A 27 base-pair deletion of the anti-Müllerian type II receptor gene is the most common cause of the persistent Müllerian duct syndrome
A 27 base-pair deletion of the anti-Müllerian type II receptor gene is the most common cause of the persistent Müllerian duct syndromeSandrine Imbeaud, Corinne Belville, Liza Messika-Zeitoun, Rodolfo Rey, Nathalie di Clemente, Nathalie Josso and Jean-Yves Picard*
Unité de Recherches sur l'Endocrinologie du Développement (INSERM), Ecole Normale Supérieure, Département de Biologie, 1 rue Maurice Arnoux, 92120 Montrouge, France
Received April 16, 1996;Revised and Accepted June 11, 1996
The persistent Müllerian duct syndrome, characterized by the lack of regression of Müllerian derivatives, uterus and tubes in otherwise normally masculinized males, is a genetically transmitted disorder implicating either anti-Müllerian hormone (AMH), a member of the transforming growth factor-[beta] superfamily, or its type II receptor, a serine/threonine kinase homologous to the receptors of other members of the transforming growth factor-[beta] superfamily. We have now performed molecular studies in a total of 38 families. The basis of the condition, namely 16 AMH and 16 AMH receptor mutations, was identified in 32 families. The type of genetic defect could be predicted from the level of serum AMH which is very low or undetectable in patients with AMH mutations and at the upper limit of normal in receptor mutations. Whereas AMH mutations are extremely diverse, patients from 10 out of 16 families with receptor mutations had a 27 bp deletion in exon 10 on at least one allele. This deletion is thus implicated in approximately 25% of patients with persistent Müllerian duct syndrome. All AMH and AMH receptor mutations were consistent with an autosomal recessive mode of transmission.
Male sex differentiation is driven by two discrete fetal testicular hormones (1 ). Testosterone, synthesized by Leydig cells, maintains the Wolffian ducts and virilizes the external genitalia. Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance (MIS) or factor (MIF), secreted by immature Sertoli cells, is responsible for regression of Müllerian ducts in male fetuses. Various mutations of the AMH gene have been identified in the persistent Müllerian duct syndrome (PMDS) (2 ), a disorder characterized by the lack of regression of Müllerian derivatives in otherwise normally virilized 46,XY subjects. However, the AMH gene and AMH serum level are normal in approximately half the PMDS patients, implicating target organ insensitivity as a possible cause of PMDS. Indeed, a mutation of the AMH type II receptor (AMH-RII), belonging to the group of type II serine/threonine kinase receptors for members of the TGF-[beta] superfamily (3 ), has already been identified in a PMDS patient (4 ). We have now performed molecular studies in a total of 38 PMDS families and have detected19 new mutations of the AMH or AMH receptor genes, including a receptor deletion which accounts for approximately 25% of PMDS cases.
PMDS patients are externally phenotypic males in whom the presence of uterus and tubes is discovered during surgical correction of cryptorchidism and/or inguinal hernia. The clinical picture in our patients conforms to this general pattern, however in several cases the diagnosis was overlooked, either because symptoms were neglected or because Müllerian derivatives were not seen or correctly identified initially (5 ). The uterus was described as hypoplastic in three patients (nos 25, 33, 68).
The testes are tightly attached to the Fallopian tubes. Their position is variable: both may be intraabdominal, in the position normally occupied by ovaries; alternatively, one testis and the Müllerian derivatives may be placed in an inguinal hernia, a condition known as hernia uteri inguinalis. The controlateral testis may also be dragged into the hernia, producing transverse testicular ectopia (6 ). According to Hutson and Baker (7 ) the anatomical abnormality common to all PMDS variants lies in the failure of the gubernaculum to anchor the testis to the base of the scrotum. Secondary migration of the gubernaculum to the scrotum creates a patent processus vaginalis into which one or both testes, accompanied by Müllerian derivatives, can herniate. Abnormal testicular mobility facilitates torsion and can lead to secondary testicular degeneration observed bilaterally in patients 22, 23 and 25 (8 ), and unilaterally in patient 18. The phenotype can differ within the same family and bears no relationship to the type of genetic defect (AMH or AMH receptor) involved.
