Advances in Molecular Diagnosis of Neurofibromatosis Type 1 Ben Shofty, MD,*,‡ Shlomi Constantini, MSc, MD,*,†,‡ and Shay Ben-Shachar, MD‡,§ Neurofibromatosis 1 (NF1) is a common neurocutaneous and tumor predisposing genetic disorder with an autosomal dominant mode of inheritance. NF1 is solely caused by mutations in the NF1 gene, and disease-causing mutations can be found in more than 95% of individuals with a clinical diagnosis. Although NF1 has a distinctive clinical phenotype, it has a highly variable expression, even among individuals from the same family. Identifying the specific mutation does not usually assist in determining disease course and severity, and relatively few genotype-phenotype correlations have thus far been found. This review discusses the basic clinical aspects of NF1 and the current explanations for the high phenotypic variability, and provides the recently detected genotype-phenotype correlations. Semin Pediatr Neurol 22:234-239 C 2015 Elsevier Inc. All rights reserved.
Introduction Historically referred to as von Recklinghausen’s disease, neurofibromatosis type 1 (NF1, MIM162200) is a genetic neurocutaneous disorder that is inherited in an autosomal dominant fashion. NF1 affects approximately 1 in 2500 newborns.1 The main disease manifestations are café-au-lait macules (CALM), neurofibromas, skinfold freckling, iris hamartomas (Lisch nodules), optic pathway gliomas (OPGs), and skeletal deformities. In addition, behavioral and cognitive impairments are common among individuals with NF1.2 Although considered benign, plexiform neurofibromas (PN) and OPG are major causes of concern, as they may produce disfigurement and blindness. The main reason for close, prolonged follow-up is the associated susceptibility to malignancies, as patients may develop malignant peripheral nerve sheath tumors, hematologic malignancies, and high-grade gliomas. Mutations in the NF1 gene that encodes the protein neurofibromin are the sole known cause of NF1. Although From the nDivision of Neurosurgery, Tel-Aviv Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. †Department of Pediatric Neurosurgery, Tel-Aviv Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. ‡Gilbert Israeli Neurofibromatosis Center, Tel-Aviv Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. §Genetic Institute, Tel-Aviv Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel. Address reprint requests to Shay Ben-Shachar, MD, The Gilbert Israeli Neurofibromatosis Center, Tel-Aviv Medical Center, 6 Weizmann St, Tel-Aviv 6423906, Israel. E-mail:
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the disease has a complete penetrance, each affected individual will eventually present with some of the widely variable disease manifestations. For that reason, the natural history cannot usually be determined, even for people from the same family or with the same specific mutation in the gene.3 Mutation analyses is important for early diagnosis of NF1 and for clarifying ambiguous clinical cases, in addition to being essential for prenatal and pre-gestational diagnoses. The few genotype-phenotype correlations that were recently established have assisted in determining disease severity in these cases.
Clinical Diagnosis and Clinical Presentation The diagnosis of NF1 had traditionally been based on clinical criteria, because of their high level of accuracy and the absence of reliable molecular tests. They were established in 1987 by the American National Institute of Health (NIH) (Table 1),4 and 2 or more are required for diagnosing NF1. The sensitivity and specificity of that system are high and robust enough to diagnose 50% of affected children with no family history by the age of 1 year and 95% by the age of 8 years.5-7 As disease manifestations present gradually, a clinical diagnosis cannot always be made in very young children, especially among those with a negative family history of first-degree relatives with NF1, which is one of the disease criteria. It has therefore been suggested that modification of
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Table 1 Clinical Diagnostic Criteria for NF144 and Associated Diagnostic and Clinical Findings Expected Age at Percentage of Presentation Patients (%)
Diagnostic Criteria
CALM (Z6, 415 mm in adults and 45 mm in children) Birth to 2 years Cutaneous neurofibromas (Z2) 5þ years One plexiform neurofibroma Birth to 3 years Axillary or inguinal freckling 3-5 years Lisch nodules (Z2) 5-10 years Optic pathway glioma 3-8 years Typical osseous lesion (sphenoid wing dysplasia or long-bone cortical thinning, 1-3 years w/wo pseudoarthrosis) Family history of NF1 (first-degree relative) – Additional common clinical findings (percentage of patients) ADHD or memory and learning difficulties (50%-75%) T2 hyper-intensities (60%-70%) Cardiovascular abnormalities (hypertension, renal artery stenosis, pulmonary artery stenosis; 2% each) Hormonal abnormalities or precocious puberty Progressive scoliosis Neuropathy ( 1%, usually distal and symmetrical) Pheochromocytoma (2%)
499 499 30-50 90 90 15-30 2% Pseudoarthrosis 1% SWD 50
Required evaluation Neuropsychological consultation None Yearly blood pressure monitoring and heart assessment, echocardiography as needed Growth curves at every routine follow-up Skeletal or bone assessment Rule out other causes for neurological changes in NF1 patients (PN, MPNST, etc.) 24 h urinary excretion of catecholamines and their metabolites
CALM, café-au-lait macules; ADHD, attention deficit hyperactivity disorder; PN, plexiform neurofibroma; MPNST, malignant peripheral nerve sheath tumor.
