724
Prenatal
sex
determination
Detection of schistosome antigen in mummies
SIR,-Dr Nakagome and colleagues (Feb 3, p 291) comment on our earlier report (Dec 9, p 1363). As we suggested, the failure by two groups to detect fetal DNA from maternal blood may be related to their use of a single amplification compared with our dual amplification system. In addition, we used extracted and partly fragmented DNA, which resulted in the dispersal into solution of the multiple copies of the target sequence whereas Adinolfi et all used whole cells. Although we cannot formally exclude autosomal sequences that show
homology to the 149 bp sequence and are amplified, as suggested by Nakagome et al, we make the following observations. First, following a single round of amplification with either pair of primers (40 cycles), we have detected no signal in any of the DNA samples from non-pregnant women tested in our hands. Second, various groups have used primers Y 1.1and Y 1.2 to sex fetal genetic materials without having difficulties in crossreactivity with autosomal sequences,3,4 though we note that Handyside et als reported an occasional faint band in some of their samples from female fetuses. All these data exclude the possibility of an exact direct copy of the 149 bp sequence on the autosome(s) but do not rule out the existence of some related but non-identical sequences that are amplified with a consequent suboptimum efficiency. These related sequences would, however, have to be identical in length to the target Y-sequence and show homology to both pairs of primers (Yl.l/Y1.2 and Y1.3/Y1.4) before a false-positive signal is produced. Data on autosomal crossreactivity of the region concerned are based on Southern blotting, in-situ hybridisation,6,7 and re-association kinetics,8 and whether results obtained with these techniques can be extrapolated to those with the polymerase chain reaction is unclear. Furthermore, the existence of crossreacting autosomal sequences does not invalidate our observations since pregnant women bearing female fetuses were our internal negative controls. Such crossreacting sequences would not account for the strong correlation between positive signal and fetal sex. However, we would emphasise that the narrow window between the level of amplification giving a genuine result and that producing a false-positive result indicates that further modifications to the method are needed. Several possible areas for development include the selection of alternative Y-specific primers, as discussed by Nakagome et al, or a more rigorous "closed" environment for blood collection and DNA extraction. New anticontamination regimens, such as the use of ultraviolet radiation for damaging contaminant DNA,9 may also contribute. The ability to select fetal cells from maternal circulation may also help in single gene diagnosis1O although our preliminary calculations show that this might be feasible without previous cell selection. University of Oxford Clinical School, Department of Haematology, and Nuffield Department of Pathology, John Radcliffe Hospital, Oxford OX3, 9DU, UK
Y-M. D. Lo P. PATEL J. S. WAINSCOAT K. .A. FLEMING
1. Adinolfi M, Camporese C, Canr T. Gene amplification to detect fetal nucleated cells in pregnant women. Lancet 1989; ii: 328-29.
2.
Schwinger E, Hillers M, Vosberg HP. No identification of Y-chromosomal DNA in blood from pregnant women bearing a male fetus? Am J Hum Genet 1989; 45: A268.
