Hum Genet (1992) 89:323-328
9 Springer-Verlag1992
Human a-globin gene expression is silenced by terminal truncation of chromosome 16p beginning immediately 3' of the -globin gene Luisa Romao 1'2, Faith Cash 1, Ingrid Weiss I, Stephen Liebhaber 1, Mario Pirastu 3, Renzo Galanello 4, Angela Loi 3, Elisabetta Paglietti 4, Panos loannou 5, and Antonio Cao 4 1Howard Hughes Medical Institute and Departments of Genetics and Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 2Laboratorio de Genetica Humana, Instituto Nacional de Saude, Lisboa, Portugal 3Istituto di Ricerca sulle Talassemie ed Anemia Mediterranee, CNR, Cagliari, Italy 4Instituto di Clinica e Biologia dell'Eta Evoluttiva, Universit~tdegli Studi, Cagliari, Italy 5Thalassemia Centre, Nicosia, Cyprus Received October 7, 1991 / Revised December 30, 1991
Summary. The high level expression of the human aglobin genes in erythroid tissue appears to require a set of DNaseI hypersensitive sites located upstream of the human a-globin gene cluster. These sequences, termed the locus control region (LCR), include two erythroid specific and a number of less restricted DNaseI hypersensitive sites. In this report we describe an individual with a-thalassemia associated with a truncation of the short arm of chromosome 16 that removes the LCR region and inactivates the adjacent intact a-globin genes. This genetic study supports the critical role of the L C R in the transcriptional activation of the human a-globin gene cluster and substantiates the importance of L C R deletions in the etiology of ~-thalassemia.
Introduction The human a-globin gene cluster, located subtelomeric on the short arm of chromosome 16, contains four functional genes: a single embryonic ~-globin, two fetal/ adult a-globin genes (al and ct2), and one poorly expressed fetal/adult 0-globin gene (Deisseroth et al. 1977; Nicholls et al. 1987a; Albitar et al. 1989). The two a-globin genes are coexpressed in the adult (Liebhaber et al. 1986). Synthesis of the normal hemoglobin tetramer, ct2132, depends on balanced expression of the ~- and [3globin gene clusters. The transcriptional activation of both of these clusters depends on the presence and activity of their respective locus control regions (LCRs) located 5' to each cluster ([~: Grosveld et al. 1987; a: Higgs et al. 1990; Jarman et al. 1991). The a-globin L C R consists of a set of two major erythroid specific, and a number of less restricted, DNaseI hypersensitive sites loCorrespondence to: S.A.Liebhaber, Room 437A, Clinical Re-
search Building, 422 Curie Boulevard, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104, USA
cated between 33kb and 55kb 5' to the a-globin gene cluster. These sites, which support high levels of position-independent expression of linked genes when expressed in stably transfected mouse erythroleukemia cells and when integrated in the genome of transgenic mice (Higgs et al. 1990), are critical to the expression of the cluster in vivo (Hatton et al. 1990; Liebhaber et al. 1990; Wilkie et al. 1990; Romao et al. 1991). Hemoglobin H disease (Hb H), the most severe form of ct-thalassemia compatible with postnatal viability, usually reflects the loss of function of three of the four ctglobin genes (Higgs et al. 1989; Liebhaber 1989). The most common molecular basis of this disease state is the deletion of both adjacent a-globin loci on one chromosome and the deletion of a single a-globin gene in trans (genotype: - - / - a ; Phillips et al. 1979, 1980; Embury et al. 1979; Orkin and Michelson 1980; Pressley et al. 1980a; Sophocleus et al. 1981; Nicholls et al. 1985). Less frequently, Hb H disease can also be due to coinheritance of deletion and non-deletion defects (genotype: - - / c t a z ; Moi et al. 1987), homozygosity for nondeletion defects (~taz/aar; Kattamis et al. 1988; Pressley et al. 1980b) or coinheritance of an a-cluster containing a deletion of a single a-globin gene on one chromosome and a deletion of the L C R in trans (Hatton et al. 1990; Liebhaber et al. 1990; Wilkie et al. 1990; Romao et al. 1991). In this report, we describe an Italian patient with Hb H disease, in whom both intact ct-globin genes are silenced by deletion in cis of the LCR. These results further substantiate the functional role of the a-globin cluster L C R and the importance of its loss in the etiology of a-thalassemia.
Materials and methods Clinical data on the patient
Diagnostic procedures including complete blood counts, red cell indices, hemoglobin electrophoresis at pH 8.6 and 7.0, demonstra-
324 tion of HbH precipitates by brilliant cresyl blue staining of peripheral blood, and determination of relative a- and 13-globin chain synthetic ratios were perfomed according to standard laboratory procedures.
