Letter to the Editor

Journal of Investigative Dermatology (2008) 128, 1050–1053; doi:10.1038/sj.jid.5701095; published online 4 October 2007

Somatic Mutation Analysis in NF1 Café au lait Spots Reveals Two NF1 Hits in the Melanocytes

Sofie De Schepper1, Ophélia Maertens2,4, Tom Callens3,4, Jean-Marie Naeyaert1, Jo Lambert1 and Ludwine Messiaen3

  1. 1Department of Dermatology, Ghent University Hospital, Ghent, Belgium
  2. 2Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium
  3. 3Department of Genetics, Medical Genomics Laboratory, University of Alabama at Birmingham, Birmingham, Alabama, USA

Correspondence: Dr Sofie De Schepper, Department of Dermatology, Ghent University Hospital, De Pintelaan 185, Ghent 9000, Belgium. E-mail: sofie.deschepper@ugent.be

4These authors contributed equally to this work.

Abbreviations:

CALM, Café au lait macule; LOH, loss of heterozygosity; LR, long range; NF1, neurofibromatosis type 1

TO THE EDITOR

Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by mutations in the NF1 gene, is characterized by presence of neurofibromas, skin-fold freckling, café au lait macules (CALMs) and iris Lisch nodules. So far, little is known about the etiopathogenesis of NF1-related CALMs. The NF1 gene, like other tumor suppressor genes, follows the Knudson two-hit hypothesis of tumor formation, implying that, apart from the germline NF1 mutation, an additional somatic inactivation of the remaining wild-type NF1 allele is needed to initiate tumor formation (Knudson, 1971). In general, somatic inactivation of a tumor suppressor gene can occur via three major mechanisms: loss of heterozygosity (LOH), somatic intragenic mutations, and promoter hypermethylation. One study addressed somatic NF1 inactivation in 11 CALM melanocyte cultures and found both NF1 alleles to be present (Eisenbarth et al., 1997). However, this study only searched for LOH, and not for minor lesion somatic mutations.

Therefore, we now performed a comprehensive search for NF1 somatic alterations in melanocytes, keratinocytes, and fibroblasts obtained from NF1 CALMs, allowing identification of LOH, copy-number changes of one to multiple exons, and minor lesion mutations. Three keratinocyte, 11 fibroblast and five melanocyte CALM-derived primary cell cultures of 13 patients with a known germline NF1 mutation were examined, in addition to six melanocyte cultures from neonatal foreskin of healthy control persons.

Patients were diagnosed according to the NIH diagnostic criteria. Skin biopsies from NF1 CALMs were taken after written informed consent, and guided by the institutional ethics committee and the Declaration of Helsinki Principles. Skin biopsies and cell cultures were performed essentially as described in De Schepper et al. (2006).

For genomic DNA (gDNA) extraction (QiaAmp, Belelux, Spoorstraat, Venlo, The Netherlands) or Gentra Puregene (Qiagen, Venlo, The Netherlands), RNA isolation (Rneasy; Qiagen), and cDNA synthesis (iScript; BioRad) the manufacturer's instructions were followed.

LOH in the NF1 gene region was evaluated by genotyping two telomeric microsatellite markers and four within the NF1 gene as described previously (Wimmer et al., 2006). Primer sequences can be found in Table 1. Fragments for all loci were compared between DNA extracted from peripheral blood leukocytes from each patient and the CALM derived cell cultures.


NF1 gene somatic mutation analysis was performed essentially as described in Vandenbroucke et al. (2004). All mutations found at the cDNA level were confirmed on gDNA. If no somatic hit was detected after sequencing of the entire coding region, multiplex ligation probe assay analysis using the P081/P082 assay (MRC Holland) was performed as described (Wimmer et al., 2006). In order to determine whether both NF1 mutations detected in melanocyte cultures derived from the CALM of patient NF-008 resided on different alleles, cloning experiments were performed using the pCR2.1-TOPO Vector (Invitrogen, Merelbeke, Belgium).

Top

LOH analysis

LOH analysis could not detect a somatic event in any of the investigated samples. All patients displaying heterozygosity for at least one microsatellite marker in blood lymphocytes did not show LOH in the CALM-derived cells (fibroblasts, keratinocytes, and melanocytes) (Table 2). Since the six polymorphic markers span the NF1 gene, this analysis excludes loss of the wild-type NF1 allele in NF1-related CALMs.


Six normal skin melanocyte cultures obtained from healthy controls and cultured in exactly the same way as the NF1 CALM melanocyte samples were subjected to microsatellite marker and multiplex ligation probe assay analysis. Two NF1 copies were present in all melanocyte cultures and no copy-number changes were detected.

