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PRENATAL DIAGNOSIS OF HAEMOPHILIA A BY FACTOR VIII GENE ANALYSIS
Saturday 22 June
PRENATAL DIAGNOSIS OF HAEMOPHILIA A BY FACTOR VIII GENE ANALYSIS
STYLIANOS E. ANTONARAKIS ROBERT J. CARPENTER, JR LEON W. HOYER JOHN J. TOOLE
KAREN L. COPELAND CARL A. CARTA C. THOMAS CASKEY HAIG H. KAZAZIAN, JR
Genetics Unit, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, Robert J. Kleberg Center for Human Genetics, Baylor College of Medicine, Houston, Texas; Department of Medicine, University of Connecticut School of Medicine, Farmington, Connecticut; and Genetics Institute Inc, Boston, Massachusetts, USA
1985
This method involves recombinant DNA technology, amniocentesis or chorionic villus sampling, and study of the entire family to establish the linkage phase between the DNA marker and the disease locus.9 The major drawback of this approach is that the accuracy of the diagnosis depends upon the recombination distance between the marker and the locus of interest. Here we describe the prenatal diagnosis of a case of haemophilia A by analysis of DNA polymorphic sites within the factor VIII gene.10 Cloned FVIII complementary DNA (cDNA) sequences were used as probes to identify and mark the defective FVIII gene. Materials and Methods
Cloned factor VIII
deoxyribose nucleic acid (DNA) sequences were used as probes in the prenatal diagnosis of haemophilia A. Fetal DNA from
Subjects
cultured amniotic fluid cells was examined for a DNA polymorphism within the factor VIII gene which marked the haemophilia A gene in the pregnant obligate carrier. The fetus was predicted to be an affected male, and the diagnosis of haemophilia A was confirmed both in utero and after termination of the pregnancy.
She is an obligate carrier of an abnormal factor VIII gene because her two sisters have haemophilic sons (fig 1).
Summary
Introduction HAEMOPHILIA A due to deficiency of coagulation factor VIII (FVIII:C) is the commonest inherited disorder of blood coagulation.l It affects approximately 1 in every 10 000 males. Although substitution therapy is reasonably satisfactory, the disease puts a considerable strain on families. Therefore, many at-risk families seek prenatal diagnosis for haemophilia A. Until recently prenatal diagnosis depended upon the measurement of the X-linked FVIII coagulant antigen (FVIII:CAg) and the autosomal FVIII related antigen
(FVIIIR:Ag)
in fetal
plasma2-5
obtainable
between the 18th and 21st weeks
only by fetoscopy
ofgestation.6
Another approach is by the study of DNA polymorphic markers genetically linked to the haemophilia locus.’,8
A 30-year-old Saudi Arabian woman married to her first cousin presented at 12 weeks of gestation in her third pregnancy. She had previously borne a son with haemophilia A and a normal daughter.
Restriction Endonuclease Analysis Nuclear DNA was isolated from individuals 111-1, III-2, IV-1, IV-2, from leucocytes of 10-15 ml of edetic acid anticoagulated
as described previously.ll Fetal DNA was obtained from two 25 ml flasks of cultured amniocytes. These cultures were established after amniocentesis at week 16 of pregnancy. Cells were harvested and DNA was isolated as described before. 12 About 60 pig of DNA was obtained from the cells in the two flasks, sufficient for several DNA analyses by restriction endonuclease digestion. 5-10 J.lg of DNA was digested with one of various endonucleases under conditions recommended by the suppliers. The methods of electrophoresis, transfer of DNA fragments to nitrocellulose filters, hybridisation of genomic fragments with radioactive probes, washing of filters, and autoradiography have been described.13,14 The following cloned FVIII DNA sequences were used as probes1o,1s,16: (i) probe A, a 1. 7 kb Kpn I cDNA fragment which spans exons 1-12, (ii) probe B, a
peripheral blood
8443
1408
Arrow shows fetus at risk of haemophilia A (individual IV-3). Presence or absence of the polymorphic Bcl site is depicted as + or - for the individuals examined.
