87
EDITORIALS
Is it
a
boy?
For social and medical reasons, people often wish to know well in advance the sex of an expected child. When a woman carries a severe X-linked disorder, it may be ethically justifiable to arrange termination of a male fetus at a very early stage. In Asian communities," the very strong cultural preference for males has sometimes encouraged ethically unjustifiable disposal of females. Until lately, karyotype analysis of a chorion villus biopsy sample taken at 8-10 weeks’ gestation provided the earliest diagnosis of fetal sex. The speed of sex determination from such samples was greatly accelerated with the application of the polymerase chain reaction (PCR) specific for Y-sequence.11 The ability of PCR to increase the sensitivity of detection of specific DNA sequences by many orders of magnitude has led to widespread applications in medical research.2,3 So powerful is the method that analysis of DNA samples as small as a single cell can be attempted.4 Application of this technology to a cell obtained from in-vitro fertilised pre-implantation human embryos has allowed the correct determination of sexs in each of 15 normal embryos in which the sex could subsequently be confirmed by in-situ hybridisation and/or fluorescent staining of the whole embryo. This approach led to the successful transfer of female embryos to mothers who were carriers of serious X-linked disorders.66 Determination of sex by PCR has been facilitated by the presence on the Y chromosome of highly repeated DNA segments with copy numbers of 800 to 5000.7 Several investigators have attempted to determine fetal sex from maternal blood samples since fetal cells are known to be present in the maternal circulation. The only source of Y-chromosomespecific material in maternal blood would be a male fetus. Thus, Lo and colleagues8 used an amplification protocol in which DNA from maternal blood was subjected to 40 rounds of amplification, then subsampled and further amplified (15-20 cycles) by use of primers located within those used for the first
round (nested primers). Success of the prediction was checked by eventual outcome and, although not perfect, was impressive. A worrying aspect was the occurrence of false-positive results if the amplification cycles went beyond 20. Our correspondence columns have lately carried a small flurry of letters about false-positive PCR amplification. This issue is important not only for Y-sequence detection but also for single-copy gene detection and procedures for determination of viral DNA sequences. Holzgreve and colleagues9 encountered difficulties when they attempted to amplify a single-copy Y-specific sequence, which has no autosomal homology, and found a detection limit corresponding to one or two male cells. Even though DNA was isolated under class II containment conditions and all PCR reagents were digested with an enzyme that cut the target sequence, they still found that DNA from 6 of 10 control non-pregnant women gave false-positive bands. Beyer-Finkler et all who encountered a high proportion of positives when screening for human papillomavirus in cervical scrapes from healthy women by means of PCR, were able to reduce the positive rate from 23 of 53 samples to 13 by use of nested primers. Lo and colleaguesll have now used nested primers to detect single-copy Y-sequence of presumed fetal origin in maternal blood of 27 women. From 12 early pregnancy samples, there were 10 correct results with 2 falsenegatives. From 15 samples taken in intermediate-tolate pregnancy, there were 10 correct determinations with 2 false-positives and 2 false-negatives. PorterJordan and Garrett12,13 suggest, on the basis of their own experience with cytomegalovirus, that an important source of contamination could be fragmented DNA. Their hypothesis is that overlapping sequences of fragmented DNA could
sequentially serve as a template to complete a mosaic sequence, which could be amplified-"jumping PCR". They suggest that the use of nested primers should eliminate false-positives generated by jumping PCR, although their reasons are not entirely clear.
88
The power of the PCR technique lies in its extraordinary sensitivity, which also gives rise to its greatest drawback. False-positive amplification is a common occurrence-from carry-over of previously amplified DNA, or from cloned DNA (which may be present in the laboratory in milligram quantities), or from contamination of the PCR reaction by fragments of genomic DNA from the skin surfaces of operators. PCR preparation and amplification should therefore be physically separated from the analysis of the PCR and from all cloned DNAs. The workers who reported sexing of single cells from pre-implantation embryos4 commented on the necessity for a strict anticontamination protocol because of the ubiquity in buffers of Y-sequences that almost certainly came from skin cells of male workers. Thus the protocol included filtration, use of an enzyme to cleave potential contaminating DNA within the target sequence (followed by boiling to destroy the restriction enzyme), and the performance of all procedures under class II containment. However, unlike the investigators attempting to determine fetal sex by analysis of maternal blood samples they did not have to isolate or purify the DNA for analysis, thereby eliminating a potentially large opportunity for contamination. The problem of contamination in PCR has exercised many workers, and other suggestions have been made on the avoidance of false-positives. Lo et ap4 advocated the use of a no-DNA negative control and a primer pair designed to detect contamination with plasmid DNA. Kitchin and colleagues15 found that certain operators readily shed specific DNA (presumably picked up from general laboratory fomites) from their face and hair and that contamination of the PCR reaction from this source could be prevented by use of simple disposable protective clothing (mob cap, face mask, goggles, gloves). Kwok and Higuchi16 list nine tips for better PCR, among which are the physical separation of PCR reactions from all other DNA manipulation, use of positive displacement pipettes, autoclaving of all solutions and disposable plastic ware, and inclusion of carefully chosen positive and negative controls.
