1 Department of Gynaecology and Obstetrics and 2 Department of Biochemistry at the University of Erlangen-Nuremberg, Erlangen, Germany
3 To whom correspondence should be addressed at: University of Erlangen-Nuremberg, Department of Gynaecology and Obstetrics, Universitätsstrasse 2123, D-91054 Erlangen, Germany. Email: reiner.strick{at}gyn.imed.uni-erlangen.de
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Abstract |
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Key words: anti-Müllerian hormone/DNA polymorphism/ionization-time-of-flight/matrix-assisted laser desorption/MayerRokitanskiKüsterHauser syndrome/promoter elements
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Introduction |
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In general, most MRKH cases are isolated among families and karyotypic analyses have demonstrated normal 46,XX profiles (Simpson, 1999). The MRKH syndrome has an incidence of 1 per 40005000 female live births, where during fetal development the Müllerian ducts fail to develop into the uterus, cervix and upper vagina (Hauser and Schreiner, 1961
; Basile and De Michele, 2001
). Most of the MRKH patients have normal morphological ovaries and fallopian tubes and due to steroid hormone production develop normal secondary sex characteristics. Primary amenorrhea is the most common symptom leading to diagnosis. The presence of only a shallow vaginal pouch can be corrected with reconstructive surgery (Vecchietti, 1979
). MRKH is classified clinically into the following subtypes: (i) typical patients have normal developed fallopian tubes, ovaries and a renal system, (ii) atypical patients have renal, ovarian or other reproductive organ abnormalities, and (iii) the most rare and severe form is known as MURCS (Müllerianrenalcervicothoracic somite abnormalities) where abnormalities of the renal system or cervical-thoracic (partial or complete absence of the spinal column, ribs, or arms; asymmetric ribs or arms; improperly developed spinal column, ribs, or arms) or somite dysplasia occurs (Duncan et al., 1979
).
The AMH (anti-Müllerian hormone, also called Müllerian-inhibiting-substance = MIS or -factor = MIF) gene, on chromosome 19p13.3, codes for a dimeric glycoprotein of 560 amino acids with a 2425 amino acid leader, and a C-terminus sharing homology to the transforming growth factor (TGF)- family. In human testis, AMH protein is secreted from Sertoli cells from the start of sex differentiation and remains at high levels until puberty (Jamin et al., 2002
; MacLaughlin and Donahoe, 2002
; Visser, 2003
). Specifically, AMH in the 6th gestational week initiates Müllerian duct regression until the 9th week by inducing ductal epithelial regression through a paracrine mechanism, which then initiates apoptosis (Taguchi et al., 1984
; Visser, 2003
). In contrast, in XX females, where no AMH is expressed during fetal development, the paramesonephric or Müllerian duct grows into the oviducts, uterus, cervix, and the upper 75% of the vagina. AMH is produced at low levels in ovarian granulosa cells during the perinatal period through menopause. Female adult AMH serum levels are normally below <75 pmol/l and decrease over time, while AMH male serum levels range from 270640 pmol/l during fetal to pre-pubertal periods (Rey et al., 1999
). In addition, abnormally low AMH serum levels can be a marker for boys with cryptorchidism and children with intersex conditions (Lee et al., 1997
), whereas high AMH serum levels can be a marker of sertoli- and granulosa-cell tumors (Rey et al., 2000
). However, the AMH blood protein levels in MRKH or in other patients with uterine malformations have not been previously determined.
The AMH gene is located downstream of the splicing factor 3a subunit 2 (SF3a2) gene, also called SAP62, a house keeping gene and a member of the U2snRNP complex (Bennett and Reed, 1993). The 3' untranslated region of SF3a2 is located only 739 bp 5' from the transcriptional start site of AMH (Dresser et al., 1995
). The occurrence of read through transcription of SF3a2 into AMH, has been hypothesized due to DNA sequence variations resulting in a failure of SF3a2 polyadenylation (Dresser et al., 1995
). If this were to occur, following RNA and protein processing AMH would be constitutively expressed along with SF3a2 and result in higher than normal AMH blood levels.
