Proc. Natl. Acad. Sci. USAVol. 88, pp. 3767-3771, May 1991Medical Sciences

Anti-Mullerian hormone Bruxelles: A nonsense mutation associatedwith the persistent Mullerian duct syndrome

(cDNA polymerase chain reaction/illegtimate transcription/Mullerian-inhibiting substance/sexual differentiation)

BERTRAND KNEBELMANN*, LAURENCE BOUSSINt, DANIEL GUERRIERt, LAURENCE LEGEAIt, AXEL KAHN*,NATHALIE JOSSOt, AND JEAN-YVES PICARDtftUnitd de Recherches sur l'Endocrinologie du DE&eloppement, Institut National de la Sante et de la Recherche MWdicale Unite U293, Ecole Normale Supdrieure,Departement de Biologie, 1 rue Maurice-Arnoux, 92120 Montrouge, France; and *Unite de Recherches en Gdndtique et Pathologie Moldculaires, Institut Nationalde la Sante et de la Recherche Mddicale Unite U129, Institut Cochin de Gdndtique Moldculaire, 24 rue du Fg St. Jacques, 75014 Paris, France

Communicated by Alfred Jost§, January 7, 1991

ABSTRACT The persistent Mullerian duct syndrome(PMDS) is characterized by the persistence of Mfillerian de-rivatives, uterus and tubes, in otherwise normally virilizedmales. In a previous study, we showed that this syndrome isheterogeneous, with lack of production of anti-Mullerian hor-mone (AMH) by testicular tissue accounting for only some,AMH-negative, cases of this disorder. We have characterizedthe point mutation responsible for an AMH-negative PMDS inthree siblings: a guanine to thymine transversion at position2096 in the fifth exon changes a GAA triplet, coding forglutamic acid, to a TAA stop codon. The mutation could alsobe recognized, using the polymerase chain reaction, on RNAproduced in trace amounts by a lymphoblastic cell line. Thetranslation product, although undetectable in testicular tissue,could be visualized in culture medium of cells transfected withthe mutant gene.

The persistent Mullerian duct syndrome (PMDS) is a rareform of inherited male pseudohermaphroditism character-ized by the presence of uterus and tubes in otherwisenormally virilized males. The condition, usually discoveredat surgery for inguinal hernia and/or cryptorchidism, hasbeen reported in approximately 150 patients to date.Regression of Mullerian ducts, the first step of male

differentiation of the internal reproductive tract, is due to theproduction by the fetal testis of anti-Mullerian hormone(AMH) (1), a glycoprotein dimer, also called Mullerian-inhibiting substance or factor. Recent data have demon-strated that not all cases ofPMDS are linked to a defect oftheAMH gene itself, with some patients expressing a normalamount of bioactive testicular AMH (2). Delineation of themolecular defect responsible for the AMH-positive type ofPMDS will probably require prior characterization of theAMH receptor.

In contrast, the molecular tools for analyzing the AMH-negative form ofPMDS are already available: AMH has beenpurified to homogeneity (3), and the cDNA (4) and genomicDNA (5) coding forAMH have been cloned. The human genecontains 2.8 kilobases (kb), arranged in five exons; the fifthone is the most conserved among mammals and showsmarked homology to the transforming growth factor ,B su-perfamily (5).Using the polymerase chain reaction (PCR) to amplify and

sequence DNA from a child with AMH-negative PMDS, wehave identified a nonsense mutation leading to the synthesisof a truncated gene product. The variant hormone, labile intesticular tissue, was detectable in the culture medium oftransfected Chinese hamster ovary (CHO) cells. The muta-tion could also be identified on the minute amount of AMH-

specific RNA "illegitimately" (6) transcribed by a lympho-blastoid cell line. We have named the mutation AMH Brux-elles, for the city in which the patients have lived.

