American Journal of Medical Genetics 43:217-223 (1992)

Molecular Studies of the Fragile X Syndrome Samantha J.L. Knight, Mark C. Hirst, Anya Roche, Zoe Christodoulou, Susan M. Huson, Robin Winter, Margaret Fitchett, Mark J. McKinley, Richard H. Lindenbaum, Yutaka Nakahori, and Kay E. Davies Molecular Genetics Group, Institute of Molecular Medicine, John Radcliffe Hospital (S.J.L.K., M.C.H., A.R., Z.C., Y.N., K.E.D.); Department of Medical Genetics, Churchill Hospital (S.M.H., M.F., M.J.M., R.H.L.), Headington, Oxford; The Kennedy-Galton Centre, North-West Thames Regional Genetics Service, Northwick Park Hospital, Waford Road, Harrow, Middlesex (R.W.),England

We have studied families segregating for the fragile X syndrome for the presence of amplification of t h e CGG repeat sequence adjacent t o the HpaII Tiny Fragment (ETF) We demonstrate island in the FMR-1 gene. that 138/143 fragile X positive, mentally retarded males show a characteristic smear of fragments corresponding to somatic variation in the amplification of the CGG sequence. In 1/8 normal transmitting males (NTM's), we show that there is a small amplification of sequence but no evidence for somatic variation. Defined mutated fragments in the size range found in NTM's are seen in daughters of NTM's. The daughters of these female carriers show either a defined fragment in the NTM size range, a defined larger fragment or a heterogeneous pattern of fragments. In the latter 2 cases t h e clinical phenotype of the females cannot easily be predicted, presumably because of variable X inactivation. In some families, the observed DNA genotype does not correlate with the phenotype; in others we demonstrate t h e occurrence of individuals with a mosaic DNA genotype. The implications of these data for diagnosis of the disease are discussed.

KEY WORDS: Fragile carriers, fragile retardation

X, mutation, diagnosis, site, X-linked mental

Received for publication September 30, 1991; revision received December 26, 1991. A d d r e s s r e p r i n t r e q u e s t s t o : D r Kay E . Davies, Mol.ecular G e n e t i c s Group, I n s t i t u t e o f Molecular Medicine, John Radclif f e Hospital, Headington, O x f o r d , OX3 9DU, E n g l a n d .

0 1992 Wiley-Liss, Inc.

INTRODUCTION The f r a g i l e X [ f r a ( X ) l syndrome i s t h e most f r e q u e n t f a m i l i a l form of mental r e t a r d a t i o n and i s t h e s e c o n d m o s t common g e n e t i c c a u s e o f m e n t a l r e t a r d a t i o n a f t e r Down s y n d r o m e [ f o r review see Fryns, 19891. I t a f f e c t s 1 i n 1 2 5 0 males a n d 1 i n 2 0 0 0 females [ K a h k o n e n e t a l . , 1 9 8 7 ; Webb e t a l . , T h e d i s e a s e s h o w s u n u s u a l X1 9 8 6 ; Webb, 1 9 8 9 1 . l i n k e d i n h e r i t a n c e e x h i b i t i n g non-penetrance in males and semi-dominance i n c a r r i e r females. A p p r o x i m a t e l y 30% of f e m a l e c a r r i e r s show some degree o f m e n t a l i m p a i r m e n t w h i l e 2 0 % o f males a r e p h e n o t y p i c a l l y normal t r a n s m i t t e r s of t h e d i s o r d e r (NTM's) ) ( s o - c a l l e d n o r m a l t r a n s m i t t i n g males [Sherman et a l . , 1984; 19851. The syndrome i s a s s o c i a t e d w i t h t h e c y t o g e n e t i c e x p r e s s i o n of a f r a g i l e s i t e a t X q 2 7 . 3 when l y m p h o c y t e s o f p a t i e n t s are grown u n d e r d e f i n e d c u l t u r e c o n d i t i o n s [Lubs, 1969; S u t h e r l a n d , 19771. T h e e x p r e s s i o n of t h e f r a g i l e s i t e h a s b e e n used a s a c y t o g e n e t i c m a r k e r f o r d i a g n o s i s , carrier d e t e c t i o n a n d p r e n a t a l d e t e c t i o n o f t h e f r a ( X ) s y n d r o m e [ f o r review see Tommerup, 19891. However, NTM's and t h e i r daughters d o not g e n e r a l l y e x p r e s s t h e f r a g i l e site making carrier s t a t u s d e t e r m i n a t i o n s i m p o s s i b l e except by linkage analysis. Recently, investigators h a v e shown t h a t m e n t a l l y r e t a r d e d males a r e f r a (X) positive, h y p e r m e t h y l a t e d a t a HpaII T i n y F r a g m e n t (HTF) i s l a n d i n Xq27.3 [ V i n c e n t e t a l . , 1991; B e l l e t a l . , 1 9 9 1 1 . A n a l y s i s o f t h e r e g i o n o f DNA a r o u n d t h i s HTF i s l a n d l e d t o t h e i d e n t i f i c a t i o n of t a n d e m l y a r r a y e d CGG t r i n u c l e o t i d e repeats w i t h i n

