Pathophysiology of Neurofibromatosis Type 1 - American

PHYSIOLOGY IN MEDICINE: A SERIES OF ARTICLES LINKING MEDICINE WITH SCIENCE Physiology in Medicine: Dennis A. Ausiello, MD, Editor; Dale J. Benos, PhD, Deputy Editor; Francois Abboud, MD, Associate Editor; William J. Koopman, MD, Associate Editor

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Annals of Internal Medicine: Paul Epstein, MD, Series Editor

Pathophysiology of Neurofibromatosis Type 1 Amy Theos, MD, and Bruce R. Korf, MD, PhD

Clinical Principles

Pathophysiologic Principles

Neurofibromatosis type 1 (NF1) was formerly known as von Recklinghausen disease.

Neurofibromatosis type 1 is a classic single-gene disorder with a high rate of new mutations.

It has autosomal dominant inheritance with complete penetrance, variable expression, and a high rate of new mutation.

The NF1 gene is located on chromosome 17.

It affects approximately 1 in 3500 individuals worldwide.

Protein includes a domain with guanosine triphosphatase (GTPase)–activating protein (GAP) function.

Diagnostic clinical signs include neurofibromas, cafe´-au-lait macules, skinfold freckling, skeletal dysplasia, Lisch nodules, and optic gliomas. Persons with NF1 are at increased risk for malignant conditions, especially malignant peripheral nerve sheath tumor (MPNST), leukemia, and rhabdomyosarcoma.

The protein product, neurofibromin, consists of 2818 amino acids.

Neurofibromin GAP regulates conversion of Ras– guanosine triphosphate (GTP) to Ras– guanosine diphosphate (GDP). NF1 gene mutations are highly diverse and are found throughout the gene. Most mutations lead to lack of expression of the gene product.

Other complications include cognitive problems and vascular dysplasias. Molecular genetic testing is available. Clinical trials of potential therapies for plexiform neurofibromas are under way.

Few genotype–phenotype correlations are known, although complete gene deletions lead to severe disease. Loss of function of both NF1 alleles in Schwann cells of neurofibromas indicates that NF1 functions as a tumor suppressor gene. Neurofibromas consist of Schwann cells with both NF1 alleles mutated, along with heterozygous fibroblasts, perineurial cells, and mast cells. Malignant tumors require loss of NF1 function, as well as additional genetic changes. Pathophysiology of nontumor manifestations is unknown; NF1 haploinsufficiency may play a role. Modifying genes probably contribute to variable expression.

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eurofibromatoses are a set of inherited disorders, designated as neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2), and schwannomatosis, that tend to result in the development of benign tumors of the nerve sheath. The 3 entities are distinguished by specific clinical features and are due to mutations in distinct genes (Table 1). Neurofibromatosis type 1 is the most common of the disorders, affecting approximately 1 in 3500 individuals

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Pathophysiology of Neurofibromatosis Type 1

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Table 1. National Institutes of Health Criteria for Neurofibromatosis Type 1* Clinical Criteria

Comments

6 or more cafe´-au-lait macules larger than 5 mm before puberty or 15 mm after puberty Skinfold freckling 2 or more neurofibromas or 1 plexiform neurofibroma 2 or more Lisch nodules Optic glioma Characteristic skeletal dysplasia Affected first-degree relative

10% of the population has 1 cafe´-au-lait macule Axillae, neck, inguinal folds, inframammary; highly specific for NF1 – Requires slit-lamp examination; highly specific for NF1 – Tibial or orbital (sphenoid wing) dysplasia Severity may vary widely among members of the same family

* Two or more criteria are needed for definitive diagnosis. NF1 ⫽ neurofibromatosis type 1.

worldwide. The hallmark lesion in NF1 is the neurofibroma, whereas schwannomas (see Glossary) are characteristic of NF2 and schwannomatosis. These tumors may be difficult to distinguish clinically, but they can be differentiated histologically. Unlike the other 2 disorders, NF1 also includes nontumor manifestations, which makes it a true multisystem disorder. The NF1 gene, which produces the NF1 phenotype, was identified in 1990, and much has been learned about the pathophysiology of the disorder since. This has resulted in the availability of molecular diagnostic testing, and clinical trials for drugs to treat neurofibromas are beginning. Our review will briefly consider the NF1 phenotype, the current understanding of basic mechanisms, and the status of translation of this knowledge into clinical application.