false-positive occurs during preimplantation diagnosis the worst result is a missed opportunity to implant a female embryo, but were it to occur during fetal sexing of an established pregnancy by analysis of maternal blood a termination might be carried out erroneously. More work is required before it would be ethically justifiable to offer such a test to aid in prenatal diagnosis of sex or of single-gene genetic disease. Confidence in any procedure or laboratory will depend upon the continuous application of strict standards. The widely canvassed view that PCR is the DNA technique that will be most widely used in the future in routine diagnosis adds weight to these concerns. The day when we can confidently say whether it is a boy, without biopsy and When
a
karyotype confirmation,
is-
near
but has
not
yet
arrived. Kogan SC, Doherty M, Gitschier J. An improved method for prenatal diagnosis of genetic disease by analysis of amplified DNA sequences. N Engl J Med 1987; 317: 985-90. 2. Editorial. DNA diagnosis and the polymerase chain reaction. Lancet 1.
1988; i: 1372-73. 3. Eisenstein BI. The polymerase chain reaction. A new method of using molecular genetics for medical diagnosis. N Engl J Med 1990; 322: 178-83. 4. Li A, Gyllensten UB, Cui X, Saiki RK, Erlich HA, Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 1988; 355: 414-19. 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. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990; 344: 768-70. 7. Nakahori Y, Mitani K, Nakagome Y. A human Y-chromosome specific repeated DNA family (DYZ1) consists of a tandem array of pentanucleotides. Nucl Acids Res 1986; 14: 7569-80. 8. Lo Y-MD, Patel P, Wainscoat JS, et al. Prenatal sex determination by DNA amplification from maternal peripheral blood. Lancet 1989; ii: 1363-65.
Holzgreve W, Gänshirt-Ahlert D, Burschyk M, et al. Detection of fetal DNA in maternal blood by PCR. Lancet 1990; 335: 1220-21. 10. Beyer-Finkler E, Pfister H, Girardi F. Anti-contamination primers to improve specificity of polymerase chain reaction in human papillomavirus screening. Lancet 1990; 335: 1289-90. 11. Lo Y-MD, Patel P, Sampietro M, Gillmer MDG, Fleming KA, Wainscoat JS. Detection of single-copy fetal DNA sequence from maternal blood. Lancet 1990; 335: 1463-64. 12. Porter-Jordan K, Rosenberg EI, Keiser JF, et al. Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA. J Med Virol 1990; 30: 85-91. 13. Porter-Jordan K, Garrett CT. Source of contamination in polymerase chain reaction assay. Lancet 1990; 335: 1220. 14. Lo Y-MD, Mehal WZ, Fleming KA. False-positive results and the polymerase chain reaction. Lancet 1988; ii: 679. 15. Kitchin PA, Szotyoni Z, Fromholc C, Almond N. Avoidance of false positives. Nature 1990; 344: 201. 16. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989; 9.
339: 237-38.
Phrenic
pacing in quadriplegia nerve
Victims of high spinal cord injury (SCI) with apnoea are often successfully resuscitated by emergency service personnel. If the lesion is complete above C3, normal function of the diaphragm will not return and the patient will remain dependent on a mechanical ventilator, usually with a tracheostomy. Some patients may learn to achieve an adequate tidal volume for part of the day by using their accessory muscles, but this technique will not serve during sleep, so these individuals are ventilator-dependent at night. Only a few patients with SCI are so unfortunate-eg, in the UK there are about 300 new spinal injuries a year and only 1 or 2 patients remain permanently dependent on a ventilator. If the phrenic nucleus (C3,4,5) is substantially undamaged by the cervical lesion rostral to it, the phrenic nerve will remain excitable; phrenic stimulation will cause inspiration, with descent of the diaphragm by at least 5 cm in an adult. Trains of stimulation pulses with suitable intervals between
EDITORIALS
Is it
a
boy?
