Am. J. Hum. Genet. 48:460-467, 1991

Genetic Mapping of New DNA Probes at Xq27 Defines a Strategy for DNA Studies in the Fragile X Syndrome G. K. Suthers,*,l J. C. Mulley,* M. A. Voelckelt N. Dahl,§ M. L. Vaisanen,II P. Steinbach,# 1. A. Glass,**,2 C. E. Schwartz,tt B. A. van Oost,$: S. N. Thibodeau,§§ N. E. Haites,IIII B. A. Oostra,## R. Gine,*** M. Carballo,ttt C. P. Morris, t J. J. Hopwood,t and G. R. Sutherland* *Department of Cytogenetics and Molecular Genetics and tLysosomal Diseases Research Unit, Department of Chemical Pathology, Adelaide Children's Hospital, Adelaide; $Centre de Diagnostic Prenatal, H6pital d'Enfants de la Timone, Marseilles; §Department of Medical Genetics, University of Uppsala, Uppsala; IlDepartment of Clinical Genetics, Oulu University Central Hospital, Oulu, Finland; #Department of Clinical Genetics, University of Ulm, Ulm; *'Department of Medical Genetics, University of Glasgow, Glasgow; ttGreenwood Genetics Center, Greenwood, SC; $Department of Human Genetics, University Hospital, Nijmegen; §§Department of Laboratory Medicine, Mayo Clinic, Rochester, MN; IllAberdeen Medical Genetics Group, Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen; ##Department of Cell Biology and Clinical Genetics, Erasmus University, Rotterdam; * * *Institute of Medical Genetics, University of Zurich, Zurich; and tttMolecular Genetics, Consejo Superior de Investigaciones Cientificas, Barcelona

Summary The fragile X syndrome is the most common cause of familial mental retardation and is characterized by a fragile site at the end of the long arm of the X chromosome. The unusual genetics and cytogenetics of this X-linked condition make genetic counseling difficult. DNA studies were of limited value in genetic counseling, because the nearest polymorphic DNA loci had recombination fractions of 12% or more with the fragile X mutation, FRAXA. Five polymorphic loci have recently been described in this region of the X chromosome. The positions of these loci in relation to FRAXA were defined in a genetic linkage study of 112 affected families. The five loci-DXS369, DXS297, DXS296, IDS, and DXS304 -had recombination fractions of 4% or less with FRAXA. The closest locus, DXS296, was distal to FRAXA and had a recombination fraction of 2%. The polymorphisms at these loci can be detected in DNA enzymatically digested with a limited number of restriction endonucleases. A strategy for DNA studies which is based on three restriction endonucleases and on five probes will detect one or more of these polymorphisms in 94% of women. This strategy greatly increases the utility of DNA studies in providing genetic advice to families with the fragile X syndrome.

Introduction

Mutations of genes on the human X chromosome are a common cause of mental retardation. The incidence of X-linked mental retardation is approximately 1 /

Received August 9, 1990; revision received October 18, 1990. Address for correspondence and reprints: Dr. G. R. Sutherland, Department of Cytogenetics and Molecular Genetics, Adelaide Children's Hospital, North Adelaide, SA 5006, Australia. 1. Present address: Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, England. 2. Present address: Queen Elizabeth Medical Centre, Birmingham Maternity Hospital, Birmingham, England. o 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4803-0005$02.00

460

600 male births (Sutherland and Hecht 1985). The fragile X syndrome is diagnosed in approximately a quarter of these boys, and it is the most common cause of familial mental retardation (Sutherland and Hecht 1985). Once a child has been diagnosed as having the fragile X syndrome, other members of the family frequently seek genetic counseling, either to determine their risk of being a carrier or to request prenatal diagnosis (Turner et al. 1986). The unusual pattern of inheritance of the syndrome (Sherman et al. 1985) and the lack of an unequivocal marker of those carrying the mutation (Nussbaum and Ledbetter 1986) have made it difficult to provide reliable risk estimates. The fragile X mutation, FRAXA, has not been isolated, but it is