MATER1ALS AND METHODSSource of Human Materials. DNA and RNA were prepared

from Epstein-Barr virus-transformed lymphocytes (7) in fivepreviously reported patients with PMDS (2). Four wereAMHnegative (numbering according to ref. 2): patient 2, of Flem-ish ancestry, aged 2 months, and patients 4-6, born inMorocco, brothers aged 5 yr, 2 months, and 7 yr, respec-tively. No bioactive or immunoreactive AMH was detectablein the testicular tissue ofthese siblings, but specific RNA wasexpressed in the amount expected for age (2). The parentswere not available for study. The fifth previously reportedpatient, no. 3, was AMH positive. DNA was prepared fromlymphocytes of one other AMH-negative PMDS patient, no.7, an 8-month-old Caucasian American infant with PMDS,whose case will be reported separately (D. Loeff, I. M.Rosenthal, and J.-Y.P., unpublished work). In this subject,no AMH could be detected in serum by ELISA (8), or in atesticular biopsy by immunocytochemistry (9), with eitherpolyclonal antibody (pAb) or monoclonal antibody (mAb).Initiation sites of RNA transcription were normal by S1nuclease protection assay; the amount of testicularRNA thatcould be prepared from the biopsy was insufficient to allowRNA-blot hybridization analysis. Control DNA was obtainedfrom subjects with normal sex differentiation.

Reagents. Human recombinant AMH (10) and mAb 10.6were generous gifts ofRichard L. Cate and R. Blake Pepinsky(Biogen). mAb 10.6, raised against human recombinantAMH, is directed against a species-specific epitope locatedon the N-terminal side of the molecule (R. B. Pepinsky,personal communication). Anti-AMH pAb was raised againstpurified bovine AMH and is not species specific in mammals(9). All other reagents were of the highest research gradecommercially available. Dulbecco's minimum essential me-dium, with and without nucleosides, was obtained fromGIBCO-BRL.PCR. PCR (11) was carried out in a DNA thermal cycler

(Perkin-Elmer/Cetus) with Thermus aquaticus (Taq) poly-merase from the same source. Oligonucleotide primers (Fig.1), designed to amplify the five exons and the exon-intronboundaries of the AMH gene, were obtained from the InstitutPasteur and purified by polyacrylamide gel electrophoresis.

Abbreviations: PMDS, persistent Mullerian duct syndrome; AMH,anti-Mullerian hormone; PCR, polymerase chain reaction; CHO,Chinese hamster ovary; mAb, monoclonal antibody; pAb, polyclonalantibody.fTo whom reprint requests should be addressed.§Deceased, February 3, 1991.

3767

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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3768 Medical Sciences: Knebelmann et al.

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FIG. 1. PCR amplification of ge-nomic DNA and cDNA. The direc-tion ofthe sense (s) and antisense (a)oligonucleotide primers is indicatedby arrows. (A) DNA amplification,using primer couples 1-5. See B forasterisk designation. (B) cDNA am-plification, first using primer couple7 and then using nested primer cou-ple 8. On the left, amplification ofcDNA is expected to yield a frag-ment of 536 base pairs (bp) betweenprimers 8s and 8a. On the right isshown the result that would havebeen obtained, had contaminatinggenomic DNA been amplified in-stead of cDNA. A 626-bp fragment,including the 90-bp intron, wouldhave been observed. Location ofthe mutation is marked by an aster-isk. (C) Sequences of oligonucleo-tides, identical (s) or complemen-tary (a) to the coding strand; num-bers refer to the location of the first5' base, as published by Cate et al.(5).

Genomic DNA was digested with EcoRI to increase ampli-fication efficiency. The PCR reaction was carried out in 50 ,AlofPCR buffer consisting of 67 mM Tris HCl (pH 8.8), 6.7 mMMgCl2, 17 mM S04(NH4)2, 10 mM 2-mercaptoethanol, 6.7,4M EDTA, 2mM (each) dNTP (dATP, dCTP, dGTP, dTTP),and 10% (vol/vol) dimethyl sulfoxide. To this buffer wereadded 2 units of Taq polymerase and 80 pmol (each) ofupstream and downstream oligonucleotide primers (Fig. 1).PCR conditions were 30 cycles of denaturation at 92TC for 1min, annealing at 55TC for 1 min, and primer extension at 720Cfor 1 min. Amplified products were analyzed on a 10%polyacrylamide gel and in some cases purified by electro-phoresis through a 2% (wt/vol) low-melting-point agarose gel(NuSieve, FMC).

