Proc. Nati. Acad. Sci. USAVol. 85, pp. 7680-7684, October 1988Genetics

Pst I restriction fragment length polymorphism of human placentalalkaline phosphatase gene: Mendelian segregation and localizationof mutation site in the geneLARISA TSAVALER, ROBERT C. PENHALLOW, AND HOWARD H. SUSSMAN*Laboratory of Experimental Oncology, Department of Pathology, Stanford University, School of Medicine, Stanford, CA 94305

Communicated by Roy Hertz, July 11, 1988

ABSTRACT The pattern of inheritance of a Pst I restric-tion fragment length polymorphism (RFLP) of the humanplacental alkaline phosphatase gene was studied in nine nuclearfamilies by Southern blot hybridization analysis of genomicDNA. The dimorphic RFLP is defined by the presence of allelicfragments 1.0 kilobase and 0.8 kilobase long. The results of thisstudy show that the two alleles ofthe Pst I RFLP of the placentalalkaline phosphatase gene segregate as codominant traitsaccording to Mendelian expectations. For a polymorphism tobe useful as a genetic marker the probability that an offspringis informative (PIC) must be at least 0.15. The allelic frequencyof the 1.0-kilobase allele is 0.21, which correlates to a proba-bility that an offspring is informative of 0.275 and is indicativeof a useful polymorphism. By using probes derived fromdifferent regions of the placental alkaline phosphatase cDNA,the mutated Pst I site causing the RFLP was located in thepenultimate intron 2497 base pairs downstream from thetranscriptional initiation site.

The alkaline phosphatases [orthophosphoric-monoester phos-phohydrolase (alkaline optimum), EC 3.1.3.1] are a family ofmetalloenzymes that hydrolyze phosphate esters at a high pHoptimum (pH 10-10.5). At least three gene loci exist for thehuman alkaline phosphatases including term placental(PLAP), intestinal, and liver/kidney/bone or tissue-unspecific isoenzymes. The cDNA of each enzyme has beencloned and sequenced (1-6). PLAP is of particular interestfrom both a genetic and medical standpoint because of itsextensive genetic polymorphism and ectopic expression insome human cancers. Electrophoretic, immunological, andgenetic studies in human populations have shown there arethree common alleles and a number of minor variants ofPLAP. Two of the common alleles and one of the minorvariants are among those cDNA clones isolated and se-quenced (1-3). We have described (7) an allelic Pst Irestriction fragment length polymorphism (RFLP) in thePLAP gene locus and provided an estimate of the frequencyof each allele based on the binomial distribution. This studydemonstrates the Mendelian segregation of the polymorphicallele and locates the mutation causing the polymorphismwithin the PLAP gene.

MATERIALS AND METHODSDNA. DNA from nine families and 10 randomly chosen

individuals, all Caucasians from the San Francisco Bay areaselected for linkage studies (8), were used in this study. Highmolecular weight DNA was obtained from peripheral bloodlymphocytes by a standard protocol (7).

Restriction Enzyme Digestion of Genomic DNA. Ten micro-grams ofDNA was digested with restriction enzymes (5 units

of enzyme per ,ug of DNA) overnight under conditionsspecified by the supplier (Bethesda Research Laboratories).The next day, an additional 2 units ofenzyme per ,4g ofDNAwas added and incubation was continued for 3-4 hr. Sampleswere ethanol-precipitated overnight at - 20TC and redis-solved in TE buffer (10 mM Tris'HCI/1 mM EDTA, pH 7.4).

Probes. The probe used in the family study was a 1.7-kilobase (kb) fragment resulting from BamHI and Kpn Irestriction enzyme digestion of the original full-length 2.7-kbcDNA of PLAP constructed by Kam et al. (1). The 1.7-kbprobe does not contain the Alu sequence present at the 3' endof the full-length 2.7-kb cDNA.