DNA Studies in the Fragile X Syndrome

possible to determine an individual's risk of being a carrier, by DNA studies in affected families. The polymorphic loci generally used in studies of fragile X families have been F9, DXS1 05, DXS98, and DXS52 (Sutherland and Mulley 1990). Each of these loci has a recombination fraction of more than 12% with FRAXA (Mandel et al. 1989). In view of these large recombination fractions, an estimate of carrier risk was ideally based on the inheritance of any two polymorphisms which flank FRAXA. However, the advantage of using flanking polymorphisms is lost if recombination has occurred in the interval between the two polymorphic loci, making it impossible to determine which of the two polymorphisms was inherited with FRAXA. The ideal polymorphic locus for investigating families with the fragile X syndrome would be very close to FRAXA and have a high probability of being informative in the women of the family. In practice it is usually necessary to examine the family at a number of polymorphic loci and to seek some compromise between testing highly polymorphic loci far from FRAXA and testing closer but relatively uninformative loci. Strategies for DNA studies in fragile X families have been proposed (Heilig et al. 1988; Sutherland and Mulley 1990) which indicate the order in which the polymorphisms should be evaluated to provide the most efficient diagnostic service for the genetic counselor. Recently, three polymorphic loci-DXS369 (Oostra et al. 1990), DXS296 (Suthers et al. 1989), and DXS304 (Vincent et al. 1989)-were reported to have recombination fractions of less than 5% with FRAXA. The locus DXS296 showed no recombination with FRAXA in the first few families studied and is the locus closest to FRAXA. A combined linkage study of these three loci in fragile X families has not been reported. Recently two more polymorphic loci-DXS297 and IDS-were mapped to Xq27-q28 in normal families (Suthers et al., in press); the role of these loci in DNA studies of fragile X families has not been defined. A collaborative genetic linkage study of FRAXA and of these five polymorphic loci was performed using data from 112 families with the fragile X syndrome. The five loci all had recombination fractions of 4% or less with FRAXA. The closest locus DXS296, which had a recombination fraction of 2% with FRAXA. More than 98% of women were heterozygous at one or more of these loci. On the basis of these results, an efficient strategy for DNA studies in families with the fragile X syndrome is presented.

461 Methods Selection of Pedigrees

Data from a total of 153 families were obtained from 13 centers around the world. As affected individuals with no affected relatives could represent new mutations (Sherman et al. 1988), families were included in the analysis only if (a) at least one family member was mentally retarded and expressed the fragile site at Xq27 and (b) at least one further family member expressed the fragile site. Expression of the fragile site was assessed by culturing peripheral lymphocytes under specific conditions (Sutherland, in press). Expression of the fragile site in 1% or more of lymphocytes was regarded as positive. There was a general selection bias in favor of families having women heterozygous at loci near FRAXA; and there was a specific bias in favor of families having women heterozygous at DXS2 96, as this polymorphic locus was the closest to FRAXA. Details regarding the pedigrees are available on request. DNA Studies

Various DNA probes were used to identify RFLPs (Gusella 1986) at nine loci near FRAXA. The approximate positions of the loci on the X chromosome are shown in figure 1. Details of the polymorphisms are summarized in table 1. DNA samples were extracted X chromosome

Polymorphic

Loci

centromere

proximal _______F9 DXS105 DXS98 DXS369 DXS297

fragile site +

DXS296

distal Order of polymorphic loci down X chromosome. Figure I The positions of the loci and of the fragile site in relation to the chromosome bands are approximate.

Suthers et al.

