620
A bolus of corticosteroids should be given before starting OKT3 treatment, to reduce the release of cytokines which could have coagulation-promoting activity.3 Before starting OKT3 treatment the graft perfusion should be evaluated either by doppler sonography or by nuclear scan. Department of Nephrology, Heinrich-Heine Universitat Dusseldorf, W4000 Dusseldorf,
Germany
PRENATAL EXPOSURE TO FOLIC ACID IN MALFORMED CHILDREN
MARKUS HOLLENBECK ANDREAS WESTHOFF DIETER BACH BERND GRABENSEE *Children with causal syndromes
Department of Vascular Surgery and Kidney Transplantation, Heinrich-Heine Universitat Dusseldorf
RALF KOLVENBACH HORST-WILHELM KNIEMEYER
and
were
excluded
very large population study with good clinical definition of would be needed to detect an association. This result should be confirmed, but nonetheless the observed negative (ie, protective) statistical association between hydranencephaly and prenatal folic acid supplementation also accords with Hook’s statement about the effectiveness of postconceptional folic acid supplementation. Since we investigated malformed children with no family history of NTD, our results also support the assumption that folic acid supplementation could prevent not only the recurrence of NTD, but also the first occurrence of this defect in a family. However, if we take into account that the prenatal mortality rates of conceptuses with NTD is about 98%,3 and that most occurred during the early stage of gestation including the prerecognition period, then there may have been a previously affected conceptus in the family that no one would have known about. a
cases
1. Hollenbeck M, Stuhrmann M, Trapp R, Grabensee B. Colour-coded doppler ultrasonography for early detection of rejection after allogenic renal transplantation. Dtsch Med Wochenschr 1991; 116: 921-27. 2. Warshauer DM, Taylor KJW, Bia MJ, et al. Unusual causes of increased vascular impedance in renal transplants: Duplex doppler evaluation. Radiology 1988; 169: 367-70. 3. Goldmann M, Abramowicz D, Depauw L, et al. OKT3-induced cytokine release attenuation by high-dose methylprednisolone. Lancet 1989; ii: 802-03.
Folic acid
supplementation
and neural tube
defects SIR,-We would like to add our experience to that of Professor (April 18, p 1000). The Spanish Collaborative Study of Congenital Malformations (ECEMC) is a hospital-based casecontrol study and surveillance system. Malformed infants are ascertained in each of the participating hospitals by examination of all livebom babies by a physician within the first three days of life. For each malformed baby, the next non-malformed infant of the same sex born in the same hospital was the control. Mothers of cases and controls were interviewed about prenatal, obstetric, and family histories, and exposures during pregnancy.l,2 From April, 1976, to September, 1990, the ECEMC surveyed 830 882 livebom infants. Of those, 16 736 were malformed and 16 574 were selected as
Hook
controls. In a continuing case-control study on multivitamins and folic acid supplementation at any time during the first trimester of pregnancy, we identified a protective effect for neural tube defects (NTD) after post conceptional folic acid (with or without other vitamins) supplementation. Cases were children with NTD and controls were those with defects other than NTD, none of whom had a previously affected individual with NDT in the family (table). The overall odds ratio (OR) was 0-69 (95 % CI 0-51-0-94; p = 0-01). 22 of 54 mothers of infants with NTD, and 1002 of 2163 mothers of children with other defects, used daily doses of folic acid 03 mg or over (OR 0-61 [0-38-ü.96]; p 0-02). The OR for those exposed to less than 0-3 mg folic acid daily was 0 76 (p = 0-2). To control for possible confounders such as voluntary interruption of pregnancy (VIP) which was legalised in Spain in December, 1985, we examined the period before VIP and that afterwards; the respective ORs were 0-66 (p=0’04) and 0.87 (p = 0-6). The OR for the second period could be biased by VIP and by raised awareness of the preventive effect of folic acid. Hook discusses neural tube closure and post-closure rupture of the neural tube in man, and he states that one should not assume that preventive strategies such as maternal folic acid supplementation will only be effective in the periconceptional period. Our results seem to support his point since our data on exposure to folic acid were postconceptional. However, Shiota3 showed that there is wide variation in development of human embryos at any gestational age, which he judged was at least in part normal biological variability that should be taken into account when estimating the teratogenic risk of environmental agents. We therefore cannot totally exclude the possibility that postconceptional folic acid supplementation could also be effective in the prevention of defects of neural tube closure. We also noted that among 15 cases with isolated hydranencephaly, none of the mothers had folic acid supplementation, whereas 28-6% (4 of 14) of controls were exposed at any time during the first trimester (p 0-04). This observation has not been previously reported, perhaps because this defect is rare =
=
ECEMC, Hospital Universitario San Carlos, Faculty of Medicine, Universidad Complutense, 28040 Madrid, Spain
MARÍA-LUÍSA MARTÍNEZ-FRÍAS ELVIRA RODRÍGUEZ-PINILLA
1. Martinez-Frías ML, Salvador J. Epidemiological aspects of prenatal exposure to high doses of vitamin A in Spain. Eur J Epidemiol 1990; 6: 118-23. 2. Martinez-Frías ML. Valproic acid and spina bifida. Lancet 1991; 338: 196-97. 3. Shiota K. Development and intrauterine fate of normal and abnormal human
conceptuses.
