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Micropenis and the AR Gene: Mutation and CAG
Repeat-Length Analysis
TOMOHIRO ISHII, SEIJI SATO, KENJIRO KOSAKI, GORO SASAKI, KOJI MUROYA,
TSUTOMU OGATA, AND NOBUTAKE MATSUO
Department of Pediatrics, Keio University School of Medicine, Tokyo 160-8582, Japan
Various mutations of the AR gene and expanded CAG repeats
at exon 1 of that gene have been reported in patients with
hypospadias or genital ambiguity. However, the role of the AR
gene has not been systemically studied in those with isolated
micropenis lacking hypospadias or genital ambiguity. We
studied 64 Japanese boys with isolated micropenis (age, 0–14
yr; median, 7 yr), whose stretched penile lengths were be-
tween ⴚ2.5 and ⴚ2.0 SD (borderline micropenis) in 31 patients
(age, 0–13 yr; median, 8 yr) and below ⴚ2.5 SD (definite mi-
cropenis) in 33 patients (age, 0–14 yr; median, 6 yr). Mutation
analysis of the AR gene was performed for exons 1– 8 and their
flanking introns, except for the CAG and GGC repeat regions
at exon 1, by denaturing HPLC and direct sequencing, iden-
tifying a substitution of cytosine to thymine at a position ⴚ3
in the 3ⴕ splice site of intron 1 in a patient with definite mi-
cropenis. CAG repeat length at exon 1 was determined by
electrophoresis with internal size markers and direct se-
quencing, revealing no statistically significant difference in
the distribution of CAG repeat lengths [median (range) and
mean ⴞ SE: total patients with isolated micropenis, 24 (14–34)
and 23.5 ⴞ 0.38; patients with borderline micropenis, 24 (15–
29) and 23.5 ⴞ 0.53; patients with definite micropenis, 23 (14–
34) and 23.5 ⴞ 0.56; and 100 control males, 23 (16 –32) and 23.5 ⴞ
0.29] or in the frequency of long CAG repeats (percentage of
CAG repeats >26 and >28: total patients with isolated micro-
penis, 17.2 and 4.7%; patients with borderline micropenis, 19.4
and 6.5%; patients with definite micropenis, 15.2 and 3.0%; and
100 control males, 21.0 and 10.0%). These results suggest that
an AR gene mutation is rare and that CAG repeat length is not
expanded in children with isolated micropenis. (J Clin Endo-
crinol Metab 86: 5372–5378, 2001)
M
ICROPENIS IS DEFINED as significantly small penis,
as compared with penile lengths of age-matched
normal males (1–3). The underlying mechanism for the de-
velopment of micropenis is either an inadequate production
of gonadal androgens for stimulation of the target organ or
an inadequate response of the target organ to stimulation
(1–3). Micropenis appears as an isolated form or occurs in
association with other genital anomalies such as hypospa-
dias. In this regard, differentiation of male external genitalia
is induced by placental human CG (hCG)-dependent go-
nadal androgens during the critical period for sex develop-
ment, and further growth of the male external genitalia is
primarily caused by fetal LH-dependent gonadal androgens
(1–5). Thus, defective androgen effect during the critical pe-
riod frequently results in micropenis with hypospadias or
genital ambiguity, whereas impaired androgen effect after
the critical period usually leads to isolated micropenis (1–3).
The AR plays a crucial role in male sexual differentiation
by mediating the biological effects of gonadal androgens (4,
5). The AR gene resides on Xq11–12 and consists of eight
exons; exon 1 encodes the transactivation domain, exons 2
and 3 encode the DNA binding domain, the 5⬘ portion of
exon 4 encodes the hinge domain, and the 3⬘ portion of exon
4 and exons 5–8 encode the ligand binding domain (6–9). In
addition, exon 1 contains a highly polymorphic CAG repeat
for the polyglutamine tract, and function studies with dif-
ferent CAG repeat numbers have indicated an inverse rela-
tionship between the CAG repeat length and transactivation
function or expression level of the AR gene (10 –13).
The AR gene has been examined in patients with defective
male sex differentiation. To date, many mutations have been
identified in patients with a wide range of clinical features
from complete female genitalia to normal male genitalia with
infertility (6 –9), including micropenis with hypospadias or
genital ambiguity (14–16). In addition, significant expansion
of the CAG repeat lengths has been reported in patients with
moderate to severe undermasculinization, most of whom
have micropenis with hypospadias or genital ambiguity (17).
