Molecular Dissection of Isolated Disease Features in Mosaic Neurofibromatosis Type 1
2007; Elsevier BV; Volume: 81; Issue: 2 Linguagem: Inglês
10.1086/519562
ISSN1537-6605
AutoresOphélia Maertens, Sofie De Schepper, Jo Vandesompele, Hilde Brems, Ine Heyns, Sandra Janssens, Frank Speleman, Eric Legius, Ludwine Messiaen,
Tópico(s)Vascular Malformations Diagnosis and Treatment
ResumoElucidation of the biological framework underlying the development of neurofibromatosis type 1 (NF1)–related symptoms has proved to be difficult. Complicating factors include the large size of the NF1 gene, the presence of several NF1 pseudogenes, the complex interactions between cell types, and the NF1-haploinsufficient state of all cells in the body. Here, we investigate three patients with distinct NF1-associated clinical manifestations (neurofibromas only, pigmentary changes only, and association of both symptoms). For each patient, various tissues and cell types were tested with comprehensive and quantitative assays capable of detecting low-percentage NF1 mutations. This approach confirmed the biallelic NF1 inactivation in Schwann cells in neurofibromas and, for the first time, demonstrated biallelic NF1 inactivation in melanocytes in NF1-related café-au-lait macules. Interestingly, both disease features arise even within a background of predominantly NF1 wild-type cells. Together, the data provide molecular evidence that (1) the distinct clinical picture of the patients is due to mosaicism for the NF1 mutation and (2) the mosaic phenotype reflects the embryonic timing and, accordingly, the neural crest–derived cell type involved in the somatic NF1 mutation. The study of the affected cell types provides important insight into developmental concepts underlying particular NF1-related disease features and opens avenues for improved diagnosis and genetic counseling of individuals with mosaic NF1. Elucidation of the biological framework underlying the development of neurofibromatosis type 1 (NF1)–related symptoms has proved to be difficult. Complicating factors include the large size of the NF1 gene, the presence of several NF1 pseudogenes, the complex interactions between cell types, and the NF1-haploinsufficient state of all cells in the body. Here, we investigate three patients with distinct NF1-associated clinical manifestations (neurofibromas only, pigmentary changes only, and association of both symptoms). For each patient, various tissues and cell types were tested with comprehensive and quantitative assays capable of detecting low-percentage NF1 mutations. This approach confirmed the biallelic NF1 inactivation in Schwann cells in neurofibromas and, for the first time, demonstrated biallelic NF1 inactivation in melanocytes in NF1-related café-au-lait macules. Interestingly, both disease features arise even within a background of predominantly NF1 wild-type cells. Together, the data provide molecular evidence that (1) the distinct clinical picture of the patients is due to mosaicism for the NF1 mutation and (2) the mosaic phenotype reflects the embryonic timing and, accordingly, the neural crest–derived cell type involved in the somatic NF1 mutation. The study of the affected cell types provides important insight into developmental concepts underlying particular NF1-related disease features and opens avenues for improved diagnosis and genetic counseling of individuals with mosaic NF1. Neurofibromatosis type 1 (NF1 [MIM 162200]) is a common autosomal dominant disorder caused by alterations in the NF1 gene. The NF1-encoded protein, neurofibromin, functions as a negative regulator of Ras-mediated signaling.1Xu GF Lin B Tanaka K Dunn D Wood D Gesteland R White R Weiss R Tamanoi F The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae.Cell. 1990; 63: 835-841Abstract Full Text PDF PubMed Scopus (531) Google Scholar, 2Martin GA Viskochil D Bollag G McCabe PC Crosier WJ Haubruck H Conroy L Clark R O’Connell P Cawthon RM et al.The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21.Cell. 1990; 63: 843-849Abstract Full Text PDF PubMed Scopus (705) Google