American Journal of Medical Genetics 98:129–136 (2001)
Role for Anti-Müllerian Hormone in Congenital
Absence of the Uterus and Vagina
Barbara L. Resendes,1,2 Sae H. Sohn,1 James R. Stelling,3 Rafael Tineo,3 Ann J. Davis,1,3
Mark R. Gray,1,2,3 and Richard H. Reindollar 1,3*
1
Department of Obstetrics and Gynecology, New England Medical Center, Tufts University School of Medicine,
Boston, Massachusetts
2
Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts
3
Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, Massachusetts
Molecular genetic techniques were used to
determine if mutations in the genes encod-
ing anti-Müllerian hormone (AMH) (also
known as Müllerian inhibiting substance
(MIS)) and its receptor (AMHR) are com-
monly present in patients with congenital
absence of the uterus and vagina (CAUV).
Twenty-two CAUV patients and 96 control
subjects from diverse ethnic groups were
studied after obtaining informed consent.
Genomic DNA samples prepared from leu-
kocytes were digested separately with sev-
eral different restriction enzymes, and the
resultant fragments were analyzed for re-
striction fragment melting polymorphisms
(RFMPs) by denaturing gradient gel electro-
phoresis (DGGE). Electrophoretic mobility
of DNA fragments which were 200–700 base
pairs in length was compared using poly-
acrylamide gels that included linear gradi-
ents of denaturing solvents designed to
separate DNA fragments according to se-
quence-dependent variation in thermal sta-
bility. Two RFMPs were found in the AMH
gene in both patients and normal control
subjects. One RFMP in the AMHR gene was
present at low frequencies in both patients
and normal control subjects. No RFMPs spe-
cific to CAUV patients were found in either
gene. Because no mutations or rare DNA se-
quence polymorphisms were detected in the
AMH and the AMHR genes in this group of
CAUV patients, it is unlikely that either
gene commonly has an etiologic role in
CAUV. © 2001 Wiley-Liss, Inc.
*Correspondence to: Richard H. Reindollar, M.D., Division of
Reproductive Endocrinology, Department of Obstetrics, Gynecol-
ogy, and Reproductive Biology, Beth Israel Deaconess Medical
Center, Harvard Medical School, 330 Brookline Avenue, Boston,
Massachusetts 02215. E-mail: rreindol@caregroup.harvard.edu
Received 5 January 2000; Accepted 18 September 2000
Published online 29 December 2000
© 2001 Wiley-Liss, Inc.
KEY WORDS: anti-Müllerian hormone;
AMH; anti-Müllerian hor-
mone receptor; AMHR; vagi-
nal agenesis; Rokitansky syn-
drome; CAUV; denaturing
gradient gel electrophoresis;
DGGE
INTRODUCTION
Congenital absence of the uterus and vagina, also
known as the Mayer-Rokitansky-Kuster-Hauser syn-
drome, is the second most common cause of pubertal
aberrancy in females, occurring once in every 4,000 to
5,000 female infants [Griffin et al., 1976; Reindollar et
al., 1981; Neinstein and Castle, 1983; Altchek, 1991].
Patients with CAUV are phenotypically female, with
normal ovaries, breast development, and female pat-
terns of body hair. In most CAUV patients, the uterus,
cervix, and upper two-thirds of the vagina is absent;
normal fallopian tubes are present and caudally at-
tached to small muscular buds. The uterine buds are
often asymmetric, with one bud slightly more devel-
oped than the other. In rare cases, functional endome-
trium is present within the uterine buds of CAUV pa-
tients [Davis et al., 1992]. Several other developmental
anomalies are frequently associated with CAUV.
Thirty percent of patients have renal malformations,
most commonly unilateral renal agenesis or ectopia of
one or both kidneys [Griffin et al., 1976; Neinstein and
Castle, 1983]. Skeletal abnormalities, including spinal
and limb defects, are present in 11–12% of CAUV pa-
tients [Griffin et al., 1976; Altchek, 1991]. Other
CAUV-associated anomalies, including cardiac defects
and hearing defects, have been described [Griffin et al.,
1976; Altchek, 1991]. CAUV patients with concomitant
extragenital anomalies have been differentiated from
those without [Strübbe et al., 1993; Strübbe et al.,
1994].
