- Email: [email protected]
Advances in Molecular Diagnosis of Neurofibromatosis Type 1
Author's Accepted Manuscript
Advances in Molecular Diagnosis of Neurofibromatosis Type 1 Ben Shofty MD, Shlomi Constantini MSc, MD, Shay Ben-Shachar MD
www.elsevier.com/locate/enganabound
PII: DOI: Reference:
S1071-9091(15)00074-1 http://dx.doi.org/10.1016/j.spen.2015.10.007 YSPEN564
To appear in: Semin Pediatr Neurol
Cite this article as: Ben Shofty MD, Shlomi Constantini MSc, MD, Shay Ben-Shachar MD, Advances in Molecular Diagnosis of Neurofibromatosis Type 1, Semin Pediatr Neurol , http://dx.doi.org/10.1016/j.spen.2015.10.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Advances in Molecular Diagnosis of Neurofibromatosis type 1 Ben Shofty, MD,1,3 Shlomi Constantini, MSc, MD,1,2,3 and Shay Ben-Shachar, MD3,4
1
Division of Neurosurgery, 2Department of Pediatric Neurosurgery, 3Gilbert Israeli Neurofibromatosis
Center, 4Genetic Institute, Tel-Aviv Medical Center, Tel-Aviv, Israel, affiliated to the Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
Correspondence: Dr. Shay Ben-Shachar The Gilbert Israeli Neurofibromatosis Center Tel-Aviv Medical Center 6 Weizmann St. Tel-Aviv, Israel, 6423906 Email: [email protected]
Key words: Neurofibromatosis, NF1, DNA, RNA, Clinical criteria, Genotype-phenotype correlations, Missense mutations, Prenatal diagnosis All authors declare no conflict of interest.
Received: August 27, 2015
2 Abstract 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. While 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 genotypephenotype correlations.
Introduction Historically referred to as von Recklinghausen’s disease, neurofibromatosis type 1 (NF1, MIM#162200) is a genetic neuro-cutaneous 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 (OPG), and skeletal deformities. In addition, behavioral and/or cognitive impairments are common among individuals with NF1 2. Although considered benign, plexiform neurofibromas (PN) and OPG are major causes of concern, since they may produce disfigurement and blindness. The main reason for close, prolonged follow-up is the associated susceptibility to malignancies, since 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. While 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 can not usually be determined, even for people from the same family or with the same specific mutation in the gene3. Mutation analyses is important for early diagnosis of NF1 and for clarifying ambiguous clinical
3 cases, in addition to being essential for prenatal and pre-gestational diagnoses. The few genotypephenotype 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, due to 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 two 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 one year and 95% by the age of eight years 5-7. Because disease manifestations present gradually, a clinical diagnosis cannot always be made in very young children, especially among these 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 these criteria may be necessary for children younger than eight years. For example, T2 hyperintensities on brain magnetic resonance imaging were suggested as a supplemental diagnostic criterion, but they are seldom used for the initial diagnosis due to 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. Plexiform neurofibromas, 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.
4
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, although 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 hyper-activated, leading to deregulated cellular proliferation and survival 12. Up-regulation of Ras triggers downstream signaling pathways, including the mitogen-activated protein kinase (MAPK) RAF/MEK/ERK pathway. Given that activation of the RAF/MEK/ERK pathway is associated with tumorigenesis, the NF1 gene is considered to be a tumorsuppressor 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 one of the two 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, since 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.
5
Variable Expression of NF1 and the “Two Hits” Theory NF1 patients harbor a heterozygous germline mutation, meaning that they have one normal DNA copy (allele) and another allele with the disease-causing-mutation in each nucleated, non-gamete cell. However, molecular testing of each lesion, e.g., CALM, neurofibromas, OPGs, detects two 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 lossof-heterozygosity (LOH). 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 optic pathway gliomas, another disease
hallmark, in murine models using bi-allele Nf1 deletion from astrocytes on an haplo insufficient background 21,22. Two large familial phenotype studies that 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
6 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. While 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. While 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, i.e., 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 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 one copy of the neurofibromin contributes to a more severe NF1related 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 5’ (proximal)
7 third of the NF1 gene had an odds ratio of 6.05 to have optic pathway gliomas 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 non-truncating 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 non-truncated mutations suggest that in-frame/ 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 plexiform neurofibromas 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 plexiform neurofibromas, 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 Noonan-like features, and pulmonary valve stenosis. Developmental delays and/or learning disabilities were reported in over 50% of these patients 34,35. Multiplicity of constitutional missense and/or 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 non-tumor-related phenotypes as well. While 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
8 cardiac defects (e.g., pulmonary stenosis). However, different non-truncating, 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.