Cong Anom 1991; 31: 67-80.
Semi-quantitative detection of syndrome with PCR
Down’s
SiR,—Fetal cells can be retrieved from maternal blood samples.’ Although this material cannot be cultured, it can be analysed by fluorescence in-situ hybridisation (FISH) with gene-specific probes.2 This approach could achieve higher rates of detection than existing methods but it is time-consuming and unsuitable for mass screening. We have pioneered a rapid, semi-quantitative, gene amplification technique. Blood samples were taken from 4 patients with Down’s syndrome and 4 controls, and DNA was isolated by standard methods. The investigators were unaware of the identity of the samples. Two sets of primers3,4 flanking a short 337 bp region of the amyloid precursor protein gene (AP) on chromosome 21 and a 330 bp region linked to the cystic fibrosis gene (CS-7) on chromosome 7 were synthesised, and the two sequences were simultaneously amplified by standard methods except for the addition of [35S]-dATP to the reaction. All reactions were in triplicate and products were resolved on thin 4% polyacrylamide gels. Product bands were visualised by silver staining, excised from the dried gels, and counted by liquid scintillation.
Expressing the results as a ratio of the counts from AP and CS-7
products allowed us to discriminate accurately between the Down’s and control samples (figure). Mean ratios were 1-69 and 1-14, respectively (p < 0-05, U test), and close to the expected values of 15 and 1 ’0. Because inter-replicate variability, especially in the Down’s group, was considerable the mean of the replicates would in practice be used. These ranged from 0-99 to 1-23 (controls) and from 1-48 to 1’84 (Down’s syndrome), respectively. The assay’s speed (one day, including DNA isolation and amplification) makes it well suited to rapid processing. The AP locus is close to the translocation site on chromosome 21 and may therefore be unreliable for detecting Down’s syndrome caused by unbalanced translocations, which could be overcome by amplifying telomeric gene sequences. The method is, however,
A bolus of corticosteroids should be given before starting OKT3 treatment, to reduce the release of cytokines which could have coagulation-promoting activity.3 Before starting OKT3 treatment the graft perfusion should be evaluated either by doppler sonography or by nuclear scan. Department of Nephrology, Heinrich-Heine Universitat Dusseldorf, W4000 Dusseldorf,
Germany
PRENATAL EXPOSURE TO FOLIC ACID IN MALFORMED CHILDREN
MARKUS HOLLENBECK ANDREAS WESTHOFF DIETER BACH BERND GRABENSEE *Children with causal syndromes
Department of Vascular Surgery and Kidney Transplantation, Heinrich-Heine Universitat Dusseldorf
RALF KOLVENBACH HORST-WILHELM KNIEMEYER
and
were
excluded
very large population study with good clinical definition of would be needed to detect an association. This result should be confirmed, but nonetheless the observed negative (ie, protective) statistical association between hydranencephaly and prenatal folic acid supplementation also accords with Hook’s statement about the effectiveness of postconceptional folic acid supplementation. Since we investigated malformed children with no family history of NTD, our results also support the assumption that folic acid supplementation could prevent not only the recurrence of NTD, but also the first occurrence of this defect in a family. However, if we take into account that the prenatal mortality rates of conceptuses with NTD is about 98%,3 and that most occurred during the early stage of gestation including the prerecognition period, then there may have been a previously affected conceptus in the family that no one would have known about. a
cases
1. Hollenbeck M, Stuhrmann M, Trapp R, Grabensee B. Colour-coded doppler ultrasonography for early detection of rejection after allogenic renal transplantation. Dtsch Med Wochenschr 1991; 116: 921-27. 2. Warshauer DM, Taylor KJW, Bia MJ, et al. Unusual causes of increased vascular impedance in renal transplants: Duplex doppler evaluation. Radiology 1988; 169: 367-70. 3. Goldmann M, Abramowicz D, Depauw L, et al. OKT3-induced cytokine release attenuation by high-dose methylprednisolone. Lancet 1989; ii: 802-03.