Testes are often not properly connected to male excretory ducts because of aplasia of the epididymis and upper part of the vas deferens or because of dissociation between the epididymis and the testis (patients 20, 27, 28, 30, 39, 45, 55, 56 and 68). The vas deferens is usually short and its lower segment is tightly associated to or even embedded in the uterine wall. Testicular histology is usually normal, with presence of spermatogonia, except in the event of longstanding cryptorchidism.
AMH serum concentration was measured in 41 PMDS patients, from 2 days to 77 years of age. AMH is detectable by ELISA in the serum of normal males from birth to puberty. After sexual maturation, the assay is no longer informative, since AMH production is normally repressed (10 ). Six of our patients were postpubertal and three had undergone fetal or postnatal testicular regression (8 ); they will not be considered further in this section. ELISA results in the 32 remaining cases are shown on Figure 1 . Ten of 11 patients with mutations of the AMH gene had abnormally low or undetectable serum AMH levels. The one exception, patient 68, with serum concentrations at the lower limit of normal, had a mutation of the last exon of the AMH gene, which codes for the C-terminal bioactive domain, whereas the monoclonal antibody used for the AMH ELISA recognizes an epitope on the N-terminal part of the protein. Serum AMH levels were at the upper limit of normal in most patients with a receptor mutation and were clearly elevated in two (nos 41 and 65). In contrast, AMH was low in the serum of patient 46, although serum AMH was normal for age in his affected older brother. Blood had been collected 4 days after attempted correction of cryptorchidism, a procedure which could have compromised testicular function. AMH measurements in patient 19 also showed a decrease from 39 ng/ml before surgery to 9 ng/ml afterwards. The mean and standard deviation of AMH serum concentration in the patients with completed molecular studies are shown in the legend to Figure 1 . Analysis of variance showed a significant difference (F = 15.12, p <0.0001) between mean AMH values according to the type of molecular defect.
The AMH gene was studied by single strand conformation polymorphism analysis (SSCP-PCR) in all PMDS patients from whom DNA was available. Fragments exhibiting abnormal migration were cloned into pGEM-T vector and sequenced. Mutations were identified in 16 families, nine of which have been previously reported (patients 1-13) (2 ). Details of new cases are shown on Table 1 ; the type and location of all AMH gene mutations are indicated on Figure 2 A. Exon 1 and the 3' half of exon 5 are the main sites of deleterious base changes, essentially short deletions and missense mutations.
Screening for mutations of AMH-RII were performed in all patients in whom a mutation of the AMH gene had been excluded. Southern blots failed to reveal large DNA rearrangements (results not shown). The AMH-RII gene was amplified by the polymerase chain reaction (PCR) using 11 primer pairs (Table 2 ). The PCR fragments were subjected to SSCP-PCR analysis. Normal results, apart from mobility shifts due to polymorphisms, were obtained in eight patients from six families; sequencing of the AMH-RII gene was not performed. One patient (no 24) had associated abnormalities of other hormonal receptors (9 ) and two others (nos 22, 23) had suffered testicular regression (8 ).
Sequences of oligonucleotides, identical (s) or complementary (a) to the coding strand, used for PCR and SSCP-PCR investigations; numbers refer to the location of the 5' base. Numbering begins at the initiation site of transcription, corresponding to base 734 in the sequence deposited in Genbank (U29700).
Fragments exhibiting abnormal migration by SSCP-PCR were subcloned into pGEM-T vector and sequenced. Significant mutations were observed in 18 patients from 16 families (Table 3 and Fig. 2 B), one of which (no 27) has been previously reported (4 ). The most frequent mutation was a 27 bp deletion ([Delta]27 bp) in exon 10 which is visible by acrylamide gel electrophoresis of PCR fragments (Fig. 3 ). This mutation ([Delta]6331-6357) leaves the reading frame intact while deleting nine amino acids from the intracellular region of the protein. It was present either in the homozygous state (patients 19, 31, 32, 40) or coupled with missense mutations (patients 14, 25, 33, 38, 45, 46, 65). In three patients, mutations truncating the extracellular domain of the receptor were restricted to a single allele, the other being normal by SSCP-PCR. These included nonsense mutations (patients 20 and 51) and a 4 bp deletion ([Delta]84-87) which disrupts the reading frame in patient 21 (Fig. 4 A).
