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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2791 CORRESPONDENCE HEMOGLOBIN CHESTERFIELD (828 LEU + ARG) PRODUCES THE PHENOTYPE OF INCLUSION BODY 8 THALASSEMIA To the Editor: Several dominant forms of p thalassemia have recently been identified that result in a thalassemia intermedia phenotype in individuals who have inherited only a single copy of the abnormal f3 gene.’’6We describe here an individual with severe heterozygous p thalassemia characterized by large inclusion bodies in the peripheral blood associated with a single-base substitution, CTG -+ CGG, in codon 28 of the p gene. This novel mutation, which is found in exon 1, leads to the synthesis of an unstable pvariant, p28 Leu + A r g (hemoglobin [Hb] Chesterfield). The patient was a 34-year-old English woman who presented at the age of 7 years with abdominal pain and was found to be anemic and jaundiced with hepatosplenomegaly. She received sporadic blood transfusions but became transfusion-dependent at the age of 10 and, because of increasing transfusion requirements, underwent splenectomy 3 years later, followed by a cholecystectomy at the age of 28. Post-splenectomy, she remained transfusion-dependent although transfusion requirements were less frequent and she was started on iron chelation therapy. Recent investigations showed: Hb 7 to 9 g/dL, MCV 86.6 fL,MCV 30.3 pg, reticulocytes lo%, Hb A, 4.0%, Hb F 3.4%, and serum ferritin 183 kg/mL. Blood film examination showed marked erythroblastosis (500 to 800 nucleated red cells per 100 white blood cells [WBCs]) with mature erythrocytes showing bizarre morphologic changes and basophilic stippling. Numerous inclusion bodies could be demonstrated in peripheral erythrocytes on incubation with methyl violet. No abnormal Hb was detected on electrophoresis of red cell lysate at pH 8.6, but separation of in vitro labeled globin chains showed an abnormal peak (p”) eluting after the normal PA chain (Fig 1A). The total radioactivity of dPA+ p” was 1.64 while dPAwas 2.47 and P” accounted for 33% of the total p chains after 60 minutes of incubation. No abnormal Hb was detected by heat denaturation or isopropanol tests. Analysis of seven restriction fragment length polymorphisms (RFLPs) in the p-globin gene cluster showed: HindII-c -/-, HindIII-‘yiAy +-/--, HindII-Jrp, 3‘Jrp ++/--, AvaII-P +/+ and BamHI-p +/-. Heterozygosity for five of these sites indicates the absence of a major rearrangement or deletion. a-globin gene mapping showed a normal a-genotype (adaa).The P-globin genes were .enzymatically amplified by the polymerase chain reaction (PCR) and directly sequenced as previously described.’ Direct genomic sequencing of 2,100 base pairs (bp) of the p-globin genes, extending from 307 bp upstream of the Cap site to 320 bp downstream from the termination site, showed a T G ( E G CGG) substitution in codon 28 (Fig 1B) of only one of the p-globin genes; DNA sequence of the other p gene was normal. The sequence modification abolishes the cleavage site for BstNI in exon 1 so that BstNI analyses of 355-bp fragment amplified using primers AP3iAP4 results in only a single 355-bp band if the mutation is present, but two fragments of 85 bp and 270 bp if the p allele is normal (Fig 1C). The results confirmed that the patient is heterozygous for the mutation. Both parents were clinically asymptomatic with normal red blood cell indices and normal levels of Hb A2 and F. The patient had six siblings, all of whom were clinically asymtomatic but none were available for investigation. Therefore, it was likely that the mutation was a de novo genetic event. Several families with a phenotypically dominant form of p -+ -+ From www.bloodjournal.org by guest on June 18, 2017. For personal use only. CORRESPONDENCE 2792 07 A 0.6 0.5 1 1 0.4 0 Q) (Y 0.3 P 0 0.2 0.1 ! Fraction number TGCAGAT C IG - B 27 A la Ala 28 Leu Arg* 29 GlY \A thalassemia have now heen reported. Unlike typical B thalassemia, which is prevalent in walaria-endemic populations. dominant B thalasemias are rare with a wider geographical distribution. At the molecular level they fall into three groups: those resulting in a highly unstable B chain as a result of a single base substitution,'" thcm with a truncated f3 chain due a premature termination codon as a result of a base suhstitution.'