Abstracts

Individuals with neurofibromatoses (NF) usually present to neurologists with one or more of the following clinical findings: cognitive and developmental deficits, macrocephaly, deafness, blindness, neuropathic pain, seizures or benign/malign tumors of the central nervous system (CNS). Also, NF affected persons may be referred to psychiatrists due to behavior disorders, including attention deficit/hyperactivity disorder (ADHD) and autism and/or learning difficulties.

There are three distinct diseases under NF denomination: neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2) and schwannomatosis (SCH). The NF share the genetic autossomal dominant predisposition to multiple tumor growth and the neural origin of tumors and cutaneous signals. They are progressive, present a great heterogeneity in phenotype expression and have unpredictable natural course. The NF differ profoundly in age of first manifestations, symptoms, evolution and prognosis ¹1 . Riccardi VM. Historical background and introduction. In: Friedman JM, Gutmann DH, MacCollin M, Riccardi VM (Eds). Neurofibromatosis - Phenotype, Natural History, and Pathogenesis. 3 ed. Baltimore: The Johns Hopkins University Press, 1999:1-28. .

NF diseases affect nearly 80 thousands of Brazilian individuals ²2 . Geller M, Bonalumi Filho A. Neurofibromatose: clínica, genética e terapêutica. 1 ed. Rio de Janeiro: Guanabara Koogan, 2004. and if NF is suspected, it is necessary to distinguish NF1 from NF2 and SCH. Figure 1 depicts a stepwise diagnosis pathway to distinguish the three NF diseases. In most cases, the differential diagnosis among the NF diseases is made with certainty. Nevertheless, special NF patients present overlap with some other diseases because of: a) cafe au lait lesions (CAL); b) localized hyper growth syndromes; c) neurofibromas-like tumors; and d) multiple endocrine neoplasia. These particular situations should deserve attention from the NF specialist.

The present Part 1 text offers step-by-step guidelines for differential diagnosis among the neurofibromatosis types and other diseases. A further Part 2 will present the NF clinical management. Basic information for patients can be found in www.amanf.org.br .

Step by step diagnosis of NF1, NF2 and SCH

Figure 1 depicts a practical step-by-step guide to differentiate NF1 from NF2 and SCH. The first two questions to be answered are: are there cafe au lait lesions (CAL)? Are there tumors?

STEP 1- Are there café au lait lesions?

Yes - CAL are usually multiple and present or develop at birth or in early infancy and are observed in 99% of people with NF1 by age of 3 years. They are macules with uniform ovoid shape and smooth outline, varying of light to dark brown color ( Figure 2A ). Its size varies from 5 mm (infancy) to 30 mm (adulthood), but they can be bigger than 20 cm and involve an entire anatomic region ⁴4 . Ferner RE. Neurofibromatosis 1. In: Ferner RE, Huson SM, Evans DG (Eds.) Neurofibromatoses in clinical practice. 1 ed. London: Springer-Verlag London Limited, 2011:1-46. . The CAL size is proportionate to body growth and they are randomly distributed, although scalp, palms, and soles are spared ⁵5 . Boyd KP, Gao L, Feng R, et al. Phenotypic variability among cafe-au-lait macules in neurofibromatosis type 1. J Am Acad Dermatol 2010;63:440-447. . Many other hyperpigmented skin lesions are commonly misdiagnosed as CAL, such as the lesion in Figure 2D .

Six or more typical CAL are a strong sign (95%) of NF1. NF1 is the most common condition associated with multiple CAL and patients with a higher number of CAL are more likely to develop NF1, but CAL can also be seen in other syndromes ( Table 5 ). On the other hand, the presence of more than three CAL is detected in only 0.3% of children with no evidence of genetic disorders ⁶6 . Nunley KS, Gao F, Albers AC, Bayliss SJ, Gutmann DH. Predictive value of cafe au lait macules at initial consultation in the diagnosis of neurofibromatosis type 1. Arch Dermatol 2009;145:883-887. .

STEP 2 – Are there axillary and inguinal freckling?