218

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t h e c o d i n g r e g i o n o f a g e n e d e s i g n a t e d FMR-1 [Yu e t a l . , 1991; O b e r l e et a l . , 1991; Verkerk et a l . , 1 9 9 1 ; Kremer e t a l . , 1 9 9 1 ; H i r s t e t a l . , 1 9 9 1 a I . Studies of f a m i l i e s i n which t h e f r a ( X ) mutation i s s e g r e g a t i n g s h o w t h a t t h e s i z e o f t h e CGG a m p l i f i c a t i o n varies from g e n e r a t i o n t o g e n e r a t i o n suggesting t h a t t h i s sequence is genetically unstable. S u c h v a r i a t i o n c a n be u s e d t o e x p l a i n the peculiar genetics. Normal i n d i v i d u a l s have up t o 5 0 c o p i e s o f t h e CGG r e p e a t , NTM's h a v e a s m a l l a m p l i f i c a t i o n a n d a f f e c t e d males h a v e a l a r g e r a m p l i f i c a t i o n [Kremer e t a l . , 19911. These changes assayed r e a d i l y by an increase i n c a n be r e s t r i c t i o n f r a g m e n t s i z e on a S o u t h e r n b l o t . NTM's a l w a y s s h o w d i s c r e t e f r a g m e n t s i z e s w h e r e a s a f f e c t e d males o f t e n h a v e m u l t i p l e f r a g m e n t s o f different sizes. P h e n o t y p i c a l l y normal d a u g h t e r s o f NTM's h a v e s m a l l i n s e r t i o n s i n t h e NTM r a n g e although t h e fragment size i s occasionally This indicates increased [Hirst et a l . , 1991al. t h a t a l t e r a t i o n s i n t h e fragment s i z e can occur i n d e p e n d e n t l y of a n o p p o r t u n i t y f o r X c h r o m o s o m e interaction i n meiosis. T h e female o f f s p r i n g o f t h e d a u g h t e r s o f NTM's c a n h a v e d e f i n e d f r a g m e n t s o r m u l t i p l e f r a g m e n t s w h i c h a p p e a r a s smears o f hybridization. I n t h i s p a p e r w e p r e s e n t a summary o f o u r d a t a a n d a more d e t a i l e d s t u d y o f s e v e r a l f r a ( X ) syndrome f a m i l i e s i n which t h e m u t a t i o n e x h i b i t s unusual o r mosaic e f f e c t s . The i d e n t i f i c a t i o n o f i n d i v i d u a l s with genotype:phenotype inconsistency i s d i s c u s s e d w i t h r e g a r d t o t h e u s e o f t h e new DNA test f o r molecular d i a g n o s i s of t h e f r a ( X ) syndrome.

MATERIALS

AND

RESULTS Molecular a l t e r a t i o n s a t t h e f r a ( X ) locus can be v i s u a l i z e d a f t e r d i g e s t i o n o f DNA s a m p l e s w i t h EcoRI, H i n d I I I o r B g l I I a n d h y b r i d i z a t i o n o f t h e S o u t h e r n b l o t s of t h e g e l s t o t h e p r o b e Ox1.9 An e x a m p l e o f t h i s t y p e [Nakahori et a l . , 19911. o f a n a l y s i s a n d t h e map p o s i t i o n s o f O x 1 . 9 a n d OxO.55 r e l a t i v e t o t h e (CGG)n repeat a r e g i v e n i n F i g u r e 1. I n l y m p h o c y t e DNA a n a l y s e s n o r m a l males s h o w a 5 . l k b f r a g m e n t ( l a n e 6), a f f e c t e d males show a l a r g e f r a g m e n t o r smear o f f r a g m e n t s ( l a n e s 3 a n d 4 ) , a NTM s h o w s a s m a l l i n c r e a s e i n f r a g m e n t s i z e (lane 5) and an a t r i s k , cytogenetically negative, f e m a l e gives a d o u b l e t ( l a n e 1 ) .

1

2

3

4

5

6

5.1 kb-

ox 1.9

2.3kb-

VK 21C

METHODS

S a m p l e s o f p e r i p h e r a l b l o o d l y m p h o c y t e s were collected for both cytogenetic and DNA investigations. T h e D N A samples were d i g e s t e d w i t h HindIII, EcoRI, B g l I I o r P s t I as d e s c r i b e d p r e v i o u s l y [ N a k a h o r i e t a l . , 1 9 9 1 ; Hirst e t a l . , 1 9 9 1 a l . H i n d I I I o r E c o R I d i g e s t e d samples were r u n o n 20cm x 25cm 0 . 8 % a g a r o s e g e l s a n d a l l o w e d t o m i g r a t e a t 65V f o r 2 0 h r s o r u n t i l t h e 5 k b m a r k e r reached t h e c e n t r e of t h e gel. B g l I I digested s a m p l e s were r u n o n 20cm x 25cm 0 . 8 % a g a r o s e g e l s and a l l o w e d t o migrate a t 65V f o r 1 8 h r s . PstI d i g e s t e d samples were r u n o n 20cm x 25cm, 1 . 5 - 2 . 0 % a g a r o s e g e l s a n d a l l o w e d t o m i g r a t e a t 60V f o r 1 4 hrs. E l e c t r o p h o r e s e d samples w e r e t r a n s f e r r e d o n t o Hybond-N m e m b r a n e ( A m e r s h a m ) a n d h y b r i d i s e d w i t h Ox1.9 f o r H i n d I I I , EcoRI a n d B g l I I digests o r OxO.55 f o r P s t I d i g e s t s . T h e DNA p r o b e s O x 1 . 9 a n d OxO.55 h a v e b e e n described p r e v i o u s l y [ N a k a h o r i e t a l . , 19911. H y b r i d i z a t i o n s o f O x 1 . 9 were c a r r i e d o u t a t 65'C i n a s o l u t i o n c o n t a i n i n g 0.25M NaH2P04 ( p H 7 . 2 ) , 0.25M N a C 1 , 1 m M EDTA, 7 % SDS a n d 5 % d e x t r a n s u l p h a t e . A h y b r i d i z a t i o n m i x c o n t a i n i n g 6% PEG, 4 X SSPE, 2X D e n h a r d t ' s r e a g e n t , 0 . 5 % SDS a n d 1 0 0 u g / m l s a l m o n sperm DNA w a s o p t i m a l f o r OxO.55.