to compression of nerves and other structures and erosion of bones. Plexiform neurofibromas can occur congenitally, although deeper tumors may not be recognized until later in life. Neurofibromas are histologically benign, but transformation to malignant peripheral nerve sheath tumors (MPNSTs) is a risk. These tumors arise predominantly from preexisting plexiform neurofibromas. Signs of malignant tumors are pain or sudden growth, although benign tumors can be associated with these signs as well. The lifetime risk for MPNST in patients with NF1 is about 10%, and prognosis is poor (3). The poor outcome may be partly due to delayed diagnosis and poor therapeutic success with sarcomas in general. The tumors tend to be treated with surgery. Whether radiation or chemotherapy markedly improves outcome is not clear.

THE NF1 PHENOTYPE Neurofibromatosis type 1 is a highly variable disorder with signs and symptoms that may begin at birth and evolve over a lifetime. Phenotypic features can be broadly divided into tumors and nontumor manifestations. Tumors

The most characteristic tumor in NF1 is the neurofibroma. Neurofibromas arise from cells of the nerve sheath and consist of a mixture of Schwann cells, fibroblasts, perineurial cells, and mast cells (1). They arise along peripheral nerves, including the nerve root; on sites along the course of nerves; or at nerve endings. Neurofibromas may be focal growths or can extend along the length of a nerve, involving several fascicles and including nerve branches. The latter are called plexiform neurofibromas (2). Dermal neurofibromas may project above the surface of the skin or may reside within the skin (Figure 1). Dermal neurofibromas usually first appear in the preadolescent years and continue to occur throughout life. Aside from puberty, pregnancy seems to be a time of growth or appearance of dermal neurofibromas. The number of dermal tumors that an individual will develop is unpredictable. Some people are covered with thousands of tumors, leading to major cosmetic burden and a reclusive life. Spinal neurofibromas can cause nerve root compression and may invade the spinal canal and compress the spinal cord. Plexiform neurofibromas can grow very large, leading to deformity, and can lead www.annals.org

Glossary Allelic heterogeneity: A single disorder caused by different mutations within a gene. Cafe´-au-lait macule: Coffee-colored skin spot that is a common clinical feature of neurofibromatosis type 1 (NF1). Conditional knockout mice: An investigative tool that allows a target gene to be removed in a specific tissue or stage of development. Guanosine triphosphatase (GTPase)–activating protein (GAP): Protein that stimulates enzyme activity to convert protein-bound GTP to guanosine diphosphate (GDP). Haploinsufficiency: Situation in which the protein produced by the remaining single copy of an otherwise normal gene is present in insufficient quantity and leads to a disease state. Heterotopia: Displacement of gray matter, usually into the deep cerebral white matter. Heterozygous: Possessing 2 different copies of a single gene. Lisch nodule: Melanocytic iris hamartoma associated with NF1. Modifying genes: Genes that, when mutated, alter the normal phenotypic expression of a disease. Mosaicism: An individual containing 2 genetically distinct cell lines; can involve only sperm or egg cells (germline mosaicism) or nongermline cells (somatic mosaicism). Protein truncation assay: Molecular test that is used to identify shortened proteins produced in vitro by an abnormal gene. Schwannoma: Nerve sheath tumor derived from Schwann cells. Splicing mutation: Change in the DNA sequence that disrupts proper cutting or processing of messenger RNA (mRNA). Stop mutation: Change in the DNA sequence that causes premature termination of translation of mRNA and a truncated protein product. Translation: Process of decoding mRNA to produce a specific protein. Tumor suppressor gene: One of a class of genes in which both copies are mutated in tumor cells; inheritance of predisposition to tumors occurs by inheritance of 1 mutant allele.

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Figure 1. Several dermal neurofibromas that are visible as raised lesions but are sometimes first detected by palpation.

The distance between each hash mark is 1 cm.

