No mutations found in candidate genes for dystocia

Michael Algovik1,2, Jacob Lagercrantz3, Magnus Westgren1 and Agneta Nordenskjöld3,4,5

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
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Dystocia is a disorder characterized by prolonged or dysfunctional labour. Delivery that starts late or not at all, leads to an increased risk for Caesarean section, infant morbidity and mortality. Familial aggregations of dystocia suggest a polygenic background. We have studied three candidate genes for dystocia, i.e. the genes for testosterone 5-{alpha} reductase type 1, prostaglandin F2{alpha} receptor and endothelin 1 and performed mutational screening in 23 women with dystocia, of which 12 have affected relatives. No mutations were found, making it unlikely that any of these genes represent a major cause of dystocia in man.

Key words: dystocia/endothelin 1 gene/mutational screening/prostaglandin F2{alpha}-receptor gene/testosterone 5 {alpha} reductase type 1 gene


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mechanisms determining initiation, progress and timing of delivery at term are, to a large extent, unknown. Dystocia represents a term that includes failure to progress in labour (prolonged or dysfunctional labour) as well as cephalopelvic disproportions. The incidence is 3–8% of all deliveries (Berg-Lekås et al., 1998Go). Dystocia has been shown to be a significant factor in causing operative deliveries (Macara and Murphy, 1994Go; Turcot et al., 1997Go). For the mother and child there is a significantly increased morbidity and mortality (Saunders et al., 1992Go; Chelmow et al., 1993Go).

In several other obstetric conditions a genetic influence has been identified, for example in pre-eclampsia (Ward et al., 1993Go) and low birth weight (Johnstone and Inglis, 1974Go). 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., 1996Go). 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., 1998Go). 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-{alpha} reductase type 1 (SRD5A1) and for the prostaglandin F2{alpha} receptor (PGF2{alpha}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., 1996Go). 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., 1991Go). Transgenic mice with a disrupted PGF2{alpha}R gene show a similar phenotype with lack of induction of labour (Sugimoto et al., 1997Go). The PGF2{alpha}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, 1997Go). 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., 1989aGo; Arinami et al., 1991Go).

Our working hypothesis initiating this study was to evaluate whether aberrations in any of the three genes SRD5A1, PGF2{alpha}R 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{alpha}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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cases
Charts for patients being delivered at Huddinge University Hospital and Västervik County Hospital between March 1, 1996 and September 30, 1997 were examined. Women who had completed 286 days of gestation and who were subject to a Caesarean section were invited to participate and 23 agreed. The majority of the patients (78%) were primiparous; the rest had undergone Caesarean section at the previous delivery. Seven of the women had undergone X-ray of the pelvis, all with normal findings. All women except one were >=155 cm and the mean height in the study group (166.5 cm) was slightly taller than the mean for all women admitted for delivery at Västervik Hospital over the period 1996–1998 (166.0 cm; n = 2413) (unpublished data). Furthermore, the mean weight of the children (3829 g) was equal to the mean weight of all children born at the same gestational age at Västervik Hospital during the same period (3856 g; n = 580). Clinical data on the 23 women who agreed to participate in the study are given in Table IGo. Among these, 12 had a familial history of dystocia (Table IIGo). In the group with a gestational time >=42 weeks five out of seven had one or more relatives with dystocia, whereas in the group with a gestation of <42 weeks, four out of 16 had a family history of dystocia.


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Table I. Clinical data on women (n = 23) taking part in mutational screening
 

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Table II. Familial associations for dystocia in the 23 subjects
 
Mutational screening with single-stranded conformation polymorphism (SSCP)
Constitutional DNA was prepared from peripheral blood according to standard protocols with phenol–chloroform extraction and ethanol precipitation. Polymerase chain reaction (PCR) products were generated by exon flanking primers according to published sequences of the SRD5A1 gene (Jenkins et al., 1992Go) and the EDN-1 gene (Inoue et al., 1989bGo) (Tables III and VGoGo). In order to analyse the PGF2{alpha} receptor gene (Abramovitch et al., 1994Go) we have characterized exon flanking intronic sequences (Betz et al., 1999Go) and exon flanking primers were then designed for the gene. Primers and PCR conditions are given in Table IVGo. Fragments with a deviant migration were sequenced. Although there was no formal control group of women with uneventful labour, for DNA sequencing the control material was normal DNA.


