Original Article

/?°-Thalassemia due to a Nonsense Mutation at /?90 (GAG->TAG) in Human Gene

Yoshinari NOMURA,Kishio NANJO, Kei MlYAMURA, Teruo HARANO*, Keiko HARANO*and Satoshi UEDA*

Westudied a patient with severe anemia and jaundice who exhibited a high hemoglobin Ai (HbAi) level secondary to an increase in HbFdespite normal glucose tolerance. The red blood cells showed anisocytosis, poikilocytosis and polychromasia; target cells, Howell-Jolly bodies, Heints bodies and punctate basophilia were observed. No defect or reduction in activity was observed in 19 red cell enzymes. A family history of similar anemia in the patient's daughter and cousins on the mother's side indicated an involvement of genetic factors. Genecloning and DNAanalysis showedthat the condition is a new type of /9°-thalassemia caused by a nonsense mutation (GAG-»TAG)in codon 90 of the ^-globin gene. Key words: ^-thalassemia, Nonsense mutation, Codon 90, Gene cloning, DNAanalysis

HbAi and HbAic levels have been studied in- anemia. She underwent splenectomy at the age of creasingly as indices of long-term blood sugar con- 27 with a diagnosis of hemolytic anemia, but this trol in diabetic patients (1) and as aids in the early diagnosis had not been confirmed. She also under- detection of diabetes (2). As a result, manytypes went cholecystectomy due to gall stones at the age of have been found by routine of 39. At the time of the examination, the patient clinical evaluation of the HbAic level (3). Ween- still showed jaundice and anemia and complained countered a patient with severe anemia and jaundice of mild malaise. There was a history of one abor- in whomthe HbAilevel was abnormally high at tion. She was 154 cm tall and weighed 48 kg. 20.1% due to an increase in HbFdespite the absence Onphysical examination, there were no marked of glucose intolerance. Bya detailed examination abnormalities except for anemia and jaundice, func- this condition was identified as /3°-thalassemia tional systolic murmur, and hepatomegaly. caused by a nonsense mutation (GAG-> TAG)in Laboratory data included RBC263 x lO4/^l, codon 90 of the /3-globin gene. Hb 6.8 g/dl, Ht 26.0%, MCV 99 fl, and MCH 26 pg, indicating severe anemia. The total nucleated cell PATIENT AND METHODS count including erythroblasts and leukocytes was Patient (Record: November, 1980) 44,700/ Ml; were increased to 32.5%. The patient was a 42-year-old female with a Total bilirubin was high at 4.5 mg/dl (direct 1.3 history of jaundice at the age of 13 and untreated mg/dl, indirect 3.2 mg/dl), but transaminase levels anemia at the age of 19. She received a transfusion were normal. However, liver biopsy suggested after giving birth to a child at the age of 24, and hemosiderosis. Renal function and electrolyte levels subsequently demonstrated severe jaundice and were normal. The results of direct and indirect From the First Department of Medicine, WakayamaMedical College, Wakayamaand *Department of Biochemistry, Kawasaki Medical School, Okayama Received for publication May2, 1989; Accepted for publication October 24, 1989 Reprint request should be addressed to Yoshinari Nomura, MD,the First Department of Medicine, Wakayama Medical College, 27-Banchi, 7-Bancho, Wakayama 640, Japan