Eight probes derived from various regions of the PLAPcDNA were used in the localization study. The probes weremade by digesting the 2.7-kb PLAP cDNA with the appro-priate restriction enzymes according to the protocol providedby the supplier (Bethesda Research Laboratories). Theresulting DNA fragments were fractionated in 0.7%-1.5%agarose gels containing ethidium bromide to aid visualizationand recovered by electroelution of excised gel bands. TheDNA was then extracted sequentially with 1-butanol, phenol,phenol/chloroform, and chloroform, precipitated with etha-nol, and redissolved in TE buffer.

32P Labeling of the Probe. The probe was labeled byincorporating deoxycytidine 5'-[a-32P]triphosphate ([32p]dCTP) by using the hexadeoxynucleotide primer methoddescribing by Feinberg and Vogelstein (9). Unincorporated[32P]dCTP was removed by the spun-column procedure (10).Southern Blot and Hybridization. Gel electrophoresis was

performed on 0.7% agarose gel. Transfer of fragments fromthe gel to a nylon sheet (Genatran 45) was done according tospecifications of the supplier (Plasco, Woburn, MA). Afterthe transfer, the membrane was briefly washed in 2 x SSC(1 x SSC = 0.15 M sodium chloride/0.015 M sodium citrate,pH 7.0), air-dried, and baked in a vacuum oven at 80°C for 2hr. The membrane was saturated in 2 x SSC buffer, placed ina plastic bag containing a prehybridization solution consistingof 2 x SSC, 5 x Denhardt's solution (1 x Denhardt's solution- 0.02% Ficoll/ 0.02% polyvinylpyrrolidone/0.02% bovineserum albumin), 1 mM sodium pyrophosphate, 4.8% (wt/vol)dextran sulphate, 1% sodium dodecyl sulfate (SDS), 50%(vol/vol) formamide, and denatured salmon sperm DNA at100 ,ug/ml. Prehybridization was done at 42°C for 12 hr.The radiolabeled probe (=0.1 tg) was then added to the

blot and hybridized for 20-36 hr at 42°C with gentle rocking.The membrane was washed once for 20 min in 2 x SSC/O.1%SDS at room temperature and then three times for 1 hr eachwash in 0.1% SDS/0. 1 x SSC at 68°C in a shaking water bath.Autoradiography was performed at - 70°C by using a singleintensifying screen for 12 hr to 5 days.

Abbreviations: RFLP, restriction fragment length polymorphism;PLAP, placental alkaline phosphatase.*To whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 85 (1988) 7681

RESULTS

Family Study. The segregation ofthe 1.0-kb DNA fragmentdefining the Pst I RFLP ofthe PLAP gene was studied in ninefamilies. The genotype of each individual was determined bycomplete digestion ofDNA with the Pst I restriction enzyme,separation of fragments by agarose gel electrophoresis, andSouthern blot hybridization analysis with a 1.7-kb PLAPcDNA probe. Genomic blots of the nine families and 10randomly chosen individuals revealed three types of restric-tion patterns as observed in our previous report (7). Thesegenotypes are designated Pst+/- Pst-U', and Pst+1+based on the presence, absence, or presence in doubleintensity, respectively, of a 1.0-kb band. In this study weused a 1.7-kb cDNA probe that lacks the Alu sequencelocated in the extreme 3' region of the full-length PLAPcDNA. The use of this truncated PLAP probe eliminatesbackground interference in the 0.8-kb region of Southernblots and reveals an allelic 0.8-kb fragment that is absent inPst+l+ homozygotes, but present in both Pst+1- andPstU samples.A genomic blot, containing DNA digests of three families

is shown in Fig. 1. The DNA blot of a heterozygous male(Pst+1 ) is shown in lane 14 and that ofa homozygous female(Pst1) in lane 15. The DNA blots of their progeny showingthe transmission of the allelic fragments at a frequencyconsistent with Mendelian expectations are seen in lanes 16-19. Two of the offspring are heterozygotes (lanes 17 and 19)and two are homozygotes (lanes 16 and 18). An identicalpattern of inheritance is seen in family 9, lanes 1-5.The family pedigree resulting from the mating of a heter-

ozygous male (Pst+/ ) and a homozygous female (Pst+/is shown in lanes 20-24. As expected, the alleles segregate sothat each of the offspring has a copy of the 1.0-kb fragment;one of the siblings (lane 22) is a homozygote (Pst+1+), andthe other two siblings are heterozygotes (Pst+1).Lanes 6-13 show the pedigree of a family resulting from

two homozygous (Pst-l-) parents. All the offspring inheritthe parental genotype.

