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THE NEUROFIBROMIN GENE (NF1)

THE NEUROFIBROMIN GENE (NF1)
 

THE NEUROFIBROMIN GENE (NF1)

Di Antonella Selvaggio

Quello che ospito è uno studio condotto da Antonella Selvaggio studentessa della Queen Margaret University di Edimburgo.

Normal gene:

The neurofibromin (NF1) gene codes for a cytoplasmic protein called neurofibrominof 2,818 amino acids. This protein is produced in many types of cells. It is primarilyexpressed in neurons and specialized cells called oligodendrocytes and Schwann cells that surround nerves. These specialized cells form myelin sheaths, which are the fatty coverings that insulate and protect certain nerve cells.

Neurofibromin is also expressed into leukocytes (white blood cells).

Neurofibromin acts as a tumor suppressor protein.

Tumor suppressors normally prevent cells from growing and dividing too rapidly or in an uncontrolled way. This protein prevents cell overgrowth by turning off another protein called ras that triggers cell growth and division.(Genetic Home Reference, 2015).

Mutated gene, conditions, signs and symptoms.

The neurofibromin gene can sometimes be altered by mutations.

Mutations in the NF1 gene lead to the production of a nonfunctional version of the protein neurofibromin whichresults in it being unable to regulate cell growth and division because levels of intracellular ras activity are elevated. Due to this,benign or non canceroustumors such as neurofibromas (figure 1) can form along nerve tissues throughout the body.

Figure 2. Figure 1. Two or more neurofibromas

One example is neurofibromatosis-1, an inherited disorder in which neurofibromas can form in the bottom layer of skin (subcutaneous tissue), into cranial nerves or into the spinal cord (spinal root nerves).

Neurofibromatosis type 1 is thought to have an autosomal dominant pattern of inheritance. People with this condition are born with one mutated copy of the NF1 gene in each cell. In about half of cases, the altered gene is inherited from an affected parent. The remaining cases result from new mutations in the NF1 gene and occur in people with no history of the disorder in their family. Neurofibromatosis type 1 occurs in 1 in 3,000 to 4000 individuals worldwide regardless of race, gender or ethnic backgrounds (Genetic Home Reference, 2015).

In neurofibromatosis type 1, two copies of the NF1 gene must be altered to generate tumor formation. A mutation in the second copy of the NF1 gene occurs during a person’s lifetime in specialized cells surrounding nerves. Almost everyone who is born with one NF1 mutation acquires a second mutation in many cells and develops the tumors characteristic of neurofibromatosis type 1.

Neurofibromatosis type 1presents different signs and symptoms among affected people.

Beginning in early childhood, almost all people with neurofibromatosis type 1 show multiple café-au-lait spots (figure 2), which are flat patches on theskin ,darker than the surrounding area.As the individual grows older these spots increase in size and number.

Figure 1. Figure 2. café-au-lait

Later in childhood, also freckles in the underarms and groin usually develop.

During childhood, benign growths called Lisch nodules appear in the iris (the colored part of the eye). Those nodules do not interfere with vision. Some affected individuals are also likely to grow tumors along the optic nerve which from the eye leads to the brain. These tumors, which are called optic gliomas, may cause a reduction in vision or total vision loss. In some cases, optic gliomas have no effect on vision.

Children with NF1 often show specific learning disabilities, attention deficit hyperactivity disorder (ADHD) andhyper intense lesion in a form of brain imaging called T2-weighted brain magnetic resonance imaging (MRI) (Arun &Gutmann, 2004). Although, deficits in spatial ability, language skills and behavioural and psychosocial problems have also been documented in NF1 individuals (North et al., 2002).

Most adults with neurofibromatosis type 1 can develop cutaneous neurofibromas which grow from nerves on or just under the skin, spinal neurofibromas which grow from nerves near the spinal cord and plexiform neurofibromas (Figure 3), that grow from nerves elsewhere in the body. These tumors,might change into a cancer called malignant peripheral nerve sheath tumors.

