- Email: [email protected]
Exclusion of the GABAA-receptor β3 subunit gene as the Angelman's syndrome gene
122
Exclusion of the GABAA-receptor &bgr;3 subunit gene as the Angelman’s syndrome gene SIR,-About 60% of patients with Angelman’s syndrome have a maternally inherited deletion of chromosome 15ql 1-ql 3. Less than 5% have uniparental disomy, and genomic imprinting has been proposed for this chromosomal region.1 Saitoh et aP reported a family in which a small molecular deletion on chromosome 15 had been inherited from the grandfather, through the healthy mother, to three sibs, all of whom had Angelman’s syndrome. Initial analysis of the deletion had shown it to encompass only the small region around the locus D15SlO (p3-21),3 although Saitoh and colleagues later
HLA-A locus allele-specific PCR (a) and HLA-A locus typing of B lymphoblastoid cell line HRC.304 by allele-specific PCR (b).
(a) Àsizemarkers (lane1 ); internal control reaction (&bgr;2 microglobulin, lane 2); positive allele-specific PCR for HLA-A1, A2, A3, A9, A11, A25(10), A26(10), A28, A29, A30, A31, A32, and A33 (lanes 3-15, respectively). (b) &lgr; size markers (lane 1), negative control (no DNA, lane 2), HLA-A locus allele-specific PCR for HLA-A1, A2, A3, A9, A10, A11, A28, A19(except A30) and A30 (lanes 3-11, respectively). Positive reactions, yielding allele-specific bands of predicted size, are seen in lanes 4 and 10, corresponding to the presence of H LA-A locus alleles A2 and A19(subtyped as HLA-A31, not shown) HLA-A locus alleles from DNA extracted from peripheral blood lymphocytes and from whole blood. Examples of allele-specific reactions and a representative HLA-A locus typing are shown in the figure. DNA-based tissue typing has been applied previously to alleles of HLA class II 10ci.3-S Our system for HLA class I DNA typing has several advantages over serological typing. In practical terms, the method uses renewable reagents and standard laboratory equipment, widening the scope of tissue typing for clinical or research purposes. The use of standard conditions and incorporation of internal controls would allow the method to be adapted as a kit. Since the method is based on genomic sequences, the typing obtained is definitive (within the confines of known HLA sequences). A further advantage is that the system does not require viable cells, and can be used to type material from cells that cannot be tissue typed by conventional means (eg, epithelial cells and cells with low or undetectable HLA expression). The method is a important advance in HLA typing that has implications for both histocompatibility testing for organ transplantation and HLAdisease association studies. ICRF Cancer Immunology Laboratory, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK, and Tissue Antigen Laboratory, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WC2
WF, Bodmer JG. Cytofluorochromasia for HLA-A, -B, -C and DR typing. NIAID manual of tissue typing techniques 1979-1980. Bethesda, Maryland: NIH, 1979. NIH publication no. 80-545. Newton CR, Graham A, Heptinstall LE, et al. Analysis of any point mutation in DNA. the amplification refractory mutation system (ARMS). Nucleic Acids Res 1989; 17: 2503-16. Carlsson B, Wallin J, Bohme J, Moller E. HLA-DR-DQ haplorypes defined by restriction fragment length analysis correlation to serology Hum Immunol 1987; 20: 95-113. Erlich HA, Sheldon EL, Horn G. HLA typing using DNA probes. Biotechnology 1986; 4: 975-81. Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequencespecific primers (SSP-PCR) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 1992; 39: 225-35.
1. Bodmer
2.
3.
4.