The etiology of CAUV has not been determined in
any patient to date. A polygenic and/or multifactorial
etiology for CAUV has been suggested, because most
130 Resendes et al.
cases are isolated with only occasional reports of famil-
ial occurrence [Carson et al., 1983]. Several cases of
concordance of CAUV between siblings have been de-
scribed [Griffin et al., 1976; Lischke et al., 1973; the
present study], and identical twins have been found
discordant for CAUV [Reindollar et al., 1981; Jones and
Mermut, 1972]. Although discordance of CAUV in
monozygotic twins might suggest that an exclusively
genetic etiology is unlikely, other genetic factors, such
as variable expressivity, penetrance, X-inactivation,
and gene imprinting all could account for phenotypic
differences between identical twins and siblings [Burn
et al., 1986; Lubinsky and Hall, 1991; Hall, 1996; Ma-
chin, 1996]. The vast majority of CAUV patients have a
normal female karyotype (46,XX); only a few have been
found with chromosomal abnormalities, such as recip-
rocal translocations [Driscoll et al., 1996; van Lingen et
al., 1997; Sarto, 1974; Kucheria et al., 1988].
CAUV patients were unable to reproduce until the
advent of assisted reproductive technology, and infor-
mation on the heritability of this disorder has been
limited only to reports of familial cases. A survey of 17
female infants born through surrogacy to CAUV
women did not reveal any similarly-affected offspring,
suggesting that CAUV may not be commonly inherited
as an autosomal dominant trait [Petrozza et al., 1997].
The lack of informative CAUV-affected families has
prevented the identification of any disease loci by con-
ventional genetic linkage analysis. Alternative ap-
proaches to identify disease loci include candidate gene
analysis and positional cloning of chromosome break-
points in patients with structural chromosomal abnor-
malities. Experiments utilizing both approaches to
identify genes underlying CAUV are underway in our
laboratory. CAUV patients do not commonly have mu-
tations or specific polymorphisms in the genes encod-
ing the transcription factor associated with Wilms tu-
mor, WT1 [van Lingen et al., 1998a], PAX2 [van Lingen
et al., 1998b], or galactose-1-phosphate uridyl transfer-
ase (GALT) [Bhagavath et al., 1998]. The rearranged
chromosomes in a CAUV patient with an apparently
balanced reciprocal translocation are being character-
ized in our laboratory [van Lingen et al., 1997].
The present study was designed to test the hypoth-
esis that mutations in the AMH or AMHR genes are a
frequent cause of CAUV. Deficiency of AMH signaling
due to either absence of AMH or its receptor, which is
caused by loss-of-function mutations in either the AMH
or AMHR genes, results in the anomalous persistence
of Müllerian duct derivatives in male vertebrates, in-
cluding humans [Imbeaud et al., 1995; Knebelmann et
al., 1991]. Although no examples of apparent gain-of-
function mutations in AMH signaling have been found,
it remains possible that inappropriate expression of the
AMH gene or constitutive activation of the AMHR gene
could potentially result in the disruption of normal
Müllerian duct development in females [Lindenman et
al., 1997]. To test this hypothesis, DNA samples from
CAUV patients and control subjects were analyzed for
single base-pair mutations and sequence polymor-
phisms using denaturing gradient gel electrophoresis.