These findings imply that the development of micropenis
with associated hypospadias or genital ambiguity can be
related to AR gene mutations or expanded CAG repeat
lengths.
However, patients with isolated micropenis have not been
systemically studied for the AR gene abnormality, although
some patients with isolated micropenis have been found in
families with AR gene mutations (18, 19). To elucidate the
role of the AR gene in the development of isolated micro-
penis, we examined 64 Japanese boys with isolated micro-
penis for mutation and expanded CAG repeat length of the
AR gene.
Subjects and Methods
Subjects
Sixty-four Japanese patients with isolated micropenis (age, 0 –14 yr;
median, 7 yr) were studied, after obtaining an appropriate written in-
formed consent that has been approved by the Institutional Review
Board Committee. All patients were seen at the outpatient clinic of
pediatric endocrinology in Keio University Hospital, from August in
Abbreviations: C3 T substitution, Cytosine to thymine substitution;
DHPLC, denaturing HPLC; hCG, human CG.
0013-7227/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism 86(11):5372–5378
Printed in U.S.A. Copyright © 2001 by The Endocrine Society
5372
1993 through December in 1999. The selection criteria included: 1)
stretched penile length below ⫺2.0 sd of the mean in age-matched
normal Japanese boys (20); 2) lack of hypospadias; 3) no gynecomastia;
4) 46,XY karyotype; 5) no demonstrable malformation syndromes
known to be associated with genital abnormalities; and 6) normal
growth and development. Stretched penile lengths were obtained by the
standard method, which included placement of a ruler against the dor-
sum of stretched penis and measurement of the distance between the tip
of glands and the pubic symphisis, with depressing the suprapubic fat
pad as completely as possible (1–3, 20). Age-appropriate pubertal de-
velopment was observed in cases 28, 29, 31, and 64 (21). Basal serum LH,
FSH, and T levels were within age-matched Japanese reference data in
all cases (22).
The stretched penile length of each patient is summarized in Table 1,
together with the effect of T treatment. Although ⫺2.5 sd has been used
as the lower limit of normal penile lengths (1–3), ⫺2.0 sd has been
regarded as the lower limit of normal variations for most quantitative
traits. Thus, the patients were divided into 2 groups: 1) borderline
micropenis between –2.0 and –2.5 sd below the mean (cases 1–31; age,
0–13 yr; median, 8 yr); and 2) definite micropenis below ⫺2.5 sd of the
mean (cases 32– 64; age, 0 –14 yr; median, 6 yr). Administration of T
enanthate (25 or 50 mg/dose, im, 1–4⫻) increased the stretched penile
lengths in all the 46 patients treated (23 with borderline micropenis and
23 with definite micropenis), although the effect was variable among the
patients.
For controls, 50 Japanese boys with normal external genitalia who
were diagnosed as having idiopathic short stature after extensive studies
(age, 3–16 yr; median, 8.5 yr) and 50 Japanese adult males with proven
fertility (age, 25– 48 yr; median, 38.5 yr) were similarly analyzed, with
permission. All the 100 control males had a 46,XY karyotype.
Mutation analysis of the AR gene
Each of the eight exons of the AR gene was amplified from genomic
DNA using primers based on the reported genomic sequence of the
human AR gene (GenBank accession number AH002607) (23, 24). PCR
was performed with a thermal cycler PTC200 (MJ Research, Inc.,
Waltham, MA) in a reaction vol of 20
l containing 0.1
g leukocyte
genomic DNA, 10 pmol primers, 5 nmol each deoxynucleotide 5⬘-
triphosphates, and 1 U AmpliTaq Gold DNA polymerase (Perkin-Elmer
Corp., Foster City, CA). The PCR conditions were: denaturation at 95 C
for 10 min; followed by 35 cycles at 95 C for 1 min, 60 C for 1 min, and
72 C for 1 min; and the final extension step at 72 C for 10 min. The PCR
product derived from a normal individual known to have wild-type
sequence and the product amplified from the sample genomic DNA
were mixed, denatured, and reannealed and subjected to denaturing
HPLC (DHPLC) (WAVE, Transgenomic, Inc., Omaha, NE) (25–27). Be-
cause of complex heteroduplex formation, the CAG and GGC repeat
regions at exon 1 were not studied. The primer sequences, the PCR
product sizes, and the PCR annealing temperatures are shown in Table
2, together with the DHPLC melting temperatures. When abnormal
chromatographic patterns were detected, the PCR products were se-
quenced on an automated sequencer ABI 310 (Perkin-Elmer Corp.).