In addition to peripheral nerve sheath tumors, tumors of the central nervous system may occur in affected individuals. Optic gliomas are low-grade pilocytic astrocytomas that occur in 15% of children with NF1 (4). Although at least 50% are asymptomatic, progressive tumors can cause loss of vision, constriction of visual fields, or neuroendocrine disturbances, such as precocious puberty. Optic gliomas progressing after 7 to 8 years of age are rare, and therefore, annual screening in adults is not usually performed. Gliomas can occur elsewhere in the central nervous system, especially in the brainstem. These can also be indolent or may progress (5). Malignant tumors seen in patients with NF1, predominantly children, include leukemia, especially juvenile myelomonocytic leukemia, and rhabdomyosarcoma (6). Individuals with NF1 are also at increased risk for pheochromocytoma.

of cells in the intima. Involvement of the renal arteries can lead to hypertension, which most often presents in childhood or in pregnancy (Figure 2). Cerebral artery stenosis can lead to strokes or to the moyamoya syndrome, where collateral vessels develop around the stenotic areas and appear as a puff of smoke on cerebral angiography. Arterial dissection and hemorrhage can be other complications of the vascular involvement of NF1. Pheochromocytoma should be considered in adult patients with NF1 who have refractory hypertension and symptoms of catecholamine excess, such as headache, palpitations, and diaphoresis (10). Neurofibromatosis type 1 has many additional clinical features (Table 2) (11). Individually, the features are relatively uncommon, but they must be considered in the management of affected individuals. Two of three individuals with NF1 are estimated to experience relatively minor problems (12). Also, many of the more severe problems, such as tibial or vertebral dysplasia or deformity due to plexiform neurofibroma, tend to occur early in life (Figure 3). Nevertheless, severity is highly variable and unpredictable. Average life expectancy is reduced, mostly due to malignant tumors and vascular complications (13).

GENETICS

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NF1

Germline and Somatic Mosaicism

Neurofibromatosis type 1 is a classic autosomal dominant, single-gene disorder. Approximately half of affected individuals have no affected parent, which is indicative of new mutation. It has the highest rate of new mutation of any known single-gene disorder. An affected individual has a 50% chance of passing NF1 to any offspring. Severity can vary from generation to generation or between affected siblings. The parents of a sporadically affected child are at

Figure 2. Angiogram showing renal artery stenosis in a patient with neurofibromatosis type 1.

Nontumor Manifestations

Major nontumor manifestations of NF1 include pigmentary features, skeletal dysplasias, learning disabilities, and vascular dysplasias. These manifestations have characteristic times of appearance. We will further discuss only the features that are salient to adults. Learning disabilities are among the most common complications of NF1 (7). While most research has focused on children, NF1 results in global cognitive impairment among adults as well. Adults with NF1 may have deficits in inductive reasoning, visuoconstructive skill, memory, logical abstraction, coordination, and mental flexibility (8). In addition to cognitive problems, affective disorders, most commonly dysthymia, occur with increased frequency (9). The vascular complications of NF1, vasculopathy and hypertension, are a major cause of premature death. Vasculopathy consists of arterial stenoses due to proliferation 844 6 June 2006 Annals of Internal Medicine Volume 144 • Number 11

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low risk for having another affected child if both parents are without signs, barring the rare possibility of germline mosaicism (see Glossary) in 1 parent (14). Some affected individuals have manifestations that are confined to a restricted region of the body (15), called segmental neurofibromatosis. At least some cases are due to somatic mosaicism (see Glossary) for an NF1 gene mutation (16), but only a few have been analyzed at the molecular level. Moreover, some instances of somatic mosaicism have been reported without segmental involvement (17). These individuals have generalized NF1, although their features may be milder because of the mosaicism.

Review

Figure 3. Skeletal dysplasias in neurofibromatosis type 1.

Guanosine Triphosphatase–Activating Protein (Neurofibromin)

The gene responsible for NF1 is located on chromosome 17 and encodes a protein called neurofibromin, which is a negative regulator of cellular proliferation and differentiation. Neurofibromin includes a guanosine triphosphatase (GTPase)–activating protein (GAP) (see Glossary) domain (18). This domain has been demonstrated to be active (19) and is the only part of the protein whose function is known. Neurofibromin is expressed in a wide variety of cell types, including (most notably) neurons, glial cells, and Schwann cells (20). The target of neurofibromin GAP activity is the intracellular signal transducer protein Ras. Ras is activated when certain cell surface receptors, including those with tyrosine kinase activity, bind ligand (21). Ras is activated by a family of exchange factors and is inactivated by a family of proteins known as GAPs, of which neurofibromin is an example. Activated Ras interacts with and alters the activity of several effector proteins, leading to various cellular responses, including changes in gene expression (Figure 4). The GAPs stimulate a GTPase activity intrinsic to Ras to inactivate the signal transduction pathway by converting Ras– guanosine triphosphate (GTP) to Ras– guanosine diphosphate (GDP). NF1 Gene Mutations