462

Table I RFLPs Used in Linkage Study

Locus (probe) and Enzyme

F9 (pVIII): TaqI ................................. DXS1O05 (cX55.7): TaqI ................................. HindIII .............................. EcoRI ............................... StuI .................................. DXS105a (55E): PstI ................................... DXS98a (4D-8): M spI................................. XmnI ................................ DXS98a (4D8-B): BgllI ................................. DXS98a (4D-81V): XmnI ................................ DXS369a (RN1): XmnI ................................ TaqI ................................. DXS297a (VK23B): HindIII .............................. XmnI ................................ DXS296a (VK21A): TaqI .................................

BclI................................... DXS296a (VK21C): M spI................................. IDS (pc2Sl5): StuI .................................. TaqI ................................. DXS304a (U6.2): Many ................................ DXS3043 (U6.2-20E): BanI.................................. DXS52 (Stl4-1): TaqI ................................. BclI................................... a

Reference

PIC

Location

.38

Xq26.3-q27.1

Kidd et al. 1989

.11 .48 .40 .41

Xq27.1-q27.2

Kidd et al. 1989

Xq27.2

Kidd et al. 1989

.48 .30 .08

Schnur et al. 1989

.15

.08 .48 .24

Xq27.2-q27.3

Oostra et al. 1990 Oberle et al., in press

.34 .49

Xq27

Suthers et al., in press

.23 .23

Xq27.3-q28

Suthers et al. 1989 Yu et al. 1990 Suthers et al. 1989

.31 .50 .08

Xq28

Suthers et al. 1991

.36

Xq28

Dahl et al. 1989 Rousseau et al. 1990

.49

.80 .32

Xq28

Kidd et al. 1989

Polymorphisms are in linkage disequilibrium.

from lymphocytes or lymphoblastoid cell lines of individuals from the families by established methods (Maniatis et al. 1982). DNA samples were digested with an appropriate restriction endonuclease, electrophoresed in agarose gels, and transferred to nylon membranes by Southern blotting. Probes were radiolabeled with 32P and hybridized to the membrane-bound DNA samples. Details of each pedigree and the genotypes at each locus were sent to one of us (G.K.S.) for analysis. Linkage Analysis

All the genotype and pedigree data were checked by hand. Pedigrees having either a single affected individ-

ual or apparent non-Mendelian inheritance of a polymorphism were excluded. A total of 1,368 individuals from 112 pedigrees were included in the analysis. The number of families informative at each locus is shown in table 2. Two-point and multipoint linkage analyses were performed using the LINKAGE package of computer programs (version 5) (Lathrop et al. 1985; Lathrop and Lalouel 1988). The genetic parameters relating to FRAXA were as follows: mutant allele frequency (.0006), mutation rate (.00024 in males and .00048 in females), and penetrance of either mental retardation or fragile site expression (.80 among males and

DNA Studies in the Fragile X Syndrome

463

Table 2 Summary of Two-Point Linkage Analysis of Fragile X Locus, FRAXA, and Nearby Loci PEAK

RECOMBINATION FRACTION

SCORE

intervala)

Nb

.190 (.12-.28) .152 (.08-.24) .058 (.01-.15) .066 (.04-.12) .042 (.005-.18) .015 (.005-.04) .089 (.02-.23) .031 (.005-.10) .126 (.09-.17)

44 37 25 45 12C 67 16c 27 89

RECOMBINATION FRACTION

.00

FRAXA vs. F9 ......... -26.29 DXS105 ..... -19.68 DXS98 ...... 3.65 DXS369 5.04 DXS297 3.13 DXS296 30.75 IDS .34 7.57 DXS304 DXS52 ...... -21.42 .....

.....

.....

.........

.....

