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From the * Unidad Investigación
Endocrinología y Nutrición Pediátricas, Hospital Vall
d'Hebron, Barcelona, Spain; the Servicio de
Anatomía Patológica and the
Servicio de Endocrinología
Pediátrica, Hospital La Paz, Madrid, Spain; and the
Servicio de Cirugía Pediátrica,
Hospital Infantil La Paz, Madrid, Spain.
| Correspondence to: Dr Laura Audí, Unidad Investigación Endocrinología y Nutrición Pediátricas, Hospital Vall d'Hebron, Paseo Vall d'Hebron 119, Barcelona 08035, Spain (e-mail: laudi{at}vhebron.net). |
| Received for publication November 7, 2003; accepted for publication December 22, 2003. |
 |
Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-reductase type 2 gene
(SRD5A2) analysis in a male pseudohermaphrodite (MPH) patient with normal
testosterone (T) production and normal androgen receptor (AR) gene coding
sequences. A patient of Chinese origin with ambiguous genitalia at 14 months,
a 46,XY karyotype, and normal T secretion under human chorionic gonadotropin
(hCG) stimulation underwent a gonadectomy at 20 months. Exons 1–8 of the
AR gene and exons 1–5 of the SRD5A2 gene were sequenced from peripheral
blood DNA. AR gene coding sequences were normal. SRD5A2 gene analysis revealed
2 consecutive mutations in exon 4, each located in a different allele: 1) a T
nucleotide deletion, which predicts a frameshift mutation from codon 219, and
2) a missense mutation at codon 227, where the substitution of guanine (CGA)
by adenine (CAA) predicts a glutamine replacement of arginine (R227Q). Testes
located in the inguinal canal showed a normal morphology for age. The patient
was a compound heterozygote for SRD5A2 mutations, carrying 2 mutations in exon
4. The patient showed an R227Q mutation that has been described in an Asian
population and MPH patients, along with a novel frameshift mutation, Tdel219.
Testis morphology showed that, during early infancy, the 5--reductase
enzyme deficiency may not have affected interstitial or tubular
development.
Key words: 5--Reductase enzyme deficiency, 5--reductase type 2 gene mutations, male pseudohermaphroditism
-reduced metabolites, namely
dihydrotestosterone (DHT). The primordial role of 5--reductase enzyme
activity was first demonstrated in animal studies. Later, a cluster of male
pseudohermaphrodite (MPH) patients were described in whom deficient
virilization was attributable to an almost complete lack of this enzyme
activity (Imperato-McGinley et al,
1974; Imperato-McGinley and
Peterson, 1976). A number of deletions and point mutations in the
5--reductase type 2 gene (SRD5A2) gene that encodes the
5--reductase type 2 enzyme (Labrie
et al, 1992) have been shown to occur in patients with various
clinical and biochemical phenotypes
(Thigpen et al, 1992; Wilson et al, 1993; Boudon et
al,
1995a,b;
Cai et al, 1996; Hochberg et al, 1996; Vilchis
et al, 1997,
2000;
Can et al, 1998;
Nordenskjold and Ivarsson,
1998; Nordenskjold et al,
1998; Chavez et al,
2000; Hiort et al,
2002; Hafez et al,
2003; Mazen et al,
2003a,b).
The phenotypes of newborn MPH patients with partial androgen insensitivity
(PAIS), 5--reductase enzyme deficiency, or 17-ß-hydroxysteroid
dehydrogenase enzyme deficiency may be indistinguishable. Measurement of the
T-to-androstendione (
4) (T/4) ratio in
peripheral blood under human chorionic gonadotropin (hCG) stimulation
facilitates a biochemical diagnosis for 17-ß-hydroxysteroid dehydrogenase
enzyme deficiency; similarly, a serum T/DHT ratio or a 5-/5-ß
urinary androgen metabolite analysis under hCG may provide evidence for the
diagnosis of a 5--reductase enzyme deficiency. However, urinary
androgen metabolites are not usually determined, and the serum T/DHT ratio may
be almost normal in male adults with 5--reductase enzyme deficiency
and, conversely, be abnormally high in adult patients with androgen
insensitivity; thus, a correct differential diagnosis between PAIS and
5--reductase enzyme deficiency can be ensured only through the use of
molecular studies, with mutations in the androgen receptor (AR) gene
being by far more frequent than mutations in the SRD5A2 gene.