Scholar, 3Ballester R Marchuk D Boguski M Saulino A Letcher R Wigler M Collins F The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins.Cell. 1990; 63: 851-859Abstract Full Text PDF PubMed Scopus (621) Google Scholar The primary clinical features of NF1 are café-au-lait macules (CALMs), freckling, and benign peripheral nerve sheath tumors or neurofibromas.4Riccardi VM Neurofibromatosis: phenotype, natural history and pathogenesis. John Hopkins University Press, Baltimore and London1992Google Scholar Patients with NF1 also have a predisposition to develop a wide spectrum of other symptoms, illustrating the critical function of neurofibromin in a variety of tissues and cell types. The biological context underlying the development of many NF1-related symptoms and complications, however, remains incompletely understood. Mosaic NF1 is caused by a postzygotic NF1 lesion5Vandenbroucke I van Doorn R Callens T Cobben JM Starink TM Messiaen L Genetic and clinical mosaicism in a patient with neurofibromatosis type 1.Hum Genet. 2004; 114: 284-290Crossref PubMed Scopus (45) Google Scholar, 6Tinschert S Naumann I Stegmann E Buske A Kaufmann D Thiel G Jenne DE Segmental neurofibromatosis is caused by somatic mutation of the neurofibromatosis type 1 (NF1) gene.Eur J Hum Genet. 2000; 8: 455-459Crossref PubMed Scopus (164) Google Scholar, 7Consoli C Moss C Green S Balderson D Cooper DN Upadhyaya M Gonosomal mosaicism for a nonsense mutation (R1947X) in the NF1 gene in segmental neurofibromatosis type 1.J Invest Dermatol. 2005; 125: 463-466Crossref PubMed Scopus (66) Google Scholar and can present as mild generalized disease, segmental disease, or gonadal mosaicism.8Ruggieri M Huson SM The clinical and diagnostic implications of mosaicism in the neurofibromatoses.Neurology. 2001; 56: 1433-1443Crossref PubMed Scopus (266) Google Scholar Revertant mosaicism, as reported to be caused by a postzygotic back mutation in some disorders,9Youssoufian H Pyeritz RE Mechanisms and consequences of somatic mosaicism in humans.Nat Rev Genet. 2002; 3: 748-758Crossref PubMed Scopus (272) Google Scholar has not yet been described in cases of NF1. The mosaic phenotype most probably reflects the timing of the somatic mutation and some of the tissues affected by it. Since segmental NF1 is characterized by the regionally limited distribution of NF1 disease signs, it provides the opportunity to study cell populations differing only with regard to the mutation(s) giving rise to mosaicism. Determining when and in what cell types inactivation of the NF1 gene occurs is critical for understanding the basic pathology of NF1-related symptoms. In this study, we investigated one patient mildly affected with NF1 and two patients with segmental NF1. The three patients had different clinical manifestations—that is, neurofibromas only, pigmentary changes only, and a combination of both neurofibromas and pigmentary changes. To elucidate the involvement of particular cell types and mutational mechanisms in the respective phenotypes, we investigated various tissues and cell types from every patient with mosaic NF1 with quantitative assays capable of detecting low-percentage NF1 mutations. Three patients with distinct NF1-associated clinical manifestations were included in the study. Patient SNF1-1 is a 46-year-old woman who has several small (1–4 mm) cutaneous neurofibromas clustered within a limited body region on her trunk and neck. At a given time, 35–40 neurofibromas were present. Several tumors have been removed during different sessions, and three were available for this study. She also underwent surgery for an intestinal ganglioneuroma (S100 and neurofilament positive; c-Kit negative). Careful examination did not reveal freckling or localized hyperpigmentation. Only three hyperpigmented spots smaller than 2 cm were present. She had no Lisch nodules, no learning disabilities, and no other signs of NF1 except for a glomus tumor at the distal phalanx of the right middle finger. Her height is 164 cm, and there was no clinical evidence of macrocrania. Her parents and her two sons (aged 20 