Molecular diagnosis of neurofibromatosis type 1: 2 years experience

Abstract Our experience of providing an NF1 gene

diagnostic mutation detection service as part of the UK

Genetic Testing Network (UKGTN) is presented. A

total of 169 unrelated individuals suspected of having

neurofibromatosis type I (NF1) were referred for NF1

diagnostic testing over a 2 year period. Mutation

analysis of the entire NF1 coding region and the

flanking splice sites was carried out, and included the

use of a combination of FISH, dHPLC and MLPA.

Possible disease causing mutations were identified in

109 (64%) cases. These comprised 88 different se-

quence alterations, of which 57 were novel. Out of the

169 cases referred, there were 102 patients with reliable

clinical data, of whom 78 satisfied the NIH diagnostic

criteria for NF1. Within this better defined cohort of

NF1 patients, NF1 mutations were identified in 61

individuals (78%), showing the importance of clinical

selection on overall test sensitivity, and highlighting

the problem of full clinical data collection in the audit

of routine services. As mutation detection technologies

advance, facilitating direct sequencing of all coding and

flanking non-coding regions of the NF1 gene, the

development of an even more cost-effective, quick and

sensitive diagnostic test for future testing of NF1 is

discussed.

Keywords Denaturing High Performance Liquid

Chromatography (dHPLC) Æ Fluorescence In-Situ

Hybridisation (FISH) Æ Multiplex Ligation-Dependent

Probe Amplification (MLPA) Æ Mutation ÆNeurofibromatosis Type 1 (NF1) Æ Polymerase chain

reaction (PCR) Æ Polymorphism Æ Sequencing ÆUK Genetic Testing Network (UKGTN)

Introduction

Neurofibromatosis type 1 (NF1) is a common familial

syndrome that afflicts all ethnic groups, equally affects

males and females, and all age groups. The disease is

transmitted in an autosomal dominant fashion, with a

prevalence of approximately one in 3,500 individuals

worldwide.

The NF1 gene is one of the largest human genes and

spans ~350 kb of 17q11.2, contains 60 exons, of which 3

are alternatively spliced and encodes a 12 kb mRNA

transcript. Intron 27 b of the NF1 gene contains three

embedded genes (OMGP, EVI2B and EVI2A), which

are all transcribed in the opposite orientation to the

NF1 gene. Neurofibromin, the NF1 gene protein

product, is ubiquitously expressed and exhibits struc-

tural and sequence similarity to an evolutionarily

conserved family of proteins, the mammalian GTPase-

activating proteins (GAP). The most highly conserved

region of neurofibromin is the GAP-related domain

(GRD) encoded by exons 21–27a [1, 2].

The germline mutation rate for the NF1 gene is

some 10-fold higher than that observed in most other

comparable inherited disease genes, and de novo

mutations are present in almost half of all index NF1

cases [3]. Approximately 800 different NF1 mutations

are reported to-date (http://www.hgmd.org), but there

is no evidence of localised clustering. Examination of

these germline mutations demonstrates that some 80%

S. Griffiths Æ P. Thompson Æ I. Frayling ÆM. Upadhyaya (&)Institute of Medical Genetics, University Hospital of Wales,Cardiff CF14 4XN, UKe-mail: [email protected]

Familial Cancer (2007) 6:21–34

DOI 10.1007/s10689-006-9001-3

123

ORIGINAL PAPER

Molecular diagnosis of neurofibromatosis type 1: 2 yearsexperience

Sian Griffiths Æ Peter Thompson Æ Ian Frayling ÆMeena Upadhyaya

Received: 23 January 2006 / Accepted: 11 July 2006 / Published online: 31 August 2006� Springer Science+Business Media B.V. 2006

are predicted to be truncating mutations, with about

30% of these expected to result in RNA splicing

abnormalities [4]. Efforts to identify and characterise

all NF1 gene mutations continue to present a consid-

erable research and diagnostic challenge due to a

combination of the large gene size, the absence of any

localised mutation clustering, little evidence of repeat

mutation and the wide diversity of mutation types

observed. Furthermore, the presence of a number of

highly homologous partial NF1 pseudogene-like se-

quences located throughout the human genome has

increased the complexity of PCR-based mutation

analysis [5–7]. Indeed, most diagnostic laboratories

were until recently only able to offer molecular testing

in families using linked markers, a strategy that is

obviously not possible in de novo cases which con-

tribute almost half of all new cases [8, 9].

NF1 is characterised by the presence of multiple

cafe-au-lait spots (CAL), benign neurofibromas, and

Lisch nodules in the eye [3]. The often extreme vari-

ability in the presentation of extent and severity of NF1

clinical signs and symptoms, even within the same

family, affected by the same mutation has meant that

establishing definitive clinical diagnostic criteria has

often been problematic. Recognition of this issue led

the National Institute of Health (NIH) in 1988 to issue

a ‘‘Consensus Statement’’ which defined the standard

diagnostic criteria for NF1 to help clinicians to be able

to distinguish it from other related disorders [10].

A number of clinical complications may also occur

in patients and these include abnormalities of the car-

diovascular, gastrointestinal, renal and endocrine sys-

tems; the presence of major orthopedic problems;

facial and body disfigurement; the presence of cogni-

tive deficit and the development of malignancy. There

is an increased risk of development of malignant tu-

mours of the peripheral nerve sheath and the central

nervous systems (CNS). About a quarter of NF1 pa-

tients may eventually develop one or more of these

various clinical complications, underlining the signifi-

cant morbidity and increased mortality associated with

this disorder. It is noteworthy that while the majority of

NF1 patients exhibit only mild symptoms, the lifetime

risk of developing a MPNST in NF1 patients is about

8–13% [11].