Four intronic mutations, observed both in PMDS and normal subjects at an incidence of 4 to 19% (Table 4 ), represent polymorphisms of the AMH receptor gene, none of which were associated with any given mutation (Table 3 ). In patient 41, a newly created restriction site for DdeIwas discovered by chance while seeking for potential polymorphisms and led to the identification of a homozygous nonsense mutation (Fig. 5 ).
AMH mutations occur predominantly in patients from Arab or Mediterranean countries, characterized by a high rate of consanguinity, 81% of patients are homozygous. In contrast, patients with AMH receptor mutations come mainly from northern France or northern Europe and only 45% are homozygous. RFLP analysis was performed in the parents and siblings of the patients whenever possible. The parents of homozygotes and compound heterozygotes for AMH or AMH receptor mutations (Table 3 ) were carriers, as expected for a recessive mutation.
PMDS is considered an extremely rare form of male pseudohermaphroditism although the increasing number of cases brought to our attention in recent years suggests that this might be a misconception. The molecular basis of the condition was identified in 32 families. In the remaining six, the sequence of the AMH gene was normal and no abnormality of the receptor gene was detected by SSCP-PCR. However, mutations can be missed by this technique, even in PCR fragments under 200 bp (11 ). Alternatively, other genes required for signal transduction could be involved. Like other members of the TGF-[beta] family, AMH probably signals through two distantly related serine/threonine kinase receptors, type I and type II (3 ,12 ,13 ); only the latter has been cloned at the present time. The significant differences in mean AMH serum concentration (legend to Fig. 1 ) and the association of other receptor defects in patient 24 (9 ) suggest that, at least in some cases, a yet unidentified link of the AMH signalling pathway is missing.
In the families with recognized molecular defects, mutations of the AMH and the AMH receptor gene were equally distributed. In a previous report, involving nine families with AMH gene defects, although no true mutational `hotspot' was identified, mutations were seen to cluster at the 5' end of the gene. Surprisingly, the GC rich 3' end of exon 5, coding for the bioactive domain of the protein (14 ), was spared. Of the seven new mutations reported here, four again strike exon 1 but two map to the bioactive domain of the protein (Fig. 2 A)
Whereas mutations of the AMH gene are extremely diverse, with only one (E382 Stop) found in two different families, patients from 10 out of 16 families with a mutation of the AMH receptor have the [Delta]27 bp mutation on at least one allele (Table 3 ). Altogether, the [Delta]27 bp mutation accounts for 25% of PMDS cases and can be diagnosed rapidly by PCR. Deletional events in human genes appear to be at least in part related to local DNA sequence environment (15 ). A sequence, TGAGGC, common to various human deletion hotspots (15 ) is present at 6321-6326. The most common cause of small recurrent deletions, DNA repeats (16 ,17 ) such as Alu sequences (18 ), is not implicated in the [Delta]27 bp mutation. This deletion may result from a founder effect, since all alleles carrying this mutation possess the same haplotype, compared to approximately 50% in the general population.
Alignment between AMH-RII, the type II TGF-[beta] receptor (TGF-[beta]-RII) and the activin type II receptor (Fig. 4 ) shows that the [Delta]27 bp mutation is located immediately upstream of insert 2, a region which in TGF-[beta]-RII is crucial for kinase activity (19 ) and does not tolerate amino acid changes (19 ,20 ). Mutation V458A is located in insert 2 itself and D491H immediately precedes a proline which plays a key role in transphosphorylation of receptor type I (21 ). Missense mutations H282Q, D491H and R504C involve amino acids conserved in the receptor family.