and 23 years) don’t show any NF1-related symptoms. Peripheral blood from the patient and her sons, as well as the patient's buccal smears, hair roots, urine, Schwann cells, and fibroblasts cultured from three neurofibromas, were available for analysis. Patient SNF1-2 is a 23-year-old man presenting with several CALMs within a background of hyperpigmented skin involving his entire right leg, hip, and lower back (fig. 1A). Inguinal freckling was present in the affected segment. No hyperpigmentation or CALMs were revealed outside the described area. Careful examination of his entire body did not reveal any neurofibromas. Ophtalmological examination findings were negative for Lisch nodules. His height is 182 cm, and his head circumference is 58 cm. He has no learning disabilities; currently, he is a high school student. He does not have children, and his parents do not show any NF1-related symptoms. Peripheral blood, buccal smears, hair roots, and urine sediment cells were available for investigation, as were fibroblasts and melanocytes cultured from normal skin, CALM, and the hyperpigmented area. Patient SNF1-3 is a 15-year-old girl with more than six CALMs scattered over her body and several small cutaneous and subcutaneous neurofibromas located on her right hand within an overlying CALM (fig. 1B). CALMs (>1.5 cm) not occurring within regions of hyperpigmented skin are located on her right lower back (two), right arm (one on the front [fig. 1B] and one on the back), left wrist (one), left thigh (one), left ankle (one), left gluteal region (one), right calf (one), right thigh (two), and right groin (two). Total-body nuclear magnetic resonance imaging (MRI) revealed a putative neurofibroma on her left shoulder. Physical examination did not reveal skinfold freckling or any other signs of NF1. Her height is 163.5 cm, and her head circumference is 55 cm. She does not have learning disabilities, and her parents do not show any NF1-related symptoms. Peripheral blood, buccal smears, hair roots, and urine sediment cells, together with Schwann cells and fibroblasts derived from one subcutaneous neurofibroma and fibroblasts and melanocytes derived from three CALMs (on her right hand, left thigh, and right lower back) and from normal skin (right buttock), were available for analysis. MRI of the patient’s right hand revealed a subcutaneous nodule with a few smaller surrounding subcutaneous satellite lesions. Histopathologically, the lesions were determined to be neurofibromas by the absence of mitotic activity and the mixture of elongated spindle-shaped Schwann cells and fibroblasts in a background of wavy collagenous fibers. This diagnosis was confirmed by immunohistochemistry (S100 positivity in the majority of spindle-shaped cells). A sample of normally pigmented and/or hyperpigmented skin was taken using 5-mm punch biopsy excision. To separate the epidermal layer (with melanocytes anchored to the basal membrane) from the underlying dermis (with fibroblasts), skin biopsy samples were incubated overnight at 4°C in dispase II (Boehringer Mannheim). Primary epidermal melanocyte cultures from the skin biopsy samples were established as described elsewhere.10Naeyaert JM Eller M Gordon PR Park HY Gilchrest BA Pigment content of cultured human melanocytes does not correlate with tyrosinase message level.Br J Dermatol. 1991; 125: 297-303Crossref PubMed Scopus (99) Google Scholar In brief, melanocytes were cultured in Ham’s F10 medium (Gibco, Invitrogen) supplemented with 2.5% fetal calf serum (FCS), 1% Ultroser, 5 ng/ml basic fibroblast growth factor, 10 ng/ml endothelin-1, 0.33 nM cholera toxin, 0.033 mM isobutyl-methyl-xanthine, 5.3 nM 12-O-tetradecanoyl phorbol-13-acetate, and 20 ng/ml stem cell factor (SCF). Dermal fibroblasts were grown in Dulbecco’s modified Eagle medium (Gibco, Invitrogen) supplemented with 10% FCS. Culture conditions for neurofibroma-derived Schwann cells and fibroblasts were as described elsewhere.11Serra E Rosenbaum T Winner U Aledo R Ars E Estivill X Lenard HG Lazaro C Schwann cells harbor the somatic NF1 mutation in neurofibromas: evidence of two different Schwann cell subpopulations.Hum Mol Genet. 