- Email: [email protected]
BLEEDING DISORDERS
1127
THE LANCET
powerfully than old photographs or verbal reminders. A hot dry summer day and the whiff of dung can immediately transport one to India. Smell is of course allied to taste. Our appetite is created by smells of anticipation, and a good dinner succeeds by offering us a wide variety so that our taste is constantly exposed to new stimuli. So, should we sacrifice smell on the altar of atmospheric purity? After all, serious atmospheric threats to our health such as carbon monoxide, asbestos, and radon are odourless. And speaking personally, I like "people smell". The smell of young babies and of small children, the smell of some men and many women (if not drowned in cheap scent). Among the worst smells are the artificial odours that are pumped into the air to overpower the wholesome smells of farts and faeces, of sweat and stout. Even the stink of silage and pig slurry are short lived in their effect and are part of the rich world of smell. There is also a question of pheromones. Would Fanger and his disciples deny me the subliminal excitement to which I may all unwittingly be exposed in my dealings with the opposite, and for all I know, the same sex? In fairness, Fanger’s concern is not with the great outdoors but the indoor office environment. I doubt whether he would deny us delicious smells such as old fashioned roses, hyacinths, new-mown hay, linseed oil, a new tennis and an old cricket ball, wet dogs and wet tweeds, salt marshes and sea. Smells are sensual and like all sensual experience a cause of intense pleasure and occasional pain. To my mind smells are not pollution. May we be preserved from those who wish to sanitise our environment on the basis of man meters.
Department of Health duly found that 40% of children at the school had the black spots, but siblings at other schools had no spots. When children graduated to the middle school, in a different building, they lost their spots. Scalp scrapings from the spots were analysed by gas-liquid chromatography and infrared spectroscopy and the pigment proved to be a complex mixture of long-chain hydrocarbons resembling tar. The school roof was in poor repair, with large exposed patches of torn tar-paper. Small black flakes eddied in the dusty wind in the playground. Chromatographic analysis of the roof tar and the black dust showed that they were identical to the tar spots on the scalps. In the best traditions of Koch, tar-dust was sprinkled on the scalp of an investigator, who then exercised vigorously. Particles trapped by the hair dissolved in the skin lipid and sweat to stain the scalp and reproduce the "disease". But why are black scalp spots apparently confined to
certain mountainous
states
in the western USA? As with so
many other
epidemic diseases, climatic factors provide an explanation. In areas with little rain, roof repairs are infrequent. High-altitude ultraviolet radiation degrades the exposed tar, and the strong and consistent winter winds of the Wyoming wind corridor then disperse the aetiological agent in the community. John L. Burton 1.
2.
Spitzer WO, Dales R, Schechter MT, Tousignant P, Hutcheon M. Subjective fears and objective data: an epidemiologic study of environmental health concerns. Trans Assoc Am Phys 1987; 100: 40-41. Cobb N, Etzel Ruth A, Hudson R. Black spots on the scalps of schoolchildren: a recurrent condition in the windy West. Western Med J 1993; 158: 139-41.
James McCormick 1. Shusterman D. Community health and odor pollution regulation. Am J Publ Health 1992; 82: 1566-67. 2. Fanger PO. Perceived quality of indoor and ambient air. Proceedings of the Indoor Ambient Air Quality Conference, London, 1988: 365-76.
