Am JHum Genet 33:752-761, 1981

Fragile X Syndrome: Search for PhenotypicManifestations at Loci for Hypoxanthine

Phosphoribosyltransferase andGlucose-6-Phosphate Dehydrogenase

CRISTINA MARENII AND BARBARA R. MIGEON2

SUMMARY

The subjects of this study were individuals with the form of X-linkedmental retardation that is associated with the presence of a cytologicallyvariant X chromosome having a secondary constriction or "fragile site" atXq 27-28 (Fra X). Studies were carried out to test the hypothesis thatdeletions or modifications at neighboring loci occur as a consequence ofevents at the fragile site. Skin fibroblasts and peripheral blood lympho-cytes from affected males were analyzed with respect to the expression oftwo X-linked enzymes: glucose-6-phosphate dehydrogenase (G6PD) andhypoxanthine phosphoribosyltransferase (HPRT); loci for these enzymesare known to be located in the region of the fragile site. Although thenumber of cells resistant to thioguanine (HPRT-deficient) obtained fromsome cultures from one Fra X male and blood cells of another was greaterthan expected, the frequency of these cells was not increased in culturesfrom other Fra X males. Furthermore, our results indicate that the G6PDactivity and electrophoretic mobility in Fra X males is similar to that innormal cells, thus providing no evidence for the loss of the long-armtelomere in the fragile X syndrome.

Received December 18, 1980; revised February 15, 1981.This work was supported by grant HD-05465 from the National Institutes of Health. C. M. is the

recipient of a Fogarty International Fellowship.Department of Hematology, University of Genoa, School of Medicine, Genoa, Italy.

2 Department of Pediatrics, CMSC 10-04, The Johns Hopkins Hospital, 601 N. Broadway, Baltimore,MD 21205. Request reprints from B. R. M.

© 1981 by the American Society of Human Genetics. 0002-9297/81/3305-0010$02.00

752

FRAGILE X SYNDROME

INTRODUCTION

The fragile X syndrome is a common form of mental retardation associated with amarker X chromosome [1]. This chromosome has an unusual secondary constric-tion at the distal end of the long arm (Xq27-28) and appears to be satellited. Themarker X is present in leukocytes of affected males and some carrier females, but itsexpression is variable depending upon the culture medium used [2-4]. Althoughthere have been difficulties in demonstrating this phenomenon in cultured fibro-blasts, it is likely that the abnormality underlying the X-linked mental retardation isnot confined to lymphocytes alone, and, in fact, the fragile site can occasionally beinduced in fibroblasts [5].

It is not clear whether the fragile site is the locus responsible for the associatedX-linked mental retardation or reflects an underlying metabolic abnormality atanother X-linked locus. The fragile site may be attributable to viral modification orabnormal chromatin structure secondary to some metabolic deficiency duringDNA synthesis [2], but no direct investigations have been carried out to test thesehypotheses. If the fragile site represents a chromosomal break or a displacement dueto uncoiling of DNA, we would expect that neighboring loci on the X might beaffected. Hypoxanthine phosphoribosyltransferase (HPRT) (E.C.2.4.2.8.) and glu-cose-6-phosphate dehydrogenase (G6PD) (E.C.1.1.1.49) have been mapped toXq26-27 [6-8] and Xq28 [8], respectively. Therefore, we have examined the pheno-typic expression at these two loci in fibroblasts as well as in blood cells derived fromindividuals with the syndrome. Our strategy was to look for variants at these lociusing selection for deficient cells at the HPRT locus and determination of activity byspectrophotometric assay, gel electrophoresis, and histochemical methods at theG6PD locus.

MATERIALS AND METHODS

Cells

Skin fibroblasts were isolated from four affected males and three carrier females whoselymphocyte cultures had previously revealed the presence of the fragile X (Fra X) chromo-some (table 1). Strains nos. 241, 242, and 337 were isolated in our laboratory; strains nos. 351,352, 353, and 354 were kindly supplied by Dr. P. Jacobs in Hawaii [9]. Fibroblast culturespreviously established in our laboratory from normal subjects were used as control cells. Skinfibroblasts carrying the Mediterranean variant of the enzyme were used for G6PD studies.Heparinized blood samples from subjects with demonstrated Fra X as well as from controlswere used to establish phytohemagglutinin (PHA)-stimulated lymphocyte cultures or forstudies of erythrocyte enzymes. Fibroblasts and blood samples generally came from differentFra X males, except for subject A. J. (337) for whom both specimens were available to us.