For social and medical reasons, people often wish to know well in advance the sex of an expected child. When a woman carries a severe X-linked disorder, it may be ethically justifiable to arrange termination of a male fetus at a very early stage. In Asian communities," the very strong cultural preference for males has sometimes encouraged ethically unjustifiable disposal of females. Until lately, karyotype analysis of a chorion villus biopsy sample taken at 8-10 weeks’ gestation provided the earliest diagnosis of fetal sex. The speed of sex determination from such samples was greatly accelerated with the application of the polymerase chain reaction (PCR) specific for Y-sequence.11 The ability of PCR to increase the sensitivity of detection of specific DNA sequences by many orders of magnitude has led to widespread applications in medical research.2,3 So powerful is the method that analysis of DNA samples as small as a single cell can be attempted.4 Application of this technology to a cell obtained from in-vitro fertilised pre-implantation human embryos has allowed the correct determination of sexs in each of 15 normal embryos in which the sex could subsequently be confirmed by in-situ hybridisation and/or fluorescent staining of the whole embryo. This approach led to the successful transfer of female embryos to mothers who were carriers of serious X-linked disorders.66 Determination of sex by PCR has been facilitated by the presence on the Y chromosome of highly repeated DNA segments with copy numbers of 800 to 5000.7 Several investigators have attempted to determine fetal sex from maternal blood samples since fetal cells are known to be present in the maternal circulation. The only source of Y-chromosomespecific material in maternal blood would be a male fetus. Thus, Lo and colleagues8 used an amplification protocol in which DNA from maternal blood was subjected to 40 rounds of amplification, then subsampled and further amplified (15-20 cycles) by use of primers located within those used for the first
round (nested primers). Success of the prediction was checked by eventual outcome and, although not perfect, was impressive. A worrying aspect was the occurrence of false-positive results if the amplification cycles went beyond 20. Our correspondence columns have lately carried a small flurry of letters about false-positive PCR amplification. This issue is important not only for Y-sequence detection but also for single-copy gene detection and procedures for determination of viral DNA sequences. Holzgreve and colleagues9 encountered difficulties when they attempted to amplify a single-copy Y-specific sequence, which has no autosomal homology, and found a detection limit corresponding to one or two male cells. Even though DNA was isolated under class II containment conditions and all PCR reagents were digested with an enzyme that cut the target sequence, they still found that DNA from 6 of 10 control non-pregnant women gave false-positive bands. Beyer-Finkler et all who encountered a high proportion of positives when screening for human papillomavirus in cervical scrapes from healthy women by means of PCR, were able to reduce the positive rate from 23 of 53 samples to 13 by use of nested primers. Lo and colleaguesll have now used nested primers to detect single-copy Y-sequence of presumed fetal origin in maternal blood of 27 women. From 12 early pregnancy samples, there were 10 correct results with 2 falsenegatives. From 15 samples taken in intermediate-tolate pregnancy, there were 10 correct determinations with 2 false-positives and 2 false-negatives. PorterJordan and Garrett12,13 suggest, on the basis of their own experience with cytomegalovirus, that an important source of contamination could be fragmented DNA. Their hypothesis is that overlapping sequences of fragmented DNA could
sequentially serve as a template to complete a mosaic sequence, which could be amplified-"jumping PCR". They suggest that the use of nested primers should eliminate false-positives generated by jumping PCR, although their reasons are not entirely clear.
88
The power of the PCR technique lies in its extraordinary sensitivity, which also gives rise to its greatest drawback. False-positive amplification is a common occurrence-from carry-over of previously amplified DNA, or from cloned DNA (which may be present in the laboratory in milligram quantities), or from contamination of the PCR reaction by fragments of genomic DNA from the skin surfaces of operators. PCR preparation and amplification should therefore be physically separated from the analysis of the PCR and from all cloned DNAs. The workers who reported sexing of single cells from pre-implantation embryos4 commented on the necessity for a strict anticontamination protocol because of the ubiquity in buffers of Y-sequences that almost certainly came from skin cells of male workers. Thus the protocol included filtration, use of an enzyme to cleave potential contaminating DNA within the target sequence (followed by boiling to destroy the restriction enzyme), and the performance of all procedures under class II containment. However, unlike the investigators attempting to determine fetal sex by analysis of maternal blood samples they did not have to isolate or purify the DNA for analysis, thereby eliminating a potentially large opportunity for contamination. The problem of contamination in PCR has exercised many workers, and other suggestions have been made on the avoidance of false-positives. Lo et ap4 advocated the use of a no-DNA negative control and a primer pair designed to detect contamination with plasmid DNA. Kitchin and colleagues15 found that certain operators readily shed specific DNA (presumably picked up from general laboratory fomites) from their face and hair and that contamination of the PCR reaction from this source could be prevented by use of simple disposable protective clothing (mob cap, face mask, goggles, gloves). Kwok and Higuchi16 list nine tips for better PCR, among which are the physical separation of PCR reactions from all other DNA manipulation, use of positive displacement pipettes, autoclaving of all solutions and disposable plastic ware, and inclusion of carefully chosen positive and negative controls.