BLEEDING DISORDERS
Haemophilia DERMATOLOGY
Epidemic black spots on the scalp All public health physicians know that clusters of an unusual disease in a small area can generate communal anxiety quite out of proportion to the morbidity of the diseased All the more so with a new condition that affects many children, and when the cause is unknown. This situation arose over a ten-year period in certain townships in the Rocky Mountain states in the USA, where epidemics of black spots developed on the scalps of schoolchildren. Several communities were affected, and in the largest epidemic 1380 cases were recorded. The lesions were black or brown macules, 1-5 mm in diameter. Those affected had from 1 to 20 spots on the scalp, but nowhere else, and they were otherwise well. Suggested diagnoses included tinea nigra, macula cerulea (due to flea bites), naevi, and "dirt", but confirmation was lacking despite examination of skin biopsy specimens (no abnormalities seen). In the latest epidemic in Green River, Wyoming, in 1989, only one school of nine in the district reported cases and parents were advised by the local health authority to keep their children at home when they had the spots. Parental pressure prompted pigmentary probing.2 The Wyoming
B and factor IX mutations
Haemophilia B or Christmas disease is an X-linked recessive disorder caused by mutations of the gene encoding coagulation factor IX, a 415 aminoacid glycoprotein. Clinically the disorder ranges from the severe form, with absent factor IX coagulant activity accompanied by recurrent spontaneous haemarthroses, to a mild variety, with excessive bleeding only after surgery.1 The heterogeneous nature of this disease reflects the vast array of causal mutations in the factor IX gene, most of which affect the aminoacid sequence of the protein.2 A few of the described mutations result in normal factor IX protein structure but abnormally low levels of expression. Haemophilia B-Leyden is one such rare variant. First identified in a Dutch family, the disease is characterised by severe haemophilia during childhood which improves to a mild symptomless condition after puberty.3 Plasma factor IX concentrations in such patients increase from low or of normal) to around 50% after undetectable ( < 1 % Twelve such mutations have been identified as puberty. causing haemophilia B-Leyden, all localised to the promoter region (nucleotides - 20, - 6, - 5, + 8, and + 13 relative to the start site of transcription) of the factor IX gene.2 Crossley et al4 lately described another Leyden-like mutation (G to C at position - 26) within this promoter region; however, this patient (haemophilia B-Brandenburg) showed no clinical improvement after puberty.
1128
THE LANCET
regions are stretches of DNA in the start-site of transcription that help to control RNA synthesis. Control is exerted by the binding of proteins (trans-activators such as transcription factors or steroid receptors) to specific recognition sequences (cisactivators) on the DNA: this process influences the ability of RNA polymerase II to initiate transcription. Numerous transcriptionally active proteins have been described, and many are able to alter expression of different genes owing to the presence of recognition sequences in the promoter regions of those genes.5 Mutations within such recognition sequences that affect the binding of the protein to the promoter have the potential to disrupt transcriptional regulation and hence gene expression. The occurrence of a range of promoter mutations within the factor IX gene with different phenotypic expression offers a rare glimpse of how normal regulation is effected in vivo. Crossley et al found that the promoter region of factor IX contains the recognition sequence for a liver-enriched transcription factor, HNF-4.6 In a series of in-vitro studies this group showed that both a Leyden mutation (T to A at position - 20 in the factor IX promoter) and the Brandenburg mutation disrupted the binding of this protein to the factor IX promoter and severely inhibited transcription from this promoter in liver-derived HepG2 cells. Close examination of the data suggests that there is a quantitative difference in the disruption caused by these two mutations (the Leyden mutation seems to reduce the affinity of the HNF-4 protein for the promoter whereas the Brandenburg mutation abolishes binding completely). The improvement of haemophilia B-Leyden (but not Brandenburg) patients after puberty, and the observed improvements of factor IX concentrations and symptoms after treatment with androgenic and anabolic steroids,3,7,7 prompted the investigators to examine the promoter sequence for an androgen