Yes - Axillary and/or inguinal freckling are hyperpigmented spot macules (1-3mm) and the second most common feature in NF1 ( Figure 2B ). Their appearance is similar to solar-induced freckling, but in NF1 they occur typically in areas with minimal to none sun exposure. Freckling generally appears between 3 and 5 years of age in the axillae and/or inguinal region. Other sites include neck and breasts, around the lips, and even the trunk in adults, but in these sites they are not a diagnostic criterion ⁶6 . Nunley KS, Gao F, Albers AC, Bayliss SJ, Gutmann DH. Predictive value of cafe au lait macules at initial consultation in the diagnosis of neurofibromatosis type 1. Arch Dermatol 2009;145:883-887. .

Until recent years, the presence of CAL associated with freckling was enough to the NF1 diagnosis. Nevertheless, the discovery of the Legius syndrome (LEGIUS), which has in common with NF1 the CAL, the freckling, macrocephaly and cognitive deficits, made required a third criterion to make the NF1 diagnosis certain ⁷7 . Brems H, Pasmant E, Van Minkelen R, et al. Review and update of SPRED1 mutations causing Legius syndrome. Hum Mutat 2012;33:1538-1546. . LEGIUS could be considered the fourth NF disease ³3 . Huson SM. The neurofibromatoses: differential diagnosis and rare subtypes. In: Ferner RE, Huson SM, Evans DG (Eds). Neurofibromatoses in Clinical Practice. 1 ed. London: Springer-Verlag London Limited, 2011:1-46. .

STEP 3 – Are there other NF1 consensus criteria?

The diagnostic criteria for NF1 have been established by a National Institute of Health consensus of experts in 1987. A person is thought to have NF1 if they have two or more of the Table 1 criteria.

STEP 4 – Established NF1 diagnosis (previous von Recklinghausen’s Disease)

NF1 is the most prevalent human monogenetic disease (incidence of 1:2,500 live births) ¹⁴14 . Huson SM, Harper PS, Compston DA. Von Recklinghausen neurofibromatosis. A clinical and population study in south-east Wales. Brain 1988;111:1355-1381. , it is an autossomal dominant disorder caused by hereditary or new mutations in the neurofibromin protein codification gene, located in Chromosome 17 (locus 17q11.2), which results in neural and other tissues dysplasia and increased tumor formation of neural origin ¹⁵15 . Ferner RE, Huson SM, Thomas N, et al. Guidelines for the diagnosis and management of individuals with neurofibromatosis 1. J Med Genet 2007;44:81-88. .

STEP 5 – Are there tumors?

Most NF cases are associated with some CAL; therefore, its absence suggests other diseases. Nevertheless, considering that some special NF patients do not have CAL, the next step is to search for tumors.

The common types of tumors associated with NF1 are neurofibromas ( nfbs ), plexiform neurofibromas (PNF), malignant peripheral nerve sheath tumors (MPNST) and optic glioma (OPG) (see below). The presence of two or more of these tumors is strongly suggestive of NF1 and other NIH criteria must be investigated (STEP 4). If no other criterion is found, this situation deserves a NF specialist support. On the other side, the presence of two or more schwannoma tumors (see below) is suggestive of both NF2 and SCH.

NF1 associated tumors

Although a specialized physician usually recognizes the different types of neurofibromas, the diagnostic certainty needs further histological studies. Below, we present a brief description of NF1 commonest tumors ²2 . Geller M, Bonalumi Filho A. Neurofibromatose: clínica, genética e terapêutica. 1 ed. Rio de Janeiro: Guanabara Koogan, 2004. ^{, 16}16 . Cunha KS, Geller M. Advances in neurofibromatosis research. 1 ed. New York, USA: Nova Science Publishers Inc, 2012:1-273. and detailed histological information can be found elsewhere ¹⁷17 . Carranza AC, Salinas Martín MV, Polo R, Córdoba JC, González-Cámpora R. Problemas diagnósticos en tumores del nervio periférico (I and II). Rev Esp Patol 2011:97-116. .

Neurofibromas (WHO grade I)

Neurofibromas, the hallmark tumors of NF1, are benign tumors of the peripheral nerve sheath. They exhibit extensive cellular heterogeneity (Schwann cells, perineural cells, mast cells, fibroblasts and axons in an extracellular matrix) and it can be classified into two major types: localized and PNF.

Localized neurofibromas are the most prevalent type of tumor in NF1. They tend to develop from skin sensory nerves and usually present as a cutaneous and/or subcutaneous tumor that remains associated with a single nerve ending. The nfb s are more commonly found on the skin, but other sites can also be affected, such as the spinal roots, heart, stomach, larynx, bladder, bowels and oral mucosa.