Folic acid
supplementation
and neural tube
defects SIR,-We would like to add our experience to that of Professor (April 18, p 1000). The Spanish Collaborative Study of Congenital Malformations (ECEMC) is a hospital-based casecontrol study and surveillance system. Malformed infants are ascertained in each of the participating hospitals by examination of all livebom babies by a physician within the first three days of life. For each malformed baby, the next non-malformed infant of the same sex born in the same hospital was the control. Mothers of cases and controls were interviewed about prenatal, obstetric, and family histories, and exposures during pregnancy.l,2 From April, 1976, to September, 1990, the ECEMC surveyed 830 882 livebom infants. Of those, 16 736 were malformed and 16 574 were selected as
Hook
controls. In a continuing case-control study on multivitamins and folic acid supplementation at any time during the first trimester of pregnancy, we identified a protective effect for neural tube defects (NTD) after post conceptional folic acid (with or without other vitamins) supplementation. Cases were children with NTD and controls were those with defects other than NTD, none of whom had a previously affected individual with NDT in the family (table). The overall odds ratio (OR) was 0-69 (95 % CI 0-51-0-94; p = 0-01). 22 of 54 mothers of infants with NTD, and 1002 of 2163 mothers of children with other defects, used daily doses of folic acid 03 mg or over (OR 0-61 [0-38-ü.96]; p 0-02). The OR for those exposed to less than 0-3 mg folic acid daily was 0 76 (p = 0-2). To control for possible confounders such as voluntary interruption of pregnancy (VIP) which was legalised in Spain in December, 1985, we examined the period before VIP and that afterwards; the respective ORs were 0-66 (p=0’04) and 0.87 (p = 0-6). The OR for the second period could be biased by VIP and by raised awareness of the preventive effect of folic acid. Hook discusses neural tube closure and post-closure rupture of the neural tube in man, and he states that one should not assume that preventive strategies such as maternal folic acid supplementation will only be effective in the periconceptional period. Our results seem to support his point since our data on exposure to folic acid were postconceptional. However, Shiota3 showed that there is wide variation in development of human embryos at any gestational age, which he judged was at least in part normal biological variability that should be taken into account when estimating the teratogenic risk of environmental agents. We therefore cannot totally exclude the possibility that postconceptional folic acid supplementation could also be effective in the prevention of defects of neural tube closure. We also noted that among 15 cases with isolated hydranencephaly, none of the mothers had folic acid supplementation, whereas 28-6% (4 of 14) of controls were exposed at any time during the first trimester (p 0-04). This observation has not been previously reported, perhaps because this defect is rare =
=
ECEMC, Hospital Universitario San Carlos, Faculty of Medicine, Universidad Complutense, 28040 Madrid, Spain
MARÍA-LUÍSA MARTÍNEZ-FRÍAS ELVIRA RODRÍGUEZ-PINILLA
1. Martinez-Frías ML, Salvador J. Epidemiological aspects of prenatal exposure to high doses of vitamin A in Spain. Eur J Epidemiol 1990; 6: 118-23. 2. Martinez-Frías ML. Valproic acid and spina bifida. Lancet 1991; 338: 196-97. 3. Shiota K. Development and intrauterine fate of normal and abnormal human
conceptuses.
Cong Anom 1991; 31: 67-80.
Semi-quantitative detection of syndrome with PCR
Down’s
SiR,—Fetal cells can be retrieved from maternal blood samples.’ Although this material cannot be cultured, it can be analysed by fluorescence in-situ hybridisation (FISH) with gene-specific probes.2 This approach could achieve higher rates of detection than existing methods but it is time-consuming and unsuitable for mass screening. We have pioneered a rapid, semi-quantitative, gene amplification technique. Blood samples were taken from 4 patients with Down’s syndrome and 4 controls, and DNA was isolated by standard methods. The investigators were unaware of the identity of the samples. Two sets of primers3,4 flanking a short 337 bp region of the amyloid precursor protein gene (AP) on chromosome 21 and a 330 bp region linked to the cystic fibrosis gene (CS-7) on chromosome 7 were synthesised, and the two sequences were simultaneously amplified by standard methods except for the addition of [35S]-dATP to the reaction. All reactions were in triplicate and products were resolved on thin 4% polyacrylamide gels. Product bands were visualised by silver staining, excised from the dried gels, and counted by liquid scintillation.
Expressing the results as a ratio of the counts from AP and CS-7
products allowed us to discriminate accurately between the Down’s and control samples (figure). Mean ratios were 1-69 and 1-14, respectively (p < 0-05, U test), and close to the expected values of 15 and 1 ’0. Because inter-replicate variability, especially in the Down’s group, was considerable the mean of the replicates would in practice be used. These ranged from 0-99 to 1-23 (controls) and from 1-48 to 1’84 (Down’s syndrome), respectively. The assay’s speed (one day, including DNA isolation and amplification) makes it well suited to rapid processing. The AP locus is close to the translocation site on chromosome 21 and may therefore be unreliable for detecting Down’s syndrome caused by unbalanced translocations, which could be overcome by amplifying telomeric gene sequences. The method is, however,
