Am. J. Hum. Genet. 58:484-490, 1996

Somatic Mosaicism in a Patient with Neurofibromatosis Type 1

Steven D. Colman,' Sonja A. Rasmussen,' Vu T. Ho,' Corinne R. Abernathy,' andMargaret R. Wallace',2'3

'Division of Genetics, Department of Pediatrics, Center for Mammalian Genetics, 2Department of Biochemistry and Molecular Biology, and3Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Gainesville

Summary

Using loss of heterozygosity analysis, a method designedto detect moderate to large gene deletions, we have iden-tified a new-mutation neurofibromatosis type 1 (NF1)patient who is somatically mosaic for a large maternallyderived deletion in the NFl gene region. The deletionextends at least from exon 4 near the 5' end of the geneto intron 39 near the 3' end. The gene-coding region is,therefore, mostly or entirely deleted, encompassing aloss of - 100 kb. We hypothesize that the deletion oc-curred at a relatively early developmental timepoint,since signs of NF1 in this patient are not confined to aspecific body region, as seen in "segmental" NF, andsince both mesodermally and ectodermally derived cellsare affected. This report provides the first molecularevidence of somatic mosaicism in NF1 and, taken to-gether with a recent report of germ-line mosaicism inNF1, adds credence to the concept that mosaicismplays an important role in phenotypic and genetic as-pects of NF1 and may even be a relatively commonphenomenon.

Introduction

Neurofibromatosis type 1 (NF1) is an autosomal domi-nant disease with an prevalence estimated at -1/3,000(Riccardi 1992). This disease is characterized by caf6-au-lait spots, cutaneous neurofibromas, axillary freck-ling, and Lisch nodules (hamartomas) of the iris. Otherfeatures may include plexiform neurofibromas, bonedysplasias, learning disabilities, seizures, and scoliosis(Riccardi 1992). The NF1 gene, which is -350 kb inlength, encodes a cytoplasmic GTPase activating protein(GAP) called neurofibromin. The GAP domain only en-compasses about one-eighth of neurofibromin, and thusthis protein may have other, yet undiscovered, functions.

Received September 29, 1995; accepted for publication December14, 1995.Address for correspondence and reprints: Dr. Margaret R. Wallace,

Department of Pediatric Genetics, Box 100296, University of Florida,Gainesville, FL 32610-0296. E-mail: peggyw@cmg.health.ufl.eduC 1996 by The American Society of Human Genetics. All rights reserved.0002-9297/96/5803-0006$02.00

The NF1 gene has a very high mutation rate; approxi-mately half of all NF1 patients have no family historyof the disease. Up to 90% of new mutations are believedto be paternal in origin (Jadayel et al. 1990; Stephenset al. 1992); however, it has recently been suggested thatlarge deletions are more commonly maternally derived(Soltan et al. 1995). Although the large gene size pro-vides an expansive target for mutation, size alone cannotaccount for the elevated mutation rate (Marchuk et al.1991). One proffered explanation for this discrepancyis that the mutation rate reflects not only germ-line mu-tations passed to patients through their parents but alsopostzygotic disease-causing somatic mutations in the pa-tients themselves (Zlotogora 1993). This occurrencewould result in mosaicism, in which at least some tissuesin an individual are composed of two (or more) geneti-cally distinct cell types.Mosaicism has been shown to have important clinical