DNA analysis" and mapping Isolation of total genomic DNA from peripheral blood leukocytes, Southern blotting, and hybridization were carried out as described (Southern 1975; Reed and Mann 1985; Church and Gilbert 1984). The probes used to map the ct-globin gene cluster itself were an aglobin gene cDNA cloned in the plasmid JW101 (Wilson et al. 1978) and a Hinfl fragment of ,~-globin gene clone, pBR~ (Proudfoot et al. 1982). The insert in the later clone represents the entire 4globin gene excluding the 5' part of the first exon. To screen for the presence of previously defined nondeletion defects in the ctland a2-globin genes, DNA from the patient was digested with Ncol to detect the AUG initiation codon mutations and Hphl to identify the IVS-I splice junction mutation (Pirastu et al. 1984; Moi et al. 1987: Orkin et al. 1981). The region 5' to the cluster was mapped with the following probes: ~,-globin: pBR, inter-~-HVR: pSG21 (Goodbourn et al. 1983), LI: pBam6.8 (Nicbolls et al. 1987b), RA0.6:pRNot2 and RA2.2: pRA5' (Nicholls et al. 1987b), and NFG400 (Wilkie et al. 1991).
Gene cloning and sequencing Total genomic DNA from the patient (IdF) was digested to completion with BamHI and size fractionated on a continuous sucrose density gradient. The fraction containing 14- to 15-kb fragments was purified and ligated to EMBL3 in the BamHI site, packaged in vitro, and propagated in Escherichia coli. The recombinant phage with the 14.5-kb fragment encompassing the two adjacent o,-globin genes was identified by hybridization to the a-globin gene probe. It was plaque purified and its structure was confirmed by restriction analysis. The fragments containing c,1- and ~.2-globin genes were subcloned from the recombinant phage in the PstI site of the Bluescript plasmid and sequenced by the dideoxy-chain termination method (Sanger et al. 1977).
Expression studies Assessment of ct-globin gene function was carried out in mouse erythroleukemia (MEL) cells by transient expression of the cloned c~2-globin gene from the transcriptionally silent chromosome. MEL cells were maintained in suspension culture in minimal essential media with 9% fetal bovine serum, 20 mM glutamine, 100 lag/ml streptomycin, and 100 units penicillin. The 1.5-kb PstI fragment containing the c~2-globin gene from the patient's silent cluster was subcloned into the EcoRI site of the expression vector pSV2Aneo as previously detailed (Weiss et al. 1990). MEL cells were co-electroporated at 800 laF with 50 lag of this plasmid and an equal quantity of pSV2Aneo.Hc~2/4. The Hc~2/4 gene contains an c~2-globin gene with a functionally silent mutation in the 3' nontranslated region that serves as a hybridization marker (Liebhaber et al. 1990). 2h after electroporation live cells were isolated on a Percoll gradient and placed in culture (Weiss et al. 1990). The total cellular RNA was isolated 48 h after transfection and analyzed by a quantitative reverse transcription/polymerase chain reaction assay as described (Weiss et al. 1990; Yamada et al. 1989).
RNA analysis Total reticulocyte RNA was isolated from peripheral blood by phenol extraction of acid precipitated polysomes (Liebhaber and Kan 1983). In vitro translations, using 1 lag of total reticulocyte RNA, were carried out in a micrococcal nuclease treated rabbit reticulocyte lysate, in a 15-lal reaction mix, in the presence of (35S) methionine (L-(35S) methionine 1400ci/mmol, Amersham), ana-
lyzed on a Triton X-100/acid/urea gel (Rovera et aI. 1978: Alter 1979) and quantitated by soft-laser densitometry, as described (Liebhaber et al. 1984; Liebhaber and Cash 1985). Analysis of the relative c~2-and c~l-globin mRNA levels was carried out by primer extension of a (32p) 5'-end-labeled primer followed by HaeIiI digestion (Moi et al. 1987; Liebhaber and Kan 1981). The digested products were analyzed on a 8% acrylamide/SM urea gel, and the band intensities on autoradiographs were quantitated as previously described (Liebhaber and Kan 1981).