response element (ARE, a DNA sequence capable of binding the androgen receptor and affecting transcription rates). The putative ARE that they found was capable of binding the androgen receptor in vitro, and its sequence partly overlapped with that of the HNF-4 recognition sequence. The sequence overlap was such that the Leyden mutation z20) did not affect androgen receptor binding, but the Brandenburg mutation z26) caused a reduction in binding. These observations accord with the view that, in prepubertal life, factor IX is driven by HNF-4, and that both the Leyden and Brandenburg mutations prevent this protein from binding to the promoter, leading to reduced transcription rates and low protein concentrations. As the Leyden mutation lies outwith the ARE, any androgenmediated increase in transcription would be unaffected. By contrast, the Brandenburg mutation, by interfering with androgen-receptor binding, could prevent any androgendriven pubertal rise. Unfortunately, to show significant androgen-driven transcription, the researchers had to use a DNA construct containing four of these AREs in series; this suggests either that the transcription assay was unreliable or that additional elements may be required for the full transcriptional response. Another report also emphasises the importance of HNF-4 in haemophilia B-Leyden. Comparing the same two mutations, Reijnen et al8 reported that HNF-4 was responsible for the tissue (liver) specific expression of factor IX (as it is for several other reported genes), and that the binding of this protein to the promoter was disrupted by these mutations. This group also reported a quantitative Gene promoter
proximity
to
difference in the effects of these two mutations on HNF-4driven transcription: the Leyden mutation caused an 80% decrease in transcriptional activity whereas the Brandenburg mutation abolished it completely. This result accords with the apparent quantitative difference in HNF-4 binding to the two mutated promoters in the report by Crossley et al. Together, these studies suggest that factor IX expression is absolutely dependent on HNF-4 transactivation, the Leyden mutation reducing this binding to such an extent that only when additional (androgen-dependent) transactivation occurs at puberty is there any significant expression. The Brandenburg mutation, by completely abolishing HNF-4 binding, and possibly also by interfering with androgen-receptor binding, prevents even
postpubertal transcription. There are clearly other transcription factors involved in factor IX expression: mutations at + 13 abolish transactivation by CCAAT/enhancer-binding protein (C/EPB) while mutations at - 6 inhibit transactivation by an as yet unknown mechanism.2,9 Knowledge of how these factors interact to control transcription will not only shed light on the aetiology of haemophilia B-Leyden but also will provide valuable insights into the interaction of tissuespecific, hormone-dependent, and constitutive control of transcription in general. David Stirling Christopher A. Ludlam 1. Brownlee GG.
Hemophilia B: a review of patient defects, diagnosis with
gene probes and prospects for gene therapy. Recent Adv Hematol 1989; 5: 251-64. 2. Giannelli F, Green PM, High KA, et al. Haemophilia B: database of point mutations and short deletions—second edition. Nucleic Acids Res 1991; 19: 2193-211. 3. Veltkamp JJ, Meilof J, Remmelts HG, Van Der Vlerk D, Loeliger EA. Another genetic variant of haemophilia B; haemophilia B Leyden. Scand J Haematol 1970; 7: 82-90. 4. Crossley M, Ludwig M, Stowell KM, De Vos P, Olek K, Brownlee GG. Recovery from hemophilia B Leyden: an androgen-responsive element in the factor IX promoter. Science 1992; 257: 377-79. 5. Alberts B. Control of gene expression. In: Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD, eds. Molecular biology of the cell. New York: Garland, 1989: 551-612. 6. Ramji DP, Tadros MH, Hardon EM, Cortese R. The transcription factor LF-a1 interacts with a bipartate recognition sequence in the promoter region of several liver specific genes. Nucleic Acids Res 1991; 19: 1139-46. 7. Briet E, Wijnands MC, Veltkamp JJ. The prophylactic treatment of hemophilia B Leyden with anabolic steroids. Ann Intern Med 1985; 103: 225-26. 8. Reijnen MJ, Sladek FM, Bertina RM, Reitsma PH. Disruption of a binding site for hepatocyte nuclear factor 4 results in hemophilia B Leyden. Proc Natl Acad Sci USA 1992; 89: 6300 - 03. 9. Crossley M, Brownlee GG. Disruption of a C/EBP binding site in the factor IX promoter is associated with haemophilia B. Nature 1990; 345: 444-46.
Prof David B. Barnett, Department of Pharmacology and Therapeutics, Leicester Royal Infirmary, UK Prof
E.
