Childs Nerv Syst DOI 10.1007/s00381-013-2352-9 ORIGINAL PAPER The mutational spectrum of the NF1 gene in neurofibromatosis type I patients from UAE Salma Ben-Salem & Aisha M. Al-Shamsi & Bassam R. Ali & Lihadh Al-Gazali Received: 6 November 2013 / Accepted: 30 December 2013 # Springer-Verlag Berlin Heidelberg 2014 Abstract Introduction Germline heterozygous mutations in the tumor suppresser NF1 gene cause a cancer predisposition syndrome known as neurofibromatosis type 1 (NF1). This disease is one of the most common multisystem disorders with an estimated incidence of 1 in 3,000 to 1 in 4,000 births. Clinically, NF1 patients are prone to develop “café au lait” spots, neurofibromas, Lisch nodules, freckling of the axillary, or inguinal region and optic nerve gliomas. Materials and methods In the present study, we report clinical and molecular findings of five unrelated patients and seven cases from four families with NF1 from UAE. To reveal the genetic defects underlying NF1 in our cohort of patients, we screened the whole coding and splice site regions of the NF1 Electronic supplementary material The online version of this article (doi:10.1007/s00381-013-2352-9) contains supplementary material, which is available to authorized users. S. Ben-Salem : B. R. Ali Department of Pathology, College of Medicine and Heath Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates S. Ben-Salem e-mail: salmabs@uaeu.ac.ae S. Ben-Salem e-mail: salma81bs@yahoo.fr B. R. Ali e-mail: bassam.ali@uaeu.ac.ae A. M. Al-Shamsi Department of Paediatrics, Tawam Hospital, Al-Ain, United Arab Emirates e-mail: aishamsi@tawamhospital.ae L. Al-Gazali (*) Department of Paediatrics, College of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al-Ain, United Arab Emirates e-mail: algazali@hotmail.com gene. In addition, MLPA or CGH array has been used to screen for structural variations including deletions, indels, and complex rearrangements. Results This resulted in the identification of five distinct novel mutations and two previously reported ones. These variations included three missense and one nonsense mutations, one single base, one dinucleotide, and one large deletion. Conclusion Four mutations were inherited, and the remaining were absent from both parents and therefore are “de novo” mutations. This analysis represents the spectrum of NF1 mutations in UAE and supports the premise of absence of hotspot mutations in the NF1 gene. Moreover, no obvious genotypephenotype correlations were observed in our patients. Keywords Neurofibromatosis type 1 . von Recklinghausen . NF1 . “de novo” mutations Introduction Neurofibromatosis is a genetically inherited disorder characterized by benign tumor proliferation and increased risk for malignancy [1]. This autosomal genetic disorder can be caused by heterozygous mutations in either NF1 gene located on chromosome 17q11.2 (type 1) or NF2 gene mapped to chromosome 22q12.2 (type 2) [2, 3]. Neurofibromatosis type 1 (NF1_MIM 162200), also known as von Recklinghausen disease, is the most common form of neurocutaneous syndromes with a prevalence of 1 in 3,000 to 4,000 people worldwide [4]. NF1 cases display “café au lait” spots, Lisch nodules in the eye, and fibromatous tumors of the skin along with high susceptibility for benign and malignant form of tumors [5, 6]. The diagnosis criteria of NF1 according to the National Institutes of Health Consensus Development Conference are (1) the presence of more than six café au lait spots (>0.5 cm in children or >1.5 cm in adults), (2) two or more Childs Nerv Syst neurofibromas or one or more plexiform neurofibromas, (3) freckling in the axilla or groin, (4) optic glioma, (5) two or more Lisch nodules (iris hamartomas), (6) distinctive bony dysplasia, and (7) first degree relative with NF1 [7]. The presence of 2 of the above criteria is sufficient to make the diagnosis. The café au lait spots generally appear at birth or arise during puberty as skin-colored either papules