British Journal of Obstetrics and Gynaecology October 1992. Vol. 99. pp. 858-860
S H 0 RT C 0 M M U N I CAT10 N S
Determination of twin zygosity by DNA hybridisation to wild type bacteriophage M13 PHILLIP BENNETT DEBORAH HENDERSON P H I L I P STANIER JANET VAUGHAN GUDRUN MOORE Institute of Obstetrics and Gynaecology Queen Charlotte's and Chelsea Hospital Goldhawk Road, London W6 OXG, UK The incidence of twin pregnancy is about 1 % (Registrar General 1979) of which two thirds are dizygotic and one third are monozygotic (Bulmer 1970). Dizygotic twins always have dichorial placentation whilst monozygotic twins may be mono- and dichorial. Antenatal ultrasound can determine zygosity when the twins are of different sex, but it is impossible to identify monozygotic twins with certainty unless they are conjoined. Techniques which may be used to identify monochorial monozygotic twins include counting of the number of layers in the septum dividing the sacs and transfusion of maternal blood into one twin followed by sampling of the other twin for a Kleihauer test to detect communication between the two circulations. Postnatal examination of the placentas and membranes will identify those monozygotic twins who have monochorial placentation but zygosity cannot be determined for same sex twins with dichorial placentation. Analysis of DNA polymorphisms can separate monozygotic from dizygotic twins with certainty. This can be performed antenatally if fetal blood or chorion villi from separate placentas is available, or postnatally using cord or neonatal blood. Zygosity testing may be of value in the diagnosis of twin-to-twin transfusion syndrome, in exclusion of monozygosity when one twin has a lethal abnormality, or prior to selective fetocide where separate placentas cannot be seen. Zygosity testing is performed probably most frequently at parental request. Zygosity testing is usually performed using the hypervariable minisatellite DNA probes described by Jeffreys et ul. (1985). D N A from each twin is digested with a frequent cutter restriction enzyme, Southern blotted and hybridised to the Jeffreys probe. The autoradiograph produced will show a complex banding pattern. Any individual fetus will inherit approximately half of the bands from each parent. Dizygotic twins will share some but not all bands. Monozygotic twins will have an identical banding pattern. Jeffreys probes are only available under licence and their diagnostic use is restricted. However, DNA from bacteriophage M13 will, under certain conditions, detect a distinct set of hypervari-
858
able regions in human D N A (Vassart etal. 1987). Bacteriophage M13 is used as a vector for DNA sequencing, is available in virtually all molecular biology laboratories, and carries no restrictions upon its use. We have therefore assessed the use of bacteriophage M13 DNA as a tool to determine zygosity in twin pregnancy.
Methods D N A was obtained, using standard techniques, from cord blood from seven pairs of same-sex twins. D N A (4 pg) was digested with the restriction endonuclease Tuq I and Southern blotted onto Hybond-N nylon filters. Filters were baked and prehybridized for 2 h at 42°C in 0.9 M sodium chloride, 0.09 M sodium citrate, 5 mM EDTA (pH 8), 0.25% dried skimmed milk (MarvelTM). Wild type bacteriophage M13 DNA was radiolabelled using '*P-labelled dCTP by random hexanucleotide priming.
Fig. 1. DNA prepared from each of 7 same-sex twin pairs has been digested with the restriction enzyme Taq I, Southern blotted and hybridized to wild type bacteriophage M13 DNA. Analysis of this autoradiograph shows that twin pairs a, b, d, f and g are monozygotic, whilst twin pairs c and e are dizygotic.
SHORT C O M M U N I C A T I O N S
Hybridisation was performed overnight at 42°C in the same solution as for prehybridisation. Filters were washed in 1 x SSC with 1% SDS (SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) and autoradiography was undertaken for 2 days at -70°C with intensifying screens.
Results and discussion Bacteriophage M13 DNA detected in excess of 30 bands in each sample (Fig. 1). Unrelated DNA samples had different patterns. Of the seven pairs of twins, five had 100% band sharing, indicating monozygosity. Two pairs of twins had about 50% sharing of polymorphic bands, indicating dizygosity. The quality of the banding pattern was similar to that which we have seen previously when using the original Jeffreys probe. The segment in M13 DNA which detects the hypervariable region is competed for by fish DNA. Herring or salmon sperm DNA is usually included in hybridisation solutions to compete out non-specific DNA binding of the probe. In these experiments we have used dried skimmed
859
milk (MarvelTM)in place of fish DNA to act as a block to nonspecific DNA hybridisation. Bacteriophage M13 DNA can therefore be used to determine zygosity in same-sex twins. This technique will allow such tests to be performed rapidly in any biochemistry or microbiology laboratory using readily available reagents.
