American Journal of Medical Genetics 3099-107 (1988)

FAXAGILE-X MUTATION AND KLINEFELTER SYNDROME :

F i l i p p i , G. , P e c i l e , V. , B i n a l d i , A

A *WAPP~pAISAL

, S i n i s c a l c o , M

C a t t e d r a d i G e n e t i c a Medica d e l l ' U n i v e r s i t a ' , I s t i t u t o p e r L ' I n f a n z i a , Trieste (F.G., P.V.); 9

I s t i t u t o d i B i o l o g i a Genera l e , F a c o l t a ' d i Medicina U n i v e r s i t a ' d i C a g l i a r i , I t a l y ( % A . 1; Memorial Sloan-Ketter ing Cancer C e n t e r , New York (S.M.)

ABST,PACT

To d a t e t h e concur ren t p re sence of t h e fragi le-X and t h e K l i n e f e l t e r syndromes i n t h e same i n d i v i d u a l has been found a t l e a s t 8 t imes e i t h e r i n t h e cour se of s c r e e n i n g f o r t h e f r a (X) c o n d i t i o n i n m e n t a l l y r e t a r d e d males o r among t h e r e l a t i v e s of f r a (X> p r o p o s i t i . Given t h e h igh frequency of bo th e v e n t s i n t h e g e n e r a l popu la t ion and t h e heterogeneous approaches w i t h which t h e above cases were a s c e r t a i n e d , i t h a s n o t been p o s s i b l e t o de t e rmine unequ ivoca l ly so f a r whether t h e f i n d i n g i s p u r e l y c o i n c i d e n t a l o r t h e expres s ion of some u n d e r l y i n g b i o l o g i c a l r e l a t i o n s h i p . To e v a l u a t e t h e i s s u e , we have sc reened a l a r g e popu la t ion of i n s t i t u t i o n - a l i z e d men ta l ly r e t a r d e d males f o r microorchidism, and submi t t ed t o a f u l l karyotype a n a l y s i s and f r a (X) t e s t i n g t h e p a t i e n t s t h a t were found t o have marked b i l a t e r a l microorchidism. Thus, i n a t o t a l of 32 mic roorch id i sm p a t i e n t s i d e n t i f i e d among 1115 menta l ly r e t a r d e d males , w e found 6 t o have a 47,XXY chromosome complement i n a l l ( o r i n most) of t h e i r c e l l s , w i t h one of them having also t h e f r a (X) marker i n 9% of t h e metaphases examined. In a d d i t i o n , ano the r b e a r e r of t h e f r a (X> marker (bu t only i n 4% of h i s metaphases) w a s found among 26 47,XXY m e n t a l l y normal ma es a s c e r t a i n e d throughout r o u t i n e c y t o g e n e t i c

Key words f r a (X> mutat i o n , 47 ,XXY non-dis junct ion

Address r e p r i n t r e q u e s t s t o Marce l lo S i n i s c a l c o , Memorial S loan-Ke t t e r ing Cancer Cen te r , 1275 York Avenue, New York, N.Y. 10021, USA

0 l9SE Alan R. Lm, Inc.

100 Filippi et al.

a n a l y s i s of males w i t h microorchidism r e f e r r e d t o our g e n e t i c c o u n s e l i n g u n i t d u r i n g t h e l a s t 10 y e a r s . In our l a b o r a t o r y t h e f r a ( X ) marker has never been observed w i t h such a f requency i n a t o t a l of s e v e r a l hundred normal XY males and XX females s t u d i e d a s c o n t r o l ca ses i n t h e cour se of p r e v i o u s l y r e p o r t e d f ami ly and popu la t ion s t u d i e s . Taken a t t h e i r f a c e v a l u e , t h e p r e s e n t f i n d i n g s sugges t t h a t t h e inc idence of t h e f r a (X) marker among men ta l ly r e t a r d e d 47,XXY males (and p o s s i b l y among 47,XXY i n d i v i d u a l s i n g e n e r a l ) i s s e v e r a l f o l d g r e a t e r than t h e inc idence r e p o r t e d . for XY males and XX f ema les . One of t h e most economical i n t e r p r e t a t i o n s of t h e s e o b s e r v a t i o n s i s t h a t h e t e r o z y g o s i t y f o r t h e f r a (X) muta t ion may be a major cause of m e i o t i c X chromosome non-d i s junc t ion . I f confirmed, t h i s a t t r a c t i v e hypo thes i s is l i k e l y t o provide new c l u e s f o r i n v e s t i g a t i n g t h e b io logy of t h e f r a (X) mutat ion and i t s r e l a t i o n s h i p - i f any - w i t h t h e mechanisms of normal and a b e r r a n t me ios i s .