FAMILY 9 FAMILY 16

The genotype ascribed to each family member used in thisstudy based on Southern blot analysis ofPst I-digested DNAis shown in Fig. 2. The cumulative results from eight of thenine families and all the random individuals are presented inTable 1. In the construction of Table 1, family 31 from Fig.2 was separated into two families to account for the fact thatthe original brother/sister siblings produced families of theirown. Family 8 was omitted from tabulation because thematernal genotype is unavailable. The data show that the1.0-kb and 0.8-kb fragments defining the Pst I polymorphismin the PLAP gene segregate according to Mendelian expec-tations in the F1 generation as co-dominant alleles.

Loclization of Pst I Polymorphism. In our previous com-munication reporting the existence ofthe Pst I polymorphism(7), we speculated that the RFLP results from a pointmutation that created or destroyed a Pst I site. To determinethe location of the mutation site, a series of probes wereconstructed and the ability of each probe to recognize PstI-generated restriction fragments of genomic DNA wasassessed by Southern blot analysis. Maps showing theregions of genomic DNA that the cDNA-derived probes willrecognize are provided in Fig. 3. The maps depict the regionsofgenomic DNA to which the probes will hybridize, based onthe exons within the genomic sequences, and do not repre-sent actual physical size of the probe. The PLAP-1 gene mapshown for comparison was provided by Brian Knoll (TexasMedical Center, Houston, TX) (11). The results of theSouthern blot analysis with each of the probes are shown inFig. 4. Probe IV, which covers 2.5 kb ofgenomic DNA at the5' end of the PLAP gene, and probe VI, which covers 0.7 kbat the 3' end of the gene did not detect the 1.0-kb and 0.8-kbbands defining the polymorphism. All other probes detectedthe polymorphic fragments. Hybridization with probe IIIproduced a weak but detectable signal with the polymorphicbands, probably because oflimited sequence overlap with theregion of the gene in question. Probes V, VII, and VIIIproduced strong hybridization signals to the polymorphicbands with minimal detection of accessory fragments. Theother bands detected may be intestinal alkaline phosphatase

FAMILY 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

kb

30- 4 _

* b,6 a04,42 0-""94646-t

1 6-

_ *'i'*s

so .40 .0 .t*"4w &

10- ' *4 *

05*- *.* _w AP.

0 5 ---

Pst 1 MALE 0 Pst 1 FEMALF

FIG. 1. Southern blot analysis of Pst I-digested DNA. The family of origin and genotype of each individual are located in the pedigree atthe top of the gel. * or e, individual homozygous for the 1.0-kb allele, o or o, homozygous for 0.8-kb allele, i or heterozygote. Fragmentsizes (in kb) are indicated at the left.

OPsl 1

Genetics: Tsavaler et al.

Proc. Natl. Acad. Sci. USA 85 (1988)

17 ~~~~~16

9 9a

*Pst1 +/, MALE 0 Pstl +/-, FEMALE

FIG. 2. Mendelian segregation of Pst I RFLP alleles in nuclear families. Genotypes were determined by Southern blot analysis. Symbolsare identical to those used in Fig. 1.

gene fragments that are also recognized by the PLAP probe.tThe ability of probes V, VII, and VIII to select for thepolymorphic bands indicates that the Pst I restriction frag-ment containing the altered site must overlap the 746-base-pair (bp) region located from 2.5 kb to 3.3 kb downstreamfrom the transcriptional start site. Inspection of the restric-tion map of PLAP-1 permits additional refinement of thisestimate. Pst I sites at bp 2312, bp 2497, and bp 3267 providefor fragments 0.2 kb and 0.8 kb long compared to 1.0-kb and0.8-kb bands visualized by Southern blot analysis with probesto this region. Based on this evidence, we conclude that thePst- genotype is in fact the wild-type PLAP gene possessingthe Pst I site at bp 2497 and the Psi' genotype is the resultof a point mutation destroying the Pst I site at bp 2497,resulting in a 1.0-kb fragment.