LOD

.01

.05

.10

.20

.30

.40

-8.85 -5.11 5.88 11.31 4.17 33.36 2.69 9.30 2.31

1.02 2.55 6.69 14.50 4.46 32.05 4.01 9.56 16.84

4.88 4.88 6.47 14.24 4.23 28.58 4.18 8.74 21.07

6.49 5.11 5.10 11.06 3.28 20.29 3.50 6.27 19.43

5.18 3.38 3.25 6.74 1.99 11.56 2.32 3.49 12.98

2.66 1.22 1.36 2.56 .68 4.08 1.02 1.09 5.43

6.49 5.40 6.70 14.62 4.46 33.45 4.19 9.67 21.45

(confidence

Approximate 90% confidence interval for the recombination fraction (Conneally et al. 1985). Number of families polymorphic at locus. c Locus was analyzed in a subset of families from Adelaide. a

b

.55 among females) (Sherman et al. 1985, 1988). If pedigree data indicated that an apparently normal individual of either sex was an obligate carrier, that individual was coded as affected for the linkage analysis. For two-point linkage analysis, LOD scores were calculated for recombination fractions between FRAXA and each of the polymorphic loci. For multipoint linkage analysis the genetic location of FRAXA was determined in relation to a known genetic map. The genetic map consisted of the positions of six polymorphic loci-DXS98, DXS369, DXS297, DXS296, IDS, and DXS304. The order of these loci down the X chromosome (fig. 1) has been determined independently by both physical mapping (Suthers et al. 1990) and genetic linkage studies (Suthers et al., in press). In the CEPH pedigrees the recombination fractions between these six loci have been estimated to be DXS98-(12.3% )-DXS369-(0% )-DXS297-(5.7% )DXS296-(0% )-IDS-(1 .2% )-DXS304 (Suthers et al., in press). The order of these loci together with the recombination fractions constituted the genetic map on which FRAXA was localized. Genotype data at one or more of these loci were available from 101 of the fragile X pedigrees.

in table 2. Recombination was observed between FRAXA and each of the loci. Details of both the pedigrees demonstrating recombination and of two-point linkage analysis of all pairs of loci are available. The locus closest to FRAXA was DXS296, which had a peak LOD score of 33.45 at a recombination fraction of 1.5% with FRAXA. This analysis incorporated the data from our earlier study (Suthers et al. 1989). Recombination between DXS2 96 and FRAXA was documented in three affected males from three different families. The adjacent locus IDS had a recombination fraction of 8.9% with FRAXA. The other locus distal to FRAXA, DXS3 04, had a recombination fraction of 3.1%. The proximal loci DXS369 and DXS297 had recombination fractions with FRAXA of 6.6% and 4.2%, respectively.

Results

This collaborative linkage study documents the genetic locations of nine polymorphic loci in relation to FRAXA. The relatively large recombination fractions noted between FRAXA and F9, DXS1 05, and DXSS2 (table 2) are similar to published values (Keats et al.

Two-Point Linkage Analysis

The results of two-point linkage analysis of FRAXA and each of the nine polymorphic loci are summarized

Multipoint Linkage Analysis

Multipoint LOD scores were calculated for various positions of FRAXA along the genetic map (fig. 2). The peak multipoint LOD score was 48.49. The corresponding location of FRAXA was 2.2 cM proximal to DXS296. Discussion

Suthers et al.

464 50

1_

45-

MULTI-

POINT LOD 40-

SCOR

35-

Ij-20 DXS98

I

T

I

-10

~

0

VI

DXS369 DXS297 DXS296 IDS DXS,04

GENETIC MAP (distances

in

cM)

Figure 2 Multipoint LOD scores for location of FRAXA that are plotted against genetic location along X chromosome. The background genetic map was derived from a linkage study of normal families (see Methods). The origin of the map was arbitrarily placed at DXS296. Distances along the map were derived from recombination fractions by using Haldane's formula (Ott 1985). DXS98 lay 20.2 cM proximal to DXS296; DXS369 and DXS297 lay 6.1 cM proximal to DXS296; IDS was placed coincident with DXS296; and DXS304 lay 1.2 cM distal to DXS296. Multipoint LOD scores for FRAXA location were calculated at 20 points in the interval DXS297-DXS296; calculations were performed at five points in each of the remaining intervals. The peak of the multipoint LOD score curve occurred 2.2 cM proximal to DXS296.

