1 Department of Obstetrics and Gynaecology, Huddinge Hospital, Stockholm, 2 Department of Obstetrics and Gynaecology, Västervik Hospital, 3 Department of Molecular Medicine, CMM 02, Karolinska Hospital, Stockholm and 4 Department of Paediatric Surgery, Astrid Lindgren Children Hospital, Karolinska Hospital, Stockholm, Sweden
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words:
dystocia/endothelin 1 gene/mutational screening/prostaglandin F2-receptor gene/testosterone 5
reductase type 1 gene
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In several other obstetric conditions a genetic influence has been identified, for example in pre-eclampsia (Ward et al., 1993) and low birth weight (Johnstone and Inglis, 1974
). In two recent studies on dystocia, a genetic influence or familial association has been implicated. In one cohort study, an intergenerational predisposition to Caesarean delivery and dysfunctional delivery was identified (Varner et al., 1996
). In another cohort study, it was shown that if a mother had dystocia when delivering her eldest daughter, this daughter had an increased risk of dystocia when delivering her first child (Berg-Lekås, et al., 1998
). The risk for an instrumental delivery is more than tripled if one sister has dystocia and for twins the risk increases >20-fold if one of the sisters had dystocia during the first delivery. One way of identifying genetic factors involved in a disease is to study mutations in candidate genes in affected individuals.
Based on animal data, two genes have been suggested as candidates for dystocia, the genes for testosterone 5- reductase type 1 (SRD5A1) and for the prostaglandin F2
receptor (PGF2
R). In a knock-out model for SRD5A1, the only abnormality observed was that 67% of female mice exhibited absence of parturition (Mahendroo et al., 1996
). The SRD5A1 gene is expressed in several tissues in mice including the uterus. The gene is fully characterized and located on human chromosome 5p (Jenkins et al., 1991
). Transgenic mice with a disrupted PGF2
R gene show a similar phenotype with lack of induction of labour (Sugimoto et al., 1997
). The PGF2
R gene is expressed in the uterus, during pregnancies at ~60% of the level in a non-pregnant uterus.
During delivery, endothelin 1 (ET-1) is known to have a uterotonic effect (Dizon-Townson and Ward, 1997). ET-1 is the strongest vasoconstricting agent known and is produced mainly in the vascular endothelium but is also found in several other tissues including the uterus and the placenta (Inoue et al., 1989a
; Arinami et al., 1991
).
Our working hypothesis initiating this study was to evaluate whether aberrations in any of the three genes SRD5A1, PGF2R and EDN-1 could completely or in part be the genetic component involved in dystocia. The basis for selecting these three genes was, as mentioned above, their clear involvement in parturition in mice (SRD5A1, PGF2
R) and strong ability to contract the human uterus (EDN-1). The design of the study was to perform mutational screening of these three genes in women with dystocia, some of whom have affected relatives.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
Successful amplification of the PCR products was checked on 1.5% agarose gels. The products were then subjected to cold SSCP after denaturation for 5 min at 96°C. The 20% polyacrylamide gels were run at 4°C and 24°C on a Novex Thermo FlowTM electrophoresis unit (Novex, San Diego, CA, USA) and the gels were silverstained (Bio-Rad Laboratories, Hercules, CA, USA)
DNA sequencing
Aberrant fragments were subject to a new round of PCR after purifying the fragment with WizardPrep (Promega), direct sequencing was performed according to standard procedures using cycle sequencing (Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit; Amersham Life Science, Solva, Sweden). The products were run on a 6% denaturing polyacrylamide gel. The gels were dried and exposed to films (Hyperfilm TM-MP; Amersham) for 1224 h at room temperature.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two polymorphisms in exon 2 of the SRD5A1 gene were detected, comparing with the normal sequence. Pro103Pro (CCGCCA) were found in homozygous form in 14 cases. Nine additional cases were heterozygous for this polymorphism and were also heterozygous for another polymorphism, Ala116Ala (GCA
GCG). This gave frequencies of 80 and 20% respectively for the two polymorphisms.
Two aberrant bands on SSCP were detected in the first part of exon 1 of the EDN-1 gene. Sequencing failed to show any DNA alterations in the exon and >60 intronic flanking bp. One polymorphism, in homozygous form, was detected in the first part of exon 5 in one case and in heterozygous form in four cases. This polymorphism was substituting Lys198 to Asn (AAGAAT). It was present in six out of 46 chromosomes analysed, giving a frequency of 15%. In the normal population (tested on 100 chromosomes) the corresponding frequency was 20%.