While the initial diagnosis is almost always made on

a clinical basis, additional specialist examination is

often needed for those NF1 children who present with

no major disease complications, and who often repre-

sent the first case in the family. It is such families who

may be especially aided by the identification of a

specific NF1 gene mutation. Early diagnosis of the

disease is considered essential so that such patients and

their families can be offered appropriate counselling,

and the affected children can be regularly monitored

for complications, such as learning difficulties, optic

glioma and hypertension.

Genetic counselling in NF1 is greatly complicated by

the marked intra- and inter-familial variation in NF1

disease expression [12]. The combination of this clini-

cal variability and the extensive mutational heteroge-

neity in NF1, necessitating labour-intensive mutation

screening, has meant that attempts to produce geno-

type–phenotype correlations for this disorder are still

very much in their infancy [13, 14].

Another consequence of the variability of clinical

expression in NF1 is that disease progression and

severity cannot be readily predicted. As a result, there

has been little demand to date for prenatal diagnosis,

possibly because most couples would want to know the

clinical severity of the disease in their baby, which

currently cannot be predicted [8]. Despite this lack of

demand, there is still an urgent need for a cost-effective,

rapid and accurate DNA-based test for NF1, and

development of a suitable preimplantation test for NF1

has been reported [15, 16].

Here we describe our experiences from 2 years of

offering an NF1 molecular diagnostic service. To date,

we have analysed the entire NF1 coding region and

the flanking splice site in 169 unrelated individuals,

suspected of having NF1. Possible disease-causing

mutations have been identified in 109 of them.

Materials and methods

Patient samples

Over a 2 year period from April 2003 to June 2005, 169

unrelated patients were referred for NF1 mutation

analysis from medical genetics centres throughout the

UK. Patients were referred for three main reasons: (1)

confirmation of a clinical diagnosis of NF1, in partic-

ular for those individuals who do not fully meet the

diagnostic criteria for NF1, or for children in whom a

precise clinical diagnosis is often difficult (115 indi-

viduals), (2) For NF1 families requesting or consider-

ing prenatal diagnosis in the near future (19

individuals), and (3) A cohort of 32 patients who all

display a severe clinical phenotype, suggesting the

presence of a large NF1 gene deletion. For the

remaining three patients, the reason for referral was

not mentioned. Full clinical details were available on

102 of these patients.

123

22 Familial Cancer (2007) 6:21–34

DNA samples

Genomic DNA was isolated from peripheral blood

cells by our previously published method [17], an

aliquot of each DNA sample being taken and stored

for later confirmation of any identified mutation.

Fluorescent in-situ hybridisation (FISH)

FISH analysis was performed using probes P1-9 and

P1-12 as previously described [17, 18].

Point mutation detection

PCR reactions

PCR reactions were set up using a Biomek FX

automated robotic platform (Beckman Coulter) in a

final volume of 25 ll, using the buffer provided with

the enzyme and containing 25 ng of genomic DNA,

10 pmol of each primer, 10 mM dNTPs, and 1U Taq

DNA polymerase or Hotstar Taq DNA polymerase

(Qiagen). The amplification cycles for reactions using

Taq DNA polymerase consisted of an initial dena-

turation at 94�C for 5 minutes, followed by 35–40

cycles of 94�C for 30 s, Ta �C for 30 s and 72�C for

30 s. Each reaction was terminated with a final

extension step at 72�C for 10 min. For those PCR

reactions using the Hotstar Taq DNA polymerase the

initial denaturation at 94�C was for 15 min followed

by 35 cycles at 94�C for 1 min, Ta �C for 1 min and

72�C for 1 min completed with a final extension of

72�C for 10 min.

Oligonucleotide PCR primers

PCR amplification of the 57 coding exons of the NF1

gene was carried out. The three alternatively spliced

exons (9b, 23a and 48a) were not analysed as no

mutations have yet been identified in these exons

despite NF1 mutational screen of over 1000 unrelated

patients [1, 19–21]. The oligonucleotide primers are

based on those previously described [22], with twenty

nine pairs being re-designed using Primer3-primer de-

sign software [http://frodo.wi.mit.edu/cgi-bin/primer3/

primer3_www.cgi] either to optimise PCR amplifica-

tion, or to extend the sequence analysed around indi-

vidual splice sites (primer sequences available on

request). For exon 19b, nested PCR primers were

designed in order to sequence the fragment. All

oligonucleotides were synthesised by MWG Biotech,

Milton Keynes UK.

Denaturing HPLC analysis

Denaturing high performance liquid chromatography

(dHPLC) was performed on a WAVE DNA fragment

analysis system (Transgenomic, Crewe, UK) using a

DNAsep column. Full diagnostic details are available

from the authors, but in brief, heteroduplex formation

was enhanced by subjecting the untreated PCR

product to denaturation at 95�C for 5 min and then

allowing the reaction mix to gradually reanneal over

30 min by decreasing the temperature to 50�C. PCR

products were analysed for heteroduplexes by sub-

jecting 5 ll of PCR product to a 2% linear acetoni-

trile gradient at a rate of 0.9 ml/min. The starting

concentrations of buffer B were selected by the

WaveMaker software version 4.1. The temperatures

for optimal heteroduplex separation were determined

from the melting profiles produced for each fragment

using the WAVEmaker software. Depending on the

melting domains predicted by the software, the use of

one, two or three different melting temperatures (Tm)

may be required to allow individual PCR fragments

to be successfully analysed.

Sequencing

PCR products were purified using the Montage PCR96

Plates (Millipore), according to the manufacturer’s

protocol, with 5 ll of each purified PCR product being

sequenced in both orientations using ABI Prism

BigDye terminator cycle sequencing kit version 3.1

(Applied Biosystems). Sequencing reactions were also

purified using the Montage SEQ96 Plates (Millipore),

with the addition of 10 ll of Hi-DI formamide and

analysis on an ABI Prism 3100 genetic analyzer.