CAG repeat-length analysis
The CAG repeat region at exon 1 was amplified by PCR with primers
flanking that region (28) and was examined for the triplet repeat number
in all patients and controls. Amplification was performed in a reaction
vol of 20
l containing 0.1
g leukocyte genomic DNA, 8 pmol fluo-
rescently labeled forward primer, 8 pmol unlabeled reverse primer, 5
nmol each deoxynucleotide 5⬘-triphosphates, and 1 U AmpliTaq Gold
DNA polymerase (Perkin-Elmer Corp.). The PCR conditions were: de-
naturation at 95 C for 10 min; followed by 35 cycles at 95 C for 1 min,
60 C for 1 min, and 72 C for 1 min; and the final extension step at 72 C
for 10 min. The primer sequences are shown in Table 2. The PCR
products were mixed with internal control size markers and were elec-
trophoresed on the autosequencer. The size of the PCR products was
determined by a GeneScan software version 3.1 (Perkin-Elmer Corp.). To
further confirm the precise CAG repeat number, a total of 20 PCR
products with different CAG repeat lengths were subjected to direct
sequencing on the autosequencer. The normality of the CAG repeat
lengths was examined by the
2
test. The statistical significance in the
median of CAG repeat lengths between different groups was analyzed
by the Mann-Whitney’s U test, and that in the frequency of long CAG
repeats (ⱖ26 or ⱖ28) was examined by the Fisher’s exact probability test.
P ⬍ 0.05 was considered significant.
Results
Mutation analysis of the AR gene
The results are shown in Table 1. Mutation screening
showed an abnormal chromatographic pattern for exon 2 and
its flanking introns in case 61 (Fig. 1A). This abnormal chro-
matographic pattern was undetected in the remaining cases
and in the 100 control males. Subsequent direct sequencing
of the region in case 61 revealed a substitution of cytosine to
thymine (C3 T) at a position ⫺3 in the 3⬘ splice site of intron
1 (Fig. 1B). There were no abnormal chromatographic pat-
terns for other exons.
Case 61 was born to nonconsanguineous parents, at 40 wk
of gestation, after an uncomplicated pregnancy and delivery.
At birth, his length was 51.0 cm (⫹0.6 sd), and his weight was
3.42 kg (⫹0.4 sd). At 11
2
⁄
12
yr, 2 months of age, he was seen
in our institution because of isolated micropenis. His height
was 152.6 cm (⫹1.6 sd), and his weight was 57.6 kg (⫹2.5 sd).
Stretched penile length was 2.5 cm (age-matched Japanese
data, 4.8 ⫾ 0.8 cm) (20), testicular volume was 1 ml bilaterally
(age-matched Japanese data, 1.5– 8.0 ml) (30), and pubic hair
development was at Tanner stage 1. His urethral meatus was
placed at the top of the glans. Bone age was 13 yr on the
Japanese standard (31). Basal serum LH was less than 0.2
IU/liter (age-matched Japanese reference value, ⬍0.2–2.83
IU/liter); FSH, 1.1 IU/liter (0.58–5.24 IU/liter); and T, less
than 0.4 nm (⬍0.4 –18.4 nm) (22). After the identification of the
C3 T substitution, a GnRH test (100
g bolus iv; blood sam-
pling at 0, 30, 60, 90, and 120 min) and an hCG test (3000
IU/m
2
im for 3 consecutive days; blood sampling on 0d and
4d) were performed at 11
6
⁄
12
yr of age. Peak serum LH was
5.4 IU/liter (age- and pubertal stage-matched Japanese ref-
erence value, 2.0–11.8 IU/liter), and FSH was 4.8 IU/liter
(5.7–16.6 IU/liter) in the GnRH test (32); and serum T was
increased to 6.3 nm (4.0 –15.0 nm) in the hCG test (33). Fa-
milial pedigree of case 61 is shown in Fig. 1C. The mother had
menarche at 11.7 yr of age (the mean age of menarche in
Japanese girls, 12.25 ⫾ 1.25 yr) (21) and, thereafter, regular
menses. A maternal cousin had micropenis and bilateral
cryptorchidism but lacked gynecomastia. The mother and
the grandmother of case 61 were found to be carriers for the
C3 T substitution identified in case 61. Unfortunately, the
maternal cousin with micropenis and cryptorchidism was
not examined because of his refusal.