or nodules with solitary multiple or segmentary distribution [8, 9]. Freckles are the same color as café au lait spots that are affecting mainly axillary and inguinal regions. Neurofibromas define a benign nerve sheath tumor in the peripheral nervous system. The Lisch nodules, which are the most common ocular manifestations in NF1 patients, are usually a clear yellow to brown melanocytic hamartomas affecting the surface of the iris. After puberty, 90 % of individuals carrying genetic defect in the NF1 gene developed most of the above clinical features [10]. Though NF1 is completely penetrant, it shows a variable clinical expressivity even among individuals with the same genetic mutations [11, 12]. Phenotypic variability ranges from extremely mild features in about 60 % of the cases to serious complications in around 40 %. These complications may include congenital defects of the bone, scoliosis, optic glioma, and neurological impairment leading to learning or intellectual disabilities [13–17]. Approximately, 10–40 % of affected children with NF1 developed optic nerve gliomas affecting optic nerve, chiasm, or optic tract. These children have increased risk for pilocytic astrocytomas and juvenile myelomonocytic leukemia which might potentially evolve to an acute myeloid leukemia due to secondary somatic mutations or loss of the second wild type allele [18–20]. The NF1 gene is a large gene spanning a region of 350 kb of genomic DNA that encompasses 60 coding exons [21]. To date, more than a thousand pathogenic mutations have been reported in the NF1 gene including point mutations, small and large deletions, duplications, and indels as well as complex rearrangements (Human Gene Mutation Database (HGMD), 2013). Most of these variations lead to the production of truncated proteins. These mutations occur throughout the gene and some mutations segregated within families [12]. There are no reports of mutations among NF1 patients from the UAE and therefore this study was undertaken to establish the clinical phenotypes and the underlying causes of this condition among residents of this country. Patients and methods Patients A total of 12 cases with neurofibromatosis were ascertained from Tawam Hospital in Al-Ain between 2008 and 2013. All patients were diagnosed to have NF1 based on standard diagnostic criteria of the US NIH diagnostic standard of the year 1987. Informed written consents were obtained from all subjects in this study before collecting the blood samples for genetic analysis. Mutational analysis Blood samples were collected in EDTA tubes, and the genomic DNA was extracted using Flexigene DNA extraction kit (Qiagen Gmbh, Germany) according to manufacturer's instructions. Primers for the FN1 gene were designed using Primer3 software version 0.4.0 (http://frodo.wi.mit.edu/) covering all the exons (1–57 including alternatively spliced exon between exon 30 and 31) and all intronic flanking regions (Supplementary Table 1). Alternatively spliced exons (9a, 10a2, and 48a) were not analyzed since no variation has ever been reported in over 500 NF1 patients screened for these exons [22]. All coding exons were amplified by PCR and further sequenced using dye-primer chemistry (Applied Biosystems, USA) on a 3130xl capillary sequencer (Applied Biosystems, USA). PCR amplifications were performed on 2720 thermal cycler in a total volume of 20 μl of PCR reactions prepared containing 1X PCR buffers (Qiagen Gmbh, Germany), 0.2 mM dNTPs, 5 μM of each forward and reverse primers, 100 ng of template DNA, and 0. 