Cloning and Sequencing of Amplified DNA. PCR productswere phosphorylated and blunt-ligated into Sma I-digestedphosphatase-treated M13 vector. Recombinant phages werescreened with oligonucleotide probes allowing identificationof sense and antisense strands. The latter were sequencedwith the dideoxynucleotide chain-termination method (12)using dATP[a-35S] and T7 DNA polymerase (Sequenase,United States Biochemical).

Southern Blotting Analysis and Gene Dosage. GenomicDNA from patients 2-6 and from normal male and femalecontrols was digested by Taq I restriction enzyme. Identicalamounts were loaded on a 1% agarose gel and transferredonto a nylon Hybond-N+ membrane (Amersham). Twoprobes labeled with [32P]dCTP by random priming were used:a 2.8-kb genomic DNA segment spanning the full AMH geneand an X chromosome-located probe, XJ2.2 (13) recognizingpart of the dystrophin gene. Hybridization was performed at65°C for 15 hr with 1.5 x 106 cpm/ml. Band intensities on theautoradiogram were measured with a Shimadzu densitome-ter. The intensity of hybridization of the AMH probe wascompared to that of the X-located one.

Allele-Specific Oligonucleotide Hybridization. Thirty-nanogram samples of heat-denatured PCR products weredot-blotted on a Hybond-N membrane (Amersham). Oligo-nucleotides wl and w2 and ml and m2 (Fig. 4), matchingeither the wild-type or the mutant AMH gene, were labeledby kination and hybridization was performed for 16 hr at 45°Cin 3x SSPE (lx SSPE = 0.18 M NaCl/0.01 M NaH2PO4/0.001 M EDTA, pH 7.4), 5x Denhardt's solution, 0.5%(wt/vol) SDS, and 150 ,g of sonicated salmon sperm DNAper ml, with 106 cpm/ml. Blots were washed in 2x SSPE atroom temperature for 15 min and then at the discriminatingtemperature for each oligonucleotide for 5 min (14).cDNA PCR Amplification. Total cellular RNA was pre-

pared from lymphoblasts according to the method of Chirg-win et al. (15). cDNA synthesis was carried out from 10 ,ugof total RNA in 20 ,ul of the PCR buffer described abovecontaining 1 mM ofeach dNTP. To this buffer were added 100pmol of random hexanucleotide primers (16), 20 units ofRNasin (Promega Biotec), and 200 units of Moloney murineleukemia virus reverse transcriptase. The reaction was al-lowed to proceed for 30 min at 42°C, before the reversetranscriptase was inactivated for 5 min at 95°C. PCR ampli-fication was carried out in the same tube after adjustment ofthe volume to 100 ,ul with PCR buffer containing oligonucle-otides 7s and 7a (Fig. 1). Cycle number was increased to 40.The PCR product was subjected to a second PCR amplifica-tion, using nested primers 8s and 8a (Fig. 1), under theconditions described for genomic DNA.

Expression Analysis of the Normal and Mutant AMH Gene.HumanAMH genomic DNA was cleaved between restrictionsites Afl II, at position -26, and Not I, at approximatelyposition 3000, and inserted into plasmid pSV2 (17). To obtainrecombinant AMH Bruxelles, the fragment of the mutantgene located between restriction sites Xho I and Sac I, atpositions 1790 and 2352, respectively, was amplified by PCR

B

Proc. Natl. Acad. Sci. USA 88 (1991)

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FIG. 2. Identification of a stopcodon mutation in the last exon ofthe AMH gene of patient 5. (Left)Comparison of normal and mutantDNA shows the guanine to thyminetransversion (closed circle), chang-ing the GAA triplet coding for Glu-358 to a TAA stop codon. (Right)This mutation is best shown (ar-rows) by migrating normal (N) andmutant (M) DNA samples side byside for each base.

