exon, or a fragment of an exon if the mutation creates a new splice site. Intronic mutations can result in the insertion of a cryptic exon, or result in exon skipping if the mutation is in the conserved 3’ or 5’ end.
NF1 encodes neurofibromin (Nf1), which is a 320-kDa protein that contains 2,818 amino acids. Neurofibromin is a GTPase-activating protein (GAP) that negatively regulates Ras pathway activity by accelerating hydrolysis of Ras-bound guanosine triphosphate (GTP). Neurofibromin localizes in the cytoplasm; however, some studies have found neurofibromin or fragments of it in the nucleus. Neurofibromin does contain a nuclear localization signal that is encoded by exon 43, but whether or not neurofibromin plays a role in the nucleus is currently unknown. Neurofibromin is ubiquitously expressed, but expression levels vary depending on the tissue type and developmental stage of the organism. Expression is at its highest level in adult neurons, Schwann cells, astrocytes, leukocytes, and oligodendrocytes.
The catalytic RasGAP activity of neurofibromin is located in a central portion of the protein, that is called the GAP-related domain (GRD). The GRD is closely homologous to RasGAP and represents about 10% (229 amino acids) of the neurofibromin sequence. The GRD is made up of a central portion called the minimal central catalytic domain (GAPc) as well as an extra domain (GAPex) that is formed through the coiling of about 50 residues from the N- and C- terminus. The Ras-binding region is found in the surface of GAPc and consists of a shallow pocket that is lined by conserved amino acid residues.
In addition to the GRD, neurofibromin also contains a Sec14 homology-like region as well as a pleckstrin homology-like (PH) domain. Sec14 domains are defined by a lipid binding pocket that resembles a cage and is covered by a helical lid portion that is believed to regulate ligand access. The PH-like region displays a protrusion that connects two beta-strands from the PH core that extend to interact with the helical lid found in the Sec14 domain. The function of the interaction between these two regions is presently unclear, but the structure implies a regulatory interaction that influences the helical-lid conformation in order to control ligand access to the lipid binding pocket.
Through its NF1-GRD domain, neurofibromin increases the rate of GTP hydrolysis of Ras, and acts as a tumor suppressor by reducing Ras activity. When the Ras-Nf1 complex assembles, active Ras binds in a groove that is present in the neurofibromin catalytic domain. This binding occurs through Ras switch regions I and II, and an arginine finger present in neurofibromin. The interaction between Ras and neurofibromin causes GAP-stimulated hydrolysis of GTP to GDP. This process depends on the stabilization of residues in the Ras switch I and switch II regions, which drives Ras into the confirmation required for enzymatic function. This interaction between Ras and neurofibromin also requires the transition state of GDP hydrolysis to be stabilized, which is performed through the insertion of the positively charged arginine finger into the Ras active site. This neutralizes the negative charges that are present on GTP during phosphoryl transfer. By hydrolyzing GTP to GDP, neurofibromin inactivates Ras and therefore negatively regulates the Ras pathway, which controls the expression of genes involved in apoptosis, the cell cycle, cell differentiation or migration.
Neurofibromin is also known to interact with CASK through syndecan, a protein which is involved in the KIF17/ABPA1/CASK/LIN7A complex, which is involved in trafficking GRIN2B to the synapse. This suggests that neurofibromin has a role in the transportation of the NMDA receptor subunits to the synapse and its membrane. Neurofibromin is also believed to be involved in the synaptic ATP-PKA-cAMP pathway, through modulation of adenylyl cyclase. It is also known to bind the caveolin 1, a protein which regulates p21ras, PKC and growth response factors.
There are currently five known isoforms of neurofibromin (II, 3, 4, 9a, and 10a-2) and these isoforms are generated through the inclusion of alternative splicing exons (9a, 10a-2, 23a, and 48a) that do not alter the reading frame. These five isoforms are expressed in distinct tissues and are each detected by specific antibodies.
Neurofibromin type II, also named GRD2 (domain II-related GAP), results from the insertion of exon 23a, which causes the addition of 21 amino acids in the 5’ region of the protein. Neurofibromin type II is expressed in Schwann cells and has reduced GAP activity.
Neurofibromin type 3 (also called isoform 3’ ALT) contains exon 48a which results in the insertion of 18 amino acids into the 3’ terminal. Neurofibromin type 4 contains exons 23a and 48a, which results in the insertion of 21 amino acids in the 5’ region, and 18 amino acids in the 3’ terminal.
Neurofibromin 9a (also referred to as 9br), includes exon 9a which results in the insertion of 10 amino acids in the 5’ region. This isoform shows little neuronal expression and may play a role in memory and learning mechanisms.