1989). The recombination fraction of 5.8% between FRAXA and DXS98 is similar to the value initially reported (Brown et al. 1987), but subsequent pooled studies have indicated that the recombination fraction is more likely to be 15% (Mandel et al. 1989). The larger value is also more consistent with the relative positions of these polymorphic loci in normal pedigrees (Suthers et al., in press). Two-point linkage analysis of FRAXA with the loci DXS369, DXS297, DXS296, IDS, and DXS304 indicated that the best estimates ofthe recombination fractions were all less than 10%. The recombination fractions between FRAXA and DXS369 and DXS304 were consistent with published values (Vincent et al. 1989; Oostra et al. 1990). The recombination fraction between FRAXA and IDS was estimated to be 8.9%. This value seems inconsistent with other data. Physical mapping studies have indicated that IDS lies between DXS296 and DXS304 (Suthers et al. 1990). However, these two loci had recombination fractions with FRAXA of only 1.5% and 3.1%, respectively

(table 2). This apparent discrepancy is probably not ofbiological significance but simply reflects a sampling fluctuation. IDS was studied in a small number of fragile X families, and the confidence interval for the recombination fraction was wide. Estimates of the recombination fractions between DXS98, DXS369, DXS297, DXS296, IDS, and DXS304, as determined by multipoint linkage analysis, do not differ in normal families versus fragile X families (authors' unpublished data). In normal families there was no recombination between IDS and DXS296 (Suthers et al., in press), and the true recombination fraction between FRAXA and IDS is likely to be less than 8.9%. Multipoint linkage analysis is statistically more efficient than two-point linkage analyses and provides a more accurate and precise genetic map (Lathrop et al. 1985). Estimates of the recombination fractions between the various loci and FRAXA were derived from figure 2 and are summarized in table 3. On multipoint linkage analysis, the five loci DXS369, DXS297, DXS296, IDS, and DXS304 all had recombination fractions of 4% or less with FRAXA. Only one other multipoint linkage study of the fragile X syndrome has used a sample of this magnitude. Brown et al. (1988) described a linkage study of 147 families. The closest polymorphic loci that were localized in that study were F9 and DXSS2, each of which has recombination fractions of more than 12% with FRAXA. This represents a major advance in the development of the genetic map near FRAXA and has immediate application in genetic counseling. Diagnosis by linkage is necessary for family members in whom the fragile X cannot be detected by cytogenetic analysis. An estimate of carrier risk based on the inheritance of any one of these polymorphic loci would be correct in at least 96% of cases. The inclusion of other pedigree or cytogenetic data in the analysis may reduce the carrier risk even further (Mulley et al. 1987; Sutherland and Mulley 1990). In the case of prenatal diagnosis, accurate classification of a fetus as a carrier by using closely linked markers does not accurately predict phenotype. The risk of mental impairment in a fetus with the fragile X is based on both the sex-dependent penetrance of mental impairment and the clinical status of the mother (Sutherland and Mulley 1990). This must be taken into account and must be carefully explained during genetic counseling. Fewer than 50% ofwomen are heterozygous at each of the five polymorphic loci close to FRAXA (table 1), and at first glance these polymorphisms might appear

DNA Studies in the Fragile X Syndrome

465

Table 3 Strategy for DNA Studies of Fragile X Families

Digest DNA with

Step 1 ..............

.......

Probe DNA witha

Taqlb StuI XmnI

Step 2 (if necessary) ............. Step 3 (if necessary) .............