INTRODUCTION

There have been 7 independent r e p o r t s on t h e concur- r ence of t h e K l i n e f e l t e r syndrome and of t h e f r a (X) marker i n t h e same i n d i v i d u a l [Wilmot e t a l , 1980; R o s t e r - I s k e n i u s e t a l , 1982; O'Brien e t a l , 1982; F i l i p p i e t a l , 1983; Fryns e t a l , 1983; Fryns e t a l , 1984a; Schnur e t a l , 19861 and one a d d i t i o n a l c a s e i s known t o u s th rough p e r s o n a l communica- t i o n s r e c e i v e d by Drs. P . J a c o b s and T . Brown. Our own c a s e [ F i l i p p i e t a l , 19831 was i d e n t i f i e d among t h e r e l a t i v e s of a p r o p o s i t u s wi th t h e f u l l complement of symptoms t h a t c h a r a c t e r i z e t h e Mart in-Bel l syndrome (MBS), i . e . men ta l r e t a r d a t i o n a s s o c i a t e d w i t h macroorchidism and an i n d u c i b l e f r a g i l e s i t e a t Xq27.3 ( f r a ( X ) ) .

Th i s 47,XXY i n d i v i d u a l w i th the f r a ( X ) , occu r red i n a f r a (X) o b l i g a t o r y h e t e r o z y g o t e who was 26 y e a r s o l d a t t h e t i m e of h i s b i r t h , has now been proven t o have been t h e r e s u l t of ma te rna l non-dis j u n c t i o n of t h e X chromosomes through t h e a n a l y s i s of t h e g e n e t i c c o n s t i t u t i o n of t h e two X chromosomes i n terms o f DNA r e s t r i c t i o n s i t e v a r i a n t s [ P u r r e l l o e t a l , 19871. Using t h e same approach, Schnur et a 1 [1986] have e s t a b l i s h e d t h a t t h e i r ca se is a c l e a r example of p a t e r n a l non-d i s junc t ion , wh i l e no d e f i n i t e i n fo rma t ion i s a v a i l a b l e about t h e o r i g i n of t h e super- numerary X i n a l l o t h e r pub l i shed c a s e s , i n c l u d i n g t h o s e found t o e x p r e s s t h e f r a ( X ) marker on b o t h X s [O'Rrien e t a l , 1982; Fryns e t a l , 1984bl s i n c e t h i s f i n d i n g by i t s e l f does no t r u l e ou t t h e p o s s i b i l i t y of pos t - zygo t i c non-dis- j u n c t i o n . In p a r t i c u l a r , g iven t h e heterogeneous approaches wi th which t h e above r e f e r r e d c a s e s have been a s c e r t a i n e d , i t i s d i f f i c u l t t o a s s e s s whether t h e concurrence of t h e 47,XXY complement and f r a (X) marker t h u s f a r r e p o r t e d i s

Fragile X and Non-Disjunction 101

purely co inc identa l o r r a t h e r the expression of some unknown common underlying b io log ica l r e l a t i o n s h i p . To eva lua te t h i s important i s sue we decided t o screen f o r t he fra(X) among 47 ,XXY ind iv idua l s a sce r t a ined on t he bas i s of adul t b i l a - t e r a l microorchidism, a c l i n i c a l s ign which - though not an exc lus ive component of t h e K l i n e f e l t e r syndrome - o f f e r s t he advantage of being cons t an t ly present among 47,XXY males and cons tan t ly absent among MBS p a t i e n t s with an XY complement. Since most 47,XXi/fra(X) males thus f a r reported were found among mentally re ta rded p a t i e n t s , we have performed our study sepa ra t e ly on 47,XXY a d u l t males with or without ment a1 r e t a r d a t ion.