4 7 kb Eco RI cDNA fragment which contains exons 14-25 and part of exon 26, and (iii) probe C, a 1 - 8 kb Eco RI cDNA which contains the remainder of exon 26 and the 3’ untranslated region of the factor VIII gene. All of these probes were radiolabelled with
32[P]dCTP (deoxycytidine triphosphate) by function of Escherichia coli DNA polymerase karyotyped according to standard protocols. 18
the nick translation 1.17 Fetal cells were
Haematological Investigations FVIIIC:Ag and factor VIII related antigen VIIIR:Ag) levels in plasma obtained from cord blood in uteroand abortus blood were measured by fluid-phase immunoradiometric assays.20,21 fetal
Results DNA from the pregnant woman (III-2), her husband (III-1), and her two offspring, IV-1and IV-2, were analysed for the presence of any gross abnormality (deletion) in the factor VIII gene. None of the molecular defects directly detectable by restriction endonuclease analysis was identified in this Saudi Arabian family. Examination of the DNA from 111-2 for heterozygosity at DNA polymorphic sites within the factor VIII gene (fig 2) showed that she was heterozygous for the presence ofa previously described BclI site in exon 18 of the FVIII gene. 10,22 Since this woman was an obligate carrier of a haemophilia A gene and her affected son (IV-2) demonstrated the presence of the Bd I polymorphic site in his defective FVIII gene, we concluded, by the use of simple linkage analysis, that the presence of the BclI site marked the maternal X chromosome which carries the haemophilia gene. In the course ofour initial evaluation of the family we found, using the same DNA polymorphism analysis, that III-2’s daughter (IV-1) also carries the
haemophilia gene.
Fig 2-Autoradiogram patterns of DNA from members of family after digestion with BclI and hybridisation with FVIII-B probe. Note the inheritance of the presence (+) or absence (-) of the Bcl I polymorphic site in different family members. Male fetus IV-3, like his affected brother IV-2, has inherited the therefore, affected with haemophilia A.
allele from his mother and is,
+
had inherited the haemophilia A gene from its mother and, therefore, was affected with haemophilia A. This suggestion was confirmed by immunoradiometric assay for FVIII:CAg and FVIIIR:Ag in fetal blood obtained by percutaneous cordblood sampling under direct visualisation of the needle by ultrasonography at week 21 of pregnancy (see accompanying table). The family elected to have the affected fetus aborted and the diagnosis of haemophilia A was verified by measuring FVIII:CAg and FVIIIR:Ag in the blood of the abortus. Discussion
prenatal diagnosis of haemophilia A based on immunoradiometric assay of FVIII:CAg and FVIIIR:Ag in fetal blood obtained by fetoscopy is made at weeks 18-21 of pregnancy. Between 1978 and 1983 only 92 pregnancies were The
evaluated in the United States, out of an estimated 500 pregnancies at risk for haemophilia A each year. The risk of spontaneous abortion after fetoscopy for prenatal diagnosis of haemophilia A was about 6% (3/52) in normal fetuses.4 In addition, it was not possible to obtain a satisfactory sample in 12 of the 92 pregnancies. Because of these factors fetoscopy has not been used much for the prenatal diagnosis of haemophilia A. Recombinant DNA technology,23 linkage analysis by the use of DNA polymorphism,24 and the cloning of the factor VIII gene’ 5,16 should make the prenatal diagnosis of IMMUNORADIOMETRIC ASSAYS OF FVIII
done in week 16 of pregnancy. Amniocentesis of the cultured amniotic fluid cells Cytogenetic analysis showed that the fetus had a normal male karyotype. When DNA was extracted from the cultured amniotic fluid cells and the Bcl I polymorphic site within the FVIII gene was examined, we found that the male fetus had inherited the FVIII gene which was marked by the presence of the BclI polymorphic site. This finding suggested that the male fetus was
I
*Data from reference 4, measurements made Values in parentheses refer to range.