Sequence analysis was performed to characterise each

identified aberrant fragment, with each of the muta-

tions identified confirmed independently by two expe-

rienced molecular genetic scientists.

Analysis for either complete gene or multi-exon

deletions

DNA samples from all patients in whom neither a

sequence variation or a mutation could be identified

were genotyped using a panel of five highly polymor-

phic extragenic and intragenic markers (from 5¢ to 3¢:pHHH202/RsaI [23], intron 12b (unpublished),

IVS27CA 28.4 [23], IVS38GT53 [23] and 3¢NF1-1 [24]).

The polymorphic markers were analysed either by

restriction fragment length polymorphism (RFLP)

analysis or microsatellite analysis using the ABI Prism

123

Familial Cancer (2007) 6:21–34 23

3100 Genetic Analyzer with ROX 500 (Applied Bio-

systems) as an internal size standard.

Multiplex Ligation Probe Amplification (MLPA)

With the availability of the NF1 MLPA commercial

probe sets, samples in which dHPLC failed to identify

a point mutation were analysed for the presence of

complete or partial NF1 gene deletions. Approxi-

mately 250 ng of DNA was used for MLPA analysis

using the SALSA P081 and P082 NF1 MLPA kits from

MRC-Holland (Amsterdam, NL), these together con-

tain probes for 51 of the 57 NF1 coding exons, as well

as a probe for a sequence located 17 kb downstream of

the NF1 gene and two probes specific for the OMG

gene located in intron 27. Information regarding the

probe sequences and ligation sites may be obtained at

www.mlpa.com. Hybridization, ligation, and amplifi-

cation of the MLPA probes was carried out as

described [25].

Results

Of the 169 patient samples referred to us for NF1

diagnostic analysis over a two year period, mutations

were identified in 109 (64%) cases (Table 1). Of the

46 sporadic NF1 patients, clinical details were avail-

able from 41, of which, 35 individuals met the diag-

nostic criteria of NF1. Mutations which could be

confidently assessed as pathogenic (large exonic

deletions, nonsenses, frameshifts and splice-site

mutations) were identified in 26/35 (74%) of these. In

the remaining six sporadic cases with clinical infor-

mation, but who did not fulfil the diagnostic criteria

for NF1, only one disease causing mutation was

identified (Table 1 2340: Ex17 c.2970_2972delATT).

Thirty three cases are known to be familial and clin-

ical details were available from 26 of these, of which,

21 individuals met the diagnostic criteria for NF1.

Disease-causing mutations were identified in 17/21

(81%). In the remaining 5 cases who did not fulfil the

diagnostic criteria, only one possible disease causing

mutation was identified (Table 1 2404: Ex21 3560

T>C L1187P).

Mutation spectrum

We have identified NF1 mutations in 109 individuals,

comprising 88 different mutations. 57 mutations are

novel (Table 1).

Twelve of the 99 mutations identified by dHPLC were

present in more than one individual:c.1383C>T (R461X)

found in three unrelated patients, whilst another eleven

mutations (c. 541C>T Q181X, c.495_498delTGTT,

c.1721+3A>G, c.2041C>T R681X, c.2540T>C L847P,

c. 5791T>C W1931R, c.2970_2972delAAT (delMet990),

c.6792C>G (Y2264X), c.3113+1G>T, c.3916C>T

(R1306X) and c.6789_6792delTTAC) being identified in

at least two unrelated patients. These may represent

recurrent de novo mutations at ‘hot spots’, or common

alleles due to some founder effect, or a combination of

both factors. We have not carried out haplotype analysis

to help establish this.

Thirty of the 109 (27.5%) possibly pathogenic se-

quence changes identified are predicted to cause

frameshift mutations, including 22 deletions, 7 inser-

tions and 1 insertion/deletion (indel). Two of these

were found in more than two unrelated individuals. Of

these, five mutations have been previously published.

The largest deletion identified by dHPLC was a 22 bp

deletion in exon 16, although the majority of the

frameshift mutations were due to single base pair

deletions or insertions. The single indel was identified

in exon 34. Two unrelated patients had an identical

in-frame deletion in exon 17 (c.2970_2972delAAT).

Nonsense mutations were identified in 30/109

(27.5%) patients and of these 20 have previously been

reported. Thus, 81 (74%) of the 109 mutations identi-

fied were predicted to result in the formation of a

premature termination codon and can be considered

highly likely to be pathogenic.

Mutations predicted to cause missense changes in the

NF1 protein accounted for 16 (15%) of the sequence

variants observed, of which ten have not been previously

described: c.667T>C (W223R), c.1658A>C (H553P),

c.1658A>G (H553R), c.2339C>G (T780R), c.2352G>T

(W784C), c.3447G>A (M1149I), c.3560T>C (L1187P),

c.3604 G>T (A1202S) c.5426G>T (R1809L) and

c.5435T>C (L1812P). Each of these sequence variants

observed is at a highly conserved amino-acid residue and

has not been observed in more than 200 normal chro-

mosomes studied. Whenever possible, parental DNA

was also obtained in order to help determine the po-

tential pathogenicity of such mutations. Two patients

could be identified as having definite de novo mutations,

these include the missense c.5793G>A (W1931R) and

c.3447G>A (M1149I) alterations. In neither case was the

sequence change identifiable in DNA from the parent,

thus implying probable pathogenicity. In another

patient with a family history of NF1, the missense

mutation c.1658A>G (H553P) identified in exon 11

segregated with 2 affected members of the family

123


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c.2

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[19]

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123


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cesi

te2

7[1

8]

23

40

E1

7N

c.2

97

0_

29

72

de

lAA

TIn

fra

me

de

leti

on

11

[19

]2

39

7E

17

Yc.

29

70

_2

97

2d

elA

AT

Infr

am

ed

ele

tio

n8

[19

]1

11

2I

18

Yc.