An isoform with insertion of exon 10a-2 has been studied introduces a transmembrane domain. The inclusion of exon 10a-2 causes the insertion of 15 amino acids in the 5’ region. This isoform is expressed in most human tissues, therefore it likely performs a housekeeping function in intracellular membranes.
It has been suggested that the quantitative differences in expression between the different isoforms may be related to the phenotypic variability of neurofibromatosis type 1 patients.
In the NF1 mRNA, there is a site within the first half of the GRD where mRNA editing occurs. Deamination occurs at this site, resulting in the conversion of cytidine into uridine at nucleotide 3916. This deamination changes an arginine codon (CGA) to an in-frame translation stop codon (UGA). If the edited transcript is translated, it produces a protein that cannot function as a tumor suppressor because the N-terminal of the GRD is truncated. The editing site in NF1 mRNA was shown to have high homology to the ApoB editing site, where double stranded mRNA undergoes editing by the ApoB holoenzyme. NF1 mRNA editing was believed to involve the ApoB holoenzyme due to the high homology between the two editing sites, however studies have shown that this is not the case. The editing site in NF1 is longer than the sequence required for ApoB mediated mRNA editing, and the region contains two guanidines which are not present in the ApoB editing site.
Mutations in NF1 are primarily associated with neurofibromatosis type 1 (NF1, also known as von Recklinghausen syndrome). NF1 is the most common single gene disorder in humans, occurring in about 1 in 2500-3000 births worldwide. NF1 is an autosomal dominant disorder, but approximately half of NF1 cases arise from de novo mutations. NF1 has high phenotypic variability, with members of the same family with the same mutation displaying different symptoms and symptom intensities. Café-au-lait spots are the most common sign of NF1, but other symptoms include lisch nodules, cutaneous neurofibromas, plexiform neurofibromas, skeletal defects, and optic nerve gliomas.
In addition to neurofibromatosis type I, mutations in NF1 can also lead to juvenile myelomonocytic leukemia, Watson syndrome, and breast cancer.
|Recessive lethal study||Abnormal|
|Glucose tolerance test||Normal|
|Auditory brainstem response||Normal|
|Peripheral blood lymphocytes||Abnormal|
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Model organisms have been used in the study of NF1 function. A conditional knockout mouse line, called Nf1tm1a(KOMP)Wtsi was generated as part of the International Knockout Mouse Consortium program, a high-throughput mutagenesis project to generate and distribute animal models of disease to interested scientists.
Male and female animals underwent a standardized phenotypic screen to determine the effects of deletion. Twenty six tests were carried out on mutant mice and four significant abnormalities were observed. Over half the homozygous mutant embryos identified during gestation were dead, and in a separate study none survived until weaning. The remaining tests were carried out on heterozygous mutant adult mice: females displayed abnormal hair cycling while males had an decreased B cell number and an increased monocyte cell number.
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 Peltonen S, Kallionpää RA, Peltonen J (July 2017). "Neurofibromatosis type 1 (NF1) gene: Beyond café au lait spots and dermal neurofibromas". Experimental Dermatology. 26 (7): 645–648. doi:10.1111/exd.13212. PMID 27622733.
- ↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 Abramowicz A, Gos M (July 2014). "Neurofibromin in neurofibromatosis type 1 - mutations in NF1gene as a cause of disease". Developmental Period Medicine. 18 (3): 297–306. PMID 25182393.
- ↑ 3.00 3.01 3.02 3.03 3.04 3.05 3.06 3.07 3.08 3.09 3.10 3.11 3.12 Scheffzek K, Welti S (2012). "Neurofibromin: Protein Domains and Functional Characteristics". In Upadhyaya M, Cooper D. Neurofibromatosis Type 1. Berlin, Heidelberg: Springer. pp. 305–326. doi:10.1007/978-3-642-32864-0_20. ISBN 978-3-642-32864-0.
- ↑ 4.00 4.01 4.02 4.03 4.04 4.05 4.06 4.07 4.08 4.09 4.10 4.11 4.12 4.13 4.14 4.15 4.16 4.17 4.18 4.19 4.20 4.21 Trovó-Marqui AB, Tajara EH (July 2006). "Neurofibromin: a general outlook". Clinical Genetics. 70 (1): 1–13. doi:10.1111/j.1399-0004.2006.00639.x. PMID 16813595.
- ↑ Peltonen S, Pöyhönen M (2012). "Clinical Diagnosis and Atypical Forms of NF1". In Upadhyaya M, Cooper D. Neurofibromatosis Type 1. Berlin, Heidelberg: Springer. pp. 17–30. doi:10.1007/978-3-642-32864-0_2. ISBN 978-3-642-32864-0.
