126
Case Reports
It is well recognized that lymphoprolierative and myeloproliferative disorders can occur simultaneously in the same patient (Camba & Joyner. 1985: Copplestoneet al, 1986) and that serum immunoglobulin abnormalities occur quite frequently in patients with lymphoproliferative disorders (Mufti et 01. 1986). Indeed. whilst gamma heavy chain disease shows a diversity of clinical manifestations and haematological features (Fermand et al. 1989), it most commonly presents as a lymphoproliferative disorder. A high prevalence ( 12.5%)of monoclonal gammopathies has also been demonstrated (Mufti et al. 1986) in patients with MDS (lymphoproliferative disorders having been excluded), but the association of gamma heavy chain disease with MDS alone has not been previously described. It has been established that the time interval between lymphoid and myeloid disorders in our patient was approximately 8 months: however, it is not possible to say categoricallywhether the gamma heavy chain disease arose prior to the development of lymphoma or as a consequence of it. The development of gamma heavy chain disease over 8 months in a patient with MDS on chronic transfusion therapy also raises the possibility that it may have resulted from a transmissable agent. No evidence of any such transmission, however, was found with serology for hepatitis A, hepatitis C and HIV being negative and serology for hepatitis B indicating a past rather than a recent infection. Douglass Laboratories Pty Ltd. P.O. Box 145, Ryde, N.S.W.. A ust ralia
V. M. ELLIS
*Department of Biochemistry. tDepartment of Haematologg, Mater Misericordiae Hospital, South Brisbane, Queensland, Australia
D. M. COWLEY' K. M. TAYLORt P. MARLToNt
REFERENCES Bergsagel, D. (1990)The heavy-chain diseases.Hematology. 4th edn (ed. by W. J. Williams, E. Beutler. A. Erslev and M. Richtman).pp. 1146-1 148. McGraw-Hill Publishing Co., U.S.A. Carnba. L. &]oyner.M.V. (1985) Refractory anaemia terminating in a combined lymphoproliferative and myeloproliferative disorder. Journal oJClinical Pathology. 38. 297-300. Copplestone, J.A.. Mufti, G.J., Hamblin. I.J. & Oscier, D.G.(1986) Immunological abnormalities in myelodysplastic syndromes. II. Coexistent lymphoid or plasma cell neoplasms:a report of 20 cases unrelated to chemotherapy. British lournal oJ Haematology. 63, 149-159. Fermand. 1.P.. Brouet, J.C.. Danon, F. & Seligmann. M. (1989) Gamma heavy chain 'disease':heterogeneityof the clinicopathologic features. Medicine. 68, 321-335. Guglielmi. P.. Bakhshi. A.. Cogne. M.. Seligmann. M. & Korsmeyer. S.J.(1988)Multiple genomic defects result in an alternative RNA splice creating a human gamma H chain disease protein. journal o/ Immunology. 141, 1762-1768. Mufti. C.J.. Figes. A., Hamblin. I.J.. Oscier, D.G.& Copplestone, 1.A. ( 1986) Immunological abnormalities in myelodysplastic syndromes. I. Serum immunoglobulins and autoantibodies. British Journal oJ Haematology, 63, 143-147.
HIGH PREVALENCE OF THE 0-THALASSAEMIA NONSENSE 37 MUTATION IN CATALONIANS FROM THE EBRO DELTA
We observed a 0-thalassaemia 3 7 nonsense mutation in both members of a heterozygous couple requesting prenatal diagnosis and originating from the Ebro delta, a small Mediterranean coastal region of the Iberian peninsula. This mutation was previously observed only once in a Saudi Arabian family, in association with the 0-globin Mediterranean haplotype I (Boehm et al, 1986). This finding prompted us to screen for this defect in 0-thalassaemia carriers originating from the Ebro delta. Surprisingly, the mutation was found in all unrelated 0-thalassaemia carriers examined ( n = 3 2 ) . and in all instances associated with the same 0globin locus haplotype I. The mutation was detected using denaturing gradient gel electrophoresis (DGGE) analysis of PCR amplificated segments of the 0-globin gene, as previously described (Goossens et al. 1990). The DGGE pattern of one of the fragments (fragment C) displayed a shift in mobility compatible with the presence of a destabilizing nucleotide substitution (Fig 1A). The mutation was subsequently characterized by DNA sequencing of the abnormal fragment (Gyllensten & Erlich, 1988). This G to A substitution in codon 3 7 (Fig 1B) abolished a cutting site for the endonuclease Ava 11. a feature which proved useful in subsequent screening experiments. The fact that the j-thalassaemia 3 7 nonsense mutation observed in the Ebro delta region is associated, as is the Saudi
Arabian defect, with the 0-globin locus haplotype I suggests a possible common origin for the two abnormalities. Whether the defect found in the Ebro delta has been imported into Spain, or exported to the Arabic peninsula through migration during the eight centuries of Moorish occupation of Spain, is difficult to assess. However, it is interesting to note that this region of Spain is an area where malaria was endemic until the begining of this century. Although the 0-thal 37 nonsense mutation has not been observed among Mediterranean, Indian and oriental fl-thalassaemia genes in spite of extensive screening, its actual frequency in Saudi Arabia is not known. Our findings which further extend the spectrum of 0thalassaemia mutations in the Mediterranean basin will be helpful in genetic counselling for this disease in Spain.