5
PETER KRAUSA JONATHAN MOSES WALTER BODMER JULIA BODMER MICHAEL BROWNING
showed it to also include the gene for the GABAA receptor &bgr;3 subunit.4 This chromosomal region was termed the deletion-critical region, since all deletions or unbalanced translocations causing Angelman’s syndrome involved at least these two loci. We report a 3-year-old boy with Angelman’s syndrome and an unbalanced chromosomal translocation in chromosomes 4 and 15, whose molecular characterisation differs from those so far reported. The boy was referred because of developmental delay and feeding problems. He presented with microcephaly with flat occiput, large mandible, and open mouth with protruding tongue, Now, at 2 years and 3 months, motor and mental development are severely retarded. He has atactic-dyskinetic movements and cannot walk unassisted. No speech has developed but he laughs easily. Skin pigmentation is normal. Electroencephalogram (EEG) revealed rhythmic 4’5-6/s activity and generalised high amplitude theta complexes. One generalised astatic seizure has been recorded. Cyotgenetic analysis revealed an unbalanced chromosomal translocation in chromosomes 4 and 15. The patient’s mother carries a balanced reciprocal translocation, with breakpoints on the long arms of chromosome 4 and 15 (46, XX,t[4;15][q35.2;q13)). This translocation transposes all but the most centromeric material of the long arm of chromosome 15 onto a slightly shortened chromosome 4, thus creating a very large derivative chromosome. The small telomeric fragment of chromosome 4 is translocated to the remaining centromeric material of chromosome 15, creating a very small derivative. The breakpoint on chromosome 15 is in the region to which the Angelman’s syndrome gene has been localised.5 The patient has inherited the large maternal translocation chromosome containing most of chromosome 4 and 15 as well as normal paternal chromosomes 4 and 15. He has not inherited the small derivative and is therefore monosomic for this region (45, XY,-4,-15,+der[4]t[4;15][q35.2;q13]). The patient’s maternal grandmother and two uncles have a normal karyotype; the maternal grandfather is dead. A healthy sib has not yet been analysed. The maternally derived deletion of the centromeric part of chromosome 15 is therefore the most probable cause of the patient’s phenotype. Molecular analysis of the patient’s DNA with probes from the centromeric part of chromosome 15 confirmed the cytogenetic analysis. The patient is monosomic for the paternal allele at the microsatellite at D 15 S 11,6 while the mother is heterozygous (table). This result confirms that the patient has not inherited maternally derived material from this chromosomal region. The family is not informative at the D 15 S 10 locust which is located more telomeric than D 15 S 11, but the patient’s DNA exhibits only half the dosage at this locus compared with his parents, confirming monosomy at this locus as well. By contrast with these loci, the patient was heterozygous at the GABAA-R[&bgr;3 locus for both the microsatellite8 and the genomic probe (p28&bgr;3).4 Since this patient has symptoms of Angelman’s syndrome, the DNA analysis indicates that the deletion-critical region does not include the GABAA -R&bgr;3locus. The absence of some characteristic DNA ANALYSIS WITH PROBES DERIVED FROM PROXIMAL CHROMOSOME 15 OF ANGELMAN’S SYNDROME PATIENT AND HIS PARENTS
123
EEG findings in this patient, by comparison with most other Angehnan’s syndrome patients, is remarkble. The course will show whether the occurrence of a generalised seizure was an isolated incident. This patient indicates that loss of the maternal copy of the gene for the GABAA receptor &bgr;3 subunit is not the cause of uncontrolled behaviour and disorders of movement and, probably, also not of the seizures in Angelman’s syndrome. Extended molecular analysis of this patient should help in further delineation of the gene(s) involved in this syndrome. We thank Dr M. Lalande, Boston, for making DNA Human Genetics Institute, Free University, D-1000 Berlin 19, Germany; Anthropology and Human Genetics Institute, Department of Clinical Genetics,
University of Tubingen, Tubingen, and Children’s Clinic, University of Tubingen
probes available.