Pathogenic mutations in the NF1 gene have been found in most of the 60 exons. They comprise a wide diversity of mutation types (22), including complete gene deletions, chromosome rearrangements that disrupt the gene, smaller deletions or insertions, stop mutations (see Glossary), amino acid substitutions, and splicing mutations (see Glossary). The latter are particularly prevalent given the many exons, and most lead to premature termination of translation (see Glossary). Indeed, most mutations result in absent or nonfunctional protein. Some are known to disrupt the Ras-GTPase–activating function, but the mechanism of action of others is unknown. Genotype–Phenotype Correlations

Genotype–phenotype correlations have been difficult to establish because of the complexity of both the phenotype and the gene. Complete gene deletions (consisting of about 1.5 Mb), including the NF1 gene and several conwww.annals.org

Top. Dysplasia of tibia and fibula. Bottom. Computed tomography scan showing dysplasia of thoracic vertebra. 6 June 2006 Annals of Internal Medicine Volume 144 • Number 11 845

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bromas but may differ in other features, such as plexiform neurofibromas (26). Identification of genetic modifiers would be of interest both to enhance the predictive value of diagnostic tests and to better understand pathogenesis. This is an area of active study.

Figure 4. Ras signaling pathway.

PATHOPHYSIOLOGY

Binding of ligand to membrane tyrosine kinase receptor results in conversion of Ras– guanosine diphosphate (GDP) to Ras– guanosine triphosphate (GTP). This leads to a cascade of activation of other proteins (“effector pathways”), which eventually results in activation of transcription of specific genes. Shc, Grb2, SoS, Raf, Mek, and Erk are additional proteins in the Ras signal transduction pathway. NF1 ⫽ neurofibromatosis type 1; P ⫽ phosphate

tiguous genes, are associated with a severe presentation, including developmental impairment, dysmorphic features, and a large neurofibroma burden, and a substantially higher lifetime risk for MPNSTs (23, 24). Another distinct phenotype, familial spinal neurofibromatosis, may also be associated with a distinct mutational spectrum, with enrichment for amino acid substitutions or stop mutations near the 3' end of the gene (25). Other genotype–phenotype correlations may emerge as more carefully phenotyped patients are studied. Modifying Genes

Although allelic heterogeneity (see Glossary) may account for some variable expressivity of NF1, modifying genes (see Glossary) are likely to also play a role. Affected identical twins have been shown to have similar overall burden of cafe´-au-lait macules (see Glossary) and neurofi-

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NF1

Any explanation of the pathophysiology of NF1 must account for the diverse phenotypic manifestations, including both tumors and nontumor features. A leading hypothesis to account for the tumors has been that the NF1 gene functions as a tumor suppressor gene (see Glossary), with tumors developing only after both NF1 alleles have been lost. Substantial evidence has accumulated that support this hypothesis: 1) Mutation of both NF1 alleles, one inherited and the other acquired, was initially demonstrated in some MPNSTs (27) and subsequently in neurofibromas (28); 2) mice with only 1 mutated murine Nf1 (Nf1 is the murine equivalent to the human NF1 gene) allele do not develop neurofibromas (29, 30); and 3) mice with both Nf1 alleles mutated die of cardiovascular anomalies in utero, but chimeras, in which only a few of their cells have lost both Nf1 alleles, survive and develop plexiform neurofibromas (31). More recently, conditional knockout technologies have been used to generate mice, called conditional knockout mice (see Glossary), with one Nf1 gene mutated in the germline and the other subject to mutation only in developing Schwann cells (32). These animals get neurofibromas, providing powerful support for the tumor suppressor hypothesis and also offering a test system for potential therapies. This evidence suggests that a neurofibroma forms after a Schwann cell, which at first is heterozygous (see Glossary) for an NF1 gene mutation, undergoes somatic mutation to lose function of the other allele. This results in loss of neurofibromin function, but although necessary for neurofibroma formation, this seems to not be sufficient (Figure 5). Neurofibromas consist of a mixture of cell types. Only Schwann cells in neurofibromas have been shown to have 2 mutated NF1 alleles. The other cells are probably induced to proliferate by cytokines. These cells are heterozygous for an NF1 mutation and may be hypersensitive to stimulation. Mast cells are prevalent in neurofibromas and may be a source of cytokines. Evidence suggests that the tumor suppressor hypothesis most likely applies to other tumor-