Taqlb (reprobe) XmnI (reprobe) BanI MspI TaqI (reprobe) TaqI (reprobe) HindIII

VK21A (DXS296) pc2S15 (IDS) VK23B (DXS297) U6.2 (DXS304) RN1 (DXS369) U6.2-20E (DXS304) VK21C (DXS296) pc2S15 (IDS) RN1 (DXS369) VK23B (DXS297)

Recombination fraction with FRAXA 2% 2% 4% 3% 4% 3% 2% 2% 4% 4%

Distal Distal Proximal Distal Proximal Distal Distal Distal Proximal Proximal

Probes available from sources given in references listed in table 1. Enzyme/probe combinations of TaqI/VK21A and TaqI/U6.2 could be replaced with MspI/VK21C and MspI/U6.2. a

b

to be of little added value in studies of fragile X families. However, two factors argue against such a pessimistic conclusion. First, all five loci are close to FRAXA, and an accurate estimate of carrier risk can be made on the basis of the inheritance of just one polymorphism. The probability that a woman would be heterozygous for at least one of the loci is high. Second, a number of the polymorphisms can be detected by using the same restriction endonuclease, and it is possible to rapidly screen the polymorphisms that are close to FRAXA. An efficient strategy for DNA studies in families having the fragile X syndrome is presented in table 3. Step 1 involves digesting the DNA samples of family members with three different restriction endonuclease and using probes which identify polymorphisms at DXS296, IDS, and DXS297. The probability of a woman being heterozygous at one or more of these loci is 80%. In the event that a woman is not informative at these loci, the digested DNA samples may be reprobed to identify polymorphisms at DXS369 and DXS304 (step 2). Polymorphisms would be detected in a further 14% of women. When just three enzymes are used in conjunction with five probes, 94% of women would be heterozygous for at least one of these polymorphisms. The probability that a woman would be heterozygous for polymorphisms which flank FRAXA is 68%. Step 3 increases the proportion of women who would be polymorphic at one or more loci to more than 98%.

In presenting this diagnostic strategy, two cautions should be noted. First, careful cytogenetic examination remains crucial for avoidance of inaccurate diagnosis. A second fragile site, in normal men and women immediately proximal to the fragile site characteristic of the fragile X syndrome, has been documented (Ledbetter and Ledbetter 1988; Sutherland and Baker 1990). If the two fragile sites are not distinguished, an individual may be incorrectly classified as either having the fragile X syndrome or being a carrier, and subsequent genetic risk estimates based on DNA studies could be incorrect. Second, the fragile X syndrome is a complex genetic disorder. In all but the simplest of counseling situations, it is advisable to use appropriate computer programs (such as LINKAGE) to integrate the pedigree, cytogenetic, and DNA polymorphism data to provide accurate genetic risk estimates (Mulley et al. 1987). It is now possible to correlate physical distances (measured as kilobases of DNA) near FRAXA with genetic distances (measured as recombination fractions). DXS296 and IDS are separated by 800 kb of DNA, and in normal families there was no recombination between them (Suthers et al., in press). IDS and DXS304 are no more than 900 kb apart and had a recombination fraction of 1.2%. If this relationship between physical and genetic distances is maintained near FRAXA, FRAXA is approximately 2,000 kb proximal to DXS296. The recent cloning of the gene responsible for cystic fibrosis (Rommens et al. 1989)

466 has demonstrated that it is feasible to cover a distance such as this -and so to isolate the fragile X mutation itself.

Acknowledgments We would like to thank the many clinicians, genetic counselors, laboratory colleagues, and forbearing families who made this study possible. We acknowledge the advice and assistance of M. C. Pellisier, I. Boccaccio, U. Pettersson, J. 0. Ulmer, A. Smits, J.C.F.M. Dreesen, and A. Schinzel. The LINKAGE programs were provided by Dr. J. Ott of Columbia University. Dr. J. Suthers critically reviewed the manuscript. This work was supported by the National Health and Medical Research Council of Australia, the Adelaide Children's Hospital Research Foundation, CNAM and Association Francaise, the Swedish Medical Research Council, the Sigrid Juselius Foundation, Deutsche Forschungsgemeinschaft CDFG, and Grampian Health Board and Scottish Hospitals Endowments Research Trust.

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