'Hospital de la Santa Creu i Sant Pau. Barcelona, Spain, lHopital Henri Mondor, Creteil, France, and 'Hopital Verge de la Cinta, Tortosa, Spain
P. GALLANO] E. GIRODON~ N. GHANEM~ LL. FONT' E. DEL RIO' J. MARTIN^ M. GOOSSENS~ M. BAIGET]
Case Reports
T
C
G
127
A
T
G -GA A C C
Fig 1A.DCGE analysis of fragment C of the /&globin gene amplified from an individual heteroqgous for P-globin gene framework 1 and 3 (lane 1 ) and an individual homozygous for framework 1 (lane 2). SamplesC (Pjymutation) and P (patient)show a band faster than the normal heteroduplex. compatible with a destabilizing mutation.
1 2 3 KEFERENCES Boehm. C.. Ilowling. C.E.. Waber. P.G., Giardina. J.V. & Kazazian. H.H.. ]r (1986) llse of oligonucleotide hybridization in the characterization of a p-thalassemia gene (/l”TGC+TCA) in a Saudi Arabian family. Blood. 67, 1185-1 188. Goossens. M..Fanen. P.. Attree. 0.& Vidaud. M. (1990) A new strategy for direct detection of b-thalassemia mutations. Annals the New York Academ!j of Science. 612, 74-80.
Fig 1B.DNA sequence autoradiograph of part of fragment C. Lane 1: patient: lane 2: control (p39heterozygous): lane 3: normal control. A G to A substitution is shown in lane 1 at the third nucleotide of codon 37. Gyllensten. U.B. & Erlich, H.A. ( 1 988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proceedings of the National AcademyofSciencesofthe UnitedStatesofAmericn. 85. 7652-7656.
TREATMENT OF SEVEKE APLASTIC ANAEMIA WITH TOTAL LYMPHOID IRRADIATION AND METHY LPKEDNISOLONE The majority of patients with severe aplastic anaemia (SAA) have T-cell-mediated bone marrow suppression (Ascensao et al, 1976). Thus while bone marrow transplantation (BMT) remains the therapy of choice for SAA, various modalities of immunosuppression such as antithymocyte globulin and corticosteroids have been tried with varying success (Bacigalupo et a / . 1988). Cyclosporine has been reported to induce a complete haematologic remission in SAA at full immunosuppressive doses (Dundar & Damirkezik, 1990) or at a low dose (Mehta et a/, 1992). Total lymphoid irradiation (TLI)results in lasting immunosuppression through a selective reduction in the number and function of helper T-cells and certain subsets of B-cells (Strober, 1987). TIJ-induced immunosuppression has
resulted in long-term survival of allografts in experimental animals (Slavin et al. 1978). and marked improvement in autoimmune diseases such as rheumatoid arthritis (Trentham et al. 198 1) and lupus nephritis (Strober et al, 1985). A 12-year-old female with known trisomy 2 1 and Down’s syndrome was admitted with a purpuric rash and vaginal bleeding of 2 d duration. She had fever 1 5 d before for which she had received empiric co-trimoxazole and chloroquine. Examination revealed fever, pallor, generalized purpuric eruption, and vaginal bleeding. There was no organomegaly or lymphadenopathy. Investigations revealed: haemoglobin 5.2 g/dl. leucocytes 1* 1 x 1 09/1(15% granulocytes), reticulocytes 0.1%.platelets 10 x 109/1,and a markedly hypocellular bone marrow on trephine biopsy. Ham’s acidified serum test
Case Reports
It is well recognized that lymphoprolierative and myeloproliferative disorders can occur simultaneously in the same patient (Camba & Joyner. 1985: Copplestoneet al, 1986) and that serum immunoglobulin abnormalities occur quite frequently in patients with lymphoproliferative disorders (Mufti et 01. 1986). Indeed. whilst gamma heavy chain disease shows a diversity of clinical manifestations and haematological features (Fermand et al. 1989), it most commonly presents as a lymphoproliferative disorder. A high prevalence ( 12.5%)of monoclonal gammopathies has also been demonstrated (Mufti et al. 1986) in patients with MDS (lymphoproliferative disorders having been excluded), but the association of gamma heavy chain disease with MDS alone has not been previously described. It has been established that the time interval between lymphoid and myeloid disorders in our patient was approximately 8 months: however, it is not possible to say categoricallywhether the gamma heavy chain disease arose prior to the development of lymphoma or as a consequence of it. The development of gamma heavy chain disease over 8 months in a patient with MDS on chronic transfusion therapy also raises the possibility that it may have resulted from a transmissable agent. No evidence of any such transmission, however, was found with serology for hepatitis A, hepatitis C and HIV being negative and serology for hepatitis B indicating a past rather than a recent infection. Douglass Laboratories Pty Ltd. P.O. Box 145, Ryde, N.S.W.. A ust ralia
