clonal determination by the fragile x (fmrj) and ... · determination of clonality by southern...

[CANCERRESEARCH54, 5212-5216, October1, 19941

ABSTRACT

The polymerase chain reaction (PCR) clonality assay based on theprinciple of random X chromosome methylation in females provides apotentially important tool in both cancer research and diagnostics. Thisassay, however, has not been compared to the standard Southern blotassay and is limited by the rate of heterozygosity of the X-linked phosphoglycerate kinase (PGK) and androgen receptor genes, the only twogenes yet described with which this technique may be used. Using 46marrow and blood specimens from females with and without hematological malignancies, the PCR and Southern blot methods of clonality werecompared. In addition, a new technique based on the highly polymorphicfragile X (FMRJ)locus was examined. The rate ofheterozygosity was 25%for the PGK gene and 45% for the FMRJ gene. In the PCR assay, 7 of 8and 11 of 14 normal control specimens showed a polyclonal methylationpaftern in the PGK and FMRJ genes, respectively. Of the malignantspecimens, 17 of 17 and 17 of 18 showed a monoclonal methylation patternin the PGK and FMR1 genes, respectively. The Southern blot and PCRassay gave similar results with regards to the PGK gene. It is concludedthat the PCR and Southern blot clonality assays are comparable withregards to the PGK gene and that both the PGK and FMR1 genes may bereliably used in the determination ofclonality. The methods, however, arelimited by the skewed methylation patterns seen in hematological specimess in a significant number of normal females.

INTRODUCTION

Although clonal analysis based on X chromosome inactivation is apotentially powerful tool in diagnostic pathology, it currently hasseveral limitations. The original method described by Linder andGartler (1) and championed by Fialkow (2) was based on expressionpatterns of the X-linked enzyme glucose-6-phosphate dehydrogenase(1, 2). This technique was laborious and required that the femaletissue be heterozygous for glucose-6-phosphate dehydrogenase isotypes; the rate of heterozygosity allowed only a small fraction offemales to be examined effectively, eliminating any practicalapplication in routine practice.

Vogelstein et a!. (3) used a modified version of this test to examineneoplasia in females. The system was based on the principle ofdifferential methylation of the active and inactive X chromosomes andrequired the performance of Southern blots. Using probes to the PGK@gene and hypoxanthine phosphoribosyltransferase gene, 50% ofAmerican females were heterozygous for restriction fragment lengthpolymorphisms and, therefore, informative to study. However, thecomplex and time-consuming nature of this Southern blot assay didnot lend itself to practical application.

Recently, investigators have introduced a clonality assay that isbased on the PCR (4, 5). This technique uses differential X chromosome methylation in conjunction with the same restriction fragment

Received 5/18/94; accepted 7/27/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Laboratory

Medicine and Pathology, University of Minnesota Hospitals, Box 198UMHC, 420Delaware St. SE, Minneapolis, MN 55455.

2The abbreviationsused are:PGK,phosphoglyceratekinase,PCR,polymerasechainreaction; HUMAR.4,human androgen receptor.

length polymorphism in the PGK gene used in the Southern blotassays. The technique is applicable in approximately 30% of femalesheterozygous for this polymorphism. This PCR assay was recentlymodified to use the highly polymorphic X-linked androgen receptor(HUMARA) gene, theoretically increasing the number of informativefemales to 90% (6, 7).

The rapidity and sensitivity of this PCR technique allows practicalapplication in diagnostic pathology; however, comparative studies ofthe Southern blot technique and the newer PCR technique have notbeen systematically performed. In addition, the PGK and HUMARA

genes remain the only two X-linked loci to be used by this technique.The present investigations compare the PCR and Southern blot methods. A new highly polymorphic X-linked gene, the fragile X (FMRJ)gene, is introduced to clonality determination by the PCR methodology, further expanding the general applicability of this test.