Table 2. Summary of Neurofibromatoses* Disorder

Prevalence

Features

Gene

Protein/Function

NF1 NF2

1:3500 people 1:40 000 people

NF1 NF2

Schwannomatosis

Unknown

Refer to Tables 1 and 3 Vestibular and other schwannoma, meningioma, ependymoma, glioma, neurofibroma, cataract Schwannoma

Neurofibromin/GAP Merlin or schwannomin/cytoskeletal protein Unknown

Unknown

* GAP ⫽ guanosine triphosphatase (GTPase)–activating protein; NF1 ⫽ neurofibromatosis type 1; NF2 ⫽ neurofibromatosis type 2. 846 6 June 2006 Annals of Internal Medicine Volume 144 • Number 11

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Table 3. Additional Clinical Features and Age of Onset* Age at Onset

Clinical Features

Congenital or infancy (age 0–2 y)

Cafe´-au-lait macules, plexiform neurofibroma, tibial or orbital dysplasia, pulmonary stenosis, aqueductal stenosis, and hypotonia Skinfold freckling, learning disability, ADD, scoliosis, optic glioma, precocious or delayed puberty, short stature, renal artery stenosis, and vascular dysplasia Neurofibromas, cognitive deficits, pheochromocytoma, MPNST, vascular dysplasia, and hypertension

Childhood (age 3–12 y) Adolescence and adulthood (age ⬎12 y)

* ADD ⫽ attention-deficit disorder; MPNST ⫽ malignant peripheral nerve sheath tumor.

related NF1 phenotypes, including MPNSTs, optic gliomas, and juvenile myelomonocytic leukemia (33–39). The pathogenesis of nontumor manifestations of NF1 is not known. Melanocytes from cafe´-au-lait macules express an NF1 allele (40), suggesting that these lesions do not directly result from a tumor suppressor mechanism, although this question is not settled. Whether homozygous NF1 mutations occur within the skeletal lesions is not known. The basis for learning disabilities is likewise unknown. Persons with NF1 may have abnormalities of cortical architecture, including heterotopias (see Glossary) that might be expected to interfere with neurologic function. Conceivably, these might arise by homozygous NF1 mutation in neurons during development, but an effect of haploinsufficiency (see Glossary) of neurofibromin might also be involved. Mice that are heterozygous for an Nf1 mutation have cognitive deficits (41). These deficits are corrected after treatment with a Ras inhibitor, suggesting that they may be Ras-dependent.

The ultimate hope for patients with NF1 is that insights into pathogenesis will lead to new treatments. Several challenges are inherent in the process of translating knowledge into therapy for the disorder. First, the diverse lesions of NF1 may not all be the result of the same pathologic mechanism and may not respond to the same treatments. Second, treatments may be more likely to be successful if preventive than if initiated after onset of symptoms. Third, well-defined end points must be identified so that success of treatment can be judged.

TRANSLATION

Potential Pharmacologic Agents and New Imaging Techniques

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RESEARCH

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CLINICAL PRACTICE

Molecular Testing

The identification of the NF1 gene and consequent insights into the pathophysiology of the disorder have raised hopes that new diagnostic tests and treatments will ensue. Improved diagnostic testing is likely to be the first clinical benefit of cloning the gene. Neurofibromatosis type 1 is currently diagnosed by using a set of clinical criteria (Table 3) (42). In principle, molecular testing could resolve diagnostic uncertainty, for example, in children who are too young to fulfill diagnostic criteria or in individuals who present atypically. It could also be used as a basis for prenatal diagnosis. The large size of the NF1 gene and the wide diversity of mutations have been the major obstacles to direct mutation testing. Use of the protein truncation assay (see Glossary) to screen for stop mutations simplified the process of screening the gene, but it is limited by detecting only mutations that lead to premature termination of translation (43). Hence, approximately 30% of mutations are missed with this approach. The current approach to clinical molecular testing of NF1 involves using several complementary techniques (22). Robust molecular diagnostic testing has only been available clinically for a short while but has been useful both in diagnosis of affected individuals and in prenatal www.annals.org