ANDRÉ REIS
JÜRGEN KUNZE LADISLAUS LADANYI HERBERT ENDERS UTE KLEIN-VOGLER GERHARD NIEMANN
1. Knoll JHM, Nicholls RD, Magenis RE, et al. Angelman and Prader-Willi syndromes share a common chromosome 15 deletion but differ m parental ongin of the deletion. Am J Med Genet 1989; 48: 285-90. 2. Saitoh S, Kubota T, Ohta T, et al. Familial Angelman syndrome caused by imprinted submicroscopic deletion encompassing GABAA receptor &bgr;3-subunit gene. Lancet
1992; 338: 366-61. Y, Imaizumi K, et al. DNA deletion and its parental origin in Angelman syndrome patients. Am JMed Genet 1991; 41: 64-68. 4. Wagstaff J, Knoll JHM, Fleming J, et al. Localization of the gene encoding the GABAA receptor &bgr;3 subunit to the Angelman/Prader-Willi region of human chromosome 15. Am J Hum Genet 1991; 49: 330-37. 5. Pembrey M, Fennell SJ, van den Berghe J, et al. The association of Angelman’s syndrome with deletion within 15q11-13. J Med Genet 1989; 26: 73-77. 6. Mutirangura A, Kuwano A, Ledbetter SA, et al. Dinucleotide repeat polymorphism at the D15S11 locus in the Angelman/Prader-Willi region (AS/PWS) of chromosome 15. Hum Molec Genet 1992; 1: 139. 7. Nicholls RD, Knoll JH, Glatt K, et al. Restriction fragment length polymorphisms within proximal 15q and their use in molecular cytogenetics and the Prader-Willi syndrome. Am J Med Genet 1989; 33: 66-77. 3. Hamabe J, Kuroki
8
Mutirangura A, Ledbetter AS, Kuwano A et al Dinucleotide repeat polymorphism at the GABAA receptor &bgr;3 (GABRB3) locus in the Angelman/Prader-Willi region
(AS/PWS) of chromosome 15. Hum Molec Genet 1992; 1: 67.
Occult HBV in NANB fulminant hepatitis SIR,-Wright et all have reported evidence for hepatitis B virus (HBV) DNA detected in liver tissue, but not serum, by the polymerase chain reaction (PCR) in 6 of 17 patients with non-A, non-B fulminant hepatic failure (NANB-FHF) who underwent orthotopic liver transplantation. Since recurrence of hepatitis B after this procedure is usual and is often associated with liver disease and early death or graft 10ss2 this finding has major implications for transplantation in patients with fulminant NANB. As part of a larger study of viruses in NANB-FHF we have looked for occult HBV in patients referred with fulminant hepatitis presumed to be NANB on the basis of negative serological markers for HBV or hepatitis A virus. 45 cases of NANB-FHF were studied. As an external control for HBV-DNA, 7 cases transplanted for cirrhosis due to chronic HBV and 2 cases of fulminant HBV were also studied. DNA was extracted from sections cut from wax-embedded, formalin-fixed liver tissue (obtained at transplantation or necropsy) by proteinase K digestion.3 Non-specific inhibitors of PCR amplification were removed by Centricon 30 ultrafiltration.4 The internal control for DNA extraction and amplification was &bgr;-globin DNA.’ For PCR we used a "hot start" protocols with primers to surface and pol regions of HBV,6 and amplified products (surface, 243 base-pairs; pol 110 base-pairs) were transferred to nylon membranes by Southern blotting and probed with 32P-labelled synthetic oligonucleotides internal to and not inclusive of the internal PCR primers. Each sample was assayed in duplicate for HBV and several negative control tissues (wax-embedded fbrmalinfixed tissue from unused donor liver) were assayed in tandem. To investigate sensitivity, a plasmid containing a single-copy, fulllength HBV DNA insert was serially diluted 1 in 10 in a digest of wax-embedded, formalin-fixed normal human liver tissue before extraction, nested amplification, and Southern blotting. 10 to 50 molecules of HBV DNA could be detected per reaction, corresponding to 40-200 molecules/10 lun liver section. In all 7 cases of chronic HBV-induced liver disease and both of
the fulminant HBV-related cases, surface and pol regions of HBV genome could readily be amplified. In no case of NANB-FHG was HBV DNA detected. Clinical and serological follow-up was available for 26 of these 45 cases (9 months to 8 years after transplantation), and only 1 developed anti-HBs and this was in association with the development of clinical hepatitis in the patient’s husband. Our findings do not support the observations of Wright et al.1 We cannot explain this disparity but differences in the background prevalence of HBV infection in the referral populations may be important. Although the catchment area of our institution is a region of high HBV prevalence within Britainthe prevalence in the San Francisco area may be even greater. The different techniques used for DNA extraction may also have contributed. In