MATERIALS AND METHODS
Patients and Control Subjects
Informed consent was obtained from all volunteer
subjects using protocols reviewed and approved by the
institutional human subjects investigational review
board. Twenty-one different affected families were in-
cluded in this study (Table 1). Twenty-two CAUV pa-
tients were analyzed. Twenty were the only affected
members in their families, and two patients were non-
twin siblings. All but three of the patients were exam-
ined by a board-certified reproductive endocrinologist
and clinical medical geneticist. The diagnosis of CAUV
for two patients referred to us for inclusion in the pre-
sent study was confirmed by us on the basis of their
medical records. One patient referred to us for this
study was diagnosed by a gynecologist who is board-
certified in medical genetics. Androgen-sensitivity syn-
drome was excluded by one of the following: 46,XX
karyotype; postpubertal androgen levels in the female
range; and/or identification of functional ovaries by ul-
trasonography and/or laparoscopy in some cases. The
results of cytogenetic chromosomal analysis performed
prior to referral to our clinic were available for nine
patients. Seven of these patients had a normal 46,XX
karyotype, and two patients had apparently balanced
autosomal reciprocal translocations (Table 1). All pa-
tients had normal female secondary sexual character-
istics and primary amenorrhea, and lacked the uterus,
cervix, and vagina. One patient reported cyclic pain
due to the presence of a small amount of functional
endometrium in an otherwise non-canalized muscular
uterine remnant [Davis et al., 1992].
Six patients had concomitant extragenital abnor-
malities (Table 1). Two patients had both renal and
skeletal anomalies; patient 687 had renal agenesis and
thoracolumbar dextroscoliosis, and patient 709 had a
horseshoe kidney with scoliosis and skeletal abnor-
malities of the thumb. Patient 524 had renal agenesis.
Two patients had skeletal anomalies: patient CH92-
138 had mild scoliosis, and patient 489 had sacraliza-
tion of L5 and absent 12th ribs. None of the patients
analyzed had spinal fusion defects similar to those ob-
served in Klippel-Feil syndrome patients. The control
subjects were women and men of diverse ethnic groups
with no known reproductive tract defects.
AMH levels were not determined in these patients,
because the finding of low or baseline AMH levels in an
adult CAUV patient cannot be correlated with the pa-
tient’s early embryonic exposure to AMH signaling.
DNA Samples
Genomic DNA was prepared from blood samples col-
lected by standard venipuncture in tubes containing
the anti-coagulant sodium EDTA. DNA was purified
using a standard nuclear lysis and phenol extraction
procedure [Gray, 1992].
Denaturing Gradient Gel Blots
Genomic DNA samples (10 ␮g) were digested with
one of several different restriction enzymes (RsaI,
DpnII, HaeIII, and AluI) that cut DNA into fragments
 AMH and AMHR Genes in CAUV 131

TABLE I. Summary of CAUV Patients Analyzed

Renal Affected
Patient system Skeleton Hearing Karyotype relatives
479a Normal Normal Normal Autosomal None
translocation
484 Normal Normal Normal 46,XX Sister
485 Normal Normal Normal 46,XX Sister
489 Normal Absent 12th ribs; Normal 46,XX None
L5 sacralization
524 Renal agenesis Normal Normal 46,XX None
575 Normal Normal Normal Unknown None
593 Unknown Normal Normal Unknown None
594 Normal Normal Normal Unknown None
595 Normal Normal Normal 46,XX None
686 Unknown Normal Normal Unknown None
687 Renal agenesis Thoracolumbar Normal 46,XX None
dextroscoliosis
688 Normal Normal Normal Unknown None
704 Normal Normal Normal 46,XX None
705 Normal Normal Normal Unknown None
706 Normal Normal Normal Unknown None
708b Normal Normal Normal 46,XX None
709 Horseshoe Scoliosis; Deaf Unknown None
kidney abnormal thumbs
710 Normal Normal Normal Unknown None
715 Unknown Normal Normal Unknown None
716 Unknown Normal Normal Unknown None
739 Normal Normal Normal Unknown None
CH92-138c Normal Mild scoliosis Normal Autosomal None
translocation
a
Described in Van Lingen et al. [1997].
b
Described in Davis et al. [1992].
c
Described in Driscoll et al. [1996].