0

P

I\

Ll"

/ \

H E . )

tCI

F i g . 1. A b o v e : A n a l y s i s o f D N A samples a f t e r HindIII d i g e s t i o n and h y b r i d i z a t i o n with t h e probes O x 1 . 9 a n d VK21C (control). L a n e 1: f r a ( X ) n e g a t i v e , c a r r i e r f e m a l e ; l a n e 2 : n o r m a l male; l a n e s 3,4: f r a ( X ) p o s i t i v e , mentally retarded males; l a n e 5 : NTM; l a n e 6 : n o r m a l f e m a l e . Blots were w a s h e d t o 0 . 1 x SSC a n d a u t o r a d i o g r a p h e d f o r 3 days. B e l o w : Map p o s i t i o n s o f Ox1.9 a n d OxO.55 i n r e l a t i o n t o t h e (CGG)n r e p e a t . OxO.55 d o e s n o t c o n t a i n t h e (CGG)n repeat b u t o v e r l a p s w i t h t h e f i r s t e x o n o f t h e FMR-1 g e n e [ N a k a h o r i e t a l . , 1 9 9 1 1 . H : H i n d I I I ; E: E c o R I ; P : P s t I ; N : N r u I ; S : S a c I I ; B: B s s H I I ; Ed: E a g I .

Molecular Studies of the Fragile X Syndrome TABLE I . A n a l y s i s o f f r a ( X ) P a t i e n t s w i t h Ox1.9

Total Analyzed 92 143

Result

Status

92 Normal s i z e f r a g m e n t s

Normal

138 Heterogeneous 4 Normal s i z e f r a g m e n t s 1 NTM s i z e f r a g m e n t

Affected Males

7

NTM

7 NTM s i z e f r a g m e n t s 1 Normal s i z e f r a g m e n t

4

Daughters of NTM ' s

4 Doublets

46

Other c a r r i e r c a r r i e r females (10 a f f e c t e d )

13

At risk females

28 D o u b l e t s 1 6 Heterogeneous 2 Normal s i z e f r a g m e n t s 6 Doublets 7 Heterogeneous

219

X q 2 7 [Nakahori e t a l . , 19911. T a b l e I summarizes a l l t h e a n a l y s e s c a r r i e d o u t u s i n g Ox1.9 on EcoRI, F r a g m e n t s i z e s were H i n d I I I and B g l I I d i g e s t s . determined f o r a subset of t h e s e i n d i v i d u a l s e i t h e r on P s t I d i g e s t s w i t h p r o b e OxO.55 o r o n EcoRI o r H i n d I I I d i g e s t s w i t h p r o b e Ox1.9 ( T a b l e 11).

Normal

Variation

At

The

Fra(X)

Locus

Forty-two normal i n d i v i d u a l s i n a d d i t i o n t o t h o s e i n c l u d e d i n T a b l e I were a n a l y z e d i n P s t I d i g e s t s w i t h OxO.55. This probe i s e s s e n t i a l l y i d e n t i c a l t o t h e p r o b e p f x a 3 [Yu e t a l . , 19911 a n d i s p r e p a r e d b y PCR a s d e s c r i b e d b y N a k a h o r i e t a l . A s t h e r e g i o n i s known t o v a r y i n s i z e [19911. [ K r e m e r e t a l . , 19911, t h e b a s e l i n e s i z e of t h e P s t I f r a g m e n t was d e t e r m i n e d f r o m t h e c o m p l e t e s e q u e n c e o f t h e r e g i o n w i t h no c o p i e s o f t h e CGG trinucleotide. T h i s s i z e , 970bp, was t h e n u s e d t o d e t e r m i n e t h e l e n g t h o f t h e CGG a r r a y o n n o n - f r a ( X ) W e have a d o p t e d t h e t e r m i n o l o g y of chromosomes. Oberle e t a l . [19911 i n d e s c r i b i n g t h e i n c r e a s e in s i z e o v e r t h e b a s e l i n e f r a g m e n t as A; t h e i n s e r t i o n

TABLE 11. I n s e r t S i z e s i n f r a ( X ) P a t i e n t s

A Range ( b p )

Status

Total analyzed

Normal

42

6-150

138 2

> 500

c a n b e d e s c r i b e d e i t h e r i n b a s e - p a i r s o r t h e number On n o n - f r a t x ) c h r o m o s o m e s , t h e o f CGG r e p e a t s . v a r i a t i o n in CGG c o p y number r a n g e d f r o m 2-50 c o p i e s w i t h a n a v e r a g e o f 2 3 c o p i e s . N T M ' s showed an i n c r e a s e i n s i z e of t h e normal fragment t o b e t w e e n 1 7 0 a n d 3 6 5 b p , c o r r e s p o n d i n g t o b e t w e e n 56 Affected males g e n e r a l l y a n d 122 c o p i e s o f CGG. h a v e a A o f g r e a t e r t h a n 500bp.