Long-range PCR and direct sequencing of the entire NF1 coding region

The germline NF1 mutation was confirmed to be present in the 11 CALM-derived fibroblast cultures, the three keratinocyte cultures, and five melanocyte cultures. In addition, an additional NF1 mutation was identified in all CALM-derived melanocyte cultures. Three were nonsense mutations: c.2269 A>T (p.K757X), c.4537 C>T (p.R1513X), and c.3721C>T (p.R1241X). One was a splice mutation (c.1721+542A>G), increasing the strength of a cryptic splice donor site in intron 11, that gets used in conjunction with a cryptic intronic splice acceptor site, leading to insertion of 176 bp from intron 11 in the mature transcript. Finally, one culture showed loss of expression of the wild-type NF1 allele at the transcript level, however, no second hit mutation could be identified. The cause of the loss of expression of the wild-type allele and/or allelic drop-out is currently under further investigation. Two of the identified somatic mutations (p.R1241X and p.R1513X) have been reported previously as a germline mutation (Fahsold et al., 2000; Messiaen et al., 2000, Origone et al., 2003; Upadhyaya et al., 2003). Cloning experiments demonstrated that for the CALM of patient NF-008 (c.3525_3526delAA and c.2269A>T), both NF1 mutations resided on different alleles.

No NF1 gene mutations were detected in the six melanocyte cultures obtained from neonatal foreskin from healthy controls using comprehensive mutation analysis, and microsatellite analysis showed presence of two NF1 copies.

The etiopathogenesis of neurofibroma formation in NF1 mouse models was recently ascribed to neurofibromin-deficient Schwann cells (NF1-/-) secreting a potent migratory stimulus (stem cell factor or Kit ligand) for NF1+/- mast cells (Ingram et al., 2000; Yang et al., 2003), implying a somatic second hit mutation of the NF1 gene in heterozygous Schwann cells as the initiating event for the development of a benign (plexiform) neurofibroma.

This study wants to address whether a similar mechanism, based on neurofibromin deficiency due to a second hit mutation, is also applicable for the NF1 CALMs. Several researchers have tried to characterize CALMs histologically, but with conflicting results (Kawamura, 1956; Benedict et al., 1968; Johnson and Charneco, 1970; Takahashi, 1976; Frenck and Marazzi, 1984; Ishida and Jimbow, 1987; Grossman et al., 1995). We recently demonstrated that the melanocyte density is increased in NF1 CALM skin compared with NF1 normal skin and healthy control CALM, and normal skin (De Schepper et al., 2006).

In line with this study, an NF1 second hit in NF1 CALM melanocytes was found in 5/5 available melanocyte cultures, but not in the keratinocytes or fibroblasts from these café au lait spots. No NF1 mutations were found in the melanocyte cultures from neonatal foreskin from healthy control persons.

This study confirms and extends the findings of a second hit mutation in melanocyte cultures from a segmental and mosaic NF patient (Maertens et al., 2007). We conclude from our study that there is a statistically significant difference (P<0.05) in the frequency of second hit mutations in CALM melanocytes (5/5) compared with CALM fibroblasts (0/11) (P=0.000, Fisher's Exact Test), and with CALM keratinocytes (0/3) (P=0.018, Fisher's Exact Test). So far no evidence points to a significant contribution of LOH to CALM formation. Six healthy control normal skin melanocyte cultures, which were analyzed as negative controls did not show any somatic NF1 alterations.

The finding of second hit mutations in NF1 CALM melanocytes improves our understanding of the etiopathogenic mechanisms underlying the formation of this hyperpigmentary disorder, and will boost further research aiming to understand the phenotypes associated with somatic mutations in melanocytes.

Top

Conflict of Interest

The authors state no conflict of interest.