and ligated to the vector digested by the same enzymes. Thesequence of the amplified fragment was checked by sequenc-ing in phage M13. pSV2 vectors carrying either the wild-typeor the mutant AMH gene were cotransfected into CHO cellstogether with dihydrofolate reductase recombinant DNAcarried in plasmid pAdD26 (18). The transfection efficiencyof pAdD26 is enhanced by the simian virus 40 enhancercarried by the pSV2 vector, allowing coamplification of thedihydrofolate reductase and the AMH genes. Transformantswere selected by culture in Dulbecco's alpha minimal essen-tial medium, without nucleosides, enriched with 1o dia-lyzed fetal calf serum. AMH production by the recombinantclones was measured by ELISA, performed as described (8)except that mAb 10.6 was used, to exclude reaction withbovine AMH contained in fetal calf serum. The normal andmutant AMH gene products were partially purified fromculture medium, loaded on a 30%/4% polyacrylamide gradi-ent gel (Pharmacia), blotted onto a nitrocellulose membrane,and revealed by silver/gold intensification (Janssen Pharma-ceutica), according to the manufacturer's instructions, afterincubation with mAb 10.6 (1 ug/ml). All samples wereelectrophoresed with and without prior reduction of disulfidebonds with 2-mercaptoethanol, as described (3).

RESULTSIdentification of an Ochre Codon, Leading to a Nonsense

Mutation in the Fifth Exon. Southern blotting of the DNA ofpatients 4-6 revealed no deletion or gene rearrangement(results not shown), suggesting that a point mutation wasresponsible for the PMDS in this family. This was confirmedby sequencing of the cloned PCR products of the genomicDNA of patient 5. A guanine to thymine transversion, atposition 2096 in the fifth exon, changes a GAA triplet, coding

for Glu-358, into a TAA ochre termination codon (Fig. 2). Inaddition, an adenine to thymine transversion at position 2191changes a GGA codon to a GGT one (data not shown), butthis mutation is silent because both triplets code for glutamicacid. Both mutations were recognized on 12 independentclones from patient 5, containing either sense or antisensestrands, statistically ruling out the possibility of a Taq poly-merase error. In contrast, the AMH gene of patient 3, with anAMH-positive PMDS, and of the control subject adhered tothe wild-type sequence (5).

Visualization of the Mutant Gene Product, AMH Bruxelles.Western blotting ofpartially purified recombinant AMH fromculture medium of CHO cells transfected with either thenormal or the mutant AMH gene revealed a striking sizedifference between the gene products (Fig. 3). AMH secretedby CHO cells transfected with the wild-type gene was similarto the positive control, whereas the product of cells trans-fected with the mutant gene was much shorter. The meanmolecular mass of mutant AMH, computed from two inde-pendent experiments (r = 0.985 and 0.988, respectively), was74 ± 5 kDa for the dimer and 35 ± 4.3 kDa for the monomer.

Establishment ofHomozygosity and Comparison with OtherAMH-Negative PMDS Patients. Compound heterozygositywas eliminated by the results of allele-specific hybridization(Fig. 4), which showed that, at the site of the mutation, thepatient's DNA hybridized only with the "mutant" oligonu-cleotide probe and not with the wild-type one. Identicalresults were obtained with DNA from the patient's twoaffected brothers. In contrast, the DNA from two unrelated

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FIG. 4. Hybridization with allele-specific oligonucleotides. Ge-nomic DNA was amplified by PCR using primers 4s and 4a, shownin Fig. 1. One microliter was dot-blotted in quadruplicate on Hy-bond-N filters and hybridized with either wild-type (w) or mutant (m)probes for the nonsense (1) and the silent (2) mutations. Washing wasperformed at appropriate temperatures as indicated on the right. Thesequence of allele-specific probes is shown, with bold letters indi-cating the mutant bases. Lane A, control; lane B, patient 3; lanesC-E, patients 4-6; lanes F and G, patients 2 and 7. DNA fromsiblings 4-6 hybridized only with the mutant probes, whereas allother samples hybridized only with the normal probes, indicating thatthe other PMDS patients do not share the same mutations.

Medical Sciences: Knebelmann et al.

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FIG. 5. Genotype analysis by Southern blotting. Four micro-grams of genomic DNA was digested by Taq I and loaded on a 1%agarose gel. Hybridization was performed as described in the text.Lanes A and B, control females; lanes C and D, control males; lanesE-I, patients 6, 4, 5, 2, and 3, respectively. Band intensity wasquantitated in a Shimadzu CS 930 densitometer. The ratio betweenthe intensity of the two main AMH bands versus the X-linked XJ2.2one is shown beneath each lane. This ratio is twice as high in malesas in females. No difference between controls and patients was seenindicating that the latter had two copies of the AMH gene.