METHODS

A l l 47,XXY adu l t males repor ted i n the present study were ascer ta ined exc lus ive ly throughout the manifestat ion of b i l a t e r a l microorchidism. The determinat ion of t he chromo- somal complement and of t h e fra(X) marker were c a r r i e d out according t o s tandard pro tocols [Glover, 1981; Sutherland and Baker, 19861 on shor t t e r m cu l tu re s of PHA-stimulated T-lymphocytes set up within 48 hours from the c o l l e c t i o n of t he blood samples. A minimum of 50 wel l spread metaphases was examined from each cu l tu re . Under these condi t ions , the threshold of fra(X) p o s i t i v e c e l l s i n normal ind iv idua l s was found t o be p r a c t i c a l l y n i l among seve ra l hundred normal a d u l t s of e i t h e r sex examined in our labora tory over t he years [ F i l i p p i e t a l , 1983, and unpublished d a t a ] . Thus, we have adopted the genera l ly accepted threshold values of 0.7% and 1.5% to d i sc r imina te between fra(X) nega t ive and p o s i t i v e males o r females, r e s p e c t i v e l y [Steinbach e t a l , 19821.

THE DATA

A t o t a l of 1115 i n s t i t u t i o n a l i z e d mentally re ta rded males (535 from Sard in i a and 580 from the region of T r i e s t e ) were screened by one of us (F.G.) f o r the presence of b i l a - t e r a l microorchidism. Thirty-two such cases were found and submitted t o a complete cy togenet ica l ana lys i s t o determine t h e i r chromosome complement and t h e i r proneness t o inducib le chromosome f r a g i l i t y a t Xq27.3 (fra-(X) marker). A t t he same time the screening fo r the fra(X) marker was c a r r i e d out on a group of 26 mental ly normal 47,XXY males, ha l f of whom had been i d e n t i f i e d during a population study of Sardinian consc r ip t s wi th microorchidism [ F i l i p p i , 19861, and the o ther ha l f among p r o p o s i t i with microorchidism r e f e r r e d t o our group for rou t ine cytogenet ic ana lys i s . The most r e l evan t f ind ings of t h i s inqui ry are:

102 Filippi et al.

( i ) 6 of t h e 32 menta l ly r e t a rded males wi th micro- orchidism were found t o have a 47,XXY complement with one of them having a l s o a c l e a r cu t f ra(X) marker i n 18 ou t of 200 metaphases examined (from 2 sepa ra t e pe r iphe ra l blood c u l t u r e s ) ; t h i s i nd iv idua l had mild mental r e t a r d a t i o n , a f r i e n d l y p e r s o n a l i t y , and t y p i c a l MBS f a c i a l changes (Fig. l a ) . Two b r o t h e r s (now deceased) were repor ted as having a l s o been i n s t i t u t i o n a l i z e d f o r behaviora l and mental problems (Table I ) ; the c l i n i c a l and labora tory f ind ings of t h i s 47,XXY/fra(X) p a t i e n t were: he igh t 176 cm, weight 55 Kg, s o f t small t e s t e s , normal penis , sparse ches t , a x i l l a r y and pubic h a i r wi th feminine d i s t r i b u t i o n , no gynecomastia, increased l e v e l s of F S H and LH, normal va lues of t e s tos - te rone and p r o l a c t i n ; sex chromosome mosaicism wi th 16% of c e l l s having a 46,XY complement and 84% 47,XXY, thus suggest ing t h a t t he o r i g i n a l zygote was 47,XXY;

( i i ) one of t he 26 47,XXY menta l ly normal p a t i e n t s w a s found t o have t h e fra(X) marker i n 8 out of 200 metaphases (from 2 s e p a r a t e c u l t u r e s . H i s c l i n i c a l and labora tory f ind ings w e r e : he igh t 193 cm, weight 93 Kg, span 190 cm, small s o f t t e s t e s wi th normal penis , absent ches t and sparse pubic h a i r , no gynecomastia, increased l e v e l s of FSH and LH, normal l e v e l s of t e s t o s t e r o n e and p r o l a c t i n ; he had 8 s i b s (4 males) and unremarkable f a c i a l t r a i t s (F ig l b ) ; t he age of the parents a t t he b i r t h of t he propos i tus was 38 years (mother) and 42 yea r s ( f a t h e r ) .

