7 Med Genet 1994;31:607-614

Parental origin of transcription from the humanGNAS1 gene

R Campbell, C M Gosden, D T Bonthron

AbstractVariation in the phenotypic expression ofAlbright's hereditary osteodystrophy(AHO) determined by the parent oftransmission, suggests that the humanGsa gene (GNAS1), in which mutationsoccur in AHO, may be under imprintedcontrol. GNAS1 is also known to map to achromosomal region (20q13.11) showingsyntenic homology with the imprintedmouse region 2E1-2H3. To establish ifGNAS1 is indeed imprinted, we have ex-amined the parental origin of GNAS1transcription in human fetal tissues. Of75 fetuses genotyped, at gestational agesranging from 6 to 13 weeks, 13 heterozy-gous for a FokI polymorphism in exon 5of GNAS1 were identified whose motherswere homozygous for one or other allele.RNA from up to 10 different tissues fromeach fetus was analysed by RT-PCR. Inall cases expression from both parentalalleles was shown by FokI digestion ofRT-PCR products and quantification ofthe resulting fragments. No tissue speci-fic pattern of expression was discerned inthese experiments.

Ifgenomic imprinting regulates the ex-pression of the human GNAS1 gene, ourdata suggest that the effect must either besubtle and quantitative, or be confined toa small subset of specialised hormoneresponsive cells within the target tissues.

(JT Med Genet 1994;31:607-614)Human Genetics Unit,University ofEdinburgh, WesternGeneral Hospital,Crewe Road,Edinburgh EH4 2XU,UKR CampbellD T Bonthron

Department ofObstetrics andGynaecology, Centrefor ReproductiveBiology, University ofEdinburgh,37 Chalmers Street,Edinburgh EH3 9EW,UKR Campbell

MRC Human GeneticsUnit, Western GeneralHospital, Crewe Road,Edinburgh EH4 2XU,UKC M Gosden

Correspondence toDr Campbell.Received 19 January 1994Accepted for publication16 March 1994

Albright's hereditary osteodystrophy (AHO)is characterised by obesity, brachydactyly,rounded facies, short stature, and subcutaneouscalcification. Two phenotypic variants exist. Inpseudohypoparathyroidism (PHP), hypocal-caemia and hyperphosphataemia, refractory tothe administration of exogenous parathormone(PTH), are seen. This results from end organresistance to the action of PTH. AHO is alsoseen without these biochemical changes, a sit-uation assigned the term pseudopseudohypo-parathyroidism (PPHP).2 It is now generallyaccepted that these two entities are variants ofthe same disorder, since both may appear in thesame family34 and a patient's biochemical statusmay even change with age.56The function of the renal PTH receptor-

adenylate cyclase signalling pathway is dis-turbed in these patients.7 Reduced expressionor altered function of an ubiquitous signallingprotein, Gs, has been shown in the erythro-cytes of patients with AHO.89 Gs, a hetero-trimer of a, 3, and 'y subunits, mediates stimu-

lation of adenylate cyclase following binding ofPTH to its receptor. Null mutations of thegene encoding the Gsa subunit (GNAS1) havebeen shown in patients with AHO,'01" whomay also show defective response to otherhormones which act via the adenylate cyclase-cAMP system. 2-14AHO is an autosomal dominant disorder,

with sex modification accounting for the 2:1female to male preponderance.'5 An explana-tion for the differing phenotypes resultingfrom GNAS1 mutation, and for the prepon-derance of maternally inherited PHP cases, hasrecently been offered. Genomic imprinting ofGNAS1 has been suggested, following a re-view of published cases in which inheritance ofthe condition from the mother was always seenin PHP, whereas paternal inheritance pro-duced PPHP.16 Imprinting of this locus wouldbe consistent with the chromosomal localisa-tion of GNAS1 at 20q13.1 1, a region showingsyntenic homology with the imprinted murineregion 2EI-2H3."7'8To date, four murine genes have been