Results The subject of this study was a 5-year-old girl (IdF) from Sardinia, who was referred for evaluation of anemia. Hematological studies were fully diagnostic of H b H athalassemia; H b of 8.6g/dl, M C V 62fl, M C H 18.5pg, H b A 2 1.62%, a/13 synthetic ratio 0.32, and a H b H level of 5.5% of total hemoglobin. Brilliant cresyl blue stain of her peripheral blood d e m o n s t r a t e d inclusion bodies in the majority of her red cells consistent with the high levels of H b H. T h e r e was no evidence by starch gel electrophoresis or globin chain c h r o m a t o g r a p h y of a b n o r m a l hemoglobins k n o w n to p r o d u c e a-thalassemia (data not shown). As an initial step in the molecular analysis of the H b H disease, S o u t h e r n blot analysis of the c~-globin genes was carried out. T h e most frequent molecular etiology of H b H disease is deletion of three of the four c~-globin genes ( - - / - c t ; Higgs et al. 1989; L i e b h a b e r 1989). Surprisingly, the m a p p i n g studies on this patient d e m o n s t r a t e d that she was h e t e r o z y g o u s for a c o m m o n 3.7-kb deletion that r e m o v e s a single c~-globin gene (-a3v/ao~; Surrey et al. 1978; Higgs et al. 1979; E m b u r y et al. 1980). With the exception of this deletion, mapping of the entire cluster with BamHI, BglII, and HindIII was entirely n o r m a l using both a- and ~,-globin probes. NcoI and HphI digestions excluded the presence of two previously described nondeletion defects in the c~-globin gene: loss of the A U G translation initiation codon (Pirastu et al. 1984; Moi et al. 1987) or the pentanucleotide deletion at the splice acceptor site of intron 2 of the ~2-globin gene (Orkin et al. 1981). Since the H b H p h e n o t y p e usually reflects the presence of only a single functional c~globin gene, this single deletion cannot fully account for her disease state. T h e substantial loss of c~-globin gene expression usually seen in H b H disease was confirmed by analysis of the patient's reticulocyte R N A . In vitro translations d e m o n s t r a t e d a severely depressed ct: [3 synthetic ratio. The translation of normal reticulocyte m R N A resulted in an ~ : [3 globin synthesis ratio of 1.77 while in the proposita the synthetic ratio was 0.22 (Fig. 1A). The loss of (t-globin gene expression was further explored by determining the relative levels of the (,1- and ~2-globin m R N A s . This was d e t e r m i n e d by a primer extension analysis (Liebhaber and Kan 1981) on reticulocyte R N A (Fig. 1B). In normal individuals, peripheral reticulocytes contain about 2.6-fold m o r e c~2- than al-globin m R N A (range: 2.6 + 0.6; L i e b h a b e r and Kan 1981: L i e b h a b e r et al. 1984, 1986). In this patient, the results d e m o n s t r a t e a complete lack of c~2-globin m R N A . This absence of c~2-globin m R N A was confirmed on extreme o v e r e x p o s u r e of
325
Fig. 1A, B. Analysis of reticulocyte mRNA. A In vitro translation of reticulocyte m R N A from a normal control (lane 2), and the proposita (lane 3). Lane 1 contains the translation of a water control. 1 gg total reticulocyte R N A was added to a translational lysate in the presence of (35S) methionine. Translation products were analyzed by Triton X-lOO/acid/urea polyacrylamide gel and autoradiographed. The positions of the 13- and ct-globins are indicated to the left of the autoradiograph. The ratios of c~: 13globin synthesis as detemined by densitometric scanning of the gel autoradiograph are shown below each of the respective lanes. B Primer extension analysis of the ct2:ctl m R N A ratio in the reticulocyte R N A of a water control (lane 1), a normal control (lane 2), and the proposita (lane 3). 1 Bg reticulocyte R N A was reverse transcribed using a (32p) Y-end-labeled primer hybridizing to the 3' terminus of the ct2- and a l - m R N A s . After synthesis, c D N A was digested with HaeIII and the end-labeled digestion products, specific for each of the two ct-globin m R N A species, were resolved on a 8 M urea/8% acrylamide gel and autoradiographed. The position of the al- and et2-globin mRNA-specific fragments are indicated to the left of the autoradiograph and the ratios of the a2-globin to ctl-globin m R N A specific bands are shown below each respective lane
the autoradiograph. The very low level of total et-globin mRNA as determined by the et: [3 globin synthesis in intact reticulocytes and in the in vitro translation, and the total absence of et2-globin mRNA as determined by the primer extension, suggests that both et-globin genes in the intact cluster (etet) are inactive. Based on this data the genotype of the propositus was tentatively described as _ et/(etet)T.