Media

Fibroblast and lymphocyte cultures were maintained at 370C in 5% CO2 in medium(Eagle's MEM, Gibco, Grand Island, N.Y.) supplemented with 15% fetal calf serum (FCS),nonessential amino acids, L-glutamine, pyruvate, penicillin-streptomycin, and fungizone(hereafter referred to as MEM). For some of the lymphocyte studies, the medium wasmodified as follows: folic acid was omitted and 5% rather than 15% FCS was added. Thismedium (hereafter referred to as MEM-FA) increases the frequency of the demonstrablefragile sites [2].

753

754 MARENI AND MIGEON

TABLE I

CHARACTERISTICS OF SUBJECTS WHOSE FIBROBLAST CULTURES WERE STUDIED

9s lymphocytesSubject Age with Fra X G6PD isozyme

241. Affected male .................................... 45 16, 11.2*B337, Affected male .................................... 2921 A351, Affected male .................................... 27 25, 11.6*B352, Affected male .................................... 32 27, 17*B242, Dull female, sister of 241 .......................... 42 1.5*B353, Dull female, sister of 351 .......................... 32 41, 21.4*B354, Dull female. sister of 351 .......................... 29 22,6.5. 12.7*B257, Control. .7 * B333, Control .12 ... B125, Control .30 - B107, Control............................................. 44 B80, Control .38 *- B

332, Control .12 ... B92. Control. .40 * Med.

* Values taken from [9].

Selection for HPRT Variants

For selection of variants in fibroblast cultures, cells were plated at two density levels (103and 5 X 103/60 mm dish) into MEM containing 6 X 105 M 6 thioguanine (6TG). After2-weeks incubation, the number of 6TG-resistant (6TGR) clones was determined. Althoughmost colonies were fixed and stained with methylene blue (MB), some of the 6TGR cloneswere isolated with cloning cylinders, transferred to individual 35-mm dishes, and propagatedin medium containing 6TG. Cloning efficiency was determined by plating small numbers offibroblasts into a series of dishes containing nonselective medium and counting the numberof macroscopic clones 10-12 days later.

Selection of variants among PHA-stimulated lymphocytes was carried out according tothe method of Strauss and Albertini [10]. Lymphocytes isolated from whole blood bycentrifugation in Fycoll Hypaque were resuspended at a density of 106/ml in 1.0 ml ofnonselective medium (MEM and MEM-FA) as well as in selective medium containing2 X 10-1 6TG. After 24-hrs incubation, I ,uCi 3H-thymidine (Amersham, 25 Ci/m mol)was added and incubation was resumed for an additional 12 hrs. Nuclei were prepared asdescribed [10]. Of the final nuclei suspension, 0.025 ml was gently added to each slide,reserving an aliquot for determination of cell number. Slides were autoradiographed andstained with 1% Giemsa. The percentage of labeled nuclei in selective and nonselectivemedium was counted under the light microscope, and the frequency of 6TGR cells wascalculated as the ratio of the percentage of labeled nuclei in selective medium to that innonselective medium. All determinations were carried out in duplicate, and values representthe mean of the two specimens.

Selection for Ouabain-Resistant (OUAR) Fibroblasts

This study was carried out in MEM containing 10-6 M ouabain [11]. Cells were plated at106/100 mm dish. Twenty days after plating, cells were fixed, stained with MB, and thenumber of colonies determined.

G6PD and 6-Phosphogluconate Dehydrogenase (6PGD) Spectrophotometric Assay

Fibroblasts were trypsinized and washed twice with 0.9% saline. The cell pellet wasresuspended in 300 pl of 10 MuM NADP and 25 mM ,f-mercaptoethanol and subjected to three

FRAGILE X SYNDROME

cycles freeze-thawing. Red blood cells, in specimens sent by mail, were washed twice with0.9% saline and hemolyzed in 5 vol 10 ,uM NADP and 25 mM f3-mercaptoethanol. The lysatewas centrifuged at 27,000 g, and the supernatant was assayed for G6PD [ 12] and 6-phospho-gluconate dehydrogenase (6PGD) (E.C. 1.1. 1.44) [13]. Assays were performed in a Beckmanspectrophotometer at 250C.