shown to be imprinted. 1922 Three of theirhuman homologues are similarly modified,H19,23 24 IGF2,25 26 and SNRPN,27 while bothIGF2R and IGF1R escape imprinting.2829 Foreach gene, allelic origin has been assigned tomRNA by the use of intragenic polymor-phisms; presence or absence of imprinting hasusually been considered as an all or noneeffect. We have now used similar methodologyto assign parental origin to GNAS1 cDNAPCR products; allelic expression was quanti-fied in three ways. Our experiments, examin-ing up to 10 different tissue types across aseven week gestational spectrum, have failed toconfirm that GNAS1 is under imprinted con-trol in early human embryogenesis. However,it has become apparent that subtle imprintingeffects, such as small quantitative differencesbetween paternal and maternal expression, orimprinting confined to a single cell type in onetissue, cannot be ruled out by this type ofanalysis.

Materials and methodsTISSUE SAMPLESWith informed maternal consent and ethicalcommittee permission, fetal tissue was col-lected from surgical terminations of pregnancyat 6 to 12 weeks' gestation, together with amaternal blood sample. Fetal tissues wereseparated from maternal blood clot and deci-dua, and individual organs isolated under thedissecting microscope. They were washed inice cold phosphate buffered saline and im-mediately homogenised in 4 mol/l guanidi-nium isothiocyanate. Peripheral blood lym-

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Campbell, Gosden, Bonthron

phocyte RNA was similarly prepared, afterseparation of 20 ml venous blood from 20 adultvolunteers on Lymphoprep (Nycomed) gradi-ents. These samples were paired with mouth-wash samples (10 ml tap water) from theirparents.

NUCLEIC ACID PURIFICATION AND cDNASYNTHESISMaternal and fetal DNA were extracted fromleucocytes and fetal tissues respectively byproteinase K/SDS digestion, phenol/chloro-form extraction, and ethanol precipitation.The acid guanidinium isothiocyanate/phenol/chloroform extraction method was used toisolate total RNA from lymphocytes and fetaltissues.30cDNA synthesis was performed using ap-

proximately 1 jg of total RNA. This was re-verse transcribed with 100 U of MMLV re-verse transcriptase (Gibco/BRL), primingwith 0 1 jig random hexanucleotides (Pharma-cia) in a 30 gl reaction volume. Other reactioncomponents were: 50 mmol/l Tris HCl pH 8-3,55 mmol/l KCI, 3 mmol/l MgCl2, 100 jmol/leach dNTP, 20 mmol/l DTT, and 10 U hu-man placental ribonuclease inhibitor (Gibco/BRL). Samples were incubated at 37°C for 60minutes and then the reverse transcriptase wasdenatured at 95°C for five minutes; 15 pl of thiscDNA mix was then used for PCR amplifica-tion. Negative controls, to which no reversetranscriptase was added, were also amplified.

POLYMERASE CHAIN REACTIONA single pair of primers was designedto amplify both genomic and cDNA. Thesequences were GNAS-4: 5'-AGAAGG-CAACCAAAGTGCAGGACA (sense) andGNAS-5: 5'-AGGGAAGTCAAAGTCAG-GCACGTT (antisense).Genomic DNA amplification was performed

using 0 5 pg DNA template, in the presence of1 x PCR buffer (50 mmol/l KCI, 10 mmol/lTris HC1 pH 8-3, 1-5 mmol/l MgCl2, 100 jig/ml gelatin), 0-25 jmol/l each primer, 0-1%Triton X- 100, 1 U Taq polymerase (Perkin-Elmer Cetus), and 250 jmol/l each dNTP.

