ANTENATAL AND PRENATAL DIAGNOSIS

1979 Brock DJ, Hayward C. Methylumbelliferyl-guanidinobenzoate reactive proteases and prenatal diagnosis of cystic fibrosis. Lancet 1979; i: 1245-1246. [PubMed]
The titration of trypsin like proteases in cell free amniotic fluid against an artificial substrate, 4methylumbelliferyl-guanidinobezoate which had been proposed as a means of antenatal diagnosis by Nadler et al (Lancet 1980; ii: 96-97; Nadler HL, Walsh MMJ. Pediatrics 1980; 66:690-692). [PubMed] looked hopeful but could not be reproduced in other centres resulting in an unacceptable number of false negative and false positive results (Tummler B et al. Clin Chem Acta 1982; 125:219-232). [PubMed]

1983 Carbens NJB, Gosden G, Brock DJH. Microvillar peptidase activity in amniotic fluid: possible use in prenatal diagnosis of cystic fibrosis. Lancet 1983; i: 329-331.[PubMed]
The activities of two amniotic fluid peptidases were significantly depressed in the second trimester amniotic fluid supernatant in the presence of a fetus affected by cystic fibrosis. Eventually David Brock, of Edinburgh, used monoclonal antibody specific for isoenzyme of alkaline phosphatase (see below). Eventually both methods were superseded by superior DNA based methods.

1983 Brock DJH. Amniotic fluid alkaline phosphatase isoenzymes in early prenatal diagnosis of cystic fibrosis. Lancet 1983; ii: 941-943. [PubMed]
Antenatal diagnosis in families with a known CF child was possible by assay of the microvillus enzymes at 17-18th week of pregnancy. In pregnancies with a CF fetus there was a profound deficiency of one form of alkaline phosphatase (the phenylalanine-inhibitable form). When phenylalanine and homoarginine were used to define the alkaline phosphatase isoenzymes in stored amniotic fluid, 9 out of 10 cases of CF were identified (Brock DH et al, Hum Genet 1984; 65: 248-251; Brock DH et al, Hum Genet 1988; 78:271-275).
David Brock later (1993) commented that assay of microvillar enzymes in the second trimester amniotic fluid supernatant had a rational physiological basis and had stood the test of time but eventually it was superseded by superior DNA-based methods.

1985 Brock DJH, Befgood D, Barron L, Haward C. Prospective prenatal diagnosis of cystic fibrosis. Lancet 1985; I: 1175-1178.
An immunoassay based on monoclonal antibodies with specificity for the three major isoenzymes of alkaline phosphatase (ALP) was used in second-trimester prenatal diagnosis of cystic fibrosis. When prospective and retrospective data were summed the sensitivity of the test was 91% (39 of 43) and the false-positive rate 6% (5 of 81). The authors concluded that this was probably an acceptable form of prenatal diagnosis of CF for the high-risk mother at the time.
David Brock of Edinburgh pioneered antenatal diagnosis prior to the identification of the probes in close proximity to the CF gene in 1985. He had previously made major contributions to the antenatal recognition of spina bifida.

1986 Farrell M, Law HY, Rodeck CH, Warren R, Stanier P, Super M, Lissens W, Scambler P, Watson E, Wainwright B, et al. First-trimester prenatal diagnosis of cystic fibrosis with linked DNA probes. Lancet 1986; i: 1402-1405.
More accurate antenatal diagnosis using the new linked probes was described in 1985. Linkage analysis with cloned gene probes has shown that the mutation causing cystic fibrosis was located in the middle of the long arm of chromosome 7. In this paper first-trimester diagnosis of cystic fibrosis is reported in four informative families and second-trimester diagnosis in one family with fetal DNA prepared from chorionic villi, hybridised with the tightly linked DNA probes, pJ3.11 and met. Risk calculations show that the expected false-negative and false-positive rates are approximately 2% and 6%, respectively, for typical nuclear families with one affected living child.
The authors considered existing probes to be sufficiently informative now to allow full diagnosis in about two-thirds of couples presenting with at least one affected child. In half of the remainder, the inheritance of one parental mutant chromosome could be deduced.