31

13

+1

G>

TS

pli

cesi

te9

[21

]2

36

2I

18

N/A

c.3

11

3+

1G

>T

Sp

lice

site

37

[21

]2

36

7E

19

bY

c.3

20

3_

32

04

insT

Fra

me

shif

t2

7N

ov

el

24

63

E1

9b

Yc.

32

10

de

lGF

ram

esh

ift

?N

ov

el

23

56

E1

9b

Yc.

32

41

_3

24

9d

elC

TG

GT

CT

CF

ram

esh

ift

2N

ov

el

23

12

E1

9b

N/A

c.3

24

6_

32

47

insT

Fra

me

shif

t3

2N

ov

el

24

47

E2

0Y

c.3

36

7G

>T

Glu

11

23

XN

on

sen

seG

AA

>T

AA

3[2

0]

21

98

E2

0N

/Ac.

34

47

G>

AM

et1

14

9Il

eM

isse

nse

AT

G>

AT

A2

6N

ov

el

23

99

I2

1Y

c.3

70

9-1

G>

CS

pli

cesi

te3

0N

ov

el

24

04

E2

1N

c.3

56

0T

>C

Le

u1

18

7P

roM

isse

nse

CT

T>

CC

T1

2N

ov

el

22

77

E2

2Y

c.3

72

1_

37

22

insA

Fra

me

shif

t2

No

ve

l2

35

9E

22

N/A

c.3

82

2_

38

23

de

lCT

Fra

me

shif

t1

7[1

9]

22

29

E2

2Y

c.3

82

6C

>T

Arg

12

76

XN

on

sen

seC

GA

>T

GA

20

[29

]2

35

5I

23

.1Y

c.3

97

5-2

A>

GS

pli

cesi

te1

7[1

9]

23

90

E2

3.1

N/A

c.3

91

6C

>T

Arg

13

06

XN

on

sen

seC

GA

>T

GA

2[3

0]

15

63

E2

3.1

Yc.

39

16

C>

TA

rg1

30

6X

No

nse

nse

CG

A>

TG

A3

5[3

0]

23

29

E2

5Y

c.4

30

8d

elA

Fra

me

shif

t2

6N

ov

el

21

33

E2

7a

N/A

c.4

54

9A

>T

Ly

s15

17

XN

on

sen

seA

AG

>T

AG

52

No

ve

l2

44

3E

27

bY

c.4

70

6T

>G

Le

u1

56

9X

No

nse

nse

TT

A>

TG

A3

1[3

2]

23

36

E2

7b

N/A

c.4

76

1T

>A

Ty

r15

87

XN

on

sen

seT

AT

>T

AA

4N

ov

el

20

13

E2

8N

/Ac.

48

20

T>

AL

eu

16

07

XN

on

sen

seT

TA

>T

AA

30

No

ve

l2

23

5E

28

Yc.

50

50

de

lAF

ram

esh

ift

29

No

ve

l2

40

5E

29

Yc.

52

42

C>

TA

rg1

74

8X

No

nse

nse

CG

A>

TG

A3

0[3

3]

21

84

E2

9N

c.5

42

6G

>T

Arg

18

09

Ieu

Mis

sen

seC

GC

>C

TC

10

No

ve

l2

44

4E

29

Yc.

54

35

T>

CL

eu

18

12

Pro

Mis

sen

seC

TG

>C

CG

20

No

ve

l2

32

7I

29

N/A

c.5

54

6+

5G

>A

Sp

lici

ng

?1

0N

ov

el

22

36

E3

0Y

c.5

71

9G

>T

Glu

19

07

XN

on

sen

seG

AA

>T

AA

28

[34

]2

38

9I

31

N/A

c.5

94

4-2

A>

GS

pli

cesi

te2

3[1

9]

21

32

E3

1Y

c.5

79

1T

>C

Trp

19

31

Arg

Mis

sen

seT

GG

>C

GG

5[3

5]

23

93

E3

1N

/Ac.

57

91

T>

CT

rp1

93

1A

rgM

isse

nse

TG

G>

CG

G1

4[3

5]

23

51

E3

1Y

c.5

79

3G

>A

Trp

19

31

Arg

No

nse

nse

TG

G>

TG

A1

0N

ov

el

22

75

E3

1Y

c.5

83

9C

>T

Arg

19

47

XN

on

sen

seC

GA

>T

GA

23

[36

]2

20

0E

31

Yc.

59

17

de

lGF

ram

esh

ift

24

No

ve

l2

34

8E

34

N/A

c.6

52

2_

65

23

de

lGA

Fra

me

shif

t2

2N

ov

el

24

00

E3

4Y

c.6

54

7_

65

49

de

lAC

Ain

sCC

ind

el

20

No

ve

l2

39

8E

35

Yc.

65

82

de

lAF

ram

esh

ift

22

No

ve

l2

40

7I

36

N/A

c.6

75

6+

3A

>G

an

dIV

S3

6+

4C

>T

Sp

lici

ng

?5

1N

ov

el

24

42

E3

7N

/Ac.

67

89

_6

79

2d

elT

TA

CF

ram

esh

ift

6[3

7]

24

36

E3

7Y

c.6

78

9_

67

92

de

lTT

AC

Fra

me

shif

t2

[37

]2

27

6E

37

Yc.

67

92

_6

79

3in

sAF

ram

esh

ift

18

[19

]2

34

3E

37

Yc.

67

92

C>

AT

yr2

26

4X

No

nse

nse

TA

C>

TA

A1

9[3

7]

23

71

E3

7N

/Ac.

67

92

C>

AT

yr2

26

4X

No

nse

nse

TA

C>

TA

A5

?[3

7]

123


Ta

ble

1co

nti

nu

ed

IDN

oL

oca

tio

nM

et

NIH

Cri

teri

aM

uta

tio

nT

riv

ial

Ty

pe

Co

do

nC

ha

ng

eA

ge

at

Re

ferr

al

Re

fere

nce

24

31

E3

7N

/Ac.