our hands, for example, amplification of hepatitis C viral RNA from formalinfixed tissue is about fivefold less efficient than amplification from fresh tissue8 (the method used by Wright et al), and we may have missed cases as a consequence. However, even from fixed tissues we could still reliably detect 10-50 copies of HBV per PCR reaction, and the clinical significance of lower levels of HBV in the context of fulminant hepatic failure is questionable. While it is conceivable that these cases represent mutant forms of HBV unable to secrete HBsAg’ and that an exaggerated immunological response is responsible for both the fulminant liver disease and the rapid clearance of viral antigens,9 the absence of HBV DNA from serum in the context of massive hepatic necrosis reported by Wright et al is surprising. A recent study from our unit has shown that some cases of NANB-FHF may be caused by hepatitis ElO but a substantial proportion, even a majority, of such cases do seem to be caused by viruses or be due to some other undefined cause. Institute of Liver Studies, King’s College School of Medicine and Dentistry, London SE5 9PJ, UK
RICHARD SALLIE ANNE RAYNER NIKOLAI NAOUMOV BERNARD PORTMANN ROGER WILLIAMS
Mamish D, Combs C, et al. Hepatitis B virus is a common cause of apparent fulminant non-A non-B hepatitis. Lancet 1992; 339: 952-55. 2. O’Grady JG, Smith HM, Davies SE, et al. Hepatitis B virus reinfection after orthotopic liver transplantation. Serological and clinical implications. J Hepatol 1.
Wnght TL,
3.
1992, 14: 104-11 Jackson DP, Lewis FA, Taylor GR, Boylston AW, Quirke P. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction. Clin Pathol 1990; J 43: 499-504
4. An
SF, Fleming KA. Removal of inhibitor(s) of the polymerase chain reaction from formalin fixed, paraffin wax embedded tissues. J Clin Pathol 1991; 44: 924-27. 5. Chou Q, Russell M, Birch D, Raymond J, Bloch W Prevention of pre-PCR mispriming and primer dimerization improves low-copy number amplifications. Nucleic Acid Res 1992; 20: 1717-23 6. Kaneko S, Feinstone S, Miller R. Rapid and sensitive detection of serum hepatitis B virus DNA using the polymerase chain reaction. J Clin Microbiol 1989; 27: 1930-33. 7. King R, Johnson PJ, White YS, Smith HM, Williams R, Frequency of hepatitis B and C in an inner city community and relation to possible risk factors. Q J Med 1991; 292: 641-49. 8. Sallie R, Rayner A, Portmann B, Eddleston A, Williams R. Detection of HCV RNA in formalin fixed, paraffin embedded liver tissue by nested polymerase chain reaction. J Med Virol 1992; 37: 310-14. 9. Gimson AE, Tedder RS, White YS, Eddleston AL, Williams R. Serological markers in fulminant hepatitis B. Gut 1983; 24: 615-17. 10. Sallie R, Silva AE, Smith H, et al. Detection of hepatitis E but not "C" in sera of patients with fulminant NANB hepatitis. Hepatology 1991, 14: 68A (abstr 81).
Transmission of Creutzfeldt-Jakob disease by handling of dura mater SIR,-Creutzfeldt- Jakob disease (CJD) can be transmitted iatrogenically by human pituitary growth hormone, corneal transplants, and dura mater grafts. Possible accidental transmission has been reported in only four people-a neurosurgeon,2 a pathologist,3 and two laboratory technicians. 4,5We have encountered an unusually rapid case of CJD probably acquired through handling of sheep and human dura mater. In May, 1992, a 55-year-old orthopaedic surgeon developed paraesthesia of the left arm. A few days later he had spatial disorientation, apraxia, and gait ataxia. In June he was admitted and a neurologist suspected CJD on the basis of the clinical signs, typical electroencephalogram (EEG) pattern, and history. An EEG in June revealed a typical pattern of periodic biphasic and triphasic sharp wave complexes. We saw the patient in July, 1992. He was awake
Exclusion of the GABAA-receptor &bgr;3 subunit gene as the Angelman’s syndrome gene SIR,-About 60% of patients with Angelman’s syndrome have a maternally inherited deletion of chromosome 15ql 1-ql 3. Less than 5% have uniparental disomy, and genomic imprinting has been proposed for this chromosomal region.1 Saitoh et aP reported a family in which a small molecular deletion on chromosome 15 had been inherited from the grandfather, through the healthy mother, to three sibs, all of whom had Angelman’s syndrome. Initial analysis of the deletion had shown it to encompass only the small region around the locus D15SlO (p3-21),3 although Saitoh and colleagues later
HLA-A locus allele-specific PCR (a) and HLA-A locus typing of B lymphoblastoid cell line HRC.304 by allele-specific PCR (b).