- ↑ 6.00 6.01 6.02 6.03 6.04 6.05 6.06 6.07 6.08 6.09 6.10 6.11 6.12 6.13 6.14 6.15 6.16 Li H, Wallace MR (2012). "NF1 Gene: Promoter, 5′ UTR, and 3′ UTR.". In Upadhyaya M, Cooper D. Neurofibromatosis Type 1. Berlin, Heidelberg: Springer. pp. 105–113. doi:10.1007/978-3-642-32864-0_9. ISBN 978-3-642-32864-0.
- ↑ 7.0 7.1 7.2 7.3 Lee TK, Friedman JM (August 2005). "Analysis of NF1 transcriptional regulatory elements". American Journal of Medical Genetics. Part A. 137 (2): 130–5. doi:10.1002/ajmg.a.30699. PMID 16059932.
- ↑ 8.0 8.1 Zou MX, Butcher DT, Sadikovic B, Groves TC, Yee SP, Rodenhiser DI (January 2004). "Characterization of functional elements in the neurofibromatosis (NF1) proximal promoter region". Oncogene. 23 (2): 330–9. doi:10.1038/sj.onc.1207053. PMID 14647436.
- ↑ 9.0 9.1 9.2 Haeussler J, Haeusler J, Striebel AM, Assum G, Vogel W, Furneaux H, Krone W (January 2000). "Tumor antigen HuR binds specifically to one of five protein-binding segments in the 3'-untranslated region of the neurofibromin messenger RNA". Biochemical and Biophysical Research Communications. 267 (3): 726–32. doi:10.1006/bbrc.1999.2019. PMID 10673359.
- ↑ 10.0 10.1 10.2 10.3 Baralle M, Baralle D (2012). "Splicing Mechanisms and Mutations in the NF1 Gene.". In Upadhyaya M, Cooper D. Neurofibromatosis Type 1. Berlin, Heidelberg: Springer. pp. 135–150. doi:10.1007/978-3-642-32864-0_11. ISBN 978-3-642-32864-0.
- ↑ Pasmant E, Vidaud D (May 2016). "Neurofibromatosis Type 1 Molecular Diagnosis: The RNA Point of View". EBioMedicine. 7: 21–2. doi:10.1016/j.ebiom.2016.04.036. PMC 4909605. PMID 27322453.
- ↑ Kaufmann D, Müller R, Kenner O, Leistner W, Hein C, Vogel W, Bartelt B (June 2002). "The N-terminal splice product NF1-10a-2 of the NF1 gene codes for a transmembrane segment". Biochemical and Biophysical Research Communications. 294 (2): 496–503. doi:10.1016/S0006-291X(02)00501-6. PMID 12051738.
- ↑ 13.0 13.1 13.2 13.3 Cappione, A. J.; French, B. L.; Skuse, G. R. (February 1997). "A potential role for NF1 mRNA editing in the pathogenesis of NF1 tumors". American Journal of Human Genetics. 60 (2): 305–312. ISSN 0002-9297. PMC 1712412. PMID 9012403.
- ↑ 14.0 14.1 14.2 14.3 Skuse, G. R.; Cappione, A. J.; Sowden, M.; Metheny, L. J.; Smith, H. C. (1996-02-01). "The neurofibromatosis type I messenger RNA undergoes base-modification RNA editing". Nucleic Acids Research. 24 (3): 478–485. doi:10.1093/nar/24.3.478. ISSN 0305-1048. PMC 145654. PMID 8602361.
- ↑ Woodrow, Christopher; Clarke, Anna; Amirfeyz, Rouin (2015). "Neurofibromatosis". Orthopaedics and Trauma. 29 (3): 206–210. doi:10.1016/j.mporth.2015.02.004.
- ↑ Williams, Virginia C.; Lucas, John; Babcock, Michael A.; Gutmann, David H.; Korf, Bruce; Maria, Bernard L. (January 2009). "Neurofibromatosis type 1 revisited". Pediatrics. 123 (1): 124–133. doi:10.1542/peds.2007-3204. ISSN 1098-4275. PMID 19117870.
- ↑ Ward, Beth Ann; Gutmann, David H. (April 2005). "Neurofibromatosis 1: from lab bench to clinic". Pediatric Neurology. 32 (4): 221–228. doi:10.1016/j.pediatrneurol.2004.11.002. ISSN 0887-8994. PMID 15797177.
- ↑ Peltonen S., Pöyhönen M. (2012) Clinical Diagnosis and Atypical Forms of NF1. In: Upadhyaya M., Cooper D. (eds) Neurofibromatosis Type 1. Springer, Berlin, Heidelberg
- ↑ "Entrez Gene: NF1 neurofibromin 1 (neurofibromatosis, von Recklinghausen disease, Watson disease)".