these criteria may be necessary for children younger than 8 years. For example, T2 hyper-intensities on brain magnetic resonance imaging were suggested as a supplemental diagnostic criterion, but they are seldom used for the initial diagnosis because of the complexity in acquiring those imaging data in young children, together with their lack of specificity.8 CALM, are usually the first sign of the disease and appear at birth or at the first few months of life, with a gradual increased in size and number during the first years of life. PN, if exist, may appear at birth, skinfold freckling typically presents around the age of 1-2 years. Other manifestations, such as Lisch nodules, usually appear later, and they are present in only approximately 40% of affected children at the age of 6 years. The last main manifestation of the disease to appear is cutaneous and subcutaneous neurofibromas, peaking at early adolescence.9
Basic Genetics and Cellular Biology The NF1 gene is located at chromosome 17q11.2 and contains 61 exons. It encodes a large intracellular protein ( 327 kDa) called neurofibromin. NF1 is caused by heterozygous, loss of function mutations in the NF1 gene.10,11 No other responsible genes have thus far been detected, whereas genetic insults causing similar changes in downstream intracellular pathways may present similar clinical manfiestations.
Normal neurofibromin acts a Ras-negative regulator (among other functions) by accelerating the hydrolysis of Ras-bound GTP.9 When there is a lack or dysfunction of neurofibromin, Ras is constantly attached to GTP and the pathway is hyperactivated, leading to deregulated cellular proliferation and survival.12 Upregulation of Ras triggers downstream signaling pathways, including the mitogenactivated protein kinase (MAPK) RAF or MEK or ERK pathway. Given that activation of the RAF or MEK or ERK pathway is associated with tumorigenesis, the NF1 gene is considered to be a tumor-suppressor gene, and its loss of function caused by an intragenic mutation or by a complete gene loss (gene deletion) is associated with tumorigenesis. Interestingly, other genetic disorders are associated with mutations in genes acting in this pathway: they share some phenotypes and are collectively termed “Rasopathies.” NF1 is an autosomal dominant disease caused by a mutation in 1 of the 2 copies of the NF1 gene, which has complete penetrance associated with a highly variable clinical presentation.13 Approximately 50% of NF1 patients have a familial disease,6 and the rest of the cases are de-novo events in the proband. Loss of function mutations that can be detected in more than 95% of individuals with a clinical diagnosis of NF1 are of the truncating type according to the NIH diagnostic criteria.14,15 However, as many of the disease-causing mutations are splicing mutations,15 DNA testing may not be able to detect a considerable number of them. RNA-based methods, which do detect both splice mutations, together with the protein-coding mutations not detected by DNA-based methods, are required to achieve a high molecular detection rate.16
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Variable Expression of NF1 and the “Two Hits” Theory NF1 patients harbor a heterozygous germline mutation, meaning that they have 1 normal DNA copy (allele) and another allele with the disease-causing mutation in each nucleated, nongamete cell. However, molecular testing of each lesion, for example, CALM, neurofibromas, OPGs, detects 2 genetic insults.16-18 One of them is the germline mutation, and the other is an additional specific event that can be either a second mutation or a loss of the expression of the normal allele, which is termed loss-of-heterozygosity. This is consistent with Knudsen’s “two-hit” hypothesis,17 initially described in another tumor-suppression gene, the RB gene, which is associated with the development of retinoblastoma. According to that theory, the germline mutation serves as a “first hit,” and another cell-specific event, the “second hit,” must occur in a specific cell in order for the clinical manifestation to develop. Given the large number of cells in the body and the high mutagenicity of the NF1 gene, such events always occur in patients, but in different cell numbers, types, locations, and ages. Therefore, it is extremely difficult to predict the natural history of the disease in a single affected individual. It has been proposed that the complex development of NF1-associated tumors, such as neurofibromas, can be modeled using the somatic loss of the normal NF1 allele from a Schwann progenitor cell on a complex background in which NF1 haploinsufficiency exists in neighboring cells.18-20 Further support of this theory arises from the modeling of OPGs, another disease hallmark, in murine models using bi-allele Nf1 deletion from astrocytes on an haplo insufficient background.21,22 Overall, 2 large familial phenotype studies, which looked at 696 families with NF1 used phenotypic correlations between family members to demonstrate that the primary NF1 mutation plays a minor role when compared to the role of modifier genes.23-25 All the investigated disease traits in those studies, except for neoplasms, were highly clustered in families, and the patterns of familial correlations suggested that there is a
strong genetic constituent unlinked to the NF1 locus, and with little or no influence of the constitutional NF1 mutation. In neoplasms, the “second hit” theory explains the high variability even within members of the same family.