Both the AMH (22 ) and the AMH receptor gene (4 ) are located on autosomes. Mutations were detectable on both alleles, except in three patients with receptor mutations (Table 3 ). The normal brother of patient 21 was heterozygous for [Delta]84-87, supporting the hypothesis that a second mutation, undetectable by SSCP-PCR, is present on the patient's other allele. In the other cases, familial studies were not informative, but prediction of the structure of the mutated protein and comparison with mutations of TGF-[beta]-RII provided indirect evidence for a recessive mode of transmission. The four nonsense mutations located in the extracellular domain (Fig. 4 A), including one present in the homozygous state, are expected to lead to early termination of translation, preventing the insertion of the protein in the membrane and interaction with downstream elements. Mutations in respectively the transmembrane (DR 26) or extracellular (DR 27) domains of TGF-[beta]-RII, which have been chemically induced in cell lines derived from mink lung cells, impair receptor function only in the homozygous state (23 ).
Dominant negative mutations, not detected in the present study, have been described for other receptors of the TGF-[beta] family. Truncation of TGF-[beta]-RII (19 ,24 ) or of the activin type II receptor (25 ), immediately after the transmembrane domain, produces dominant negative molecules which bind to ligand and type I receptor, but do not mediate antiproliferative or transcriptional responses. Binding of AMH to recombinant mutated AMH-RII can now be tested in vitro (26 ) but until a convenient in vitro assay for AMH transduction becomes available, the clinical repercussions of mutations of the AMH receptor remain the only way of approaching the AMH transduction pathway.
Genomic DNA from patients was digested by HindIII restriction enzymes. Identical amounts (10 [mu]g) were loaded on a 0.8% agarose gel (SeaKem GTG, FMC) and transferred onto a nylon Hybond-N+tmmembrane (Amersham). Two Megaprimetm probes (Amersham) labeled with [[alpha]32P]dCTP were used: one corresponding to the whole AMH gene and the other to full length AMH-RII cDNA. Hybridization was performed at 42oC for 15 h in 6* SSC, 5* Denhart, 10% (w/v) PEG, 1% (w/v) SDS and 50% (v/v) formamide containing 100 [mu]g/ml sonicated denatured herring sperm DNA and 3 * 106 c.p.m./ml probes. Membrane was washed three times 15 min in 2* SSC, 0.1% (w/v) SDS at 65oC (1* SSC is 150 mM NaCl and 15 mM sodium citrate).
DNA from patients and family members was extracted from peripheral blood lymphocytes. Amplification of the five exons and exon-intron boundaries of the AMH gene was performed as previously described (27 ). The AMH-RII gene was amplified by PCR using 11 pairs of oligonucleotides primers (Table 2 ). These primers were used in the conditions previously described (27 ) except that annealing was performed at 55oC. Amplified products were analysed by electrophoresis on 10% polyacrylamide gel (acrylamide/bisacrylamide: 39/1) containing TBE buffer (*1: 90 mM Tris-borate, 2 mM EDTA), run with TBE buffer and stained with ethidium bromide (0.5 [mu]g/ml).
All fragments were purified by electrophoresis through a 1.5% (w/v) low melting agarose gel (SeaPlaque GTG, FMC) run with TAE buffer (*1: 40 mM Tris, 20 mM Sodium acetate, 1 mM EDTA) and recovered by centrifugation through SpinXtm (Costar).
Table 4 . Polymorphisms of the AMH-R11 geneNucleotides are numbered from the initiation site of transcription corresponding to base 734 in the human receptor gene sequence, accessible via Genbank (#U29700). The incidence of AMH-R11 gene polymorphisms was studied on 130 independent alleles from PMDS families and normal population. Mutations either create (+) or destroy (-) the relevant restriction site.