R.
te
Velde,
Gynaecology,
Department of Obstetrics and Hospital Utrecht, the
University
Netherlands
Prof James McCormick, Department of Community Health and General Practice, Trinity College, Dublin, Ireland
Department of Dermatology, Bristol Royal Infirmary, Bristol, UK David Stirling and Dr Christopher A. Ludlam, Department of Haematology, Royal Infirmary, Edinburgh, UK
Dr John L. Burton,
Dr
THE LANCET
powerfully than old photographs or verbal reminders. A hot dry summer day and the whiff of dung can immediately transport one to India. Smell is of course allied to taste. Our appetite is created by smells of anticipation, and a good dinner succeeds by offering us a wide variety so that our taste is constantly exposed to new stimuli. So, should we sacrifice smell on the altar of atmospheric purity? After all, serious atmospheric threats to our health such as carbon monoxide, asbestos, and radon are odourless. And speaking personally, I like "people smell". The smell of young babies and of small children, the smell of some men and many women (if not drowned in cheap scent). Among the worst smells are the artificial odours that are pumped into the air to overpower the wholesome smells of farts and faeces, of sweat and stout. Even the stink of silage and pig slurry are short lived in their effect and are part of the rich world of smell. There is also a question of pheromones. Would Fanger and his disciples deny me the subliminal excitement to which I may all unwittingly be exposed in my dealings with the opposite, and for all I know, the same sex? In fairness, Fanger’s concern is not with the great outdoors but the indoor office environment. I doubt whether he would deny us delicious smells such as old fashioned roses, hyacinths, new-mown hay, linseed oil, a new tennis and an old cricket ball, wet dogs and wet tweeds, salt marshes and sea. Smells are sensual and like all sensual experience a cause of intense pleasure and occasional pain. To my mind smells are not pollution. May we be preserved from those who wish to sanitise our environment on the basis of man meters.
Department of Health duly found that 40% of children at the school had the black spots, but siblings at other schools had no spots. When children graduated to the middle school, in a different building, they lost their spots. Scalp scrapings from the spots were analysed by gas-liquid chromatography and infrared spectroscopy and the pigment proved to be a complex mixture of long-chain hydrocarbons resembling tar. The school roof was in poor repair, with large exposed patches of torn tar-paper. Small black flakes eddied in the dusty wind in the playground. Chromatographic analysis of the roof tar and the black dust showed that they were identical to the tar spots on the scalps. In the best traditions of Koch, tar-dust was sprinkled on the scalp of an investigator, who then exercised vigorously. Particles trapped by the hair dissolved in the skin lipid and sweat to stain the scalp and reproduce the "disease". But why are black scalp spots apparently confined to
certain mountainous
states
in the western USA? As with so
many other
epidemic diseases, climatic factors provide an explanation. In areas with little rain, roof repairs are infrequent. High-altitude ultraviolet radiation degrades the exposed tar, and the strong and consistent winter winds of the Wyoming wind corridor then disperse the aetiological agent in the community. John L. Burton 1.
2.
Spitzer WO, Dales R, Schechter MT, Tousignant P, Hutcheon M. Subjective fears and objective data: an epidemiologic study of environmental health concerns. Trans Assoc Am Phys 1987; 100: 40-41. Cobb N, Etzel Ruth A, Hudson R. Black spots on the scalps of schoolchildren: a recurrent condition in the windy West. Western Med J 1993; 158: 139-41.
James McCormick 1. Shusterman D. Community health and odor pollution regulation. Am J Publ Health 1992; 82: 1566-67. 2. Fanger PO. Perceived quality of indoor and ambient air. Proceedings of the Indoor Ambient Air Quality Conference, London, 1988: 365-76.