GNAS-4IVS4 83 bp

4z. ~~~_

PCR cycling was as follows: 94°C/30 seconds,65°C/30 seconds, 72°C/60 seconds, for 35cycles, and final extension at 72°C/10 minutes.This amplified a fragment of 256 bp (fig 1).cDNA amplification was performed by adding15 gil of the cDNA synthesis product above(including dNTPs) to 35 gl of a mix composedof 1 x PCR buffer, primers, Taq polymerase,and gelatin, as above. PCR cycling was as forgenomic DNA but for 30 cycles only, andgenerated a 173 bp product (fig 1). End label-ling of primer GNAS-4 was performed with7y32P-ATP (5000 Ci/mmol, Amersham Inter-national plc) in the presence of T4-polynuc-leotide kinase before addition to the cDNAPCR mix.

ANALYSIS OF ALLELIC EXPRESSIONA Fok I polymorphism in exon 53132 was usedto genotype DNA and to assign parental originto each allele in heterozygotes; 3 pl of PCRproduct was digested with 10 U Fok I (NewEngland Biolabs) at 37°C for six hours. Pre-dicted cleavage product sizes were 205/51 bpfor genomic DNA and 122/51 for cDNA. On3% agarose gels the small cleavage fragment(51 bp) cannot be resolved from primer-dimercomplexes.

32p labelled fragments were separated on10% polyacrylamide gels and visualised byethidium bromide staining. The bands wereexcised and added to 10 ml Scintran EX(BDH) for direct scintillation counting. Alter-natively, the intact gel was fixed in 10% meth-anol, 10% acetic acid, and 5% glycerol, driedonto Whatman 3MM paper, and scanned on aMolecular Dynamics model 425E-130 phos-phorimager after 20 minute and 40 minuteexposures. The band densities were then com-puter analysed (Imagequant, Molecular Dyna-mics).

In addition, PCR primer GNAS-4 wasfluorescein labelled during synthesis, usingFluorprime (Pharmacia). FokI digestion pro-ducts were then analysed on an automatedlaser fluorescence sequencer (ALF, Pharma-cia) and peak areas representing each allelewere integrated using the manufacturer's soft-ware.

GNAS-5FokI -

5 I

Genomic product 256 bp

cDNA product 173 bp

Figure 1 Diagram of part of the GNASl gene, showing the positions of the polymorphic FokI site and the PCRprimers GNAS-4/GNA S-5. Exons 4 and 5 are indicated by rectangles: Genomic (256 bp) and cDNA (173 bp)PCR products are easily distinguished by 3% agarose gel electrophoresis.

GNAS-4

4 T .5 1

GNAS-5FokI -

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Parental origin of transcription from the human GNASI gene

IMPRINTING OF THE IGF2 GENE

To provide a positive control, the allelic originof IGF2 expression was also studied, using anApaI polymorphism (allele frequencies 0 47/0-53); PCR primers and conditions were aspreviously reported.26 33 RT-PCR product(3 p1) was digested for six hours with 10 UApaI (Gibco/BRL).

ResultsDISCRIMINATION BETWEEN PARENTAL ALLELES

OF GNAS1Genomic DNA from 75 fetuses was typed for a

C/T polymorphism in exon 5 of GNAS1 byFok I digestion of the 256 bp PCR product (fig1). Thirty-nine fetuses were heterozygous. Toassign a parental identity to each allele, mater-nal genomic DNA, obtained from peripheralblood, was typed. Since paternal DNA was notavailable, the only informative situation forthese experiments was one in which themother was homozygous at the Fok I site.Thirteen such mother-fetus pairs were identi-fied and analysed further.

BIALLELIC EXPRESSION OF GNAS1Total RNA was isolated from available fetalorgans and reverse transcribed before PCRamplification. Up to 10 tissue types were ana-

lysed from each fetus (table 1), the materialspanning 6 to 13 weeks of gestation. The RT-PCR product (173 bp) was easily distinguish-able from the 256 bp genomic DNA product(figs 1 and 2A), providing a control for any

genomic DNA contamination of the RNA.Such contamination was in fact never seen.