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/regional NF1 as manifested by features of NF1 restricted to one 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. While mosaic NF1 usually has a mild phenotype, the mutations may affect the gametes, leading to an affected offspring with non-mosaic NF1 41. Because 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 NF1-related phenotypes, such as Lisch nodules, neurofibromas, optic pathway gliomas, 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
9 Rasopathies may share some characterizations with NF1, such 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 pre-implementation 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.
Conclusions NF1 is a relatively common genetic disease. While 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.
10
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Evans, D. G. et al. Birth incidence and prevalence of tumor-prone syndromes: estimates from a UK family genetic register service. American journal of medical genetics. Part A 152A, 327-332, (2010). Hyman, S. L., Shores, A. & North, K. N. The nature and frequency of cognitive deficits in children with neurofibromatosis type 1. Neurology 65, 1037-1044, (2005). Pasmant, E. et al. Characterization of a 7.6-Mb germline deletion encompassing the NF1 locus and about a hundred genes in an NF1 contiguous gene syndrome patient. Eur J Hum Genet 16, 1459-1466, 134 (2008). NIH Consensus Development Conference. Neurofibromatosis. Conference statement. Arch Neurl, 575–578. (1988). Ferner, R. E. et al. Guidelines for the diagnosis and management of individuals with neurofibromatosis 1. Journal of medical genetics 44, 81-88, (2007). Huson, S. M., Harper, P. S. & Compston, D. A. Von Recklinghausen neurofibromatosis. A clinical and population study in south-east Wales. Brain : a journal of neurology 111 ( Pt 6), 1355-1381 (1988). DeBella, K., Szudek, J. & Friedman, J. M. Use of the national institutes of health criteria for diagnosis of neurofibromatosis 1 in children. Pediatrics 105, 608-614 (2000). DeBella, K., Poskitt, K., Szudek, J. & Friedman, J. M. Use of "unidentified bright objects" on MRI for diagnosis of neurofibromatosis 1 in children. Neurology 54, 1646-1651 (2000). Williams, V. C. et al. Neurofibromatosis type 1 revisited. Pediatrics 123, 124-133, (2009). Cawthon, R. M. et al. A major segment of the neurofibromatosis type 1 gene: cDNA sequence, genomic structure, and point mutations. Cell 62, 193-201 (1990). Wallace, M. R. et al. Type 1 neurofibromatosis gene: identification of a large transcript disrupted in three NF1 patients. Science 249, 181-186 (1990). Trovo-Marqui, A. B. & Tajara, E. H. Neurofibromin: a general outlook. Clinical genetics 70, 1-13, (2006). Pasmant, E., Vidaud, M., Vidaud, D. & Wolkenstein, P. Neurofibromatosis type 1: from genotype to phenotype. Journal of medical genetics 49, 483-489, (2012). Ars, E. et al. Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients. Journal of medical genetics 40, e82 (2003). Ars, E. et al. Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1. Human molecular genetics 9, 237-247 (2000). Messiaen, L. et al. Clinical and mutational spectrum of neurofibromatosis type 1like syndrome. JAMA 302, 2111-2118, (2009). Knudson, A. G., Jr. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci U S A 68, 820-823 (1971). Gutmann, D. H. et al. Haploinsufficiency for the neurofibromatosis 1 (NF1) tumor suppressor results in increased astrocyte proliferation. Oncogene 18, 4450-4459, (1999).