averaging 200–700 bp in length. Digested DNA was
electrophoresed at 75 volts in 6.5% polyacrylamide de-
naturing gradient gels with a denaturant concentra-
tion range of 20–80% or 50–90% (100% ⳱ 40% form-
amide + 7 M urea) in E buffer (40 mM tris-acetate, 20
mM sodium acetate, 1 mM EDTA, pH 8.0) at 60°C for
20 hr. After electrophoresis, DNA was electro-
transferred to nylon membranes using Biodyne B (Pall
Biosupport Division, East Hills, NY) in TE buffer (10
mM Tris-HCl, 1 mM EDTA, pH 8.0) for 2 hr at 80 volts,
as previously described [Gray, 1992]. Following electro-
transfer, the DNA was denatured and neutralized by
soaking the blots in 0.5 M NaOH for 30 min, then in 0.5
M Tris-HCl, pH 8.0 for 5 min, and finally, in 6X SSC for
5 min. The denaturing gradient gel blots were baked
for 2 hr at 80°C.
AMH/AMHR Gene Probes and Hybridization of
DGGE Blots
The plasmid pBG311 (generously provided by Rich-
ard Cate) contains DNA encoding the entire mRNA
sequence of the AMH gene, including the four introns
and ∼1400 bp of DNA downstream from the polyade-
nylation signal and mRNA cleavage site [Fig. 1; Cate et
al., 1986]. Although this probe includes only 29 bp of
DNA upstream from the mRNA 5⬘ end, it detects over-
lapping fragments of genomic DNA that extend 100–
200 bp further upstream into the AMH gene promoter
region. The AMHR cDNA clone 4-2 (generously pro-
vided by Nathalie Josso and Jean-Yves Picard) in-
cludes an EcoRI fragment with 1635 bp of DNA of the
AMHR coding region starting in exon 2 at nucleotide
position 212 [Imbeaud et al., 1995]. The AMHR cDNA
probe was used to detect fragments that included the
protein-coding exons and intron splice junctions, but
not the introns, 5⬘ promoter region, or 3⬘ downstream
sequence.
DNA fragments were labeled with 32-P using the ran-
dom oligonucleotide priming method [Feinburg and Vo-
gelstein, 1983]. Hybridizations with blots were per-
formed as previously described [Gray, 1992].
The hybridization mixture consisted of 0.5 M
NaHPO4 7% sodium dodecyl sulfate (SDS), and 1 mM
EDTA. After overnight incubation at 65°C, the blots
were washed for 30 min at 65°C in each of the following
solutions: 2X SSC + 0.5% SDS; 1X SSC + 0.5% SDS;
and 0.1X SSC + 0.5% SDS. Blots were air-dried,
wrapped in plastic wrap, and exposed to X-ray film
(Kodak, Rochester, NY) for up to two weeks at –80°C.
DNA Sequencing
RFMP#1 was mapped to an AluI fragment down-
stream from the AMH gene polyadenylation signal and
mRNA cleavage site by successive hybridizations of the
original denaturing gradient gel blot with probes made
from short DNA fragments derived from the full-length
AMH genomic clone by digestion with restriction en-
zymes (Fig. 1). A 616 bp DNA fragment that included
RFMP#1 was amplified by PCR, using DNA from con-
trol subject #366 as the template, and the following
primers: “C” 5⬘-CCA GGC GTA AAG GAG CAG GTG-3⬘
and “D” 5⬘-TCG TCG CCC CTG GTA AGA GCT-3⬘. The
132 Resendes et al.

Fig. 1. A schematic map of the AMH gene. The positions of the AflII, AluI, PstI, and SacI restriction sites are drawn to scale. Primers “C” and “D” are
shown on the AluI restriction site map; the arrowhead indicates position of RFMP#1. The 1809 bp SacI fragment includes both RFMPs, and the 1135 bp
PstI fragment includes only RFMP#1.