Genotype-Phenotype Correlations NF1 has a highly variable and unpredictable expression, even among affected individuals with the same mutation and within the same family.26 It has been thought that the “second hit” basis of the tumor-related phenotype contributes to the highly variable expression since the development of some of the manifestations results from a second random event. Although numerous mutations in the NF1 gene have been reported, given the variable expression nature of the disease, multiple cases with the same mutation are usually required in order to estimate a genotype-phenotype correlation of a specific mutation type. Nevertheless, a few such correlations have been recently detected in NF1, assisting in predicting the natural history and in performing direct surveillance in specific cases. These are summarized in Table 2. Whereas some of the reported NF1 genotypes are associated with more severe phenotypes, most of the reported correlations involve specific mutations associated with a milder disease expression. Large complete gene deletions, ie, those encompassing 5%-10% of pathogenic NF1 mutations, are associated with a more severe clinical phenotype that includes intellectual disability, dysmorphism, cardiovascular malformations, childhood overgrowth, higher benign tumor burden, and a higher incidence of malignant nerve sheath tumors.3,27,28 The size of these deletions is typically 1.2-1.4 Mb. It is believed that the severe phenotype is related, at least in part, to the contiguous gene syndrome generated by a deletion of additional genes, specifically, at least 14 protein-coding genes in the larger more common 1.4 Mb deletion.3 For example, the deletion of the RNF135 gene located in the
Table 2 Genotype-phenotype Correlation Genotype
Phenotype
Reference #
Large or complete gene deletion
Severe disease manifestation Cognitive Cardiovascular Benign and malignant tumors OPG (OR ¼ 6.05) No cutaneous neurofibromas
3,25,26,27
31,32 33
Spinal NF1
36,37
No cutaneous manifestations, Noonan like dysmorphic features, Cardiac defect—pulmonary stenosis Cardiac defect—pulmonary stenosis Elephantiasis, solid malignancies
34,35
Upper 50 third mutation Deletion of 3-bp (c2970-2972 delAAT) Constitutional missense and splicing mutations p. Arg1809 missense mutations Nontruncating mutations Truncating mutations OPG, optic glioma; OR, odds ratio.