implications for a number of disorders (Hall 1988; Ber-nards and Gusella 1994) and evidently also plays animportant role in some cases of NF1. For example, therehave been reports of phenotypically normal individualshaving more than one child affected with NF1 (Riccardiand Lewis 1988; Lazaro et al. 1994). One explanationfor this occurrence is gonadal mosaicism in the unaf-fected parent, and, in one case, a molecular genetic ap-proach has proven this supposition (Lazaro et al. 1994).Gonadal mosaicism has been thought to be an unusualphenomenon in NF1, in light of the infrequency withwhich cases of unaffected parents having multiple af-fected offspring have been reported. However, gonadalmosaicism may occur more frequently in NF1 than iscurrently recognized; male germ-line mosaicism may ex-plain the predominance of paternally derived alleles insporadic NF1 cases. In addition, "segmental" NF (orNF-5) patients, whose symptoms are confined to a spe-cific portion of their bodies, are also believed to be so-matically mosaic (Riccardi 1992). Consistent with theidea that at least some of these individuals are gono-somic mosaics (mosaic in both somatic and germ-linecells), there are a number of reports of apparently seg-mental patients having children with full (nonsegmental)NF1 (Rubenstein et al. 1983; Riccardi and Lewis 1988;Boltshauser et al. 1989; Huson 1994). Although themost obvious explanation for these occurrences is mosa-

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icism, no molecular evidence for this phenomenon hasyet been reported.

In this article, we report a mosaic individual with arelatively late-onset case of NF1, whose leukocytes arecomprised primarily of cells that have a large deletionin the NF1 region but also include a small percentageof normal cells. Since there is no localization of signs ofNF1 to a particular body region in this patient, thismutation appears to have occurred early enough in de-velopment not to result in a segmental phenotype. Fur-ther evidence for an early developmental mutation isthe involvement of both mesodermally and ectodermallyderived tissues.

Patient and Methods

Case HistoryPatient UF161 is a 31-year-old female who was diag-

nosed with NF1 at age 27 years after pathological exam-ination of an excised neurofibroma. UF161 is evidentlya new mutation patient; her parents, ages 25 and 27years at her birth, reportedly have no signs of NFL.The patient recalls development of caf&-au-lait spots andaxillary freckling at the age of 7 or 8 years. Her firstneurofibroma appeared on her back at age 26 years afterher second pregnancy, and she subsequently developedneurofibromas on her chest, face, and, most recently,her extremities. Several of these have been removed forcosmetic reasons. Her early development was normal,and her school performance was described as average.Brain magnetic resonance imaging at age 28 years wasread as normal.On recent physical examination (at age 31 years), pa-

tient UF161 is of normal stature with a head circumfer-ence of 57.5 cm (2 SD above the mean). She has sevencaf&-au-lait macules on her trunk and extremities, withthe largest on the left side of her abdomen (1 cm X 3cm). She has axillary freckling bilaterally, with the leftside significantly more affected than the right (extendingto under her left breast). In addition, she has frecklingin the skin folds of her buttocks and on her left innerthigh. She has extensive freckling in sun-exposed areas,appearing in an irregular swirling pattern, particularlyon her back. She has multiple small cutaneous neurofi-bromas on her face, trunk, and extremities; there is noevidence of segmental distribution. Her neurologicalexam was unremarkable, and her blood pressure waswithin normal limits. Ophthalmological slit-lamp exam-ination of her eyes demonstrated multiple Lisch nodulesbilaterally.The patient has two sons, ages 7 (UF394) and 5 years

(UF395). Both boys were noted to be mildly delayed inspeech, on a prekindergarten evaluation, but are other-wise developmentally normal. A head-computerized to-mography exam performed on UF394 detected no ab-

normalities. On physical examination, both boys wereof normal stature but were macrocephalic (it should benoted that the unaffected father's head circumferencewas also above the 98th centile). UF394 has a singlecaf&-au-lait macule (0.5 cm X 0.7 cm) on his right wristbut no other signs of NF1. UF395 had no signs of NF1at all. Both boys had normal neurological examinationsand normal blood pressure. Slit-lamp examination didnot reveal Lisch nodules on the irides of either boy.