Affected males

NTM's

Daughters of NTM's Other females with doublets

I

105, 125 330

5 1

170-365 85

4

170-300

33

200-2500

T h e summary o f o u r a n a l y s e s t o d a t e a r e p r e s e n t e d i n T a b l e s I a n d 11. I n t h e s e s t u d i e s , w e have d e f i n e d o b l i g a t e carrier females a s f u l f i l l i n g a t l e a s t one of t h e f o l l o w i n g c r i t e r i a : t h e y a r e phenotypically affected, they express t h e f r a g i l e s i t e i n >2% of t h e i r lymphocytes, t h e y are d a u g h t e r s o f N T M ' s o r m o t h e r s w i t h o n e o r more W e have d e f i n e d females a t affected offspring. r i s k a s t h o s e members o f f r a ( X ) f a m i l i e s who a r e p h e n o t y p i c a l l y normal w i t h no a f f e c t e d o f f s p r i n g a n d h a v e unknown, low ( < 2 % ) o r n e g a t i v e c y t o g e n e t i c A l l the families expression of t h e f r a ( X ) s i t e . p r e s e n t e d have i n d i v i d u a l s with t h e t y p i c a l p a t t e r n of f r a ( X ) m u t a t i o n . F a m i l i e s i n w h i c h n o m u t a t i o n was f o u n d i n a n y i n d i v i d u a l were e x c l u d e d a s t h e s e may b e a t y p i c a l c a s e s w i t h a m u t a t i o n e l s e w h e r e i n

Daughters of NTM's

showed a d e f i n e d d o u b l e t o f A170-300bp ( t h a t i s i n t h e normal t r a n s m i t t i n g range) whereas o t h e r female c a r r i e r s showed e i t h e r a h e t e r o g e n e o u s s m e a r of f r a g m e n t s , a d e f i n e d d o u b l e t o f A200-2,500bp o r n o a b n o r m a l i t y d e t e c t e d o n t h e m u t a t e d X chromosome. Of t h e 1 0 a f f e c t e d f e m a l e s , 4 p o s s e s s a m u t a t e d X chromosome w i t h A600-2,200bp a n d t h e o t h e r 6 set of fragments i n possess a heterogeneous Two o b l i g a t e c a r r i e r l y m p h o c y t e DNA s a m p l e s . females have a p p a r e n t l y normal fragments in A l l 13 o f t h e HindIII, B g l I I and P s t I a n a l y s e s . f e m a l e s a t r i s k o f c a r r y i n g a m u t a t e d X chromosome gave p a t t e r n s t y p i c a l of c a r r i e r females. Of t h e s e , 4 were f r a ( X ) n e g a t i v e , 2 e x p r e s s e d t h e f r a ( X ) s i t e a t a l o w l e v e l (1%)a n d 7 h a d n o available cytogenetic results. T h e r e a r e s e v e r a l c a s e s i n Table I w h e r e t h e genotype d i d n o t c o r r e l a t e with t h e phenotype i n a p r e d i c t a b l e manner. These c a s e s occurred i n p e d i g r e e s where t h e c h a r a c t e r i s t i c i n c r e a s e i n f r a g m e n t s i z e was e v i d e n t i n o t h e r f a m i l y members. The f o u r a f f e c t e d males w i t h a p p a r e n t l y n o r m a l f r a g m e n t s a r e f r a ( X ) p o s i t i v e c y t o g e n e t i c a l l y and show n o a b n o r m a l i t y w i t h H i n d I I I , B g l I I o r P s t I digests. Three o t h e r a t y p i c a l c a s e s a r e d i s c u s s e d i n d e t a i l below.

Family

1

T h i s f a m i l v ( F i s . 2 ) was a s c e r t a i n e d t h r o u q h

220

Knight et al.

I

q-?

12%

AYe:ew

m 2

1.5%

A27533

7 3

4

20%m

AHeterO

5

0%

rn A200 b

6

o % o %

6

AHetero

A-iexero

3

2.7 %

0%

~900bp

Fig. 2. A n a l y s i s o f a f a m i l y w i t h p r o b e s Ox1.9 a n d VK21C ( c o n t r o l ) . Samples w e r e digested w i t h HindIII. T h e n u m b e r s on t h e l a n e s c o r r e s p o n d t o t h e n u m b e r s on t h e p e d i g r e e . Lane 1 i s a normal f e m a l e c o n t r o l . ?: u n c e r t a i n a f f e c t e d s t a t u s .