DISCUSSIONSouthern blot analysis with a PLAP cDNA probe to examinePst I-digested human DNA isolated from members of nuclearfamilies and random individuals reveals that the previouslydescribed (7) Pst I RFLP consists of two co-dominant allelesthat segregate through the generations according to Mende-lian expectations. The probe used in this study is a truncatedversion of the PLAP cDNA constructed by removing 800 bp,including an Alu sequence, from the 3' end of the full-lengthcDNA. Hybridization with this shortened probe results inautoradiograms with greatly reduced lane backgrounds com-pared to previous attempts, by eliminating the Alu repeats. Italso results in the exclusion of a constant gel band in the0.8-kb region revealing the Pst I RFLP to be a two-allelesystem. Individuals previously assigned a Pst'I- genotypebased on a single copy of the 1.0-kb fragment were found to

have a 0.8-kb fragment as well. Pst+1+ individuals have thedouble-intensity 1.0-kb band, but no 0.8-kb band andPst-samples have only the 0.8-kb band (Fig. 1).The second goal of this study was to localize and determine

the nature of the change in the gene resulting in the poly-morphism. It was possible to assign the site of the suspectedmutation to a region centered about 1.4 kb downstream on thefull-length cDNA by using discrete probes derived fromvarious regions of the cDNA. Inspection of the completegenomic nucleotide sequence and map of exon-intron struc-ture of the PLAP-1 gene provided by Brian Knoll allowedfurther refinement of this estimate. Three Pst I sites arepresent in, or border, the region identified by probes V, VII,and VIII and would result in fragments of 185 bp and 770 bplong. We believe that the PLAP-1 clone isolated by Knoll etal. (11) is representative of the Pst- allele and the Pst+ alleleis a product of a mutation destroying the Pst I site at bp 2497at the 3' terminus of intron I. The elimination of this sitewould lead to the production of a 1.0-kb fragment, consistentwith our findings. The inability to detect the 0.2-kb fragmentin Pst- individuals also conforms to this theory because it iscomposed entirely of intronic sequences, which are notrecognized by the cDNA-derived probes.A variant PLAP gene, the expression ofwhich is enhanced

in seminomas, has been isolated and sequenced (14). The

Table 1. Segregation of the PLAP marker in nuclear familiesidentified with Pst I

No. of offspring with

No. of genotypefamilies Pst-r- Pst+l- Pst+l+

Parental genotypesPst+l- x Pst-1- 4 9 5 0Pst+l- x Pst+l+ 2 0 4 2Pst-1- x Pst-1- 3 17 0 0

Randomly chosenindividuals* 8 1 1

*n= 10.

Q Psti-/-

tThe PLAP gene appears to have evolved from a duplication of theintestinal alkaline phosphatase gene. The nucleotide sequences inthe coding regions of PLAP and intestinal alkaline phosphataseshow 89.5% identity (5), and both genes are located on chromosome2 at q34-37 (12, 13).

7682 Genetics: Tsavaler et al.

31

1D- -0

t

Proc. NatL. Acad. Sci. USA 85 (1988) 7683

0 0.5 1.0 1.5 2.01a X

2.5 3.0 3.5 4.0 4.5

?~~~~~~~~~11 i I II I I f p

A -B C D E F UG H II1 2 3 4 5 6 7 8 9 ' 10 il

0.2 0.81.0 *

Pstl

Pst II l

+

+

A valSiniSmal'

A val

SmaI

PLAP

Sial+

Smal PstI

+ PstI Pstlva i _I

Sia l SiaiVIIl -I

+ AvaI PstI

VIII I-+I

FIG. 3. Restriction map of the PLAP-1 gene. The exon-intron structure of the gene is displayed, along with a scale calibrated in kb. Theexons are the solid regions distinguished by number; the introns are the open regions designated by letter. Selected restriction sites are indicatedabove exon-intron map. The Pst I restriction fragments defining the polymorphism are shown below the map, with the mutated Pst I sitehighlighted with a bold arrow. The probes used to locate the polymorphic region (I-VIII) are diagramed at the bottom of the figure. The diagramsrepresent the area of genomic DNA recognized by the cDNA probes, not their actual size. The ability of each probe to hybridize to thepolymorphic bands is indicated by a + or - below the probe.