11 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
34 35
Pemov, A., Park, C., Reilly, K. M. & Stewart, D. R. Evidence of perturbations of cell cycle and DNA repair pathways as a consequence of human and murine NF1haploinsufficiency. BMC Genomics 11, 194, (2010). Serra, E. et al. Confirmation of a double-hit model for the NF1 gene in benign neurofibromas. Am J Hum Genet 61, 512-519, (1997). Bajenaru, M. L. et al. Astrocyte-specific inactivation of the neurofibromatosis 1 gene (NF1) is insufficient for astrocytoma formation. Mol Cell Biol 22, 5100-5113 (2002). Bajenaru, M. L. et al. Optic nerve glioma in mice requires astrocyte Nf1 gene inactivation and Nf1 brain heterozygosity. Cancer research 63, 8573-8577 (2003). Easton, D. F., Ponder, M. A., Huson, S. M. & Ponder, B. A. An analysis of variation in expression of neurofibromatosis (NF) type 1 (NF1): evidence for modifying genes. Am J Hum Genet 53, 305-313 (1993). Szudek, J., Joe, H. & Friedman, J. M. Analysis of intrafamilial phenotypic variation in neurofibromatosis 1 (NF1). Genet Epidemiol 23, 150-164, (2002). Sabbagh, A. et al. Unravelling the genetic basis of variable clinical expression in neurofibromatosis 1. Human molecular genetics 18, 2768-2778, (2009). Ko, J. M., Sohn, Y. B., Jeong, S. Y., Kim, H. J. & Messiaen, L. M. Mutation spectrum of NF1 and clinical characteristics in 78 Korean patients with neurofibromatosis type 1. Pediatr Neurol 48, 447-453, (2013). Tonsgard, J. H., Yelavarthi, K. K., Cushner, S., Short, M. P. & Lindgren, V. Do NF1 gene deletions result in a characteristic phenotype? American journal of medical genetics 73, 80-86 (1997). Mautner, V. F. et al. Clinical characterisation of 29 neurofibromatosis type-1 patients with molecularly ascertained 1.4 Mb type-1 NF1 deletions. Journal of medical genetics 47, 623-630, (2010). Douglas, J. et al. Mutations in RNF135, a gene within the NF1 microdeletion region, cause phenotypic abnormalities including overgrowth. Nat Genet 39, 963-965, (2007). Ponti, G. et al. NF1 truncating mutations associated to aggressive clinical phenotype with elephantiasis neuromatosa and solid malignancies. Anticancer Res 34, 3021-3030 (2014). Sharif, S. et al. A molecular analysis of individuals with neurofibromatosis type 1 (NF1) and optic pathway gliomas (OPGs), and an assessment of genotypephenotype correlations. Journal of medical genetics 48, 256-260, (2011). Bolcekova, A. et al. Clustering of mutations in the 5' tertile of the NF1 gene in Slovakia patients with optic pathway glioma. Neoplasma 60, 655-665, (2013). Upadhyaya, M. et al. An absence of cutaneous neurofibromas associated with a 3bp 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 80, 140-151, (2007). Pinna, V. et al. p.Arg1809Cys substitution in neurofibromin is associated with a distinctive NF1 phenotype without neurofibromas. Eur J Hum Genet, 2014.243 (2014). Rojnueangnit, K. et al. High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype-Phenotype Correlation. in pres Hum Mut (2015).
12 36 37 38 39 40 41 42 43 44
Kluwe, L., Tatagiba, M., Funsterer, C. & Mautner, V. F. NF1 mutations and clinical spectrum in patients with spinal neurofibromas. Journal of medical genetics 40, 368-371 (2003). Wimmer, K. et al. A patient severely affected by spinal neurofibromas carries a recurrent splice site mutation in the NF1 gene. Eur J Hum Genet 10, 334-338, (2002). Ben-Shachar, S. et al. Increased rate of missense/in-frame mutations in individuals with NF1-related pulmonary stenosis: a novel genotype-phenotype correlation. Eur J Hum Genet 21, 535-539, (2013). Ruggieri, M. & Huson, S. M. The clinical and diagnostic implications of mosaicism in the neurofibromatoses. Neurology 56, 1433-1443 (2001). Messiaen, L. et al. Mosaic type-1 NF1 microdeletions as a cause of both generalized and segmental neurofibromatosis type-1 (NF1). Hum Mutat 32, 213219, (2011). Consoli, C. et al. Gonosomal mosaicism for a nonsense mutation (R1947X) in the NF1 gene in segmental neurofibromatosis type 1. J Invest Dermatol 125, 463466, (2005). Brems, H. et al. Review and update of SPRED1 mutations causing Legius syndrome. Hum Mutat 33, 1538-1546 (2012). Spits, C. et al. Preimplantation genetic diagnosis for neurofibromatosis type 1. Molecular human reproduction 11, 381-387, (2005). Chen, Y. L. et al. Successful application of the strategy of blastocyst biopsy, vitrification, whole genome amplification, and thawed embryo transfer for preimplantation genetic diagnosis of neurofibromatosis type 1. Taiwanese journal of obstetrics & gynecology 50, 74-78, (2011).