CAG repeat-length analysis
CAG repeat length of each patient is shown in Table 1, and
the data are summarized in Table 3. There was no significant
difference in the median of CAG repeat lengths between total
patients with isolated micropenis, patients with borderline
micropenis, patients with definite micropenis, and control
males. Furthermore, although case 39 had a long CAG repeat
number of 34, which was undetected in the 100 control males,
there was no significant difference in the frequency of long
CAG repeats (ⱖ26 or ⱖ28) between total patients with iso-
Ishii et al. • Micropenis and AR Gene J Clin Endocrinol Metab, November 2001, 86(11):5372–5378 5373
TABLE 1. Summary of patients examined in the present study
Patient Genital findings Testosterone enanthate therapy AR gene analysis
Case Age (yr)
Penile length
CYO Dosage (mg)
Increment in
penile length (cm)
Nucleotide
substitution
CAG repeat number
at exon 1
(cm) (SDS)
Borderline micropenis
1 0 2.3 ⫺2.5 50 (25 ⫻ 2) 1.0 No 23
2 0 2.5 ⫺2.1 B No 29
3 3 2.3 ⫺2.2 50 (25 ⫻ 2) 0.8 No 26
4 4 2.3 ⫺2.5 75 (25 ⫻ 3) 0.8 No 17
5 4 2.5 ⫺2.2 No 25
6 4 2.5 ⫺2.2 50 (25 ⫻ 2) 1.2 No 25
7 4 2.5 ⫺2.2 50 (25 ⫻ 2) 1.0 No 24
8 4 2.5 ⫺2.2 No 25
9 4 2.5 ⫺2.2 50 (25 ⫻ 2) 1.0 No 26
10 5 2.5 ⫺2.4 50 (25 ⫻ 2) 1.5 No 23
11 6 3.0 ⫺2.2 25 (25 ⫻ 1) 0.5 No 18
12 6 3.0 ⫺2.2 25 (25 ⫻ 1) 0.7 No 23
13 6 3.0 ⫺2.2 R 75 (25 ⫻ 3) 1.3 No 23
14 6 3.0 ⫺2.2 25 (25 ⫻ 1) 0.4 No 26
15 8 2.8 ⫺2.3 No 25
16 8 2.8 ⫺2.3 75 (25 ⫻ 3) 1.4 No 23
17 8 3.0 ⫺2.3 B 50 (25 ⫻ 2) 0.8 No 20
18 9 3.0 ⫺2.1 75 (25 ⫻ 3) 1.2 No 21
19 9 3.0 ⫺2.1 50 (25 ⫻ 2) 1.0 No 24
20 9 3.0 ⫺2.1 L No 29
21 10 3.0 ⫺2.1 No 23
22 10 3.0 ⫺2.1 50 (25 ⫻ 2) 0.8 No 24
23 10 3.0 ⫺2.1 50 (25 ⫻ 2) 1.0 No 24
24 10 3.0 ⫺2.1 50 (50 ⫻ 1) 1.0 No 22
25 10 3.0 ⫺2.1 No 23
26 10 3.0 ⫺2.1 75 (25 ⫻ 3) 1.0 No 15
27 11 3.0 ⫺2.3 50 (50 ⫻ 1) 1.5 No 24
28 12 2.5 ⫺2.1 50 (25 ⫻ 2) 1.1 No 23
29 12 2.8 ⫺2.3 150 (50 ⫻ 3) 1.7 No 26
30 13 3.0 ⫺2.2 25 (25 ⫻ 1) 0.5 No 24
31 13 3.0 ⫺2.2 B No 25
Definite micropenis
32 0 1.5 ⫺4.5 100 (25 ⫻ 4) 2.0 No 20
33 0 2.0 ⫺3.3 75 (25 ⫻ 3) 0.8 No 25
34 0 2.0 ⫺3.3 75 (25 ⫻ 3) 1.1 No 14
35 0 2.2 ⫺2.8 B 75 (25 ⫻ 3) 0.6 No 26
36 1 2.2 ⫺2.8 50 (25 ⫻ 2) 0.8 No 23
37 3 2.0 ⫺2.8 R 50 (25 ⫻ 2) 0.9 No 20
38 3 2.0 ⫺2.8 50 (25 ⫻ 2) 0.9 No 25
39 3 2.0 ⫺2.8 50 (25 ⫻ 2) 0.6 No 34
40 3 2.0 ⫺2.8 50 (25 ⫻ 2) 1.0 No 25
41 5 1.5 ⫺4.4 B 50 (25 ⫻ 2) 0.8 No 23
42 6 2.0 ⫺4.2 No 22
43 6 2.5 ⫺3.2 50 (25 ⫻ 2) 0.8 No 24
44 6 2.5 ⫺3.2 75 (25 ⫻ 3) 1.2 No 21
45 6 2.8 ⫺2.6 25 (25 ⫻ 1) 0.5 No 25
46 6 2.8 ⫺2.6 75 (25 ⫻ 3) 0.7 No 25
47 6 2.8 ⫺2.6 75 (25 ⫻ 3) 0.9 No 20
48 6 2.8 ⫺2.6 75 (25 ⫻ 3) 1.2 No 25
49 7 2.5 ⫺3.6 No 27
50 7 2.5 ⫺3.6 No 23
51 7 3.0 ⫺2.6 50 (25 ⫻ 2) 0.5 No 24
52 7 3.0 ⫺2.6 L No 23
53 7 3.0 ⫺2.6 50 (25 ⫻ 2) 1.3 No 22
54 8 2.5 ⫺2.8 25 (25 ⫻ 1) 0.4 No 21
55 8 2.5 ⫺2.8 B No 21