I
on
blood obtained
b- fetcscor’._:
1409
haemophilia A safer. Highly informative DNA polymorphisms have been associated with two cloned random DNA Sequences which have been mapped very close to the haemophilia A locus on the long arm of chromosome X. The cloned
COXSACKIE B, MUMPS, RUBELLA, AND CYTOMEGALOVIRUS SPECIFIC IgM RESPONSES IN PATIENTS WITH JUVENILE-ONSET INSULIN-DEPENDENT DIABETES MELLITUS IN BRITAIN, AUSTRIA, AND AUSTRALIA
DNA sequence DX13 shows no recombination
with the haemophilia A locus;the Bgl II site associated with this sequence (DX13) has been used for carrier detection of haemophilia A in a pregnant woman.9 In addition, the cloned DNA sequence Stl4 shows no recombination with the A locus and the Taq I and Msp I polymorphic sites associated with it can be used for prenatal diagnosis of this disease.8 Although prenatal diagnosis by the use of anonymous DNA fragments linked to the haemophilia locus does not incur the hazards associated with fetoscopy (the fetal cells being obtained by amniocentesis25 or chorionic villus sampling 26), there is a low risk of both false-positive and falsenegative predictions because of possible recombination between the DNA polymorphic site of interest and the FVIII gene. (The 90% confidence limits for the recombination fraction between DX13 and the locus for haemophilia A are 0-9 centimorgans,’ while those between Stl4 and the haemophilia A locus are 0-5 centimorgans.8) We have used DNA polymorphic sites within the factor VIII gene and, therefore, the error rate due to recombination between the marker DNA polymorphic site and the mutant FVIII gene should be negligible. Another advantage of methods based on DNA analysis is that carrier females can also be diagnosed prenatally. The disadvantage is that family studies are necessary in order to link the DNA polymorphic marker with the defective FVIII gene in a family,24 and Hoyer et al have reported that for many women (25 of 80) who request prenatal diagnosis for haemophilia A a male with haemophilia A is not available for the test.4 Moreover, some women present so late in pregnancy that diagnosis by DNA polymorphisms is not practical before the fetus is at 21 weeks of gestation, and fetoscopy becomes necessary for a rapid diagnosis. We think, however, that the study of the FVIII gene defects or DNA polymorphisms within this gene after’ amniocentesis or chorionic villus sampling will become the method of choice for prenatal diagnosis of haemophilia A. If adequate information cannot be obtained by the use of FVIII gene sequences, DNA polymorphisms associated with DNA sequences Stl4 and/or DX13 may be useful.
J. E. BANATVALA JENNIFER BRYANT G. SCHERNTHANER M. BORKENSTEIN E. SCHOBER D. BROWN L. M. DE SILVA M. A. MENSER M. SILINK
haemophilia
We thank Mrs Emily Pasterfield for secretarial help; Dr David Ledbetter and Dr Gail Stetten for culturing the amniotic fluid cells; and Ms Margaret Norman for technical help. The study was supported by grants from the National Institutes of Health to S. E. A., H. H. K., and L. W. H. C. T. C. is a Howard Hughes investigator.
Correspondence should be addressed to S. E. A., Department of Pediatrics, CMSC 1004, Johns Hopkins Hospital, 600 N Wolfe Street, Baltimore, Maryland 21205, USA. REFERENCES 1. McKee PA
Hemostasis and disorders of blood coagulation. In: Stanbury JB, Friedrickson DS, Goldstein JL, Brown MS, eds. The metabolic basis of inherited diseases, 5th ed. New York. McGraw-Hill, 1983: 1531-60. Firshein SI, Hoyer LW, Lazarchick J, et al. Prenatal diagnosis of classic hemophilia. N Engl J Med 1979; 300: 937-41. Mibashan RS, Rodeck CH, Furlong RA, et al. Dual diagnosis of prenatal hemophilia A by measurement of fetal factor VIIIC and VIIIC antigen(VIIICAg) Lancet 1980; ii. 994-97. Hoyer LW, Carta CA, Golbus MS, Hobbins JC, Mahoney MJ. Prenatal diagnosis of classic hemophilia (hemophilia A) by immunoradiometric assays. Blood (in press). Mibashan RS, Thumpston JK, Singer JD, et al. Plasma assay of fetal factors VIIIC and IX for prenatal diagnosis of hemophilia. Lancet 1979; i: 1309-11. Hobbins JC, Mahoney MJ. In utero diagnosis of hemoglobinopathies; technique for obtaining fetal blood. N Engl J Med 1974; 290: 1065-67. Harper K, Winter RM, Pembrey ME, Hartley D, Davies KE, Tuddenham EGD. A clinically useful DNA probe closely linked to hemophilia A. Lancet 1984; ii: 6-8.
