Genotype/phenotype correlations 2 neurofibromatosis (NF2

40MedGenet 1998;35:450-455

Genotype/phenotype correlations in type 2neurofibromatosis (NF2): evidence for moresevere disease associated with truncatingmutations

D G R Evans, L Trueman, A Wallace, S Collins, T Strachan

AbstractBlood samples from 125 unrelated fami-lies with classical type 2 neurofibromato-sis (NF2) with bilateral vestibularschwannomas have been analysed formutations in the NF2 gene. A further 17families fulfilling modified criteria forNF2 have also been analysed. Causativemutations have been identified in 54 (43%)classical families and six (35%) of thosefulfilling modified criteria. Forty-twocases from 38 families with truncatingmutations had an average age at onset ofsymptoms of 19 years and diagnosis at 22.4years. Fifty-one cases from 16 familieswith splice site mutations (15 from six),missense mutations (18 from six), andlarge deletions (18 from five) had an aver-age age of onset of 27.8 years and at diag-nosis of 33.4 years. Subjects withtruncating mutations were significantlymore likely to have symptoms before 20years of age (p<0.001) and to develop atleast two symptomatic CNS tumours inaddition to vestibular schwannoma before30 years (p<0.001). There were also sig-nificantly fewer multigenerational fami-lies with truncating mutations. Fourfurther truncating mutations were inmosaic form and were associated withmilder disease than other similar muta-tions.This large study has confirmed the pre-

vious impression that truncating muta-tions are associated with severe disease,but caution has to be exercised in usingmutation type to predict disease course.(7Med Genet 1998;35:450-455)

Keywords: neurofibromatosis type 2; somatic mosai-cism; mutation; vestibular schwannoma

Type 2 neurofibromatosis (NF2) is an auto-somal dominantly inherited condition charac-terised by the development of bilateral vestibu-lar schwannomas, schwannomas of othercranial, spinal, and cutaneous nerves, and cra-nial and spinal meningiomas.' 3 In 1987, theNational Institutes of Health Consensus Meet-ing set out firm diagnostic criteria for NF2.'These criteria meant that any person withbilateral vestibular schwannomas was assumedto have the disease and that 50% of offspringwould be predicted to be affected. The NF2gene was isolated in 19935 6 and there have

been a number of reports of germline muta-tions in large series of affected cases since thattime.` Although detection rates using SSCPor DGGE have in general been disappointinglylow, even in classically affected people, someevidence for a genotype/phenotype correlationappears to be emerging.8'- We report here forthe first time the largest single study of correla-tions between mutational type and diseasephenotype in NF2.

Patients and methodsSubjects with classical NF2 (bilateral VS) havebeen ascertained by our centres since 1989.Blood samples have been obtained fromaffected subjects from 125 unrelated families.Clinical data have been meticulously soughtand most families have been seen by the corre-sponding author (91/125). Full informationincluding scan data was available on 201subjects from the 125 families. Patients whohad died before the study were excluded fromthe main analysis. Data specifically recordedwere: age at onset of the first clearly relatedsymptom from a CNS tumour, age at diagnosisof bilateral vestibular schwannomas (VS), orNF2 and the presence of other non-VStumours either symptomatic or on scan.Although age at onset of cutaneous tumourswas sought, this was not included as represent-ing a first symptom. Although cutaneoustumours are associated with more severedisease,2 they may occur many years beforethe onset of significant CNS tumours and veryrarely contribute in themselves to diseaseseverity. Many patients also find it hard to re-collect accurately when they first noticed a skintumour. Ophthalmic data were also collected;however, age at onset of a cataract was alsoexcluded as representing a first symptom.Cataracts again are rarely a cause of seriousmorbidity in NF2 and are often found inciden-tally at routine eye checks. Disease severity inNF2 is related to early onset, number, andgrowth rate of CNS tumours. Therefore thefollowing details were analysed. (1) Age at

Table 1 Diagnostic criteria for NF2

Bilateral vestibular schwannomas orfamily history ofNF2 plus(1) Unilateral VS or

(2) Any two of: meningioma, glioma, neurofibroma,schwannoma, posterior subcapsular lenticular opacities

Additional criteriaUnilateral VS + (2)Multiple meningioma (2 or more) + unilateral VS or any 2 of:

glioma, neurofibroma, schwannoma, cataract

Department ofMedical Genetics, StMary's Hospital,Manchester M13 OJH,UKD G R EvansA Wallace

UniversityDepartment ofHumanGenetics, NewcastleUniversity, Newcastleupon Tyne, UKL TruemanS CollinsT Strachan

Correspondence to:Dr Evans.