a s p e c i a l school and i n d i v i d u a l 3 who a t t e n d e d Her expressed t h e f r a ( X ) i n 20% of h e r cells. m u t a t i o n i s v i s u a l i z e d as a h e t e r o g e n e o u s smear o f f r a g m e n t s a b o v e t h e n o r m a l X chromosome f r a g m e n t a t 5.lkb (lane 3 ) . H e r m o t h e r , who i s a t t h e l o w e r end of t h e normal i n t e l l e c t u a l r a n g e s u b j e c t i v e l y , expressed t h e f r a g i l e site i n 12% of cells and shows a s i m i l a r h e t e r o g e n e o u s smear o f f r a g m e n t s individual 4, i s more ( l a n e 7 ) . Her b r o t h e r , s e v e r e l y retarded. H e e x p r e s s e s t h e f r a ( X ) i n 20% o f c e l l s a n d h a s t h e smear of f r a g m e n t s t y p i c a l o f On H i n d I I I a n a l y s i s t h e an a f f e c t e d male ( l a n e 4 ) . smear i s v e r y d i f f u s e a n d may be b e t t e r v i s u a l i z e d by compressing t h e f r a g m e n t s u s i n g a B g l I I d i g e s t . The o t h e r s i b s h a v e n o t h a d m a j o r i n t e l l e c t u a l problems. I n d i v i d u a l 5 ( f e m a l e ) a n d 6 ( m a l e ) were f r a ( X ) n e g a t i v e a n d gave n o r m a l DNA p a t t e r n s . I n d i v i d u a l 2 (male) e x p r e s s e d t h e f r a g i l e site i n a s m a l l p r o p o r t i o n of cells ( 3 / 1 0 7 ) . Phenotypically, t h e o n l y a b n o r m a l i t y d o c u m e n t e d was a s t u t t e r a n d t h a t h e was s l i g h t l y l e s s a b l e t h a n i n d i v i d u a l 6 . On DNA a n a l y s i s , h e p r e s e n t s a p a t t e r n t y p i c a l o f an a f f e c t e d male, b u t w i t h more d i s c r e t e fragments.

/ 4

25 %

~250bp

AHetero

F i g . 3 . A n a l y s i s o f a f a m i l y w i t h p r o b e s Ox1.9 a n d VKZlC (control). S a m p l e s were d i g e s t e d w i t h HindIII. The numbers on t h e l a n e s c o r r e s p o n d t o t h e n u m b e r s o n t h e pedigree. Lanes 1 i s a normal male control, l a n e 7 i s a fra(X) p o s i t i v e female from a n u n r e l a t e d f a m i l y . ?: u n c e r t a i n a f f e c t e d status.

The s i z e o f range

his

(A275bp).

smallest The

fragment

mosaicism

i s i n t h e NTM seen

in

the

l y m p h o c y t e DNA p a t t e r n o f t h i s i n d i v i d u a l may a c c o u n t f o r h i s m i l d phenotype and low e x p r e s s i o n of t h e f r a g i l e site.

Family

2

T h i s f a m i l y ( F i g . 3 ) was a s c e r t a i n e d t h r o u g h i n d i v i d u a l 4 who p r e s e n t e d w i t h m o d e r a t e m e n t a l r e t a r d a t i o n and n o s p e c i f i c p a t t e r n of anomalies; h e e x p r e s s e d t h e f r a g i l e s i t e i n 25% o f h i s c e l l s . H e shows c o m p l e t e a b s e n c e o f a normal fragment a t 5 . l k b and a f a i n t smear of h i g h m o l e c u l a r weight fragments (lane 4 ) . On c l i n i c a l e v a l u a t i o n it was d i f f i c u l t t o e s t a b l i s h whether h i s two brothers were o r were n o t a f f e c t e d . I n d i v i d u a l 3 had no major problems i n motor development but s l i g h t d e l a y i n speech development and problems i n learning t o write; both had resolved with

Molecular Studies of the fiagile X Syndrome

221

appropriate therapy. I n d i v i d u a l 3 has been f r a ( X ) n e g a t i v e on t w o a n a l y s e s ( t o t a l 0 / 1 5 0 c e l l s ) a n d shows a f r a g m e n t i n t h e t h e r a n g e o f a n NTM

The p r o b a n d , i n d i v i d u a l 5 ( l a n e 5 ) , p r e s e n t e d w i t h M a r t i n - B e l l Syndrome a n d e x p r e s s e s t h e f r a g i l e s i t e i n 30% o f c e l l s . A n a l y s i s o f l y m p h o c y t e DNA

(A250bp).

r e v e a l s a f r a g m e n t (A330bp) i n t h e r a n g e o f a n NTM. T h i s i n d i v i d u a l i s m e t h y l a t e d a t t h e CpG i s l a n d . H i s m e n t a l l y r e t a r d e d s i s t e r ( l a n e 6) i s f r a ( X ) p o s i t i v e (33%) a n d shows a f r a g m e n t o f i n c r e a s e d

The o t h e r b r o t h e r ,

individual

6, was

born a t 31 weeks a f t e r a pregnancy c o m p l i c a t e d by He pre-eclampsia b u t d i d n o t require v e n t i l a t i o n . had s l i g h t motor and speech d e l a y and i s i n s p e c i a l H i s f i r s t c y t o g e n e t i c e x a m i n a t i o n was school. n e g a t i v e ( 0 / 5 0 ) b u t on a s e c o n d o c c a s i o n h e showed 4/100 p o s i t i v e c e l l s . H e h a s a d i s c r e t e fragment of i n c r e a s e d s i z e ( A 9 0 0 b p ) a n d may a l s o h a v e a f a i n t smear of f r a g m e n t s of h i g h e r m o l e c u l a r weight. The mother ( i n d i v i d u a l 2 ) a t t e n d e d a s p e c i a l s c h o o l and on s u b j e c t i v e assessment had d u l l normal i n t e l l i g e n c e . She h a s been f r a ( X ) n e g a t i v e on t w o o c c a s i o n s ( t o t a l 0/150 c e l l s ) a n d c a r r i e s a m u t a n t f r a g m e n t of 200bp ( n o t v i s i b l e o n this gel, which was run t o compress the heterogeneous fragments i n o t h e r i n d i v i d u a l s ) .

