418
These results confirm that in the blood of most CML patients can still be detected even 6 years after bone marrow transplantation. The clinical significance of this finding is not yet known. Experiments are in progess to find out in which cells (myeloid or lymphoid) the residual transcripts are expressed. It is possible to use PCR to evaluate residual bcrlabl transcripts provided strict precautions are taken to avoid contamination.
bcrjabl transcripts
J. GABERT Department of Cell and Molecular Biology, and Department of Haematology,
I. THURET Y. CARCASSONNE D. MARANINCHI P. MANNONI
Institut Paoli-Calmettes, 13273 Marseilles, France, and INSERM Unit 119, Marseille
JA, Thuret I, Lafage M, Carcassonne Y, Maraninchi D, Mannoni P. Detection of residual bcr/abl translocation by polymerase chain reaction in chronic myeloid leukaemia patients after bone-marrow transplantation. Lancet 1989; i: 1125-28. 2. Lange W, Snyder DS, Castro R, et al. Detection by enzymatic amplification of bcr/abl mRNA in peripheral blood and bone marrow cells of patients with chronic myelogenous leukemia. Blood 1989; 73: 1735-41. 3. Morgan GJ, Hughes T, Janssen JWG, et al. Polymerase chain reaction for the detection of residual leukaemia. Lancet 1989; i: 928. 1. Gabert
"Founder" effect in different families with haemophilia B mutation mutation is found in the factor IX genes of different families with haemophilia B, nearly all have occurred as C to T or G to A transitions within CG dinucleotides.1 A notable exception is a T to C mutation at codon 397 resulting in an Ile to Thr substitution, first identified in a patient in VancouverThis defect has been reported in eleven distinct families and four more are added in this report. As an alternative to multiple mutations, inheritance of the same mutation, especially in clinically milder, sex-linked defects, might be due to a common, prolific, distant ancestor. By silent passage through carrier women, the family histories could be negative. Haplotype analysis of affected members from seven families has been performed and the defect has been demonstrated in the mother of an affected member in which the history was of a sporadic occurrence. The four new patients (A-D) are compared with the original one (E2) and two others (Fand G4) in the table. No patient was known to be related; none exhibited spontaneous bleeding episodes; and all have "moderately" severe haemophilia B clinically. Baseline clotting activities and antigen levels vary by as much as twofold, presumably reflecting variable expression of the factor IX gene or whatever accounts for the threefold range of levels found among normal subjects. All seven patients have the common polymorphic haplotypes that are in strong linkage disequilibrium1—namely, TaqI-(intron 4), DdeI-(intron 1), XmnI-(intron 3), and the Thr type from codon 148 (exon 6). The four tested also share the common HhaI + restriction site (5 prime to exon 8), which is not linked to the above markers.5 Of greater significance, all seven have the uncommon BamHI + cleavage site 3’ to exon 1, as in the Vancouver patient." The BamHI + allele is only present in 3-5%
SIR,-When the
same
of factor IX genes in white populations. Thus, the combined probability of the BamHI marker occurring in seven different families is less than one in 109. It is most likely that the Ile 397 to Thr mutation occurred in a common ancestor in all seven families. Four additional families have been described with identical haplotypes, although BamHI was not studied.6 Furthermore, the study stated that the Ala-148 allele was found in all four. Since Ala-148 is less common than the Thr type, and since only the more common polymorphic types seem to have been found, it is r -)t clear whether or not these patients share the haplotype found in our series. Since the BamH-I cleavage site is rare, it will be of greater significance to see if these four6,’ or the three French patients8 share this cleavage site. In any event, a founder effect explains at least seven occurrences of moderately severe haemophilia B due to an Ile 397 to Thr substitution.
Supported by National Institutes of Health (HL-31193), American Heart Association (88-0805), and clinical research grant (6-463) from March of Dimes Birth Defects Foundation. Puget Sound Blood Center, and Department of Medicine, University of Washington, Seattle, Washington 98104, USA
ARTHUR R. THOMPSON
Department of Medicine, St Louis University
S. PAUL BAJAJ
Department of Pediatrics, University of Washington
SHI-HAN CHEN
Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada
R. T. A. MACGILLIVRAY
Thompson AR. Molecular biology of the hemophilias. Prog Hemostas Thrombos (in press). 2. Geddes VA, Le Bonniec BF, Louie GV, et al. A moderate form of hemophilia B is caused by a novel mutation in the protease domain of factor IX-Vancouver. J Biol 1.