Testicular morphology has rarely been described in patients with
5--reductase enzyme deficiency, and reports on postpubertal patients'
spermiograms have yielded varying results
(Imperato-McGinley et al,
1980; Cai et al,
1994). A patient with suspected PAIS, based on clinical and biochemical studies, is described in whom the testicular morphology during the neonatal period was normal and whose molecular diagnosis was heterozygous compound mutations in the SRD5A2 gene.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The patient was classified as having PAIS.
Molecular Study
Genomic DNA was isolated from blood leukocytes by standard procedures.
AR gene exons 1–8 and SRD5A2 gene exons 1–5 were
amplified by polymerase chain reaction (PCR). The primers used in the
amplification of the SRD5A2 gene have been described by Vilchis et al
(1997). PCR amplification of
exon 1 was carried out in a volume of 12.5 µL containing 50 ng of genomic
DNA, 0.5 mM of each primer, 0.375 U of FailSafe PCR Enzyme Mix, and Premix D
(EPICENTRE, Madison, Wis). PCR amplification of exons 2–5 was carried
out in a volume of 12.5 µL containing 50 ng of genomic DNA, 0.3 mM of each
primer, 0.05 mM of each deoxynucleotide triphosphate (dNTP), 1.5 mM
MgCL2, and 0.25 U Taq polymerase (ECOGEN, Barcelona,
Spain). Reactions were first denatured at 94°C for 5 minutes and then
subjected to 40 cycles of amplification with denaturation at 94°C for 1
minute, annealing at 60°C for 30 seconds (65°C for 1 minute for exon 1
amplification), and elongation at 72°C for 1 minute. A final extension at
72°C for 7 minutes was employed. After PCR, the products were analyzed in
ethidium bromide–stained agarose gels and were found to be single band
with the expected size. These products were sequenced in an automated
sequencer (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems, Foster City,
Calif) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied
Biosystems) according to the directions provided by the manufacturer. Primers
used in the sequencing were the same as those used in the PCR
amplification.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Testis Pathology
The left and right testis morphologies were similar: the albuginea were
normal, with 325 and 330 µm of width, respectively; the mean seminiferous
tubule diameters were 68 and 61.2 µm, respectively; the tubular fertility
indices were 64% and 70%, respectively; the mean germinal cells per tubular
section were 1.3 and 1.4, respectively, with abundant hypertrophic and
multinucleated spermatogonia in the left testis; and the mean Sertoli cell
numbers per cross-sectioned tubule were 25.5 and 24.5, respectively
(Figure 1). All quantitative
parameters were normal for the age of this patient. The interstitial tissue
showed undifferentiated fibroblast cells. The epidydimides and initial segment
of ductus deferens were normal for the patient's age.
|
Molecular Study
AR gene analysis showed no abnormality in the entire coding
region. SRD5A2 gene analysis revealed 2 consecutive mutations in exon
4 (Figure 2), each located in a
different allele as follows: 1) a T nucleotide deletion at position 1998
(GenBank: 338466, L03843), which predicts a frameshift mutation from codon 219
and a longer protein with 277 amino acids and an anomalous sequence from
position 219; and 2) a single base substitution of guanine (CGA) by adenine
(CAA) at codon 227, predicting a glutamine replacement of arginine at amino
acid 227 (R227Q). The patient was a compound heterozygote for 2 mutations in
the SRD5A2 gene, each located in a different allele, as shown by
sense and antisense sequences (Figure
2). The biologic parents were not available for study.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The R227Q variant has been observed only among Far East Asians, and these
patients have presented with significantly reduced in vitro
5--reductase activity (3.2% of normal activity)
(Makridakis et al, 2000). In a
population-based case-control study conducted in China that focused on
polymorphic markers in the SRD5A2 gene and prostate cancer risk, the
allele R227Q was considered a polymorphism, even though the heterozygous form
was present only in 1 of 304 controls and in 2 of 191 patients
(Hsing et al, 2001). The
latter report confirmed the presence of the mutated allele in the Chinese
population. Two brothers of Vietnamese origin, both of whom were homozygous
for the Q allele, have been described by Hiort et al
(1996) and Sinnecker et al
(1996): one presented with
scrotal hypospadias with a bifid scrotum and a small penis and thus could be
considered an MPH patient, whereas the other presented with a small penis but
otherwise normal male genitalia; in both patients, the T/DHT ratio was
abnormally high after prolonged hCG stimulation. The mutated allele has also
been described in the Japanese population: in 2% of normal male controls (1 of
50 normal boys and 1 of 50 fertile male adults) and in 3 of 81 males with a
micropenis but otherwise normal genitalia (1 was homozygous for R227Q and the
2 others were compound heterozygotes with another mutation in the
SRD5A2 gene) (Sasaki et al,
2003b). This would mean that SRD5A2 mutations might
present a prevalence of approximately 3.7% in Japanese patients with a
micropenis (penile length, <–2 SDs below the mean).