Figure 3. Figure 3. Plexiform neurofibromas

People with neurofibromatosis type 1 also have an increased risk of developing other cancers, including brain tumors and cancer of blood-forming tissue (leukemia).

Additional signs and symptoms of neurofibromatosis type 1 include high blood pressure (hypertension), short stature, an unusually large head (macrocephaly), and skeletal abnormalities such as an abnormal curvature of the spine (scoliosis).

Mutation in the NF1 gene can also cause Watson syndrome which is an autosomal dominant disorder characterized by pulmonic stenosis, café-au-lait spots, decreased intelectual ability (Watson, 1967), and short stature (Partington et al., 1985). Most affected individuas have relative macrocephaly and Lish nodules and about one-thirs of those affected have neurofibroma (Allanson et al., 1991).

In rare cases, inactivation of one copy of the NF1 gene in each cell increases the risk of developing juvenile myelomonocytic leukemia (JMML). Juvenile myelomonocytic leukemia is a cancer of blood-forming tissue that usually occurs in children younger than 2. This condition causes the bone marrow to make an excessive number of immature white blood cells that cannot carry out their normal infection-fighting functions. (Figure 4).These abnormal cells can build up in the blood and bone marrow, leaving less room for healthy white blood cells, red blood cells, and platelets.

http://www.bloodmed.org/contentimage/slide/3532.jpg Figure 4. JMML

If there are not enough normal blood cells the body cannot work normally. This can cause quite severe symptoms in children with JMML including:

  • Being tired and lethargic
  • Bruising easily
  • Nosebleeds and bleeding gums
  • Fever
  • Getting lots of infections
  • An enlarged liver and spleen
  • Swollen lymph nodes
  • Skin rashes
  • Small yellowish skin tumors. (Cancer Research UK, 2015)

Gene Map.

The neurofibromin gene can be found on the long (q) arm of chromosome 17 at position 11.2 as illustrated in (figure 5).

Genomic Location for NF1 Gene

Figure 5: (GeneCards). Illustration of Chromosome 17. The red line shows the position 17q11.2 of the NF1 gene.

The NF1 gene is located from base pair 31,045,488 from pter (locus) and ends at 31,382,116 base pair. Its size is 336,629 bases.

The NF1 gene spans 280kb of genomic DNA, it has 61 exons of which four are alternatively spliced. The large intron associated withexon 27b (61 kb) of the NF1 gene contains three unrelatedgenes, EV12A, EV12B, and OMG in which no mutationshave been identified in NF1 patients.

Summary of a recent paper:

Neurofibromatosis type 1 alternative splicing is a key regulator of Ras signalling in neurons.

Neurofibromatosis type 1 (Nf1) is a Ras GTPase activating protein (Ras-GAP) that acts as a tumor suppressor by increasing the rate at which the active GTP-bound form of Ras is converted to the inactive GDP-bound form. Ras-GTP is important as it adjusts different cellular functions, such as proliferation, death, migration and differentiation. Nf1 GAP domain is situated in the middle of the protein neurofibromin (Nf1) and it mediates its Ras-regulatory activity.

Nf1 also increases the generation of cyclic AMP (cAMP).

Changes in both Ras and cAMP signalling appear to play roles in diverse nervous system-related phenotypes of Nf1 mutant mice.

The expression of Nf1 is adjusted by the inclusion of three alterative exons. One of these is the exon 23a which is within the GAP domain coding sequence of Nf1. In neurons Nf1 mRNAs lack exon 23a and express the type 1 Nf1 protein isoform, whereas other cell types have higher levels of exon 23a inclusion and express type 2 Nf1 protein isoform.

When the two isoforms are overexpressed as truncated proteins in either mammalian or yeast cells, the Nf1 GAP domain lacking exon 23a has up to 10-fold higher Ras-GAP activity than the isoform containing exon 23a.

The aim of this study was to examine the biological importance of regulated Nf1 exon 23a inclusion in cells.