Wyngaarden JB,
2. 3.
4. 5
6. 7
Department of Virology, St Thomas’ Hospital, London; Department of Medicine II, University of Vienna, Austria; Department of Paediatrics, University of Graz, Austria; Department of Paediatrics, University of Vienna, Austria; Virus Reference Laboratory, Central Public Health Laboratory, Colindale, London; and Children’s
Hospital, Camperdown, Sydney, Australia
Patients from England, Austria, and Australia with recently diagnosed juvenileonset insulin-dependent diabetes (type 1) mellitus (IDDM) and matched controls were tested for specific IgM responses to Coxsackie B1-5 viruses. 37 of 122 (30%) patients aged <15, but only 15 of 204 (6%) controls, were positive (p<0·005). Differences in Coxsackie B virus specific IgM responses between patients and controls were statistically significant for patients in England and Austria (p<0·005). Coxsackie B virus specific IgM responses were detected in only 3 of 31 patients aged >16. Virus-specific IgM responses were directed against a single serotype, usually Coxsackie B4
Summary
I, Camerino G, Heilig R, et al. Genetic screening for hemophilia A with a polymorphic DNA probe N Engl J Med 1985; 312: 682-86. Tonnessen T, Sondergaard F, Mikkelsen M, et al. X chromosome specific probe DX13 for carrier detection and first trimester prenatal diagnosis of hemophilia A. Lancet
8. Oberle 9.
1984; ii:
1269-70.
SE, Waber PG, Kittar SD, et al. Hemophilia A. Molecular defects and detection by DNA analysis. N Engl J Med (in press). 11. Kunkel LM, Smith KD, Boyer SH, et al. Analysis of human Y chromosome specific reiterated DNA in chromosome variants. Proc Natl Acad Sci USA 1977, 74: 1245-49 12. Kan YW, Dozy AM. Antenatal diagnosis of sickle cell anemia by DNA analysis of amniotic fluid cells. Lancet 1978; ii: 910-12 13. Southern EM. Gel electrophoresis of restriction fragments. Meth Enzymol 1979; 68: 10 Antonarakis carrier
152-76. 14. Scott AF, Phillips
JA, Migeon BR DNA restriction endonuclease analysis for localization of human 6 and &bgr; globin genes on chromosome 11. Proc Natl Acad Sci USA 1979; 76: 4563-65 15. Gitschier J, Wood WI, Goralka TM, et al. Characterization of the human factor VIII gene. Nature 1984; 312: 326-30. 16 Toole JJ, Knopf JL, Wozney JM, et al Molecular cloning of a cDNA encoding human antihemophilic factor. Nature 1984; 312: 342-47 17. Maniatis T, Kee GS, Efstradiatis A, Kafatos FC. Amplification and characterization of a &bgr;-globin gene synthesized in vitro. Cell 1976; 8: 163-92. 18. Schwarzacher HC, Wolf U, eds Methods in human cytogenetics. Berlin: SpringerVerlag, 1974 19. Daffos R, Capella-Pavlosky I, Forestier F. Fetal blood sampling via the umbilical cord using a needle guided by ultrasound; report of 60 cases. Prenatal Diag 1983; 3: 271-77. 20. Lazarchick J, Hoyer LW. Immunoradiometric measurement of the factor VIII procoagulant antigen J Clin Invest 1978; 62: 1048-52. 21. Hoyer LW, Trabold NC Immunoradiometric assays for factor VIII antigens: coagulant protein (antihemophilic factor) and factor VIII-related protein (von Willebrand factor) Meth Enzymol 1982; 84: 51-60. 22. Gitschier J, Drayna D, Tuddenham EGD, White RL, Lawn RM. Genetic mapping and diagnosis of haemophilia achieved A through a Bclpolymorphism in the factor VIII gene. Nature 1985; 314: 738-40. 