PCR fragment was ligated into the plasmid pCRII (In- Table 2). A higher percentage of patients (19.0%) than
vitrogen, Carlsbad, CA) and the DNA sequences of the controls (5.2%) was heterozygous for the alleles of
inserts in the recombinant plasmid clones were deter- RFMP#1. The fifth AluI band from the top of the gel
mined using the dideoxynucleotide chain termination was also polymorphic (RFMP#2) and bi-allelic, with
method (Sequitherm, Epicentre Technologies, Madi-
son, WI). DNA sequencing gels were fixed in 5% metha- TABLE II. Summary of Polymorphisms Found in the AMH
nol + 5% acetic acid for 30 min, vacuum-dried, and Gene by DGGE
exposed to X-ray film for 1–3 days. A. Allele frequencies of AMH gene RFMPs found in
AluI-digested DNA of CAUV families. Sibling patients
RESULTS #484 and #485 are counted once in the table.
Denaturing Gradient Gel Electrophoresis Band from
Control Patient
chromosomes chromosomes
DNA samples were digested with one of four restric- the top
of the gel Allele Number % Number %
tion enzymes (AluI, DpnII, HaeIII, or RsaI) and elec-
trophoresed in either 20–80% or 50–90% denaturing RFMP #1 #2 H 5/192 2.6 4/42 9.5
M 187/192 97.4 38/42 90.5
gradient gels. DNA blots made from these gels were
RFMP #2 #5 H 1/188 0.5 1/42 2.4
hybridized with radioactively labeled probes made M 187/188 99.5 41/42 97.6
from the full-length AMH gene and the AMHR cDNA
B. Genotype frequencies of AMH gene RFMP alleles of CAUV
sequence. families. Sibling patients #484 and #485 are counted once
DGGE Analysis of the AMH Gene in the table.

CAUV
Restriction fragment melting polymorphisms Band from
Controls Patients
(RFMPs) in the AMH gene were identified among AluI the top
DNA fragments (Table 2). Eight bands were detected of the gel Genotype Number % Number %
on 50–90% DGGE blots of AluI-digested DNA; the sec- RFMP#1 M/H 5/96 5.2 4/21 19.0
ond AluI band from the top of the gel was polymorphic M/M 91/96 94.8 17/21 81.0
(RFMP#1) and bi-allelic, with middle (M) and high (H) RFMP#2 M/H 1/94 1.1 2/21 9.5
M/M 93/94 98.9 19/21 90.5
melting variant alleles separated by 2.5 mm (Figure 2,

Fig. 2. AMH gene restriction fragment melting polymorphisms
(RFMPs) detected by DGGE. DNA samples digested with AluI were elec-
trophoresed in a 50–90% denaturing gradient gel, transferred to a nylon
blot, and hybridized with the AMH gene probe. Arrows indicate RFMP#1
(lane 2) and RFMP#2 (lane 7). DNA samples from patients are indicated by
numbers, and that from the normal control subject is indicated by the
letter C.
melting variant alleles (M and H) separated by 2.5 mm
(Figure 2, Table 2). One patient (#524) and one male
control were heterozygous for RFMP#2.
Through sequential hybridization with probes made
from short AMH gene fragments, both RFMPs were
mapped to DNA downstream from the polyadenylation
signal and mRNA cleavage site of the AMH gene (Fig-
ure 1). An 1135 bp PstI fragment of the AMH gene that
contains 500 bp of exon V and 635 bp of downstream
sequence detects RFMP#1 but not RFMP#2 when used
as a probe on DGGE blots of AluI-digested DNA, indi-
cating that RFMP#2 is downstream from RFMP#1. An
1809 bp SacI fragment which includes 395 bp of exon V
and 1414 bp of downstream sequence detects both
RFMPs, further confirming that RFMP#2 is down-
stream from RFMP#1 (Figure 1).
HaeIII-digested genomic DNA did not reveal any
bands when analyzed by DGGE, because the AMH
gene sequence contains many HaeIII sites, resulting in
numerous DNA fragments that were too short (<100
bp) to be retained in denaturing gradient gels after 20
hours of electrophoresis. No RFMPs were detected in
RsaI and DpnII genomic DNA fragments. Patient-
specific polymorphisms in the AMH gene were not
found using any of the restriction enzymes.