BLEEDING DISORDERS
Haemophilia DERMATOLOGY
Epidemic black spots on the scalp All public health physicians know that clusters of an unusual disease in a small area can generate communal anxiety quite out of proportion to the morbidity of the diseased All the more so with a new condition that affects many children, and when the cause is unknown. This situation arose over a ten-year period in certain townships in the Rocky Mountain states in the USA, where epidemics of black spots developed on the scalps of schoolchildren. Several communities were affected, and in the largest epidemic 1380 cases were recorded. The lesions were black or brown macules, 1-5 mm in diameter. Those affected had from 1 to 20 spots on the scalp, but nowhere else, and they were otherwise well. Suggested diagnoses included tinea nigra, macula cerulea (due to flea bites), naevi, and "dirt", but confirmation was lacking despite examination of skin biopsy specimens (no abnormalities seen). In the latest epidemic in Green River, Wyoming, in 1989, only one school of nine in the district reported cases and parents were advised by the local health authority to keep their children at home when they had the spots. Parental pressure prompted pigmentary probing.2 The Wyoming
B and factor IX mutations
Haemophilia B or Christmas disease is an X-linked recessive disorder caused by mutations of the gene encoding coagulation factor IX, a 415 aminoacid glycoprotein. Clinically the disorder ranges from the severe form, with absent factor IX coagulant activity accompanied by recurrent spontaneous haemarthroses, to a mild variety, with excessive bleeding only after surgery.1 The heterogeneous nature of this disease reflects the vast array of causal mutations in the factor IX gene, most of which affect the aminoacid sequence of the protein.2 A few of the described mutations result in normal factor IX protein structure but abnormally low levels of expression. Haemophilia B-Leyden is one such rare variant. First identified in a Dutch family, the disease is characterised by severe haemophilia during childhood which improves to a mild symptomless condition after puberty.3 Plasma factor IX concentrations in such patients increase from low or of normal) to around 50% after undetectable ( < 1 % Twelve such mutations have been identified as puberty. causing haemophilia B-Leyden, all localised to the promoter region (nucleotides - 20, - 6, - 5, + 8, and + 13 relative to the start site of transcription) of the factor IX gene.2 Crossley et al4 lately described another Leyden-like mutation (G to C at position - 26) within this promoter region; however, this patient (haemophilia B-Brandenburg) showed no clinical improvement after puberty.
1128
THE LANCET
regions are stretches of DNA in the start-site of transcription that help to control RNA synthesis. Control is exerted by the binding of proteins (trans-activators such as transcription factors or steroid receptors) to specific recognition sequences (cisactivators) on the DNA: this process influences the ability of RNA polymerase II to initiate transcription. Numerous transcriptionally active proteins have been described, and many are able to alter expression of different genes owing to the presence of recognition sequences in the promoter regions of those genes.5 Mutations within such recognition sequences that affect the binding of the protein to the promoter have the potential to disrupt transcriptional regulation and hence gene expression. The occurrence of a range of promoter mutations within the factor IX gene with different phenotypic expression offers a rare glimpse of how normal regulation is effected in vivo. Crossley et al found that the promoter region of factor IX contains the recognition sequence for a liver-enriched transcription factor, HNF-4.6 In a series of in-vitro studies this group showed that both a Leyden mutation (T to A at position - 20 in the factor IX promoter) and the Brandenburg mutation disrupted the binding of this protein to the factor IX promoter and severely inhibited transcription from this promoter in liver-derived HepG2 cells. Close examination of the data suggests that there is a quantitative difference in the disruption caused by these two mutations (the Leyden mutation seems to reduce the affinity of the HNF-4 protein for the promoter whereas the Brandenburg mutation abolishes binding completely). The improvement of haemophilia B-Leyden (but not Brandenburg) patients after puberty, and the observed improvements of factor IX concentrations and symptoms after treatment with androgenic and anabolic steroids,3,7,7 prompted the investigators to examine the promoter sequence for an androgen response element (ARE, a DNA sequence capable of binding the androgen receptor and affecting transcription rates). The putative ARE that they found was capable of binding the androgen receptor in