The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 9 3147-3150
Copyright © 2000 by The Endocrine Society
Uniparental Disomy in Steroid 5
-Reductase 2 Deficiency
Bertha Chávez,
Evangelina Valdez and
Felipe Vilchis
Department of Reproductive Biology, Instituto Nacional de la
Nutrición Salvador Zubirán (B.C., F.V.), and Servicio de
Endocrinología, C.M.N. 20 de Noviembre ISSSTE (E.V.),
México D.F., México
Address correspondence and requests for reprints to: Bertha Chávez, Department of Reproductive Biology, Instituto Nacional de la Nutrición Salvador Zubirán, Vasco de Quiroga #15, México 14000 D.F., México.
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Abstract
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Steroid 5
-reductase 2 deficiency is an autosomal recessive form of
male pseudohermaphroditism caused by mutations in the SRD5A2 gene. In
this study, we performed DNA analyses in two unrelated subjects bearing
the enzyme deficiency and found differences in the mode of transmission
for the disease. The data showed that in both families the fathers were
carriers for an E197D mutation, whereas the mothers were carriers for a
P212R mutation. Patient 1 was identified as compound heterozygote
because he had both alterations (E197D/P212R). On the contrary, patient
2 was found to be homozygous, but only for the paternal mutation.
Because this finding could not be explained on the basis of
nonpaternity or a chromosomal abnormality, the presence of uniparental
disomy was suggested. The reduction to homozygosity for the E197D
mutation, as confirmed by restriction analysis, supported this view.
The results of our study give evidence of the first case of
5-reductase deficiency resulting from uniparental disomy and also
disclose an alternate mechanism whereby this enzymatic disorder can
derive from a single parent.
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Introduction
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STEROID 5-REDUCTASE 2 deficiency
(5-SR2D) is a rare autosomal recessive disorder that leads to a
specific form of male pseudohermaphroditism (1). Affected
subjects are 46,XY males with ambiguous external genitalia. They
usually present perineoscrotal hypospadias with pseudovagina,
microphallus, cryptorchid testes, and rudimentary prostate but normal
Wolffian derivatives (2, 3). Studies from molecular
genetics have shown that 5-SR2D is caused by either single-base
mutations or deletion of the steroid 5-reductase type 2 (SRD5A2)
gene (4, 5, 6). Most patients with 5-SR2D have homozygous
mutations, and the remainder (about 40%) are compound heterozygotes or
presumed compound heterozygotes (7, 8). Here, we report
studies of the SRD5A2 gene in two unrelated patients with 5-SR2D
whose parents are heterozygous carriers for two different, although
identical, mutations. Molecular studies of one of these patients
revealed homozygosity, but only for the paternal mutation. Because the
defective mutation was carried by the patient’s father but not by his
mother, who was carrier for another missense mutation, uniparental
disomy (UPD) was suspected.
The concept of UPD, originally introduced in 1980 by Engel
(9), has allowed to explain the inheritance of two copies
of a genetic locus from only one parent. To date, UPD has been
recognized to occur in various hereditary diseases like Prader-Willi
syndrome, cystic fibrosis, hemophilia A, Duchenne muscular dystrophy,
pycnodysostosis, and the Angelman syndrome, among others
(10). Results from our study provide evidence to document
the first case of paternal UPD in 5-SR2D.
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Subjects and Methods
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Subjects
Patient 1 (P1) is a 23-yr-old 46,XY individual who was raised as
a male. When evaluated at age 17, he had penoscrotal hypospadias with a
phallus of 3.0 cm long and 1.0 cm wide. The gonads located in the
scrotum had volumes of 22.5 and 27.0 mL, and no gynecomastia was
present. This patient has two healthy sisters, normal in
appearance.
Patient 2 (P2) is a 14-yr-old individual with a female gender identity.
This patient was referred at age 13 because of primary amenorrhea,
clitoridal enlargement, lack of breast development, and deepened voice.
Physical examination revealed a painful mass (2.5 x 3.0 cm) in
the left labium majus, a phallus of 3.0 cm, extruding gonads, and a
vestibular introitus of 2–3 cm deep. The karyotype was 46,XY. This
patient was raised as a girl, and bilateral orchidectomy was performed
before the molecular studies.
In these patients the diagnosis of 5-SR2D was established on
clinical and endocrinological criteria and karyotype. There was no
known consanguinity or family history of the disease in any of the
families, which are of Mexican-mestizo ethnic origin. This study was
carried out with the informed consent of the patient’s parents and the
approval of the ethical committee of our Institute.
DNA studies
Genomic DNA was isolated from blood leukocytes by standard
methods. Coding sequence abnormalities in the SRD5A2 gene were
ascertained by exon-specific PCR, single-stranded conformational
polymorphism (SSCP), and sequencing analysis (8, 11). SSCP
was performed according to the method of Orita et al.
(12), using [-32P]dCTP as described
elsewhere (13, 14). Mutant and control PCR products were
sequenced using the Thermosequenase ([-33P]ddNTP)
terminator cycle sequencing kit (Amersham Life Sciences Inc., Cleveland
OH), as described previously (8).