Chem 1989; 264: 4689-97 3. Ware J, Davis L, Frazier D, Bajaj
SP, Stafford DW. Genetic defect responsible for the dysfunctional protein: factor IX-Long Beach. Blood 1988; 72: 820-22. 4. Spitzer SG, Warn-Cramer BJ, Kasper CK, Bajaj SP. Replacement of 397 Ile by Thr in the clotting protease factor IXa (Los Angeles and Long Beach variants) affects macromolecular catalysis but non L-tosylarginine methyl ester hydrolysis. Lack of correlation between the ox-brain prothrombin time and the mutation site in the variant proteins. Biochem J (in press). 5. Winship PR, Rees DJG, Alkan M. Detection of polymorphisms at cytosine phosphoguanidine dinucleotides and diagnosis of haemophilia B carriers. Lancet 1989; i: 631-34. CDK, Koeberl DD, Sommer SS. Direct carrier testing in 14 familis with haemophilia B. Lancet 1989; ii: 526-29 7. Koeberl DD, Bottema CDK, Beurstedde J-M, Sommer SS. Functionally important regions of the factor IX gene have a low rate of polymorphism and a high rate of mutations m the dinucleotide CpG. Am J Hum Genet 1989; 45: 448-57. 8. Attree O, Vidaud D, Vidaud M, Amselem S, Lavergne J-M, Goossens M. Mutations in the catalytic domain of human coagulation factor IX: rapid characterization by 6. Bottema
direct genomic sequencing of DNA fragments displaying an altered melting behavior. Genomics 1989; 4: 266-72. 9. Thompson AR, Chen S-H, Smith KJ. Diagnostic role of an immunoassay-detected polymorphism of factor IX for potential carriers of hemophilia B. Blood 1988; 72: 1633-38. 10. Hay CW, Robertson KA, Young SL, Thompson AR, Growe GH, MacGillivray RTA. Use of BamHI polymorphism in the factor IX gene for the determination of hemophilia B carrier status. Blood 1986; 67: 1508-11.
PATIENTS FROM SEVEN FAMILIES WITH HAEMOPHILIA B AND AN ILE 397 TO THR SUBSTITUTION
Ages at time of blood sample
Factor IX levels previously descri’bedlfor parents A-E. Polymorph isms were distinguished by Southern blotting polymerase chain reaction,’ insertion for Codon 148 The middle as
+=presence and - =absence of restriction
or
sites are in strong linkage (or Ddel) type was distinguished by Immunoassay.9 disequilibrium, the other two are in equilibrium To identify defects in patients A-D a 3’ portion of coding region of exon 8 was amplified and sequenced *Patient E shown by Southern blotting to have BamHI site,10 other patients’ DNAs tested for this site by amplrficanon. tSporadic=no previously known affected member for at least two generations (except C, adopted), patient B has two affected brothers (one a twm) but his mother’s family history is negative, including her grandmother’s family Patient D’s mother was available for testing and she was heterozygous for both the codon 397 defect and theBamHl polymorphism site
These results confirm that in the blood of most CML patients can still be detected even 6 years after bone marrow transplantation. The clinical significance of this finding is not yet known. Experiments are in progess to find out in which cells (myeloid or lymphoid) the residual transcripts are expressed. It is possible to use PCR to evaluate residual bcrlabl transcripts provided strict precautions are taken to avoid contamination.
bcrjabl transcripts
J. GABERT Department of Cell and Molecular Biology, and Department of Haematology,
I. THURET Y. CARCASSONNE D. MARANINCHI P. MANNONI
Institut Paoli-Calmettes, 13273 Marseilles, France, and INSERM Unit 119, Marseille
JA, Thuret I, Lafage M, Carcassonne Y, Maraninchi D, Mannoni P. Detection of residual bcr/abl translocation by polymerase chain reaction in chronic myeloid leukaemia patients after bone-marrow transplantation. Lancet 1989; i: 1125-28. 2. Lange W, Snyder DS, Castro R, et al. Detection by enzymatic amplification of bcr/abl mRNA in peripheral blood and bone marrow cells of patients with chronic myelogenous leukemia. Blood 1989; 73: 1735-41. 3. Morgan GJ, Hughes T, Janssen JWG, et al. Polymerase chain reaction for the detection of residual leukaemia. Lancet 1989; i: 928. 1. Gabert
"Founder" effect in different families with haemophilia B mutation mutation is found in the factor IX genes of different families with haemophilia B, nearly all have occurred as C to T or G to A transitions within CG dinucleotides.1 A notable exception is a T to C mutation at codon 397 resulting in an Ile to Thr substitution, first identified in a patient in VancouverThis defect has been reported in eleven distinct families and four more are added in this report. As an alternative to multiple mutations, inheritance of the same mutation, especially in clinically milder, sex-linked defects, might be due to a common, prolific, distant ancestor. By silent passage through carrier women, the family histories could be negative. Haplotype analysis of affected members from seven families has been performed and the defect has been demonstrated in the mother of an affected member in which the history was of a sporadic occurrence. The four new patients (A-D) are compared with the original one (E2) and two others (Fand G4) in the table. No patient was known to be related; none exhibited spontaneous bleeding episodes; and all have "moderately" severe haemophilia B clinically. Baseline clotting activities and antigen levels vary by as much as twofold, presumably reflecting variable expression of the factor IX gene or whatever accounts for the threefold range of levels found among normal subjects. All seven patients have the common polymorphic haplotypes that are in strong linkage disequilibrium1—namely, TaqI-(intron 4), DdeI-(intron 1), XmnI-(intron 3), and the Thr type from codon 148 (exon 6). The four tested also share the common HhaI + restriction site (5 prime to exon 8), which is not linked to the above markers.5 Of greater significance, all seven have the uncommon BamHI + cleavage site 3’ to exon 1, as in the Vancouver patient." The BamHI + allele is only present in 3-5%
SIR,-When the
same
of factor IX genes in white populations. Thus, the combined probability of the BamHI marker occurring in seven different families is less than one in 109. It is most likely that the Ile 397 to Thr mutation occurred in a common ancestor in all seven families. Four additional families have been described with identical haplotypes, although BamHI was not studied.6 Furthermore, the study stated that the Ala-148 allele was found in all four. Since Ala-148 is less common than the Thr type, and since only the more common polymorphic types seem to have been found, it is r -)t clear whether or not these patients share the haplotype found in our series. Since the BamH-I cleavage site is rare, it will be of greater significance to see if these four6,’ or the three French patients8 share this cleavage site. In any event, a founder effect explains at least seven occurrences of moderately severe haemophilia B due to an Ile 397 to Thr substitution.