Received 5 September 1997Revised version accepted forpublication12 December 1997

450

on June 7, 2022 by guest. Protected by copyright.

http://jmg.bm

j.com/

J Med G

enet: first published as 10.1136/jmg.35.6.450 on 1 June 1998. D

ownloaded from

http://jmg.bmj.com/

NF2 genotypelphenotype correlations

onset of first CNS tumour related symptom.(2) Age at onset of hearing loss. (3) Age atdiagnosis of NF2 (excluding DNA diagnosisalone). (4) Number of symptomatic tumours.(5) Presence of 10 or more tumours >0.5 cmon MRI scan.

Families were arbitrarily categorised as mild,intermediate, or severe on the basis of havingpresented after 30 years, between 20 and 30years, or before 20 years. Those subjectspresenting before 30 years, but who had fewerthan three other non-VS symptomatic tumoursby 30 years of age, were considered mildlyaffected. These criteria have been modifiedslightly from our previous publications2 3 andsome families on re-evaluation have beenreclassified (for example, family 60). Allsubjects had undergone cranial imaging withMRI and the majority had undergone full spi-nal imaging. Any person with more than 10tumours >0.5 cm on scan was consideredseverely affected. As previous classificationshave been arrived at before the ability to detecttiny (1-2 mm) tumours, some modification isnecessary if scan data are to be included. Cur-rent evidence would suggest that 75-90% ofNF2 cases will have evidence of spinal tumourson MRI.8R l Many of these tumours will remainsmall and never become symptomatic. As ameasure of severity, only tumours >0.5 cmhave therefore been included. The great major-ity of patients have had some form of ophthal-mological assessment. While this involved slitlamp examination, in most cases full assess-ment for retinal hamartomas and other featuressuch as epiretinal membranes was sparse.

In addition to the 125 classical families,samples from 17 subjects fulfilling modifiedcriteria for NF2 (table 1)2 and 87 subjectsthought to be at risk of NF2, but not fulfillingcriteria, were analysed as below.

MUTATION ANALYSISGenomic DNA samples prepared from periph-eral blood were amplified with primers for all17 exons of the NF2 gene, using primers previ-ously described.5 12 PCR reactions were per-formed in a total reaction volume of 25 gl using100 ng genomic DNA, 20 pmol of each primer,200 gm of deoxyribonucleoside triphosphate,0.5 U Thr polymerase (Northumbria Biologi-cals Ltd), and 1 x reaction buffer (Northum-bria Biologicals Ltd) overlaid with mineral oil.Amplification was performed on a PCR 480Perkin Elmer Cetus machine (35 cycles of95°C for 90 seconds, 50°C for 90 seconds, and72°C for 30 seconds followed by a final elonga-tion step of 72°C for 10 minutes).SSCP analysis was carried out by Hydrolink-

MDE gel (AT Biochem) electrophoresis ac-cording to the supplier's instructions. Samplesfor SSCP analysis (3 ,ul of PCR product) werediluted with 3 ,ul of a solution containing 95%formamide, 20 mmol/l EDTA, 20 mmol/lNaOH, 0.05% bromphenol blue, and 0.05%xylene cyanol, and denatured at 95°C for threeminutes. Electrophoresis was carried out on 24cm vertical and 1 mm thick gels at a constantvoltage of 250 V for 13-16 hours. Gels were sil-ver stained by sequential incubations in 10%

ethanol and 0.5% acetic acid (2 x 3 minutes),0.1% silver nitrate (15 minutes), 1.5% NaOHand 0.15% formaldehyde (20 minutes), and0.75% sodium carbonate (10 minutes), andphotographed as appropriate.The last 13 mutations have been identified

using a modification of the above technique inthe Manchester laboratory.13

Patients homozygous for two polymorphicmarkers within intron 14 15 were investigatedfor deletions by hybridising EcoRI and NotI(pulsed field) genomic Southern blots withNEFH (neurofilament heavy chain gene), LIF(leukaemia inhibitory factor gene), and NF2gene probes (Trueman et al, in preparation).