Cleavage of genomic DNA as well as Southern blotting with conventional
markers were performed by standard techniques (15). Four
variable number tandem repeat probes [D1S7/MS1, D4S139/PH30,
D5SS110/LH1, and D17S79/VI (Life Technologies, Inc., Grand Island,
NY)] for chromosomes 1, 4, 5, and 17, respectively, were used to
validate paternity. The marker D1S80 (16) was assessed as
well. Polymorphic markers D2S162, D2S117, D2S347, D2S305, D2S151,
D2S126, D2S131, D2S396, and D2S142 from chromosome 2 were PCR amplified
using oligonucleotide primers described previously (17).
PCR was performed in 25-µL reactions, and each contained 500 ng
genomic DNA, 10 mmol/L Tris-HCl, 1.5 mmol/L MgCl2, 50
mmol/L KCl, 1.25 µmol/L of each primer, 2.5 U Taq DNA polymerase, 100
µmol/L of each dNTP, and 5 µCi [-32P]dCTP (S A.
3000 Ci/mmol; NEN-DuPont, Boston, MA).
Restriction analysis
Exon 4 of the SRD5A2 gene was amplified by PCR in the presence
of [-32P]dCTP and purified as described above.
PCR-labeled products were incubated for 2 h at 37 C with 5 U EcoR
V (New England Biolabs Inc., Beverly, MA) in a final volume of 20 µL.
Three microliters of each sample were loaded on neutral 8%
polyacrylamide gels. After electrophoresis at 500 V for 2 h, the
gels were dried and autoradiographed for 1–2 h at -70 C.
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Results
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SSCP analysis of the SRD5A2 gene of the patients and their parents
revealed no variations in exons 1, 2, 3, and 5 (data not shown). As can
be seen in Fig. 1
, the exon 4 DNAs of
both families displayed altered electrophoretic mobility as compared
with those of the control subjects. Direct sequencing of these
fragments showed that P1 was heterozygous for GAG/GAT at codon 197 and
for CCA/CGA at codon 212 (Fig. 2). The
father was carrier for a G
T transversion at nucleotide 591
(nucleotide position is given relative to the first ATG codon), which
changes codon 197 from glutamic acid to aspartic acid (E197D), whereas
the mother was carrier for a CG transversion at nucleotide 635,
which changes codon 212 from proline to arginine (P212R). This patient
was considered as compound heterozygous by having both mutations (Fig. 2).
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Figure 1. SSCP analysis of exon 4 of the SRD5A2 gene from
patients 1 and 2 (arrows) and their respective parents. F,
Father; M, mother. The DNAs of two unrelated normal subjects served as
control (C).
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Figure 2. Partial nucleotide sequence of the SRD5A2 gene
from family 1, showing two different mutations within exon 4. Patient 1
is a compound heterozygote for a Glu(GAG) to Asp(GAT) missense mutation
at codon 197 [carried by his father (F1)] and for a
Pro(CCA) to Arg(CGA) missense mutation at codon 212 [carried by his
mother (M1)]. Exon 4 DNA from an unrelated normal subject
served as reference (Control).
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As depicted in Fig. 3, the parents
of P2 were carriers for the same mutations; thus, the father was
heterozygous for the mutation E197D, and the mother was heterozygous
for the mutation P212R, however, this patient presented (exclusively)
homozygosity for the paternal mutation with absence of the maternal
mutation. Sequencing analysis (performed on both strands), using three
different DNA preparations, confirmed that P2 was, indeed, homozygote
(GAT/GAT) for the paternal mutation (Fig. 3). Additionally, we noted
that nucleotide substitution at codon 197 introduced an EcoR V
restriction site. This was confirmed by enzymatic cleavage of the exon
4 DNA fragments (Fig. 4). In P2, the
labeled PCR product (232 bp) was completely digested into 158-bp and
74-bp fragments. Likewise, digestion of the 232-bp amplimer from his
father revealed three bands, the 232-bp undigested fragment and the
158-bp/74-bp digested fragments. In contrast, the normal PCR product
and that of the mother showed only the 232-bp undigested fragment (Fig. 4). Because the mode of inheritance in P2 did not fit with the
autosomal recessive pattern of the disease, a paternity testing was
performed. The DNA markers used in this study were informative, and the
results were in agreement with paternity. Because chromosomal studies
revealed a normal diploid karyotype and no mosaicism was observed, the
overall results were interpreted as demonstrating the presence of UPD.
To verify this view, several short tandem repeats spread along
the short and long arms of chromosome 2 were evaluated. As shown in
Fig. 5, analysis of P2, with different
markers, suggested inheritance of two paternal alleles in at least five
(D2S162, D2S117, D2S305, D2S131, and D2S126) of the nine markers
examined. Other markers (D2S151, D2S142, and D2S396) exhibited a
banding pattern very similar among all the family members and,
therefore, were considered uninformative. Finally, analysis of the
D2S347 marker showed typical Mendelian inheritance with both paternal
alleles detected in the propositus (Fig. 5).