Jpn J Med Vol 29, No 1 (January, February 1990) /3°-Thalassemia

Coomb's tests were both negative. Other findings (6) were performed. were: Fe 287jug/dl, Cu 81 ^g/dl, Mg 2.0 mEq/1, tests: Osmotic fragility tests 10 mg/dl or less, vitamin B12 600 against various concentrations of NaClwere per- pg/ml, folic acid 2.3 ng/ml, total iron binding formed according to the method of Parpart et al (7). capacity (TIBC) 247.8 //g/dl, unsaturated iron Red cell enzymes: The activities of 19 red cell binding capacity (UIBC) 33.6 jug/dl, red cell iron enzymes were examined. (59Fe) utilization 78.7% , plasma iron disappearance Hb synthesis: Hb synthesis was induced by rate (PID) T1/2 48 minutes. The HbAi level deter- allowing reticulocytes to incorporate 3H-Leu in a mined with Quik-Sep (Isolab Inc., Ohio, USA) was synthetic medium. The hemolysate was treated with 20.1%, and the HbAia+b and HbAic levels deter- HCl-acetone to obtain globin, and a- and /3- mined according to the method of Trivelli et al globins were separated by CM-cellulose (CM-52; (macro-column method) (4) were 4.33% and Whatman Co., NewJersey, USA) column chro- 15.24%, respectively. These high values were, matography (eluant: 8Murea-sodium phosphate however, inconsistent with the results of 50g-OGTT buffer, pH 6.8; Na ion concentration 5 mMto 35 (plasma glucose; O'-76, 30'-105, 60'-135, mM).The radioisotope uptake was evaluated in each 90'-71, 120'-70 mg/dl). fractio n. The patient's family consisted of herself, her DNAanalysis: DNAwas extracted from leuko- husband, and a daughter, who was found to have cytes of the patient by the method of Poncz et al jaundice and anemia from about the age of six. Her (8). Genomic Southern-blot-hybridization was per- father and two brothers were alive, but her mother formed as previously described (9). The probe used had died of pancreatic cancer with jaundice at the here was the BamHI-Eco RI fragment (0.9 Kb) con- age of 47. Jaundice had been noted in 2 of her taining the intervening sequence (IVS) II of the cousins on her mother's side (Fig. 1). fi-globin gene which was radiolabeled by the pro- Meth ods cedure of nick translation. A combinant library was Blood was collected using EDTA-2Na and constructed by ligating Hind Ill-digested genomic heparin as anticoagulants, and used for the DNAfragment (ca. 7.5 Kb) to Charon 28 which had following evaluations. been digested with Hind III and subsequently de- Red cell morphology: The morphology of red phosphorylated. The ligated mixture was packaged blood cells was studied under light and scanning in the phage particles using the in vitro packaging electron microscopy using the peripheral blood and extract (Packagene, Promega Biotech, Co., USA). Recombinant phage was screened using the /3-IVS bone marrowaspirate from the iliac crest. II probe. A 2.8 Kb Sph I-Pst I fragment was Hemoglobinanalysis: Abnormalhemoglobinwas examined by isoelectric focusing. HbA2was deter- subjected to subcloning to M13mpl8phage and to mined by cellulose-acetate membrane electro- nucleotide dequencing (Dideoxy method) using a phoresis. HbF was measured by Betke's alkali universal primer as well as several synthetic primers. denaturation method (5). RESULTS Erythrocyte degeneration tests: Isopropanol tests Red blood cells showed anisocytosis,

1 poikilocytosis, and polychromasia (Fig. 2A, Fig. j^jQ ^]C1 A LhO 0t& 2B); target cells, Howell-Jolly bodies, Heints bodies, Ii'iiii'iijrh m-\ and punctate basophilia were observed. Onbone OBQOOOBDOOO marrowpuncture, the nucleated cell count was 44.9 X 104/ fitI, and M/E ratio was 0.15; an increase 1 & in the cell count in erythrocytic series was observed. Fig. 1. Pedigree of patient with /S°-thalassemia trait. The HbF and HbA2 levels were elevated at 14.1% (8) ; propositus O; female å¡;male and 4.84%, respectively. No bands of abnormal 91 ; female and male with ^-thalassemia trait hemoglobin were observed by cellulose-acetate mem- 0001 deceased brane electrophoresis or isoelectric focusing (pH

Jpn J Med Vol 29, No 1 (January, February 1990) Nomuraet al

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Fig. 2B. The morphology of red blood Fig. 2A. The morphology of red blood cells studied under scanning electron cells studied under light microscopy (peripheral microscopy (peripheral blood) blood, May-Griinwald-Giemsa stain)

6.7-7.7). in the region surrounded and within the /S-globin During the erythrocyte degeneration tests, gene, and gave the presence of polymorphism at the gradual precipitation was observed from immedi- Ava II site in the ^-globin gene. Two types of ately after the initiation of the depletion of distilled fi-globin gene clones possessing plus and minus water, and osmotic fragility tests using various con- polymorphismsat AvaII site were obtained from centrations of saline showedan increase in resistance about 5 x 105 gene library. Nucleotide sequencing of the erythrocyte membrane.Nodefect or reduc- of the relevant clones from the 5 ' flanking region tion of activity was observed in the following 19 red Optical density cell enzymes examined: hexokinase, glucosephos- (X=280nm) phate isomerase, phosphofructokinase, aldolase, 1.0 triosephosphate isomerase, glyceraldehyde 3- 3H-Leu uptake a-globin dpm phosphate dehydrogenase, phosphoglycerate kinase, xiO1 monophosphoglyceromutase, enolase, pyruvate 100 kinase, lactate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, glutathione reductase, glutathione peroxidase, adenylate kinase, adenosine deaminase, acetylcholin- esterase, and pyrimidine 5 ' -nucleotidase. 70 No. Analysis of Hbsynthesis showed suppression of Fig. 3. The measurement of radioisotope uptake in a- synthesis of /3-globin as compared with a-globin and ^-globins after hemoglobin synthesis induced by allowing reticulocytes to incorporate 3H-Leu in a synthetic ( fi/.a ratio = 0.35) (Fig. 3). The findings resulting medium. from the digest of the DNAwith several restriction oo optical density à"-à"3H-Leu uptake (total activity; endonucleases and hybridization with a ^3-IVS II /3-globin 215xlO3 dpm a-globin 620xlO3 dpm p/a probe did not show a deletion over at least 100 bp 0.35).