AMH-negative PMDS patients, no. 2 and no. 7, hybridizedonly with the wild-type probe at this location, indicating thatthe genetic abnormalities responsible for AMH-negativePMDS are heterogeneous.Homozygosity for the variant gene was established by

comparative Southern analysis and by allele-specific hybrid-ization. When identical amounts of DNA were electropho-resed and hybridized with a full-length AMH probe and an Xchromosome-linked one, the ratio of the intensity of theAMH versus the X-linked gene was twice as high in controland in PMDS males compared to females (Fig. 5), thus rulingout the possibility that the second AMH allele was deletedinstead of mutated.The Mutation Can Be Shown Using "Illegitimate" Tran-

scripts in Lymphoblasts. Two rounds of amplification of theDNA complementary to RNA extracted from lymphoblas-toid cells, which do not normally express AMH, led to thevisualization of a band whose BamHI restriction pattern wasthat expected for an AMH cDNA fragment (results notshown). Furthermore, the size of the fragment is in good

Proc. Natl. Acad. Sci. USA 88 (1991)

agreement with the 536-bp size expected if the cDNA lackingthe 90-bp intron has been amplified (and not possibly con-taminating DNA) (Fig. 1). This band was eluted, cloned in

- M13 phage, and sequenced. The cDNA carried the twomutations characteristic of AMH Bruxelles (Fig. 6).

DISCUSSIONThe results of this study indicate that a nonsense mutation inthe fifth exon of the AMH gene is associated with PMDS inthree siblings. Nonsense mutations create a stop codon thatinterrupts gene translation. Characteristically, as in our ownpatients (2), RNA accumulation is normal-this, however, isnot always the case (19-23). The truncated product of non-sense gene mutations may (24) or may not (25-28) be detect-able. In the present case, the variant AMH lacks 178 aminoacids on the C-terminal side, out of a total number of 535.Although the portion of the molecule responsible for bioac-tivity has not yet been conclusively mapped, the C-terminalend is the best candidate, because it is the part ofthe moleculemost conserved during evolution and the one showing ho-mology to the active portion of the transforming growthfactor P precursor (10). Although immunocytochemicallyundetectable in testicular tissue, AMH Bruxelles was readilysecreted by transfected CHO cells. Truncated gene productsare expected to exhibit severe alterations in tertiary struc-ture, making them more susceptible to posttranslationaldegradation by intracellular proteolytic enzymes (29), whichare probably of different specificity in human testicular andhamster ovarian cells. Biochemical study of purified AMHBruxelles will shed more light on this problem in the future.The mutation involved in the creation of the stop codon

does not affect a CpG site, the most frequent target ofspontaneous mutation (30). Guanine to thymine transver-sions have been reported relatively rarely-for instance, theyaccounted for only 3 of >30 3-globin mutations investigatedby Kazazian and his colleagues (31, 32). The mutation wasnot shared by two unrelated cases of AMH-negative PMDS,from different ethnic backgrounds (Fig. 4). Its autosomicrecessive mode of transmission is in keeping with the local-ization of the AMH gene on chromosome 19 (33). Althoughthe parents denied being related, consanguinity remains apossibility in this Moroccan family originating from a smallvillage.The AMH gene is normally expressed only in immature

Sertoli cells; in boys, serum levels begin to decline as earlyas 2 yr postnatally (8). It is therefore difficult to obtain tissuesamples for isolation of specific cDNA. Chelly et al. (6) haverecently shown that any gene is transcribed in any cell type,albeit in very low abundance. We took advantage of this"illegitimate transcription" to test whether it was possible toidentify a mutation on RNA by this technique. This indeedproved feasible (Fig. 6), opening up new possibilities for the

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Proc. Natl. Acad. Sci. USA 88 (1991) 3771

detection by PCR of mutations occurring in huge genes withnumerous exons.AMH, in addition to its undisputed role in triggering

Mullerian regression, has been shown to masculinize fetalovaries (34). Unregulated production of AMH induces tran-sient differentiation of seminiferous tubules in the ovaries oftransgenic mice, leading Behringer and his colleagues (35) tosuggest that AMH plays a role in normal testicular differen-tiation. Although, obviously, the AMH gene is a temptingtarget for the newly isolated SRY gene (36, 37), the demon-stration that a mutation leading to the synthesis ofa labile andinactive AMH does not block the differentiation of functionaltesticular tissue conclusively proves that AMH is not for-mally required for male gonadal organogenesis.