(a) Àsizemarkers (lane1 ); internal control reaction (&bgr;2 microglobulin, lane 2); positive allele-specific PCR for HLA-A1, A2, A3, A9, A11, A25(10), A26(10), A28, A29, A30, A31, A32, and A33 (lanes 3-15, respectively). (b) &lgr; size markers (lane 1), negative control (no DNA, lane 2), HLA-A locus allele-specific PCR for HLA-A1, A2, A3, A9, A10, A11, A28, A19(except A30) and A30 (lanes 3-11, respectively). Positive reactions, yielding allele-specific bands of predicted size, are seen in lanes 4 and 10, corresponding to the presence of H LA-A locus alleles A2 and A19(subtyped as HLA-A31, not shown) HLA-A locus alleles from DNA extracted from peripheral blood lymphocytes and from whole blood. Examples of allele-specific reactions and a representative HLA-A locus typing are shown in the figure. DNA-based tissue typing has been applied previously to alleles of HLA class II 10ci.3-S Our system for HLA class I DNA typing has several advantages over serological typing. In practical terms, the method uses renewable reagents and standard laboratory equipment, widening the scope of tissue typing for clinical or research purposes. The use of standard conditions and incorporation of internal controls would allow the method to be adapted as a kit. Since the method is based on genomic sequences, the typing obtained is definitive (within the confines of known HLA sequences). A further advantage is that the system does not require viable cells, and can be used to type material from cells that cannot be tissue typed by conventional means (eg, epithelial cells and cells with low or undetectable HLA expression). The method is a important advance in HLA typing that has implications for both histocompatibility testing for organ transplantation and HLAdisease association studies. ICRF Cancer Immunology Laboratory, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK, and Tissue Antigen Laboratory, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WC2
WF, Bodmer JG. Cytofluorochromasia for HLA-A, -B, -C and DR typing. NIAID manual of tissue typing techniques 1979-1980. Bethesda, Maryland: NIH, 1979. NIH publication no. 80-545. Newton CR, Graham A, Heptinstall LE, et al. Analysis of any point mutation in DNA. the amplification refractory mutation system (ARMS). Nucleic Acids Res 1989; 17: 2503-16. Carlsson B, Wallin J, Bohme J, Moller E. HLA-DR-DQ haplorypes defined by restriction fragment length analysis correlation to serology Hum Immunol 1987; 20: 95-113. Erlich HA, Sheldon EL, Horn G. HLA typing using DNA probes. Biotechnology 1986; 4: 975-81. Olerup O, Zetterquist H. HLA-DR typing by PCR amplification with sequencespecific primers (SSP-PCR) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens 1992; 39: 225-35.
1. Bodmer
2.
3.
4.