diagnosis. For the most part, however, molecular testing does not predict disease severity or specific complications. We have noted exceptions where genotype–phenotype correlations have been established. Whether additional genotype–phenotype correlations will emerge when testing is done on a wider scale is unknown. If modifying genes also contribute to the NF1 phenotype, molecular testing of the NF1 gene alone is unlikely to be fully predictive. Challenges to Therapy

Plexiform neurofibromas can be very large and irregularly shaped, making them surgically intractable and mak-

Figure 5. Formation of neurofibroma.

A Schwann cell that is heterozygous for an NF1 mutation (⫹/⫺) undergoes mutation to ⫺/⫺. This cell proliferates and also attracts other cells, including fibroblasts, perineurial cells, and mast cells, to proliferate in the lesion. 6 June 2006 Annals of Internal Medicine Volume 144 • Number 11 847

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ing it difficult to monitor rate of growth and to assess the efficacy of potential therapies. Several clinical trials (www .ctf.org/clinical_trials/list.htm) are under way to treat plexiform neurofibromas. Experimental agents include pirfenidone, an antifibrotic drug, and tipifarnib, a farnesyltransferase inhibitor that targets Ras. Moreover, an ongoing trial is using volumetric magnetic resonance imaging to measure plexiform neurofibroma growth rate and may provide a useful outcome measure (44). Treatment of malignant conditions is another attractive therapeutic target. Better approaches for managing MPNSTs are needed. Early diagnosis is particularly problematic since tumors usually arise from preexisting benign lesions and individuals with NF1 are accustomed to having lesions for which they do not seek care. Fluorodeoxyglucose positron emission tomography has shown some promise in distinguishing benign from malignant lesions and deserves further study (45). For any of these lesions, various approaches to therapy might be considered. These include improvements in “conventional” approaches, such as imaging and surgery, but also the use of new pharmacologic agents. The latter might be developed specifically for use in NF1 on the basis of knowledge of the aberrant signaling pathway or might be borrowed from efforts to develop treatments for other disorders. Various Ras inhibitors or other effectors of Ras function have been developed because of the importance of Ras signaling in common types of cancer (46). Other drugs might target intercellular signaling or angiogenesis required to feed growing tumors. Since neurofibromas tend to grow during puberty and pregnancy, antagonists of hormone action might also be considered.

CONCLUSION Neurofibromatosis type 1 has been recognized as a clinical entity for more than a century, but it is only since the NF1 gene was identified in 1990 that we have begun to understand the pathophysiology of the disorder. It is clear that NF1 has tumor suppressor function, which is involved in the formation of neurofibromas, and that it also has other important functions in the pathogenesis of nontumor manifestations. Genetic testing was a long time coming, due to the complexity of the gene, but it is now available on a clinical basis. Clinical trials have begun with various drugs, suggesting that medical treatment of NF1 may become a reality. Achieving this hope, however, will require additional insights into pathogenesis and efforts to translate this knowledge into new methods of therapy. From the University of Alabama at Birmingham, Birmingham, Alabama. Potential Financial Conflicts of Interest: Grants received: B.R. Korf

(National Institutes of Health, U.S. Army). Requests for Single Reprints: Bruce R. Korf, MD, PhD, Department

of Genetics, University of Alabama at Birmingham, 1530 3rd Avenue 848 6 June 2006 Annals of Internal Medicine Volume 144 • Number 11

South, Birmingham, AL 35294; e-mail, [email protected]. Current author addresses are available at www.annals.org.

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6 June 2006 Annals of Internal Medicine Volume 144 • Number 11 849

Annals of Internal Medicine Current Author Addresses: Dr. Theos: Department of Dermatology, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham, AL 35294.

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Dr. Korf: Department of Genetics, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham, AL 35294.

6 June 2006 Annals of Internal Medicine Volume 144 • Number 11 W-207