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,
From the * Department of Reproductive Genetics,
Reproductive Biomedicine Research Center of Royan Institute, Tehran, Iran; the
Genetic Research Center of Social Welfare and
Rehabilitation Sciences University, Tehran, Iran; the
Department of Nanotechnology, Avesina Research
Institute, Beheshti University, Tehran, Iran; and the
Department of Urology, Biomedical Research
Center of Military University of Medical Sciences, Tehran, Iran.
| Correspondence to: Ramin Radpour, Department of Reproductive Genetics, Reproductive Biomedicine Research Center of Royan Institute, PO Box 19395-4644, Tehran, Iran (e-mail: rradpour{at}royaninstitute.org). |
| Received for publication June 7, 2006; accepted for publication August 28, 2006. |
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Abstract |
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Key words: CAG repeats, testicular dysgenesis syndrome, molecular diagnosis
The presence of androgens and androgen receptor (AR) together with insulin-like peptide 3 (INSL3) are thought to be important in testicular descent, inducing the involution of the cranial suspensory ligament and the second migration step from the groin to the scrotum (transinguinal descent) (Ivell and Hartung, 2003). The human AR gene is located on chromosome Xq11-12 (Lubahn et al, 1988). This gene exhibits 2 polymorphic sites in exon 1 characterized by different numbers of CAG and GGN repeats. The AR is highly polymorphic due to a glutamine repeat encoded by (CAG)nCAA and a glycine repeat encoded by (GGT)3(GGG)(GGT)2(GGC)n. Abnormal expansion of the CAG segment to 44 repeats, which is known to reduce AR function both in vivo and in vitro (La Spada et al, 1991; Tut et al, 1997), has been found in 1 patient with hypospadias (Ogata et al, 2001). A more modest expansion of CAG repeat lengths, although within the normal range (approximately 10–30), has also previously been reported in 78 males with varying under-masculinization, including hypospadias (Lim et al, 2000). The CAG repeat length has also been assessed in males with cryptorchidism, but no association between CAG repeat length and undescended testes has been found (Sasagawa et al, 2000; Lim et al, 2001). Although the polymorphic GGN region of the AR also plays a role in the receptor function (Gao et al, 1996), studies on this polymorphism in relation to hypospadias or cryptorchidism are still not clear.
The purpose of this study was to examine the association between cryptorchidism or hypospadias with polymorphisms in the AR gene involved in the androgen pathway, the CAG/GGN repeat length polymorphisms. Therefore, we conducted a case-control study of 76 cryptorchid and 92 hypospadiac patients. To eliminate other mutations in the AR gene that might be implicated in cryptorchidism and hypospadias, exons 2–8 of the AR gene were screened.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Genotyping of CAG/GGN Trinucleotide Repeats
The genomic DNA samples of all the subjects were prepared from peripheral
blood lymphocytes according to standard protocols, and CAG/GGN repeat motifs
of exon 1 in the AR gene were determined by analyzing the size of a
polymerase chain reaction (PCR) product containing the polymorphic
microsatellites. The AR exon 1 was amplified from genomic DNA in 2
different PCR reactions, giving overlapping amplicons by previously reported
primers A2, A5, A8, and A10 (Lubahn et al,
1989) and primers A0 and A3n
(Ferlin et al, 2005). Both
reactions were performed with the same cycle (94°C for 1 minute, 58°C
for 1 minute, and 72°C for 1 minute, repeated 37 times). The CAG repeat
was amplified with the primers A0 and A5
(Table 2). The GGN repeat is
amplified with the primers A3 and A10
(Table 2). The reaction
components for each PCR consisted of a total volume of 50-µL reaction
mixture containing 100 ng DNA, PCR buffer (Applied Biosystems, Norwalk, CT),
1.5 mM MgCl2 (Applied Biosystems), 200 µmol deoxynucleotide
triphosphate (Roche Diagnostics, Vienna, Austria), 20 pmol of each primer, and
1.5 units of Taq Polymerase (Applied Biosystems).