The spatial and temporal transcription of AMH depends upon a series of essential transcription factors binding to the promoter, especially the steroidogenic factor (SF)-1 at position 92 and 218, SOX9 at 141 and GATA4 at 75 and 168 (Morikawa et al., 2000; Rey et al., 2003
l; Figure 1). SF1 and GATA4 DNA binding sites are essential for AMH expression in vitro (Arango et al., 1999
). Transgenic mice studies involving AMH promoter mutations, demonstrated that the SF1 binding site is a quantitative regulator of AMH, but is not essential for Müllerian duct regression (Arango et al., 1999
). These same mice studies showed that the SOX9 binding site at AMH position 141 was essential for Müllerian duct regression. Interestingly, human XY male patients with SOX9 gene mutations develop campomelic dysplasia where most exhibit sex reversal (Foster et al., 1994
).
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Several examples in the literature describe DNA promoter sequence variations stimulating gene expression. For example, a DNA polymorphism at position + 331G/A in the progesterone receptor promoter creates a new transcription start site for the B isoform and appears to be associated with an increased risk for endometrial cancer (De Vivo et al., 2002). DNA polymorphisms within the interleukin-10 (IL-10) gene promoter associate with high IL-10 expression associated with systemic lupus erythematosus (Lazarus et al., 1997
). The present study hypothesized that DNA polymorphisms could induce aberrant AMH expression during fetal development leading to Müllerian duct regression and the MRKH syndrome. Thirty MRKH patients including three MURCS phenotypes were analyzed for DNA sequence variations in the entire AMH gene promoter and downstream regulatory elements of the SF3a2 gene, which could be involved in aberrant AMH gene activation and protein production as detected by blood plasma levels.
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Materials and methods |
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DNA, plasma and peritoneal fluid collection and processing
All blood samples were collected in EDTA or CPDA tubes for preservation of the lymphocyte and plasma blood fractions. After an initial centrifugation at 1300 g, plasma was aliquoted and stored at 80°C. DNA from 8 ml of blood was extracted using a genomic DNA purification kit (Puregene) with modifications. Briefly, after initial centrifugation, the white blood cell layer was removed and added to red blood cell lysis buffer, pH 7.3, containing 0.15 M NH4Cl, 0.01 M K2CO3 and 0.1 mM Na-EDTA. After a 10 min incubation the cells were centrifuged at 2000 g and incubated with 3 ml of cell lysis buffer containing 20 mM Tris, 15 mM Na-EDTA, 1% sodium dodecyl sulphate and treated with RNase A and proteinase K. The proteins were precipitated with 1 ml of protein precipitation solution (Puregene) and the DNA precipitated by the addition of isopropanol, washed with 70% ethanol, dried, solubilized with a TrisEDTA buffer, pH 7.5, quantitated using a spectrophotometer and stored at 80°C. Using this methodology an average of 70100 µg of DNA per patient was obtained. Several peritoneal fluids were collected from MRKH patients during a laparoscopic exam.
PCR amplification of the entire AMH promoter and DNA sequencing
The complete gene sequence was extracted from the gene data bank at NCBI for chromosome 19 (NT_011255) and for AMH (NT_011255) and SF3a2 (NM_007165). A standard 100 µl PCR reaction was performed to amplify the entire AMH promoter (1066 bp DNA fragment) spanning from the last exon of SF3a2 to the first exon of AMH: 200 ng of total genomic DNA was incubated with 100 ng of primers SAP-TF (5'-GTCCACCCTCAGCCTCCGG GAGTTCACC) and MIS-BR (5'-GATGCCTGGAGGCCAGTCCAAGTCTTCTCG), 200 µM dNTP, PCR buffer containing MgCl2 and 2.5 U Taq DNA polymerase for 30 cycles (3 min 95°C initial, 90 s 95°C, 90 s 57°C, 3 min 72°C). The MRKH patient genomic AMH promoter was 5' and 3' DNA sequenced [in part by GATC-Biotech (Konstanz, Germany) and at the Department of Biochemistry, University of Erlangen (Germany)] using the primers SAP-TF and MIS-BR and in addition MISMP-Seq: 5'-AGGCCATCTCCAAGGTACTG with dye termination (Pharmacia Amersham) and then analysed on an ABI377. All DNA alignments and analyses were performed using MacVector 7.11 (Accelrys, Inc.).
The AMH specific enzyme-linked immunosorbent assay (Beckman-Coulter) uses monoclonal antibodies for detecting AMH in serum or plasma and was performed according to the manufacturer's instructions in the ultrasensitive mode with a detection limit of 0.7 pM. No cross-reactivity with TGF- was noted.