Sample Acquisition and ProcessingBlood was initially obtained from UF161 and her par-

ents (UF160 and UF162). A second sample of bloodfrom UF161 was subsequently acquired. Finally, bloodsamples were obtained from UF161's sons (UF394 andUF395) and their father (UF393). In all cases, DNA wasextracted according to the method of Madisen et al.(1987). Neurofibromas (confirmed by pathological ex-amination) were kept in cell culture media and processedthe day after surgery. Any tissue identifiable as "nor-mal," such as skin, was carefully dissected away, so thatthe remaining tissue appeared to be as homogenous aspossible; DNA was then extracted by standard methods(Sambrook et al. 1989).A 3-mm skin sample was obtained from an apparently

unaffected area of the patient's right forearm. The tissuewas divided in half and processed in parallel. Tissuesamples were initially placed in Leibowitz L-15 media(Sigma, L4386) supplemented with 100 U/ml penicillin-G, 100 jg/ml streptomycin sulfate, 50 jg/ml kanamycinsulfate, 1.25 U/ml dispase (Collaborative BiomedicalProducts), and 0.05% collagenase (Sigma, type 1A), in-cubated 2 h at 37°C with mild agitation, and then centri-fuged at 500 x g for 5 min. Cell pellets were then washedin 3 ml of Iscove's modified Dulbecco's medium(IMDM) (Gibco, 12200-069) supplemented with 2 mML-glutamine, 100 U/ml penicillin-G, 100 jg/ml strepto-mycin sulfate, and 20% fetal bovine serum and recentri-fuged. The supernatants and cell pellets (which had beenresuspended in 3 ml of the supplemented IMDM) wereplaced in separate T-25 tissue culture flasks and incu-bated at 37°C. After expansion of these four indepen-dent cultures, cells were harvested, and DNA extracted,as previously described.

Loss of Heterozygosity (LOH) AnalysisAll of the markers listed in figure 1 were scored for

patient UF161 and her parents by using conditions speci-fied in the references, with the exception of the Alu tetra-nucleotide (AAAT)n polymorphism (Xu et al. 1991).This tetranucleotide repeat was typed nonradioactivelywith a modification to simplify the procedure: the PCRproduct was digested with BfaI, which cuts the productinto a constant 268-bp fragment, and a polymorphicfragment, which varies in size from 127 bp to 139 bp.

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p11.2

pl1.1

q1.1

qll.2

ql2

HHH202UT172GE2 (Exons 1-4)E5 RFLP (Exon 5)AIu (AAAT) n (127b)EVI2A (Intron 27b)EVI2B (Intron 27b)IVS 24.8 (Intron 27b)AE25 (Exons 27b-38)Scal RFLP (Intron 39)Pad RFLP (Intron 41)lF1o

UFI610

00.00.00

.

Figure 1 LOH within the NF1 gene. Black circles representdeleted regions, while open circles represent undeleted regions. Graycircles indicate uninformative loci.

These alleles were resolved on 10% native polyacryl-amide gels and visualized with ethidium bromide stain-ing. DNA from UF161's children (UF394 and -395) andtheir father (UF393) was assayed with the UT172, exon5, IVS 24.8 (intron 27b), and intron 39 polymorphisms.

Cloning and Screening of PCR ProductsTo confirm the finding of somatic mosaicism and to

provide information regarding the ratio of mutant tonormal leukocytes, exon 39 PCR products were clonedinto a T-vector (Novagen) and transfected into Esche-richia coli DH5a cells (Gibco-BRL) by using conditionsrecommended by the manufacturers. Insert-containingcolonies were used directly as templates for PCR usingexon 39 primers and conditions as described elsewhere(Martin-Gallardo et al. 1992). These PCR products weredigested with ScaI, and the alleles were typed as de-scribed by Abernathy et al. (1994).

Results

Patient UF161 showed LOH of the maternally derivedallele for every informative intragenic NF1 polymor-phism tested in leukocytes, including GE2/EcoRI (Rey-niers et al. 1993), exon 5/RsaI (Hoffmeyer and Assum1994), intron 27b IVS 24.8 (Lazaro et al. 1994), Alu(AAAT)n (Xu et al. 1991), and intron 39/ScaT (Aberna-thy et al. 1994) (fig. 1). VNTR analysis showed normalMendelian inheritance at other loci in the genome forthis family (data not shown). At every NF1 marker listedabove, there was a weak signal from the "missing" ma-ternal allele. This included several cases where incom-plete digestion of DNA could not account for the tracesignal. To assure that these results were not due to con-tamination or mix-up of the DNA sample, we obtaineda new blood sample from UF161 (3 years subsequent

to the first sample) and repeated the experiments. Therewas identical representation of the normal maternal al-lele in this sample.