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Sequencing was carried out by using A2 for CAG and A8 (Table 2) for GGN repeats by VBC-Genomics (VBC-Genomics Bioscience Research, Vienna, Austria) using 50 ng (2 µL) of PCR product and 4 pM (1 µL) of nonfluorescent primer (forward and reverse separately), 4 µL of BigDye Terminator ready reaction kit (Perkin Elmer, Wellesley, Mass), and 3 µL of double-distilled water to adjust the volume to 10 µL. Genotyping was done blinded to case-control status, and 25% of the samples were randomly repeated for quality control, where no discrepancies were observed. Sequencing results were analyzed with DNASIS MAX software version 2.6 (Hitachi Software Engineering Co, Tokyo, Japan).
AR Gene Point Mutation Screening
The remaining coding sequences of AR gene (exons 2–8) were
examined by PCR–single-strand conformation polymorphism (PCR-SSCP) using
new primers (Table 2) to screen
for associated subtle mutations. Each primer was localized in intron sequence,
allowing amplification of the corresponding exon coding sequence and the
associated intron-exon junctions. A positive control of mutations was
artificially created by using a primer containing a point mutation
(Table 3). This mutated control
was performed for each exon by using DNA of a participant belonging to the
control population. For SSCP, the PCR products were heated at 95°C for 10
minutes and placed on ice. Single-strand DNA separation was done by using
12.5% polyacrylamide gels according to the size of the amplified fragments.
For each exon, SSCP was performed at 4°C and compared with the mutated
control. This was done to ensure that our data were not confounded by exonic
mutations that were linked to any particular trinucleotide repeat allele.
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Statistical Analysis
Means, standard deviations, and standard errors were calculated for both
the patients and healthy fertile groups. The mean number of CAG/GGN repeats
from patients (cryptorchidism and hypospadias) was compared with those in
healthy fertile controls by 2-sample independent t-test (using the
computer software SPSS for Windows version 13.0; SPSS Inc, Chicago, Ill).
Subsequently, multiple comparisons were performed comparing the mean number of
CAG/GGN repeats in cryptorchid and hypospadiac patients with fertile controls
by analysis of variance. Logistic regression analysis was performed using the
number of CAG/GGN repeats as the exposure and clinical infertility as the
outcome. All P values were 2-sided; P less than .05 was
considered statistically significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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GGN numbers were found to be significantly higher (median, 24 vs 22; Table 3) among both subjects with penile hypospadias (P = .018) and those with a history of cryptorchidism (P = .001) compared with controls. In addition, the GGN numbers among subjects with penile hypospadias were significantly different compared with the 2 other subgroups of hypospadias combined (P = .001). The highest mean length of the AR gene GGN number in patients was observed in the cryptorchid group (24.5), but there were no significant differences between the 2 subgroups (bilateral and unilateral) (Table 3). In addition, the frequency of having a GGN repeat number (at least 22) was 85.5% in healthy infertile men and 33.7% in fertile controls (P = .001) (Figure). Also, our findings showed no significant differences between CAG repeat numbers in patients and control groups.
The distribution of GGN allele frequencies (Figure) was different between
cryptorchid men and controls, and there was an apparent trend toward a shift
to a GGN of 22 or a GGN of more than 22 in males with cryptorchidism, but no
significant difference was observed with the hypospadiac group with respect to
controls. We were able to identify 12 different CAG alleles and 8 different
GGN alleles in the cryptorchid group, with a range of 17–30 CAG repeats
corresponding and a range of 18–26 GGN repeats. In the hypospadiac
group, the CAG repeat range was 16–29 and the GGN repeat range was
13–25. The most common alleles in the cryptorchid group consisted of 24
GGN repeats (25.1%) corresponding to 24 glycine residues and 23 GGN repeats
(19.6%) corresponding to 23 glycine residues in the hypospadiac group, whereas
the most common alleles in fertile control group consisted of 22 GGN repeats
(14.8%) corresponding to 22 glycine
residues.