5 U Taq DNA polymerase (Qiagen Gmbh, Germany). The PCR products were purified using ExoSAP-IT (USB Inc.) followed by DNA Sanger cycle sequencing using the BigDye Terminator kit v3.1 (Applied Biosystems, USA) as detailed by manufacturer's instructions. DNA chromatograms were inspected and analyzed based on cDNA sequence in accordance with the Ensembl entries ENST00000356175 (note that this reference sequence misses an exon between 30 and 31, analyzed separately) using the Sequencing Analysis® 5.3 software (Applied Biosystems, USA) and clustalW2 (http:// www.ebi.ac.uk/Tools/msa/clustalw2/). In the absence of point mutations, MLPA (Kit P081/P082) or CGH array analyses were performed to screen for deletions and/or duplications in the NF1 gene [23, 24]. Results Clinical data A total of twelve affected individuals from UAE were collected, among seven from four different families, and five were sporadic cases (Fig. 1). Clinical features of all affected subjects are summarized in Table 1. Molecular analysis Genomic DNA from 12th patient, who fulfilled the clinical criteria of NF1, were inspected for mutations screening using Childs Nerv Syst Fig. 1 Pedigree of the NF1 cases from UAE. Pedigree showing Emirati cases with NF1 and the corresponding identified mutations. Circles and squares denote females and males, respectively; filled symbols represent affected members; double lines denote consanguineous marriage; Roman numbers indicates the first generation until their offspring; Arabic numbers depict individuals; and asterisk symbol denotes participants in this study PCR-directed sequencing of whole coding sequences and flanking splice site regions of the NF1 gene. Mutations analysis revealed a spectrum of heterozygous mutations distributed across intron 12 to exon 54 of the NF1 gene (Table 2, Fig. 2a). Nine mutations were found: two missense mutations c.2540T>C (p.Leu847Pro) and c.6374T>C (p.L2125P); one nonsense mutation c.6546C>G (p.Tyr2182X); one splice site mutation in exon 9 (c.1062+2T>C); three single nucleotide Table 1 Clinical features of Emirati NF1 patients Patient ID FH CA lesion or spots Freckling Lisch nodule Neurofibroma Plexiform neurofibroma Scoliosis Brain MRI F1-3 F1-4 F1-5 F2-3 F2-1 F3-3 F4-3 F5-3 F6-3 F7-3 F8-3 F9-3 + + + + + + − ? − − − − + + + + + + + + + + + + − − − − ? − + + − ? − − + + − − − − + + − + − − + − − − + − − − − ? − − − − Neurofibroma of eyelids − + − − + − ? − − + − − − − + + + − ? ND + ? − + + + − Axillary+ − − Inguinal+ − FH family history, CA café au lait, MRI magnetic resonance imaging, + present, − absent, ? unknown, ND not determined Childs Nerv Syst Table 2 Summary of NF1 mutations detected in Emirati samples Patients Gender Exons ID DNA level Alleles F1-3 F1-4 F1-5 F2-3 F F M M x13-14del x13-14del x13-14del x42 9.2 kb deletion 9.2 kb deletion 9.2 kb deletion c.6374T>C F2-1 F3-3 F4-3 F5-3 F6-3 F7-3 F8-3 F9-3 M F M M M M F F x42 x17 x30 x43 x21 x37 x25 x9 c.6374T>C c.1846delC c.4065_4066delAG c.6546C>G c.2540T>C c.5346delT c.3291delA c.1062+2T>C Protein level Type of mutation Type Country References Heterozygous Heterozygous Heterozygous Heterozygous p.Leu2125Pro Large deletion Large deletion Large deletion Missense Familial Familial Familial Familial UAE UAE UAE UAE This study This study This study This study Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Missense Frameshift Frameshift Nonsense Missense Frameshift Frameshift Splice site Familial Familial Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic UAE UAE UAE UAE UAE UAE UAE UAE This study This study This study Ars et al. [39] Fahsold et al. [10] This study This study Bausch et al. [40] p.Leu2125Pro p.Gln616ArgfsX15 p.Glu1356IlefsX17 p.Tyr2182X p.Leu847Pro p.Ile1782MetfsX60 p.Ala1098ProfsX14 F female, M male, x exon, kb kilo base, del deletion, fs frameshift, X stop codon, UAE United Arab Emirate deletions affecting exons 17 (c.1846delC/p.Q616ArgfsX15), exon 25 (c.3291delA/p.Ala1098ProX14), and exon 37 (c.5346delT/p.I1782MfsX60); and one dinucleotide deletion (c.4065_4066delAG/p.E1356IfsX17) in exon 30. Moreover, a Fig. 2 Distribution of the identified mutations in the NF1 gene. a Schematic representation of neurofibromin and the identified mutations in this study. CSRD cysteine/serine-rich domain, GRD GAP-related domain, TBD tubulin-binding domain, Sec14-PH Sec14-homologous domain and pleckstrin homology domain, NLS nuclear localization