DISCUSSION

Since the re probably i s no 47,XXY male of meio t ic o r i g i n ( i . e . r e s u l t i n g from non-dis junct ion i n maternal or pa te rna l gametogenesis) without microorchidism, we can assume t h a t t h e 6 47,XXY males found among t h e 32 i n s t i t u - t i o n a l i z e d males wi th microorchidism rep resen t t he t o t a l number of 47,XXY a c t u a l l y r e s i d i n g i n t he populat ion of mental ly r e t a rded males analyzed. Thus, t he frequency of 47,XXY males i n t h i s populat ion i s .006, a f i g u r e which i s f u l l y compatible wi th the estimates repor ted by more com- p l e t e s t u d i e s based on the d i r e c t cy togenet ic sc reening of l a rge samples of mental ly r e t a rded males [Fryns e t a l , 1984al. The frequency of MBS a t b i r t h has been es t imated t o be of t he o rde r of 1:2000, bu t has been found t o be a s high as .06 among i n s t i t u t i o n a l i z e d mental ly re ta rded males [Fros te r - I skenius e t a l , 19831. It i s f o r t h i s reason t h a t t he f ind ing of 47,XXY/fra(X) males among mental ly r e t a rded males has thus f a r not been considered a s an except iona l one. Given t h e apparent absence of microorchidism i n a d u l t MBS 46,XY males, t he p r o b a b i l i t y of f ind ing the fra(X) marker i n a sample o f 47,XXY K l i n e f e l t e r p a t i e n t s should no t be g r e a t e r than the expected frequency of heterozygotes f o r

Tab

le I

. C

lin

ical

and

Cy

tog

enet

ical

F

indi

ngs

on

six

Kli

ne

felt

er

Pa

tie

nts

Fou

nd A

mon

g th

e 32

Pat

ien

ts w

ith

Mic

roor

chid

ism

A

scer

tain

ed

in a

Po

pu

lati

on

of

1115

Men

tall

y R

etar

ded

Mal

es*

Pro

po

situ

s P

aren

tal

age

Vol

ume

of

Kar

yoty

pe

% (a

nd

tota

l #

ce

lls

Ad

dit

ion

al f

ind

ing

s a

t b

irth

of

test

es

(ml)

of

c

ell

s w

ith

pr

opos

i tu

s an

alyz

ed )

fra(

X)

Cod

e #

Age

M

othe

r F

ath

er

Lef

t R

ight

%

(#)

KL3

55

37

50

10.6

KL6

80

24

27

8.9

KL8

75

33

36

4.9

KL2

2 77

42

43

1

.5

KL3

0 57

20

32

2 .o

KL3

1

71

40

45

4.7

9.8

47 ,X

XY

2.5

47 ,xXY

5.7

47 ,m

1.4

47,m

48 ,x

xxY

unde

- sc

ende

d 47

,XXY

47 ,x

xY

10.1

46

, XY

100

(50)

100

(51)

100

(51)

100

(51)

83

17

(100

)

a4

(200)

16

0 0 0 0 0 0 17 1

Two

norm

al

sist

ers

Unr

emar

kabl

e fa

mil

y h

isto

ry

Seve

n no

rmal

si

bs

(3 m

ales

)

Thr

ee n

orm

al

sib

s (o

ne f

emal

e)

One

hea

lth

y b

roth

er

Thr

ee n

orm

al

sib

s (2

mal

es)

Two

bro

ther

s (n

ow d

ecea

sed)

in

stit

uti

on

ali

ze

d for

men

tal

reta

rdat

ion

* T

he t

hre

sho

ld v

olum

e us

ed

for

the

cla

ssif

ica

tio

n o

f m

icro

orch

idis

m w

as 1

2 m

l.,

i.e

. tw

o st

and

ard

dev

iati

on

s be

low

th

e no

rmal

mea

n v

alu

e (32.9

2 9.5)

esti

mat

ed i

n a

co

ntr

ol

grou

p of

XY

mal

es

of

the

sam

e ag

e gr

oup.