38 30
Advances in molecular diagnosis deleted region is associated with the increased rate of overgrowth seen in those cases.29 Similarly, truncating mutations are related to a severe phenotype that manifests as elephantiasis and solid malignancies.30 In addition, the complete loss of 1 copy of the neurofibromin contributes to a more severe NF1-related phenotype. Similarly, it was found that truncating NF1 gene mutations located in proximal exons were associated with an increased rate of OPG: patients with mutations in the 50 (proximal) third of the NF1 gene had an odds ratio of 6.05 to have OPGs,31,32 most likely due to the shorter length of the remaining protein. Given that missense mutations, which cause amino acid substitution, and in-frame mutations may retain some of the gene function, in contrast to truncating mutations, it is expected that these mutations may be associated with a milder effect. Numerous different nontruncating mutations have been reported in NF1. However, given the fact that none of these mutations is common, and the variable expression nature of NF1, it is difficult to detect clear genotype-phenotype correlations associated with specific mutations. The few described genotype-phenotype correlations related to nontruncated mutations suggest that inframe and missense mutations may be associated, indeed, with a milder phenotype. The in-frame deletion of 3-bp (c2970-2972 delAAT) from axon 17 was described to be associated with lacking cutaneous neurofibromas or clinically obvious PN.33 Recently, different missense mutations of the Arginine located at codon 1809 were described.34,35 These missense mutations (including p. Arg1809Cys, p.Arg1809Leu, p.Arg1809Pro, p. Arg1809Ser, and p.Arg1809Gly) are found in about 1.2% of patients with NF1. These mutations are associated with multiple CALM, and skinfold freckling, but lack discrete cutaneous or PN, Lisch nodules, typical NF1 osseous lesions and symptomatic optic gliomas. Interestingly, patients with missense mutations in codon 1809 have some specific characteristic not commonly associated with truncating mutations, such as an increased Noonanlike features, and pulmonary valve stenosis. Developmental delays and learning disabilities were reported in over 50% of these patients.34,35 Multiplicity of constitutional missense and splicing mutations is associated with the unique phenotype spinal neurofibromatosis. Affected individuals harbor multiple spinal neurofibromas but display very few, if any, other disease manifestations.36,37 Genotype-phenotype correlation is associated with nontumor-related phenotypes as well. Although NF1 is a Rasopathy by definition, most patients with NF1 do not present with the hallmarks of the typical Rasopathies (prototyped by Noonan syndrome), such as distinct facial dysmorphism and distinct cardiac defects (eg, pulmonary stenosis). However, different nontruncating, disease-causing mutations in the NF1 gene were found to be specifically associated with pulmonary stenosis type of heart defect, a classic finding in Noonan syndrome.35,38
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NF1 Somatic Mosaicism Somatic mosaicism in which a mutation can be detected in only some of the cells has been reported in NF1. These patients typically have segmental or regional NF1 as manifested by features of NF1 restricted to 1 part of the body, and both of their parents are unaffected.39 Most individuals with mosaicism for an NF1 pathogenic variant have mild, but not segmental, neurofibromatosis.40 Whereas mosaic NF1 usually has a mild phenotype, the mutations may affect the gametes, leading to an affected offspring with nonmosaic NF1.41 As somatic mutations may be overlooked in genetic tests that are based on blood leukocytes, testing for somatic NF1 may be based on detecting the same disease-causing mutation in different affected tissues, such as CALM.
Legius Syndrome: NF1-Like Disease Heterozygous mutations in the SPRED1 gene typically cause Legius syndrome, a Rasopathy characterized by multiple CALM, skinfold freckling and lipomas, but not other NF1related phenotypes, such as Lisch nodules, neurofibromas, OPGs, and malignant peripheral nerve sheath tumors.42 Given these clinical characterizations together with the autosomal dominant mode of inheritance, individuals with SPRED1 mutations are occasionally diagnosed as having NF1 according to the NIH diagnostic criteria.4 Indeed, mutations in the SPRED1 gene were found in about 3% of NF1 mutation-negative individuals. The detection rate of SPRED1 mutations is higher among families with an autosomal dominant phenotype of CALM with or without freckling and no other NF1 feature.16 Therefore, Legius syndrome can be described as an “NF1-like disease.” While other Rasopathies may share some characterizations with NF1, such as hyperpigmented skin lesions, there have been no descriptions of mutations in other genes as causing NF1 disease.
Genetic Counseling Offspring of affected individuals have a 50% chance to inherit NF1. Genetic counseling is therefore warranted for individuals with NF1 who are of child-bearing age. Prenatal diagnosis can be offered when the mutation responsible for the disease has been identified. Alternatively, individuals with NF1 and their partners can benefit from preimplementation genetic diagnosis,43,44 which facilitates the decision-making process by providing the ability to predict disease severity based on family history as well as on the specific familial mutations in most cases. When indicated, the recently established genotype-phenotype correlations may now be used in genetic counseling.
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Conclusions NF1 is a relatively common genetic disease. Although our ability to detect mutations in clinically affected individuals is high, data on gene function are incomplete. Its highly variable expression makes the course of NF1 difficult to predict. The increased number of genotype-phenotype correlations that have recently been detected may not only enable a better understanding of the disease processes and the physiological effect of the NF1 gene, but they may have some practical aspects related to improvement in patient care and surveillance as well. These new findings are crucial in providing more accurate genetic counseling.
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