1989 Handyside AH, Pattinson JK, Penketh RJ, Delhanty JD, Winston RM, Tuddenham EG. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1989; 1 (8634):347-349. [PubMed]
The first biopsy of a rabbit embryo had been performed in 1968 (Edwards RG & Gardner RL New Scientist 1968; 38:218-2). In this present paper Handyside and colleagues from the Hammersmith Hospital, London, reported the first unaffected child born following preimplantation genetic diagnosis for an X-linked disorder. A single cell was removed through a hole in the zona pellucida from each of 30 human embryos at the 6-10 cell stage three days after in vitro fertilisation. A normal proportion developed (37%) to the blastocyst stage and six hatched from the zona. Each male embryo was sexed from DNA amplification of a repeated sequence specific for the Y chromosomes. In 15 embryos with the normal two pronuclei the sex was also determined by in situ hybridisation.
Although at this stage not performed for CF, this technique of pre-implantation genetic diagnosis, was to prove a major advance for some CF carrier couples at high risk of having a CF infant, as it provided an alternative to prenatal diagnosis and termination if the fetus was affected – a course of action which was understandably unacceptable to many couples. The technique was first used for CF in 1992 (Handyside et al, 1992 below).

1992 Mennie ME, Gilfillan A, Compton M, Curtis L, Liston WA, Pullen I, Whyte DA, Brock DJH. Prenatal screening for cystic fibrosis. Lancet 1992; 340:214-216. [PubMed]
This is the first report of antenatal couple screening for CF in the Edinburgh maternity hospitals. Of 4348 women, 14% declined prenatal screening and 13% were not screened for other reasons. Amongst 3165 women there were 111 carriers detected of whom four had carrier partners and all 4 couples opted for prenatal diagnosis. One pregnancy with an affected fetus was terminated. The importance of adequate counselling was stressed.
Antenatal screening for CF became routine in Edinburgh but was eventually discontinued in 2005 for various reasons including the improving prognosis for CF and also the introduction of neonatal screening in Scotland (also Brock 1985 above; Livingstone et al, 1994 below). National antenatal CF carrier screening had not been introduced in the UK by 2011 although accepted in principle by the UK National Screening Committee

1994 Super M, Schwarz MJ, Malone G, Roberts T, Haworth A, Dermody G. Active cascade screening for carriers of cystic fibrosis gene. BMJ 1994; 308:1462-1467. [PubMed]
Dr Maurice Super (1936-2006) (figure 30) first encountered CF in Windhoek in South West Africa (Namibia) in 1967 where he started a CF clinic. He subsequently became a leading geneticist and paediatrician in the UK working in Manchester. He was a major protagonist of carrier screening in the extended families of people with cystic fibrosis – so-called “cascade screening”. The present paper describes 15 carrier couples detected out of 1563 relatives of people with CF who were tested; eight had prenatal tests and three pregnancies were terminated. An average of 16 people per family had been tested. Cascade screening was acceptable to relatives, particularly on the mother’s side of the family and 10 times more successful in detecting carrier couples than unfocused screening.
The genetic testing of all child-bearing relatives of a person with CF is now provided by the UK NHS if the individuals wish to be tested.

1994 Livingstone J, Axton RA, Gilfillan A, Mennie M, Compton M, Liston WA, Liston WA, Calder AA, Gordon AJ, Brock DJ. Antenatal screening for cystic fibrosis: a trial of the couple model. BMJ 1994; 308:1459-1462. [PubMed]
The second report of antenatal screening of 8536 couples in Edinburgh. 8.4% were “ineligible”, 1900 declined screening for various reasons and 5922 (69.4%) were screened. There were four positives (i.e. both partners were CF carrier heterozygotes) and all four couples opted for prenatal diagnosis. There were three terminations where the fetus was affected and one couple elected to have the CF infant. There was 99% satisfaction by those screened.

Antenatal CF screening was pioneered in Edinburgh by David Brock and his colleagues and this is one of the first reports (also Mennie et al, 1992 first report above). Screening was introduced into the two Edinburgh trial hospitals following this report. However, the service was eventually discontinued in 2005 soon after neonatal CF screening was introduced into Scotland. As the outlook for CF improved parental attitudes changed to antenatal diagnosis and termination, also the mutations tested differed from the neonatal ones, and finally funding for both antenatal and neonatal screening was inadequate. It has been estimated from various studies that for every CF fetus detected by antenatal screening the cost is between £50K and £100K.

1996 Brock DJH. Prenatal screening for cystic fibrosis: 5 years’ experience reviewed. Lancet 1996; 347:148-150. [PubMed]
Antenatal screening had been available at two maternity clinics in Edinburgh, UK, since January, 1992, first on a research basis and then routinely. 25,000 couples had been screened. The take-up rates for the two-step and couple models of delivery were very similar at about 70%. Of 22 high-risk couples identified entirely through screening, 20 (91%) opted for prenatal diagnosis. Four couples returned for second and two for third monitored pregnancies. In all eight cases where affected fetuses were identified, pregnancy was terminated.
David Brock concluded that “these data remove one of the few remaining obstacles to a general implementation of prenatal screening for CF”. However, although prenatal screening was recommended in the UK by a Health Technology Assessment (Murray et al, 1999) and after this was accepted in principle by the National Screening Committee, prenatal screening had not been introduced in the UK by 2011. Furthermore, antenatal CF screening was discontinued in the Edinburgh hospitals in 2005 on grounds of both cost and also the introduction of neonatal screening and the evidence of improving prognosis for infants with CF diagnosed soon after birth (also Mennie et al, 1992 above; Livingstone et al, 1994 above).