Sequencing of AMH Gene RFMP#1
A 616 bp DNA fragment that included RFMP#1 was
amplified by PCR using genomic DNA from control
subject #366 as template and oligonucleotide primers
that flank the ends of the AluI DNA fragment that
included the polymorphism. The 616 bp amplified DNA
fragment was ligated with a plasmid and used to gen-
erate recombinant bacterial clones. Since control sub-
AMH and AMHR Genes in CAUV 133
ject #366 was heterozygous for RFMP#1, individual re-
combinant plasmid clones could have had either the H
or M allele (Fig. 2). DNA sequencing of the coding
strand of the AMH gene inserts from 13 clones revealed
an adenosine nucleotide (A) in place of the cytosine (C)
at position 3865 in 10 of them; the remaining 3 clones
contained cytosine at position 3865 [Cate et al., 1986].
The C/A polymorphism is 734 nucleotides downstream
from the AMH gene poly(A) addition site at nucleotide
position 3131. Because the inter-strand DNA base-
pairing interaction between C and G is much more
thermo-stable than that of an A/T base pair, the pres-
ence of an A/T base pair results in a lower melting
stability of this region of DNA in comparison to the
same sequence with a G/C base pair. This difference
would cause the fragment to stop migrating at a higher
position in a denaturing gradient gel, where the physi-
cal environment is analogous to a lower temperature.
The DNA sequences with either the M allele (C at
nucleotide position 3865) or the H allele (A at nucleo-
tide position 3865) were compared using a computer
program that predicts the melting temperature (Tm) of
short portions of DNA fragments [MeltMap; Behrens et
al., 1990]. The sequence of the M allele was predicted to
have a melting temperature (Tm) of 79.8°C, in contrast
to a Tm of 79.0°C for the H allele, suggesting that the
C/A base difference accounted for the DGGE band mo-
bility difference.
DGGE Analysis of the AMHR Gene
Four bands were detected on 20–80% denaturing
gradient gel blots of DpnII-digested DNA when hybrid-
ized with an AMHR cDNA probe. The fourth DpnII
band from the top of the gel was polymorphic, with two
alleles, middle (M) and low (L), separated by 2 mm
(Fig. 4; Table 3). Most patients (95.0%) and controls
(96.9%) were homozygous for the M allele. One of 20
Fig. 3. Sequence analysis of the AMH gene RFMP#1 using DNA from a
control subject (#366). The position of the C/A base difference at position
3865 is denoted by the asterisk [Cate et al., 1986].
134 Resendes et al.
Fig. 4. AMHR gene RFMP detected by DGGE. DNA samples digested
with DpnII were electrophoresed in a 20–80% denaturing gradient gel,
transferred to a nylon blot, and hybridized with the AMHR cDNA probe.
Four bands were detected with the AMHR cDNA probe; the third and
fourth bands from the top of the gel have co-migrated. The arrow indicates
the position of the L RFMP allele. Most samples were homozygous for the
M allele; patient 709 and one control subject were heterozygous.
different patients (5.0%) and 2 of 64 controls (3.1%)
were heterozygous for both alleles. Since the two con-
trol subjects carrying the L allele were both fertile fe-
males, it was most likely a single nucleotide polymor-
phism and not a mutation. No further AMHR gene
RFMPs were detected in HaeIII, RsaI, and AluI DNA
fragments. No patient-specific RFMPs were detected
with the AMHR cDNA probe, suggesting that there is
little DNA sequence variability in the coding region of
the AMHR gene.