56 9 2.0 ⫺3.6 50 (25 ⫻ 2) 0.9 No 25
57 9 2.5 ⫺2.9 100 (25 ⫻ 4) 1.5 No 21
58 10 2.5 ⫺2.8 50 (25 ⫻ 2) 0.8 No 24
59 10 2.5 ⫺2.8 No 25
60 11 2.0 ⫺3.5 No 23
61 11 2.5 ⫺2.9 Yes
a
23
62 11 2.5 ⫺2.9 100 (50 ⫻ 2) 0.6 No 26
63 11 2.5 ⫺2.9 B No 27
64 14 4.5 ⫺4.8 No 22
SDS,
SD score; CYO, cryptorchidism; B, bilateral; R, right; L, left.
a
Having a C 3 T substitution at a position ⫺3 in the 3⬘ splice site of intron 1.
5374 J Clin Endocrinol Metab, November 2001, 86(11):5372–5378 Ishii et al. • Micropenis and AR Gene
lated micropenis, patients with borderline micropenis, pa-
tients with definite micropenis, and control males.
Discussion
Mutation analysis failed to unequivocally identify a caus-
ative mutation in the AR gene of our patients. It may be
possible that such a mutation remained undetected as an
abnormal chromatographic pattern by the DHPLC analysis,
because the sensitivity of DHPLC analysis is between 95 and
100% (27, 34). It may also be possible that a mutation existed
in an unexamined region, such as the promoter, the intron,
or the triplet repeat regions. The results, nevertheless, sug-
gest that a mutation of the AR gene is rare in children with
isolated micropenis, and this is consistent with the previous
report by Lee et al. (2) that there was only 1 patient with
androgen insensitivity syndrome found in 45 patients with
isolated micropenis, though the diagnosis was based on en-
docrine studies and family history.
AC3 T substitution was identified in the consensus se-
quence of the splice acceptor site in case 61. In this regard,
this substitution was not detected in the 100 control males,
and the heterozygosity for the C3 T substitution in the
mother and the grandmother would raise the possibility that
micropenis of the maternal cousin could also be attributable
to the same substitution. In addition, cytosine accounts for
approximately 74% of the nucleotides at position ⫺3inthe
3⬘ splice site consensus sequence (35), and the same splice
acceptor site substitution (a C3 T change at position ⫺3in
the 3⬘ splice site) in the intron 6 of the lipoprotein lipase gene
has been suggested as a causative mutation in patients with
familial lipoprotein lipase deficiency (36). However, endo-
crine studies in case 61 failed to show results consistent with
androgen insensitivity syndrome, such as elevated serum LH
level; and the occurrence of an abnormal splicing has not
been demonstrated by expression studies using genital skin
fibroblasts in case 61. In addition, there has been no report
documenting the same substitution in patients with andro-
gen insensitivity syndrome. Thus, it is uncertain, at present,
whether the C3 T substitution in the consensus sequence of
the splice acceptor site in case 61 is a true mutation or a rare
polymorphism.