Supported by National Institutes of Health (HL-31193), American Heart Association (88-0805), and clinical research grant (6-463) from March of Dimes Birth Defects Foundation. Puget Sound Blood Center, and Department of Medicine, University of Washington, Seattle, Washington 98104, USA
ARTHUR R. THOMPSON
Department of Medicine, St Louis University
S. PAUL BAJAJ
Department of Pediatrics, University of Washington
SHI-HAN CHEN
Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada
R. T. A. MACGILLIVRAY
Thompson AR. Molecular biology of the hemophilias. Prog Hemostas Thrombos (in press). 2. Geddes VA, Le Bonniec BF, Louie GV, et al. A moderate form of hemophilia B is caused by a novel mutation in the protease domain of factor IX-Vancouver. J Biol 1.
Chem 1989; 264: 4689-97 3. Ware J, Davis L, Frazier D, Bajaj
SP, Stafford DW. Genetic defect responsible for the dysfunctional protein: factor IX-Long Beach. Blood 1988; 72: 820-22. 4. Spitzer SG, Warn-Cramer BJ, Kasper CK, Bajaj SP. Replacement of 397 Ile by Thr in the clotting protease factor IXa (Los Angeles and Long Beach variants) affects macromolecular catalysis but non L-tosylarginine methyl ester hydrolysis. Lack of correlation between the ox-brain prothrombin time and the mutation site in the variant proteins. Biochem J (in press). 5. Winship PR, Rees DJG, Alkan M. Detection of polymorphisms at cytosine phosphoguanidine dinucleotides and diagnosis of haemophilia B carriers. Lancet 1989; i: 631-34. CDK, Koeberl DD, Sommer SS. Direct carrier testing in 14 familis with haemophilia B. Lancet 1989; ii: 526-29 7. Koeberl DD, Bottema CDK, Beurstedde J-M, Sommer SS. Functionally important regions of the factor IX gene have a low rate of polymorphism and a high rate of mutations m the dinucleotide CpG. Am J Hum Genet 1989; 45: 448-57. 8. Attree O, Vidaud D, Vidaud M, Amselem S, Lavergne J-M, Goossens M. Mutations in the catalytic domain of human coagulation factor IX: rapid characterization by 6. Bottema
direct genomic sequencing of DNA fragments displaying an altered melting behavior. Genomics 1989; 4: 266-72. 9. Thompson AR, Chen S-H, Smith KJ. Diagnostic role of an immunoassay-detected polymorphism of factor IX for potential carriers of hemophilia B. Blood 1988; 72: 1633-38. 10. Hay CW, Robertson KA, Young SL, Thompson AR, Growe GH, MacGillivray RTA. Use of BamHI polymorphism in the factor IX gene for the determination of hemophilia B carrier status. Blood 1986; 67: 1508-11.
PATIENTS FROM SEVEN FAMILIES WITH HAEMOPHILIA B AND AN ILE 397 TO THR SUBSTITUTION
Ages at time of blood sample
Factor IX levels previously descri’bedlfor parents A-E. Polymorph isms were distinguished by Southern blotting polymerase chain reaction,’ insertion for Codon 148 The middle as
+=presence and - =absence of restriction
or
sites are in strong linkage (or Ddel) type was distinguished by Immunoassay.9 disequilibrium, the other two are in equilibrium To identify defects in patients A-D a 3’ portion of coding region of exon 8 was amplified and sequenced *Patient E shown by Southern blotting to have BamHI site,10 other patients’ DNAs tested for this site by amplrficanon. tSporadic=no previously known affected member for at least two generations (except C, adopted), patient B has two affected brothers (one a twm) but his mother’s family history is negative, including her grandmother’s family Patient D’s mother was available for testing and she was heterozygous for both the codon 397 defect and theBamHl polymorphism site