67

92

C>

GT

yr2

26

4X

No

nse

nse

TA

C>

TA

G1

0[3

8]

21

85

E3

7Y

c.6

83

3d

elC

Fra

me

shif

t1

9N

ov

el

24

58

I3

8N

/Ac.

70

00

-2A

>G

Sp

lice

site

8N

ov

el

24

08

I3

9Y

c.7

12

7-1

G>

TS

pli

cesi

te?

No

ve

l2

44

0E

41

Yc.

72

85

C>

TA

rg2

42

9X

No

nse

nse

CG

A>

TG

A7

[19]

23

34

E4

1Y

c.7

38

0_

73

81

insT

Fra

me

shif

t2

2N

ov

el

23

70

E4

2Y

c.7

41

1C

>T

Gln

24

71

XN

on

sen

seC

AG

>T

AG

30

[39]

23

26

I4

2Y

c.7

55

2+

1G

>A

Sp

lice

site

?N

ov

el

23

57

E4

4N

c.7

69

7C

>G

Se

r25

66

XN

on

sen

seT

CA

>T

GA

72

No

ve

l2

27

8E

45

Yc.

78

92

_7

89

3d

elA

AF

ram

esh

ift

41

No

ve

l2

29

2N

/AW

ho

leg

en

ed

ele

tio

n(M

LP

A)

24

No

ve

l2

33

7Y

Wh

ole

ge

ne

de

leti

on

(ML

PA

)3

4N

ov

el

23

38

N/A

66

kb

de

leti

on

(ML

PA

)3

5N

ov

el[

40

]2

46

7N

/AW

ho

leg

en

ed

ele

tio

n(M

LP

A)

6N

ov

el

24

60

N/A

Wh

ole

ge

ne

de

leti

on

(ML

PA

)1

0N

ov

el

22

85

N/A

Wh

ole

ge

ne

de

leti

on

(FIS

H)

2[2

4]

22

86

N/A

Wh

ole

ge

ne

de

leti

on

(FIS

H)

2[2

4]

21

31

N/A

Wh

ole

ge

ne

de

leti

on

(FIS

H)

16

[24]

21

48

N/A

Wh

ole

ge

ne

de

leti

on

(FIS

H)

12

[24]

22

68

N/A

Wh

ole

ge

ne

de

leti

on

(FIS

H)

4[2

4]

Re

curr

en

tm

uta

tio

ns

=E

xo

n4

b5

41

C>

TQ

18

1X

,E

xo

n4

b4

95

_4

98

de

lTG

TT

,E

xo

n1

0a

13

81

C>

TR

46

1X

,In

tro

n1

1IV

S1

1+

3A

>G

,E

xo

n1

32

04

1C

>T

R6

81

X,

Ex

on

16

25

40

T>

CL

84

7P

,E

xo

n1

72

97

0_

29

72

de

lAA

T,

Intr

on

18

IVS

18

+1

G>

T,

Ex

on

23

.13

91

6C

>T

R1

30

6X

,E

xo

n3

15

79

1T

>C

W1

93

1R

,E

xo

n3

76

78

9_

67

92

de

lTT

AC

,E

xo

n3

76

79

2C

>G

Y22

64

X,

wh

ole

ge

ne

de

leti

on

ide

nti

fie

db

yF

ISH

,w

ho

leg

en

ed

ele

tio

nid

en

tifi

ed

by

ML

PA

.I=

Intr

on

,E

=E

xo

n,

Y=

Ye

s,N

=N

o,

N/A

=N

ocl

inic

al

de

tail

ssu

pp

lie

d,

?D

ate

of

bir

tho

fp

ati

en

tn

ot

sup

pli

ed

123


provided for analysis, again providing good evidence for

its probable pathogenicity.

Fifteen sequence variants occurred at splice donor

or acceptor sites, and a further 6 sequence changes

were detected in the adjacent intronic sequences,

within five base pairs of the intron/exon boundary. Five

of these previously reported variants include, 4

involving the donor or acceptor splice sites

(c.2850+1G>A [21], c.3113+1G>T [21], c.3975-2A>

G[19], c.5944-2A>G [19]) and one in the adjacent in-

tronic sequence of exon 11 (c.1721+3A>G [5]). Two

variants c.6756+3A>G and c.6756+4C>T (both within

the accepted consensus region of the donor splice site)

occurred simultaneously in one individual. On further

investigation it was determined that both variants

segregated with two affected family members provid-

ing evidence that they probably are pathogenic.

The spectrum of different mutation types identified

in this study is illustrated in Fig. 1. Of all the point

mutations identified, 74% are predicted to result in

truncated mRNA transcript.

Table 2 displays the 23 sequence variants detected

in this study. Variants were classed as polymorphisms if

they were either found in association with a pathogenic

mutation, or if they have been detected in unaffected

individuals, or have been established as such in previ-

ous publications [19, 22, 27].

Table 3 shows the seven sequence variants for which

pathogenicity has yet to be definitively established.

However, none of these sequence changes were found

in 200 normal chromosome 17.