th

e fr

a(X

) ph

enot

ype

was

ca

rrie

d o

ut u

sing

th

e p

roto

cols

of

bo

th G

love

r [1

981]

and

Su

ther

lan

d a

nd B

aker

11

9861

. T

he d

eter

min

atio

n o

f

104 Filippietal.

Fig. 1. The f a c i a l f e a t u r e s of the 2 f ra(X) p o s i t i v e 47,XXY i nd iv idua l s : t he menta l ly r e t a rded one (marker 's inc idence 16%) has a t y p i c a l MBS f a c i e s ( l a ) , whereas the mental ly normal (marker 's inc idence 4%) has an unremarkable f a c i e s ( l b ) . Thus, both mental r e t a r d a t i o n and expression of t he f a c i a l MBS phenotype appear t o be c o r r e l a t e d with the marker 's incidence i n XXY c a r r i e r s of t he fra(X) mutation.

Fragile X and Non-Disjunction 105

t he fra(X) mutant a t b i r t h , i . e . approximately 1:1000, i f the normal and the mutant genes a r e i n a Hardy-Weinberg equi l ibr ium. Thus, t h e f ind ing of 2 f ra(X) p o s i t i v e ind iv i - duals among the 32 47,XXY p r o p o s i t i examined (one among the 6 mental ly re ta rded and one among t h e 26 mental ly normal 47,XXY) i n d i c a t e t h a t t h e two events a re not independent of one another. I n add i t ion , the apparent preponderance of the fra(X) marker observed among the mental ly r e t a rded 47 ,XXY males suggests t ha t t he concurrence of the two condi t ions i n the same indiv idua l may exacerbate the expression of the mental r e t a rda t ion .

The conclusion proposed is t h a t the incidence of meio- t i c X chromosome non-disjunction is higher than usual i n t h e heterozygotes for t he fra(X) mutation. This conclusion i s s t rong ly supported by cases of 47 ,XXY/fra(X) ind iv idua l s de tec ted without ascer ta inment b i a s ( i . e . among X-related males of t he fra(X) p r o p o s i t i ) , and d i r e c t l y proven t o be t h e r e s u l t of maternal non-disjunction. Our own case [ F i l i p p i e t a l , 1983 and Fig. 21 f a l l s i n t o t h i s category a s unequivocally proven by molecular s tud ie s ca r r i ed out wi th DNA probes t h a t i d e n t i f y common X-linked r e s t r i c t i o n fragment length polymorphisms [ P u r r e l l o e t a l , 19871.

Fig. 2. Facies of 47,XXY/fra(X) pa t i en t der ived from maternal non-disjunction occurred i n a heterozygote f o r t he fra(X) mutation.

106 Filippietal.

A s f a r as we can ga the r from the published r e p o r t s [Wilmot e t a l , 19801 and personal communications [P . Jacobs and T. Brown], a t least another 2 cases of 47,XXY/fra(X) have been i d e n t i f i e d i n pedigrees a sce r t a ined through an MBS propo- s i t u s . I n t e r e s t i n g l y , a l s o a case of a tr iplo-X female (without t he fra(X) marker has been repor ted i n the of f - sp r ing of an ob l iga to ry heterozygote f o r t he fra(X) mutation [Kshkgnen, 19831. I f these a d d i t i o n a l cases a r e a l s o proven t o be the r e s u l t of maternal non-dis junct ion, t h e r e would be no escaping the conclusion t h a t t he fra(X) mutation i s a major cause of f a i l u r e i n the r egu la r s epa ra t ion of X chromosomes a t meios is .

We t r u s t t h a t t hese cons idera t ions and the observat ions repor ted w i l l s t i m u l a t e the quick c o l l e c t i o n of s i m i l a r d a t a on a world wide s c a l e s ince the confirmation of our f ind ings beyond reasonable doubt would o f f e r an important c l u e toward the understanding of t he na tu re of t he fra(X) mutation and of i t s poss ib l e i n t e r f e r e n c e wi th the r egu la r mechanism of meio t ic s epa ra t ion of X chromosomes i n heterozygous c a r r i e r s .