2000; 9: 3055-3064Crossref PubMed Scopus (185) Google Scholar, 12Rosenbaum T Rosenbaum C Winner U Muller HW Lenard HG Hanemann CO Long-term culture and characterization of human neurofibroma-derived Schwann cells.J Neurosci Res. 2000; 61: 524-532Crossref PubMed Scopus (42) Google Scholar The presence of forskolin (F+) in the Schwann cell medium promotes proliferation of cells bearing only the first hit (NF1+/−, SC F+). Replacement of proliferation medium by serum-free N2 medium and, subsequently, by proliferation medium without forskolin (F−) promotes proliferation of cells containing both hits (NF1−/−). To estimate the purity of Schwann cell and fibroblast cultures derived from the neurofibroma of patient SNF1-3, immunofluorescence staining with rabbit S100 primary antibody (Dako) was performed as described elsewhere.13Maertens O Brems H Vandesompele J De Raedt T Heyns I Rosenbaum T De Schepper S De Paepe A Mortier G Janssens S et al.Comprehensive NF1 screening on cultured Schwann cells from neurofibromas.Hum Mutat. 2006; 27: 1030-1040Crossref PubMed Scopus (86) Google Scholar For one SC F− culture (patient SNF1-1, neurofibroma 3), Schwann cells were separated from contaminating fibroblasts by the use of p75 (nerve growth factor receptor)–coupled Magnetic Cell Sorting Microbeads (Miltenyi Biotec), which magnetically label the Schwann cell target population. Subsequent growth of recovered cells in Schwann cell medium resulted in a highly pure Schwann cell culture (>95%, estimated by S100 staining). NF1 mutation analysis (GenBank reference sequence NM_000267) was performed essentially as described elsewhere.14Messiaen LM Callens T Mortier G Beysen D Vandenbroucke I Van Roy N Speleman F De Paepe A Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects.Hum Mutat. 2000; 15: 541-555Crossref PubMed Scopus (382) Google Scholar In brief, genomic DNA (gDNA) was extracted from melanocyte cultures with the QiaAmp procedure (Qiagen) and from all other cell cultures with the Puregene procedure (Gentra). Cultures were treated with puromycin (200 μg/ml for 4–6 h) before RNA extraction (RNeasy kit [Qiagen]). The entire NF1 cDNA was sequenced using the ABI3730XL genetic analyzer (Applied Biosystems). All mutations found at the cDNA level were confirmed in gDNA by cycle sequencing. Multiplex ligation-dependent probe amplification (MLPA) analysis was performed using the SALSA NF1 area kit (MRC Holland), in accordance with the manufacturer’s instructions, to detect deletions. The genomic deletions detected by MLPA analysis were evaluated in further detail. The location of the NF1 microdeletion breakpoints was determined by aspecifically amplifying and sequencing paralogous sequence variants (PSVs) in the low copy repeats (LCRs) flanking the NF1 microdeletion region.15De Raedt T Stephens M Heyns I Brems H Thijs D Messiaen L Stephens K Lazaro C Wimmer K Kehrer-Sawatzki H et al.Conservation of hotspots for recombination in low-copy repeats associated with the NF1 microdeletion.Nat Genet. 2006; 38: 1419-1423Crossref PubMed Scopus (70) Google Scholar By scoring the relative intensity of both nucleotides of the PSV, the location of the breakpoint was determined to be centromeric (higher relative intensity of the nucleotide specific to the telomeric LCR) or telomeric (higher relative intensity of the nucleotide specific to the centromeric LCR) of the PSV investigated. Loss of heterozygosity (LOH) in the NF1 gene region was evaluated by genotyping two microsatellite markers telomeric of (3′ NF1-3 and 3′ NF1-1)16Lopez-Correa C Brems H Lazaro C Estivill X Clementi M Mason S Rutkowski JL Marynen P Legius E Molecular studies in 20 submicroscopic neurofibromatosis type 1 gene deletions.Hum Mutat. 1999; 14: 387-393Crossref PubMed Scopus (51) Google Scholar and four within the NF1 gene (Alu, IVS27AC33.1, IVS38GT53.0, and IVS27TG24.8).17Xu GF Nelson L O’Connell P White R An Alu polymorphism intragenic to the neurofibromatosis type 1 gene (NF1).Nucleic Acids Res. 1991; 19: 3764Crossref PubMed Scopus (71) Google Scholar, 18Lazaro C Gaona A Xu G Weiss R Estivill X A highly informative CA/GT repeat polymorphism in intron 38 of the human neurofibromatosis type 1 (NF1) gene.Hum Genet. 