DISCUSSION
To determine whether or not CAUV patients have
frequent mutations in the AMH or AMHR genes, we
analyzed patient genomic DNA using DGGE, a com-
monly-used method for detecting and mapping gene
mutations. DGGE separates DNA fragments on the ba-
sis of distinctive melting behavior, a property that de-
pends on the fragment’s DNA sequence. When heated,
nearly all double-stranded DNA fragments melt into
single strands stepwise in distinct 50–150 bp domains
in a progression that is extremely sensitive to subtle
DNA sequence differences. DGGE is commonly used to
detect differences in the melting behavior of small
PCR-amplified DNA fragments (200–700 bp) that dif-
fer by as little as a single base substitution [Fischer
and Lerman, 1983]. For large regions of DNA sequence
for which optimal PCR primer sequences are not
readily available, such as genes for which only the
cDNA sequence is known, DGGE can be used to detect
DNA sequence differences in genomic DNA fragments
without prior PCR amplification by transferring DNA
from the gels to nylon blots and hybridizing them with
specific gene probes [Gray, 1992; Gray et al., 1991].
DGGE can detect between 75–100% of all mutations
in a gene [Myers et al., 1985; Gray et al., 1991]. In the
DGG blot strategy used in these experiments, muta-
tions in the most thermo-stable regions of the gene can
theoretically escape detection, since the last melting
domains of DNA fragments do not denature in a se-
quence-dependent manner. We increased the extent of
DNA sequence analyzed by DGGE by digesting each
genomic DNA sample with four different restriction en-
zymes. By analyzing sets of overlapping DNA frag-
ments with different endpoints and melting domain
structures, mutations within a high melting domain of
one restriction fragment may be detected by moving
this portion of the sequence into a lower melting do-
main of an overlapping fragment produced by digestion
using a different restriction enzyme. In a previous
DGG blot-based search for mutations in Drosophila,
80% of mutations were detected by analyzing five dif-
ferent restriction enzyme digests prepared from each
genomic DNA sample [Gray et al., 1991]. We have also
used the DGG blot method to successfully detect and
map single base change mutations in the human factor
VIII gene in 82% of a group of hemophilia A patients
[Laprise et al., 1998].
DGGE analysis of CAUV patient DNA samples re-
vealed no sequence differences in the protein-coding
portions of the AMH and AMHR genes, nor in the four
AMH gene introns. Two DNA sequence polymorphisms
(RFMPs) downstream from the 3⬘ end of the AMH gene
were detected in both patients and normal controls.
Because both mapped downstream from the mRNA
cleavage and polyadenylation signals, it is unlikely
that they adversely affect the translation or processing
of AMH mRNA. RFMP#1 was a C/A single base pair
difference 734 bp downstream from the AMH gene
polyadenylation site. RFMP#2 was even further down-
stream, and its exact sequence difference was not de-
termined. RFMP#1 was more frequent in CAUV pa-
tients (9.5%) than in normal controls (2.6%), and
RFMP#2 was present in one CAUV patient and one
male control. Although these polymorphisms could be
benign single nucleotide differences and not mutations
that alter gene expression, it remains possible that
they could change as yet unidentified enhancer se-
quences, or could in some way alter mRNA stability. In
vitro expression experiments specifically designed to
analyze AMH genes with each of these polymorphisms
would be necessary to determine whether or not they
affect AMH gene expression.
TABLE III. Summary of Polymorphisms Found in the AMHR
Gene Using DGGE
A. Allele frequencies of the AMHR DpnII band #4 RFMP in
CAUV families. Sibling patients #484 and #485 are counted
once in the table.
Control Patient
chromosomes chromosomes
Allele Number % Number %
M 126/128 98.4 39/40 97.5
L 2/128 1.6 1/40 2.5
B. Genotype frequencies of AMHR DpnII band #4 RFMP
alleles in CAUV families. Sibling patients #484 and
#485 are counted once in the table.