MATERIALS AND METHODS

Control and Patient Specimens. With the approval of the University ofMinnesota Human Subjects in Research Committee, peripheral blood or bonemarrow samples were obtained from a total of 175 females. The controlspecimens were normal blood or marrow from volunteers and patients andcontained less than 20% malignant cells by morphology. The malignantspecimens were from female patients with various hematological malignancies; all of these specimens contained greater than 50% malignant cells bymorphology (Table 1).

Peripheral blood and bone marrow cells were lysed in ThE [10 mMTris-CI(pH 8.0), 100 mM NaCl, and 1 mM EDTAJ buffer in the presence of 1% sodiumdodecyl sulfate. High molecular weight DNA from the cells was furtherpurified by standard proteinase K treatment (Boehringer-MannheimBiochemicals) at a final concentration of 0.1 mg/ml. The specimens were precipitated inethanol after several phenol-chloroform extractions. RNase treatment wasfollowed by several more phenol-chloroform extractions and a final ethanolprecipitation.

Determination of Clonality by Southern Hybridization. DNA speci

mens were analyzed by Southern blot as described by Vogelstein et al. (3).Briefly, 10 ,.tg of DNA were digested with 50 units of PstI, according to themanufacturer's recommendations, for 4 h, followed by an overnight digestionwith BstXl (50 units) at 55Â°C.The digests were then ethanol precipitated,lyophilized, and redissolved in 50 @.dof TE [10 mM Tris (pH 7.5)-0.1 mMEDTA]. One-half of this DNA aliquot was further digested with 30 units ofHpaII at 37Â°C for 2 h. The resulting restriction fragments from each aliquot

were then electrophoresed on 1.5% agarose gels, transferred to nylon membranes, prehybridized, and hybridized to the 32P-labeled PGK probe accordingto the manufacturer's recommendations (GeneScreen Plus; Dupont, Inc.). Theprobe in this study is an 800-base pair BamHh/EcoRIfragment from the 5' endof the PGK gene cloned into the BamHIJEcoRIsites of pSP64 (8). Followinghybridization, the filter was washed with 2X standard saline citrate-0.1%sodium dodecyl sulfate and 0.7X standard saline citrate-0.1% sodium dodecylsulfate at 65Â°Cfor 20 mm each. The filters were exposed to KOdakXAR-5 filmat â€”85Â°Cfor 24 h to several days.

Determination of Clonality by the PCR. Fig. 1 and 2 illustrate the

principle of the PCR test in clonality determination using the PGK and FMRIgene, respectively.

PGK Gene. The procedure of Gilliland et aL (5) was followed. Briefly, theDNA samples were first screened for heterozygosity of the BstXI polymorphism in the PGK gene. This was accomplished by a two-stage PCR, followedby Bs:XI digestion of the amplified product. In the first stage, 0.05 @gof DNA

5212

Clonal Determination by the Fragile X (FMRJ) and Phosphoglycerate Kinase (PGK)Genes in Hematological Malignancies

Siew-Teh Lee, Ronald C. McGlennen, and Craig E. Litz1Departments of Laboratory Medicine and Pathology (S-T. L, R. C. M., C. E. LI and Medical Technology (S-T. LI, University of Minnesota Hospital, Minneapolis, Minnesota

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Table 1 Patient and controlspecimensHematological

diagnosisNo.of

casesPGK

geneFMRIgeneControls814Chronic

myeloid leukemiaChronicphase76Acute

myeloid leukemiaWithout differentiation(Ml)With differentiation (M2)Monoblastic (M5a)1

311

2Acute

lymphoblasticleukemia12Chronic

lymphocyticleukemia2Multiple

myeloma11Myelodysplastic

syndrome34Total2532

CLONAL DETERMINATION BY FMRI AND PGK GENES

To stop the reaction, 6@ of the amplified products were added with 4 piof loading buffer containing formamidelEDTA/xylene cyanol/bromophenolblue (Sequanase sequencing kit; U.S. Biochemicals, Inc.). The sample DNAwas heated for 3 mm at 95Â°Cimmediately before loading onto a 5% denaturingpolyacrylamide gel (40-cm length). Electrophoresis was carried out at a constant voltage of 40 V/cm for 3.5 h. The gel was dried and autoradiographed onX-ray film for 12-48 h at room temperature.