signal, FAK focal adhesion kinase. b Black color denotes previously reported mutations; red color indicates the identified mutations in this study. c Amino acid conservation was compiled using Consurf online website (http:// consurf.tau.ac.il/), and logos were generated using WebLogo v3.3 (weblogo.berkeley.edu) [57]. Red arrows represent the studied mutation d pie chart showing the percentage of total mutations entries in the NF1 gene according to Human Gene Mutation Database (HGMD) professional as for September 2013 Childs Nerv Syst large deletion of approximately 9.2 kb encompassing exons 13 and 14 was revealed by MLPA analysis, and the exact breakpoints were determined using CGH array. This deletion is located at genomic positions g.29538981-g.29548220 in introns 12 and 14, respectively. Computational prediction softwares SIFT, PROVEAN, PolyPhen-2, and Mutation taster showed that the two non-synonymous substitutions (p.L847P and p.L2125P) were found to be pathogenic and highly conserved (Fig. 2b–c) [25–30]. All the identified nonpathogenic polymorphisms in this study are listed in Supplementary Table 2. A novel intronic polymorphism c.7869+22G>A was found in individual F6-3. Discussion von Recklinghausen disease is a common autosomal dominant disorder caused by mutations in the NF1 gene. This gene encodes a 2,818 polypeptide named neurofibromin which is a tumor suppressor protein involved in the downregulation of the RAS-mitogen-activated protein kinase (MAPK) pathway. About 50 % of NF1 patients had a family history of neurofibromatosis, giving a total of approximately 15 million patients worldwide [31, 32]. About 50 % of NF1 cases are affected by new mutations along with germline mosaicism in some cases [33]. Among them, 90 % of these mutations were inherited from a paternal chromosome, while large deletions were of maternal origin [34–37]. In this report, four families (F1–F4 in Fig. 1) had a family history of NF1 disorder, three from the paternal side and one from the maternal side. The severity of clinical manifestations varies widely among patients carrying mutations within NF1 gene. This variability on numbers and locations of neurofibromas throughout patient's bodies can be attributed mainly to implication of other factors such the modifier genes [38]. To date, nearly 1,500 mutations and complex rearrangements have been identified in the NF1 gene. Based on HGMD database (2013), most of these DNA changes are small deletions (27 %), missense/nonsense (24 %), and splicing mutations (23 %) (Fig. 2d). In this study, we identified three single nucleotide deletions: c.1846delC/p.Gln616ArgfsX15, c.3291delA/ p.Ala1098ProX14, and c.5346delT/p.Ile1782MetfsX60 affecting (1) exon 17 in patient F3-3, (2) exon 25 in the affected female F8-3, and (3) exon 37 in individual F7-3, respectively (Figs. 1 and 2a). The fourth mutation is a dinucleotide deletion (c.4065_4066delAG/p.Glu1356IlefsX17) identified in exon 30 in patient F4-3 (Figs. 1 and 2a). All these deletions resulted in frameshifts and subsequently led to the creation of premature stop codons (PTCs) in one allele of the NF1 gene. A previously reported nonsense mutation c.6546C>G leading to p.Tyr2182X have been identified in exon 43 in patient NF1-07 (Figs. 1 and 2a) [39]. A splice mutation (c.1062+2T>C) previously reported by Bausch et al. has been identified in sporadic case from F9. This mutation will abolish the authentic splice site of exon 9 which results in truncated NF1 transcripts [40]. Since all mutations are positioned more than 55 nucleotides before exon-exon boundaries, the truncated mRNA are most probably eradicated by NMD leading to haplo insufficiency of normal neurofibromin proteins in the cells. Based on HGMD database, large deletions represent 9 % of total NF1 reported mutations (Fig. 2d). In family 1, a large novel deletion of 9.2 kb encompassing exon 13 and 14 has been identified in all affected individuals F1-3, F1-4, and F15 using MLPA