Family

3

W e w e r e a b l e t o f o l l o w t h e i n h e r i t a n c e of t h e f r a g i l e site i n t h i s family (Fig. 4 ) through 3 generations.

size

(A2050bp)

with

an

additional

smear

of

H i s p h e n o t y p i c a l l y normal fragments above it. sister g i v e s a normal p a t t e r n w i t h only a fragment a t 5.1 k b ( l a n e 7 ) . T h e i r mother, i n d i v i d u a l 4 , i s fra(X) positive ( 1 4 % ) a n d shows a smear of fragments i n a d d i t i o n t o h e r normal fragment. The shows a g r a n d m o t h e r , who i s f r a ( X ) n e g a t i v e , Her a b n o r m a l t y p i c a l c a r r i e r d o u b l e t ( l a n e 1). f r a g m e n t (A330bp) i s a p p r o x i m a t e l y t h e same s i z e a s t h a t s e e n i n h e r a f f e c t e d g r a n d s o n ( i n d i v i d u a l 5; A330bp). Thus, i n t h i s f a m i l y t h e f r a ( X ) m u t a t i o n d e v e l o p s i n s u c h a way t h a t a g r a n d m o t h e r w i t h a defined doublet passes on t o her daughter t h e mutated chromosome which then exhibits a The m u t a t e d X h e t e r o g e n e o u s set o f f r a g m e n t s . chromosome s u b s e q u e n t l y i n h e r i t e d by t h e a f f e c t e d g r a n d s o n shows a s i n g l e f r a g m e n t i n t h e s i z e r a n g e t y p i c a l o f a n NTM ( A 3 3 0 b p ) . It is possible t h a t t h e p a t t e r n observed i n t h e lymphocytes of t h i s male does n o t r e f l e c t t h e p a t t e r n of t h e mutated chromosome i n o t h e r t i s s u e s s u c h a s t h e b r a i n . Such mosaicism i n t h e f r a ( X ) m u t a t i o n h a s been s u g g e s t e d p r e v i o u s l y [ O b e r l e e t a l . , 19911 a n d w e have demonstrated v a r i a t i o n between somatic t i s s u e s i n s a m p l e s from a n a f f e c t e d f e t u s [ W o h r l e e t a l . , 19921.

DISCUSSION

1 8 3 4 5 6 7 8 9 1 O l l 1 2

5.lkb-

0Sl.S

2.3kb-

VK2lC

Fig. 4 . A n a l y s i s o f a f a m i l y w i t h p r o b e s Ox1.9 a n d VK21C (control). Samples w e r e d i g e s t e d w i t h HindIII. T h e n u m b e r s on t h e l a n e s c o r r e s p o n d t o t h e numbers on t h e p e d i g r e e . Lane 1 2 i s a n o r m a l female c o n t r o l .

The data presented here confirm the o b s e r v a t i o n s made i n o u r l a b o r a t o r y a n d by o t h e r i n v e s t i g a t o r s [Yu e t a l . , 1991; O b e r l e e t a l . , 1991; Verkerk e t a l . , 1991; K r e m e r et a l . , 1991; 1991; Hirst e t a l . , 1991aI. Nakahori et a l . , C l e a r l y t h e p r e d i c t i o n of an i n d i v i d u a l ' s phenotype b y d i r e c t DNA t e s t i n g i s p a r t i c u l a r l y u s e f u l f o r t h e i d e n t i f i c a t i o n o f t h e c a r r i e r s t a t u s of N T M ' s a n d t h e i r d a u g h t e r s who a r e n o r m a l l y f r a ( X ) negative, and f o r t h e i d e n t i f i c a t i o n of a f f e c t e d males. The p r e s e n c e o f a f r a g m e n t w i t h an i n c r e a s e i n s i z e of g r e a t e r t h a n 5 0 0 b p i s u s u a l l y i n d i c a t i v e of an a f f e c t e d male. However, t h e d a t a p r e s e n t e d h e r e show t h a t t h e r e a r e some e x c e p t i o n s t o t h i s which would l e a d t o b o t h f a l s e p o s i t i v e and f a l s e n e g a t i v e p r e d i c t i o n s of phenotype. For e x a m p l e , t h e DNA a n a l y s i s o f i n d i v i d u a l 2 i n f a m i l y 1 p r e d i c t s a t y p i c a l a f f e c t e d phenotype; i n f a c t t h i s male p r e s e n t s o n l y v e r y m i l d l y . By c o n t r a s t , i n d i v i d u a l 5 i n f a m i l y 3 , i s a f f e c t e d b u t would h a v e b e e n p r e d i c t e d t o b e a n NTM on t h e b a s i s of DNA a n a l y s i s . We also report 4 fra(X) positive, m e n t a l l y r e t a r d e d males who a p p e a r t o h a v e n o a b n o r m a l f r a g m e n t s on H i n d I I I , B g l I I o r P s t I analyses. I n view of t h e e x c e p t i o n a l c a s e s p r e s e n t e d i n t h i s a n a l y s i s our c u r r e n t assessment o f t h e DNA t e s t f o r t h e p r e d i c t i o n o f a f f e c t e d i s t h a t i t i s 9 9 % a c c u r a t e when a males

222

Knight et al.