SSCP-PCR analysis (28 ) was performed as previously described (2 ). Restriction enzymes and sizes of the digested DNA fragments, already described for the AMH gene (2 ), are shown in Table 5 for the AMH-RII gene. One or two additional digestions were necessary to analyse DNA subfragments over 300 bp obtained in the first digestion (fragments 2-5, Table 5 ). Thus, most subfragments were under 200 bp, the longest studied was 264 bp. The reaction product was loaded onto a 0.4* Hydrolink MDEtm (Bioprobe) gel (31 * 38 cm) containing TBE buffer. Electrophoresis was performed at 15 W in 0.6* TBE, at room temperature. The gel was dried and exposed to X-ray film.
. Restriction enzymes used in the SSCP-PCR study of the AMH-RII gene
Fragment
Size (bp)
Enzyme
Restriction fragments (bp)
0
667
HaeIII
122, 208, 231, 106
1
383
DdeI
44, 192, 147
2
937
BanII
218, 252, 386, 81
AluI
63, 387, 21, 10, 204, 252
BsmAI
99, 501, 205, 132
3
731
RsaI
287, 328, 113
Bsp1286I
141, 169, 191, 227
4
468
BanII
353, 116
StyI
81, 181, 207
5
495
BglI
157, 338
RsaI
213, 94, 188
6
236
HhaI
155, 81
7
368
PvuII
201, 167
8
408
BanII
162, 151, 95
9
382
HhaI
227, 155
10
394
HhaI
130, 264
Digested DNA fragments are indicated by order of the restriction site position. Fragments 2-5 were digested by several enzymes to obtain smaller fragments.
PCR products purified on 1.5% SeaPlaque GTG agarose gel (FMC), were directly ligated into pGEM-T vector using the AT-cloning System (Promega). Clones were sequenced by the dideoxynucleotide chain termination method with [[alpha]35S]dATP and the Sequenasetm version 2.0 sequencing kit (United State Biochemicals). In some cases the purified PCR products were directly sequenced by the Thermo Sequenasetm cycle sequencing kit used according to the manufacturer (Amersham). Electrophoresis was performed at 44 W in 0.6* TBE buffer on a 0.6* Hydrolink long rangertm, 7 M urea, 0.6* TBE sequencing gel (31 * 38 cm). The gel was dried and exposed to X-ray film.
Allele-specific-oligonucleotide hybridization was performed as previously described (27 ). Wild type and mutated sense oligonucleotides and washing temperatures were: 5'-CAGTGTCCCCAGCAGGTAAT-3' (58oC) and 5'-CAGTGTCCCTAGC- AGGTAAT-3' (56oC) to detect the C -> T transition at position 1827.
Serum AMH was assayed as previously described (29 ) by a sandwich ELISA using one monoclonal (a generous gift of Dr R. L. Cate, Biogen Inc, Cambridge, MA, USA) and one polyclonal antibody, both raised against human recombinant AMH.
We are grateful to the following physicians for providing us with blood samples from their patients: Drs Peter Blümel (Austria), Jean-Marie van der Winden, Lionel van Maldergen (Belgium), Jörn Müller (Denmark), Philippe Bouchard, Sylvie Cabrol, Jean-Louis Chaussain, Maguelone G. Forest, Alain Fourmaintraux, Abdul Lababidi, Bruno Leheup, Marc Nicolino, Gilles Pierre (France), Ekhard Körsch, Bärbel Töpke (Germany), Christopher Bennett, Ieuan Hughes, Eleni Pelekouda, Jeremy F.K. Wales (UK), Marco Cappa, Gianni Forti (Italy), Jan Maarten (Netherlands), Martin Ritzén (Sweden), Primus Mullis (Switzerland), Aysehan Akinci, Nurçin Saka (Turkey), Joseph M. Gertner, Robert H. Lustig, Maria New, Ira Rosenthal (USA). Jean-Claude Barbot provided DNA samples for analysis of polymorphisms. Sandrine Imbeaud and Rodolfo Rey are recipients of postdoctoral fellowships of the Association Française contre les Myopathies, France and the CONICET, Argentina, respectively. The Association Française contre les Myopathies also provided financial support for this project.
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