A distant gene deletion affects beta-globin gene function in an atypical gamma delta beta-thalassemia. P Curtin, … , A D Stephens, H Lehmann J Clin Invest. 1985;76(4):1554-1558. https://doi.org/10.1172/JCI112136. Research Article We describe an English family with an atypical gamma delta beta-thalassemia syndrome. Heterozygosity results in a beta-thalassemia phenotype with normal hemoglobin A2. However, unlike previously described cases, no history of neonatal hemolytic anemia requiring blood transfusion was obtained. Gene mapping showed a deletion that extended from the third exon of the G gamma-globin gene upstream for approximately 100 kilobases (kb). The A gamma-globin, psi beta-, delta-, and beta-globin genes in cis remained intact. The malfunction of the beta-globin gene on a chromosome in which the deletion is located 25 kb away suggests that chromatin structure and conformation are important for globin gene expression. Find the latest version: https://jci.me/112136/pdf A Distant Gene Deletion Affects ,8-Globin Gene Function in an Atypical '6y5-Thalassemia Peter Curtin, Mario Pirastu, and Yuet Wai Kan Howard Hughes Medical Institute and Department ofMedicine, University of California, San Francisco, California 94143 John Anderson Gobert-Jones Department ofPathology, West Suffolk County Hospital, Bury St. Edmunds IP33-2QZ, Suffolk, England Adrian David Stephens Department ofHaematology, St. Bartholomew's Hospital, London ECIA-7BE, England Herman Lehmann Department ofBiochemistry, University ofCambridge, Cambridge CB2-lQW, England Abstract tologic picture of f3-thalassemia minor in adult life. Globin syn- thetic studies reveal a ,3 to a ratio of -0.5, but unlike the usual We describe an English family with an atypical 'yS6-thalassemia fl-thalassemia heterozygote, the levels of HbA2 (and HbF) are syndrome. Heterozygosity results in a ft-thalassemia phenotype normal. To date, the molecular defects responsible for this dis- with normal hemoglobin A2. However, unlike previously de- order have all involved extensive deletions of most or all of the scribed cases, no history of neonatal hemolytic anemia requiring f3-globin gene cluster. In three of the previously described cases, blood transfusion was obtained. Gene mapping showed a deletion the deletion encompasses the entire fl-globin gene cluster, in- that extended from the third exon of the G-yglobin gene upstream cluding all or part of the f3-globin gene itself, and inactivates all for -100 kilobases (kb). The Ay-globin, #(3-, 5-, and fl-globin the f3-globin-like genes in cis (9, 10, 14, 15). In a fourth and genes in cis remained intact. The malfunction of the fl-globin perhaps most interesting case, the E-, G7_, Ay-, and 6-globin genes gene on a chromosome in which the deletion is located 25 kb are deleted, whereas the fl-globin gene and 2.5 kilobases (kb) of away suggests that chromatin structure and conformation are 5' flanking sequence remain intact. Although the fl-globin gene important for globin gene expression. is present and structurally normal, it is not expressed (11, 12). This report describes an English family with a syndrome Introduction resembling y6fl-thalassemia. Restriction endonuclease mapping Studies with recombinant DNA technology have uncovered revealed a large deletion that included the E-globin gene and part many different molecular mechanisms that can cause defective of the Gy-globin gene, but spared the At_, 5-, and fl-globin genes. globin chain synthesis in the thalassemia syndromes. The com- Although the breakpoint of the deletion was located more than mon defects in ,B-thalassemia are point mutations that result in 25 kb from the f3-globin gene, the gene malfunctioned. abnormal globin mRNA transcription or processing, and in nonsense or frameshift mutations affecting globin chain trans- Methods lation (1-3). Less commonly, ,B-thalassemia is caused by gene deletion. Deletions affecting the ,B-globin gene cluster result in Hematologic values were determined by routine methods (16, 17). The the syndromes of (3-thalassemia (4), hereditary persistence of Aty and Gt ratios were determined by high pressure liquid chromatography fetal hemoglobin (Hb)' (5-8), 6fl-thalassemia (7, 8), and (18). DNA was prepared from peripheral blood lymphocytes from two -ybf- of the subjects heterozygous for the disorder, and from normal controls. thalassemia (9-15). The structural genes are usually inactivated Extensive restriction mapping was performed in one subject (I-1), whereas the an by deletion, although exception has been described in selected enzymes were used in the second (II-1) to ascertain whether the one case of y6yl-thalassemia (11, 12). same lesion was inherited in the family. The DNA was digested as rec- 'y6f3-Thalassemia is a rare syndrome that has been described