Plexiform neurofibromas are almost always associated with NF1 and they are classified as benign peripheral nerve sheath tumors that involve multiple nerve fascicles or large branches of a major nerve. PNF are the first and foremost source of morbidity in NF1, due to their tendency to grow to large sizes and their capacity to cause significant deformity and compression of adjacent structures. Some may be present at birth, but most of them usually become apparent during the first two years of life and, if not present by then, rarely develop after adolescence. The growth rate associated with these neoplasms is unpredictable, with periods of rapid growth followed by periods of relative inactivity.

Malignant peripheral nerve sheath tumor (WHO grade II, III or IV)

MPNST commonly derive from pre-existing PNF. The NF1 patient lifetime risk of developing MPNST is 8-13% (being the double in the NF1 microdeletion subtype). Because MPNST develop from malignant progression of pre-existing PNF, the risk of developing MPNST increases to almost 50% in patients with NF1 and PNF ¹⁸18 . Park SJ, Sawitzki B, Kluwe L, Mautner VF, Holtkamp N, Kurtz A. Serum biomarkers for neurofibromatosis type 1 and early detection of malignant peripheral nerve-sheath tumors. BMC Med 2013;11:109. . MPNST occurrence in childhood and adolescence is uncommon. Incidence is higher among adults in the third to sixth decade of life. MPNST should be suspected when the patient develops persistent pain with no other explanation and/or unexplained neurological deficit associated with changes in texture and enlargement of the PNF. Histological differentiation between a PNF with atypical features and a low grade MPNST is difficult, since these lesions actually represent a histological continuum and it is possible to find lesions with areas of atypical features adjacent to areas of frank MPNST.

Gliomas

Gliomas are the predominant CNS tumor type associated with NF1 and they may occur in all parts of the nervous system, with a preference for the optic pathways, brainstem and cerebellum. Frequent NF1-associated gliomas include optic pathway gliomas (OPG), other pilocytic astrocytomas, diffuse astrocytomas and glioblastomas.

OPG (WHO grade 1)

The majority of OPG in NF1 patients are pilocytic astrocytomas that are located within the optic nerve. Macroscopically most pilocytic astrocytomas are a relatively circumscribed, slowly growing soft-grey mass. Intra or paratumoural cyst formation is common. Optic nerve tumors also often show collar-like involvement of the subarachnoid space. Histopathologically, this astrocytic tumor of low to moderate cellularity exhibits an often biphasic pattern with varying proportions of compacted bipolar cells with Rosenthal fibers and loose-textured multipolar cells with microcysts and granular bodies/hyaline droplets. Rare mitosis, hyperchromatic and pleomorphic nuclei, glomeruloide vascular proliferation, infarct-like necrosis and infiltration of leptomeninges are compatible with the diagnosis of pilocytic astrocytoma and are not signs of malignancy ¹⁹19 . HO Classification of Tumours of the central nervous system. 4 ed: WHO; 2007:1-312. . Bilateral growth, when present, is characteristic of NF1.

Diffuse astrocytomas (WHO grade II)

That is a diffusely infiltrating astrocytoma that typically affects young adults and is characterized by a high degree of cellular differentiation and slow growth; the tumor occurs throughout the CNS but is preferentially supratentorial and has an intrinsic tendency for malignant progression to anaplastic astrocytoma and, ultimately, glioblastoma.

Glioblastoma (WHO grade IV)

The most malignant neoplasm with predominant astrocytic differentiation; histological features include nuclear atypia, pleomorphic cells, mitotic activity, vascular thrombosis, microvascular proliferation and necrosis. It typically affects adults and is preferentially located in the cerebral hemispheres. Most glioblastomas manifest rapidly de novo, without recognizable precursor lesions (primary glioblastoma). Secondary glioblastomas develop slowly from diffuse WHO grade II astrocytoma or anaplastic astrocytoma (WHO grade III). Due to their invasive nature, glioblastomas cannot be completely removed and, despite progress in radio/chemotherapy, less than half of patients survive more than one year, with older age being the most significant adverse prognostic factor.