STATISTICAL ANALYSISAnalyses were undertaken using methodsappropriate to dependent data, accounting forwithin family correlation."6 For the comparisonof proportions, intracluster correlation coeffi-cients were estimated for each end point and anadjusted chi squared statistic calculated; p val-ues were calculated from tables. Age distribu-tions and mean numbers were compared usingWilcoxon rank sum test, with family specificmean ages as the unit of analysis. Two sided pvalues are reported.

ResultsA total of 54 causative mutations have beenidentified from the 125 classically affectedunrelated subjects (43%). These consist of 22nonsense mutations, 11 small frameshift dele-tions, one in frame deletion, three frameshiftinsertions, five missense mutations, six splicesite mutations, one 3' untranslated regionmutation, and five large deletions (table 2).Index (first affected) cases were unavailable foranalysis in 20 families. In the 46 families thathave had an affected second generation, 26mutations (57%) have been identified. Of the79 sporadically affected subjects, mutationshave only been identified in 28 (35%). Thisdifference reaches statistical significance(X2=4.16, df 1, p<0.05). Four of the truncatingmutations have been found to be in mosaicform, present in 10-38% oflymphocytes." Oneof these mutations (family 10) was transmittedin full form to an affected daughter, althoughthe son who had inherited the high riskchromosome 22 had not inherited themutation." Several of the mutations reportedhere have been previously published.". 17-21However, 30/60 are reported here for the firsttime. The five large deletions are the subject ofa further detailed report (Trueman et al, inpreparation).

Six causative mutations (35%) have beenidentified in the 17 cases fulfilling modifiedcriteria (two nonsense mutations, twoframeshift deletions, one frameshift insertion,and one missense mutation). Four of 13 caseswith unilateral VS + two other NF2 featureshad mutations, while 2/4 (50%) with multiplemeningiomas + two other NF2 features weremutation positive. No mutations were found insamples from 87 other patients with featuressuggestive of NF2, but not fulfilling modifiedcriteria. These varied from unilateral VS at a

451


http://jmg.bm

j.com/

J Med G


ownloaded from

http://jmg.bmj.com/

Evans, Trueman, Wallace, et al

Table 2 NF2 mutations identified in 125 classical and 17 atypical unrelated NF2families

No of affected Ages (y) at

Family Mutation Exon T Cases Generations Onset HL Diagnosis

Families fulfilling classical criteriaNonsense mutations1 G1 22>A, Trp4 1 >stop'2 C169>T, Arg57>stopl'6]3 C169>T, Arg57>stop'16'4 C169>T, Arg57>stop"`15 C331>T, GlnlI 1 >stop6 C586>T, Argl96>stop'717 C586>T, Argl96>stop8 C586>T, Argl96>stop9 C592>T, Argl98>stop10 C784>T, Arg262>stopt`'411 G949>T, Glu317>stop'12 C1021>T, Arg341>stop[6'13 C 1021 >T, Arg341 >stop14 C 1021 >T, Arg341 >stop15 C1021>T, Arg341>stop16 C1 357>T, Gln453>stop17 C1396>T, Arg466>stop1'7118 C1408>T, Gln470>stopl"7'19 C1606>T, Gln536>stop20 C1606>T, Gln536>stop21 C1612>T, Gln538>stop'71Frameshifting deletions22 39 delT'23 40 delCT24 40 delCTI 17125 523 delAA'26 571 delT'27 713 delC28 770 deIC'29 855 delTn30 953 delT'6IFrameshifting insertions31 183 insCAAA[16132 1206 insCn33 1333 insG'In frame deletion34 355 delTTC'7'Missense35 T185>C, Phe62>Ser[16]

36 A229>G, Met77>Valn

37 C1055>T, Thr352>Metl'6138 A1237>G, Lys4l3>Glu17139 T1604>C, Leu535>Pro'71

Splice site40 448-lagTA>aaTA

41 599-lAGgt>AGatl"7'

42 600-2agGG>ggGGI'7'

43 600-3cagGG>gagGG

44 1 123-2agAT>ggAT^'7'45 1447-lagCC>aaCCn3' untranslated region46 C>T at 1788n

Large deletions47 650kb (whole gene)"'I48 5kb (intron 1)