Jpn J Med Vol 29, No 1 (January, February 1990) jS-°-Thalassemia

GT AC logical abnormalities of erythrocytes, increases in HbA2 and HbF, and the appearance of Howell- Jolly bodies and Heintz bodies. Therefore, suspecting unstable hemoglobin or thalassemia, we carried out amino acid analysis and DNAgene analysis of hemoglobin, which demonstrated a nonsense mutation (GAG-> TAG)in codon 90 of /3-globin gene. However, due to the precipitation which gradually increased from immediately after the start of distilled water depletion, erythrocyte degeneration tests and amino acid analysis were difficult to perform. In fact, the identification of Normal amino acid abnormalities was impossible in the hemolysate after removal of the sediment. Variations of ^°-thalassemia due to nonsense Sly Thr Pile Ala Tftr Leu Ser Glu L«u Hi5 Cys Normal Seq. - &- GGC -ACC -TTT^CC-ACA-CTS~AST~ 6AG -CTG« CAC -TGT~ mutations such as substitution of AAGwith stop TAG Abnormal Seq. (Ter) codon TAGat £17 (10), substitution of GAGwith TAGat ft39 (ll) and substitution of GAGwith Fig. 4. Autoradiographs of portion of the sequencing TAGat /343 (12) have been reported. Deletions gels showing the partial nucleotide sequence of both normal and mutant alleles for this case in the area of the point have also been shownto cause /3°-thalassemia in mutation. Nonsense mutation (GAG-> TAG) at codon 90 a Turkish patient (1 1) and in Indian patients in the is shown. (Ter), termination codon. areas of Sind and Gujarat (13). More recently, /3°-thalassemia due to a nonsense mutation in codon 121 was reported (14). However, there have through the 3' flanking region of the gene was been no reports of a nonsense mutation with the carried out. There was no abnormal sequence in substitution of GAGwith stop codon TAGat j9 ^ the clone possessing plus Ava II polymorphism, but as was observed in the present case. This patient was another clone showedthat the first nucleotide Gof a native of the place of her present residence the codon GAGencoding 90th amino acid (Glu) in (Wakayama), but there have been no reported in- the ExsonII segment of the /3- globin gene was stances of /3°-thalassemia in or near this region. substituted with T, forming the termination codon Regional screening of /S°-thalassemia of the TAG (Fig. 4). sametype as this patient is anticipated. In this DISCUSSION patient, who exhibited a heterozygous mutation, the ft chain is considered to be complete as far as the In this patient the high HbAi level, despite the 89th amino acid, but to lack the 92nd amino acid absence of glucose intolerance, prompted closer (histidine) which connects with Fe of heme. examination. The macro-column method using Bio- Therefore, the molecule, which is incomplete as Rex 70 showed that the increased HbFinfluenced hemoglobin, is considered to have been present as HbAia+ b and HbAic fractions. a very labile peptide. Characteristics of clinical features in this patient As a matter of convenience, we propose that this were: 1) hemolytic anemia with primary manifesta- type of ^-thalassemia should be named tions of anemia and jaundice (especially an increase ^-thalassemia Wakayama, since thalassemia in indirect bilirubin), 2) a family history of similar identical to the type found in this case has not been anemia in the patient's daughter and cousins on the reported in other regions. mother's side, indicating an involvement of genetic ACKNOWLEDGMENTS:We wish to thank Dr. Shiro factors, 3) exclusion of autoimmune diseases and Miwa of the Okinaka Adult Disease Institution for the assay abnormalities in red cell enzymes, and 4) morpho- of red cell enzymes.

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