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Table 1 Clinical Diagnostic Criteria for NF144, and Associated Diagnostic and Clinical Findings Expected Age at
Percentage of
Presentation
Patients
Birth to 2 years
>99%
Cutaneous neurofibromas (≥2)
5+ years
>99%
One plexiform neurofibroma
Birth to 3 years
30%-50%
Axillary or inguinal freckling
3-5 years
90%
Lisch nodules (≥2)
5-10 years
90%
Optic pathway glioma
3-8 years
15%-30%
Typical osseous lesion (sphenoid wing
1-3 years
2% pseudoarthrosis
Diagnostic Criteria
CALM (≥6, >15 mm in adults and >5 mm in children)
dysplasia/long-bone cortical thinning, w/wo
1% SWD
pseudoarthrosis) Family history of NF1 (first-degree relative)
-
Additional Common Clinical Findings (% of
Required Evaluation
Patients) ADHD/memory and learning difficulties (50-75%)
Neuropsychological consultation
T2 hyper-intensities (60-70%)
None
Cardiovascular abnormalities (hypertension, renal
Yearly blood pressure
artery stenosis, pulmonary artery stenosis; 2%
monitoring and heart
each)
assessment,
50%
14 echocardiography as needed Hormonal abnormalities/precocious puberty
Growth curves at every routine follow-up
Progressive scoliosis
Skeletal/bone assessment
Neuropathy (~1%, usually distal and symmetrical)
Rule out other causes for neurological changes in NF1 patients (PN, MPNST, etc.)
Pheochromocytoma (2%)
24h 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
15 Table 2 Genotype-phenotype Correlation Genotype
Phenotype
Reference #
Large/complete gene deletion
Severe disease manifestation
3,25,26,27
Cognitive Cardiovascular Benign and malignant tumors Upper 5` third mutation
OPG (OR = 6.05)
31,32
Deletion of 3-bp (c2970-2972 delAAT)
No cutaneous neurofibromas
33
Constitutional missense and/or splicing
Spinal NF1
36,37
No cutaneous manifestations, Noonan
34,35
mutations p. Arg1809 missense mutations
like dysmorphic features, Cardiac defect - pulmonary stenosis Non-truncating mutations
Cardiac defect - pulmonary stenosis
38
Truncating mutations
Elephantiasis, solid malignancies
30
OPG = optic glioma; OR =odds ratio
Advances in Molecular Diagnosis of Neurofibromatosis Type 1 Ben Shofty MD, Shlomi Constantini MSc, MD, Shay Ben-Shachar MD
www.elsevier.com/locate/enganabound
PII: DOI: Reference:
S1071-9091(15)00074-1 http://dx.doi.org/10.1016/j.spen.2015.10.007 YSPEN564
To appear in: Semin Pediatr Neurol
Cite this article as: Ben Shofty MD, Shlomi Constantini MSc, MD, Shay Ben-Shachar MD, Advances in Molecular Diagnosis of Neurofibromatosis Type 1, Semin Pediatr Neurol , http://dx.doi.org/10.1016/j.spen.2015.10.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Advances in Molecular Diagnosis of Neurofibromatosis type 1 Ben Shofty, MD,1,3 Shlomi Constantini, MSc, MD,1,2,3 and Shay Ben-Shachar, MD3,4
1
Division of Neurosurgery, 2Department of Pediatric Neurosurgery, 3Gilbert Israeli Neurofibromatosis
Center, 4Genetic Institute, Tel-Aviv Medical Center, Tel-Aviv, Israel, affiliated to the Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
Correspondence: Dr. Shay Ben-Shachar The Gilbert Israeli Neurofibromatosis Center Tel-Aviv Medical Center 6 Weizmann St. Tel-Aviv, Israel, 6423906 Email: [email protected]
Key words: Neurofibromatosis, NF1, DNA, RNA, Clinical criteria, Genotype-phenotype correlations, Missense mutations, Prenatal diagnosis All authors declare no conflict of interest.
Received: August 27, 2015
2 Abstract 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. While 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 genotypephenotype correlations.
Introduction Historically referred to as von Recklinghausen’s disease, neurofibromatosis type 1 (NF1, MIM#162200) is a genetic neuro-cutaneous 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 (OPG), and skeletal deformities. In addition, behavioral and/or cognitive impairments are common among individuals with NF1 2. Although considered benign, plexiform neurofibromas (PN) and OPG are major causes of concern, since they may produce disfigurement and blindness. The main reason for close, prolonged follow-up is the associated susceptibility to malignancies, since 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. While 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 can not usually be determined, even for people from the same family or with the same specific mutation in the gene3. Mutation analyses is important for early diagnosis of NF1 and for clarifying ambiguous clinical
3 cases, in addition to being essential for prenatal and pre-gestational diagnoses. The few genotypephenotype 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, due to 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 two 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 one year and 95% by the age of eight years 5-7. Because disease manifestations present gradually, a clinical diagnosis cannot always be made in very young children, especially among these 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 these criteria may be necessary for children younger than eight years. For example, T2 hyperintensities on brain magnetic resonance imaging were suggested as a supplemental diagnostic criterion, but they are seldom used for the initial diagnosis due to 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. Plexiform neurofibromas, 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.