In addition to the mutated R227Q allele that has been described to date only in Asian populations, the patient of our study presented with a single nucleotide deletion in the other allele. Because the 2 mutations are located very near to each other, this could be clearly demonstrated by analysis of the sense and antisense exon 4 sequences. The deletion affects codon 219 and predicts an anomalous protein with an abnormal sequence from amino acid 219 as well as a longer sequence of 277 amino acids. Unfortunately, no genital skin fibroblasts were available from this patient; hence, a determination of the SRD5A2 messenger RNA (mRNA) sequence could not be conducted.
The degree of external genitalia virilization at birth in SRD5A2 mutations has been described as extremely variable, ranging from a simple micropenis to an enlarged clitoris, labioscrotal fusion, single urethral meatus, and palpable gonads in the more highly affected cases. The less severe forms seem to carry mutations such as R227Q, for which residual enzyme activity has been detected (Hiort et al, 1996; Sasaki et al, 2003b). Our patient of Chinese origin presented with a severely affected phenotype, although a small prostatic utricle was detected by cystoscopy. This R227Q mutation was combined with a novel mutation in the other allele, which predicted that an abnormal protein would be produced. Female gender was assigned in China and was maintained in Spain for 3 reasons: 1) because of legal adoption requirements, 2) because of the severely affected phenotype, and 3) because of the molecular diagnosis, which had been performed only after an orchidectomy and a genitoplasty.
Testicular morphology has rarely been described in patients with
5--reductase enzyme deficiency, because a considerable percentage of
patients were not diagnosed until puberty and male gender assignment changed
at that age. Various reports on spermiogram and fertility analyses have
yielded varying results and, although the volume was very low in all reports,
one patient from an initial Dominican pedigree presented with a normal total
sperm concentration count, motility, and morphology, but others from the same
pedigree presented with oligospermia and azoospermia
(Cai et al, 1994). Probably,
DHT-deficient local formation more greatly affected seminal plasma formation,
which requires development and function of prostate and seminal vesicles, than
spermatogenesis. Paternity by intrauterine insemination with sperm from one
such patient was achieved (Katz et al,
1997), and fertility in 2 brothers from a Swedish family harboring
2 mutations in exon 4 has also been demonstrated
(Nordenskjold and Ivarsson,
1998). Whether abnormal spermatogenesis in these patients is the
consequence of SRD5A2 gene mutations or is secondary to the position
of the cryptorchid testis that was uncorrected during early infancy has not
been clarified. Testis morphology has been described in few of these patients
at pubertal and postpubertal ages, and most reports have described abnormal
Sertoli cell maturation and absent or incomplete spermatogenesis
(Imperato-McGinley et al,
1980; Okon et al,
1980; Johnson et al,
1986; Steger et al,
1999). A recent report described an adult Japanese patient who was
homozygous for the Q6X mutation in whom an inguinal testis presented a giant
seminoma (Sasaki et al,
2003a). In the patient of our study, the testicular morphology was
almost completely normal at 20 months of age, presenting a normal tubular
diameter, spermatogonial index, and Sertoli cell number, as did the testis
morphology at 4 months of age in another compound heterozygous patient for
whom the SRD5A2 mutations were present (personal data). This would
mean that early testis descent is advisable when the male gender is officially
assigned and that gonadectomy is to be performed as soon as possible when the
female gender is chosen in order to avoid both virilization at puberty and
testicular malignancy at postpubertal ages.
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Acknowledgments |
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Footnotes |
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