After Fok I digestion, visual assessment of eth-idium stained 3% agarose gels indicatedroughly equivalent biallelic expression in allfetal tissues at all gestational ages (fig 2A).IGF2 expression by comparison, in the same

tissues from fetuses DM09 and LR19, showedexclusive paternal transcription, consistentwith previous reports2526 (fig 2B).

QUANTIFICATION OF ALLELIC EXPRESSION

Three methods were used to quantify the rela-tive expression of each parental allele. Firstly,cDNA-PCR was performed in the presence of32P end labelled oligonucleotide primer GNAS-4 (fig 1). The relative expression of each allelewas then quantified by excision of the bandsfrom an acrylamide gel and scintillation count-ing, or by phosphorimager analysis of the fixeddried gel (fig 3).

Finally, an alternative non-radioactive tech-nique, using a fluorescein labelled primer(again GNAS-4) and automated laser fluores-cence (ALF), was used. The amounts ofdigested and undigested cDNA-PCR productwere estimated by computer integration of thepeak areas corresponding to each fragment (fig4).As shown in detail in tables 2 to 4, all three

analytical methods suggested a slight predom-inance (ratio - 1-2) of expression from thematernal GNAS1 allele. However, this effectdid not reach statistical significance for thedata generated from fluorescein labelling andautomated peak integration (table 4). It isfurther discussed below.

GNAS1 EXPRESSION BY PERIPHERAL BLOOD

LYMPHOCYTESFollowing the above experiments, a secondsource of RNA was studied. In this experi-ment, DNA and RNA were extracted fromperipheral blood from persons in one genera-

tion, and mouthwash DNA was later obtainedfrom the parental generation. This was

designed to eliminate any bias introduced bypossible maternal blood/tissue contaminationof our fetal samples. Twenty persons were

genotyped and in those heterozygous for theFok I polymorphism (n= 13), the relative ex-

pression of each parental allele in lymphocyteRNA was measured as above. Only then were

the parents of these 13 persons genotyped.This showed seven informative persons inwhom one or both parents were homozygous,

Table 1 Range of tissues examined in 13 informative fetuses. Gestational ages are indicated in weeks. The fetuses are separated into two groups toallow for the detection of possible systematic errors owing to incomplete restriction enzyme digestion; their mothers are homozygous either for presence(n = 7) or absence (n = 6) of the FokI restriction site

Fetus no Gestation Tissues

(weeks) Heart Sp cord Muscle Kidney Lung Gut Eye Brain Adrenal Stomach Gonad

Maternal allele upper(uncut) (n= 6)MC2601 10 + + + + + +CA2505 6 ± +LF0101 10 + + + + + +FBI103 12 + + + + + + + +SN3010 8 + + +LR1907 13 + + + + + + + + + +

Maternal allele lower (cut)(n= 7)NG1003 6 +DB2807 8 + + +HW3004 11 + + + +EH1311 8 + + +DM0909 9 + + + +MT1509 7 + +HM1709 10 + + + + +

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Figure 2 (A) Biallelic expression of GNAS1, shown by FokI digestion of RT-PCR products derived from 10different tissue types. Both 173 bp (undigested) and 122 bp (digested) fragments are present. Both of these fetuses wert

heterozygous for the FokI polymorphism, the maternal allele being the upper (LR 19) or lower (DM 09) bandrespectively. An apparent increase in density of the upper band is artefactual owing to counter migration of ethidiumbromide. Genomic DNA PCR controls, showing homozygosity for both lower (205 bp) and upper (256 bp) bands, andheterozygosity are shown for comparison. The marker is a 123 bp DNA ladder. FokI possesses an asymmetricalrecognition site (GGA TG) and cleaves the ds-DNA 9 base pairs downstream.4' This suggests the possibility that thisenzyme may be capable of cleaving one or other of the heteroduplexes generated by PCR in heterozygotes. In support ofthis, a consistent bias in favour of the upper (uncut) fragment was not encountered suggesting complete digestion. (B)Monoallelic expression of IGF2, shown by ApaI digestion of RT-PCR products. Fetuses LR 19 and DM 09 were