Figure 2 shows results from the exon 5/RsaI and in-tron 39/ScaI polymorphism assays, which indicate bothLOH and mosaicism. Since the exon 5 and GE2 poly-morphisms are near the 5' end of the coding regionand intron 39 is nearer the 3' end, these experimentsdemonstrate that most (and possibly all) of the mater-nally derived NF1 allele is deleted in affected cells.Therefore, in these cells, UF161 is functionally hemizy-gous for the NF1 locus. To delineate the extent of thisdeletion, the family was typed for the UT172 (Shannonet al. 1994) and HHH202 polymorphisms (Hoff et al.1988), 0.6 cM and 1.5 Mb upstream of NF1, respec-tively. While UT172 was uninformative, UF161 wasfully heterozygous for HHH202 (data not shown), indi-cating that the proximal endpoint of this deletion is be-tween exon 4 of the NF1 gene (the GE2 probe includespart of exon 4) and HHH202. The polymorphic locusiFlO (Jorde et al. 1993), -100 kb downstream from theNF1 gene, was not informative in this family; however,densitometric analysis of the Southern blot results (com-paring signal intensity from iF10 and a chromosome 7probe) indicated that UF161 has two copies of iF10(data not shown). Thus, the distal breakpoint of thisdeletion lies between intron 39 and iF10, as indicatedin figure 1.

3 UF160 UF161 UF16218180 r _

b

Figure 2 LOH and somatic mosaicism of the maternally derivedallele. a, LOH of exon 5 within the NF1 gene. The presence of thepolymorphic RsaI site allows digestion of the 180-bp band into a 130-bp and 50-bp band (50-bp band not shown). UF161 is the proband,UF160 is her father, and UF162 is her mother. The residual 180-bpmaternally derived band is the result of somatic mosaicism. b, LOHof exon 39 within the NFl gene. The presence of the polymorphicScaI site allows digestion of the 285-bp band into a 208-bp and 77-bp band (77-bp band not shown). The residual 208-bp maternallyderived band is the result of somatic mosaicism.

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0c

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UF16O UF162

UF161 2 2 UF393

UF394 UF395

bp285

208

77

Figure 3 Inheritance of NF1 exon 39 alleles in the family ofUF161.

UF161/exon 39 PCR products (which include intron39 sequences) were subcloned and analyzed to confirmmosaicism and to provide information regarding the ra-

tio of mutant to normal leukocytes. Sixty-nine of thesesubclones were analyzed for the intron 39 polymor-phism; one clone retained the normal maternally derivedallele, while the other 68 contained the paternally de-rived allele (data not shown). This analysis, added to

densitometric studies of the RFLPs, suggests that a smallpercentage of UF161's lymphoid cells contain the mater-nal allele.We acquired skin fibroblasts and two cutaneous neu-

rofibromas from UF161 and blood samples fromUF161's children and their father. DNA from these sam-ples was analyzed with the UT172, exon 5, IVS 24.8(intron 27b), and intron 39 polymorphisms. The intron39 polymorphism demonstrated that one of UF161'ssons (UF394) received her normal maternally derivedNF1 allele (fig. 3), while the IVS 24.8 polymorphismshowed that her other son (UF395) received her pater-

nally derived NF1 allele (data not shown). It is alsoapparent (both visually and densitometrically) that bothof the neurofibroma samples have skewed ratios of NF1alleles. This indicates that at least some of the tissueswithin these tumors (neurofibromas are composed of anadmixture of cell types) are mosaic (fig. 4, T1 and T2).The skin fibroblasts, however, appear to have normal

amounts of both alleles (as seen in four independent celllines established from the same skin biopsy) (fig. 4, SF1and SF2). Results from the exon 5 and IVS 24.8 poly-morphism assays paralleled the intron 39 results (datanot shown).