Matrix-assisted laser desorption/ionization-time-of-flight mass spectronomy (MALDI-TOF-MS based genotyping)
Aliquots of AMH promoter PCR products for 639C/T using 639TF: CAGCAAGCCCAGCGCCAGGTGCTCTTGCCT and 639BR: CAGGCCATCTCCAAGGTACAGCCATCT and for 210 G/A using 210TF: CAGCGCTGTCTAGTTTGGTTGCCTGGCCGTCA and 210BR: GCCTGCCTTAAGTGAGCCGAGTGGAAGGTG were purified using magnetic beads (genopure dsTM) as specified by the supplier (Bruker Daltonik, Bremen, Germany). Purified PCR DNA was used for the primer extension reactions after addition of 10 µl of extension mix, containing 12 pmol extension primer (639C/T forward TGATGTCCGCAGCGC or 210G/A reverse CCTGATGTGTCAACATGC), 1U Thermosequenase, reaction buffer (Pharmacia, Germany), 2 nmol ddCTP, ddGTP and dTTP. Extension reactions were performed for 45 cycles (2 min 96°C initial, 30 s 96°C, 30 s 53°C, 3 min 72°C). To confirm the MALDI-TOF-MS based genotyping, samples without DNA were used as negative and samples with known genotypes as positive controls. Primer extension products were purified with magnetic beads (genopure oligoTM) and then aliquots spotted onto matrix crystals of 3-hydroxypicolinic acid on an anchor targetTM (Bruker Daltonik) and air dried. Mass determinations were performed on an AutoflexTM MALDI-TOF mass spectrometer (Bruker Daltonik) equipped with a nitrogen laser (=337 nm) and delayed extraction. Laser-desorbed positive ions were evaluated following acceleration by 20 kV in the linear mode. External calibration was performed using a standard oligonucleotide mixture. Generally, 20 individual spectra were averaged to produce a mass spectrum. Genotypes were determined by analyzing the signals observed in the spectra: the forward extension primer for nucleotide position 639 C/T had a theoretical molecular mass of 4570 Da and was designated to anneal to DNA adjacent to the polymorphic C/T side. In case of the C-allele, the primer was terminated directly by a ddCTP, resulting in a mass of 4843 Da. When the allelic C to T exchange was present a dTTP was incorporated while the following ddGTP terminated the primer extension reaction yielding an extended primer of 5187 Da. The reverse extension primer used to analyze the 210 G/A single nucleotide polymorphism possessed a molecular mass of 5475 Da. When the G-allele was present, the primer was extended by ddC resulting in a mass of 5748 Da, while it was elongated by dTTP and terminated by ddG yielding a mass of 6092 Da in case of the A-allele.
Statistical methods
The SPSS 11.1 (SPSS Inc., Chicago, IL) package was used for all statistical analysis. The HardyWeinberg agreement was proven by a 2 test.
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Results |
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AMH and SF3a2 DNA sequencing of 30 MRKH patients and MALDI-TOF MS of 48 controls for detection of DNA sequence variations
From 30 MRKH patients, genomic sequencing of the entire AMH promoter including parts of the last SF3a2 exon and first exon of AMH resulted in several genotype distributions (Table II). Two main DNA polymorphisms were detected in MRKH patients: 22 (73%) were both homozygous for a DNA base change from C to T at position 639 (639 C/T) and homozygous for another DNA base change from G to A at position 210 (210 G/A). Six patients (20%) were both heterozygous for 639 C/T and homozygous for 210 G/A (Table II and IIITable II and III).
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Interestingly, in seven of 30 MRKH patients DNA polymorphisms were also detected in the coding region of the last exon of SF3a2 resulting in specific amino acid exchanges (Table II). For example, the codon at position (+1321)(+1323) ATG (Met) of SF3a2 either changed from an A to C at + 1321 (Met to Leu) for patients 155, 288, 331 and 390; a T to C at position + 1322 (Met to Pro) for patient 390, or a G to C at position + 1323 (Met to Ile) for patients 205 and 368. These base changes did not associate with any specific MRKH phenotype. In addition, no DNA sequence variations were detected in the transcriptional stop site or the poly A regulatory signal element of SF3a2, thus by DNA sequence analysis the possibility for read through transcripts containing both SF3a2 and AMH cannot be a possibility in MRKH patients.