5
PETER KRAUSA JONATHAN MOSES WALTER BODMER JULIA BODMER MICHAEL BROWNING
showed it to also include the gene for the GABAA receptor &bgr;3 subunit.4 This chromosomal region was termed the deletion-critical region, since all deletions or unbalanced translocations causing Angelman’s syndrome involved at least these two loci. We report a 3-year-old boy with Angelman’s syndrome and an unbalanced chromosomal translocation in chromosomes 4 and 15, whose molecular characterisation differs from those so far reported. The boy was referred because of developmental delay and feeding problems. He presented with microcephaly with flat occiput, large mandible, and open mouth with protruding tongue, Now, at 2 years and 3 months, motor and mental development are severely retarded. He has atactic-dyskinetic movements and cannot walk unassisted. No speech has developed but he laughs easily. Skin pigmentation is normal. Electroencephalogram (EEG) revealed rhythmic 4’5-6/s activity and generalised high amplitude theta complexes. One generalised astatic seizure has been recorded. Cyotgenetic analysis revealed an unbalanced chromosomal translocation in chromosomes 4 and 15. The patient’s mother carries a balanced reciprocal translocation, with breakpoints on the long arms of chromosome 4 and 15 (46, XX,t[4;15][q35.2;q13)). This translocation transposes all but the most centromeric material of the long arm of chromosome 15 onto a slightly shortened chromosome 4, thus creating a very large derivative chromosome. The small telomeric fragment of chromosome 4 is translocated to the remaining centromeric material of chromosome 15, creating a very small derivative. The breakpoint on chromosome 15 is in the region to which the Angelman’s syndrome gene has been localised.5 The patient has inherited the large maternal translocation chromosome containing most of chromosome 4 and 15 as well as normal paternal chromosomes 4 and 15. He has not inherited the small derivative and is therefore monosomic for this region (45, XY,-4,-15,+der[4]t[4;15][q35.2;q13]). The patient’s maternal grandmother and two uncles have a normal karyotype; the maternal grandfather is dead. A healthy sib has not yet been analysed. The maternally derived deletion of the centromeric part of chromosome 15 is therefore the most probable cause of the patient’s phenotype. Molecular analysis of the patient’s DNA with probes from the centromeric part of chromosome 15 confirmed the cytogenetic analysis. The patient is monosomic for the paternal allele at the microsatellite at D 15 S 11,6 while the mother is heterozygous (table). This result confirms that the patient has not inherited maternally derived material from this chromosomal region. The family is not informative at the D 15 S 10 locust which is located more telomeric than D 15 S 11, but the patient’s DNA exhibits only half the dosage at this locus compared with his parents, confirming monosomy at this locus as well. By contrast with these loci, the patient was heterozygous at the GABAA-R[&bgr;3 locus for both the microsatellite8 and the genomic probe (p28&bgr;3).4 Since this patient has symptoms of Angelman’s syndrome, the DNA analysis indicates that the deletion-critical region does not include the GABAA -R&bgr;3locus. The absence of some characteristic DNA ANALYSIS WITH PROBES DERIVED FROM PROXIMAL CHROMOSOME 15 OF ANGELMAN’S SYNDROME PATIENT AND HIS PARENTS
123
EEG findings in this patient, by comparison with most other Angehnan’s syndrome patients, is remarkble. The course will show whether the occurrence of a generalised seizure was an isolated incident. This patient indicates that loss of the maternal copy of the gene for the GABAA receptor &bgr;3 subunit is not the cause of uncontrolled behaviour and disorders of movement and, probably, also not of the seizures in Angelman’s syndrome. Extended molecular analysis of this patient should help in further delineation of the gene(s) involved in this syndrome. We thank Dr M. Lalande, Boston, for making DNA Human Genetics Institute, Free University, D-1000 Berlin 19, Germany; Anthropology and Human Genetics Institute, Department of Clinical Genetics,
University of Tubingen, Tubingen, and Children’s Clinic, University of Tubingen
probes available.