Other NF1 associated tumors

NF1 patients present with a higher risk of developing other tumors such as:

Sarcomas

Gastrointestinal stromal tumor (GIST)

Neuroendocrine/neuroectodermal tumours

Pheochromocytomas

Carcinoid tumor

Medullar thyroid carcinoma

C-cell hyperplasia

Hematopoietic tumors

Juvenile chronic myeloid leukemia

Juvenile xanthogranuloma

Acute lymphocytic leukemia

Non Hodgkin lymphoma

STEP 6 – Are there bilateral vestibular schwannomas (BVS)?

Yes – The presence of BVS is a hallmark of NF2. NF2 is an autossomal dominant disorder caused by hereditary or new mutations in the merlin protein codification gene located in chromosome 22 (locus 22q12.2). NF2 presents with BVS, multiple meningiomas, juvenile subcapsular cataract and peripheral schwannomas and other neural tumors.

Schwannomas (WHO grade I)

Schwannomas are benign true nerve sheath neoplasms composed entirely of Schwann cell proliferation, well circumscribed, nodular or ovoid, usually encapsulated dermal, subcutaneous and central nerves tumors ²2 . Geller M, Bonalumi Filho A. Neurofibromatose: clínica, genética e terapêutica. 1 ed. Rio de Janeiro: Guanabara Koogan, 2004. .

The prevalence of NF2 vary from 1:25,000-33,000 individuals ²⁰20 . Blakeley JO, Evans DG, Adler J, et al. Consensus recommendations for current treatments and accelerating clinical trials for patients with neurofibromatosis type 2. Am J Med Genet A 2012;158:24-41. and it presents with deafness and/or tinnitus (50%), balance dysfunction (5%), focal weakness or sensory change (18%), seizure (5%), ocular symptoms (7%) and genetic or radiographic diagnosis of an asymptomatic family member (10%), during the second decade or early adulthood ²¹21 . MacCollin M. Neurofibromatosis 2: clinical aspects. In: Friedman JM, Gutmann DH, MacCollin M, Riccardi VM (Eds). Neurofibromatosis - phenotype, natural history, and pathogenesis. 3 ed. Baltimore: The Johns Hopkins University Press, 1999:299-326. .

Nevertheless, some special cases of NF2 are less evident. The Manchester (modified NIH) diagnostic criteria for NF2 have been expanded to include patients with no family history who have multiple schwannomas and or meningiomas, but who have not yet developed bilateral vestibular nerve tumors ²²22 . Baser ME, Friedman JM, Wallace AJ, Ramsden RT, Joe H, Evans DG. Evaluation of clinical diagnostic criteria for neurofibromatosis 2. Neurology 2002;59:1759-1765. . The diagnosis is best confirmed using high quality MRI of the brain.

If there are two or more painful schwannomas, the present step is to complete the diagnosis of SCH with the following criteria ²³23 . MacCollin M, Chiocca EA, Evans DG, et al. Diagnostic criteria for schwannomatosis. Neurology 2005;64:1838-1845. . Table 2 presents the consensus criteria for NF2 and SCH diagnosis.

SCH typically becomes symptomatic during the third decade of life and presents a prevalence of 1:40,000 individuals. The molecular biology of SCH has not been well established yet and a candidate gene for SCH, called INI1, was identified, but the NF2 gene and possibly other genes may also be involved. Unlike NF1 and NF2, SCH does not have a clear pattern of inheritance.

The Table 3 summarizes the main clinical differential characteristics of NF1, NF2 and SCH.

NF1 variations

There are genetically related (allelic) disorders, where apparently pathogenic NF1 mutations have been demonstrated in a few individuals or families who do not have NF1 according to the NIH Diagnostic Criteria ⁴4 . Ferner RE. Neurofibromatosis 1. In: Ferner RE, Huson SM, Evans DG (Eds.) Neurofibromatoses in clinical practice. 1 ed. London: Springer-Verlag London Limited, 2011:1-46. . They have specific phenotype and clinical course and are presented in Table 4 and its management will be discussed in Part 2 of this text.

Mosaicism or segmental NF

Occasionally NF1, NF2 (rarely) and schwannomatosis (often) occur in mosaic forms. Mosaicism in NF1 is observed more frequently and results from somatic mutations. Early somatic mutations cause generalized disease, clinically indistinguishable from no mosaic forms. Later somatic mutation gives rise to localized disease often described as segmental. In individuals with mosaic or localized manifestations of NF1 (segmental NF1), disease features are limited to the affected area, which varies from a narrow strip to one quadrant and occasionally to one half of the body. Distribution is usually unilateral but can be bilateral, either in a symmetric or asymmetrical arrangement.