Kogan SC, Doherty M, Gitschier J. An improved method for prenatal diagnosis of genetic diseases by amplified DNA sequences. N Engl J Med 1987; 317: 985-90. 4. Pinckert TL, Lebo RV, Golbus MS. Rapid determination of fetal sex by deoxyribonucleic acid amplification of Y chromosome specific sequences. Am J Obstet Gynecol 1989; 161: 693-98. 5. Handyside AH, Pattinson JK, Penketh RJA, Delhanty JDA, Winston RML, Tuddenham EGD. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1989; i: 347-49. 6. Burk RD, Szabo P, O’Brien S, Nash WG, Yu L, Smith KD. Organization and chromosomal specificity of autosomal homologs of human Y chromosome repeated
SIR,-We have applied an ELISA that is both sensitive to and highly specific for circulating antigens during active schistosoma infections,l,2 to the detection of the schistosome circulating anodic antigen (CAA) in Egyptian mummies. This proteoglycan from the syncytium lining the schistosome gut is regurgitated and released into the bloodstream of the host, where it can be detected in circulation.3 Three samples from unembalmed, desiccated Egyptian mummies were tested-namely, cheek and colon from an adolescent male, Nakht (Thebes, 1198-1150 BC), in which Schistosoma haematobium eggs had been found in gut, kidney, and liver,4 and shin tissue from a late predynastic (3200 BC) adolescent, coded BM 32753.5 As negative control samples we used gut tissue from five unembalmed, desiccated mummies from the Atacama desert near Arica, northern Chile (CAM9 T12, CAM9 T13, AZ6 T6, MO 1-6 T-U7, Pircas 2 T6) dated from 2000 BC or earlier to 600 AD. Specimens were homogenised in an all-glass homogeniser in 7-5% trichloroacetic acid to remove proteins and dissociate immune complexes and then spun down (25 000 g, 15 min); the supemates were dialysed for 24 h against distilled water (4°C) and
lyophilised. In the
negative controls no CAA could be detected even at very
high sample concentrations (ie, at a cut-off value of 0-5 pg/mg no positive results were detected). With the Egyptian samples 123 pg CAA/mg and 13 pg/mg, respectively, were found in the cheek and gut of Nakht and 8 pg/mg in the shin of BM 32753. It is thus possible to detect CAA after 3200-5000 years in desiccated tissue preserved under ideal conditions in a desert environment not subject to post mortem leaching from weathering or embalming. With a ten-fold reduction in the weight of dried tissue and 10% blood content of cheek tissue, the CAA determined in Nakht’s cheek would correspond to a serum concentration of about 25 ng/ml. This accords well with CAA levels in contemporary S haematobium infected individuals; 10-40 ng CAA/ml serum has been demonstrated in patients with heavy infectionadegree of infection indicated by the pathological studies of Nakht.’ Schistosoma infestation has previously been diagnosed in mummies either by the demonstration of eggs in viscera4 or by radiological detection of bladder wall calcification.s The nondestructive but sensitive and specific technique described here could be used on a larger scale in fragments of mummy tissue where viscera are not preserved or are inaccessible. The discovery of active Schistosoma infestation in a predynastic adolescent (BM 32753) significantly extends the history of the disease.
Supported by the Science and Technology for Development Programme of European Community and by the Wellcome Trust. We thank F. Nowell-Smith, Academy of Medicine, Toronto; M. Bierbrier and V. Davies, British Museum, London; T. Holden, Institute of Archaeology, London; and A. C. Aufderheide, University of Minnesota, Duluth, for providing samples. the
Laboratory of Parasitology, University of Leiden, Leiden 2300 RC, Netherlands
College, Cambridge, UK
R. L. MILLER
Laboratory of Parasitology, University of Leiden
N. DE JONGE F. W. KRIJGER
3.
DNA. Chromosoma 1985; 92: 225-33. KD, Young KE, Talbot CC Jr, Schmeckpeper BJ. Repeated DNA of the human Y chromosome. Development 1987; (suppl): 77-92. 8. Kunkel LM, Smith KD. Evolution of human Y-chromosome DNA. Chromosoma 1982; 86: 209-28. 9. Sarkar G, Sommer SS. Shedding light on PCR contamination. Nature 1990; 343: 27. 10. Yeoh SC, Sargent IL, Redman CWG, Thein SL. Detecting fetal cells in maternal circulation. Lancet 1989; ii: 869-70. 7. Smith
A. M. DEELDER
Darwin
1. Deelder AM, de Jonge N, Boerman OC, Fillié YE, Hilberath GW, Rotmans JP, etal Sensitive determination of circulating anodsic antigen in Schistosoma mansoni
infected individuals by an enzyme-linked immunosorbent assay using monoclonal antibodies. Am J Trop Med Hyg 1989; 40: 268-72. 2. De Jonge N, De Caluwé P, Hilberath GW, Krijger FW, Polderman AM, Decider AM. Circulating anodic antigen levels in serum before and after chemotherapy with praziquantel in schistosomiasis mansoni. Trans R Soc Trop Med Hyg 1989; 83: 368-72. 3. Nash TE. Factors and modulate clearance and ultimate fate of a specific schistosome antigen (GASP) in schistosome infections. J Immunol 1982; 128: 1608-13.
4. Millet NB, Hart GD, Reyman TA, Zimmerman MR, Lewin PK. ROMI mummification for the common people. In: Cockburn A, Cockburn E, eds Mummies, disease and ancient cultures. Cambridge: Cambridge University Press, 1980: 71-84.