Data Analysis. Band intensities from the autoradiographs and the photographs of the ethidium bromide-stained agarose gels from both the FMRJ andPGK gene were quantitatedand analyzed densitometncally. Densitometry wasperformed on a digitahized video image of the illuminated autoradiograms orphotos using the Quick Capture frame grabber board and software (DataTranslation, Inc., Marlboro, MA) in conjunction with a MacIntosh hIcicomputer.

The values were expressed as the log of an allelic ratio (high-molecularweight band/low-molecular-weight band) from the HpaII-digested samplenormalized to the allelic ratio of the non-HpaII-digested sample, i.e. , R log[(high-molecular-weight HpaII/low-molecular-weight HpahI)/(high-molecular-weight No Hpalh/low-molecular-weight No Hpall)].

R reflects the change in allelic intensities between the HpaII and non-HpaIIdigested samples. This change is an indication of the methylation status of theX chromosomes in the specimen. Polyclonal patterns of X inactivation have Rs

close to 0. â€œSignificantdeviationâ€• from this value indicates a shift in allelicband intensity between the HpaII and non-HpaII-digested samples and amonoclonal pattern of X inactivation. Significant deviation is defined as Rsgreater or less than the 95% confidence interval of the Rs for the polyclonalcontrol samples; polyclonal control samples exhibiting extreme skewing werenot included in the confidence interval determination (10). The confidenceinterval was determined on the pre-log ratio values.

RESULTS

Plots of the Rs in all cases for the PGK and FMRJ loci are shownin Fig. 3 and 4, respectively. The polyclonal control values define the95% confidence interval for a polyclonal R defined in â€œDataAnalysis.â€•

PGK Gene. Of 108 specimens from females examined, 27 wereheterozygous for the BstXl polymorphism in the PGK gene; the rateof the heterozygosity was 25%. Two of the specimens contained aninadequate amount of DNA for study by both Southern blot and PCR;these were not examined further.

By Southern blot, the Rs of the polyclonal control samples variedfrom â€”0.12 to 0.22 (Fig. 3, â€¢,cases 1â€”8).The 95% confidenceinterval excluding the single skewed sample was â€”0.18to 0.10. Rsabove and below this range were considered to demonstrate a monoclonal pattern of X chromosome methylation (inactivation). The R ofone of the eight control specimens was outside of this range (Fig. 3,., case5); this specimen was from the peripheral blood of a 24-yearold healthy adult female. The Rs of the specimens defined as morphologically malignant (see above) ranged from â€”2.52to 3.10 (Fig.3, ., cases 9â€”25).All 17 of these specimens showed a R outside ofthe 95% confIdence interval for the polyclonal specimens (monoclonal X chromosome methylation pattern).

Results from the PCR method (Fig. 3, El) were comparable to theSouthern blot method. The PCR R values of the polyclonal controlsamples ranged from â€”0.13 to 2.00 (Fig. 3, El, cases 1â€”8).Thepolyclonal 95% confidence interval excluding the single skewed valuewas â€”0.27to 0.25; this was a larger confidence interval than that ofthe Southern blot assay. The PCR R from one of the eight controlspecimens was outside of this range (Fig. 3, El, case 5); this was thesame specimen that was outside the polyclonal range with the Southern blot assay. The Rs of the morphologically malignant specimensranged from â€”1.46 to 2.64 (Fig. 3, El, cases 9â€”25).All 17 malignantspecimens showed monoclonal Rs.