1993; 92: 429-430Crossref PubMed Scopus (65) Google Scholar, 19Lazaro C Gaona A Ravella A Volpini V Casals T Fuentes JJ Estivill X Novel alleles, hemizygosity and deletions at an Alu-repeat within the neurofibromatosis type 1 (NF1) gene.Hum Mol Genet. 1993; 2: 725-730Crossref PubMed Scopus (38) Google Scholar, 20Lazaro C Gaona A Estivill X Two CA/GT repeat polymorphisms in intron 27 of the human neurofibromatosis (NF1) gene.Hum Genet. 1994; 93: 351-352Crossref PubMed Scopus (55) Google Scholar gDNA from paired melanocyte/Schwann cell and lymphocyte cultures from the same patient was investigated by touchdown PCR for the microsatellite markers (PCR program starting at 62°C, gradually reduced [1°C/cycle] to 50°C for an additional 25 cycles) and was subsequently analyzed on the ABI3130XL genetic analyzer (Applied Biosystems) with GeneMapper software version 3.7 (Applied Biosystems). To determine the extent of LOH in different samples from patient SNF1-3, additional SNPs proximal (rs6505129, rs6505165, and rs8071580) and distal (rs9904537 and rs753750) to the NF1 microdeletion region were evaluated as described elsewhere.21De Raedt T Maertens O Chmara M Brems H Heyns I Sciot R Majounie E Upadhyaya M De Schepper S Speleman F et al.Somatic loss of wild type NF1 allele in neurofibromas: comparison of NF1 microdeletion and non-microdeletion patients.Genes Chromosomes Cancer. 2006; 45: 893-904Crossref PubMed Scopus (46) Google Scholar LOH for a SNP was scored when the average ratio (SNP nucleotide:control nucleotide) of the two alleles in the tested tissue fell outside the 95% CI of the ratios observed in control blood DNA of the same patient and when the average ratios in tested tissue versus control blood were at least 20% different. The mechanism underlying LOH (deletion vs. mitotic recombination) was evaluated by semiquantitative PCR, which took advantage of the amplification of NF1 exon 22 (103 bp) together with the corresponding fragment of its pseudogene located on chromosome 15 (107 bp), as described elsewhere.21De Raedt T Maertens O Chmara M Brems H Heyns I Sciot R Majounie E Upadhyaya M De Schepper S Speleman F et al.Somatic loss of wild type NF1 allele in neurofibromas: comparison of NF1 microdeletion and non-microdeletion patients.Genes Chromosomes Cancer. 2006; 45: 893-904Crossref PubMed Scopus (46) Google Scholar To determine whether both NF1 mutations detected in SC F− cultures derived from the neurofibromas of patient SNF1-1 resided on different alleles, cloning experiments were performed. For neurofibroma 1 (NF1 c.2041C→T and c.1655T→G), a fragment containing both alterations and an additional SNP in exon 13 (rs2285892) was amplified (516 bp), cloned in the pCR2.1-TOPO Vector (Invitrogen), and sequenced. For neurofibroma 2 (NF1 c.2041C→T and c.603_621delinsC), a fragment containing the deletion and a SNP in exon 5 (rs1801052) was amplified (286 bp), cloned, and sequenced. Since rs1801052 and rs2285892 are in complete linkage disequilibrium, information on the genotype of the SNP in exon 13 linked with the first hit (cloning experiment neurofibroma 1) and information on the genotype of the SNP in exon 5 linked with the second hit (cloning experiment neurofibroma 2) provide information regarding whether both mutations reside on the same (exon 5/exon13: A/G or G/A) or different (exon5/exon13: A/A or G/G) haplotypes. To detect low-percentage mosaicism for NF1 point mutations against a background of normal and pseudogene alleles, a nested real-time quantitative PCR (qPCR) assay was developed.22Maertens O Legius E Speleman F Messiaen L Vandesompele J Real-time quantitative allele discrimination assay using 3′ locked nucleic acid primers for detection of low-percentage mosaic mutations.Anal Biochem. 