V. M. ELLIS
*Department of Biochemistry. tDepartment of Haematologg, Mater Misericordiae Hospital, South Brisbane, Queensland, Australia
D. M. COWLEY' K. M. TAYLORt P. MARLToNt
REFERENCES Bergsagel, D. (1990)The heavy-chain diseases.Hematology. 4th edn (ed. by W. J. Williams, E. Beutler. A. Erslev and M. Richtman).pp. 1146-1 148. McGraw-Hill Publishing Co., U.S.A. Carnba. L. &]oyner.M.V. (1985) Refractory anaemia terminating in a combined lymphoproliferative and myeloproliferative disorder. Journal oJClinical Pathology. 38. 297-300. Copplestone, J.A.. Mufti, G.J., Hamblin. I.J. & Oscier, D.G.(1986) Immunological abnormalities in myelodysplastic syndromes. II. Coexistent lymphoid or plasma cell neoplasms:a report of 20 cases unrelated to chemotherapy. British lournal oJ Haematology. 63, 149-159. Fermand. 1.P.. Brouet, J.C.. Danon, F. & Seligmann. M. (1989) Gamma heavy chain 'disease':heterogeneityof the clinicopathologic features. Medicine. 68, 321-335. Guglielmi. P.. Bakhshi. A.. Cogne. M.. Seligmann. M. & Korsmeyer. S.J.(1988)Multiple genomic defects result in an alternative RNA splice creating a human gamma H chain disease protein. journal o/ Immunology. 141, 1762-1768. Mufti. C.J.. Figes. A., Hamblin. I.J.. Oscier, D.G.& Copplestone, 1.A. ( 1986) Immunological abnormalities in myelodysplastic syndromes. I. Serum immunoglobulins and autoantibodies. British Journal oJ Haematology, 63, 143-147.
HIGH PREVALENCE OF THE 0-THALASSAEMIA NONSENSE 37 MUTATION IN CATALONIANS FROM THE EBRO DELTA
We observed a 0-thalassaemia 3 7 nonsense mutation in both members of a heterozygous couple requesting prenatal diagnosis and originating from the Ebro delta, a small Mediterranean coastal region of the Iberian peninsula. This mutation was previously observed only once in a Saudi Arabian family, in association with the 0-globin Mediterranean haplotype I (Boehm et al, 1986). This finding prompted us to screen for this defect in 0-thalassaemia carriers originating from the Ebro delta. Surprisingly, the mutation was found in all unrelated 0-thalassaemia carriers examined ( n = 3 2 ) . and in all instances associated with the same 0globin locus haplotype I. The mutation was detected using denaturing gradient gel electrophoresis (DGGE) analysis of PCR amplificated segments of the 0-globin gene, as previously described (Goossens et al. 1990). The DGGE pattern of one of the fragments (fragment C) displayed a shift in mobility compatible with the presence of a destabilizing nucleotide substitution (Fig 1A). The mutation was subsequently characterized by DNA sequencing of the abnormal fragment (Gyllensten & Erlich, 1988). This G to A substitution in codon 3 7 (Fig 1B) abolished a cutting site for the endonuclease Ava 11. a feature which proved useful in subsequent screening experiments. The fact that the j-thalassaemia 3 7 nonsense mutation observed in the Ebro delta region is associated, as is the Saudi
Arabian defect, with the 0-globin locus haplotype I suggests a possible common origin for the two abnormalities. Whether the defect found in the Ebro delta has been imported into Spain, or exported to the Arabic peninsula through migration during the eight centuries of Moorish occupation of Spain, is difficult to assess. However, it is interesting to note that this region of Spain is an area where malaria was endemic until the begining of this century. Although the 0-thal 37 nonsense mutation has not been observed among Mediterranean, Indian and oriental fl-thalassaemia genes in spite of extensive screening, its actual frequency in Saudi Arabia is not known. Our findings which further extend the spectrum of 0thalassaemia mutations in the Mediterranean basin will be helpful in genetic counselling for this disease in Spain.