Prenatal
sex
determination
Detection of schistosome antigen in mummies
SIR,-Dr Nakagome and colleagues (Feb 3, p 291) comment on our earlier report (Dec 9, p 1363). As we suggested, the failure by two groups to detect fetal DNA from maternal blood may be related to their use of a single amplification compared with our dual amplification system. In addition, we used extracted and partly fragmented DNA, which resulted in the dispersal into solution of the multiple copies of the target sequence whereas Adinolfi et all used whole cells. Although we cannot formally exclude autosomal sequences that show
homology to the 149 bp sequence and are amplified, as suggested by Nakagome et al, we make the following observations. First, following a single round of amplification with either pair of primers (40 cycles), we have detected no signal in any of the DNA samples from non-pregnant women tested in our hands. Second, various groups have used primers Y 1.1and Y 1.2 to sex fetal genetic materials without having difficulties in crossreactivity with autosomal sequences,3,4 though we note that Handyside et als reported an occasional faint band in some of their samples from female fetuses. All these data exclude the possibility of an exact direct copy of the 149 bp sequence on the autosome(s) but do not rule out the existence of some related but non-identical sequences that are amplified with a consequent suboptimum efficiency. These related sequences would, however, have to be identical in length to the target Y-sequence and show homology to both pairs of primers (Yl.l/Y1.2 and Y1.3/Y1.4) before a false-positive signal is produced. Data on autosomal crossreactivity of the region concerned are based on Southern blotting, in-situ hybridisation,6,7 and re-association kinetics,8 and whether results obtained with these techniques can be extrapolated to those with the polymerase chain reaction is unclear. Furthermore, the existence of crossreacting autosomal sequences does not invalidate our observations since pregnant women bearing female fetuses were our internal negative controls. Such crossreacting sequences would not account for the strong correlation between positive signal and fetal sex. However, we would emphasise that the narrow window between the level of amplification giving a genuine result and that producing a false-positive result indicates that further modifications to the method are needed. Several possible areas for development include the selection of alternative Y-specific primers, as discussed by Nakagome et al, or a more rigorous "closed" environment for blood collection and DNA extraction. New anticontamination regimens, such as the use of ultraviolet radiation for damaging contaminant DNA,9 may also contribute. The ability to select fetal cells from maternal circulation may also help in single gene diagnosis1O although our preliminary calculations show that this might be feasible without previous cell selection. University of Oxford Clinical School, Department of Haematology, and Nuffield Department of Pathology, John Radcliffe Hospital, Oxford OX3, 9DU, UK
Y-M. D. Lo P. PATEL J. S. WAINSCOAT K. .A. FLEMING
1. Adinolfi M, Camporese C, Canr T. Gene amplification to detect fetal nucleated cells in pregnant women. Lancet 1989; ii: 328-29.
2.
Schwinger E, Hillers M, Vosberg HP. No identification of Y-chromosomal DNA in blood from pregnant women bearing a male fetus? Am J Hum Genet 1989; 45: A268.
Kogan SC, Doherty M, Gitschier J. An improved method for prenatal diagnosis of genetic diseases by amplified DNA sequences. N Engl J Med 1987; 317: 985-90. 4. Pinckert TL, Lebo RV, Golbus MS. Rapid determination of fetal sex by deoxyribonucleic acid amplification of Y chromosome specific sequences. Am J Obstet Gynecol 1989; 161: 693-98. 5. Handyside AH, Pattinson JK, Penketh RJA, Delhanty JDA, Winston RML, Tuddenham EGD. Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 1989; i: 347-49. 6. Burk RD, Szabo P, O’Brien S, Nash WG, Yu L, Smith KD. Organization and chromosomal specificity of autosomal homologs of human Y chromosome repeated