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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, although 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 hyper-activated, leading to deregulated cellular proliferation and survival 12. Up-regulation of Ras triggers downstream signaling pathways, including the mitogen-activated protein kinase (MAPK) RAF/MEK/ERK pathway. Given that activation of the RAF/MEK/ERK pathway is associated with tumorigenesis, the NF1 gene is considered to be a tumorsuppressor 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 one of the two 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, since 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 one normal DNA copy (allele) and another allele with the disease-causing-mutation in each nucleated, non-gamete cell. However, molecular testing of each lesion, e.g., CALM, neurofibromas, OPGs, detects two 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 lossof-heterozygosity (LOH). 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 optic pathway gliomas, another disease
hallmark, in murine models using bi-allele Nf1 deletion from astrocytes on an haplo insufficient background 21,22. Two large familial phenotype studies that 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
6 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. While 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. While 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, i.e., 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 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 one copy of the neurofibromin contributes to a more severe NF1related 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 5’ (proximal)
7 third of the NF1 gene had an odds ratio of 6.05 to have optic pathway gliomas 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 non-truncating 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 non-truncated mutations suggest that in-frame/ 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 plexiform neurofibromas 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 plexiform neurofibromas, 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 Noonan-like features, and pulmonary valve stenosis. Developmental delays and/or learning disabilities were reported in over 50% of these patients 34,35. Multiplicity of constitutional missense and/or 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 non-tumor-related phenotypes as well. While 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
8 cardiac defects (e.g., pulmonary stenosis). However, different non-truncating, 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.
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/regional NF1 as manifested by features of NF1 restricted to one 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. While mosaic NF1 usually has a mild phenotype, the mutations may affect the gametes, leading to an affected offspring with non-mosaic NF1 41. Because 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 NF1-related phenotypes, such as Lisch nodules, neurofibromas, optic pathway gliomas, 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
9 Rasopathies may share some characterizations with NF1, such 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 pre-implementation 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.
Conclusions NF1 is a relatively common genetic disease. While 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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References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
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34 35
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Table 1 Clinical Diagnostic Criteria for NF144, and Associated Diagnostic and Clinical Findings Expected Age at
Percentage of
Presentation
Patients
Birth to 2 years
>99%
Cutaneous neurofibromas (≥2)
5+ years
>99%
One plexiform neurofibroma
Birth to 3 years
30%-50%
Axillary or inguinal freckling
3-5 years
90%
Lisch nodules (≥2)
5-10 years
90%
Optic pathway glioma
3-8 years
15%-30%
Typical osseous lesion (sphenoid wing
1-3 years
2% pseudoarthrosis
Diagnostic Criteria
CALM (≥6, >15 mm in adults and >5 mm in children)
dysplasia/long-bone cortical thinning, w/wo
1% SWD
pseudoarthrosis) Family history of NF1 (first-degree relative)
-
Additional Common Clinical Findings (% of
Required Evaluation
Patients) ADHD/memory and learning difficulties (50-75%)
Neuropsychological consultation
T2 hyper-intensities (60-70%)
None
Cardiovascular abnormalities (hypertension, renal
Yearly blood pressure
artery stenosis, pulmonary artery stenosis; 2%
monitoring and heart
each)
assessment,
50%
14 echocardiography as needed Hormonal abnormalities/precocious puberty
Growth curves at every routine follow-up
Progressive scoliosis
Skeletal/bone assessment
Neuropathy (~1%, usually distal and symmetrical)
Rule out other causes for neurological changes in NF1 patients (PN, MPNST, etc.)
Pheochromocytoma (2%)
24h 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
15 Table 2 Genotype-phenotype Correlation Genotype
Phenotype
Reference #
Large/complete gene deletion
Severe disease manifestation
3,25,26,27
Cognitive Cardiovascular Benign and malignant tumors Upper 5` third mutation
OPG (OR = 6.05)
31,32
Deletion of 3-bp (c2970-2972 delAAT)
No cutaneous neurofibromas
33
Constitutional missense and/or splicing
Spinal NF1
36,37
No cutaneous manifestations, Noonan
34,35
mutations p. Arg1809 missense mutations
like dysmorphic features, Cardiac defect - pulmonary stenosis Non-truncating mutations
Cardiac defect - pulmonary stenosis
38
Truncating mutations
Elephantiasis, solid malignancies
30
OPG = optic glioma; OR =odds ratio