AMH protein levels in blood and peritoneal fluid of MRKH patients and controls
In a series of control individuals AMH blood plasma levels were determined in adult males, females and cord blood samples from newborn babies (Figure 3). AMH concentration in 10 adult males (age 2239 years, mean 28.7 years) averaged 97.85 pM (range 41.6206.7 pM). In cord blood from females, AMH averaged 9.95 pM and in boys 267.98 pM, a difference of 27-fold. These AMH protein blood levels are in accordance with reports from normal individuals in the literature (Rey et al., 1999
). AMH protein plasma analysis of the 30 MRKH patients in this study (age 1749 years, mean 27.6 years) resulted in a mean concentration of 44.44 pM (range 15.2138.9 pM), compared with 45.08 pM (range 14.964.15 pM) in 10 normal ovulatory control women (age 1842 years, mean 27.5 years) (Figure 3). However, MRKH patients 205, 331 and 1740 had relatively high AMH levels of 87.8, 138.9 and 121.5 pM, respectively.
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Discussion |
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LH, FSH, prolactin, E2 and progesterone levels have previously been determined to be normal in MRKH patients (Carranza-Lira et al., 1999). In the present study AMH has been measured and determined as generally normal in blood (Figure 3) and peritoneal fluid of MRKH patients. This finding reflects normally regulated AMH gene promoters, transcription and AMH protein production in adult MRKH patients. Normal female blood AMH levels decrease over time and associate with the number of remaining antral follicles (Fanchin et al., 2003
), therefore AMH represents a specific marker for ovarian egg age and could be helpful for predicting successful IVF or surrogacy pregnancies. AMH serum concentrations of non-ovulatory women in the reproductive years were increased 3-fold when compared to normal-ovulatory women of the same age (Laven et al., 2004
). In this study, MRKH patients 205 had a 2-fold, 331 and 1740 an
3-fold higher AMH concentration (Figure 3), suggesting a non-ovulating state, whereas all other MRKH patients most likely have normal ovulation according to the AMH and other hormones, but no menstrual cycle. In addition, MRKH patients 751 and 1239 with unilateral ovarian agenesis and patients 1665 and 1832 with rudimentary and streak ovaries, respectively, support that these abnormal ovaries are sufficient for AMH production. Taken together the findings of normal hormone levels in MRKH individuals supports normal pituitary gland and ovarian function with ovulation.
Maternalships of MRKH patients through 34 successful surrogacy pregnancies have been described and resulted in 17 male and 17 normal female babies (Petrozza et al., 1997). Besides normal hormone levels and egg development among MRKH women, these maternalships strongly support a multifactorial inheritance that cannot be explained by autosomal or X-linked dominant genes. Further support for a multifactorial inheritance comes from a study of monozygotic twins where one of the twins had a uterus and vagina aplasia and the other twin had severe skeletal defects (Steinkampf et al., 2003
), however another report described only one discordant monozygotic twin with the MRKH phenotype (Lischke et al., 1973
). In contrast, Shokeir, (1978)
described the MRKH syndrome inherited as sex-linked dominant, while other studies demonstrated a more recessive inheritance (Jones and Mermut, 1972
). In the present investigation none of the MRKH patients had affected siblings with MRKH, indicating no clear inheritance pattern.
In this investigation we determined that the AMH promoter and SF3a2 downstream regulatory elements of MRKH patients are similar to control individuals indicating that aberrant developmental AMH gene activation or constitutive expression of AMH cannot be due to DNA sequence variations (Tables II and III). However, some DNA base changes were observed in the AMH promoter and in the last exon of SF3a2. For example, in the AMH promoter the DNA base change at 639 C/T alters the GC-Box 5'-GGGCGG to 5'-GGGCAG destroying the SP1 transcription factor binding site, thus only two partial GC-boxes remain at positions 111 and 370. Variation in the number of promoter SP1 sites appears to be common in the population and could cause differences in gene transcription levels. All other DNA polymorphisms did not occur in any known transcription factor binding sites. Interestingly, the 639 C/T and the 210 G/A polymorphisms represent the prominent AMH genotypes in the general population (Table II). Therefore, the DNA sequence in the NCBI databank depicts the rare polymorphic DNA sequence. In addition, one AMH promoter polymorphism at 380 found in the Japanese population (Haga et al., 2002) was neither detected in the MRKH patients nor in the control population, probably representing a race specific polymorphism. The finding of single nucleotide base changes in SF3a2 exon 8 alters the amino acids in the codon, however, would not influence activation of AMH.