." and those with an abnormal f3 globin that is elongated with an altered carhoxy-terminal end as a result of a frameshift mutation."." These mutations result in the production of unstable B chains that are not capable of forming viahle tetramers with a chains and, thus. are not only nonfunctional. hut prove to he an additional burden to the cell's proteolytic machinery. The single base suhstitution C G -+ e G in d o n 28. results in BWLeu Arg. Although there i s an increase i n the net positive charge of the I l b molecule, no abnormal Hb could he detected by - 5 . 1 . (A) Chromatography of the I a M e d globin chaim. The peripheral blood takul0cyt.r of the patient were incubated inthe presence of 'H-leucine for 60 minutes at 37C. The labeled globin chains were analyzed by CMcellulo8echromatography. r e p resents the abnormal peak corresponding t o the expected position of the abnormal p chain. ( 8 )DNA sequence of part of the p-globin gene in exon 1. Eruymatically amplified p-globin genes were purified and sequenced directly using the dideoxynucleotide chain termination method.' The sequence reaction was loaded in the order TGCA for the first four lanes and repeated in the order GATC for the second four lanes. The PCR rimultanbourly amplifies the wild-type and the p-thalasuemia alleles so that both alleles are sequenced. In this case. if both alleles had been normal, only a T should be present in the second position of codon 28. The presence of a G as well as a T in this position (orrowed) indicates a G substthction in the mutant allele, altering Leu for Arg (*I. electrophoresis. CM-cellulosc chromatography demonstrated an abnormal peak in the position expected for the abnormal f3 chain but with no detectable corresponding protein peak, suggesting the production of a Bthain variant that is highly unstable and rapidly degraded after synthesis. The clinical severity of unstable Hb variants presumahly depends on the degree of instahility of the abnormal chain. The most severely unstable variants produce the picture of dominant B thalassemia with a clinical picture of marked ineffective erythropoiesis and thalassemia intermedia. as seen in the current case. Those with a lesser degree of instahility that are still capable of forming viable tetramers may give rise to less inefTective erythropoiesis and a more typical picture of hemolytic anemia. Two other p variants due to amino acid substitutions involving g2S Leu have been descrihed. H b Genova (g28 Leu + Pro)' is an unstable Hb producing a phenotype of severe congenital hemolytic anemia associated with Heinz b d i e s in the From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2793 CORRESPONDENCE erythrocytes. I t appears that the replacement of 828 leucine by a proline residue, although causing a profound distortion o f the B helix, seems less destructive than the replacement 828 Leu to Arg, because 828 Pro is stable enough to allow the formation of viable tetramers (Hb Genova). which then fall apart in the peripheral circulation, causing a hemolytic anemia. Hb St Louis (828 Leu +Gin)" is also an unstable Hb but produces a distinctly different clinical phenotype of severe hemolytic anemia associated with methemoglobinemia. In this case, 828 (BIO) glutamine and the distal histidine (histidine E7) swing towards each