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Figure 3. Partial nucleotide sequence of the SRD5A2 gene
from family 2, showing two different missense mutations within exon 4.
Sequence analysis indicated that the father (F2) was
carrier for a Glu197-Asp mutation and the mother (M2) was
carrier for a Pro212-Arg mutation. The patient (P2) was
found to be homozygous, but only for the paternal mutation (E197D).
Exon 4 DNA from an unrelated normal subject served as reference
(Control).
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Figure 4. Autoradiograph of restriction enzyme analysis of
exon 4 of the SRD5A2 gene. Exon 4 and its flanking intronic sequences
were PCR amplified from DNA of patients 1 (P1) and 2
(P2), their respective parents (M, Mother; F, father) and a
normal individual (C). The [32P]-labeled PCR products
were cleavaged with EcoR V and separated on a neutral 8.0%
polyacrylamide gel. Exon 4 of the mutant SRD5A2 gene was cut into 158
and 74 bp, but that of the wild-type gene was not (232 bp).
Heterozygote carriers for this mutation exhibit wild-type and mutant
bands.
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Figure 5. Genetic marker analysis in patient 2 and his
parents. Autoradiograms of several microsatellite markers of chromosome
2 amplified from genomic DNA of the father (F), the mother (M), and the
affected subject (P). The banding patterns for D2S117, D2S162, D2S126,
D2S305, and D2S131 were similar in the patient and his father. The
markers D2S151, D2S396, and D2S142 were not informative, but the
migration pattern for D2S347 in the affected subject suggested
inheritance from the two paternal alleles.
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Discussion
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It is well established that mutations of the SRD5A2 gene in
karyotypic males result in a spectrum of external genitalia phenotypes
ranging from complete female to nearly complete males (1, 14). In this study, we performed molecular analyses of the
SRD5A2 gene from two unrelated patients, born to nonconsanguineous
parents who were carriers of two distinct missense mutations, and found
differences in the mode of transmission of the disease. Sequencing
analyses showed that in these families the fathers were heterozygous
carriers of an E197D mutation and the mothers were heterozygous
carriers of a P212R mutation. Both changes are found at exon 4, very
close to each other. In our population, this region of the gene seems
particularly mutation prone, because in 10 of 12 pedigrees so far
examined the molecular lession has been detected in exon 4 (11, 13). As such, the mutations Glu197-Asp and Pro212-Arg have
already been described; the former was present in heterozygous form in
a Russian-American patient (5), and the second one (P212R)
was previously detected in various affected subjects, most of them of
Mexican origin (8, 13). Molecular studies of site-directed
mutagenesis, in which the mutant cDNAs are expressed in mammalian
cells, have shown that both mutations (E197D and P212R) completely
inactivate the enzyme, perhaps by affecting its Vmax, or
else, the half-life of the protein (1, 18, 19). Although
in these two families the parents carry identical mutations, the
results suggested two distinct patterns of transmission for this
deficiency. Thus, P1, who was reared as a male and presented a more
masculine phenotype, was identified as a compound heterozygote by
harboring the two parental mutations. By contrast, DNA sequence
analysis showed P2 to be homozygote for the E197D mutation, with a T at
nucleotide position 591 in both alleles.
Restriction-enzyme analysis confirmed that P2 was homozygous for this
mutation. The absence of maternal mutation, together with homozygosity
for the paternal mutation found in this patient, indicated an abnormal
pattern of inheritance. Because the reduction to homozygosity for the
E197D mutation could not be explained on the basis of nonpaternity or a
chromosomal abnormality, the presence of UPD was considered. UPD seems
to be a well recognized phenomenon that can give rise to congenital
disorders by mechanisms other than classical Mendelian genetics
(9, 20, 21). As in the case of P2, UPD has been recognized
in other diseases because of anomalous patterns of transmission from
recessive genes. Thus, steroid 21-hydroxylase deficiency
(22), pycnodysostosis, complement (C4A + C4B)
deficiency, cystic fibrosis, cartilage/hair hypoplasia, ß-thalassemia
major, and methylmalonic acidemia are some of the 18 recessive
disorders in which UPD has been documented (10).
To our knowledge, P2 represents the first case of 5SR2D resulting
from UPD and also the first case of paternal UPD involving chromosome
2, because the three cases of UPD for chromosome 2 previously described
were of maternal origin (23, 24, 25). We believe that P2
represents a particular case of this disease, especially if we consider
the low frequency of UPD and the relative rarity of 5-SR2D in the
population. In summary, these studies have identified the first case of
paternal UPD that was detected in a patient who inherited at least two
identical regions of a paternal chromosome 2 carrying a SRD5A2 gene
mutation, resulting in 5-SR2D. This observation states an alternate
mechanism whereby 5-SR2D can derive from a single parent.
Received December 2, 1999.
Revised April 6, 2000.
Accepted May 11, 2000.
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Abstract
Introduction