Differential diagnosis

The main characteristics of other diseases, which present differential diagnosis with NF1, are summarized in Table 5 .

When ordering genetic tests

Approximately 50% of individuals with NF1 or NF2 have not inherited the disease from a parent and are result of a de novo mutation. Individuals who meet the clinical diagnosis criteria for NF1, NF2 and SCH are diagnosed with confidence in most cases, and genetic tests are not required for diagnostic confirmation. Therefore, genetic testing may be helpful in specific cases. The decision to order or not a genetic test remains a physician/patient (family) discussion. Genetic testing for NF is clinically available in just a few laboratories around the world. For an updated list of laboratories that offers genetic tests for NF, please refer to www.genetests.org .

NF1 versus LEGIUS Syndrome

The NF1 gene is large (280 kb and 60 exons) and most mutations are unique to a particular family, what turns the molecular diagnostic testing laborious and expensive. A definite diagnosis of NF1 can be made in most children by age four using only the NIH criteria ²⁴24 . Friedman JM. Neurofibromatosis 1. Seattle (WA): University of Washington, Seattle; 1993-2013; 1998 (Updated 2012) [cited 2013]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK1109/.
http://www.ncbi.nlm.nih.gov/books/NBK110... . Confirmatory diagnostic testing can detect the mutation in 95% of cases and is indicated for suspected NF1 individuals, which do not fulfill the NIH diagnostic criteria, most often in young children, if it can affect clinical management.

The presence of only pigmented lesions at early age is a diagnostic dilemma between NF1 and LEGIUS (caused by SPRED1 gene mutations). The distinction between NF1 and LEGIUS in young children, in the absence of NF1 family history and neoplastic or osseous manifestations can only be achieved by genetic testing. It is cost saving and easier to start by investigation of SPRED1 gene than by NF1, because SPREAD1 contains only 8 exons ⁷7 . Brems H, Pasmant E, Van Minkelen R, et al. Review and update of SPRED1 mutations causing Legius syndrome. Hum Mutat 2012;33:1538-1546. .

It is important to emphasize that currently there are only two important genotypes that correlate to phenotype in NF1. One of these is the microdeletion syndrome, in which a large part of or the whole NF1 gene is absent and is associated with a more severe phenotype ²⁵25 . Pasmant E, Sabbagh A, Spurlock G, et al. NF1 microdeletions in neurofibromatosis type 1: from genotype to phenotype. Hum Mutat 2010;31:1506-1518. . The other genotype is the mutation involving the deletion of three base pairs in exon 17, which correlates with a mild phenotype ²⁶26 . Upadhyaya M, Huson SM, Davies M, et al. An absence of cutaneous neurofibromas associated with a 3-bp inframe deletion in exon 17 of the NF1 gene (c.2970-2972 delAAT): evidence of a clinically significant NF1 genotype-phenotype correlation. Am J Hum Genet 2007;80:140-151. . Therefore, discarding these two specific genotypes, it is not possible to predict the severity of the disease based on NF1 genetic testing.

NF2

It is expected that 25% to 33% of those who have NF2 and are a single occurrence in the family (simplex cases) are mosaic for NF2 mutation. The mutation detection rate by sequencing and deletion/duplication analysis is around 92% for familial cases and 72% in simplex cases. NF2 genetic testing may be helpful in specific cases and, although some genotype-phenotype correlations in NF2 are known, it cannot yet predict the severity of the disease ²⁷27 . Nunes F. Research advances in mutational analysis of the NF2 gene. In: Cunha K, Geller M, editors. Advances in neurofibromatosis research. 1 ed. New York, USA: Nova Science Publishers Inc, 2012:165-80. .

a. Pre-symptomatic testing of at-risk patients

Because detection of tumors at an early stage is effective in improving the clinical management of NF2 patients, the screening for NF2 in children of affected patients can start at birth with the search of cataracts ²⁸28 . Evans DG, Raymond FL, Barwell JG, Halliday D. Genetic testing and screening of individuals at risk of NF2. Clin Genet 2012;82:416-424. . If no cataracts are found, pre-symptomatic genetic testing, if possible, should be considered.