In contrast to the bovine, rat and mouse AMH gene, the human AMH gene promoter only partly has a consensus to the TATAA-box element (CTTAA) at position 27. In vitro studies have demonstrated that human AMH transcription initiation is directed by an initiator region at 10 to + 10 bound by transcription factor (TF) II-I (Morikawa et al., 2000). Other regulatory elements also mapped in the AMH promoter including possible protein DNA binding sites for NFKB, ATF, nuclear factor (NF)1/CTF, SP1, activator protein (AP)1, serum response factor, as well as for ER (Figure 1). All of these DNA elements were normal in MRKH patients. In previous studies, DNA sequence variations were screened for in a variety of genes to determine an association with MRKH. AMH and AMHR2 gene coding regions have been analysed for polymorphisms in over 37 MRKH patients (Resendes et al., 2001
; Zenteno et al., 2004
). The identified polymorphisms in AMH and AMHR2 genes stated above were different between both patient groups in nucleotide position, but were not significant when compared to the control population. The present and above mutation analyses of the AMH promoter and gene strongly support that there is no link between the MRKH syndrome and AMH at the genomic level. Analysis of the Wilms Tumor (WT) 1 gene and cystic fibrosis transmembrane conductance regulator gene also demonstrated no significant mutations in MRKH patients (Van Lingen et al., 1998
; Timmreck et al., 2003
). In addition, a DNA polymorphism in the galactose-1-phosphate-uridyl transferase (GALT) gene appears to associate with MRKH (Cramer et al., 1996
), but is not proven as yet (Klipstein et al., 2003
; Zenteno et al., 2004
).
Other candidate genes than AMH or AMHR2 in the AMH signaling pathway may play a role in the molecular etiology of the MRKH syndrome. Mutations in the receptor AMHR1 genes (ALK2 and 3), especially the kinase domain could lead to constitutive self-phosphorylation activity. However, a deletion of AMHR1 cannot be responsible for the MRKH syndrome, due to knock-out AMHR1 mice experiments, which was embryonic lethal (Goumans and Mummery, 2000). Interestingly, ALK6 knock-out mice showed Müllerian duct regression and developed bone defects (Clarke et al., 2001
).
Alternative mechanisms for the MRKH syndrome could be due to maternal AMH or environmental sources, which have to cross the placental barrier. For example, higher than normal maternal AMH protein levels during pregnancy could be responsible for the degradation of female fetal Müllerian ducts during development. Other substances influencing the steroid hormone synthesis have been implicated in AMH expression and sex reversal phenotypes. E2 has been shown to induce AMH expression in cell lines (Chen et al., 2003). It could be possible that high E2 or high amounts of estrogen-like bioflavonoids found in foods and dietary supplements, like genistein (Strick et al., 2000
) during early pregnancy could induce AMH and result in Müllerian duct regression. Interestingly, one putative ER binding element (75% match to the consensus) was identified in the AMH promoter (Figure 1).
Finally, it will be important to compare the genetic etiologies in other syndromes as they become known, which share similar phenotypes with MRKH. For example, the rare Winter's syndrome, KlippelFeil syndrome, and the vaginal aplasia syndrome. The KlippelFeil syndrome, a rare skeletal disorder that affects both males and females, can show a familial dominant or recessive inheritance pattern (Clarke et al., 1995) and where some females with normal secondary sex characteristics also have abnormalities of the fallopian tubes, uterus and/or vagina (Park et al., 1971
). Other syndromes include the urogenital adysplasia syndrome, showing an autosomal dominant pattern of inheritance with incomplete penetrance and is also associated with Müllerian defects. Thus, determining the molecular cause for the MRKH syndrome may also help to discern if the other syndromes described above have different molecular etiologies, or more importantly, share overlaps with some of the involved genes.
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Acknowledgements |
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Notes |
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References |
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Submitted on May 4, 2004; resubmitted on July 8, 2004; accepted on September 10, 2004.