vitro, and its sequence partly overlapped with that of the HNF-4 recognition sequence. The sequence overlap was such that the Leyden mutation z20) did not affect androgen receptor binding, but the Brandenburg mutation z26) caused a reduction in binding. These observations accord with the view that, in prepubertal life, factor IX is driven by HNF-4, and that both the Leyden and Brandenburg mutations prevent this protein from binding to the promoter, leading to reduced transcription rates and low protein concentrations. As the Leyden mutation lies outwith the ARE, any androgenmediated increase in transcription would be unaffected. By contrast, the Brandenburg mutation, by interfering with androgen-receptor binding, could prevent any androgendriven pubertal rise. Unfortunately, to show significant androgen-driven transcription, the researchers had to use a DNA construct containing four of these AREs in series; this suggests either that the transcription assay was unreliable or that additional elements may be required for the full transcriptional response. Another report also emphasises the importance of HNF-4 in haemophilia B-Leyden. Comparing the same two mutations, Reijnen et al8 reported that HNF-4 was responsible for the tissue (liver) specific expression of factor IX (as it is for several other reported genes), and that the binding of this protein to the promoter was disrupted by these mutations. This group also reported a quantitative Gene promoter
proximity
to
difference in the effects of these two mutations on HNF-4driven transcription: the Leyden mutation caused an 80% decrease in transcriptional activity whereas the Brandenburg mutation abolished it completely. This result accords with the apparent quantitative difference in HNF-4 binding to the two mutated promoters in the report by Crossley et al. Together, these studies suggest that factor IX expression is absolutely dependent on HNF-4 transactivation, the Leyden mutation reducing this binding to such an extent that only when additional (androgen-dependent) transactivation occurs at puberty is there any significant expression. The Brandenburg mutation, by completely abolishing HNF-4 binding, and possibly also by interfering with androgen-receptor binding, prevents even
postpubertal transcription. There are clearly other transcription factors involved in factor IX expression: mutations at + 13 abolish transactivation by CCAAT/enhancer-binding protein (C/EPB) while mutations at - 6 inhibit transactivation by an as yet unknown mechanism.2,9 Knowledge of how these factors interact to control transcription will not only shed light on the aetiology of haemophilia B-Leyden but also will provide valuable insights into the interaction of tissuespecific, hormone-dependent, and constitutive control of transcription in general. David Stirling Christopher A. Ludlam 1. Brownlee GG.
Hemophilia B: a review of patient defects, diagnosis with
gene probes and prospects for gene therapy. Recent Adv Hematol 1989; 5: 251-64. 2. Giannelli F, Green PM, High KA, et al. Haemophilia B: database of point mutations and short deletions—second edition. Nucleic Acids Res 1991; 19: 2193-211. 3. Veltkamp JJ, Meilof J, Remmelts HG, Van Der Vlerk D, Loeliger EA. Another genetic variant of haemophilia B; haemophilia B Leyden. Scand J Haematol 1970; 7: 82-90. 4. Crossley M, Ludwig M, Stowell KM, De Vos P, Olek K, Brownlee GG. Recovery from hemophilia B Leyden: an androgen-responsive element in the factor IX promoter. Science 1992; 257: 377-79. 5. Alberts B. Control of gene expression. In: Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD, eds. Molecular biology of the cell. New York: Garland, 1989: 551-612. 6. Ramji DP, Tadros MH, Hardon EM, Cortese R. The transcription factor LF-a1 interacts with a bipartate recognition sequence in the promoter region of several liver specific genes. Nucleic Acids Res 1991; 19: 1139-46. 7. Briet E, Wijnands MC, Veltkamp JJ. The prophylactic treatment of hemophilia B Leyden with anabolic steroids. Ann Intern Med 1985; 103: 225-26. 8. Reijnen MJ, Sladek FM, Bertina RM, Reitsma PH. Disruption of a binding site for hepatocyte nuclear factor 4 results in hemophilia B Leyden. Proc Natl Acad Sci USA 1992; 89: 6300 - 03. 9. Crossley M, Brownlee GG. Disruption of a C/EBP binding site in the factor IX promoter is associated with haemophilia B. Nature 1990; 345: 444-46.
Prof David B. Barnett, Department of Pharmacology and Therapeutics, Leicester Royal Infirmary, UK Prof
E.
R.
te
Velde,
Gynaecology,
Department of Obstetrics and Hospital Utrecht, the
University
Netherlands
Prof James McCormick, Department of Community Health and General Practice, Trinity College, Dublin, Ireland
Department of Dermatology, Bristol Royal Infirmary, Bristol, UK David Stirling and Dr Christopher A. Ludlam, Department of Haematology, Royal Infirmary, Edinburgh, UK
Dr John L. Burton,
Dr