American Journal of Medical Genetics 43312-319 (1992)

Linkage and Risk Assessment in Fragile X Families Using New DNA Probes at Xq27 Nancy J. Carpenter, Jennifer Swartz-Boyd,Jane K. Prichard, and Tan Lam H A . Chapman Research Institute of Medical Genetics, Children’s Medical Center, Tulsa, OK

Until recently few polymorphic loci had been genetically mapped close to the fragile X syndrome locus [FRAXA]. Six polymorphic loci, DXS369, DXS297, DXS296, DXS304, IDS and DXS374, have now beenmappedclosertothe fragile X FRAXA than in the present study. We report the results of genetic linkage analysis of 32 fragile X [ fra (X)] families using 12 polymorphic loci including these new markers. Cytogenetic and molecular data were combined in two-point linkage analysis for the estimation of lod scores and carrier probabilities in potential carriers. Combined with results from previous studies, recombination fractions (0)

corresponding to the maximum lod scores (Z max) were obtained for each of the 12 loci versus FRAXA. Recombination fractions between marker loci in the families were also calculated. The data were evaluated to determine the efficacy of using the strategy suggested by Suthers et al. (1991a) for molecular studies in fra(X) families. The large proportion of females heterozygous for at least one locus (83%) and of females heterozygous for flanking loci (60%) indicate that this is a very useful diagnostic strategy. Use of these new marker loci substantially changed the carrier

Received for publication October 1,1991;revision received January 16,1992.

Address reprint requests to: Dr. Nancy J. Carpenter, H.A. Chapman Research Institute of Medical Genetics, Children’s Medical Center, 5300 East Skelly Drive, T u l s a , OK 74135. 0 1992 Wiley-Liss, Inc.

DNA Studies in Fragile X Syndrome r i s k estimates f o r members o f 7 o f t h e 32 families from t h e r i s k

estimates p r e v i o u s l y c a l c u l a t e d on t h e b a s i s o f less c l o s e l y l i n k e d p r o b e s available p r i o r t o 1989. KEY WORDS: Fragile X syndrome, DNA markers, l i n k a g e a n a l y s i s

INTRODUCTION The polymorphic l o c i u s e d p r i o r t o 1989 f o r l i n k a g e s t u d i e s o f f r a ( X ) f a m i l i e s were F9, D X S 1 0 5 , DXS98,'DXS52 and D X S 1 5 . However, t h e recombinat i o n f ract i o n s f o r t h e s e markers w i t h FRAXA a r e l a r g e (12-20%) [Mandel e t a l . , 19893 and t h e c a r r i e r r i s k estimates a r e t h e r e f o r e less r e l i a b l e t h a n t h e y would have been had t i g h t l y l i n k e d markersbeenavailable f o r study. R e c e n t l y , 5 polymorphic l o c i DXS369, DXS296, DXS297, DXS304 and I D S - were r e p o r t e d t o have r e c o m b i n a t i o n f r a c t i o n s o f 5%o r less w i t h FRAXA [ O o s t r a e t a l . 1990; S u t h e r s e t a l . , 1989; S u t h e r s e t a l . , 1991b; V i n c e n t e t al., 19891. DXS374 was a l s o r e p o r t e d t o be p r o x i m a l t o DXS52 [ P a t t e r s o n e t a l . , 19893. One combined l i n k a g e s t u d y o f t h e s e n e w l o c i h a s b e e n r e p o r t e d [Suthers e t a l . , 1991aI. S t r a t e g i e s f o r molecular d i a g n o s t i c s t u d i e s i n f r a ( X ) f a m i l i e s have been proposed which recommend t h e o r d e r i n which polymorphic l o c i s h o u l d be s t u d i e d f o r l o c i available p r i o r t o 1989 and f o r t h e new l o c i [ S u t h e r l a n d and Mulley, 1990;

Suthers et al., The

313

1991aI.

present

study of

32

f r a ( X ) f a m i l i e s w a s performed t o provide l i n k a g e data f o r 5 o f t h e 6 l o c i t i g h t l y l i n k e d t o FRAXA,

t o r e e v a l u a t e r i s k estimates f o r c a r r i e r s t a t u s i n r e l a t i v e s , and t o assess t h e u t i l i t y o f t h e s t r a t e g y proposed by S u t h e r s e t a l . E1991aI f o r d i a g n o s t i c t e s t ing. MATERIALS AND METHODS P e d i g r e e and C y t o g e n e t i c Analysis T h e f r a ( X ) syndrome w a s as-

c e r t a i n e d i n 32 families by pedigree a n a l y s i s , by c l i n i c a l f i n d i n g s and by d e m o n s t r a t i o n o f

fragilesiteexpressionatXq27.3. The p e d i g r e e s o f 26 o f t h e f a m i l i e s have been r e p o r t e d [ C a r p e n t e r , 1991; Johnson e t a l . , 19911. C y t o g e n e t i c a n a l y s e s were performed on 1 4 7 i n d i v i d u a l s f r o m p e r i p h e r a l lymphocytes grown i n m o d i f i e d R P M I 1640 w i t h o u t f o l i c acid, R P M I supplemented with 5-fluorodeoxyuridine M) and R P M I supplemented w i t h t h y m i d i n e ( l ( r 4M) F i f t y t o 1 0 0 c e l l s i n males and a t l e a s t 1 0 0 c e l l s i n females w e r e examined.