The CAG repeat length was not expanded in patients with
isolated micropenis, nor was the frequency of long CAG
repeats increased. In addition, the CAG repeat number of 34
in case 39, though it was absent in the 100 control males
examined in this study, has been reported in normal Japanese
males (37). The results suggest that the genotype of CAG
repeat length has no discernible effect on the development of
isolated micropenis in our patients. However, this would not
necessarily imply that CAG repeat lengths are not expanded
in other patient populations with isolated micropenis. For
example, previous reports on azoospermia have shown both
positive and negative results for the association between
expanded CAG repeat lengths and infertility (13, 38– 41). It
is likely that the CAG repeat length in the AR gene consti-
tutes one of multiple genetic factors relevant to the devel-
TABLE 2. The primer sequences, the product sizes, and the PCR annealing and DHPLC melting temperatures
Location
Forward primer
Reverse primer
Product size (bp) PCR temp. (C) DHPLC temp. (C)
Mutation analysis
Exon 1-a 5⬘-CGGGGTAAGGGAAGTAGGTGGAAG-3⬘⬃356 60
5⬘-TAGCCTGTGGGGCCTCTACGATG-3⬘ (n ⫽ 20)
Exon 1-b 5⬘-CAGCAAGAGACTAGCCCCAGGC-3⬘ 314 60 63, 64
5⬘-GGATACTGCTTCCTGCTGCTGTTGC-3⬘
Exon 1-c 5⬘-CTTAAGCAGCTGCTCCGCTGACC-3⬘ 599 60 62, 63
5⬘-CGGCCAGAGCCAGTGGAAAGTTG-3⬘
Exon 1-d 5⬘-GTCTACCCTGTCTCTCTACAAGTC-3⬘ 320 60 62, 63
5⬘-GTCCATACAACTGGCCTTCTTCG-3⬘
Exon 1-e 5⬘-TGCAGCGGGACCCGGTTCTGGGTCACC-3⬘⬃413 65
5⬘-ACTCTGCCCTGGGCCGAAAGGCGACATTTC-3⬘ (n ⫽ 18)
Exon 2 5⬘-GCCTGCAGGTTAATGCTGAAGACC-3⬘ 379 60 58, 59, 60
5⬘-CCTAAGTTATTTGATAGGGCCTTG-3⬘
Exon 3 5⬘-GTTTGGTGCCATACTCTGTCCACT-3⬘ 413 60 57, 58, 59
5⬘-CTGATGGCCACGTTGCCTATGAAA-3⬘
Exon 4 5⬘-AATGGTGATTTTCTTAGCTAGGGC-3⬘ 394 60 57, 59, 61
5⬘-TTACCAGGCAAGGCCTTGGCCCAC-3⬘
Exon 5 5⬘-CAACCCGTCAGTACCCAGACTGACC-3⬘ 285 60 60, 62
5⬘-AGCTTCACTGTCACCCCATCACCA-3⬘
Exon 6 5⬘-CTCTGGGCTTATTGGTAAACTTCC-3⬘ 294 60 58, 59, 60
5⬘-GTCCAGGAGCTGGCTTTTCCCTAA-3⬘
Exon 7 5⬘-CTTTCAGATCGGATCCAGCTATC-3⬘ 416 60 59, 60, 61
5⬘-CTCTATCAGGCTGTTCTCCCTGAT-3⬘
Exon 8 5⬘-GAGGCCACCTCCTTGTCAACCCTG-3⬘ 347 60 59, 60
5⬘-GGAACATGTTCATGACAGACTGTA-3⬘
CAG repeat
length analysis
CAG repeat 5⬘-TCCAGAATCTGTTCCAGAGCGTGC-3⬘⬃282 60
region 5⬘-GCTGTGAAGGTTGCTGTTCCTCAT-3⬘ (n ⫽ 20)
The primer sequences are based on Allen et al. (28) (the CAG repeat region), Lubahn et al. (29) (exon 2 forward, exon 6 forward, exon 7 reverse,
and exon 8 forward primers), and our design (the remaining primers). Exon 1-a to 1-e indicate five primer sets covering the long exon 1; the
region amplified with exon 1-a contains the CAG repeat region, and that amplified with exon 1-e contains the GGC repeat region.