2006; 359: 144-146Crossref PubMed Scopus (12) Google Scholar In brief, the region spanning the NF1 point mutation was amplified (primer sequences available on request), and equimolar dilutions of cloned PCR fragments (wild-type and mutant alleles) were used to generate standard curves of 5 orders of magnitude. For actual quantification, allele-specific 3′ locked nucleic acid primers (Eurogentec) were used. Since both somatic NF1 point mutations appeared to be present in several NF1 pseudogenes, samples were first amplified with NF1 specific primer pairs and were diluted prior to nested real-time qPCR. Real-time qPCR reactions were performed on an iCycler iQ instrument (Bio-Rad). In each experiment, duplicates of a standard dilution series of specific PCR fragments for each allele variant (wild-type and mutant) and triplicates of 10 ng DNA of unknown samples (different tissues from patient with mosaic NF1 under study and non-NF1 control sample) were amplified in a 15-μl reaction containing 1× SYBR Green I Master Mix (Eurogentec) and 250 nM of allele-specific primers. The thermal profile consisted of 1 cycle at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and at 61°C or 63°C for 1 min. Each experiment was performed twice, and data acquisition and automated analysis was done by the iCycler iQ software version 3.1 (Bio-Rad). The relative number of molecules of each allele was determined by interpolating the threshold cycle values of the unknown samples to each standard curve, followed by the determination of the fraction of mutant alleles (number of mutant molecules divided by the sum of the number of wild-type and mutant molecules). Submicroscopic NF1 deletions were scored using dual-color FISH23Van Roy N Laureys G Cheng NC Willem P Opdenakker G Versteeg R Speleman F 1;17 translocations and other chromosome 17 rearrangements in human primary neuroblastoma tumors and cell lines.Genes Chromosomes Cancer. 1994; 10: 103-114Crossref PubMed Scopus (130) Google Scholar with PAC clones 22 (RP5-926B9; 5′ NF1) and 13 (RP5-1002G3; 3′ NF1).16Lopez-Correa C Brems H Lazaro C Estivill X Clementi M Mason S Rutkowski JL Marynen P Legius E Molecular studies in 20 submicroscopic neurofibromatosis type 1 gene deletions.Hum Mutat. 1999; 14: 387-393Crossref PubMed Scopus (51) Google Scholar To investigate mosaicism, at least 400 interphase nuclei were evaluated. By analysis of control samples derived from patients not affected with NF1, the sensitivity of the assay was estimated at 1% for lymphocytes and 7% for fibroblasts. NF1 mutation screening of selectively cultured Schwann cells (SC F−) revealed an identical mutation (c.2041C→T [p.R681X]) in two different neurofibromas. In addition, two tumor-specific alterations (c.1655T→G [p.L552R] and c.603_621delinsC) were detected. Cloning experiments demonstrated that both NF1 mutations (common and tumor specific) resided on different alleles. Quantification of mutant transcripts in the presence of the wild-type form was tested by mixing an excess (5×105 molecules) of wild-type allele with a 5-point 10-fold dilution series of the mutant form (5×105—50 molecules). As a control, a standard curve containing only the mutant transcript was used. Ideally, both series should result in overlapping amplification plots. We observed, however, that, at low levels of mutant transcript (<2,500 molecules), the presence of the wild-type transcript (5×105 molecules) significantly impaired accurate quantification of the low-abundance mutant transcript. Therefore, the sensitivity of the quantitative assay is estimated at 1/200. Real-time qPCR demonstrated the presence of the first hit (mutant allele percentage±SEM) in Epstein Barr virus–transformed white blood cells (3.7%±.4%), hair follicles (1.8%±0.3%), fibroblasts derived from both neurofibromas (8.4%±0.5% and 19.4%±5.5%), and selectively cultured Schwann cells from a third, smaller neurofibroma (6.7%±1.0%). Findings from buccal smears, urine sediment cells, and fibroblasts derived from the third neurofibroma were negative or below the detection limit, as were findings from blood from both children of the patient and the control sample (table 1).Table 1Overview of NF1 Mutations Revealed by Routine Mutation-Detection Techniques and Real-Time qPCR in Different Tissues Derived from Segmental Patient SNF1-1, Presenting with Neurofibromas Only, and from Two Sonsc.2041C→T (p.R681X)Patient and SampleaSC F− = Schwann cells grown without forskolin.RoutineqPCRbPercentage of mutant allele±SEM determined by real-time qPCR.Second HitSNF1-1: Neurofibroma 1: SC F−+41.4%±6.4%c.1655T→G (p.L552R) Fibroblasts−8.4%±.5%− Neurofibroma 2: SC F−+47.4%±8.6%c.603_621delinsC Fibroblasts−19.4%±5.5%− Neurofibroma 3: SC F−−6.7%±1.0%− Fibroblasts−−− Blood−3.7%±.4%− Buccal smear−−− Urine−−− Hair−1.8%±.3%−Child 1: Blood−−−Child 2: Blood−−−Note.