References Bulmer M. G. (1970) The Biology of Twinning in Man. Clarendon Press, Oxford. Jeffreys A. J., Wilson V. & Thein S. L. (1985) Hypervariable minisatellite regions in human DNA. Nature 31467. Registrar General (1979) Birth Statistics, Series 6, OPCS, HMSO London. Vassart G., Georges M., Monsieur R., Brocas H., Lequarre A. S. & Christophe D. (1987) A sequence in M13 phage detects hypervariable minisatellite in human and animal DNA. Science 235, 683.
Received 16 March I992 Accepted 16 April I992
British Journal of Obstetrics and Gynaecology October 1992, Vol. 99, pp. 859460
Measurement of amniotic fluid volume in early labour is a useful admission test T. G . T E O H Registrar
R . P. G L E E S O N Senior Registrar
M . R . N. D A R L I N G
gestation 237 completed weeks; singleton pregnancy; intact membranes; cephalic presentation; no significant vaginal bleeding. One hundred and twenty women were included in the study.
Master
The Rotunda Hospital, Dublin, Ireland
Methods
A portable real-time scanner (General Electric, RT50) was Ingemarsson et al. (1986) have proposed that a cardiotocograph record, made on admission to the labour ward as an ‘Admission Test’ may be useful for identifying fetuses at risk of developing distress. Despite the well-acknowledged role of antenatal measurement of amniotic fluid volume (AFV), there have been no reports of the use of AFV estimation in early labour as an ‘Admission Test’.
Subjects and methods Subjects
AFV estimation was performed during a three-month period on a random selection of women who presented to the labour ward with uterine contractions and met the following criteria:
Table 1. Labour outcome in relation to amniotic fluid pool depth.
Mode of delivery
Combined depth of two largest liquor pools (cm)
5
72
1
1
5
3
3
13
1
3 1 2
7
13
94
S H 0 RT C 0 M M U N I CAT10 N S
Determination of twin zygosity by DNA hybridisation to wild type bacteriophage M13 PHILLIP BENNETT DEBORAH HENDERSON P H I L I P STANIER JANET VAUGHAN GUDRUN MOORE Institute of Obstetrics and Gynaecology Queen Charlotte's and Chelsea Hospital Goldhawk Road, London W6 OXG, UK The incidence of twin pregnancy is about 1 % (Registrar General 1979) of which two thirds are dizygotic and one third are monozygotic (Bulmer 1970). Dizygotic twins always have dichorial placentation whilst monozygotic twins may be mono- and dichorial. Antenatal ultrasound can determine zygosity when the twins are of different sex, but it is impossible to identify monozygotic twins with certainty unless they are conjoined. Techniques which may be used to identify monochorial monozygotic twins include counting of the number of layers in the septum dividing the sacs and transfusion of maternal blood into one twin followed by sampling of the other twin for a Kleihauer test to detect communication between the two circulations. Postnatal examination of the placentas and membranes will identify those monozygotic twins who have monochorial placentation but zygosity cannot be determined for same sex twins with dichorial placentation. Analysis of DNA polymorphisms can separate monozygotic from dizygotic twins with certainty. This can be performed antenatally if fetal blood or chorion villi from separate placentas is available, or postnatally using cord or neonatal blood. Zygosity testing may be of value in the diagnosis of twin-to-twin transfusion syndrome, in exclusion of monozygosity when one twin has a lethal abnormality, or prior to selective fetocide where separate placentas cannot be seen. Zygosity testing is performed probably most frequently at parental request. Zygosity testing is usually performed using the hypervariable minisatellite DNA probes described by Jeffreys et ul. (1985). D N A from each twin is digested with a frequent cutter restriction enzyme, Southern blotted and hybridised to the Jeffreys probe. The autoradiograph produced will show a complex banding pattern. Any individual fetus will inherit approximately half of the bands from each parent. Dizygotic twins will share some but not all bands. Monozygotic twins will have an identical banding pattern. Jeffreys probes are only available under licence and their diagnostic use is restricted. However, DNA from bacteriophage M13 will, under certain conditions, detect a distinct set of hypervari-
858
able regions in human D N A (Vassart etal. 1987). Bacteriophage M13 is used as a vector for DNA sequencing, is available in virtually all molecular biology laboratories, and carries no restrictions upon its use. We have therefore assessed the use of bacteriophage M13 DNA as a tool to determine zygosity in twin pregnancy.