1998 Cunningham S, Marshall T. Influence of five years of antenatal screening on the paediatric cystic fibrosis population in one region. Arch Dis Child 1998; 78:345-348. [PubMed]
The incidence of CF in the five years before and after antenatal screening was introduced in Edinburgh decreased from 4.6 to 1.6 infants per year – a reduction greater than could be accounted for by prenatal diagnosis and termination.
Much of the early work on antenatal screening during the Eighties was done in Edinburgh by David Brock and his colleagues (Brock 1992; Livingstone et al. 1994; Brock, 1996 all above). It is disappointing that the antenatal screening which Brock pioneered was eventually abandoned in Edinburgh and has not been introduced elsewhere in the UK. The introduction of neonatal CF screening in Scotland and the steady improvement in prognosis, being two reasons given for withdrawal of the antenatal screening in Edinburgh. Financial reasons prevented introduction in England.

2003 Simon-Bouy B. Satre V. Ferec C. Malinge MC. Girodon E. Denamur E. Leporrier N. Lewin P. Forestier F. Muller F. French Collaborative Group. Hyperechogenic fetal bowel: a large French collaborative study of 682 cases. Am J Med Genet 2003; Part A. 121A:209-213. [PubMed] Hyperechogenic fetal bowel is detected in 0.1-1.8% of pregnancies during the second or third trimester. This 1997-1998 multicenter study in 22 molecular biology laboratories identified 682 cases of hyperechogenic fetal bowel detected by routine ultrasound examination during the second (86%) or third trimester. The fetal bowel was considered hyperechogenic when its echogenicity was broadly similar to, or greater than, that of the surrounding bone. Karyotyping, screening for viral infection, and screening for cystic fibrosis mutations were performed in all cases. Pregnancy outcome and postnatal follow-up were obtained in 656 of the 682 cases (91%). In 447 cases (65.5%), a normal birth was observed. Multiple malformations were observed in 47 cases (6.9%), a significant chromosomal anomaly was noted in 24 (3.5%), cystic fibrosis in 20 (3%), and viral infection in 19 (2.8%). In utero unexplained fetal death occurred in 1.9% of cases, toxemia in 1.2%, IUGR in 4.1%, and premature birth in 6.2%.

This study demonstrates that this ultrasound sign is potentially associated with medically significant outcomes. Having established that the bowel is hyperechogenic, recommended investigations should include a detailed scan with Doppler measurements, fetal karyotyping, cystic fibrosis screening, and infectious disease screening. After birth, newborns require pediatric examination because a surgical treatment may be necessary. This should be combined with clear counseling of the parents. This is a very clear and practically useful paper from a large multicentre French study which indicates the significance of hyperechogenic bowel during pregnancy.

2006 Saker A, Benachi A, Bonnefont JP, Munnich A, Dumez Y, Lacour B, Paterlini-Brechot P. Genetic characterisation of circulating fetal cells allows non-invasive prenatal diagnosis of cystic fibrosis. Prenatal Diagnosis 2006; 26:906-916. [PubMed]
The purpose of this study from Paris was to develop a molecular method to characterise both paternal and maternal CFTR alleles in DNA from circulating fetal cells (CFCs) isolated by ISET (isolation by size of epithelial tumour/trophoblastic cells). This protocol was validated in 12 pregnant women, at 11 to 13 weeks of gestation, whose offspring had a 1 in 4 risk of CF. Results showed that one fetus was affected, seven were heterozygous carriers of a CFTR mutation, and four were healthy homozygotes. These findings were consistent with those obtained by chorionic villus sampling (CVS). This test affords a reliable method prenatal diagnosis for high risk couples and avoids the risks associated iatrogenic miscarriage with chorionic biopsy (also note Fetal DNA detected at 13 weeks of a Q890X carrier fetus by Gonzalez-Gonzalez MC et al. Prenatal diagnosis 2002; 22:946-948. [PubMed]