G6PD Electrophoresis

This assay was performed on cellucose acetate gel (Titan III plates, Helena Labs) inTris-glycine buffer, pH 9.2, 365 V for 25 min. Cells were removed from dishes with a rubberpoliceman in 50 Ml of 10 MM NADP, sonicated, and the extract applied to the gel [14].

G6PD Histochemical Staining

Fibroblasts were plated at 2 X( 102/60 mm dish. After 12 days, clones were stained in thedark at 370C with a staining mixture containing NADP, glucose-6-phosphate, nitro bluetetrazolium, and phenazine methosulfate, according to Wajntal and DeMars [15]. Afterfixation in formalin, dishes were scored under the microscope for staining characteristics ofthe colonies.

HPRT Autoradiography

6TGR clones were exposed to medium containing 5 ,uCi of 3H-hypoxanthine (New EnglandNuclear, Boston, Mass., 3.6 Ci/m mol). Autoradiographs were obtained, and clones werescored for the incorporation of labeled hypoxanthine [16].

Chromosome Studies

Chromosome preparations were stained for Q banding [17], C banding [18], and Rbanding [19].

RESULTS

Studies of HPRT Locus

To determine the frequency of mutants at the HPRT locus, we have selectedHPRT-deficient variants using 6TG, a purine analog that inhibits the proliferationof cells with normal enzyme activity [20]. To avoid contact feeding that interfereswith the recovery of mutants, we plated fibroblasts at low density [21]. Table 2shows the results of6TG selection in fibroblasts. No 6TGR clones were obtained indishes plated with cells from nonaffected individuals. On the other hand, clonesresistant to 6TG were obtained in cultures from three affected males and one carrierfemale. In some experiments, the frequency of 6TGR clones was greater than 10-6,that expected for normal fibroblasts [22]. However, the results varied considerably.Frequencies greater than 10-6 were not observed in all Fra X males nor were resultsreproducible in repeat experiments from the same Fra X male. Autoradiographs of6TGR clones from one subject showed lack of incorporation of 'H-hypoxanthineand, therefore, absence of HPRT activity.

Similar results were obtained when 6TG selection was carried out in PHA-in-duced cultures of blood lymphocytes. Table 3 shows that there was little differencebetween affected males and controls, except for values from one Fra X male. Theresults were the same even when cells were cultured in medium without folic acid.

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MARENI AND MIGEON

TABLE 2

FREQUENCY OF 6TGR CLONES IN FIBROBLASTS FROM AFFECTEDMALES, CARRIER FEMALES, AND CONTROLS

6TG-RESISTANT CLONESSUB- No. CELLS CLONING

SUBJECT CULTURE PLATED EFFICIENCY* No. Frequency

Affected mal241 At

B

C

337 AB

351

352

[es:................ .....3................ .....4................ 12

................ .....45

7................ .....3

34

................ 3.............

................ .....79

................ . 12

1314

Dull females:353

354242 A

B

Controls:257

333

125

................ 9

11................ .. 7

3

3

5X5X3X5XIX8X6X5X5X5X9X3X4X3X4X5X

34366

3

4

5

67

................ .... 3

4

4

X

X

x

X

X

3X2XIX8X4X5X5X5X5X

10410'10410'

10'

10'

104

10'

10'105104104104104104

10-

.14

.03

.11

.02

.13

.19

.20

.30

.09

.14

.15

10410410410410'

.04

.01

.34

10410410'10'

10410410'

10'10'

0

0

0

-1000200160

0

0

0

0

20

0

40

0

0

0

.40

.40

.34

.09

.21

.15

.15

2 X 10-'2 X 10-'2 X 10--

1.1 X 10'5

6.7 X 10-'

1.3 X 10-4

0

0

0

0

0

0

0

0

0

* Clones obtained/no. cells plated.t Cultures established from different pieces ot original biopsy.

