Molecular Human Reproduction, Vol. 9, No. 3, 171-174,
March 2003
© 2003 European Society of Human Reproduction and Embryology
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The N314D polymorphism of the GALT gene is not associated with congenital absence of the uterus and vagina
Submitted on July 29, 2002; resubmitted on November 7, 2002. accepted on November 26, 2002
1 Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Room KS-322, Boston, MA 02215 and Harvard Medical School Boston, MA 02215 and 2 Department of Obstetrics and Gynecology, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA and 3 Department of Obstetrics and Gynecology, Jetanin Hospital, No. 5 Soi Chidlom, Ploenchit Road, Lumpini, Pathumwan, Bangkok, 10330, Thailand
4 To whom correspondence should be addressed. e-mail: sklipste{at}caregroup.harvard.edu
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The aetiology of anomalous embryonic and fetal development of the female reproductive tract, ranging from common uterine abnormalities to the somewhat rare congenital absence of the uterus and vagina (CAUV), is unknown. Some have proposed that abnormal galactose metabolism might cause CAUV. An association between CAUV and the N314D allele of the galactose-1-phosphate uridyl transferase (GALT) gene has been proposed as aetiological. We tested this hypothesis further by performing a case–control molecular study analysing 32 patients with CAUV for the presence of the N314D allele. These patients were compared with 138 normal controls. No association between CAUV and the N314D polymorphism was found (P = 0.32). It is unlikely that either maternal or fetal GALT enzyme activity could affect paramesonephric duct development, because neither galactosaemic subjects nor their children have an increased incidence of uterine anomalies.
Key words: CAUV/galactosaemia/GALT/Mayer–Rokitansky–Küster–Hauser syndrome/N314D
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Introduction |
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Congenital absence of the uterus and vagina (CAUV), also known as Mayer–Rokitansky–Küster–Hauser syndrome, occurs in
1 in 5000 female births (Griffin et al., 1976). It is caused by a failure of development of the caudal portion of the embryonic paramesonephric (Müllerian) ducts, the anlage of the isthmus of the Fallopian tubes, the uterus, cervix, and the upper two-thirds of the vagina (Ludwig, 1998). CAUV is typically diagnosed after puberty, initially presenting with primary amenorrhoea. Almost all CAUV patients have a normal 46,XX female karyotype. Physical findings include an absent or hypoplastic vagina, absence of the cervix, replacement of the normal uterus with bilateral uterine remnants usually lacking a cavity and endometrium, and a high incidence of abnormally developed Fallopian tubes. Approximately half of CAUV patients have concomitant congenital renal, skeletal, cardiac, and hearing abnormalities. The aetiology of CAUV remains unknown. Although most cases are sporadic and occur in all ethnic groups, some familial examples have been described (Shokeir, 1978). The pattern of inheritance in some affected families seems to be autosomal recessive (Sarto et al., 1978). Identical twins concordant and discordant for CAUV have been described, suggesting that a causative gene for CAUV might have variable expressivity or penetrance (Heidenreich et al., 1977; Reindollar et al., 1981).
We have been studying the genetic basis of CAUV by analysing candidate genes for germline mutations in CAUV patients (Lindenman et al., 1997). These genes have included those encoding the anti-Müllerian hormone (AMH) (Resendes et al., 2001), anti-Müllerian hormone receptor (AMHR) (Resendes et al., 2001), HOXA10 (Lalwani et al., 2001), HOXA13 (Karnis et al., 2000), cystic fibrosis transmembrane conductance regulator (CFTR) (Timmreck et al., 2003), Wilms’ tumour transcription (WT1) factor (Driscoll et al., 1996; Van Lingen et al., 1997, 1998a), PAX 2 (Van Lingen et al., 1998b) and WNT7A (L.Timmreck, personal communication). No mutations or polymorphisms specific to CAUV patients have been found. An association between the N314D polymorphism of the galactose-1-phosphate uridyl transferase (GALT) gene and CAUV has been reported (Cramer et al., 1987, 1996). This association has been widely quoted and considered by some to be definitive. We questioned this association because the rationale for the prior studies was based on an animal model that is not analogous to CAUV (Chen et al., 1981; Rivest et al., 1985), and also because CAUV has not been found in galactosaemic patients or their daughters. The purpose of the present study was to test this hypothesis further by analysing a larger group of CAUV patients than was previously tested for this polymorphism.