other. stablizing a water molecule in the normally hydrophobic heme pocket. which results in thermal instability and the high rate of methemoglobin formation. These findings illustrate that the nature of the amino acid substitution as well as its position in the globin chain i s a critical determinant of the resulting phenotype. ACKNOWLEDGMENT We thank Liz Rose and Linda Roberts for preparation of the manuscript, Drs Max Perutz, Bill Wood, and John Clegg for helpful criticisms. and Prof Sir D.J. Weatherall for encouragement and support. S.L. THElN S. BEST J. SHARPE MRC Molecular Haematology Unit Institute of Molecular Medicine John Radclife Hospital Headington. Oxford B. P A U L Fig. 1. (cant'd) (C) IdentHkatlon of the CTG -L CGG mutation in codon 28 of the p-globin gene by BstN1 rertriction analysis of AP3/AP3 amplified DNA. BstNl cuts the 355 bp resulting in two fragments of 85 and 270 bp; presence of the mutation removes the BstNl cleavage site. The sequences of the primers are 5' 3': AP3: ATGGTGCACCTG ACTCCTGAGG. AP4-GCCATCACTAAAGGCACCGAGC. Lanes a represent amplified DNA cleaved with BstNI, and lanes b, the corresponding un-cleaved DNA; 1, uncharacterized p-thalassaemia heterozygote; 2, mother of the patient; 3, patient; 4, normal individual. The lanes M represent d q Hae 111 size markers. Presence of the three bands of size 355,270. and 85 bp in lane a, no. 3, after digestion with BstNl indicates that the patient is heterozygous for the T G mutation in codon 28. - D.J. CLARK kpanment of Haematology Chesterjieldand North Derbyshire Royal Hospital Calow, Chesterjield M.J. BROWN Depanment of Haematology Nonhem General Hospital Shefield, UK -. REFERENCES 1. Thein SL, Hesketh C, Taylor P, Temperley IJ, Hutchinson RM, Old JM, Wood WG, Clegg JB. Weatherall DJ: Molecular basis for dominantly inherited inclusion body 8-thalassemia. Proc Natl Acad Sci USA 873924,1990 2. Fucharoen S , Kobayashi Y, Fucharoen G, Ohba Y, Miyazono K, Fukumaki Y, Takaku F A single nucleotide deletion in codon 123 of the f3-globin gene causes an inclusion body f3-thalassaemia Br J Haematol75:393, trait: A novel elongated globin chain pWab". 1990 3. Fei YJ, Stoming TA, Kutlar A, Huisman THJ. Stamatoyannopoulos G: One form of inclusion body 8-thalassemia i s due to a GAA + TAA mutation at codon 121of the 8 chain. Blood 73:1075, 1989 4. Kazazian HH Jr, Dowling CE, H u w i t z RL, Coleman M, Adams JG 111: Thalassemia mutations in exon 3 of the &globin gene often cause a dominant form of thalassemia and show no predilection for malarial-endemic regions of the world. Am J Hum Genet 29950, 1989 (suppl) 5. Beris P, Miescher PA, Diaz-Chico JC, Han I-S, Kutlar A, H u H, Wilson JB, Huisman THJ: Inclusion body 8-thalassemia trait in a Swiss family i s caused by an abnormal hemoglobin (Geneva) with an altered and extended 8 chain carboxy-terminus due to a modification in codon 81 14. Blood 72:801. 1988 6. Kobayashi Y, Fukumaki Y, Komatsu N. Ohba Y. Miyaji T, Miura Y: A novel globin structural mutant, Showa-Yakushiji (f31 IO Leu-Pro) causing a 8-thalassemia phenotype. Blood 7031688, 1987 7. Sansone G . Carrel1 RW, Lehmann H: Haemoglobin Genova: 828 (BIO) Leucine 4 Proline. Nature 214:877, 1967 8. Thillet J, Cohen-Sola1 M, Seligmann M, Rosa J: Functional and physicochemical studies of hemoglobin St. Louis 828 (BIO) Leu -t Gln. A variant with ferric 8 heme iron. J Clin Invest 58:1098,1976 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1991 77: 2791-2793 Hemoglobin Chesterfield (beta 28 Leu----Arg) produces the phenotype of inclusion body beta thalassemia [letter] SL Thein, S Best, J Sharpe, B Paul, DJ Clark and MJ Brown Updated information and services can be found at: http://www.bloodjournal.org/content/77/12/2791.citation.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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