'Hospital de la Santa Creu i Sant Pau. Barcelona, Spain, lHopital Henri Mondor, Creteil, France, and 'Hopital Verge de la Cinta, Tortosa, Spain
P. GALLANO] E. GIRODON~ N. GHANEM~ LL. FONT' E. DEL RIO' J. MARTIN^ M. GOOSSENS~ M. BAIGET]
Case Reports
T
C
G
127
A
T
G -GA A C C
Fig 1A.DCGE analysis of fragment C of the /&globin gene amplified from an individual heteroqgous for P-globin gene framework 1 and 3 (lane 1 ) and an individual homozygous for framework 1 (lane 2). SamplesC (Pjymutation) and P (patient)show a band faster than the normal heteroduplex. compatible with a destabilizing mutation.
1 2 3 KEFERENCES Boehm. C.. Ilowling. C.E.. Waber. P.G., Giardina. J.V. & Kazazian. H.H.. ]r (1986) llse of oligonucleotide hybridization in the characterization of a p-thalassemia gene (/l”TGC+TCA) in a Saudi Arabian family. Blood. 67, 1185-1 188. Goossens. M..Fanen. P.. Attree. 0.& Vidaud. M. (1990) A new strategy for direct detection of b-thalassemia mutations. Annals the New York Academ!j of Science. 612, 74-80.
Fig 1B.DNA sequence autoradiograph of part of fragment C. Lane 1: patient: lane 2: control (p39heterozygous): lane 3: normal control. A G to A substitution is shown in lane 1 at the third nucleotide of codon 37. Gyllensten. U.B. & Erlich, H.A. ( 1 988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proceedings of the National AcademyofSciencesofthe UnitedStatesofAmericn. 85. 7652-7656.
TREATMENT OF SEVEKE APLASTIC ANAEMIA WITH TOTAL LYMPHOID IRRADIATION AND METHY LPKEDNISOLONE The majority of patients with severe aplastic anaemia (SAA) have T-cell-mediated bone marrow suppression (Ascensao et al, 1976). Thus while bone marrow transplantation (BMT) remains the therapy of choice for SAA, various modalities of immunosuppression such as antithymocyte globulin and corticosteroids have been tried with varying success (Bacigalupo et a / . 1988). Cyclosporine has been reported to induce a complete haematologic remission in SAA at full immunosuppressive doses (Dundar & Damirkezik, 1990) or at a low dose (Mehta et a/, 1992). Total lymphoid irradiation (TLI)results in lasting immunosuppression through a selective reduction in the number and function of helper T-cells and certain subsets of B-cells (Strober, 1987). TIJ-induced immunosuppression has
resulted in long-term survival of allografts in experimental animals (Slavin et al. 1978). and marked improvement in autoimmune diseases such as rheumatoid arthritis (Trentham et al. 198 1) and lupus nephritis (Strober et al, 1985). A 12-year-old female with known trisomy 2 1 and Down’s syndrome was admitted with a purpuric rash and vaginal bleeding of 2 d duration. She had fever 1 5 d before for which she had received empiric co-trimoxazole and chloroquine. Examination revealed fever, pallor, generalized purpuric eruption, and vaginal bleeding. There was no organomegaly or lymphadenopathy. Investigations revealed: haemoglobin 5.2 g/dl. leucocytes 1* 1 x 1 09/1(15% granulocytes), reticulocytes 0.1%.platelets 10 x 109/1,and a markedly hypocellular bone marrow on trephine biopsy. Ham’s acidified serum test