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Molecular A n a l y s i s A t o t a l o f 326 r e l a t i v e s w a s s t u d i e d . Genomic DNA w a s ext r a c t e d from 20 m l o f whole b l o o d

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or EBV-transformed lymphoblastoid lines using the method of Aldridge et al. [19841 or a nucleic acid extractor (Applied Biosystems) DNA was digested with restriction endonucleases according to the manufacturer's instructions. The cleaved DNA was size fractionated by electrophoresis using 0.7-1.0% agarose gels and Southernblots were madeusing Genescreenplus orMSI managraph nylon membranes. DNA probes were radiolabelled using 32P-dCTP by nick translation. The membranes were hybridized under standard conditions and exposed for autoradiography at -7OOC. The 12 probes used to detect restriction fragment length polymorphisms (RFLPs) are shown in Table I.

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Linkage Analysis The MLINK program of the LINKAGE computer program was used to calculate two-point LOD scores [Lathrop and Lalouel, 19841. Individuals were entered as affected if they expressed the fragile site at Xq27.3 in 1% or more of the cells or if they were mentally impaired.Penetrances of 56% in females and 80% in males, a gene frequency of 6 X l U 4 ,and a mutation rate of 2.4 X l ( r 4 were used.

RESULTS Therewere 67 affectedmales and 24 males at 20% risk. There were also 57 obligate carrier

TABLE I . R e s t r i c t i o n F r a g m e n t L e n g t h P o l y m o r p h i s m s Used i n t h e L i n k a g e S t u d y Locus

Probe

Enzyme

F9 DXS105

Pl

Taq I Taq I Pst I MspI XmnI Hind1I I XmnI TaqI MspI TaqI

DXS98 DXS369 DXS297 DXS296 DXS304 DXS374 DXS52 DXS134 DXS15 F8C

cx55.7 55E 4D-8 RN1 VK23B VK2 1 A VK21C U6.2 1Al

St14 cpX67 DX13 p114.12 p482.6

Taq Taql

MspI BglII BclI Xba I

A l l e l e s (kb)

1.8/1.3 4.5/3.2 16/10 25/7.8 1.25/1.1 10.5/9.5 10.3/6.6 10.9/9.9 12.7/9.9 7.0/3.3 4.5/4.0 Many 3.7/3.4 5.8/2.8 1.2/0.9 6.2/4.8

PIC

Reference

0.33 0.11 0.48 0.30 0.48 0.34 0.49 0 -23 0.31 0.36 0.26 0.80 0.49 0.47 0.41 0.48

Camerino e t a l . [1984] H o f k e r e t a1.119871 B o g g s and N u s s b a u m [ 1 9 8 4 ] Oostra et a l . [1990] Hyland e t a l . [ 1 9 8 9 ] S u t h e r s e t a l . [19891 Dahl e t a l . [ 1 9 8 9 ] P a t t e r s o n et a1.[1989] Oberlg e t a 1 . [ 1 9 8 5 ] Hofker et a1.[1987] Drayna e t a 1 . [ 1 9 8 4 1 G i t s c h i e r e t al. [ 1 9 8 5 1 Wion e t a l . [ 1 9 8 7 ]

DNA Studies in Fragile X Syndrome

females and 66 females at 20-40% risk. Of the males at risk, 18 were found to have a low risk, 3 had a high risk (>15%) of being non-penetrant, and 3 were inconclusive. Of the females at risk, 44 were determined to have a low risk (88%), and 6 were inconclusive. Prenatal diagnosis was carried out for 4 male fetuses and 3 female fetuses. One male fetus and one female fetus had high risks to have the fra(X) mutation and were cytogenetically positive. The remainder had low risks and were negative. The use of these new loci substantially changed the carrier risk estimates for 13 females in 6 families from the risks previously calculated on the basis of less closely linked probes prior to

1989 (Table 11). In such cases, the new loci provided informative flanking loci allowing the recognition of or the localization of a recombination event with respect to the FRAXA locus.

As shown in Table 111, twopoint analysis produced recombination fractions of 6% or less for each of the five new marker loci with FRAXA. The results of two-point analysis for all polymorphic loci are presented in Table IV and show no substantial deviation from those reported by Suthers et al. [1991al. An evaluation of the strategy of Suthers et al. [1991al for linkage analysis usingthemarker loci DXS369, DXS297, DXS296 and DXS304 showed that 88 of 116 females (76%)were heterozygotes

TABLE 11. Comparison of Carrier Risks Based on 6 Loci vs 12 Loci

Previous risk 16 loci)

Current risk (12 1 0 c a

Females with increased risk FX19.5 FX26.6 FX30.7 FX39.3

.44 .05 .06 .06

.77 .87 1.00 .16

.88 -80 .44 .22 .22 .10 * 12

.06

Linkage and risk assessment in fragile X families using new DNA probes at Xq27.

Until recently few polymorphic loci had been genetically mapped close to the fragile X syndrome locus [FRAXA]. Six polymorphic loci, DXS369, DXS297, D...
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