Fragile X (FMRJ) Gene. Of 78 females examined, 35 were heterozygotes for the FMRI gene as defined by the presence of two

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was added to a 100-g.tlaliquot containing 1X PCR Taq buffer [10 mMTris(pH8.3), 50 mM KC1, and 1.5 mM MgCl2], 20 pmol of each deoxynucleotide

triphosphate, 2.5 units ofTaq polymerase, and 20 pmol ofeach XL-l and XL-4oligonucleotide primer (Table 2). Mter an initial denaturation of 94Â°Cfor 3mm, PCR amplification was carried out by using the following parameters:94Â°Cfor 1 mm, 63Â°Cfor 45 s, and 72Â°Cfor 1 mm (35 cycles). This wasfollowed by an extension step of 72Â°Cfor 10 mm. For the second-stage PCR,5 pi of the first stage-amplified PCR product were mixed in the same reactionmixture, except two different primers, XL-2 and XL-3, were added in place ofXL-! andXL-4 (Table2). The amplificationsteps were repeatedas describedabove. After the PCR, the amplified DNA was ethanol precipitated andresuspended in 30 @.dof water. To screen for BstXI heterozygosity, 8 @lof theamplified PGK gene product were added to a total volume of 20 pi containing20 units of BstXI, 1 X BstXI reaction buffer, and 250 ng of 4174 RF DNA(Bethesda Research Laboratories, Bethesda, MD). 4,174 RF DNA is a 5386.-base pair supercoiled circular DNA having three BsIXI restriction sites andserves as an internal control to confirm the completion of BstXI digestion byproducing three restriction fragments of 962, 2179, and 2245-base pairs.Completeness of digestion was assumed when less than 5% of the intact 4174RF DNA was present. Enzyme digestion was carried out overnight at 55Â°C.Samples were electrophoresed on 1.5% agarose gels stained with ethidiumbromide. Photographs were taken of gels exposed to UV light.

To determine clonality by the PCR technique, two aliquots of 0.1 @.tgofgenomic DNA from BsIXI heterozygotes were digested with and without 20units HpaII overnight in a 20-pi reaction volume at 37Â°C.Heat inactivation ofHpahIwas then carriedout at 95Â°Cfor 10 mm. Five pi from the HpaII-digestedand undigested DNA aliquots (0.025 @tg)were then used in the PCR amplification, and BstXI digestion was as described above. Analysis of the sampleswas on 1.5% agarose gels as described above.

Fragile X (FMRJ) Gene. To determine clonality, 0.4 @gof genomic DNA

was incubated in the presence or absence of 20 units HpaLI in reaction bufferin a total reaction volume of 20 p.1at 37Â°Cfor 4 h. After heat inactivation ofHpaII at 95Â°Cfor 10 mm, the specimens were precipitated in ethanol andresuspended in 20 @.dof water.

PCRamplificationwas performedaccordingto Fu et a!. (9). Briefly, 5 pi(0.1 @g)of the HpaIh-digested or undigested genomic DNA samples werebrought up to 15 @lwith a fmal concentration of 10 mr.t Tris-HC1 (jH 8.3), 50mM KC1, 2 mM MgCl2, 0.2 @Mof each dATP, dTTP, and dCTP, 0.05 mMdGTP, 0.15 mM7-deaza-dGTP, 10% dimethyl sulfoxide, 0.2 mMof primers Cand F (Table 2). [32P]dCTP (3.5 DCl) and 0.75 units of Taq polymerase wereadded into this volume. The mixture was overlaid with 20 g.tlof mineral oil.PCR amplification followed. One cycle of DNA denaturation at 95Â°Cfor 10mm preceded 25 cycles of primer annealing at 65Â°Cfor 1 mm, primerextension at 72Â°Cfor 2 mm, and denaturation at 95Â°Cfor 1 mm 30 s. A finalcycle of primer annealing at 65Â°Cfor 1 mm and primer extension at 72Â°Cfor10 mm was performed.