Normal CAUV
controls patients
Genotype Number % Number %
M/M 62/64 96.9 19/20 95.0
M/L 2/64 3.1 1/20 5.0

The AMHR gene has been previously characterized
in detail and shown to be expressed in mesenchymal
cells adjacent to the fetal Müllerian ducts in both males
and females, in fetal and postnatal granulosa cells, and
in postnatal Sertoli cells [Imbeaud et al., 1995;
Baarends et al., 1994]. Numerous male patients with
persistent Müllerian duct syndrome (PMDS) with both
decreased and normal AMH levels have been described
and examined for mutations and sequence polymor-
phisms in the AMH gene that would explain the appar-
ent absence of normal AMH-mediated function [Kne-
belman et al., 1991; Baarends et al., 1994; Imbeaud et
al., 1994, 1995; Carre-Eusebe et al., 1992]. A large
study of PMDS patients from Mediterranean countries
revealed numerous different point mutations and three
sequence polymorphisms in the AMH gene [Imbeaud et
al., 1994]. In the present report, DGGE analysis dem-
onstrated two low-frequency DNA sequence polymor-
phisms in both CAUV patients and normal controls; no
patient-specific AMHR gene RFMPs were found.
Two unrelated patients with Müllerian duct failure
have been reported to have chromosomal transloca-
tions near the cytogenetic location of the AMHR gene
[Kucheria et al., 1988]. Mutations in the AMH and
AMHR genes have been suggested previously as poten-
tial genetic causes of CAUV [Lindenman et al., 1997].
This hypothesis would require that the following two
conditions would have to converge in order to produce
CAUV: (1) a dominant gain-of-function mutation in ei-
ther the AMH or AMHR gene that would cause preco-
cious AMH signaling in female embryos, and (2) the
caudal portion of the Müllerian duct that forms the
uterus and vagina would be sensitive to AMH signal-
ing, and the cranial portion that forms the fallopian
tubes would be resistant to AMH [Stelling et al., 1997].
This hypothesis also presumes that during early em-
bryogenesis, complete Müllerian ducts form in CAUV
patients before precocious AMH expression mediates
the removal of the caudal portions that develop into the
uterus and the upper two-thirds of the vagina. In ad-
dition, expression of AMH in female embryos, presum-
ably from ovarian granulosa cells, would most likely
result in abnormal ovarian development (early meiotic
arrest and premature loss of oocytes) similar to that
found in murine mutants engineered to overexpress
AMH [Behringer et al., 1990]. These mutant animals
lack all Müllerian duct derivatives, including the fallo-
pian tubes, in contrast to CAUV in humans. Complete
absence of all Müllerian duct derivatives, including the
fallopian tubes, uterus, and upper vagina, also occurs
in freemartin cattle, because of the presence of AMH
from the male embryo’s testes in the circulation shared
between male and female embryos [Jost et al., 1972].
Activation of AMH signaling in female embryos
should result in complete Müllerian aplasia, and the
presence of only the Müllerian duct remnants observed
in normal male development. Because the observed
CAUV phenotype is inconsistent with the complete
Müllerian aplasia expected from the inappropriate ac-
tivation of AMH signaling, and no AMH/AMHR gene
mutations or DNA sequence polymorphisms specific for
patients were found, the present data suggest that it is
AMH and AMHR Genes in CAUV 135
unlikely that either the AMH gene or the AMHR gene
has a major role in CAUV. Although one of the AMH
gene sequence polymorphisms was found more fre-
quently in CAUV patients than in normal control sub-
jects, it is not possible to establish its significance with-
out analyzing many more patients. It is possible that,
in combination with differences in the levels and tim-
ing of expression of other gene products that direct nor-
mal Müllerian duct development, AMH/AMHR gene
sequence polymorphisms might contribute to the
CAUV phenotype.
ACKNOWLEDGMENTS
We thank our study patients and control subjects for
their willingness to participate in this study. We thank
Dr. Deborah Driscoll and Dr. Gita Gidwani for patient
blood samples, and Dr. Andrea Zuckerman for assis-
tance in preparation of the manuscript. We thank Dr.
Richard Cate for the AMH gene plasmid pBG311, and
Dr. Nathalie Josso and Dr. Jean-Yves Picard for the
AMHR gene plasmid 4-2.
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