49 1OOkb

50 60kb

51 40kbMosaic casesNonsense52 C169>T, Arg57>stop""510§ C784>T, Arg262>stop '4]

2 S 2 22 S 2 22 S 2 22 S 1 13 S 1 16 I 1 16 S 2 26 I 1 16 S 1 18 S 1 110 I 1 111 S 1 111 S 1 111 M 1 111 S 1 113 S 1 113 S 1 113 S 1 115 M 1 115 S 2 215 S 1 1

1 S 1 11 M 1 11 M 1 16 I 2 26 S 1 18 S 2 28 S 1 19 I 2 310 S 2 2

2 S 2 212 S 1 112 S 1 1

3 I 1 1

2 M 3

2 I 3

11 S 212 I 115 M 5

5 I 3 4

6 M 2

7 I 2

7 M 3

12 M 114 M 3

M 1

M 2

M 5

M 3

I 2

S 1

6 M 18 I 2

22,11-,22-,2017172022,918131910131740141817275416,AS6

19302828,-21-,222024,2 1-'3

-,3 -,1119 1918 22

18 18

4 4040,43AS25t

4 -1824,21

2 17,141 1710 ,

61,4352,30AS30AS 12,AS I6t

2614,21AS625AS16t,AS 13

20,19562,303547

35 35

3 2323,AS

10 _,_

47,2927,2324,28AS,AS

5 -

29,3325,AS

3 -

19,251 2

41 4123 27

22,15-,22-,2218172022,918161910161740141935275416,AS17

19302828,-21-,222424,2 1-,3

29,11-,23-,2617182826,921162031161840162030325422,2*23

20303232,823-,232426,27-,4

-,52022

21

6643,4323*-4824,2125,1418

69,5872,33301 1*,15*

2620,244*2614*,10*

21,195620,303961

38

3822,12

66,5132,2324,2813*,9*

31,3325,6*

20,3015

4040,43

-,1824,2117,1417

61,5752,30AS30

14,24

26

20,1956-,303547

3524,AS16

47,3827,2324,28AS16t,AS12

31,3325,AS6

19,2813

4947

452


http://jmg.bm

j.com/

J Med G


ownloaded from

http://jmg.bmj.com/


Table 2 Continued

No of affected Ages (y) at

Family Mutation Exon T Cases Generations Onset HL Diagnosis

Frameshift deletions53 896 delTATGI"I 10 M 1 1 21 21 2354 1632 delAG&"I 15 M 1 1 28 28 34Families ftdfilUing modified criteriaNonsense55 G100>T, Glu34>stop' 1 S 1 1 18 AS 1956 C586>T, Argl96>stop 6 I 1 1 18 - 22

Frameshifting deletion57 40 delCT 1 M 1 1 28 28 3258 887 delT' 10 S 1 1 3 - 4

Frameshifting insertion59 1191 insT' 12 I 1 1 20 20 24

Missense60 A317>G, Glu106>Gly"161 3 M 1 1 20 20 39

T, type of NF2; HL, hearing loss; AS, asymptomatic.*DNA diagnosis alone. tDNA diagnosis later found to have VS on scan. tDNA diagnosis full neuroaxis MRI imaging normal.§Family with mosaic affected mother and daughter with germline mutation 'Novel mutation.Ages at onset for the various variables are set out in generation order. Where possible these are shown vertically generation by gen-eration, but for simplicity some generations are shown on the same level. Missing values are shown by -, where information on theperson is not considered secure or they died before the study.

young age to patients with meningiomatosis or

schwannomatosis with some subjects nearlymeeting modified criteria.The clinical data are summarised in table 3.