analysis. The aCGH defined the exact breakpoints from intron 12 to intron 14 on genomic locations g.29,538,981-29,548,220 in chromosome 17. Moreover, two missense mutations c.2540T>C and c.6374T>C were identified in exons 21 and 42, resulting in amino acids changes p.Leu847Pro and p.Leu2125Pro, respectively (Figs. 1 and 2a). The p.Leu847Pro mutation has been previously reported by Fahsold et al. 2000 [10]. This mutation affects the cysteine/ serine-rich domain (CSRD) of neurofibromin which has been suggested to be the second functional domain after GAP-related domain (GRD) [10]. Neurofibromin, a large cytosolic protein, contains several domains, most notably, a central domain named GRD, similar to Ras-GTPase-activating (GAP) proteins that function as negative regulator of Ras [41, 42]. The N-terminal region of the GRD domain defines the tubulin-binding domain (TBD), followed by a CSRD [43]. This latter controls the association of neurofibromin to microtubules via cAMP-mediated pathway [10, 44]. The C-terminal region consists of Sec14homologous domain and pleckstrin homology domain (Sec14PH) which might be implicated in protein and lipid trafficking [45]. Functional study showed that p.Leu847Pro might affect the localization and regulation of neurofibromin rather than its RasGAP activity [46]. The second mutation p.Leu2125Pro is a novel mutation not listed in the NHLBI exome variant database (http:// evs.gs.washington.edu/EVS/) nor in the HGMD professional databases 2013. This mutation is located between the Sec14PH and focal adhesion kinase (FAK) domains in the C-terminal end of neurofibromin. Since proline is recognized to weaken helices and beta sheets, the p.Leu2125Pro might affects the correct tertiary structure and normal folding of neurofibromin [47]. Both non-synonymous mutations are therefore pathogenic since they are affecting highly conserved residue of the neurofibromin according to ConSurf analysis (Fig. 2b–c). In this report, the majority of these mutations are truncating mutations identified in exons 9, 17, 25, 30, 37, 43, and 45 with equal distribution in the NF1 gene, which result in PTCs leading to truncated or shortened NF1 protein product. Therefore, loss of neurofibromin function or NF1 deficiency is associated with elevated Ras activity and high rate of proliferation [41]. Deficiency will increase RAS-GTP level leading to hyperactive RAS signaling in the cells and uncontrolled mitogenic signals to the nucleus, especially for neuronal cells [48, 49] [41]. Thus, the amplified RAS activity will increase the susceptibility to develop juvenile myelomonocytic leukemia (JMML) and chronic Childs Nerv Syst myelomonocytic leukemia which can progress to acute myeloid leukemia (AML) once accompanied with other genetic alterations in RAS genes [50, 51]. The multifunctional aspect of neurofibromin shed light on its vital role during cell and nervous system development and tumor predisposition which might explain the clinical variability and severity among NF1 patients [46]. Furthermore, we noticed that the following SNPs: rs2952976, rs1801052, rs2905876, rs71142036, rs2905880, rs9894648, rs2285894, rs7405740, and rs964288 were found to be the most common polymorphisms in Emirati populations (Supplementary Table 1). Noteworthy, the mutational spectrum of the NF1 gene tends to be similar to other tumor suppressor genes such as BRCA1, TSC1, and APC genes [52–54]. This gene exhibits one of the highest rates of mutations among human disorders [55]. The highest mutation rate is due to its large size, similarly to the FBN1 gene with approximately 110 kb, which is affecting 1/3,000 to 1/4,000 individuals with Marfan syndrome. Moreover, no obvious correlation could be detected between the identified mutations and clinical features of NF1 patients. This is consistent with previous studies that failed to find any phenotype-genotype correlation for the NF1 gene [10, 56]. 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