SIR,-We have applied an ELISA that is both sensitive to and highly specific for circulating antigens during active schistosoma infections,l,2 to the detection of the schistosome circulating anodic antigen (CAA) in Egyptian mummies. This proteoglycan from the syncytium lining the schistosome gut is regurgitated and released into the bloodstream of the host, where it can be detected in circulation.3 Three samples from unembalmed, desiccated Egyptian mummies were tested-namely, cheek and colon from an adolescent male, Nakht (Thebes, 1198-1150 BC), in which Schistosoma haematobium eggs had been found in gut, kidney, and liver,4 and shin tissue from a late predynastic (3200 BC) adolescent, coded BM 32753.5 As negative control samples we used gut tissue from five unembalmed, desiccated mummies from the Atacama desert near Arica, northern Chile (CAM9 T12, CAM9 T13, AZ6 T6, MO 1-6 T-U7, Pircas 2 T6) dated from 2000 BC or earlier to 600 AD. Specimens were homogenised in an all-glass homogeniser in 7-5% trichloroacetic acid to remove proteins and dissociate immune complexes and then spun down (25 000 g, 15 min); the supemates were dialysed for 24 h against distilled water (4°C) and
lyophilised. In the
negative controls no CAA could be detected even at very
high sample concentrations (ie, at a cut-off value of 0-5 pg/mg no positive results were detected). With the Egyptian samples 123 pg CAA/mg and 13 pg/mg, respectively, were found in the cheek and gut of Nakht and 8 pg/mg in the shin of BM 32753. It is thus possible to detect CAA after 3200-5000 years in desiccated tissue preserved under ideal conditions in a desert environment not subject to post mortem leaching from weathering or embalming. With a ten-fold reduction in the weight of dried tissue and 10% blood content of cheek tissue, the CAA determined in Nakht’s cheek would correspond to a serum concentration of about 25 ng/ml. This accords well with CAA levels in contemporary S haematobium infected individuals; 10-40 ng CAA/ml serum has been demonstrated in patients with heavy infectionadegree of infection indicated by the pathological studies of Nakht.’ Schistosoma infestation has previously been diagnosed in mummies either by the demonstration of eggs in viscera4 or by radiological detection of bladder wall calcification.s The nondestructive but sensitive and specific technique described here could be used on a larger scale in fragments of mummy tissue where viscera are not preserved or are inaccessible. The discovery of active Schistosoma infestation in a predynastic adolescent (BM 32753) significantly extends the history of the disease.
Supported by the Science and Technology for Development Programme of European Community and by the Wellcome Trust. We thank F. Nowell-Smith, Academy of Medicine, Toronto; M. Bierbrier and V. Davies, British Museum, London; T. Holden, Institute of Archaeology, London; and A. C. Aufderheide, University of Minnesota, Duluth, for providing samples. the
Laboratory of Parasitology, University of Leiden, Leiden 2300 RC, Netherlands
College, Cambridge, UK
R. L. MILLER
Laboratory of Parasitology, University of Leiden
N. DE JONGE F. W. KRIJGER
3.
DNA. Chromosoma 1985; 92: 225-33. KD, Young KE, Talbot CC Jr, Schmeckpeper BJ. Repeated DNA of the human Y chromosome. Development 1987; (suppl): 77-92. 8. Kunkel LM, Smith KD. Evolution of human Y-chromosome DNA. Chromosoma 1982; 86: 209-28. 9. Sarkar G, Sommer SS. Shedding light on PCR contamination. Nature 1990; 343: 27. 10. Yeoh SC, Sargent IL, Redman CWG, Thein SL. Detecting fetal cells in maternal circulation. Lancet 1989; ii: 869-70. 7. Smith
A. M. DEELDER
Darwin
1. Deelder AM, de Jonge N, Boerman OC, Fillié YE, Hilberath GW, Rotmans JP, etal Sensitive determination of circulating anodsic antigen in Schistosoma mansoni
infected individuals by an enzyme-linked immunosorbent assay using monoclonal antibodies. Am J Trop Med Hyg 1989; 40: 268-72. 2. De Jonge N, De Caluwé P, Hilberath GW, Krijger FW, Polderman AM, Decider AM. Circulating anodic antigen levels in serum before and after chemotherapy with praziquantel in schistosomiasis mansoni. Trans R Soc Trop Med Hyg 1989; 83: 368-72. 3. Nash TE. Factors and modulate clearance and ultimate fate of a specific schistosome antigen (GASP) in schistosome infections. J Immunol 1982; 128: 1608-13.
4. Millet NB, Hart GD, Reyman TA, Zimmerman MR, Lewin PK. ROMI mummification for the common people. In: Cockburn A, Cockburn E, eds Mummies, disease and ancient cultures. Cambridge: Cambridge University Press, 1980: 71-84.