h e t e r o g e n e o u s set o f f r a g m e n t s i s o b s e r v e d a n d 95% a c c u r a t e when a f r a g m e n t i n t h e n o r m a l o r NTM range i s observed [Hirst et a l . , 1991bl. The carrier s t a t u s i s clear f o r t h o s e f e m a l e s i n w h i c h a d e f i n e d m u t a n t f r a g m e n t o r smear o f T h o s e females w i t h d e f i n e d fragments i s observed. fragments of increased s i z e i n t h e range found i n t h e NTM a r e p h e n o t y p i c a l l y n o r m a l [ t h i s paper; Snow et a l . , 19911. However, f e m a l e s w i t h a m u t a t i o n v i s u a l i z e d as a smear o f f r a g m e n t s may be a f f e c t e d o r unaffected, presumably due t o t h e degree o f The i n a c t i v a t i o n o f t h e m u t a t e d X chromosome. s m e a r o f fragments a b o v e t h e n o r m a l f r a g m e n t i s s o m e t i m e s d i f f i c u l t t o v i s u a l i z e as it i s o f t e n extremely diffuse. I n s u c h cases, t h e u s e o f B g l I I t o compress t h e mutant fragments i s useful. N e v e r t h e l e s s , of t h e 50 o b l i g a t e carrier f e m a l e s a n a l y z e d i n t h e s e s t u d i e s 2 showed n o a b n o r m a l i t y Linkage with HindIII, B g l I I o r P s t I digests. a n a l y s i s i n f a m i l i e s u s i n g h i g h l y polymorphic l o c i i s t h e best l y i n g w i t h i n lOkb o f t h e m u t a t i o n a v a i l a b l e method f o r d e t e r m i n i n g carrier s t a t u s i n t h o s e cases w h e r e n o m u t a t i o n i s v i s i b l e [ R i c h a r d s et a l . , 19911. I n s u m m a r y , t h e DNA t e s t s f o r p r e d i c t i o n o f There t h e f r a ( X ) p h e n o t y p e are e x t r e m e l y u s e f u l . a r e , however, a small number o f males i n w h i c h t h e c o r r e l a t i o n of genotype t o phenotype is not consistent. F u r t h e r a n a l y s i s of t h e s e exceptions may p r o v i d e v a l u a b l e i n f o r m a t i o n r e g a r d i n g t h e development o f t h e f r a (X) phenotype.

ACKNOWLEDGMENTS W e are g r a t e f u l t o H e l e n B l a b e r f o r a s s i s t a n c e We i n t h e preparation of t h e manuscript. a c k n o w l e d g e t h e Medical R e s e a r c h C o u n c i l a n d A c t i o n Research f o r f i n a n c i a l support.

REFERENCES 3ell

MV, H i r s t MC, N a k a h o r i Y , MacKinnon RN, R o c h e F l i n t T J , J a c o b s PA, Tommerup N , T r a n e b j a e r g L, F r o s t e r - I s k e n i u s U, Kerr B, T u r n e r G , L i n d e n b a u m RH, W i n t e r R, P e m b r e y M , T h i b o d e a u S , Davies KE ( 1 9 9 1 ) : P h y s i c a l mapping a c r o s s t h e f r a g i l e X: Hypermethylation and c l i n i c a l e x p r e s s i o n of the C e l l 64:861-866. f r a g i l e X syndrome. F r y n s J-P (1989) : X-linked m e n t a l r e t a r d a t i o n a n d the f r a g i l e X Syndrome: A c l i n i c a l a p p r o a c h . I n Davies KE ( e d ) : "The F r a g i l e X S y n d r o m e " . O x f o r d : O x f o r d U n i v e r s i t y P r e s s , pp 1 - 3 9 . l i r s t MC, N a k a h o r i Y , K n i g h t SJL, S c h w a r t z C, T h i b o d e a u SN, R o c h e A, F l i n t TJ,MC, T h i b o d e a u SN, R o c h e A , , F r y n s J - P , D a v i e s KE ( 1 9 9 1 a ) : G e n o t y p e p r e d i c t i o n i n t h e f r a g i l e X s y n d r o m e . J Med G e n e t 28:824-829. H i r s t M, K n i g h t S , C r o s s G , O ' C r a f t K , R a e b u r n S, H e e g e r S , E u n p u D , J e n k i n s E, L i n d e n b a u m R , Davies K (1991b): P r e n a t a l Diagnosis of t h e F r a g i l e X Syndrome. L a n c e t 338:956-957. A,