—+ = Mutation detectable; − = mutation not detectable.a SC F− = Schwann cells grown without forskolin.b Percentage of mutant allele±SEM determined by real-time qPCR. Open table in a new tab Note.— + = Mutation detectable; − = mutation not detectable. Combined NF1 cDNA sequencing and MLPA analysis revealed an NF1 microdeletion exclusively present in the melanocytes derived from CALMs and the background hyperpigmentation area. A second alteration (c.1226_1227del) was detected only in the melanocytes of the CALM. Further characterization of the microdeletion revealed an atypical deletion, with the proximal breakpoint residing within the centromeric LCR flanking the NF1 gene and the distal breakpoint located centromeric of the telomeric LCR flanking the NF1 gene before JJAZ1 exon 7 (maximum size of the deletion 1.30 Mb). FISH analysis of skin fibroblasts (CALM and hyperpigmented and unaffected skin) with NF1-specific probes did not show evidence of low-level mosaicism when the detection limit of 7% was taken into account (table 2). FISH analysis of blood lymphocytes revealed a slightly increased number of nuclei with evidence of NF1 microdeletion (8 [2%] nuclei with one NF1 locus detected by two-color FISH in a total of 400 evaluated nuclei) (table 2), as compared with the non-NF1 control lymphocyte sample (0.01; P=.050; binomial test).Table 2Overview of NF1 Mutations Revealed by Routine Mutation-Detection Techniques and FISH Analysis in Different Tissues Derived from Segmental Patient SNF1-2, Presenting with Pigmentary Defects OnlyNF1 MicrodeletionSampleRoutineFISHaPercentage of 400 interphase nuclei with NF1 microdeletion determined by FISH analysis. ND = no data available.Second HitCALM: Melanocytes+NDc.1226_1227del Fibroblasts−<7%−Hyperpigmentation: Melanocytes+ND− Fibroblasts−<7%−Unaffected skin: Melanocytes−ND− Fibroblasts−<7%−Blood−2%−Buccal smear−ND−Urine−ND−Hair−ND−Note.—+ = Mutation detectable; − = mutation not detectable.a Percentage of 400 interphase nuclei with NF1 microdeletion determined by FISH analysis. ND = no data available. Open table in a new tab Note.— + = Mutation detectable; − = mutation not detectable. Analysis of neurofibroma-derived Schwann cells (SC F+ and SC F−) and melanocytes derived from both the CALM on the right hand and two CALMs remote from that zone revealed an identical NF1 mutation (c.2325+1G→A) leading to out-of-frame skipping of exon 14, as well as a deletion of the other NF1 allele. For all samples, the proximal deletion breakpoint resided between rs6505129 (chromosome 17 reference position 24777972 [National Center for Biotechnology Information build 36.2]) and rs6505165 (position 25598975), whereas the distal deletion breakpoint resided between the most distal PSV in the telomeric NF1 LCR (position 27439522) and rs9904537 (position 27579216) (minimum/maximum size of deletion 1.84 Mb/2.80 Mb). To test the influence of excess wild-type transcript on detection of the mutant transcript (c.2325+1G→A), a 5-point 10-fold dilution series of the mutant form (5×105—50 molecules) was mixed with excess wild-type (5×105 molecules). Equal amounts of water (instead of wild-type transcript) were added to a second series of dilutions. Comparison of both amplification plots revealed that, at low levels of mutant transcript ( 95%), SC F+ (>95%), and fibroblast (∼10%) cultures derived from the neurofibroma.Table 3Overview of NF1 Mutations Revealed by Routine Mutation-Detection Tec
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