Discussion

In this investigation, LOH studies determined that, insome cell types, patient UF161 carries a large deletionin the NF1 region of her maternally derived allele. Al-though the endpoints of this deletion have not been de-termined, LOH at all informative loci inclusive of andbetween exon 4 and intron 39 indicates that the deletionmust be ¢100 kb. The deletion does not include locusHHH202 (0.6 cM proximal) or iF10 (-100 kb down-stream). The precise size of UF161's deletion might bededuced from pulsed-field gel experiments (such as seenin NF1 deletion patients studied by Viskochil et al.[1990]); however, the probe would have to lie outsidethe deletion to detect an abnormally sized fragment, andunfortunately very few probes are currently availablethat lie close to, but outside, the NF1 gene.

Five very large deletions (including the entire NF1locus and beyond) have been documented in NF1 pa-tients (Kayes et al. 1994). These five patients have dele-tions of >700 kb, and, of the three de novo mutations,two were paternal in origin, and one was maternallyderived. All of these patients are remarkable for havingdysmorphic features and an unusually large number ofcutaneous neurofibromas. Furthermore, all of the pa-tients have cognitive deficits; three are mentally retarded,and the other two have learning disabilities. The authors

X B Ti T2 SF1 SF2 C285

208 l ; ;

Figure 4 Exon 39 analysis of somatic mosaicism in various tis-sues. DNA in the lanes was derived from UF161's blood (B), neurofi-bromas (Ti and T2), and cultured skin fibroblasts (SF1 and SF2). Thelane entitled "C" is control DNA derived from blood of an unrelated,unaffected heterozygous individual.

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of this study hypothesized that the deletions in thesepatients encompass other genes flanking and/or withinthe NF1 gene, the loss of which contributes to the com-plex phenotype (Kayes et al. 1994), as seen in contiguousgene syndromes (Schmickel 1986). While neitherUF161's deletion or the deletions identified by Kayeset al. (1994) encompass HHH202, all of the deletionsdescribed by Kayes et al. extend beyond 1F10. There-fore, it is possible that the much milder phenotype seenin UF161 is the result of a deletion that affects only theNF1 gene (and embedded genes) and does not removeputative downstream flanking genes.Of particular interest is that, in all informative assays

performed on DNA purified from leukocytes, there wasa small but readily detectable signal from the maternallyderived NF1 allele, indicating somatic mosaicism. This,therefore, is the first report that provides molecular evi-dence for somatic mosaicism in NFL. This large deletionpresumably occurred early during embryonic develop-ment, since both mesodermally and ectodermally de-rived tissues appear affected (the deletion was detectedin her mesodermally derived leukocytes, and she hastypical NF1 features, which imply involvement of themutation in the ectodermal neural crest). Furthermore,the deletion occurred early enough during developmentto preclude confinement of her NF features to certainbody regions, as in segmental NF. The identification ofonly 1 maternally derived allele in a set of 69 allelesderived from leukocytes further supports the hypothesisthat the mutation occurred relatively early on. It is possi-ble, however, that the mutation occurred later and thatthe deletion conferred a selective growth advantage (atleast in leukocytes). Why skin fibroblasts, which are alsomesodermally derived, do not seem to show mosaicism,is unclear. It is conceivable that the level of mosaicismin these tissues is simply too low to be detectable orthat, in these cells, this large deletion confers a selectivegrowth disadvantage. This wide variation in degree ofmosaicism in different tissues has been noted elsewhere(Wallis et al. 1990; Kontusaari et al. 1992).