Forty-two cases from 38 families with truncat-ing mutations (nonsense, small frameshiftinginsertions, and deletions, excluding mosaics)had an average age at onset of symptoms of 19years and diagnosis at 22.4 years (mosaic cases

were excluded). Fifty-one cases from 17 fami-lies with splice site mutations (15 from six),missense mutations (18 from six), and largedeletions (18 from five) had a later average ageof onset of 27.8 years (p<0.01) and at diagno-sis of 33.4 years (p<0.05). Subjects with trun-cating mutations were significantly more likelyto have symptoms before 20 years of age(p<0.001) and develop at least two sympto-matic CNS tumours in addition to vestibularschwannoma before 30 years (p<0.001). Sig-nificant results were also obtained for indi-vidual differences between missense, splicesite, and large deletion families and truncatingmutations for all these parameters (p<0.0 125),except for causing symptoms <20 years(p=0.02(missense)-0.06(splice site)). How-ever, four asymptomatic splice site mutationcarriers were excluded as they had not reached20 years of age. There were also significantlyfewer multigenerational families with truncat-ing mutations. Only 9/38 non-mosaic familieshad resulted in a second affected generation

compared to 13/17 of the other mutationaltypes. The four further truncating mutations inmosaic form were associated with milderdisease than other similar mutations. As onlyone case from one family had an in frame dele-tion, this was excluded from the analysis.Nine affected subjects from families with

nonsense and frameshifting mutations havedied. Six died before the study at 37,39,34,35,46, and 30 years of age. Three have died sincethe start of the study at 23, 32, and 42 years ofage. Five subjects with splice site mutationshave died at 20, 26, 29, 34, and 46 years of ageand four with missense mutations have died at45, 64, 72, and 78 years of age. The latter threecases died in old age, deaf but with no diagno-sis of NF2 or eighth nerve tumours duringlife.22 Five subjects with large deletions have

died at 34,37,34,36, and 52 years of age. Twowith truncating mutations have been diagnosedpresymptomatically, one on DNA testing at 2years of age and the other with bilateralvestibular schwannomas on scan at 9 years ofage. Five subjects with splice site mutationswere diagnosed without symptoms at 6, 9, 10,and 14 years of age on DNA testing and one onscan at 19 years of age. The DNA testedsubjects remain CNS tumour free one to twoyears later. Four with missense mutations havebeen diagnosed on DNA testing alone aged 10,

13, 23, and 30 years of age. Two of these havehad subsequent negative complete neuroaxis

Table 3 Analysis of clinicalfindings in NF2 patients with frameshift versus other mutation types

Nonsense or Splice site Missense LargeFeature frameshift mutations (M) mutations mutation deletions p value*

Families 38 4 6 6 5Patients 42 4 15 18 18Current mean age (y) 24 41 29 39 28Mean age at onset (y) 19 28 26 32 25 0.0072Mean age at hearing loss 23 29 30 36.5 28 NS% presenting before 20 years 68 0 36 27 21 <0.001Mean age at diagnosis 22.5 37 31 40 30 0.0313% with >1 non-VS symptomatic tumour <30 years 67 0 11 10 14 <0.001% cataract 60 25 45 42 27 NS% with meningioma (and mean No) 68 (2) 25 (1) 21 (0.7) 28 (0.5) 17 (0.3) 0.0043% with spinal tumours (mean) 70 (3.3) 50 (2) 23 (0.3) 28 (0.5) 28 (0.4) 0.0026% with skin tumours (mean) 90 (5.8) 0 30 (0.5) 20 (2) 47 (1.2) 0.0045

(M)=mosaic.*Truncating versus non-truncating mutations allowing for intrafamilial variation.

453


http://jmg.bm

j.com/

J Med G


ownloaded from

http://jmg.bmj.com/

Evans, Trueman, Wallace, et al

MRI at 16 and 25 years of age, although MRIin a third showed a small unilateral vestibularschwannoma at 30 years of age. Seven subjectswith large deletions were diagnosed presymp-

tomatically, four on MRI scans at 12, 22, 25,and 29 years of age and three on DNA alone at

6, 9, and 13 years of age. The 13 year old hashad a cranial MRI at 16 years which showedbilateral <1 cm asymptomatic vestibularschwannomas. Thus, only 2/18 subjects diag-nosed presymptomatically had nonsense or

frameshifting mutations even though these are

the most common abnormality.Mean age at onset of symptoms, hearing

loss, and diagnosis in the 104 classicallyaffected NF2 cases with no identified mutationwas 23.4, 25.5, and 28 years respectively. Thisis not fundamentally different from the non-

truncating mutations, but significantly laterthan for frameshift and nonsense mutations.