Kahkonen M, A l i t a l o T, A i r a k s i n e n E , M a t i l a i n e n R, L u a n i a l a K, A u t i o S , L e i s t i J ( 1 9 8 7 ) : P r e v a l e n c e of t h e f r a g i l e X syndrome i n f o u r b i r t h c o h o r t s o f c h i l d r e n o f s c h o o l age. Hum G e n e t 7 7 : 8 5 - 8 7 . Kremer E J , P r i t c h a r d M, L y n c h M, Yu S, Holman K, B a k e r E , W a r r e n ST, S c h l e s s i n g e r D, S u t h e r l a n d GR, Richards RI ( 1 9 9 1 ) : Mapping o f DNA i n s t a b i l i t y at t h e f r a g i l e X t o a trinucleotide repeat s e q u e n c e p ( C C G ) n . S c i e n c e 2 5 2 : 1 7 1 1 - 1 7 1 8 . Am J Hum L u b s HA ( 1 9 6 9 ) : A m a r k e r X-chromosome. G e n e t 21:231-244. N a k a h o r i Y , K n i g h t S J L , H o l l a n d J, S c h w a r t z C, R o c h e A, T a r l e t o n J, Wong S , F l i n t T J , F r o s t e r I s k e n i u s U, B e n t l e y D , Davies KE, H i r s t MC (1991) : Molecular h e t e r o g e n e i t y of t h e f r a g i l e X syndrome. N u c l A c i d R e s 19:4355-4359. O b e r l e I , R o u s s e a u F , H e i t z D, K r e t z C, D e v y s D , H a n a u e r A, Boue J, B e r t h e a s M, M a n d e l J L ( 1 9 9 1 ) : A m a z i n g i n s t a b i l i t y o f a 5 5 0 b p DNA s e g m e n t a n d abnormal m e t h y l a t i o n i n f r a g i l e X syndrome. S c i e n c e 252:1097-1102. R i c h a r d s R I , Holman K, Kozman H , K r e m e r E , Lynch M, P r i t c h a r d M, Yu S , M u l l e y J , S a t h e r l a n d G R ( 1 9 9 1 ) : F r a g i l e X Syndrome: g e n e t i c l o c a l i s a t i o n by l i n k a g e mapping o f two m i c r o s a t e l l i t e r e p e a t s FRAXACl a n d FRAXAC2 w h i c h i m m e d i a t e l y f l a n k t h e f r a g i l e s i t e . J Med G e n e t 2 8 : 8 1 8 - 8 2 3 . S h e r m a n SL, M o r t o n NE, J a c o b s PA, T u r n e r G ( 1 9 8 4 ) : The m a r k e r ( X ) syndrome: A c y t o g e n e t i c and genetic analysis. Ann Hum G e n e t 4 8 : 2 1 - 3 7 . S h e r m a n SL, J a c o b s PA, M o r t o n N E , F r o s t e r - I s k e n i u s U, H o w a r d - P e e b l e s PN, N i e l s e n KB, P a r t i n g t o n MW, S u t h e r l a n d GR, T u r n e r G , W a t s o n M ( 1 9 8 5 ) : Further segregation a n a l y s i s of t h e f r a g i l e X syndrome w i t h special r e f e r e n c e t o t r a n s m i t t i n g males. Hum G e n e t 6 9 : 2 8 9 - 2 9 9 . Snow K, Doud L , H a g e r m a n R , H u l l C , H i r s t MC, Davies KE, T h i b o d e a u SN ( 1 9 9 1 ) : A n a l y s i s o f m u t a t i o n s a t t h e f r a g i l e X l o c u s u s i n g t h e DNA p r o b e Ox1.9. Am J Med G e n e t , s u b m i t t e d . S u t h e r l a n d GR ( 1 9 7 7 ) : F r a g i l e s i t e s o n human chromosomes: D e m o n s t r a t i o n o f t h e i r dependence on t h e t y p e o f t i s s u e c u l t u r e medium. Science 197:256-266. Tommerup N ( 1 9 8 9 ) : C y t o g e n e t i c s o f t h e f r a g i l e s i t e a t Xq27. I n Davies KE ( e d ) : "The F r a g i l e X Syndrome". Oxford: Oxford U n i v e r s i t y Press, pp102-135. V e r k e r k A J M H , P i e r e t t i M , S u t c l i f f e J S , F U Y-H, K u h l DPA, P i z z u t i A , R e i n e r 0, R i c h a r d s S , V i c t o r i a MF, Z h a n g F, E u s s e n BE, v a n Ommen G-JB, B l o n d e n L A J , R i g g i n s G J , C h a s t a i n J L , K u n s t C, G a l j a a r d H , C a s k e y CT, N e l s o n DL, O o s t r a BA, W a r r e n ST ( 1 9 9 1 ) : I d e n t i f i c a t i o n o f a g e n e (FMR1) c o n t a i n i n g a CGG r e p e a t c o i n c i d e n t w i t h a breakpoint c l u s t e r region exhibiting length X syndrome. Cell variation in fragile 65:905-914. V i n c e n t A, H e i t z D. P e t i t C, K r e t z C, Oberle I , Mandel J - L ( 1 9 9 1 ) : Abnormal p a t t e r n detected i n pulsed f i e l d gel f r a g i l e X p a t i e n t s by electrophoresis. N a t u r e 329:624-626.

Molecular Studies of the Fragile X Syndrome

Webb T, Bundey S, Thake J, Todd A (1986): The frequency of the fragile X chromosome among schoolchildren in Coventry. J Med Genet 23:396399. Webb T (1989): The epidemiology of the fragile X syndrome. In Davies KE (ed): “The Fragile X Syndrome”. Oxford: Oxford University Press, pp40-55.

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Wohrle D , Hirst MC, Kennerknecht I, Davies KE, Steinbach P (1992): Genotype mosaicism in fragile X fetal tissues. Hum Genet, in press. Yu S, Pritchard M, Kremer E, Lynch M, Nancarrow J, Baker E, Holman K, Mulley JC, Warren S T , Schlessinger D , Sutherland GR, Richards RI (1991): Fragile X genotype characterized by an unstable region of DNA. Science 252:1179-1181.

Molecular studies of the fragile X syndrome.

We have studied families segregating for the fragile X syndrome for the presence of amplification of the CGG repeat sequence adjacent to the HpaII Tin...
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