Analysis of DNA derived from UF161's neurofibro-mas suggests that, because of skewed ratios of maternalto paternal alleles, these tissues are mosaic also. Sinceneurofibromas are composed of a variety of cell types,mosaic status in these tumors would presumably resultfrom the inclusion of affected cells carrying the maternaldeletion and a subpopulation of normal cells with twointact NF1 alleles. The exact percentage of affected cellsin a given neurofibroma is unpredictable and most likelyvariable, which could explain the observations. Also,neurofibromas in NF1 patients are, at least in somecases, associated with somatic deletions of the remainingNF1 allele, supporting the "two-hit hypothesis" in theetiology of these tumors (Colman et al. 1995). There-fore, it is possible that these neurofibromas arose from

cells carrying the maternally derived deletion subsequentto a second inactivating NF1 mutation on the paternallyderived allele. If the second hit was a relatively largedeletion, such that the affected cells did not contributeany NF1 alleles, mosaicism in these tumors would arisefrom a mixture of cells with deletions on both alleles,normal cells, and cells carrying only the maternal dele-tion.

Also of note is the relatively late-onset NF1 pheno-type in patient UF161. She did not develop neurofibro-mas until the age of 26 years, following her second preg-nancy. This is in contrast to most NF1 patients; forexample, in one population study of 155 individuals, allof the adults over the age of 20 years had neurofibromas(Huson 1994). The severity of the NF1 phenotype andthe age at onset may have been mediated by the presenceof a population of normal cells in this individual. Thiseffect has been suggested to operate in many other disor-ders, including NF2 (Bourn et al. 1994), osteogenesisimperfecta (Wallis et al. 1990), and Down syndrome(Fishler and Koch 1991). Alternatively, the less severephenotype seen in this patient (particularly the late-onsetof neurofibroma formation) may simply reflect the factthat there are fewer cells that are targets for a secondmutation. It is also possible that a null NF1 allele mayresult in a milder phenotype than a mutation that resultsin a mutant transcript/protein. Such a "dominant nega-tive" effect would be similar to that seen in other domi-nant disorders such as osteogenesis imperfecta, in whichinclusion of a mutant peptide (not usually truncated) ina multimer is more deleterious than half dosage of thegene (Byers 1995). However, since Western blot studiesof neurofibromin do not suggest that multimers are pres-ent, and since the majority of NF1 mutations are pre-dicted to result in truncated proteins (which are oftenunstable and not generally associated with dominantnegative effects), this phenomenon appears unlikely tooccur in NF1.

In conclusion, it should be emphasized that mosaicismmay be a relatively frequent occurrence in NFL. A betterunderstanding of the frequency of mosaicism may proveto be an important consideration in predicting recur-rence risk, and any mediating affect conferred by thepresence of even a small population of normal cells maybe an important parameter for predicting phenotype andprognosis. Germ-line mosaicism has elsewhere beenshown to be a relatively frequent phenomenon in otherheritable diseases, such as muscular dystrophy (Bakkeret al. 1989; van Essen et al. 1992), and has been recentlydocumented in NF1 (Lazaro et al. 1994). Somatic mosa-icism has also been reported in a variety of diseases (i.e.,Wallis et al. 1990; Hirschhorn et al. 1994; Saito et al.1995); however, the importance of somatic mosaicismin NF1 has yet to be determined. Information gleanedfrom cases such as UF161 will eventually illuminate this

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Colman et al.: Somatic Mosaicism in NF1 489

phenomenon and may also prove to be important inidentifying affected cell types in NFL.

AcknowledgmentsThe authors wish to thank the patient and her family, the

Florida Chapter of the National Neurofibromatosis Founda-tion, and Drs. C. Williams, D. Byrd, K. Kato, and D. Driscoll.Additional help was provided by the genetics clinic staff at theUniversity of Florida and B. Gray at the R. C. Philips Unit,State of Florida. This work was funded by grants to M.R.W.from the Hayward Foundation, the National Neurofibro-matosis Foundation, the American Cancer Society (Florida Di-vision), and the Children's Miracle Network. Further supportincluded grants from the National Institutes of Health toM.R.W. (R29 NS31550) and S.D.C. (T32 CA09126), the Ray-mond C. Philips Research and Education Contract, Children'sMedical Services, and the Department of Health and Rehabili-tative Services, State of Florida.

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