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Materials and methods |
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DNA samples
Thirty-two patients with CAUV and 138 controls were analysed. All but two (484 and 485) were the only affected members of their families. These two were non-twin siblings concordant for CAUV. With the exception of one Asian patient (928), all patients were Caucasian. Informed consent was obtained from each patient prior to entry into the study. Ethical approval for the study was obtained from the institutional review boards of both New England Medical Center and Beth Israel Deconess Medical Center. The presence of CAUV was established by normal pubertal development, primary amenorrhoea, and absence of the vagina or the presence of a small vaginal pouch. The clinical diagnosis was confirmed by pelvic/abdominal ultrasonography or laparoscopic observation of normal ovaries with either absence of the uterus or the presence of a rudimentary uterus. In addition, the history and physical examination targeted the renal, skeletal and auditory systems to uncover defects. The diagnostic pelvic/abdominal ultrasound also included a search for renal agenesis. Venous blood was collected under protocols approved by the hospital human investigational review board for protection of clinical research subjects. DNA was extracted from leukocytes using a standard method (Gray, 1992). The clinical phenotype of each patient, including renal, skeletal, hearing and family history, are listed in Table I .
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PCR amplification of the N314D site
The following primer set was used to amplify a 311 bp DNA fragment that includes all of exon 10 of the GALT gene: 10–5' (5'-GGGTTTGGGAGT AGGTGCT-3') and 10–3' (5'-GGGCAACAGAAGTATCAGGT-3') (Lin et al., 1994). The 50 µl PCR amplification mixture included 50 ng of template genomic DNA, 100 µmol/l dNTP (New England Biolabs, Beverly, MA, USA), 15 mmol/l MgCl2, and 0.5 IU of Taq DNA polymerase (Life Technologies, Gaithersburg, MD, USA). The reactions were incubated in a PE Applied Biosystems 9700 thermal cycler as follows: 94°C for 3 min, followed by 40 cycles of 94°C for 1 min, 57°C for 1 min and 72°C for 1 min.
Restriction enzyme assay for detection of N314D allele
A sample of 19.75 µl of PCR-amplified DNA was digested with the restriction enzyme AvaII according to the manufacturer’s directions (New England Biolabs). The digested DNA samples were electrophoresed in a 10% polyacrylamide/TBE gel (37.5:1 acrylamide:bisacrylamide) for 165 min at 285 V. These gels were then stained in 0.1 mg/l ethidium bromide for 15 min, and photographed over a long-wave UV light. The normal allele of the 311 bp exon 10 fragment is digested by AvaII to produce two fragments of 215 and 96 bp. The exon 10 fragment with the N314D polymorphism has an additional AvaII site in the normal 215 bp subfragment. AvaII digestion of the polymorphic 311 bp fragment produces three fragments of 111, 104 and 96 bp (Figure 1).
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Results |
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DNA samples from 32 patients with CAUV were analysed by electrophoresis of AvaII-digested PCR fragments that include exon 10 of the GALT gene. Eight of 32 (25%) patients with CAUV were heterozygous for the N314D polymorphism; none was homozygous (Table II). Among the 138 control subjects, 22 (15.9%) were heterozygous for N314D, and two (1.5%) were homozygous (Table II). All of the male control subjects and most of the female controls were known to be fertile.
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Although the frequency of the N314D allele was higher in CAUV patients than in control subjects, the difference between the two groups was not statistically significant (P = 0.32). The 95% confidence intervals for the CAUV patients (0.12, 0.43) were very similar to those for the control patients (0.12, 0.28).
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Discussion |
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Previous studies (Cramer et al., 1987, 1996) implicating the N314D polymorphism of the GALT gene as aetiological for CAUV have been quoted in several authoritative texts (Yen et al., 1999; Scriver et al., 2001). In humans, galactosemia is most commonly caused by mutations in the gene encoding GALT. The hypothesis for these previous studies was based on a rat model for a galactosaemia that is not analogous to human CAUV (Chen et al., 1981; Rivest et al., 1985). Vaginal development in these animals is normal (Chen et al., 1981). In the rat, vaginal opening is not part of Müllerian duct development but is an estrogen-mediated event regulated by the hypothalamic–pituitary hormonal axis (Rivest et al., 1985). Furthermore, the absence of reports of galactosaemic women or their daughters with CAUV has led us to further question this association. In the present study, which tested more than twice the number of CAUV patients previously analysed (n = 32), we found no significant difference between women with CAUV and controls in the presence of the N314D allele.