Top

References

  1. Benedict PH, Szabo G, Fitzpatrick TB, Sinesi SJ (1968) Melanotic macules in Albright's syndrome and neurofibromatosis. JAMA 205:618–626 | Article | PubMed | ChemPort |
  2. De Schepper S, Boucneau J, Vander Haeghen Y, Messiaen L, Naeyaert JM, Lambert J (2006) Café au lait spots in neurofibromatosis type 1 and in healthy control individuals: hyperpigmentation of a different kind? Arch Dermatol Res 297:439–449 | Article | PubMed | ISI |
  3. Eisenbarth I, Assum G, Kaufmann D, Krone W (1997) Evidence for the presence of the second allele of the neurofibromatosis type 1 gene in melanocytes derived from café au lait macules of NF1 patients. Biochem Biophys Res Commun 237:138–141 | Article | PubMed | ISI | ChemPort |
  4. Fahsold R, Hoffmeyer S, Mischung C, Gille C, Ehlers C, Kücükceylan N et al. (2000) Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain. Am J Hum Genet 66:790–818 | Article | PubMed | ISI | ChemPort |
  5. Frenck E, Marazzi A (1984) Neurofibromatosis of von recklinghausen: a quantitative study of the epidermal keratinocyte and melanocyte populations. J Invest Dermatol 83:23–25 | Article | PubMed | ISI | ChemPort |
  6. Grossman MC, Anderson RR, Farinelli W, Flotte TJ, Grevelink JM (1995) Treatment of café au lait macules with lasers: a clinicopathologic correlation. Arch Dermatol 131:1416–1420 | Article | PubMed | ISI | ChemPort |
  7. Ingram DA, Yang FC, Travers JB, Wenning MJ, Hiatt K, New S et al. (2000) Genetic and biochemical evidence that haploinsufficiency of the Nf1 tumor suppressor gene modulates melanocyte and mast cell fates in vivo. J Exp Med 191:181–188 | Article | PubMed | ISI | ChemPort |
  8. Ishida O, Jimbow K (1987) A computed image analyzing system for quantitation of melanocyte morphology in café au lait macules of neurofibromatosis. J Invest Dermatol 88:287–291 | Article | PubMed | ISI | ChemPort |
  9. Johnson BL, Charneco DL (1970) Café au lait spot in Neurofibromatosis and in normal individuals. Arch Dermatol 102:442–446 | Article | PubMed | ISI | ChemPort |
  10. Kawamura VT (1956) Über die herkunft der naevuscellen and die genetische verwandtschaft zwischen pigmentzellnaevus, blauem naevus und Recklinghauser phakomatose. Hautartzt 7:7–14
  11. Knudson AG Jr (1971) Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci USA 68:820–823 | Article | PubMed |
  12. Maertens O, De Schepper S, Vandesompele J, Heyns I, Janssens S, Speleman F et al. (2007) Molecular dissection of isolated disease features in neurofibromatosis type 1. Am J Hum Genet 81:243–251 | Article | PubMed | ISI | ChemPort |
  13. Messiaen L, Callens T, Mortier G, Beysen D, Vandenbroucke I, Van Roy N et al. (2000) Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Hum Mutat 15:541–555 | Article | PubMed | ISI | ChemPort |
  14. Origone P, Bellini C, Sambarino D, Banelli B, Morcaldi G, La Rosa C et al. (2003) Neurofibromatosis type 1 (NF1): identification of eight unreported mutations in NF1 gene in Italian patients. Hum Mutat 22:179–180 | Article | PubMed | ChemPort |
  15. Takahashi M (1976) Studies on café au lait spots in neurofibromatosis and pigmented macules of nevus spilus. Tohoku J Exp Med 118:255–273 | PubMed | ISI |
  16. Upadhyaya M, Majounie E, Thompson P, Han S, Consoli C, Krawczak M et al. (2003) Three different pathological lesions in the NF1 gene originating de novo in a family with neurofibromatosis type 1. Hum Genet 112:12–17 | Article | PubMed | ISI | ChemPort |
  17. Vandenbroucke I, Van Doorn R, Callens T, Cobben JM, Starink TM, Messiaen L (2004) Genetic and clinical mosaicism in a patient with neurofibromatosis type 1. Hum Mutat 114:284–290
  18. Wimmer K, Yao S, Claes K, Kehrer-Sawatzki H, Tinschert S, De Raedt T et al. (2006) Spectrum of single and multiexon NF1 copy number changes in a cohort of 1100 unselected NF1 patients. Genes Chromosomes Cancer 45:265–276 | Article | PubMed | ISI | ChemPort |
  19. Yang FC, Ingram DA, Chen S, Hingtgen CM, Ratner N, Monk KR et al. (2003) Neurofibromin-deficient Schwann cells secrete a potent migratory stimulus for NF1+/- mast cells. J Clin Invest 112:1851–1861 | Article | PubMed | ISI | ChemPort |
Top

Acknowledgments

We thank Martine De Mil for assisting with the cell culture and Marie-Chantal Herteleer and Joachim Boucneau for technical assistance. SDS is a research fellow of the Fund for Scientific research-Flanders (grant number G.0292.02). We thank the Wilderman family for their support (to LM) of research on CALMs in NF1 patients. This work is supported by an Interuniversitary Attraction Poles grant from the Federal Office for Scientific, Technical and Cultural Affairs, Belgium (2002–2006; P5/25), and by a Concerted Action Grant from the UGent (OM). This work was a collaboration between the Ghent University (Belgium) and the University of Alabama at Birmingham (USA).

MORE ARTICLES LIKE THIS

These links to content published by NPG are automatically generated.

RESEARCH

Neurofibromatosis Type 1 Protein and Amyloid Precursor Protein Interact in Normal Human Melanocytes and Colocalize with Melanosomes

Journal of Investigative Dermatology Original Article

Mechanisms of Loss of Heterozygosity in Neurofibromatosis Type 1-Associated Plexiform Neurofibromas

Journal of Investigative Dermatology Original Article

Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1?like phenotype

Nature Genetics Letter (01 Sep 2007)

Mitotic recombination effects homozygosity for NF1 germline mutations in neurofibromas

Nature Genetics Letter (01 Jul 2001)

Induction of Melanogenic Abnormalities in NF1 + /? Mutant Mice by DMBA

Journal of Investigative Dermatology Letter

Extra navigation

.
ADVERTISEMENT