ANDRÉ REIS
JÜRGEN KUNZE LADISLAUS LADANYI HERBERT ENDERS UTE KLEIN-VOGLER GERHARD NIEMANN
1. Knoll JHM, Nicholls RD, Magenis RE, et al. Angelman and Prader-Willi syndromes share a common chromosome 15 deletion but differ m parental ongin of the deletion. Am J Med Genet 1989; 48: 285-90. 2. Saitoh S, Kubota T, Ohta T, et al. Familial Angelman syndrome caused by imprinted submicroscopic deletion encompassing GABAA receptor &bgr;3-subunit gene. Lancet
1992; 338: 366-61. Y, Imaizumi K, et al. DNA deletion and its parental origin in Angelman syndrome patients. Am JMed Genet 1991; 41: 64-68. 4. Wagstaff J, Knoll JHM, Fleming J, et al. Localization of the gene encoding the GABAA receptor &bgr;3 subunit to the Angelman/Prader-Willi region of human chromosome 15. Am J Hum Genet 1991; 49: 330-37. 5. Pembrey M, Fennell SJ, van den Berghe J, et al. The association of Angelman’s syndrome with deletion within 15q11-13. J Med Genet 1989; 26: 73-77. 6. Mutirangura A, Kuwano A, Ledbetter SA, et al. Dinucleotide repeat polymorphism at the D15S11 locus in the Angelman/Prader-Willi region (AS/PWS) of chromosome 15. Hum Molec Genet 1992; 1: 139. 7. Nicholls RD, Knoll JH, Glatt K, et al. Restriction fragment length polymorphisms within proximal 15q and their use in molecular cytogenetics and the Prader-Willi syndrome. Am J Med Genet 1989; 33: 66-77. 3. Hamabe J, Kuroki
8
Mutirangura A, Ledbetter AS, Kuwano A et al Dinucleotide repeat polymorphism at the GABAA receptor &bgr;3 (GABRB3) locus in the Angelman/Prader-Willi region
(AS/PWS) of chromosome 15. Hum Molec Genet 1992; 1: 67.
Occult HBV in NANB fulminant hepatitis SIR,-Wright et all have reported evidence for hepatitis B virus (HBV) DNA detected in liver tissue, but not serum, by the polymerase chain reaction (PCR) in 6 of 17 patients with non-A, non-B fulminant hepatic failure (NANB-FHF) who underwent orthotopic liver transplantation. Since recurrence of hepatitis B after this procedure is usual and is often associated with liver disease and early death or graft 10ss2 this finding has major implications for transplantation in patients with fulminant NANB. As part of a larger study of viruses in NANB-FHF we have looked for occult HBV in patients referred with fulminant hepatitis presumed to be NANB on the basis of negative serological markers for HBV or hepatitis A virus. 45 cases of NANB-FHF were studied. As an external control for HBV-DNA, 7 cases transplanted for cirrhosis due to chronic HBV and 2 cases of fulminant HBV were also studied. DNA was extracted from sections cut from wax-embedded, formalin-fixed liver tissue (obtained at transplantation or necropsy) by proteinase K digestion.3 Non-specific inhibitors of PCR amplification were removed by Centricon 30 ultrafiltration.4 The internal control for DNA extraction and amplification was &bgr;-globin DNA.’ For PCR we used a "hot start" protocols with primers to surface and pol regions of HBV,6 and amplified products (surface, 243 base-pairs; pol 110 base-pairs) were transferred to nylon membranes by Southern blotting and probed with 32P-labelled synthetic oligonucleotides internal to and not inclusive of the internal PCR primers. Each sample was assayed in duplicate for HBV and several negative control tissues (wax-embedded fbrmalinfixed tissue from unused donor liver) were assayed in tandem. To investigate sensitivity, a plasmid containing a single-copy, fulllength HBV DNA insert was serially diluted 1 in 10 in a digest of wax-embedded, formalin-fixed normal human liver tissue before extraction, nested amplification, and Southern blotting. 10 to 50 molecules of HBV DNA could be detected per reaction, corresponding to 40-200 molecules/10 lun liver section. In all 7 cases of chronic HBV-induced liver disease and both of