ommended by the manufacturers with the restriction endonucleases ApaI, only in the heterozygous state. The carriers have a clinical course AvaII, BamHI, BclI, BglII, EcoRI, HindIIl, Hinfl, HpaI, MspI, TaqI, marked by neonatal hemolytic anemia, followed by the hema- and XbaI (Bethesda Research Laboratories, Gaithersburg, MD; New En- gland Biolabs, Beverly, MA; Boehringer Mannheim Biochemicals, In- Professor Lehmann died 13 July 1985. dianapolis, IN). Digested DNAs were size-fractionated on horizontal 0.8- This work was presented in part at the annual meeting ofthe American 1% agarose gels, transferred to nitrocellulose filters, and hybridized to Society of Clinical Investigation, Washington, DC, May 1984. specific 32P-radiolabeled probes. Bound radioactivity was visualized by Dr. Pirastu's present address is Istituto di Ricerca sulle Talassamie autoradiography on X-ray film (19). ed Anemia Mediterranee CNR, 09100 Cagliari, Sardinia, Italy. Dr. Kan Two fl-globin gene probes derived from the plasmid H(#-IS were is an Investigator ofthe Howard Hughes Medical Institute. Address reprint used. One was the 0.9-kb BamHI-EcoRI fragment that contains the sec- requests to Dr. Kan. ond intervening sequence of the fl-globin gene. The other was the 1.8- Received for publication 1 November 1984 and in revised form 20 kb BamHI fragment containing the 5' portion and flanking sequence of May 1985. the fl-globin gene. The 4,# probe was the XbaI-BglII fragment of plasmid Al 1-5 which contains the 4,#-globin gene. The -y-complementary DNA 1. Abbreviations used in this paper: Hb, hemoglobin; kb, kilobase. (cDNA) probe was from plasmid JWI5 1. The 3' 'y-globin gene probe was a 1.6-kb EcoRI fragment excised from a cloned Ge-globin gene. The J. Clin. Invest. e-globin gene probe was the EcoRI-BamHI fragment of plasmid pe 1.3. © The American Society for Clinical Investigation, Inc. Two cloned unique sequence probes flanking the fl-globin gene cluster 0021-9738/85/10/1554/05 $1.00 were used. One was derived from the '#-I plasmid, which is homologous Volume 76, October 1985, 1554-1558 to a locus more than 90 kb upstream from the ,3-globin cluster, and was 1554 Curtin et al. isolated from the breakpoint of a large deletion in a case of y5b-thalas- Hinc II Hinf I semia (1 1). The other was from the pRK28 plasmid, which is homologous Ni N2 P N P to a locus 17 kb downstream from the fl-globin gene and was isolated a _ _ kb kb b from a Charon 4A library (20). -7.6 -60 1.0- Results 0.7 - Clinical and hematologic data. A 20-yr-old pregnant English woman (II-1 in Table I) was found to have a microcytic hy- pochromic anemia in the absence of iron deficiency. The HbA2 - 3.0 level was normal and the HbF was slightly elevated. Her father pp probe ,3 1.8-kb probe (I-1) and one sibling (II-3) also have hypochromic microcytic anemia with normal HbA2 and HbF. Her husband is hemato- Bam HI Hind III logically normal. A male infant (III-1) was delivered sponta- N P N P neously and experienced an uneventful neonatal period, ac- kb kb C A d cording to the history. Hematologic data were not obtained until -22.0 15.5. the infant was 1 yr old, when he was found to have a hypochromic 13.5 and microcytic anemia. The HbA2 level was normal and the HbF was elevated. Similar values were obtained at age 2 yr. At 2 yr the Gy to Ay ratio of the fetal hemoglobin was 1 to 1. We also determined the Gy and Ay ratios for subjects 1-1 and II-1, both of whom had lower fetal hemoglobin levels. The high pres- sure liquid chromatography showed predominantly Ayglobin P 0.9kb probee pRK28 probe chains. Intact map of the 6-,5-, and gene loci. Extensive Figure 1. Autoradiogram of restriction analyses of DNA digested with fl-globin with a 1.8-kb 5' restriction endonuclease mapping was performed on DNA from (a) HincII and hybridized with 4,0 probe, (b) Hinfl probe, (c) BamHI with 0.9-kb P probe, and (d) HindI11 with pRK28 subject 1-1. Digestion with the enzymes AvaII, BamHI, BclI, probe located 3' to the P gene. The lengths of the fragments in kilo- BglII, EcoRI, HindIII, Hinfl, HpaI, MspI, TaqI, and XbaI, and bases (kb) are shown. In this and subsequent figures, N indicates nor- hybridization with f3-globin gene probe produced normal-sized mal DNA control (I and 2 when two normals are used), and P is restriction fragments. The fragments were also of normal inten- DNA from the subject I-1. sity, suggesting that the fl- and 6-globin loci were intact on both chromosomes. To confirm this, we digested the DNA with five restriction enzymes known to result in polymorphic fragments. were present, indicating the presence of a 3' HincIH site on one The presence of two fragments of different lengths with a given chromosome, and its absence on the other and confirming the enzyme would indicate the existence of two loci.