2007 Massie J. Forbes R. Dusart D. Bankier A. Delatycki MB. Community-wide screening for cystic fibrosis carriers could replace newborn screening for the diagnosis of cystic fibrosis. J Paediatr Child H 2007; 43:721-723. [PubMed]
Most babies with cystic fibrosis (CF) are born to parents who did not know they were carriers until their baby was diagnosed with CF, usually by newborn screening. It is only after the birth of their first child with CF that couples are offered genetic counselling and reproductive choices. Most use this information for prenatal testing of subsequent pregnancies. With the high uptake of first trimester screening for Down syndrome (80% in Victoria, Australia) most couples have had screening during the CF affected pregnancy. Yet screening for CF carrier status is available, costs are similar to that for Down syndrome screening and CF carrier screening only ever needs to be done once. Waiting for couples to have a baby with CF before they are identified as carriers denies them choice. A national policy on CF carrier screening in Australia, and determination to equitably fund such a programme, is required.

Although neonatal screening for CF has been introduced into many countries. There is now the means of identifying CF carrier parents which has been available for the last two decades and the feasibility of antenatal CF screening has been shown by the first studies from Edinburgh in the UK where such screening was pioneered by the late David Brock (Mennie ME et al. Lancet 1992; 340:214-216). [PubMed] Also antenatal screening was recommended in a UK Health Technology Assessment in 1999 (Murray J, Cuckle H, Taylor G, Littlewood J, Hewison J. Screening for cystic fibrosis. Health Technol Assess 1999;3(8)).[PubMed] and even agreed by the UK National Screening Committee but not introduced because of the cost (£50K - £100K) for each affected fetus detected and the lack of the necessary counselling services. 2009 Massie J, Petrou V, Forbes R, Curnow L, Ioannou L, Dusart D, Bankier A, Delatycki M. Populations based carrier screening for cystic fibrosis in Victoria: the first three years experience. Aus NZ J Obstet Gynaecol 2009; 49:484-489.[PubMed]
CF carrier screening was offered to 3020 women and couples planning a pregnancy, or in early pregnancy, through obstetricians and general practitioners in Victoria, Australia from January 2006 to December 2008. Of the nine carrier couples, six were pregnant at the time of screening (five natural conception and one in vitro fertilisation) and all had CVS examination performed (mean gestation 12.5 weeks). Two fetuses were affected, three were carriers and one was not a carrier. Termination of pregnancy was undertaken for the affected fetuses. The authors concluded that carrier screening for CF by obstetricians and general practitioners by cheek swab sample can be successfully undertaken prior to pregnancy or in the early stages of pregnancy.

Carrier screening was pioneered by David Brock in Edinburgh but abandoned there apparently as a result of the introduction of neonatal screening becoming available and the prognosis improving. However, antenatal screening has been recommended by a UK Health Technology Assessment report, by the UK National Screening Committee but not implemented on the grounds of expense. Indeed it certainly seems to be an approach which should be available to future parents should they wish it. A downward trend in the incidence of CF has been noted in northeastern Italy where antenatal screening is available (Castellani et al, 2009.[PubMed] ).

2009 Christie LM. Ingrey AJ. Turner GM. Proos AL. Watts GE. Outcomes of a cystic fibrosis carrier testing clinic for couples. M J Australia 2009; 191:499-501. [PubMed]
To review the outcomes of offering carrier testing for cystic fibrosis (CF) to couples considering pregnancy, and to women in early pregnancy and their partners. An after-hours clinic was established in Newcastle for discussion of issues related to prenatal testing. Couples were offered CF carrier testing by extracting DNA from a mouthwash sample. An expanded one-step model was used with both partners being tested initially for the p.F508del cystic fibrosis transmembrane conductance regulator gene (CFTR) mutation. If one partner was a p.F508del carrier, the other partner was tested for an additional 28 CFTR mutations. Of 1000 individuals who were offered CF carrier testing, none declined. No re-collections of mouthwash samples were required, and results were available within 14 days. There were 730 individuals who had no family historyof CF(73%); 27 were carriers (4%; 95% CI, 2.4%-5.3%), and there were two high-risk couples where both partners were carriers of p.F508del. There were 270 individuals who had an affected family member with CF or a child identified as a CF carrier through newborn screening; 126 were carriers (46%; 95% CI, 40.6%-52.8%), and there were two high-risk couples - one couple where both partners were carriers of p.F508del, and another couple where the woman was homozygous for p.F508del and the man was a p.F508del carrier. The information on carrier status led the four high-risk couples to change their reproductive decisions to avoid having a child with CF.

The authors concluded that CF carrier testing for couples using an expanded one-step model will detect about 80% of high-risk couples and enables various reproductive choices. They believe that all couples considering pregnancy, and women in early pregnancy and their partners, should be offered CF carrier testing.