Methods D N A was obtained, using standard techniques, from cord blood from seven pairs of same-sex twins. D N A (4 pg) was digested with the restriction endonuclease Tuq I and Southern blotted onto Hybond-N nylon filters. Filters were baked and prehybridized for 2 h at 42°C in 0.9 M sodium chloride, 0.09 M sodium citrate, 5 mM EDTA (pH 8), 0.25% dried skimmed milk (MarvelTM). Wild type bacteriophage M13 DNA was radiolabelled using '*P-labelled dCTP by random hexanucleotide priming.
Fig. 1. DNA prepared from each of 7 same-sex twin pairs has been digested with the restriction enzyme Taq I, Southern blotted and hybridized to wild type bacteriophage M13 DNA. Analysis of this autoradiograph shows that twin pairs a, b, d, f and g are monozygotic, whilst twin pairs c and e are dizygotic.
SHORT C O M M U N I C A T I O N S
Hybridisation was performed overnight at 42°C in the same solution as for prehybridisation. Filters were washed in 1 x SSC with 1% SDS (SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) and autoradiography was undertaken for 2 days at -70°C with intensifying screens.
Results and discussion Bacteriophage M13 DNA detected in excess of 30 bands in each sample (Fig. 1). Unrelated DNA samples had different patterns. Of the seven pairs of twins, five had 100% band sharing, indicating monozygosity. Two pairs of twins had about 50% sharing of polymorphic bands, indicating dizygosity. The quality of the banding pattern was similar to that which we have seen previously when using the original Jeffreys probe. The segment in M13 DNA which detects the hypervariable region is competed for by fish DNA. Herring or salmon sperm DNA is usually included in hybridisation solutions to compete out non-specific DNA binding of the probe. In these experiments we have used dried skimmed
859
milk (MarvelTM)in place of fish DNA to act as a block to nonspecific DNA hybridisation. Bacteriophage M13 DNA can therefore be used to determine zygosity in same-sex twins. This technique will allow such tests to be performed rapidly in any biochemistry or microbiology laboratory using readily available reagents.
References Bulmer M. G. (1970) The Biology of Twinning in Man. Clarendon Press, Oxford. Jeffreys A. J., Wilson V. & Thein S. L. (1985) Hypervariable minisatellite regions in human DNA. Nature 31467. Registrar General (1979) Birth Statistics, Series 6, OPCS, HMSO London. Vassart G., Georges M., Monsieur R., Brocas H., Lequarre A. S. & Christophe D. (1987) A sequence in M13 phage detects hypervariable minisatellite in human and animal DNA. Science 235, 683.
Received 16 March I992 Accepted 16 April I992
British Journal of Obstetrics and Gynaecology October 1992, Vol. 99, pp. 859460
Measurement of amniotic fluid volume in early labour is a useful admission test T. G . T E O H Registrar
R . P. G L E E S O N Senior Registrar
M . R . N. D A R L I N G
gestation 237 completed weeks; singleton pregnancy; intact membranes; cephalic presentation; no significant vaginal bleeding. One hundred and twenty women were included in the study.
Master
The Rotunda Hospital, Dublin, Ireland
Methods
A portable real-time scanner (General Electric, RT50) was Ingemarsson et al. (1986) have proposed that a cardiotocograph record, made on admission to the labour ward as an ‘Admission Test’ may be useful for identifying fetuses at risk of developing distress. Despite the well-acknowledged role of antenatal measurement of amniotic fluid volume (AFV), there have been no reports of the use of AFV estimation in early labour as an ‘Admission Test’.
Subjects and methods Subjects
AFV estimation was performed during a three-month period on a random selection of women who presented to the labour ward with uterine contractions and met the following criteria:
Table 1. Labour outcome in relation to amniotic fluid pool depth.
Mode of delivery
Combined depth of two largest liquor pools (cm)
5
72
1
1
5
3
3
13
1
3 1 2
7
13
94