Table 2 PCRprimersPGK

geneXL-l

XL-2@(j@3

XL-'15'-CTG

5'-AGC5-TAC5'-ACGTTC

TGGTCCCCT@TG

cccACG TTATGA AGTGTT ACGGCG

AAG

TAATAAc@

TGTGGA AGCATC AACGCT CTGTcC

GGG

ATCCAGGCA

TCGCTCGCCTrc-3'

TTA-3'TTG-3'

TCC-3'Fragile

X(FMRI)genePrimer

CPrimer F5'-GCT 5'-AGCcAGccccTc

CGTGCA CTTTTCccaGGT

TTCcca ccaACTGC!@TCCCCTGGT-3'CCA-3'

CLONAL DETERMINATION BY FMRJ AND PGK GENES

MONOCLONALMONOCLONALPOLYCLONALe@e@I

HpalIIHpaUIXp

@ PCRXm415CRXpXmXpXmXpXmXpXmXpXmXpXmXp

@XmXpXmXpXmI

BSIXIBSIXI;

Fig. 1. Schematic diagram of the PCR-basedclonality assay using the PGK gene. Cells must be ______heterozygous for the BstXJ polymorphism in thePGK gene represented as a white line through the ______bars. Left and center panels illustrate monoclonalcell populations (circles) with either the paternal(Xp) or maternal (Xm) X chromosome methylated(thick bar), respectively. The right panel illustratespolyclonal cell populations composed of equal numhers of cells with the maternal and paternal X chromosome methylated (inactive). DNA from thesecells is digested with HpaII eliminating amplifiabletemplates from the unmethylated allele (thin bar).Only methylated alleles remain and are amplified inthe PCR. Subsequent digestion with BstXI and agarose gel electrophoresis of the PCR product identifythe relative allelic composition ofthe inactive (methylated) X chromosomes. The monoclonal cells showeither a 433 or 530-base pair fragment; the polyclonal cells show both fragments. Â°,gels where the97-base pair fragment is present but not shown. bp,base pair(s).

distinguishable clusters of bands on the autoradiogram of the denaturing polyacrylamide gel (Fig. 2, Ref. 9). Eleven of these cases wereheterozygous for the BstXI polymorphism of the PGK gene and wereincluded in the previous section. The rate of heterozygosity for theFMRJ gene was 45%. Morphological data were not available on threecases; these specimens were not examined further.

The Rs for the polyclonal control samples varied from â€”1.871 to1.022 (Fig. 4, cases 1â€”14);3 of the 14 controls showed extremeskewing (Fig. 4, cases 8â€”10).Two of these specimens were from theblood of normal female volunteers; one was the same specimen thatshowed skewed methylation of the PGK gene. The third controlsample with a skewed fragile X pattern was from the marrow of awoman with immune thrombocytopenic purpura. The 95% confidenceinterval for the polyclonal R for the eleven cases was â€”0.68to 0.84;this was the largest interval of these investigations. Rs above or belowthis 95% confidence interval were considered a monoclonal pattern ofX chromosome methylation. The Rs for the morphologically malignant specimens ranged from â€”3.9to 3.15. Seventeen of 18 sampleswith hematological malignancies (>50% malignant cells by morphology) showed monoclonal patterns (Fig. 4, cases 15â€”23and 25â€”32).The one malignant specimen that was in the 95% confidence intervalfor a polyclonal R was from a patient with multiple myeloma and hadthe lowest number of malignant cells by morphology (59%, Fig. 4,case 24).

I 433bpAGAROSEGEL* AGAROSE GEL*AGAROSE GEL

5214

CLONALITYBYPGKGENE

DISCUSSION

The principle of differential X chromosome inactivation/methylation in females provides a biologically useful marker of clonality (1).Although the theory has been established for decades, the laborintensive and time-consuming procedures associated with this marker,as well as the small number of informative patients, has all butrelegated it to the research laboratory (2, 11). Recently, a rapid andsimple PCR-based clonal assay has been introduced which allowspotential diagnostic application; however, several aspects and limitstions of this new test have remained unexamined (4, 5, 7, 12, 13). Theintent of this study was to examine the use of the PCR assay withrespect to the Southern blot test and to introduce a second highlypolymorphic X-linked gene to the PCR method.