b. Determining the diagnosis of patients who fall short of the Manchester criteria, including mosaic patients

In cases of patients that fall short of the Manchester criteria, genetic testing may be helpful in establishing the diagnosis. The diagnosis of NF2 is usually a challenge when mosaicism is present. Mutations with mosaicism levels greater than 10% can be detected in blood DNA, otherwise its identification requires testing of tumor material. In these cases, the diagnosis is established if two NF2 identical mutations are found in separate tumors, but is not present in blood ²⁶26 . Upadhyaya M, Huson SM, Davies M, et al. An absence of cutaneous neurofibromas associated with a 3-bp inframe deletion in exon 17 of the NF1 gene (c.2970-2972 delAAT): evidence of a clinically significant NF1 genotype-phenotype correlation. Am J Hum Genet 2007;80:140-151. .

c. Estimating the transmission risk for mosaic patients

The transmission rate of NF2 is 50% in the second generation, but the risk of transmission in apparently isolated patients with NF2 is less than 50% due to mosaicism. Transmission risk of NF2 decreases significantly after the proband has had negative genetic testing using a blood sample ( Table 6 ) ²⁷27 . Nunes F. Research advances in mutational analysis of the NF2 gene. In: Cunha K, Geller M, editors. Advances in neurofibromatosis research. 1 ed. New York, USA: Nova Science Publishers Inc, 2012:165-80. .

Schwannomatosis (SCH)

Currently, it is known that germline mutations in SMARCB1 (or INI1 ) gene are associated to SCH. Nevertheless, they are found in only 40-50% of the familial cases and in 8-10% of the sporadic cases of SCH. For SCH cases unrelated to SMARCB1 germline mutations, the genetic alterations are not known ²⁷27 . Nunes F. Research advances in mutational analysis of the NF2 gene. In: Cunha K, Geller M, editors. Advances in neurofibromatosis research. 1 ed. New York, USA: Nova Science Publishers Inc, 2012:165-80. .

SMARCB1 gene testing may be ordered for individuals with multiple schwannomas without evidence of vestibular tumor and no NF2 mutation detected in the blood. Nevertheless, there is a great chance of not detecting a SMARCB1 genetic alteration and the diagnosis of SCH cannot be ruled out. SMARCB1 gene testing is also indicated for early detection of at-risk patients (because of family history) for management reasons.

Tumorigenesis of schwannomas associated to SCH is believed to occur through a four-hit model, involving not only mutations in SMARCB1 alleles (hits 1 and 2), but also mutations in NF2 alleles (hits 3 and 4) ³⁰30 . Plotkin SR, Blakeley JO, Evans DG, et al. Update from the 2011 International Schwannomatosis Workshop: From genetics to diagnostic criteria. Am J Med Genet A 2013;161:405-416. . Differently from NF2-associated schwannomas, SCH-associated schwannomas of the same individual usually carry different somatic NF2 mutations. Therefore, mutational analysis of schwannoma cells is also highly relevant for the diagnosis, even in the absence of SMARCB1 mutation in blood. The finding of different NF2 somatic mutations in different schwannomas of the same individual, although not indicative of the presence of a germline SMARCB1 , at least rules out the diagnosis of NF2 mosaicism ³¹31 . Papi L. Schwannomatosis: a recently recognized form of neurofibromatosis. In: Cunha K, Geller M (Eds). Advances in Neurofibromatosis Research. 1 ed. New York, USA: Nova Science Publishers Inc, 2012:258-267. .

Genetic Testing for Preimplantation Genetic Diagnosis (PGD) of NF

Another application of NF genetic test is for the PGD. It comprises a set of procedures to test a specific genetic alteration and selecting unaffected embryos for transfer to the uterus. It is a highly specialized method performed in a few centers and is available for couples when the parental mutation has already been identified. It has been shown as a promising and useful method, being the only one that prevents transmission of the mutated gene from one parent to their children ³²32 . Bonalumi Filho A, Darrigo Jr L, Oliveira L, Azulay D, Cunha K. Pre-implantation genetic diagnosis of neurofibromatosis. In: Cunha K, Geller M (Eds). Advances in Neurofibromatosis Research. 1 ed. New York, USA: Nova Science Publishers Inc, 2012:258-267. . For an updated list of centers that perform PGD for NF, please refer to www.genetests.org

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