23 Orkin SH. Prenatal diagnosis of hemoglobin disorders by DNA analysis. Blood 1984; 63: 249-53 24. Boehm CD, Antonarakis SE, Phillips JA, Stetten G, Kazazian HH Jr. Prenatal diagnosis using DNA polymorphisms. Report on 95 pregnancies at risk for sickle cell disease or &bgr;-thalassemia N Engl J Med 1983, 308: 1054-58 25. NICHD, National Registry for Amniocentesis Study Group. Midtrimester amniocentesis for prenatal diagnosis Safety and accuracy JAMA 1976; 236: 1471-76 26. Williamson R, Eskdale J, Coleman DV, Niazi M, Loeffler FE, Modell BM. Direct gene analysis of chorionic villi: A possible technique for first trimester antenatal diagnosis of hemoglobinopathies. Lancet 1981; ii: 1125-27
PRENATAL DIAGNOSIS OF HAEMOPHILIA A BY FACTOR VIII GENE ANALYSIS
STYLIANOS E. ANTONARAKIS ROBERT J. CARPENTER, JR LEON W. HOYER JOHN J. TOOLE
KAREN L. COPELAND CARL A. CARTA C. THOMAS CASKEY HAIG H. KAZAZIAN, JR
Genetics Unit, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, Robert J. Kleberg Center for Human Genetics, Baylor College of Medicine, Houston, Texas; Department of Medicine, University of Connecticut School of Medicine, Farmington, Connecticut; and Genetics Institute Inc, Boston, Massachusetts, USA
1985
This method involves recombinant DNA technology, amniocentesis or chorionic villus sampling, and study of the entire family to establish the linkage phase between the DNA marker and the disease locus.9 The major drawback of this approach is that the accuracy of the diagnosis depends upon the recombination distance between the marker and the locus of interest. Here we describe the prenatal diagnosis of a case of haemophilia A by analysis of DNA polymorphic sites within the factor VIII gene.10 Cloned FVIII complementary DNA (cDNA) sequences were used as probes to identify and mark the defective FVIII gene. Materials and Methods
Cloned factor VIII
deoxyribose nucleic acid (DNA) sequences were used as probes in the prenatal diagnosis of haemophilia A. Fetal DNA from
Subjects
cultured amniotic fluid cells was examined for a DNA polymorphism within the factor VIII gene which marked the haemophilia A gene in the pregnant obligate carrier. The fetus was predicted to be an affected male, and the diagnosis of haemophilia A was confirmed both in utero and after termination of the pregnancy.
She is an obligate carrier of an abnormal factor VIII gene because her two sisters have haemophilic sons (fig 1).
Summary
Introduction HAEMOPHILIA A due to deficiency of coagulation factor VIII (FVIII:C) is the commonest inherited disorder of blood coagulation.l It affects approximately 1 in every 10 000 males. Although substitution therapy is reasonably satisfactory, the disease puts a considerable strain on families. Therefore, many at-risk families seek prenatal diagnosis for haemophilia A. Until recently prenatal diagnosis depended upon the measurement of the X-linked FVIII coagulant antigen (FVIII:CAg) and the autosomal FVIII related antigen
(FVIIIR:Ag)
in fetal
plasma2-5
obtainable
between the 18th and 21st weeks
only by fetoscopy
ofgestation.6
Another approach is by the study of DNA polymorphic markers genetically linked to the haemophilia locus.’,8
A 30-year-old Saudi Arabian woman married to her first cousin presented at 12 weeks of gestation in her third pregnancy. She had previously borne a son with haemophilia A and a normal daughter.