Studies of the G6PD Locus

Values for G6PD activity of fibroblasts and erythrocytes from affected males andcontrols are presented in tables 4 and 5. Specific activity of the enzyme in red cellsand fibroblasts from Fra X males was identical with that in normal cells andcontrasted with the low activity in fibroblasts with the Mediterranean variant of theenzyme. The ratio between specific activities of X-linked G6PD and autosomal6PGD is also similar between Fra X males and normal controls. Thirty-one clones

756

FRAGILE X SYNDROME

from two Fra X males (241 and 337), selected on the basis of 6TG resistance, wereanalyzed by electrophoresis for G6PD activity and showed normal enzyme activityand electrophoretic mobility (fig. 1). In addition, approximately 300 6TGR fibro-blast clones (10 dishes) from Fra X male 241 were analyzed histochemically forG6PD. Each of the colonies was stained, thus none was obviously deficient inG6PD.

Studies of Autosomal Locus Resulting in Ouabain Resistance

To determine if there was an increased frequency of variants at a non-X locus, weselected for variants at the autosomal locus associated with ouabain resistance andvariant Na'-K'-ATPase activity. OUAR variants are expressed following single-stepselection [24].The results of ouabain selection in two affected males are reported in table 6. No

OUAR clones were obtained in 2 X 107 cells. This observation is consistent withfrequencies of spontaneous variants of less than 5 X 10-7 reported for normal cells[11, 24].

DISCUSSION

The basis for the fragile X associated with X-linked mental retardation has notbeen elucidated. We have carried out studies to test the hypothesis that the continui-ty of the marker chromosome has been disrupted at the level of the fragile site. Weexpected that breakage induced at the fragile site might affect the expression ofneighboring loci. Our studies were not confined to blood cells in which the Fra Xhas been observed. We also examined fibroblasts in which the Fra X is only rarelydetected to determine if cells expressed the mutation in absence of the cytologicalmarker.

TABLE 3

FREQUENCY OF 6TGR LYMPHOCYTES IN FRA X MALES AND CONTROLS

RELATIVE* FREQUENCY% LABELED NUCLEI IN % LABELED NUCLEI IN OF 6TGRNONSELECTIVE MEDIUM 6TG MEDIUM CELLS X 10-4

SUBJECT MEM MEM-FA MEM MEM-FA MEM MEM-FA

Fra X males:W. G. ................... 5.1 1.8 .006 0 12G. S ................... 25.5 17.9 .108t .110 42 61J. S ................... 19.9 21.6 .003 .017 1.5 7.9H. G ................... 13.2 12.7 .009 .006 6.8 4.7A. J. (337) ............... 23.9 14.3 .007 .009 2.9 6.3R. C. ................... 23.2 21.6 .009 0 3.9K. C ................... 22.4 14.9 .004 .009 1.8 6.0

Controls:J. N ................... 20.8 24.8 .035 .011 17 4.4L. S ................... 18.4 17.4 .003 .004 1.6 2.3B. 0 ................... 16.6 12.6 0 0 ... ...

* %o labeled nuclei in 6TG/%o labeled nuclei in nonselective medium.t Duplicates were .130 and .087.

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MARENI AND MIGEON

TABLE 4

SPECIFIC ACTIVIY OF G6PD AND 6PGD IN SKIN FIBROBLASTS FROM FRA X MALES AND CONTROLS

G6PDSubject Subculture G6PD U*/mg protein 6PGD U/mg protein 6PGD

Fra X males:241 A ... 5 .174 .016 10.9

6 .129 ... ...

B 6 .187 .015 12.57 .159 .018 8.88 .133 .019 7.0

C ... 5 .163 .017 9.66 .142 .023 6.2

337 4 .179 .029 6.2352 17 .198 .020 9.9

Mean + SD ..................... .163 + .024 .020 + .004 8.9 + 2.3

Controls:G6PD B 107 44 .192

5 .156 .029 5.46 .171 .014 12.2

80 6 .145 .034 4.37 .128 .030 4.3

332 4 .178 .034 5.2333 44 .218 .020 10.9257 5 .149

Mean ± SD ..................... .167 ± .029 .027 + .008 7.0 + 3.5

G6PD Med. 92 4 .026 .023 1.16 .031 .017 1.8

Mean ..028 .020

* pmol/min.