Criteria for designating samples as monoclonal are ill defined (14).For the Southern blot assay, Vogeistein et aL (3) accepted a greaterthan 3:1 ratio of the allelic band intensities in HpaII-digested specimens as a monoclonal pattern of X chromosome methylation. Anyratio lower than this was considered a polyclonal pattern. This valuewas selected with consideration for the technical inaccuracies of theSouthern blot procedure, such as differences in transfer of the vanously sized restriction fragments to the membrane, hybridizationefficiency of the probe to each of the fragments, and contamination ofthe tumor DNA specimen by benign reactive polyclonal cells. Nospecific criteria have been suggested for the PCR assay system.

The present study uses a ratio modified from Gale et a!. (15). Thisratio consists of the allelic band intensities of the HpaH-digestedspecimen normalized to those of the undigested specimen. For graphic

purposes, the log of this normalized ratio is used. The resulting R is ameasure of the degree of shift in allelic band intensities produced by

HpaII digestion. Values of 0 indicate no shift in allelic band intensitybetween HpaII-digested and undigested specimens; this reflects a50:50 polyclonal distribution in X chromosome methylation. Valuessignificantly greater or lower than 0 indicate a significant shift inallelic intensity and a monoclonal distribution in X chromosomemethylation. The monoclonal pattern of X chromosome methylation isdefined in these investigations relative to the 95% confidence interval



020a

a 0@

a0a@m

aB0@-200@

BB-4.

I

0 105. .@

30 40

CLONAL DETERMINATION BY FMRJ AND PGK GENES

Fig. 2. Schematic diagram of the PCR-basedclonality assay using the FMRJ gene. Cells must beheterozygous for the alleles in the FMRJ gene. Allelic differences are based on variable nucleotidetandem repeats represented by triangles. These differences are common in the general female population. Left and center panels illustrate monoclonalcell populations (circles) with either the paternal(Xp) or maternal (Xm) X chromosome methylated(thick bar), respectively. The right panel illustratespolyclonal cell populations composed of equal numbers of cells with the maternal and paternal X chromosome methylated (inactive). DNA from thesecells is digested with HpaII eliminating ampliflabletemplates from the unmethylated allele (thin bar).Only methylated alleles remain and are amplified inthe PCR. The products are electrophoresed on adenaturing polyacrylamide gel. As in Fig. 1, monoclonal cells show a predominance of one or the otherallele amplified; Polyclonal cells show both alleles.Differences in amplification efficiency of the different alleles are controlled for by normalizing theresults from the HpaII-digested sample with theresults from the undigested sample (see text).

BstXI

of Rs for a selected population of control polyclonal specimens fromsimilar tissues. This ratio controls for the methodological differencessuggested by previous investigators (3). In addition, heteroduplexformation and differences in allelic amplification efficiency by PCRare also controlled for. The ratio does not control for contamination ofthe tumor by reactive polyclonal cells.

The Southern blot and PCR assays give comparable results withrespect to the PGK gene when specimens are analyzed according tothe guidelines in this study. All specimens designated monoclonal orpolyclonal on the basis of skewed PGK gene methylation by Southernblot showed the same designation by PCR. It is important to note thatthe malignant specimens had substantial involvement by morphologically malignant cells and that the polyclonal control specimens wererelatively free of such cells. The PCR and Southern methods may notbe comparable when malignant specimens with low percentages ofmonoclonal cells are examined, due to the greater variability inpolyclonal PCR Rs (12). Therefore, careful selection of a relatively

Clonality Determination by PGK Gene

4 n LogPCRratlo

. LogSBratio ...

0a

homogeneous population of cells is necessary before meaningfulinterpretation can be performed with the PCR assay.