false-positive occurs during preimplantation diagnosis the worst result is a missed opportunity to implant a female embryo, but were it to occur during fetal sexing of an established pregnancy by analysis of maternal blood a termination might be carried out erroneously. More work is required before it would be ethically justifiable to offer such a test to aid in prenatal diagnosis of sex or of single-gene genetic disease. Confidence in any procedure or laboratory will depend upon the continuous application of strict standards. The widely canvassed view that PCR is the DNA technique that will be most widely used in the future in routine diagnosis adds weight to these concerns. The day when we can confidently say whether it is a boy, without biopsy and When
a
karyotype confirmation,
is-
near
but has
not
yet
arrived. Kogan SC, Doherty M, Gitschier J. An improved method for prenatal diagnosis of genetic disease by analysis of amplified DNA sequences. N Engl J Med 1987; 317: 985-90. 2. Editorial. DNA diagnosis and the polymerase chain reaction. Lancet 1.
1988; i: 1372-73. 3. Eisenstein BI. The polymerase chain reaction. A new method of using molecular genetics for medical diagnosis. N Engl J Med 1990; 322: 178-83. 4. Li A, Gyllensten UB, Cui X, Saiki RK, Erlich HA, Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 1988; 355: 414-19. 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. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990; 344: 768-70. 7. Nakahori Y, Mitani K, Nakagome Y. A human Y-chromosome specific repeated DNA family (DYZ1) consists of a tandem array of pentanucleotides. Nucl Acids Res 1986; 14: 7569-80. 8. Lo Y-MD, Patel P, Wainscoat JS, et al. Prenatal sex determination by DNA amplification from maternal peripheral blood. Lancet 1989; ii: 1363-65.
Holzgreve W, Gänshirt-Ahlert D, Burschyk M, et al. Detection of fetal DNA in maternal blood by PCR. Lancet 1990; 335: 1220-21. 10. Beyer-Finkler E, Pfister H, Girardi F. Anti-contamination primers to improve specificity of polymerase chain reaction in human papillomavirus screening. Lancet 1990; 335: 1289-90. 11. Lo Y-MD, Patel P, Sampietro M, Gillmer MDG, Fleming KA, Wainscoat JS. Detection of single-copy fetal DNA sequence from maternal blood. Lancet 1990; 335: 1463-64. 12. Porter-Jordan K, Rosenberg EI, Keiser JF, et al. Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA. J Med Virol 1990; 30: 85-91. 13. Porter-Jordan K, Garrett CT. Source of contamination in polymerase chain reaction assay. Lancet 1990; 335: 1220. 14. Lo Y-MD, Mehal WZ, Fleming KA. False-positive results and the polymerase chain reaction. Lancet 1988; ii: 679. 15. Kitchin PA, Szotyoni Z, Fromholc C, Almond N. Avoidance of false positives. Nature 1990; 344: 201. 16. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989; 9.
339: 237-38.
Phrenic
pacing in quadriplegia nerve
Victims of high spinal cord injury (SCI) with apnoea are often successfully resuscitated by emergency service personnel. If the lesion is complete above C3, normal function of the diaphragm will not return and the patient will remain dependent on a mechanical ventilator, usually with a tracheostomy. Some patients may learn to achieve an adequate tidal volume for part of the day by using their accessory muscles, but this technique will not serve during sleep, so these individuals are ventilator-dependent at night. Only a few patients with SCI are so unfortunate-eg, in the UK there are about 300 new spinal injuries a year and only 1 or 2 patients remain permanently dependent on a ventilator. If the phrenic nucleus (C3,4,5) is substantially undamaged by the cervical lesion rostral to it, the phrenic nerve will remain excitable; phrenic stimulation will cause inspiration, with descent of the diaphragm by at least 5 cm in an adult. Trains of stimulation pulses with suitable intervals between