DiscussionSince the first reports of germline mutations inNF2, there have been attempts to establish a

link between the specific NF2 mutation andthe disease phenotype.7 1 1 22 A generalimpression was that protein truncating muta-

tions produced a more severe phenotype whilecertain splice site and missense mutations were

associated with mild disease.7 18 19 22 Two recentpapers have confirmed this impression on

larger surveys of mutation proven subjects.8 9

However, clear statistical evidence for this hasonly been provided for data on age at onset,diagnosis, and hearing loss between nonsense

and frameshifting mutations and splice sitemutations.8 The present study confirms the age

related differences between splice site andtruncating mutations as well as providingstatistical evidence for the first time for a

milder disease course in families with missensemutations and large deletions. While some

splice site mutations could lead to the produc-tion of a truncated protein by allowing readthrough of intronic sequence and introductionof a premature stop codon, and nonsense

mutations could be ameliorated by skipping ofthe affected exon, we believe the subdivision isin general accurate. The weighted analysisallowing for intrafamilial correlation also pro-vided significant results for presence andnumber of meningiomas, spinal tumours, andskin tumours in contrast to the previousreport.8 Although statistical evidence for a dif-ference between 28 cases with protein truncat-ing mutations and 16 cases from three familieswith single amino acid changes was found inone study,9 this was based on classification intodisease type alone. No specific data on age andtumour differences were given. Theclassification of subjects into mild and severe

phenotypes is somewhat arbitrary and in manycases not possible, thus the further breakdowninto intermediate or moderate cases.9 Nonethe-less the data from the three families with singleamino acid changes appears convincing withmany asymptomatic mutation carriers, includ-ing one with negative full neuroaxis imaging intheir thirties.9 This is comparable with our own

findings of MRI negative missense mutation

carriers. The large deletions are likely to lead tocomplete absence of protein product as theyaffect the 5' end of the gene. We have previouslyreported 1/5 of these as being mildly affected2'and the original report on cloning the NF2gene relied on a 40 kb deletion in a mildlyaffected family.6 8 This latter deletion causes anin frame deletion of two exons and couldtherefore produce a stable protein product. It ispossible to hypothesise that mutations/deletions which give rise to a near normal pro-tein product or no protein product areassociated with mild disease, whereas thosethat cause a truncated protein cause severe dis-ease. This hypothesis has previously been putforward for familial adenomatous polyposis.23Whether abnormal binding of the wild typeprotein by a truncated protein could cause asimilar pattern in NF2 remains to be proven.

In addition to the data on age at onset thereappears to be a clear effect on genetic fitness;only 9/38 families with nonsense or frameshift-ing mutations had resulted in a second affectedgeneration, whereas 13/17 of the other muta-tional types resulted in multigenerational fami-lies. This difference was not seen in a report of21 mutations,8 but 8/8 splice site or missensemutations where family history was knownwere multigenerational, compared to only 6/22truncating mutations in another report.9 Thisdifference was not specifically referred to in thereport although comments were made aboutthe small number of subjects from families withtruncating mutations who were diagnosedpresymptomatically.9 This is very similar to ourown findings where only 2/18 presymptomaticdiagnoses were made in the truncating muta-tion families. This could of course be partlyexplained by the smaller family sizes, butearlier age of onset would be another majorcontributing factor.This report documents the NF2 phenotype

in 60 unrelated, mutation proven familiescompared to a combined 57 from the two pre-vious largest reports.8 9 One report did not giveclinical data on six further families.9 However,the mutation detection rate in the presentreport is low (43%) compared to 66% and 56%in the two previous large studies,8 9 althoughbetter than in two others.7 18 It is not clear whythis should be the case as similar techniqueswere used. However, the present study has ahigh number of sporadic cases which couldrepresent low level mosaicism."3 Detectionrates in our familial cases (56%) were compa-rable to the other studies.8 9 We have previouslyreported the four cases of provenmosaicism13 178 and it does appear that theseare associated with a milder phenotype. It isalso possible that the failure to detect muta-tions in more mildly affected cases in aprevious study8 could have been partly ex-plained by mosaicism. There is currently noevidence to suggest that there are NF2 casescaused by mutations at another locus and allour families with no proven mutation are con-sistent with linkage to NF2.A similar proportion of sporadic patients

(35%) fulfilling modified NF2 criteria com-pared to sporadic classical disease were found