Restriction Endonuclease Analysis Nuclear DNA was isolated from individuals 111-1, III-2, IV-1, IV-2, from leucocytes of 10-15 ml of edetic acid anticoagulated
as described previously.ll Fetal DNA was obtained from two 25 ml flasks of cultured amniocytes. These cultures were established after amniocentesis at week 16 of pregnancy. Cells were harvested and DNA was isolated as described before. 12 About 60 pig of DNA was obtained from the cells in the two flasks, sufficient for several DNA analyses by restriction endonuclease digestion. 5-10 J.lg of DNA was digested with one of various endonucleases under conditions recommended by the suppliers. The methods of electrophoresis, transfer of DNA fragments to nitrocellulose filters, hybridisation of genomic fragments with radioactive probes, washing of filters, and autoradiography have been described.13,14 The following cloned FVIII DNA sequences were used as probes1o,1s,16: (i) probe A, a 1. 7 kb Kpn I cDNA fragment which spans exons 1-12, (ii) probe B, a
peripheral blood
8443
1408
Arrow shows fetus at risk of haemophilia A (individual IV-3). Presence or absence of the polymorphic Bcl site is depicted as + or - for the individuals examined.
4 7 kb Eco RI cDNA fragment which contains exons 14-25 and part of exon 26, and (iii) probe C, a 1 - 8 kb Eco RI cDNA which contains the remainder of exon 26 and the 3’ untranslated region of the factor VIII gene. All of these probes were radiolabelled with
32[P]dCTP (deoxycytidine triphosphate) by function of Escherichia coli DNA polymerase karyotyped according to standard protocols. 18
the nick translation 1.17 Fetal cells were
Haematological Investigations FVIIIC:Ag and factor VIII related antigen VIIIR:Ag) levels in plasma obtained from cord blood in uteroand abortus blood were measured by fluid-phase immunoradiometric assays.20,21 fetal
Results DNA from the pregnant woman (III-2), her husband (III-1), and her two offspring, IV-1and IV-2, were analysed for the presence of any gross abnormality (deletion) in the factor VIII gene. None of the molecular defects directly detectable by restriction endonuclease analysis was identified in this Saudi Arabian family. Examination of the DNA from 111-2 for heterozygosity at DNA polymorphic sites within the factor VIII gene (fig 2) showed that she was heterozygous for the presence ofa previously described BclI site in exon 18 of the FVIII gene. 10,22 Since this woman was an obligate carrier of a haemophilia A gene and her affected son (IV-2) demonstrated the presence of the Bd I polymorphic site in his defective FVIII gene, we concluded, by the use of simple linkage analysis, that the presence of the BclI site marked the maternal X chromosome which carries the haemophilia gene. In the course ofour initial evaluation of the family we found, using the same DNA polymorphism analysis, that III-2’s daughter (IV-1) also carries the
haemophilia gene.
Fig 2-Autoradiogram patterns of DNA from members of family after digestion with BclI and hybridisation with FVIII-B probe. Note the inheritance of the presence (+) or absence (-) of the Bcl I polymorphic site in different family members. Male fetus IV-3, like his affected brother IV-2, has inherited the therefore, affected with haemophilia A.