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In summary, we report that the mean GGN repeat length increased with the cryptorchidism and penile hypospadias. However, statistical analysis showed no significant differences between the cryptorchidism subgroups (bilateral or unilateral) (P > .05).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We found a significant increase (P = .001) in the mean length of the GGN trenucleotide repeat (24.5 ± 2.4) in cryptorchid males and males with penile hypospadias (23.9 ± 1.9) compared with healthy fertile controls (22.3 ± 2.1). Whereas a GGN length of 22 is the most prevalent in men from the general population, most individuals with a history of cryptorchidism presented with GGN numbers of 24 or more. The same allele distribution was also found in patients treated for penile hypospadias. Until now, few studies have examined the CAG repeat length in men with cryptorchidism (Sasagawa et al, 2000; Lim et al, 2001; Aschim et al, 2004; Ferlin et al, 2005), and they found no difference with respect to controls. The functional consequences of variations in the GGN repeat are even less clear, and epidemiologic investigations of the association between the number of GGN repeats and prostate cancer risk (Hakimi et al, 1997; Irvine et al, 1997) or male infertility (Tut et al, 1997; Lundin et al, 2003; Ferlin et al, 2004) produced inconsistent results. However, we showed that an increased number of GGN repeats significantly linked with cryptorchidism and some forms of hypospadias, and our findings confirmed previous data (Aschim et al, 2004), but these data should be further confirmed in other populations. This finding is in agreement with the available data on the embryologic development of external male genital organs.
Androgens are known to play a crucial role for the development of male external genital organs as well as the testicular descent, and previous studies have pointed to partly shared etiology of these 2 conditions (Akre et al, 1999). The finding of an identical switch in the GGN repeat lengths in patients with penile hypospadias as well as in those with a history of cryptorchidism indicates that our results point to true biological associations rather than being chance findings. Despite the alteration in the distribution of the GGN lengths among subjects, they were still within the range found in controls. Therefore, it can be hypothesized that any increase in GGN length above the most common length of 22 causes a slight impairment of AR function, which, combined with insufficient function of the Leydig cells and/or increased exposure to environmentally derived antiandrogens, leads to undervirilization of male genital organs. The significance of the GGN repeats on the function of the AR has been demonstrated previously (Gao et al, 1996).
Generally, the estimated effects of CAG or GGN repeats on infertility risk in most of studies are relatively small and reached significance only in subgroups with higher grade of disease. But different analyses make these studies difficult to compare. Additionally, the studied populations of these studies vary in size. Another point that must be considered is the population heterogeneity, where disease incidence varies with ethnic background. In this Iranian population study, this problem can be excluded because the study population consisted exclusively of Iranian males. Furthermore, the selection of a control group is a critical point for the final outcome of molecular epidemiologic studies. In our study, the control group consisted of fertile men with a normal range of sperm count.
In this case-control study, our data provides an association between cryptorchidism and GGN repeat length and confirmed 1 similar study suggested that the chance to find an association between the polyglycine tract length and the cryptorchidism in a given population. It is obvious that studies from a variety of different ethnic and genetic backgrounds using comparable patient subgroups are extremely valuable to further evaluate this association. The stability of the CAG/GGN repeats is still unknown. It has been shown that about 5% of daughters conceived through intracytoplasmic sperm injection (ICSI) have an inherited AR allele with either contraction or expansion up to 8 bp (Cram et al, 2000). Because expansions of CAG or GGN repeats can be deleterious, it should be further considered in ICSI candidates for cryptorchidism, hypospadias, or idiopathic male infertility. The possible relationship of specific CAG/GGN haplotypes to cryptorchidism suggests that some combinations of CAG and GGN may modulate AR function, but this needs to be verified in other studies.
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Acknowledgments |
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Footnotes |
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