Note. This arcicle is dedicated to the memory of Prof. Alfred Jost,who died shortly after communicating it to Proc. Natl. Acad. Sci.USA. This great scientist, the first to demonstrate the existence of adiscrete testicular factor responsible for the regression of fetalMullerian ducts, had taken an active interest in the pursuit ofAMHresearch, and his encouragement was of invaluable help to us.

We are grateful to Drs. Said Akli, Jamel Chelly, Aldna Leroux, andDominique Recan for their help and advice and to Drs. R. L. Cateand R. B. Pepinsky for their generous gift of purified recombinantAMH and mAb 10.6 and for guidance in recombinant proteintechnology. We are indebted to Drs. D. Loeff and I. M. Rosenthalfor allowing us access to their patient, to Isabelle Lamarre for experttechnical assistance, and to Michel Cometto and the Banque deCellules de l'Association Frangaise contre la Myopathie for thelymphoblast cell culture.

1. Jost, A. (1953) Rec. Progr. Horm. Res. 8, 379-418.2. Guerrier, D., Tran, D., VanderWinden, J. M., Hideux, S., Van

Outryve, L., Legeai, L., Bouchard, M., VanVliet, G., DeLaet,M. H., Picard, J. Y., Kahn, A. & Josso, N. (1989) J. Clin.Endocrinol. Metab. 68, 46-53.

3. Picard, J. Y. & Josso, N. (1984) Mol. Cell. Endocrinol. 34,23-29.

4. Picard, J. Y., Benarous, R., Guerrier, D., Josso, N. & Kahn,A. (1986) Proc. Natl. Acad. Sci. USA 83, 5464-5468.

5. Cate, R. L., Mattaliano, R. J., Hession, C., Tizard, R., Farber,N. M., Cheung, A., Ninfa, E. G., Frey, A. Z., Gash, D. J.,Chow, E. P., Fisher, R. A., Bertonis, J. M., Torres, G., Wall-ner, B. P., Ramachandran, K. L., Ragin, R. C., Manganaro,T. F., MacLaughlin, D. T. & Donahoe, P. K. (1986) Cell 45,685-698.

6. Chelly, J., Concordet, J. P., Kaplan, J. C. & Kahn, A. (1989)Proc. Natl. Acad. Sci. USA 86, 2617-2621.

7. Neitzel, H. (1986) Hum. Genet. 73, 320-326.8. Josso, N., Legeai, L., Forest, M. G., Chaussain, J. L. &

Brauner, R. (1990) J. Clin. Endocrinol. Metab. 70, 23-27.9. Tran, D., Picard, J. Y., Campargue, J. & Josso, N. (1987) J.

Histochem. Cytochem. 35, 733-743.10. Pepinsky, R. B., Sinclair, L. K., Chow, E. P., Mattaliano,

R. J., Manganaro, T. F., Donahoe, P. K. & Cate, R. L. (1988)J. Biol. Chem. 263, 18%1-18%5.

11. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi,R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science239, 487-491.

12. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl.Acad. Sci. USA 74, 5463-5467.

13. Thompson, M. W., Ray, P. N., Belfall, B., Duff, C., Logan,C., Oss, I. & Worton, R. G. (1986) J. Med. Genet. 23, 548-555.

14. Conner, B. J., Reyes, A. A., Morin, C., Itakura, K., Teplitz,R. L. & Wallace, R. B. (1983) Proc. Natl. Acad. Sci. USA 80,278-282.

15. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter,W. J. (1979) Biochemistry 18, 5294-5299.