the fulminant HBV-related cases, surface and pol regions of HBV genome could readily be amplified. In no case of NANB-FHG was HBV DNA detected. Clinical and serological follow-up was available for 26 of these 45 cases (9 months to 8 years after transplantation), and only 1 developed anti-HBs and this was in association with the development of clinical hepatitis in the patient’s husband. Our findings do not support the observations of Wright et al.1 We cannot explain this disparity but differences in the background prevalence of HBV infection in the referral populations may be important. Although the catchment area of our institution is a region of high HBV prevalence within Britainthe prevalence in the San Francisco area may be even greater. The different techniques used for DNA extraction may also have contributed. In our hands, for example, amplification of hepatitis C viral RNA from formalinfixed tissue is about fivefold less efficient than amplification from fresh tissue8 (the method used by Wright et al), and we may have missed cases as a consequence. However, even from fixed tissues we could still reliably detect 10-50 copies of HBV per PCR reaction, and the clinical significance of lower levels of HBV in the context of fulminant hepatic failure is questionable. While it is conceivable that these cases represent mutant forms of HBV unable to secrete HBsAg’ and that an exaggerated immunological response is responsible for both the fulminant liver disease and the rapid clearance of viral antigens,9 the absence of HBV DNA from serum in the context of massive hepatic necrosis reported by Wright et al is surprising. A recent study from our unit has shown that some cases of NANB-FHF may be caused by hepatitis ElO but a substantial proportion, even a majority, of such cases do seem to be caused by viruses or be due to some other undefined cause. Institute of Liver Studies, King’s College School of Medicine and Dentistry, London SE5 9PJ, UK
RICHARD SALLIE ANNE RAYNER NIKOLAI NAOUMOV BERNARD PORTMANN ROGER WILLIAMS
Mamish D, Combs C, et al. Hepatitis B virus is a common cause of apparent fulminant non-A non-B hepatitis. Lancet 1992; 339: 952-55. 2. O’Grady JG, Smith HM, Davies SE, et al. Hepatitis B virus reinfection after orthotopic liver transplantation. Serological and clinical implications. J Hepatol 1.
Wnght TL,
3.
1992, 14: 104-11 Jackson DP, Lewis FA, Taylor GR, Boylston AW, Quirke P. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction. Clin Pathol 1990; J 43: 499-504
4. An
SF, Fleming KA. Removal of inhibitor(s) of the polymerase chain reaction from formalin fixed, paraffin wax embedded tissues. J Clin Pathol 1991; 44: 924-27. 5. Chou Q, Russell M, Birch D, Raymond J, Bloch W Prevention of pre-PCR mispriming and primer dimerization improves low-copy number amplifications. Nucleic Acid Res 1992; 20: 1717-23 6. Kaneko S, Feinstone S, Miller R. Rapid and sensitive detection of serum hepatitis B virus DNA using the polymerase chain reaction. J Clin Microbiol 1989; 27: 1930-33. 7. King R, Johnson PJ, White YS, Smith HM, Williams R, Frequency of hepatitis B and C in an inner city community and relation to possible risk factors. Q J Med 1991; 292: 641-49. 8. Sallie R, Rayner A, Portmann B, Eddleston A, Williams R. Detection of HCV RNA in formalin fixed, paraffin embedded liver tissue by nested polymerase chain reaction. J Med Virol 1992; 37: 310-14. 9. Gimson AE, Tedder RS, White YS, Eddleston AL, Williams R. Serological markers in fulminant hepatitis B. Gut 1983; 24: 615-17. 10. Sallie R, Silva AE, Smith H, et al. Detection of hepatitis E but not "C" in sera of patients with fulminant NANB hepatitis. Hepatology 1991, 14: 68A (abstr 81).
Transmission of Creutzfeldt-Jakob disease by handling of dura mater SIR,-Creutzfeldt- Jakob disease (CJD) can be transmitted iatrogenically by human pituitary growth hormone, corneal transplants, and dura mater grafts. Possible accidental transmission has been reported in only four people-a neurosurgeon,2 a pathologist,3 and two laboratory technicians. 4,5We have encountered an unusually rapid case of CJD probably acquired through handling of sheep and human dura mater. In May, 1992, a 55-year-old orthopaedic surgeon developed paraesthesia of the left arm. A few days later he had spatial disorientation, apraxia, and gait ataxia. In June he was admitted and a neurologist suspected CJD on the basis of the clinical signs, typical electroencephalogram (EEG) pattern, and history. An EEG in June revealed a typical pattern of periodic biphasic and triphasic sharp wave complexes. We saw the patient in July, 1992. He was awake