again informative, the paternal allele in both cases being represented by an undigested product. They were used to showexclusive paternal expression (236 bp), with absence of a maternal RT-PCR product (173 bp) in all 10 tissue typesexamined. Genomic DNA controls as in fig 2A are shown for comparison. Digestion ofgenomic DNA PCR productsfrom heterozygotes consistently showed greater intensity of the uncut product suggesting failure of ApaI (with a

symmetrical recognition site, GGGCCC) to digest heteroduplexes.3542 This experiment indicates evidence of imprintingin a wider selection of human fetal tissues and at earlier gestational ages than has previously been reported.

Table 3 Ratio of expression of GNAS1 alleles (maternal/paternal) in adult lymphocytes. Seven informative persons are represented. Scintillationcounting (single experiment), phosphorimager analysis, and ALF (mean of six experiments) showed no significant predominance of maternal topaternal expression

MB2906 _70603 LC1303 EG3011 CM3110 KM0903 LF0105

Scintillation:Ratio mat/pat 0-87 1-16 1-26 1-41 0-85 1.1 1-88

Phosphorimaging:Mean ratio mat/pat (SEM) 1-23 (0-073) 0-95 (0-066) 1-04 (0 089) 1-24 (0-19) 1-08 (0-061) 1-0 (0-102) 1-1 (0-13)

ALF:Mean ratio mat/pat (SEM) 0-99 (0-062) 0-87 (0-075) 1-35 (0-106) 1-4 (0-122) 1-5 (0-101) 1-07 (0-095) 1-15 (0-069)

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Figure 3 Phosphorimager analysis of a 10% polyacrylamide gel, showingexpression of GNAS1 following FokI digestion of R T-PCR products fromLR 19. Ten tissue types are shown. Below each lane are the values obtaineaquantification of each band, expressed as a ratio of maternal (upper): pate.(lower) allele.

allowing assignment of the origirallele. No statistically significant diiparental allelic expression could bescintillation counting, phosphorimautomated laser fluorescence (table.

DiscussionThe pattern of inheritance of Albrigditary osteodystrophy has suggesform of parental control of GNAS1tion."6 From the available evidence itcipated that were this gene imprintcnal monoallelic expression m

demonstrable in one or more tissues.our three analytical methods showeexpression in all fetal tissue typesand at all gestations.

Campbyll, Gosden, Bonthron

For some imprinted genes, privileged tis-sues may fail to silence the imprinted allele.2128This implies that control of imprinting canoperate in a tissue specific manner. Further-more, in individual tissues, some imprintedgenes may also show incomplete transcriptio-nal repression.313' The mechanism underlying

173 bp the latter observation could be either biallelicexpression by certain cell types within a tissue,

122 [ or alternatively partial silencing of one allele inall cells of such tissues. Both of these observa-tions suggest that imprinting is not necessarilyan "all or nothing" phenomenon. An analogywhich may be drawn is that of partial escape

biallelic from X inactivation by certain genes, includ-fetus ing the human steroid sulphatase gene.39 Arnal measure of the ratio of female to male steroid

sulphatase activity (XX dosage:XY dosage)yields a range of 1A42, 1 7, or 1-8: 1343540 ratherthan the 1:1 or 2:1 values expected for com-plete inactivation or non-inactivation of this

a of each gene respectively.fference in If GNAS 1 is under imprinted control, suchshown by control is unlikely to operate in a simple globallaging, or way. In erythrocytes of patients with PHP3). type la, Gs activity is reduced to about half

normal, rather than being 0 or 100% asexpected for a completely imprinted gene.This suggests the possibility of cell type speci-