- ↑ Cancer Genome Atlas (October 2012). "Comprehensive molecular portraits of human breast tumours". Nature. Nature Publishing Group. 490 (7418): 61–70. Bibcode:2012Natur.490...61T. doi:10.1038/nature11412. PMC 3465532. PMID 23000897.
- ↑ "Dysmorphology data for Nf1". Wellcome Trust Sanger Institute.
- ↑ "Peripheral blood lymphocytes data for Nf1". Wellcome Trust Sanger Institute.
- ↑ "Salmonella infection data for Nf1". Wellcome Trust Sanger Institute.
- ↑ "Citrobacter infection data for Nf1". Wellcome Trust Sanger Institute.
- ↑ 25.0 25.1 25.2 25.3 Gerdin AK (2010). "The Sanger Mouse Genetics Programme: High throughput characterisation of knockout mice". Acta Ophthalmologica. 88 (S248): 0. doi:10.1111/j.1755-3768.2010.4142.x.
- ↑ Mouse Resources Portal, Wellcome Trust Sanger Institute.
- ↑ "International Knockout Mouse Consortium".
- ↑ "Mouse Genome Informatics".
- ↑ Skarnes WC, Rosen B, West AP, Koutsourakis M, Bushell W, Iyer V, Mujica AO, Thomas M, Harrow J, Cox T, Jackson D, Severin J, Biggs P, Fu J, Nefedov M, de Jong PJ, Stewart AF, Bradley A (June 2011). "A conditional knockout resource for the genome-wide study of mouse gene function". Nature. 474 (7351): 337–42. doi:10.1038/nature10163. PMC 3572410. PMID 21677750.
- ↑ Dolgin E (June 2011). "Mouse library set to be knockout". Nature. 474 (7351): 262–3. doi:10.1038/474262a. PMID 21677718.
- ↑ Collins FS, Rossant J, Wurst W (January 2007). "A mouse for all reasons". Cell. 128 (1): 9–13. doi:10.1016/j.cell.2006.12.018. PMID 17218247.
- ↑ van der Weyden L, White JK, Adams DJ, Logan DW (June 2011). "The mouse genetics toolkit: revealing function and mechanism". Genome Biology. 12 (6): 224. doi:10.1186/gb-2011-12-6-224. PMC 3218837. PMID 21722353.
- Upadhyaya M, Shaw DJ, Harper PS (1994). "Molecular basis of neurofibromatosis type 1 (NF1): mutation analysis and polymorphisms in the NF1 gene". Human Mutation. 4 (2): 83–101. doi:10.1002/humu.1380040202. PMID 7981724.
- Shen MH, Harper PS, Upadhyaya M (January 1996). "Molecular genetics of neurofibromatosis type 1 (NF1)". Journal of Medical Genetics. 33 (1): 2–17. doi:10.1136/jmg.33.1.2. PMC 1051805. PMID 8825042.
- Feldkamp MM, Gutmann DH, Guha A (August 1998). "Neurofibromatosis type 1: piecing the puzzle together". The Canadian Journal of Neurological Sciences. Le Journal Canadien Des Sciences Neurologiques. 25 (3): 181–91. PMID 9706718.
- Hamilton SJ, Friedman JM (November 2000). "Insights into the pathogenesis of neurofibromatosis 1 vasculopathy". Clinical Genetics. 58 (5): 341–4. doi:10.1034/j.1399-0004.2000.580501.x. PMID 11140831.
- Baralle D, Baralle M (October 2005). "Splicing in action: assessing disease causing sequence changes". Journal of Medical Genetics. 42 (10): 737–48. doi:10.1136/jmg.2004.029538. PMC 1735933. PMID 16199547.
- Mensink KA, Ketterling RP, Flynn HC, Knudson RA, Lindor NM, Heese BA, Spinner RJ, Babovic-Vuksanovic D (February 2006). "Connective tissue dysplasia in five new patients with NF1 microdeletions: further expansion of phenotype and review of the literature". Journal of Medical Genetics. 43 (2): e8. doi:10.1136/jmg.2005.034256. PMC 2603036. PMID 16467218.
- Trovó-Marqui AB, Tajara EH (July 2006). "Neurofibromin: a general outlook". Clinical Genetics. 70 (1): 1–13. doi:10.1111/j.1399-0004.2006.00639.x. PMID 16813595.
- GeneReviews/NCBI/NIH/UW entry on Neurofibromatosis 1
- Human Gene NF1 (uc002hgf.1)
- neurofibromin 1 from GeneCards
- Database of RNA editing