454


http://jmg.bm

j.com/

J Med G


ownloaded from

http://jmg.bmj.com/


to have mutations. Although the numbers weresmall this would suggest that modified criteriaare not overinclusive. The presence of a unilat-eral VS would make schwannomatosis notcaused by NF2 very unlikely24 and the presenceof at least two NF2 manifestations in patientswith multiple meningiomas would also dis-count overlap with meningiomatosis. However,it is likely that these criteria will identify anumber of mosaic cases, as has been predictedfor classical sporadic disease."A number of mutations are reported here for

the first time. Fifteen are novel, but we havefound three further mutations each of theC586>T and C1021>T nonsense mutationsmaking these "warm" spots in the NF2 gene.The C1021>T mutation has been reported inat least 10 further families7'9 making it the mostcommon NF2 mutation. We have also con-firmed the high frequency of C>T transitionsat CGA codons." These occurred in 15/125(12%) classically affected cases, with otherC>T transitions occurring in a further 4% (5).There is no evidence that these are foundermutations, with most cases being de novo. Allthe other nonsense and frameshifting muta-tions would be predicted to give rise to a trun-cated protein product and it is of note that ourmost 3' truncating mutation in exon 15 givesrise to classical disease. There is no realevidence to suggest that the more 3' mutationsproduce a milder form of NF2. The only newmissense mutation reported here (A229>G)segregates with disease and has not been foundin any normal samples tested or reported as apolymorphism elsewhere. The same is true forthe exon 5 and 7 splice site mutations reportedhere for the first time. There was one mutationidentified in the 3' untranslated region. Theeffect of this mutation is unknown. In contrastto a previously reported mutation in thisregion,9 the phenotype was mild. The largedeletions will be reported in detail elsewhere(Trueman et al, in preparation).This report has confirmed the previously

described genotype/phenotype correlations inNF2. In addition these have been extended toinclude families with large deletions which, ingeneral, have a milder disease than truncatingmutations. However, caution must be exercisedin giving advice to families based on the identi-fication of a truncating or non-truncatingmutation in a single person. Even in establishedfamilies there is still significant intrafamilialvariability in disease course. Indeed mono-zygotic twins with NF2 can have somevariation in tumour burden.26 Nonetheless theidentification of missense mutations, splice sitemutations, and large deletions in some familiesis clearly consistent with a milder phenotypeand could lead to different managementdecisions.27

We would like to thank all the clinicians who have informed usof their families with NF2, especially Dr S Huson, Mr T King,Professor R Ramsden, and Mr R Lye. Previous laboratory workhas been carried out by Dr D Bourn, S Mason, and S Tekes.

This work has been supported by grants from the MedicalResearch Council, Cancer Research Campaign, and theNational Neurofibromatosis Association.

1 Kanter WR, Eldridge R, Fabricant R, Allen JC, Koerber T.Central neurofibromatosis with bilateral acoustic neuroma.Genetic, clinical and biochemical distinctions from periph-eral neurofibromatosis. Neurology 1980;30:851-9.

2 Evans DGR, Huson SM, Donnai D, et al. A clinical study oftype 2 neurofibromatosis. QJfMed 1992;84:603-18.

3 Parry DM, Eldridge R, Kaiser-Kupfer MI, Bouzas EA,Pikus A, Patronas N. Neurofibromatosis 2 (NF2): clinicalcharacteristics of 63 affected individuals and clinicalevidence for heterogeneity. Am J Med Genet 1994;52:450-1.

4 National Institutes of Health Consensus DevelopmentConference Statement on Neurofibromatosis.Neurofibromatosis Res Newsl 1987;3:3-6.

5 Rouleau GA, Merel P, Lutchman M, et al. Alteration in anew gene encoding a putative membrane-organizingprotein causes neurofibromatosis type 2. Nature 1993;363:515-21.

6 Troffater JA, MacCollin MM, Rutter JL, et al. A novelmoesin-, ezrin-, radixin-like gene is a candidate for theneurofibromatosis 2 tumor suppressor. Cell 1993;72:791-800.

7 Merel P, Hoang-Xuan K, Sansom M, et al. Screening forgerm-line mutations in the NF2 gene. Genes Chrom Cancer1995;12:117-27.