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Table III. Primers and polymerase chain reaction (PCR) conditions for the EDN-1 gene. DNA sequence from Inoue et al. (1989b)
 

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Table V. Primers and polymerase chain reaction (PCR) conditions for the SRD5A1 gene. DNA and primer sequences from Jenkins et al. (1992)
 

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Table IV. Primers and polymerase chain reaction (PCR) conditions for the PGF2{alpha}R gene. DNA sequences were from Abramovitch et al. (1994) and intron sequences from Betz et al. (1999)
 
PCR reactions were performed in 50 µl, containing 200 ng of template DNA, 0.2 mmol/l dNTPs, 10–50 pmol of each primer, 1.25 IU Taq DNA polymerase with the appropriate buffer and overlaid by 20 µl mineral oil. The thermal cycling was performed in a Perkin-Elmer Cetus Thermocycler (Perkin-Elmer, Stockholm, Sweden) (details in Table IIIGo).

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 12–24 h at room temperature.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
No clearly disease-causing mutations were detected in this selected group of 23 women (Caesarean section after >286 days gestation). A number of polymorphisms were found. The results are presented separately for each gene. DNA alterations and base pair position related to the sequences deposited in Genebank (Homo sapiens endothelin-1 accession J05008, Human steroid 5-alpha-reductase accession M32313 and M68882; Homo sapiens prostanoid FP receptor accession L24470).

Two polymorphisms in exon 2 of the SRD5A1 gene were detected, comparing with the normal sequence. Pro103Pro (CCG->CCA) 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 (AAG->AAT). 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 PGF2{alpha}R 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we present the results of our efforts to screen a selected group of 23 women, with a history of dystocia, for the presence of mutations in three candidate genes. The most likely reason for dystocia in these cases was dysfunctional uterine contractions or lack of ripening of the cervix since there was no major evidence of cephalopelvic disproportion.

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 PGF2{alpha}R, as candidates for dystocia (Mahendroo et al., 1996Go; Sugimoto et al., 1997Go). 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 70–80% of mutations. The first part of the 5-{alpha} 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
 
We wish to thank the women for participating in the study. We also thank Margareta Tapper-Persson and Desirée von Tell for excellent technical assistance. This study was supported by grants from the Swedish Medical Research Council, Allmänna BB:s minnesfond, Lars Hiertas Minne, Svenska Läkaresällskapet, Märta och Gunnar V.Philipssons Stiftelse and Gunvor and Josef Anérs Foundation.


    Notes
 
5 To whom correspondence should be addressed at: Department of Molecular Medicine, CMM 02, Karolinska Hospital, S-171 76 Stockholm, Sweden Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Abramovitch, M., Boie, Y., Nguyen, T. et al. (1994) Cloning and expression of a cDNA for the human prostanoid FP receptor. J. Biol. Chem., 269, 2632–2636.[Abstract/Free Full Text]

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, 990–996.[ISI][Medline]

Berg-Lekås, M.-L., Högberg, U. and Winkvist, A. (1998) Familial occurrence of dystocia. Am. J. Obstet. Gynecol., 179, 117–121.[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, 413–416.[ISI][Medline]

Chelmow, D., Kilpatrick, S.J. and Laros, R.K. Jr. (1993) Maternal and neonatal outcomes after prolonged latent phase. Obstet. Gynecol., 81, 486–491.[Abstract]

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Submitted on March 31, 1999; accepted on July 15, 1999.





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