Ishii et al. • Micropenis and AR Gene J Clin Endocrinol Metab, November 2001, 86(11):5372–5378 5375
opment of androgen-related disorders and that expansion of
the CAG repeat length can be detected as a positive modi-
fying factor in some patient populations but not in other
patient populations.
In contrast to our results, Lim et al. (17) have reported that
both the median of the CAG repeat lengths and the frequency
of long CAG repeats are significantly larger in 78 males, most
of whom had micropenis with hypospadias or genital am-
FIG. 1. A, Chromatographic patterns for exon 2 and its flanking introns of the AR gene, obtained under the melting temperature of 60 C in
DHPLC; left, A normal chromatographic pattern in a control male, indicating a single homoduplex peak (arrowhead); right, an aberrant
chromatographic pattern in case 61, demonstrating both homoduplex (arrowhead) and heteroduplex (arrow) peaks. B, DNA sequences of the
boundary of intron 1 and exon 2 of the AR gene. Capital and small letters indicate exon and intron sequences, respectively. Case 61 has a C3 T
substitution at a position ⫺3 in the 3⬘ splice site of intron 1 (arrow). C, Familial pedigree of case 61. Case 61 is indicated by an arrow. A maternal
cousin (III-6) has micropenis and undescended testes. Mutation analysis of the AR gene has been performed in three persons in this family (II-8,
III-3, and IV-1), revealing that the grandmother (II-8) and the mother (III-3) of case 61 are heterozygotes for the C3 T substitution at the intron
1 of the AR gene. Asterisks represent individuals with micropenis, and solid symbols indicate those with the C3 T substitution.
5376 J Clin Endocrinol Metab, November 2001, 86(11):5372–5378 Ishii et al. • Micropenis and AR Gene
biguity, although their CAG repeat lengths remain within the
normal range. Indeed, the median of CAG repeat lengths in
patients reported by Lim et al. (17) (median, 25; range, 14–31;
mean ⫾ se, 24.5 ⫾ 0.38) is significantly larger than that in our
patients (P ⫽ 0.02 by the Mann-Whitney’s U test). The dis-
cordance may be attributable to the difference in a genetic
and environmental background between the patient popu-
lations analyzed. However, because the extent of undermas-
culinization is more severe in patients described by Lim et al.
(17) than in those examined in this study, the genotype of
long CAG repeat may be more prevalent in patients with
micropenis with associated hypospadias or genital ambigu-
ity than in those with isolated micropenis.
The present study, therefore, implies that structural
changes of the AR gene are very rare, if any, in patients with
isolated micropenis. This would not be surprising, because
isolated micropenis is a highly heterogeneous condition, sub-
ject to various genetic and environmental factors. For exam-
ple, a mutation of any gene involved in the androgen pro-
duction or action could result in isolated micropenis (42–44).
In this regard, because detailed endocrine studies including
GnRH and hCG tests were not performed, several disorders
affecting androgen production in Leydig cells (such as de-
creased LH signaling and defective steroidogenesis) and
those impairing androgen action in the external genitalia
(such as reduced 5
␣
-reductase activity and defective post AR
signal transduction) may have remained undetected in the
present study. Similarly, various endocrine disruptors are
known to have a deleterious effect on the differentiation of
male external genitalia during fetal development (45). In this
context, because some endocrine disruptors impair the func-
tion of hormone receptors [including AR and LH receptor
(45–47)], functional rather than structural abnormality of the
AR gene may be relevant to the development of isolated
micropenis.
In summary, the present study suggests that an AR gene
mutation is rare and CAG repeat length at exon 1 is not
expanded in children with isolated micropenis. Further
studies will permit a better clarification on the relevance of
the AR gene abnormalities to the development of isolated
micropenis.
Acknowledgments
We thank Dr. Mike J. McPhaul (University of Texas Southwestern
Medical Center at Dallas, TX) for reviewing the manuscript.
Received January 12, 2001. Accepted July 11, 2001.
Address all correspondence and requests for reprints to: Tomohiro
Ishii, M.D., Department of Pediatrics, Keio University School of Med-
icine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail:
tomishii@mac.com.
This work was supported by a grant for Pediatric Research from the
Ministry of Health and Welfare, a grant from Keio University Medical
Science Fund, and a grant from Pharmacia Fund for Growth and De-
velopment Research.
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TABLE 3. The results of the CAG repeat-length analysis
Control subjects Patients with micropenis
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(n ⫽ 50)
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