16. Kawasaki, E. S. (1990) in PCR Protocols, eds. Innis, M. A.,Gelfand, D. H., Sninski, I. I. & White, T. J. (Academic, NewYork), 1st Ed., pp. 21-27.

17. Subramani, S., Mulligan, R. & Berg, P. (1981) Mol. Cell. Biol.1, 854-864.

18. Kaufman, R. J. & Sharp, P. A. (1982) J. Mol. Biol. 159,601-621.

19. Urlaub, G., Mitchell, P. J., Ciudad, C. J. & Chasin, L. A.(1989) Mol. Cell. Biol. 9, 2868-2880.

20. Marcelli, M., Tilley, W. D., Wilson, J. E., Griffin, J. E., Wil-son, J. D. & McPhaul, M. J. (1990) Mol. Endocrinol. 4, 1105-1116.

21. Atweh, G. F., Brickner, H. E., Zhu, X. X., Kazazian, H. H. &Forget, B. G. (1988) J. Clin. Invest. 82, 557-561.

22. Orkin, S. H. & Kazazian, H. H., Jr. (1984) Annu. Rev. Genet.18, 131-171.

23. Globerman, H., Amor, M., Parker, K. L., New, M. I. & White,P. C. (1988) J. Clin. Invest. 82, 139-144.

24. Ware, J., Russell, S. R., Vicente, V., Scharf, R. E., Tomer, A.,McMillan, R. & Ruggeri, Z. M. (1990) Proc. Natl. Acad. Sci.USA 87, 2026-2030.

25. Chang, J. C. & Wai Kan, Y. (1979) Proc. Natl. Acad. Sci. USA76, 2886-2889.

26. Safaya, S. & Rieder, R. F. (1988) J. Biol. Chem. 26, 4328-4332.27. Lichter-Konecki, U., Konecki, D. S., DiLella, A. G., Brayton,

K., Marvit, J., Hahn, T. M., Trefz, F. K. & Woo, S. L. C.(1988) Biochemistry 27, 2881-2885.

28. Attree, O., Vidaud, D., Vidaud, M., Amselem, S., Lavergne,J. M. & Goossens, M. (1989) Genomics 4, 266-272.

29. Adams, J. G., Steinberg, M. H. & Kazazian, H. H. (1990) Br.J. Haematol. 75, 561-567.

30. Green, P. M., Montandon, A. J., Bentley, D. R., Ljung, R.,Nilsson, I. M. & Giannelli, F. (1990) Nucleic Acids Res. 18,3227-3231.

31. Antonarakis, S. E., Kazazian, H. H., Jr., & Orkin, S. H.(1985) Hum. Genet. 69, 1-14.

32. Kazazian, H. H., Jr., Orkin, S. H., Boehm, C. D., Goff, S. C.,Wong, C., Dowling, C. E., Newberger, P. E., Knowlton,P. G., Brown, V. & Donis-Keller, H. (1986) Am. J. Hum.Genet. 38, 860-867.

33. Cohen-Haguenauer, O., Picard, J. Y., Mattei, M. G., Serero,S., Nguyen, V. C., de Tand, M. F., Guerrier, D., Hors-Cayla,M. C., Josso, N. & Frezal, J. (1987) Cytogenet. Cell. Genet. 44,2-6.

34. Vigier, B., Forest, M. G., Eychenne, B., Bezard, J., Garrigou,O., Robel, P. & Josso, N. (1989) Proc. Nat!. Acad. Sci. USA86, 3684-3688.

35. Behringer, R. R., Cate, R. L., Froelick, G. J., Palmiter, R. D.& Brinster, R. L. (1990) Nature (London) 345, 167-170.

36. Sinclair, A. H., Berta, P., Palmer, M. S., Hawkins, J. R.,Griffiths, B. L., Smith, M. J., Foster, J. W., Frischauf, A. M.,Lovell-Badge, R. & Goodfellow, P. (1990) Nature (London)346, 240-244.

37. Gubbay, J., Collignon, J., Koopman, P., Capel, B., Economou,A., Munsterberg, A., Vivian, N., Goodfellow, P. & Lovell-Badge, R. (1990) Nature (London) 346, 245-250.

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