protein coding regions of the variant gene possesses 98%similarity to native PLAP, but the restriction maps of the twogenes are considerably different. The variant PLAP genedoes not have restriction fragments corresponding to poly-morphic bands of the native gene.The allelic frequency reported here from the randomly

chosen individuals [1 Pst+/+ (10%), 1 Pst1/- (10%6), and 8Pst-/- (80%); n = 10] is in agreement with the largersampling reported (7) [1 Pst+/+ (3%), 12 Pst+/ - (33%), and23 Pst-/- (64%); n = 36]. Based on this frequency it isprobable that a clone picked out of a single randomlyproduced genomic library would be a Pst- allele. Such is thecase of the PLAP-1 gene provided by Knoll et al. (11).A comparison ofthe region ofcoverage ofprobe VI and the

gene map results in another interesting finding. Probe VI isderived from the 3' end of the full-length cDNA and is

11 III IV

essentially the portion removed in the construction of the1.7-kb probe used in the family study. The DNA sequence ofprobe VI hybridizes to a 777-bp fragment (Fig. 3) that isindistinguishable in size on 0.7% agarose gels from thesmaller (0.8 kb) polymorphic allele. Consequently, when thefull-length 2.7-kb probe was used in the initial study (7), theregion identified as probe VI hybridized to this 777-bpfragment and masked the second allele.The location of the PLAP gene on chromosome 2 at band

q34-37 (12) makes this RFLP potentially useful for linkagemapping of other gene loci on chromosome 2. Skolnick andWhite (15) suggest that in a general sense, for a polymorphismto be useful as a genetic marker, the probability that anoffspring is informative (PIC value) must be at least 0.15. ThePst I RFLP at PLAP satisfies this criterion; the allelicfrequency for Pst'is 0.21 corresponding to a PIC value of

V VI vil Villkb

gIs0 -3.0-2.0-1.6

,a 40 M -1.0

-0.5

~~.i ftrW '|so I"

_ -m h--lo

FIG. 4. Localization of polymorphic fragment. Southern blots of Pst I-digested DNA were probed with discrete PLAP cDNA fragments.Blots I, III, IV, VII, and VIII are the same blot rehybridized with different probes and have examples of heterozygotes in the two left lanesand Pst' homozygotes in the two right lanes. Blots II, V, and VI are also the same blot hybridized with different probes and contain left toright Pst+/-, Pst+/+, and Pst-/- alleles. The roman numeral above each panel refers to the probe used in the hybridization (see Fig. 3). Thesize of the fragments is indicated by the scale in kb to the right. The 1.0-kb and 0.8-kb bands defining the polymorphism are designated by arrowson the left border.

Pst I11Fi

Pst IIV i

_az3I

Genetics: Tsavaler et al.

I

Proc. Natl. Acad. Sci. USA 85 (1988)

0.275. The probability of missing the polymorphism in anine-member screening panel is <0.02 making this a veryuseful RFLP. In addition to the Pst I polymorphism, varia-tion in banding pattern among various individuals has beendemonstrated with enzymes Hha I, Sac II, and Taq I (16),further increasing the potential utility of the PLAP locus forlinkage mapping studies.

We thank Dr. Cavalli-Sforza, Department of Genetics, StanfordUniversity, for providing the DNA blots used in the family study andfor his valuable discussions. We thank Dr. Brian Knoll, Departmentof Pathology and Laboratory Medicine, University of Texas HealthCenter at Houston, for generously providing the complete nucleotidesequence of the human PLAP-1 gene prior to publication. We alsoacknowledge Lucille Weiss for her skillful preparation of the manu-script. This work was supported by Grants CA13533 and CA09151from the National Institutes of Health.

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9. Feinberg, A. P. & Vogelstein, R. (1983) Anal. Biochem. 132, 6-13.

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