Distribution of NF1 mutations

The distribution of mutations across the NF1 gene is

illustrated in Fig. 2. Most mutations detected were

located in exons 4b,10a, 13, 16, 31 and 37 and no

27%

15%

27%

19%

2%

1%

9%

Nonsense

Missense

Frameshift

Splicing

In frame deletion

Indel

Large deletion

Fig. 1 NF1 mutational spectrum derived from 109 NF1 patients.Indel = Insertion and deletion

Table 2 Summary of non pathogenic sequence variants identified

Location Mutation Trivial Pathogenic mutation foundin association

Reference

5¢ UTR c.1-22G>C Mattocks et al. [27] Found with definite mutation and in unaffected [27]Intron 3 c.288+41G>A Exon 28, c.5050delA This ReportExon 4b c.528T>A D176E Exon 23.1, c.3916C>T R1306X This Report and [21]Intron 4c c.655-83T>C Exon 10a, c.1318C>T R440X This ReportExon 5 c.702G>A L234L Exon 28, c.5050delA This Report and [41]Exon 6 c.846G>A Q282Q Exon 17, c.2970_2973delAAT This ReportIntron 10b c.1528-35delT Exon 30, c.5719G>T E1907X This ReportExon 12a c.1810T>C L604L Exon 16, c.2433_2456del22 This ReportExon 13 c.2034G>A P678P Exon 31, c.5917_delG This ReportIntron 16 c.2851-16T>C Exon 28, c.4820T>A L1607X This ReportIntron 17 c.2991-96T>C Exon 30, c.5719G>T E1907X This ReportExon 21 c.3564A>G Q1188Q Exon 21, c.3560T>C L1187P This ReportExon 22 c.3867C>T F1289F Mattocks et al. [27] Found with definite mutation [27]Exon 23.2 c.4020T>C L1340L IVS23.1-2G>A This ReportIntron 23.2 c.4111-7delTTG Exon 7, 1017_1019delA This ReportIntron 25 c.4368-46 Exon 13, 2050delC fs687X This ReportIntron 28 c.5205+23T>C Exon 19b, 3241_3249delCTGGTCTC This ReportIntron 29 c.5546+19T>A Exon 44, 7697C>G S2566X This ReportIntron 32 c.6084+8C>G Mattocks et al. [27] Found with definite mutation[27]Intron 39 c.7126+37G>C Exon 28, c.4820T>A L1607X This ReportIntron 40 c.7258+19delCAAA Exon 19b, 3241_3249delCTGGTCTC This ReportIntron 41 c.7395-17C>T Exon 30, c.5719G>T E1907X This ReportExon 46 c.7978A>G I2660V Exon 23.1, c.3916C>T R1306X This Report

Twenty three non pathogenic sequence variants were identified, 19 of these were detected with a definite mutation in this study andanother with a possible missense mutation, the remaining 3 have been established as non pathogenic in previous studies

123


mutations were found in exons 2, 8, 9, 14, 19a, 23.2, 24,

26, 32, 33, 40, 43 and 46–49. The most common muta-

tion, occurring three times (3%) in our series, was the

nonsense mutation, c.1381 (R461X) in exon 10a. A

direct comparison of the number of mutations detected

in each exon compared to the physical size of that exon

is shown in Fig. 3 and illustrates that for its size, exon

37 has the most mutations.

Complete or partial NF1 gene deletions

On the basis of severe clinical presentation of NF1

disease, 32 samples were tested by FISH for large

deletions encompassing NF1, in 5 cases a whole NF1

gene deletion was identified. The remaining 27 samples

were screened by DHPLC and mutations were identi-

fied in 23 of them. Therefore, in total 164 samples were

screened by dHPLC, 25 of which failed to display ei-

ther a polymorphism or a definitive mutation, indicat-

ing the possibility of either a whole or partial NF1 gene

deletion. These samples were genotyped with a panel

of 5 highly polymorphic markers. Patients 2292, 2337

and 2363 were apparently homozygous for all of these

markers, suggesting a whole gene deletion, sample

2338 was apparently homozygous for two markers,

IVS27AC and IVS38. With the introduction of MLPA

kits P081 and P082 for NF1, confirmation of a whole

gene deletion in two of the samples (2292, 2337) and a

multi exon deletion extending exons 2–10a in the other

(2338) was possible. Furthermore, MLPA analysis was

carried out on samples without point mutations pro-

vided that the DNA quantity was sufficient and of

adequate quality. To date, 33 cases out of 63 have been

analysed and deletions identified in five (2292, 2337,

2338, 2467, 2460) (Table 1). High resolution CGH

micro-array analysis has also confirmed that samples

2292 and 2337 harbour complete gene deletions, and

that sample 2338 has a 66 kb genomic deletion [40].

Discussion

Definition of the complete sequence of the human NF1

gene has provided the means to offer both presymp-

tomatic and prenatal diagnosis, either via linkage or by

direct mutation analysis of the NF1 gene. In practice,

however, it has been observed that despite this very

few families express a wish to pursue either of these

options. This may be because they either do not per-

ceive NF1 as a serious debilitating illness, or it may be

more related to the issue that even successful mutation

Table 3 Summary ofsequence variants ofunknown pathogenicityidentified

In eight NF1 patients,sequence variants wereidentified but thepathogenicity of thesechanges is not defined

ID No. Location Sequence Change Reference

2147 Intron 10c c.1641+39C>T Novel2354 Intron 12a c.1845+45G>A Novel2295 Intron 22 c.3871-39G>A Novel2192 Intron 26 c.4514+11C>G Novel2134 Intron 26 c.4514+12C>T Novel2134 Intron 34 c.6579+45T>A [19]2333 Intron 43 c.7675+46G>A Novel

0

1

2

3

4

5

6

7

8

1 4B 5 10B 11 12B 14 16 18 19B 21 24 26 27B 29 31 33 35 37 39 41 43 45 47 49

Exon

Nu

mb

er o

f m

uta

tio

ns

3 7 9 23.1

Fig. 2 Histogram representing the distribution of NF1 mutations identified in 99 patients by dHPLC. The exons or their consensussplice sites in which the mutations were identified are represented on the X axis and the number of mutations identified on the Y axis

123


analysis is often of little help in accurately predicting

the disease severity expected in any future offspring.

Insensitivity at detecting mutations may also have been

a factor in the past, but this has become less of an issue

with the recent introduction of more efficient mutation

detection techniques. Whether the patient without an

identifiable NF1 mutation may have a mutation in

some other gene(s) is also a slight possibility.