;ht's here- fic or subtle quantitative imprinting effects. It;ted some was for this reason that we surveyed a widetranscrip- range of different tissues and quantitated rela-t was anti- tive levels of expression as far as was possible.ed, mater- Both of our 32P based assays suggested aiight be small preponderance of maternal over paternalHowever, expression (ratio 1 2) in virtually all tissuesd biallelic examined (tables 2 and 3). A similar but non-examined significant trend was seen using the fluorescein

labelling protocol; this assay disappointingly

1Homozygote91.. It\

Lane 1

HomozygoteLane 2 ______________(uncut)

il. ~~~~~~~~~~~~~LR19 kidneyLane 3 -^ w¢i.9..^^V!;DR

4

LK 19 muscleLane 4 K jN4eSN _

Lane 5 ,, , I LR 19 brain

Lane 6 ._ , LR 19 gut

Lane 7 , P irannl

30 451 122 bp 1 173 bp 960 75 90

Figure 4 Analysis of allelic expression of GNAS1 by automated laser fluorescence.Peaks 1-4 represent the fluorescent primer peak, primer-dimer complexes, and digested(122 bp) and undigested (173 bp) GNAS1 products respectively. Lanes 1 and 2 showresults obtained from fetuses homozygous for cut and uncut fragments. Lanes 3-7represent the heterozygous fetus LR 19 (kidney, muscle, brain, gut, adrenal) and showthe equivalence of maternal and paternal peak sizes.

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Parental origin of transcription from the human GNASI gene

displayed a rather greater degree of randomvariability. Although a slight maternal pre-dominance of transcription would provide apossible explanation for the more severephenotype observed on maternal transmissionof PHP/PPHP, we do not consider that ourresults prove this point. There is a risk thatcontamination of the fragmented fetal tissuesby maternal blood or tissue could give rise to asimilar effect, despite the careful washingwhich was performed. To address this possib-ility, the analysis of adult peripheral bloodlymphocyte GNAS 1 expression was under-taken. Not only was this experiment free frompossible maternal contamination effects, but itwas also performed blinded, the RT-PCRquantification being performed before the par-ental genotypes were revealed. Table 3 showsthat a reproducible difference in expressionlevels between the maternal and paternalalleles was not obtained in this experiment.

If GNAS1 imprinting is a tissue specificphenomenon, it should be possible from thephenotypic features of AHO to predict whichtissues might be involved. These would in-clude PTH target tissues and other endocrineorgans in which adenylate cyclase was thesecond messenger and whose function is dis-rupted in AHO, namely kidney (proximaltubule), thyroid, gonad, and bone. Why suchembryologically diverse tissues should share acommon transcriptional control mechanism is,however, difficult to explain. We have beenunable to show a target tissue specific effect atthe whole organ level, having examined kid-neys from three fetuses and one pair of fetalovaries. It remains possible, though, thatimprinting is limited to a specific subset ofhormone sensitive cells. If so, examining thewhole fetal kidney might not show such aneffect, as the PTH sensitive proximal renaltubular epithelial cells would form a tiny pro-portion of the total tissue.

In summary, we have found no compellingexperimental evidence that GNAS1 is underimprinted control, despite the strong clinicalevidence supporting such an effect. Prospect-ive study of further AHO pedigrees, combinedwith mutational analysis, may shed furtherlight on the underlying postulate of imprintedinheritance of PHP and PPHP.

We thank Alison Rae for operation of the ALF machine, andacknowledge the help with specimen collection of our clinicalcolleagues at the Royal Infirmary of Edinburgh. Ethical Com-mittee permission and supervision of the fetal work was by theLothian Health Board Medicine/Oncology and ReproductiveMedicine/Paediatric Ethical Committees. We thank ProfessorD J H Brock in whose unit the experimental work was per-formed. This work was jointly supported by the University ofEdinburgh (Cancer Research Fund) and the Medical ResearchCouncil. RC is an MRC research training fellow.

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