At first, using a splicing reporter system obtained from previous studies, 23aIN mutations were identified to be effective at abolising Nf1 exon 23a skipping in cell types in which there is a strong negative regulation of inclusion.

Then, using gene targeting, a targeting vector was constructed to introduce the 23aIN mutations into the endogenous Nf1 locus in mouse ES cells obtained from the Case Transgenic and Targeting Facility.

The end result was the creation of two ES cell lines: Nf1 23aΔ/23aΔ, in which both copies of exon 23a are deleted, and Nf1 23aIN/23IN, with exon 23a inclusion, in which the splicing signals surrounding exon 23a are mutated to more closely match consensus sequences.

To determine whether the inclusion of exon 23a diminishes the ability of Nf1 to inactivate Ras, a well-established active Ras pulldown and detection kit were used. The level of active Ras was much lower in Nf1 23aΔ/23aΔ ES cells than in Nf1 23aIN/23aIN cells. Therefore this suggested that the inclusion of exon 23a decreases the Ras-GAP activity of endogenous Nf1 protein in ES cells.

cAMP levels were also measured in the mutant ES cells. A significant difference wasn’t observd in intracellular cAMP levels in ES cells when Nf1 exon 23a inclusion was manipulated. These results suggest that Nf1 exon 23a inclusion specifically inhibits the Ras-GAP activity of Nf1 in ES cells without influencing its ability to regulate cAMP levels.

The role of Nf1 alternative splicing was also investigated into neurons. Using an established protocol, the Nf1 mutant ES cell lines were differentiated into CNS-like neurons. Nf1 23aIN/23aIN ES cell-derived neurons showed no Nf1 exon23a inclusion.

Nf1 exon 23a skipping wasn’t required for neuronal differentiation.

Measuring active Ras-GTP levels in WT and mutant ES cell-derived neurons also showed that Nf1 exon 23a inclusion correlates with Ras-GTP levels in both ES cells and ES cell-derived neurons, providing evidence that exon 23a inclusion decreases the Ras-GAP activity of Nf1.

Finally, there was no significant difference in intracellular cAMP level among ES cell-derived neurons with different levels of Nf1 exon 23a inclusion. No change was observed in neurite length in the Nf1 23aIN/23aIN ES cell-derived neurons, which showed elevated Ras signaling but no change in cAMP levels. These results suggest that Nf1 exon 23a inclusion regulates Ras signaling but not cAMP signaling also in neurons.

Also, phospho-ERK1/2 levels downstream of Ras in neurons is increased by Nf1 exon 23a inclusion.

To conclude, this study is important as it showed for the first time that exon 23a inclusion decreases the regulation of Ras activity by the endogenous, full- lenght Nf1 protein in cells. This study could help further studies in order to identify the process by which Nf1 exon 23a inclusion actually decreases the Ras-GAP activoty of Nf1. Also, the approach used in this study, could be applied to the research of other alternative exons.

References:

http://omim.org/entry/613113

http://ghr.nlm.nih.gov/gene/NF1

http://ghr.nlm.nih.gov/condition/neurofibromatosis-type-1

https://www.nlm.nih.gov/medlineplus/ency/article/000847.htm

http://www.ncbi.nlm.nih.gov/books/NBK1109/

http://www.cancerresearchuk.org/about-cancer/cancers-in-general/cancer-questions/juvenile-myelomonocytic-leukaemia

http://www.bloodmed.org/contentimage/slide/3532.jpg

http://i.dailymail.co.uk/i/pix/2013/08/20/article-2397816-1B602AC1000005DC-926_306x423.jpg

http://www.oadd.org/publications/journal/issues/vol11no2/download/kumar.pdf

http://www.genecards.org/cgi-bin/carddisp.pl?gene=NF1&keywords=NF1,gene,mapping

http://www.ncbi.nlm.nih.gov/gene/4763

http://omim.org/entry/193520

http://omim.org/geneMap/17/314?start=-3&limit=10&highlight=314

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