TABLE 5

SPECIFIC ACTIVITY OF G6PD IN ERYTHROCYTES FROM FRA X MALES AND CONTROLS

G6PD

Subject G6PD U*/g Hb 6PGD U/g Hb 6PGD

Fra X males:A. J. (337) 5.1 3.3 1.5K. C. ............5.4 3.6 1.5R. C ................. 5.2 3.7 1.4

W. G ................. 6.3 4.4 1.4G. S ................. 5.9 4.6 1.3J. S ................. 7.0 6.0 1.2H. G ................. 6.3 4.9 1.3Mean + SD ....... 5.9 + .7 4.3 + .9 1.4 ± .1

Controls:B. 0 ................. 7.2 4.3 1.7T. H ................. 6.3T.U ................. 7.4J. V. ................. 6.1 4.6 1.3L. S ................. 6.2 5.0 1.2Mean ± SD 6.6 ± .6 4.6 + .3 1.4 + .3

* pmol/min.

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FRAGILE X SYNDROME

Fic. 1.-Cellulose acetate electrophoresis of fibroblast extracts. Upper, G6PD: lower. LDH [23].G6PD B control (+); G6PD Mediterranean variant (-); 6TGH clones from 241 B (V).

The variability observed in visualizing the fragile site in lymphocyte culturesmight, in fact, be attributable to loss of the X chromosome telomere. Because theG6PD locus is distal to the fragile site, it seemed likely that a break at the fragile siteresulting in loss of the telomere could eliminate the G6PD locus as well. If this weretrue, then the activity of G6PD in these cells might be significantly reduced.The sensitivity of the assays used for G6PD studies is not sufficient to detect small

reductions in enzyme activity, but should be able to detect the loss of l/3 or moreactivity. The results of the assays carried out in fibroblasts and erythrocytes show nodifference and no greater variability in activity from Fra X cells than in controls(tables 4 and 5).

Fibroblast clones selected in 6TG from two Fra X males were assayed for theirG6PD activity because chromosome breakage might have been responsible for theHPRT deficiency in these cells. R-banded karyotypes of these clones did not showany obvious abnormalities of the X telomere; however, the quality of the prepara-tions made a definitive interpretation difficult. The presence of normal G6PD

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MARENI AND MIGEON

TABLE 6OUABAIN SELECTION IN FIBROBLASTS FROM FRA X MALES AND CONTROLS

Subject Subculture No. cells plated Cloning efficiency No. OUAR clones

Fra X males:337 ........ 6 2 X 107 .04 0241B ........ 7 2 X 107 .14 0

Controls:333 ........ 6 2 X 107 .06 0125 ........ 6 2 X 107 .13 0

activity in 6TGR clones from two individuals suggests that the segment of the Xdistal to the fragile site is still present in these cells. Although our observations donot eliminate the possibility that the telomere has been lost from the Xq in somecells, they do suggest that this event is not a frequent one.The increased frequency of6TGR variants observed in some subjects remains to

be explained. Not only is there variability between the subjects examined, but alsobetween different cultures of the same individual. HPRT-deficient clones wereobtained consistently at high frequency from one of the cultures established from aFra X male but not from the others established from the same biopsy. Q- andC-band polymorphisms in this male were used to determine the origin of thevariants and indicated that the 6TGR clones were not contaminants, but derivedfrom his own cells.The variability in our observations at the HPRT locus is reminiscent of the

variability in the frequency of the Fra X itself. It is possible that Fra X cells arepredisposed to an increased frequency of mutational events. However, we wereunable to demonstrate an increased frequency of mutation to ouabain resistance infibroblasts from two affected males, including the culture with the high frequency ofHPRT variants (241 B). In any event, increased susceptibility to mutations stillmerits consideration. Our results, while providing little evidence for consistentabnormalities at G6PD and HPRT loci, do not exclude the possibility that modifica-tions are taking place at other neighboring loci. It should be of interest to compareDNA from males with Fra X to that of normal males when DNA probes specific tothis region are available.

ACKNOWLEDGMENTS

We are indebted to Drs. Lawrence R. Shapiro and Patricia Howard-Peebles for providingblood for enzyme studies.

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