Note added i n proof: Af te r t he submission and acceptance of t h i s r epor t we became aware of a new case of a 47,XXY/fra(X) p a t i e n t born t o an obl iga tory heterozygote f o r t he fra(X) mutation [J.P. E'ryns, personal communication].

ACKNOWLEDGEMENTS

This work was supported by NIH g r a n t s GM 37090 and CA 08748 ( t o M.S.), and by P roge t to F i n a l i z z a t o Ingegneria e Basi Molecolar i d e l l e Mala t t i e E r e d i t a r i e of CNR, Rome (to A.R. and G.F.).

ARE FE RENCE S

F i l i p p i G (1986): K l i n e f e l t e r ' s syndrome i n Sa rd in i a .

F i l i p p i G , R ina ld i A , Archidiacono N , Rocchi M , Balazs I , Cl in Genet 30:276-284.

S in i sca l co M (1983): Linkage between G6PD and Fragile-X syndrome. Am J Med Genet 15:113-119.

(1983): Screening fo r f r a (X>(q> i n a populat ion of mental ly r e t a rded males. Hum Genet 60:153-157.

(1982): Repl ica t ion p a t t e r n i n XXY ce l l s wi th fra(X). Hum Genet 60:278-280.

Lindemans I, Van den Berghe H (1983): XY/XXY mosaicism and f r a g i l e X syndrome. Ann G&t 26:251-253.

Froster-Iskenius U, Felsch G, Schi r ren C, Schwinger E

Fros te r - I skenius U, Schwinger E , Weigert M , Fonatsch C

Fryns J P , Kleczkowska A, Kubie6 E , P e t i t P , Haspeslagh M a

Fragile X and Non-Disjunction 107

Fryns J P , Kleczkowska A, Kubie6 E , Van den Berghe H (1984a): Cytogenetic f ind ing i n moderate and severe mental r e t a r d a t i o n . Acta Paed ia t r Scand Suppl 313:2-23.

Fryns J P , Kleczkowska A , Wolfs I , Van den Berghe H (1984b): K l i n e f e l t e r syndrome and two f r a g i l e X chromosomes. C l i n Genet 26:445-447.

Glover TW (1981): FUdR induct ion of the X-chromosome f r a g i l e s i t e : Evidence f o r the mechanism of f o l i c acid and thymidine i n h i b i t i o n . Am J Hum Genet 33:234-242.

Jacobs PA, Brown WT (1987): Personal communication. Kgkhb'nen M (1983): Marker-X-associated mental r e t a r d a t i o n .

Observation of t h e X-chromosome. C l in Genet 23:234- 235 A.

O'Brien MM, Padre-Mendoza T , Pueschel SM (1982) : Maternal nondis junct ion of f r a g i l e X chromosome r e s u l t i n g i n K l i n e f e l t e r syndrome. Am J Hum Genet 35: 146 A.

P u r r e l l o M, F i l i p p i G, Rocchi M, Archidiacono N, Rinaldi A, Romani M, Kekish 0, Moncrieff P , S in i sca l co M (1987): Screening for s i t e s of meio t ic recombination along X-chromosome p a i r s der ived from maternal non-disjunc- t i o n . Hum Gene Mapping I X , i n p ress .

47,XXY plus f r a g i l e X a t Xq27.3 due t o pa t e rna l non- d i s junc t ion , Am J Hum Genet Supplement 39: A100.

Steinbach P , Barbi G, Bo l l e r TH (1982): On t he frequency of te lomeric chromosomal changes induced by c u l t u r e condi t ions s u i t a b l e fo r f r a g i l e X expression. Hum Genet 61:160-162.

Suther land GR, Baker E (1986): Induct ion of f r a g i l e s i t e s i n f i b r o b l a s t s . Hum Genet 38:573-575.

Wilmot PL, Shapiro LR, Duncan PA (1980): The Xq27 f r a g i l e s i t e and 47,XXY. Am J Hum Genet 32:94 A.

Schnur RE, Ledbet ter D H , Nussbaum RL (1986): A family with a

Received for publication August 3, 1987; revision received October 22, 1987.