allele from his mother and is,
+
had inherited the haemophilia A gene from its mother and, therefore, was affected with haemophilia A. This suggestion was confirmed by immunoradiometric assay for FVIII:CAg and FVIIIR:Ag in fetal blood obtained by percutaneous cordblood sampling under direct visualisation of the needle by ultrasonography at week 21 of pregnancy (see accompanying table). The family elected to have the affected fetus aborted and the diagnosis of haemophilia A was verified by measuring FVIII:CAg and FVIIIR:Ag in the blood of the abortus. Discussion
prenatal diagnosis of haemophilia A based on immunoradiometric assay of FVIII:CAg and FVIIIR:Ag in fetal blood obtained by fetoscopy is made at weeks 18-21 of pregnancy. Between 1978 and 1983 only 92 pregnancies were The
evaluated in the United States, out of an estimated 500 pregnancies at risk for haemophilia A each year. The risk of spontaneous abortion after fetoscopy for prenatal diagnosis of haemophilia A was about 6% (3/52) in normal fetuses.4 In addition, it was not possible to obtain a satisfactory sample in 12 of the 92 pregnancies. Because of these factors fetoscopy has not been used much for the prenatal diagnosis of haemophilia A. Recombinant DNA technology,23 linkage analysis by the use of DNA polymorphism,24 and the cloning of the factor VIII gene’ 5,16 should make the prenatal diagnosis of IMMUNORADIOMETRIC ASSAYS OF FVIII
done in week 16 of pregnancy. Amniocentesis of the cultured amniotic fluid cells Cytogenetic analysis showed that the fetus had a normal male karyotype. When DNA was extracted from the cultured amniotic fluid cells and the Bcl I polymorphic site within the FVIII gene was examined, we found that the male fetus had inherited the FVIII gene which was marked by the presence of the BclI polymorphic site. This finding suggested that the male fetus was
I
*Data from reference 4, measurements made Values in parentheses refer to range.
I
on
blood obtained
b- fetcscor’._:
1409
haemophilia A safer. Highly informative DNA polymorphisms have been associated with two cloned random DNA Sequences which have been mapped very close to the haemophilia A locus on the long arm of chromosome X. The cloned
COXSACKIE B, MUMPS, RUBELLA, AND CYTOMEGALOVIRUS SPECIFIC IgM RESPONSES IN PATIENTS WITH JUVENILE-ONSET INSULIN-DEPENDENT DIABETES MELLITUS IN BRITAIN, AUSTRIA, AND AUSTRALIA
DNA sequence DX13 shows no recombination
with the haemophilia A locus;the Bgl II site associated with this sequence (DX13) has been used for carrier detection of haemophilia A in a pregnant woman.9 In addition, the cloned DNA sequence Stl4 shows no recombination with the A locus and the Taq I and Msp I polymorphic sites associated with it can be used for prenatal diagnosis of this disease.8 Although prenatal diagnosis by the use of anonymous DNA fragments linked to the haemophilia locus does not incur the hazards associated with fetoscopy (the fetal cells being obtained by amniocentesis25 or chorionic villus sampling 26), there is a low risk of both false-positive and falsenegative predictions because of possible recombination between the DNA polymorphic site of interest and the FVIII gene. (The 90% confidence limits for the recombination fraction between DX13 and the locus for haemophilia A are 0-9 centimorgans,’ while those between Stl4 and the haemophilia A locus are 0-5 centimorgans.8) We have used DNA polymorphic sites within the factor VIII gene and, therefore, the error rate due to recombination between the marker DNA polymorphic site and the mutant FVIII gene should be negligible. Another advantage of methods based on DNA analysis is that carrier females can also be diagnosed prenatally. The disadvantage is that family studies are necessary in order to link the DNA polymorphic marker with the defective FVIII gene in a family,24 and Hoyer et al have reported that for many women (25 of 80) who request prenatal diagnosis for haemophilia A a male with haemophilia A is not available for the test.4 Moreover, some women present so late in pregnancy that diagnosis by DNA polymorphisms is not practical before the fetus is at 21 weeks of gestation, and fetoscopy becomes necessary for a rapid diagnosis. We think, however, that the study of the FVIII gene defects or DNA polymorphisms within this gene after’ amniocentesis or chorionic villus sampling will become the method of choice for prenatal diagnosis of haemophilia A. If adequate information cannot be obtained by the use of FVIII gene sequences, DNA polymorphisms associated with DNA sequences Stl4 and/or DX13 may be useful.
J. E. BANATVALA JENNIFER BRYANT G. SCHERNTHANER M. BORKENSTEIN E. SCHOBER D. BROWN L. M. DE SILVA M. A. MENSER M. SILINK
haemophilia
We thank Mrs Emily Pasterfield for secretarial help; Dr David Ledbetter and Dr Gail Stetten for culturing the amniotic fluid cells; and Ms Margaret Norman for technical help. The study was supported by grants from the National Institutes of Health to S. E. A., H. H. K., and L. W. H. C. T. C. is a Howard Hughes investigator.