Nonmalignant specimens generally show a nonskewed pattern ofX-linked gene methylation and malignant specimens a skewed pattern; however, frequent exceptions occur. In the present investigation,the PGK gene revealed a skewed pattern of methylation in 1 of 8(12.5%) and the FMRJ gene in 3 of 14 (21%) normal control specimens. Previous studies have documented significant skewing in asimilar percentage (4â€”23%)of various normal tissues (3, 14, 15). Ina similar light, X-linked gene methylation may not skew in malignantspecimens; the rate varies with the locus examined. The M27(3 locusin the majority of malignancies (60%) does not show skewed methylation (14). In contrast, the PGK gene demonstrates a similar patternin only 4% of tumors (3). One exception was present in the currentstudy; the malignant specimen with the lowest percentage of tumorcells (59%) showed insignificant skewing in methylation of the FMR1gene. These observations suggest that nonskewed patterns of X chromosome methylation as determined by the PGK and FMR1 assays

4

0a

a

20Number of Case

0

â€”.3

0

5,.0 0

0ri@ .@â€˜Oaa -2

c)@M @3 . . . U

0 10 20 30

Number of Case

Fig. 3. Plot of the ratio (R) values of the control and malignant specimens from thePCR and Southernblot (SB) assay using the PGK gene. Sample numbers 1 to 8 arepolyclonal control samples; samples 9 to 25 are malignant. The solid and dotted linesindicate the polyclonal 95% confidence interval for the PCR and Southern blot results,respectively. â€¢,Southern blot results; I@PCR results.

Fig. 4. Plot of the ratio (R) values of the control and malignant specimens from thePCR assay using the FMR1 gene. Sample numbers 1 to 14 are polyclonal controlssamples; samples 15 to 32 are malignant. The solid lines indicate the polyclonal 95%confidence interval.

5215

CLONALITY BY FMR1GENEMONOCLONAL

XpSNpN@

Xmâ€”@--.

IXp NN

@ PCR

Xp@

Xp â€¢@Ã˜

Xp pflp

Xp â€¢p@N

PAGE GEL

MONOCLONAL

Xp _@p@â€”

@ Hpall

.N_ Xm

.,,_ Xm

.,,_ Xm

.,,_ Xm

.N_ Xm

ILMWPAGE GEL

POLYCLONAL

@ Hpall

Xp â€¢N0 â€¢$@ Xm

4,Xp@@ Xm

Xp â€¢pg@@ Xm

Xp@ â€¢P@@Xm

Xp@ â€¢N@ Xm

II HMW

LMW

PAGE GEL

Clonality Determination by Fragile X (FMR1) Gene

. Bm@

.



CLONAL DETERMINATIONBY FMRJ AND PGK GENES

indicate polyclonal cell populations; skewed patterns, however, arenot absolutely specific for monoclonal cell populations.

This investigation expands the useful repertoire of polymorphicX-linked genes that may be used in the PCR-based assay. The FragileX (FMRJ) gene has been shown to be methylated on the inactive Xchromosome and unmethylated on the active X chromosome (16).This property indicates that the FMRJ gene is a likely candidate to besuccessfully used in clonality determination based on differential Xchromosome methylation. In the current study, the FMR1 gene demonstrates a skewed pattern of X chromosome methylation in most

morphologically malignant tissues and a nonskewed pattern of methylation in most normal control samples. The rate of heterozygosity forthe FMRJ gene in our study is 45%; however, rates as high as 63%have been reported with this locus (9). The only other highly polymorphic gene examined by this methodology is the androgen receptor(HUMARA) gene (6, 7). Using this gene, Mashal et a!. (7) successfullystudied 11 of 15 females (73%). The combined rates of heterozygosityof the PGK, FMR1, and HUMARA gene loci should allow successfulstudy of greater than 90% of females patients by PCR techniques.

REFERENCES

1. Linder, D. and Gartler, S. M. Glucose-6-phosphate dehydrogenase mosaicism: utilization as a cell marker in the study of leiomyomas. Science (Washington DC), 150:67â€”69,1965.