8 Parry DM, MacCollin M, Kaiser-Kupfer MI, et al.Germ-line mutations in the neurofibromatosis 2 gene: cor-relations with disease severity and retinal abnormalities.AmJ7Hum Genet 1996;59:529-39.

9 Ruttledge MH, Andermann AA, Phelan CM, et al. Type ofmutation in the neurofibromatosis type 2 gene (NF2)frequently determines severity of disease. Am J Hum Genet1996;59:331-42.

10 Kluwe L, Bayer S, Baser ME, et al. Identification of NF2germline mutations and comparison with neurofibromato-sis 2 phenotypes. Hum Genet 1996;98:534-8.

11 Mautner VF, Lindenau M, Baser ME, et al. The neuroimag-ing and clinical spectrum of neurofibromatosis 2. Neurosur-gery 1996;38:880-5.

12 Jacoby LB, MacCollin MM, Louis DN, et al. Exon scanningfor mutation of the NF2 gene in schwannomas. Hum MolGenet 1994;3:413-19.

13 Evans DGR, Trueman L, Wallace A, Strachan T. Mosaicismin classical NF2: a common mechanism for sporadicdisease in tumour prone syndromes? Am J7 Hum Genet(submitted).

14 Bourn D, Strachan T. Highly polymorphic dinucleotiderepeat at the NF2 gene. Hum Genet 1995;95:712.

15 Evans DGR, Mason S, Huson SM, Ponder M, Harding AK,Strachan T. Spinal and cutaneous schwannomatosis is avariant form of type 2 neurofibromatosis: a clinical andmolecular study. Jf Neurol Neurosurg Psychiatry 1997;62:361-6.

16 Donner A, Klar N. Methods for comparing event rates inintervention studies when the unit of allocation is a cluster.Am J Epidemiol 1994;140:279-89.

17 Biljsma EK, Wallace AJ, Evans DGR. Misleading linkageresults in an NF2 presymptomatic test owing to mosaicism.JMed Genet 1997;34:934-6.

18 Bourn D, Carter SA, Evans DGR, Goodship J, Coakham H,Strachan T. A mutation in the neurofibromatosis type 2tumor-suppressor gene, giving rise to widely different clini-cal phenotypes in two unrelated individuals. Am Jf HumGenet 1994;55:69-73.

19 Bourn D, Carter SA, Mason S, Evans DGR, Strachan T.Germline mutations in the neurofibromatosis type 2tumour suppressor gene. Hum Mol Genet 1994;3:813-16.

20 Bourn D, Evans G, Mason S, Tekes S, Trueman L, StrachanT. Eleven novel mutations in the NF2 tumour suppressorgene. Hum Genet 1995;95:572-4.

21 Watson C, Gaunt L, Evans DGR, Harris R, Strachan T. Adisease associated germline deletion maps the type 2neurofibromatosis (NF2) gene between the Ewing sarcomaregion and the leukaemia inhibitory factor (LIF) locus.Hum Mol Genet 1993;2:701-4.

22 Evans DGR, Bourn D, Wallace A, Ramsden RT, MitchellJD, Strachan T. Diagnostic issues in a family with late onsettype 2 neurofibromatosis. 7Med Genet 1995;32:470-4.

23 Su LK, Johnson KA, Smith KJ, et al. Association betweenwild-type and mutant APC gene products. Cancer Res1993;53:2728-31.

24 MacCollin M, Woodfin W, Kronn D, Short MP.Schwannomatosis: a clinical and pathological study.Neurology 1996;46:1072-9.

25 Sainz S, Figuero K, Baser ME, Mautner VF, Pulst S. Highfrequency of nonsense mutations in the NF2 gene causedby C to T transitions in five CGA codons. Hum Mol Genet1995;4: 137-9.

26 Baser ME, Ragge NK, Riccardi V,Janus T, Gantz B, Pulst S.Phenotypic variability in monozygotic twins with neurofi-bromatosis 2. AmJ Med Genet 1995;57:A54.

27 Evans DGR, Ramsden R, Huson SM, et al. Type 2neurofibromatosis: the need for supraregional care. JLaryngolOtol 1993;107:401-6.

455


http://jmg.bm

j.com/

J Med G


ownloaded from

http://jmg.bmj.com/

Documents

Genotype/phenotype correlations 2 neurofibromatosis (NF2