Gross DNA re-arrangements and multi-exon dele-

tions account for between 5% and 10% [1, 19, 42, 43]

to 15 [44] of all NF1 mutations. In this series we

identified complete gene deletions in 9/169 (5 %)

individuals, while a multi-exon deletion was identified

in only 1 patient (0.6%).

The absence of any obvious clustering of mutations

within the NF1 gene clearly necessitates screening of

the entire gene for mutations. Furthermore, no single

mutation detection test system is currently able to

identify the entire spectrum of NF1 mutations, which

includes everything from point mutations through sin-

gle and multiple exon lesions up to 1.5 Mb genomic

deletions [19–21, 44, 45]. In addition, some 30% of

mutations in the NF1 gene are predicted to cause

aberrant splicing [4]. While the majority of mutations

affect consensus acceptor and donor splice site se-

quences, a number of missense, nonsense and silent

mutations may also result in splicing abnormalities,

either by creating a novel donor or acceptor site, or by

their direct effect on exonic splicing enhancer (ESE) or

exonic splicing silencer (ESS) regulatory sequences

[34, 42, 45].

No mutations were identified in exon 19b in a pre-

vious study which analysed 500 unrelated NF1 patients

[19]. In this study, nested primers were designed to

sequence exon 19b to overcome the difficulty in

sequencing this exon caused by the tract of 26 T’s in

intron 19a before the beginning of exon 19b. Using

nested PCR primers, however, we identified three

disease-causing mutations in this study. Fahsold and

colleagues found, multiple NF1 mutations in exon 4b,

in this study we identified three different mutations in

exon 4b, two of the mutations were recurrent, occur-

ring in two unrelated individuals. ‘Warm’ hot spots for

mutations in the NF1 gene have been reported in exons

31 and 37 [1], this is also confirmed in this series:

mutations in exon or intron 31 were identified in six

individuals (one of which is a previously described

recurrent nonsense mutation, c.5839C>T R1947X,

whilst another c.5791 T>C Trp1931Arg occurred in two

unrelated individuals in this study), and seven indi-

viduals harboured a mutation in exon 37 (one of which

is novel c.6833delC, another two were recurrent

c.6792C>A Y2264X and c.6789_6792delTTAC) even

though this exon represents just over 1.2% of the

coding region.

It is often difficult to judge the diagnostic signifi-

cance of missense mutations without some level of

functional analysis [45]. However, all mutations iden-

tified in this series predict changes in evolutionarily

conserved aminoacid residues in mouse v. human, and

none of the sequence alterations has been seen in at

least 200 normal chromosomes 17. Without suitable

family histories, and thus the possibility of segregation

studies to assist in deciding pathogenicity, either RNA-

based analysis or the use of a minigene splicing assay

might be usefully applied in such situations [20, 42, 45,

46]. However, the problems involved in collecting and

analysing mRNA on a routine diagnostic basis are not

to be underestimated, as are the potential issues with

the testing of illegitimate transcripts derived from

lymphocytes [47].

Ideally, diagnostic mutation screening tests should

have high sensitivity, preferably approaching 100%,

although any such estimates are inherently dependent

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

1 4B 5 9 10B 11 12B 18 2123.1 24 26 27B 31 33 37

Exon

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e m

uta

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n f

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cy (

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3 7 14 16 19B 29 35 39 41 43 45 47 49

Fig. 3 Weighted distributionof mutations over the NF1gene, calculated for each exonby dividing the number ofpossible pathogenic mutationsidentified in that exon, by thenumber of base-pairscomprising the exon, plus tenbase pairs added to allow forsplice-site mutations. Thevalues are ratios between theexon-specific mutationdensities and the averagemutation density for thewhole gene (99/9054bp)

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on the accuracy of relevant clinical diagnosis. It is

therefore encouraging that NF1 mutation detection

rates of 89–95% have been reported [20, 27]. Auto-

mated comparative sequence analysis identifies muta-

tions in 89% of NF1 patients and confirms a mutation

cluster in exons 11–17 distinct from the GAP related

domain [27]. Messiaen and colleagues [20] used a

combination of techniques to maximise sensitivity:

inherently, the more techniques that are applied, then

the greater will be the yield of mutations. However,

especially in a publicly-funded healthcare system such

as the UK National Health Service, resources are fi-

nite, and we feel that our approach is more cost-

effective for a routine diagnostic service. Any reduc-

tion in sensitivity, however, has potentially important

implications for NF families. Given the high pene-

trance of NF1 mutations, coupled with less than 100%

sensitivity of the test, then any negative mutation result

will always be difficult to interpret in individual cases,

and in series such as this audit of activity. The

mutation detection rate of 78% achieved in this audit

is lower than that obtained in other centres as our

service involves only DNA analysis, and no RNA

work. Therefore, the techniques we use will obviously

not identify ‘deep’ intronic and some splicing changes

which are located outside the region of sequence

analysed. It is also possible that methylation of the

NF1 gene promoter region may be involved in the

inactivation of NF1 gene in some patients, although

this feature has not been reported to be a common

mechanism of NF1 gene inactivation [48–51]. An

additional factor reducing the apparent sensitivity of

our analytical technique is likely to be clinical ascer-

tainment bias, in that clinicians often ask for NF1

molecular testing in circumstances where the pheno-

type is less obvious, i.e. in cases where the prior

probability of a mutation is lower, and they are

seeking the laboratory’s help in making a firm diag-

nosis. Inevitably, this will lower the overall percentage

yield of mutations, and it should be noted that this is

a report of an audit of activity, not a research study

designed to demonstrate the maximum possible

mutation detection rate in clinically definite cases,

useful though it is to know what that figure is.