2. Fialkow, P. J. Clonal origin of human tumors. Biochim. Biophys. Acts, 458: 283â€”321,1976.

3. Vogelstein, B., Fearon, E. R., Hamilton, S. R., Preisinger, A. C., Willard, H. F.,Michelson, A. M., Riggs, A. D., and Orkin, S. H. Clonal analysis using recombinantDNA probes from the X-chromosome. Cancer Res., 47: 4806â€”4813,1987.

4. Van Kamp,H., Jansen,R., Willemze, R., Fibbe,W. E., andLandegent,J. E. Studieson clonality by PCR analysis of the PGK-1 gene. Nucleic Acids Rca., 19: 2794, 1991.

5. Gilliland, D. G., Blanchard, K. L, Levy, J., Perrin, S., and Bunn, H. F. Clonality inmyeloproliferative disorders: analysis by means of the polymerase chain reaction.Proc. Natl. Acad. Sci. USA, 88: 6848-6852, 1991.

6. Allen, R. C., Zoghbi, H. Y., Moseley, A. B., Rosenblatt, H. M., and Belmont, J. W.Methylation of HpaH and HhaI sites near the polymorphic CAG repeat in the humanandrogen-receptor gene correlates with X chromosome inactivation. Am. J. Hum.Genet., 51: 1229â€”1239, 1992.

7. Mashal, R. D., Lester, S. C., and Sklar, J. Qonal analysis by study of X chromosomeinactivation in formalin-fixed paraffin-embedded tissue. Cancer Res., 53: 4676â€”4679, 1993.

8. Keith, D. H., Singer-Sam, J., and Riggs, A. D. Active X-chromosome DNA isunmethylated at eight CCGG sites clustered in a guanine-plus-cytosine.rich island atthe 5' end of the gene for phosphoglycerate kinase. Mol. Cell. Biol., 6: 4122â€”4125,1986.

9. Fu, Y. H., Kuhi, D. P. A., Pizzuti, A., Pieretti, M., Sutcliffe, J. S., Richards, S.,Verkerk, A. M. H., Holden, J. I. A, Fenwick, R. G., Warren, S. T., Oostra, B. A.,Nelson, D. L., and Caskey, C. T. Variation of the CGG repeat at the Fragile X siteresults in genetic instability: resolution ofthe Sherman paradox. Cell, 67: 1047â€”1058,1991.

10. Werner, M., Marsh, W. L, and Aryan, D. A. Normal values: theoretical and practicalaspects. CRC Crit. Rev. Chin.Lab. Sci., 6: 81â€”101,1975.

11. Beutler, E., Collins, Z., and Irwin, L E. Value of genetic variants of gjucose-6-phosphate dehydrogenase in tracing the origin of malignant tumors. N. Engl. J. Med.,276: 389â€”391,1967.

12. Noguchi, S., Motomura, K.-,Inaji, H., Imaoka, S., and Koyama, H. Clonal analysis ofhuman breast cancer by means of the polymerase chain reaction. Cancer Res., 52:6594â€”6597,1992.

13. Noguchi, S., Motomura, K., Inaji, H., Imaoka, S., and Koyama, H. Clonal analysis offibroadenoma and phyllodes tumor of the breast. Cancer Res., 53: 4071â€”4074,1993.

14. Fey, M. F., Liechti-Gallati, S., von Robe,A., BOriSCh,B., Theilkas, L, Schneider, V.,Oestreicher, M., Nagel, S., Ziemiecki, A., and Tobler, A. Qonalit@,and X-inactivationpatterns in hematopoietic cell populations detected by the highly informative M27@DNA probe. Blood, 83: 931â€”938,1994.

15. Gale, R. E., Vlheadon, H., and Linch, D. C. X-chromosome inactivation patternsusing HPRT and PGK polymorphisms in haematologically normal and postchemotherapy females. Br. J. Haematol., 79: 193â€”197,1991.

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1994;54:5212-5216. Cancer Res Siew-Teh Lee, Ronald C. McGlennen and Craig E. Litz Malignancies

) Genes in HematologicalPGKPhosphoglycerate Kinase () andFMR1Clonal Determination by the Fragile X (

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