The majority of human females with galactosaemia have hypergonadotrophic hypogonadism, often presenting as secondary amenorrhoea. Although they are often diagnosed with premature ovarian failure, none of these women has been reported also to have CAUV (Waggoner et al., 1990). There have even been cases of pregnancy reported in women with classic galactosaemia, implying that they have normal reproductive tracts (Kaufman et al., 1986).
The 4300 bp long GALT gene has 11 exons and 10 introns, and has been mapped to chromosome 9 (p13–21). The mRNA is 1295 nucleotides in length, and encodes a polypeptide of 379 amino acids. The active enzyme is an 88 kDa dimer (Tyfield et al., 1999). Surprisingly, gene knockout mice that lack GALT transferase activity do not express any abnormal phenotype (Leslie et al., 1992). The complete lack of galactosaemia symptoms in these animals demonstrates that galactose metabolic pathways are controlled differently in rodents and humans.
The N314D polymorphism in exon 10 is an A/G polymorphism at nucleotide 968 that changes amino acid 314, an asparagine codon, to an aspartic acid codon (Reichardt et al., 1988, Reichardt and Woo 1991; Leslie et al., 1992; Elsas et al., 1994). The N314D allele exists in two variant alleles, Los Angeles and Duarte, or D1 and D2, although both are identical at amino acid 314 (Greber et al., 1995; Podskarbi et al., 1996; Langley et al., 1997). The population frequency of the N314D polymorphism is 11.3–20.2% (Morland et al., 1998; Hadfield et al., 1999; Stefansson et al., 2001). Neither N314D-containing allele has been implicated in any disease phenotype, except for the previous CAUV study (Cramer et al., 1987, 1996; Elsas et al., 1994; Lin et al., 1994; Stefansson et al., 2001).
Cramer and colleagues hypothesized that excessive galactose interferes with Müllerian duct development, and that the N314D polymorphism of the GALT gene may be associated with CAUV (Cramer et al., 1987). In their study, the N314D polymorphism was found in two of four women with CAUV, and one of four mothers of affected daughters. A second study by the same group examined 13 mother–daughter pairs in which the daughters had CAUV. This study revealed four pairs in which both the mother and the daughter possessed the N314D polymorphism. Overall, 46% of the mothers and 46% of the daughters were heterozygous for the N314D polymorphism. It was concluded that a potential link exists between vaginal agenesis and errors of galactose metabolism (Cramer et al., 1996).
We tested the hypothesis that the N314D polymorphism is associated with CAUV by surveying a larger group of CAUV patients. In contrast to the finding of a 46% incidence of N314D in 13 women with CAUV, we found a 25% incidence (8/32 patients). In addition, we found a 17.4% carrier frequency in a normal control group of 138 subjects, a result similar to that found in other studies (11.3–20.2%) of the N314D polymorphism (Morland et al., 1998; Hadfield et al., 1999; Stefansson et al., 2001). In the present study, there was no significant difference in N314D carrier frequency between patients and controls (P = 0.32). The high incidence found in Cramer’s group of patients may be the result of a small sample size.
A mechanism that could explain an association between the N314D GALT gene polymorphism and CAUV is that of co-segregation. If a CAUV causative mutant gene were tightly linked to a GALT allele marked genetically by the N314D polymorphism, then CAUV would demonstrate linkage with N314D. This is unlikely, however, because the prevalence of N314D in our CAUV patients is not different from control subjects. Also, such a linkage would result in recessive inheritance patterns for CAUV. Only a few examples of families with an apparent autosomal recessive pattern of inheritance of CAUV have been found (Sarto et al., 1978). Although some examples of siblings with CAUV have been found, three sets of discordant monozygotic twins have been reported (Sarto et al., 1978; Reindollar et al., 1981). Most cases of CAUV are sporadic.
In summary, despite previous studies linking the N314D polymorphism of the GALT gene with CAUV, we have been unable to demonstrate a correlation between this polymorphism and CAUV.
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