Publication of the complete human genome

sequence has led to the identification of a number of

additional NF1-like genomic sequences located on

several other chromosomes [6]. Their potential inter-

ference in any diagnostic NF1 test is significant and

thus secure PCR strategies are warranted. As a result

we have re-designed new primers for twenty nine exons

in order to make this test more robust. We have also

developed an RNA-based diagnostic test which

permits the analysis of the entire NF1 transcript in a

series of 24 overlapping amplimers [17].

No obvious genotype–phenotype relationships have

yet been found in NF1 [12, 13]. Different mutations in

the NF1 gene are associated with Neurofibromatosis-

Noonan syndrome (NFNS) [52, 53]. The general lack

of a genotype–phenotype relationship in NF1 is

somewhat intriguing. It may be due to modifier loci

[12], or a subtle effect in terms of the nature of the

germline mutation determining the nature of the so-

matic mutations in NF1 tumours, as has been seen in

Familial Adenomatous Polyposis [54]. The pathogenic

and non-pathogenic variants identified in this study will

add to the body of data able to contribute to any such

understanding of genotype phenotype relationship in

NF1.

There are, nonetheless, some interesting observa-

tions that can be made from this data set. Firstly, two

unrelated patients, both with the same in-frame dele-

tion in exon 17 (c.2970_2972delAAT), have only CAL

spots without any obvious dermal neurofibromas. This

preliminary finding has been supported by similar

clinical findings in 18 unrelated NF1 patients with this

mutation (Upadhyaya et al. submitted). Secondly,

while a relationship between the presence of large NF1

gene deletions and dysmorphic features, learning dis-

ability and developmental delay has previously been

described [55], we were unable to confirm that all such

patients with NF1 deletions have this specific pheno-

type [56]. It is difficult to comment on a genotype-

phenotype correlation with certainty in this report in

view of the often limited clinical data given to us by

referring clinicians. These findings may indicate that

unlinked modifying genes and the normal NF1 allele

may well be involved in the development of particu-

lar clinical features in NF1, and that their relative

contributions may vary for different features.

The lack of definitive clinical information on some

67 patients included in this study, almost 40% of all

referrals, made it difficult to assess accurately the

mutation detection rate in this cohort of patients.

Recalculation of our results based only on those pa-

tients with a definitive clinical diagnosis, demonstrates

that of the 102 patients with reliable clinical data, 78

patients satisfied the NIH diagnostic criteria for NF1.

Within this defined cohort of NF1 patients, mutations

were characterised in 61 cases (78%), an increase over

the 64% in unselected cases.

The 57 novel NF1 mutations identified in this study

will expand the germline mutational spectrum of the

NF1 gene. In contrast to earlier findings [21], however,

our series would indicate that there are only a small

number of recurrent mutations in NF1 (Fig. 2). There is

123


no clustering of mutations in either the Cys-Ser rich

domain encoded by exons 11–17 [19, 27], or within the

GAP-related domain encoded by exons 21–27a. We

have found that exons 4b, 10a, 13, 16, 31 and 37 do appear

to harbour more mutations than do any other exons.

A sensitive genetic test for NF1 is warranted in or-

der to define better the clinical status of those patients

who fail to satisfy the diagnostic criteria for NF1, which

is often the case with young patients and atypical cases.

Given that almost half of all NF1 patients represent

new NF1 gene mutations, the possible presence of

germline mosaicism often makes it difficult to predict

the risk to offspring of inheriting NF1 mutations. Some

de novo patients will undoubtedly be somatic mosaics,

but we have not observed any cases of even possible

mosaicism in this series. While it is possible to detect

mosaicism by careful sequencing, the detection of

somatic mosaicism will always be challenging.

Over the two years that we have offered a complete

NF1 mutational screening service as part of the UK

Genetic Testing Network (UKGTN: http://www.ukgt-

n.org/index.html ), we have continued to modify and

improve our mutation detection technique. Our

reporting time for a complete NF1 gene mutational

screen ranges from 3 weeks up to 3 months for the

more complex mutations, however, our average

reporting time is currently about 6 weeks. The test

performance data we present here is of relevance in the

audit and development of genetic tests, as exemplified

by the requirements of the UKGTN in its gene dossier

system (http://www.ukgtn.org/dossier.html), which in-

volves assessment of genetic tests against set criteria.

The sensitivity of genetic testing is increasingly also

becoming a factor in establishing guidelines for clinical

genetic care, e.g. familial breast cancer (NICE Guide-

line CG014, May 2004, England and Wales: http://

www.nice.org.uk ) which states ‘‘A search/screen for a

mutation in a gene should aim for as close to 100%

sensitivity as possible for detecting coding alterations

and the whole gene should be searched.’’ In addition,

there is pressure on laboratory and clinical genetics

services to reduce the time spent in the diagnostic

process, as well as the public health considerations of

equity of testing. Thus, while we acknowledge that our

current system for NF1 testing could be improved, it

does go a considerable way to addressing such issues.

With the application of robotisation to increase labo-

ratory throughput, as we have started to use, it is

envisaged that turnaround times will be further

reduced. With the advancement of mutation detection

technologies including direct sequencing of all coding

and flanking non-coding regions of the NF1 gene, it

should be possible to develop a quicker and even more

sensitive diagnostic test for NF1, however, any such

testing will always be a compromise based on cost-

effectiveness.

Acknowledgments We thank all the clinicians for their help inproviding the clinical information. We are grateful to the WalesGene Park for their financial support, and the staff of theMolecular Genetics Laboratory of the All-Wales Genetics Lab-oratory Service. We thank Dr Nick Thomas for his help with thesplice prediction program.We wish to thank Dr Susan Huson forher support in initially setting up a diagnostic service, LindaSelwood for her administrative support and Andrew Wallace fordeveloping MLPA data analysis spreadsheets.

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Molecular diagnosis of neurofibromatosis type 1: 2 years experience