In the PGF2R gene a total of three different polymorphisms were found. In the non-coding part of exon 1 a polymorphism, in heterozygous form, was detected in one case (2%). This polymorphism was substituting 104C
T. In exon 2, a polymorphism, in homozygous form, was detected in two cases (9%), altering the last base of codon Thr 21 (ACC
ACT). In the non-coding part of exon 3, a polymorphism was detected, at position 1490, in four cases. It was present in heterozygous form in three cases and in homozygous form in one case (11%).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All of these three genes are expressed in the uterus, but their mutational frequency has not been well studied. Animal data have strongly suggested two of them, SRD5A1 and PGF2R, as candidates for dystocia (Mahendroo et al., 1996
; Sugimoto et al., 1997
). The third gene, EDN-1, has been shown to act directly on the myometrium, causing muscle contractions. Many studies have presented evidence of a genetic component in the risk of developing dystocia. A certain number of genes can therefore be expected to be involved. At the time of initiating this study the three genes analysed here represented good candidates for being involved in the development of dystocia.
There could be several possible explanations to the lack of mutations in this study. The material may be too small and not selected enough towards a familial aggregation of dystocia. The SSCP screening method probably detects only 7080% of mutations. The first part of the 5- reductase type 1 gene was not analysed due to failure in amplifying this segment. However, the results presented here strongly suggest that the genes analysed in this work do not commonly cause dystocia in humans. Finally, it is worth questioning whether animals represent a good model to find genes involved in disease in humans. The mechanism of parturition in man is highly complex and the problem of dystocia is probably multifactorial in its cause. It might also very well be that the mechanisms initiating the process of delivery are somewhat different in man and mice.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arinami, T., Ishikawa, M., Inoue, A. et al. (1991) Chromosomal assignments of the human endothelin family genes: the endothelin-1 gene (EDN1) to 6p23-p24, the endothelin-2 gene (EDN2) to 1p34, and the endothelin-3 gene (EDN3) to 20q13. 2-q13.3. Am. J. Hum. Genet., 48, 990996.[ISI][Medline]
Berg-Lekås, M.-L., Högberg, U. and Winkvist, A. (1998) Familial occurrence of dystocia. Am. J. Obstet. Gynecol., 179, 117121.[ISI][Medline]
Betz, R., Lagercrantz, J., Kedra, D. et al. (1999) Genomic structure, 5' flanking sequences and precise localization in 1p31.1 of the human prostaglandin F receptor gene. Biochem. Biophys. Res. Com., 254, 413416.[ISI][Medline]
Chelmow, D., Kilpatrick, S.J. and Laros, R.K. Jr. (1993) Maternal and neonatal outcomes after prolonged latent phase. Obstet. Gynecol., 81, 486491.[Abstract]
Dizon-Townson, D. and Ward, K. (1997) The genetics of labor. Clin. Obstet. Gynecol., 40, 479484.[ISI][Medline]
Inoue, A., Yanagisawa, N., Kimura, S. et al. (1989a) The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc. Natl. Acad. Sci. USA, 86, 28632867.[Abstract]
Inoue, A., Yanagisawa, M., Takuwa, Y. et al. (1989b) The human preproendothelin-1 gene, complete nucleotide sequence and regulation of expression. J. Biol. Chem., 264, 1495414959.
Jenkins, E.P., Hsieh, C.-L., Milatovich, A. et al. (1991) Characterization and chromosomal mapping of a human steroid 5 reductase gene and pseudogene and mapping of the mouse homologue. Genomics, 11, 11021112.[ISI][Medline]
Jenkins, E.P., Anderson, S., Imperato, J. et al. (1992) Genetic and pharmacological evidence for more than one human steroid 5 reductase. J. Clin. Invest., 89, 293300.[ISI][Medline]
Johnstone, F. and Inglis, L. (1974) Familial trends in low birth weight. Br. Med. J., 3, 659661.[ISI][Medline]
Macara, L.M. and Murphy K.W. (1994) The contribution of dystocia to the cesarean section rate. Am. J. Obstet. Gynecol., 171, 7177.[ISI][Medline]
Mahendroo, M.S., Cala, K.M. and Russell, D.W. (1996) 5 alpha-reduced androgens play a key role in murine parturition. Mol. Endocrinol., 10, 380392.[Abstract]
Saunders, N.S., Paterson, C.M. and Wadsworth, J. (1992) Neonatal and maternal morbidity in relation to the length of the second stage of labour. Br. J. Obstet. Gynecol., 99, 381385.[ISI][Medline]
Sugimoto, Y., Yamasaki, A., Segi, E. et al. (1997) Failure of parturition in mice lacking the prostaglandin F receptor. Science, 277, 681683.
Turcot, L., Marcoux, S. and Fraser, W.D. (1997) Multivariate analysis of risk factors for operative delivery in nulliparous women. Am. J. Obstet. Gynecol., 176, 395402.[ISI][Medline]
Varner, M.W., Fraser, A.M., Hunter, C.Y. et al. (1996) The intergenerational predisposition to operative delivery. Obstet. Gynecol., 87, 905911.
Ward, K., Hata, A., Jeuenemaitre, X. et al. (1993) A molecular variant of angiotensinogen associated with preeclampsia. Nature Genet., 4, 5961.[ISI][Medline]
Submitted on March 31, 1999; accepted on July 15, 1999.