Correspondence should be addressed to S. E. A., Department of Pediatrics, CMSC 1004, Johns Hopkins Hospital, 600 N Wolfe Street, Baltimore, Maryland 21205, USA. REFERENCES 1. McKee PA
Hemostasis and disorders of blood coagulation. In: Stanbury JB, Friedrickson DS, Goldstein JL, Brown MS, eds. The metabolic basis of inherited diseases, 5th ed. New York. McGraw-Hill, 1983: 1531-60. Firshein SI, Hoyer LW, Lazarchick J, et al. Prenatal diagnosis of classic hemophilia. N Engl J Med 1979; 300: 937-41. Mibashan RS, Rodeck CH, Furlong RA, et al. Dual diagnosis of prenatal hemophilia A by measurement of fetal factor VIIIC and VIIIC antigen(VIIICAg) Lancet 1980; ii. 994-97. Hoyer LW, Carta CA, Golbus MS, Hobbins JC, Mahoney MJ. Prenatal diagnosis of classic hemophilia (hemophilia A) by immunoradiometric assays. Blood (in press). Mibashan RS, Thumpston JK, Singer JD, et al. Plasma assay of fetal factors VIIIC and IX for prenatal diagnosis of hemophilia. Lancet 1979; i: 1309-11. Hobbins JC, Mahoney MJ. In utero diagnosis of hemoglobinopathies; technique for obtaining fetal blood. N Engl J Med 1974; 290: 1065-67. Harper K, Winter RM, Pembrey ME, Hartley D, Davies KE, Tuddenham EGD. A clinically useful DNA probe closely linked to hemophilia A. Lancet 1984; ii: 6-8.
Wyngaarden JB,
2. 3.
4. 5
6. 7
Department of Virology, St Thomas’ Hospital, London; Department of Medicine II, University of Vienna, Austria; Department of Paediatrics, University of Graz, Austria; Department of Paediatrics, University of Vienna, Austria; Virus Reference Laboratory, Central Public Health Laboratory, Colindale, London; and Children’s
Hospital, Camperdown, Sydney, Australia
Patients from England, Austria, and Australia with recently diagnosed juvenileonset insulin-dependent diabetes (type 1) mellitus (IDDM) and matched controls were tested for specific IgM responses to Coxsackie B1-5 viruses. 37 of 122 (30%) patients aged <15, but only 15 of 204 (6%) controls, were positive (p<0·005). Differences in Coxsackie B virus specific IgM responses between patients and controls were statistically significant for patients in England and Austria (p<0·005). Coxsackie B virus specific IgM responses were detected in only 3 of 31 patients aged >16. Virus-specific IgM responses were directed against a single serotype, usually Coxsackie B4
Summary
I, Camerino G, Heilig R, et al. Genetic screening for hemophilia A with a polymorphic DNA probe N Engl J Med 1985; 312: 682-86. Tonnessen T, Sondergaard F, Mikkelsen M, et al. X chromosome specific probe DX13 for carrier detection and first trimester prenatal diagnosis of hemophilia A. Lancet
8. Oberle 9.
1984; ii:
1269-70.
SE, Waber PG, Kittar SD, et al. Hemophilia A. Molecular defects and detection by DNA analysis. N Engl J Med (in press). 11. Kunkel LM, Smith KD, Boyer SH, et al. Analysis of human Y chromosome specific reiterated DNA in chromosome variants. Proc Natl Acad Sci USA 1977, 74: 1245-49 12. Kan YW, Dozy AM. Antenatal diagnosis of sickle cell anemia by DNA analysis of amniotic fluid cells. Lancet 1978; ii: 910-12 13. Southern EM. Gel electrophoresis of restriction fragments. Meth Enzymol 1979; 68: 10 Antonarakis carrier
152-76. 14. Scott AF, Phillips
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