To characterize the presumed thalassemic defect(s) on the (etet)T chromosome, both of the et-globin genes were structurally analyzed. Both the etl- and et2-globin genes in the (etet)T cluster were cloned, and the two 1.5kb PstI fragments, extending from 570 base pairs (bp) 5' to the cap site to 84bp 3' to the poly A addition site were sequenced in their entirety. Both genes had a normal structure when compared to previously reported sequences (data not shown; Orkin and Micholson 1980; Liebhaber et al. 1981; Higgs et al. 1981). To confirm the normal structure and function of the a-globin gene on the (eta) T chromosome, we assessed the function of the et2-globin gene in transfected cells. The 1.5-kb PstI genomic fragment containing this gene was electroporated into mouse erythroleukemia (MEL) cells along with an equal quantity of the normal a2-globin gene containing a functionally silent" hybridization marker (Liebhaber et al. 1990). The relative levels of mRNA from the normal et2- and et2T-globin gene as determined by a quantitative reverse transcriptase/polymerase chain reaction assay (Weiss et al. 1990) yielded a
ratio of et2T to et2 normal of 1.1 (data not shown). This demonstrates that the et2T gene, although transcriptionally silent in vivo, is structurally normal and encodes normal levels of mRNA when expressed as the isolated gene. The above results demonstrate that the Hb H phenotype in the proposita results from coinheritance of a _et3.7 deletion and a nonfunctional (etet):r gene cluster that contains structurally and functionally (in vitro) normal et-globin genes. The possibility of a deletion in the region upstream to the cluster was next investigated. The hypervariable locus 5'HVR (Nicholls et al. 1987b; Rao.6, Fig. 2) 90 kb upstream of the et-globin genes was first analyzed. As expected, based upon the highly polymorphic nature of this site (Jarman and Higgs 1988), both of the normal controls had two distinct alleles. The proposita has only a single allele, which is also present as one of two distinct alleles in both normal controls (N1 and N2, Fig. 2D,E). The equivalent intensity of the single allele present in the patient to the same size allele in the control lanes (DNA loading is balanced - see [3globin control; Fig. 2F, G) indicates that this locus is present in only one copy in the patient. This would be consistent with deletion of the region encompassing this locus in the (eta) a" chromosome. To further define this deletion, an XbaI digest of the patient's DNA was hybridized with L1 probe (Nicholls et al. 1987b; coordinate, -10.5 kb; Fig. 2). The single band detected had an intensity one-half that of the corresponding band in the normal controls (N1 and N2: Fig. 2I) when normalized for the intensity of the [3-globin gene signal (Fig. 2G). This indicated that the patient was hemizygous for the region encompassing this locus as well. Further analysis by an EcoRI digestion hybridized to the same probe (Fig. 2H), again demonstrated a band that was half the intensity of that in the normal controls when normalized to [3-globin gene hybridization on the same filter (Fig. 2F). A similar half intensity band was detected in an XbaI digest analyzed with the RA 2.2 probe (coordinate, -87; Nicholls et al. 1987b; Fig. 2C). These results document a deletion extending from at least - 8 7 to - 1 0 kb 5' to the cluster. Such a deletion would include the et-globin cluster LCR (Higgs et al. 1990). To localize the 5' breakpoint of the deletion, we used the most upstream available probe: NFG400 (Wilkie et al. 1991). This probe recognizes a set of loci, one of which is located in the subtelomeric region of chromosome 16 (coordinate, -112kb; Fig. 2). Hybridization of this probe to an XbaI digest results in the identification of five hybridizing fragments in the normal control and only four in the patient (IdF; Fig. 2A). Similarly, the EcoRI analysis (Fig. 2B) shows the loss of a single allele in the patient compared to the control. The isolated loss of a single band in each of these digest hybridizations and the identical intensity of the remaining bands between the patient and controls indicates that the deletion includes this subtelomeric locus located 112kb 5' to the cluster. To determine more accurately the position of the 3' breakpoint, we determined whether the ~-globin gene located at the 5' terminus of the cluster was present or
326 deleted. This was done by hybridizing an E c o R I digest of the patient's D N A with a ~,-globin gene probe (Proudfoot et al. 1982). The 23-kb band containing the pseudo-~, gene is intact and of normal intensity (data not shown) indicating that the deletion does not extend 5' of the E c o R I site at +3.5 kb. The 4.5-kb allele in Fig. 2J represents the region containing the ~-globin gene. In the proposita (IdF, Fig. 2J), this allele is half the intensity of the 13-globin gene hybridization on the same filter (Fig. 2F). These data indicate that the 3' terminus of the deletion is positioned between the E c o R I site at coordinate +3.5 kb and the ~,-globin gene. To identify the b r e a k p o i n t in this region, we hybridized X b a I , BgIII, and E c o R I digests (Fig. 2K, L, M) with the I ~,HVR probe (Goodbourn et al. 1983), which detects a hypervariable locus between the ~,-globin gene and the pseudo-r locus ( G o o d b o u r n et al. 1983). The E c o R I digest revealed two alleles in the proposita: an 18.0-kb fragment containing the c~2- and a l globin genes in the -c~37 chromosome, and a 23-kb allele containing the corresponding region on the (cm) T chromosome. The hybridization to a normally sized E c o R I fragment from the (ac0 T chromosome with this probe confirms that the deletion begins 5' to the E c o R I site at coordinate + 3 . 5 k b (Fig. 2). Surprisingly, however, hybridization with the same probe to XbaI and BglII digests failed to generate discrete junction fragments; instead they generated smeared bands beginning at 13.2kb in XbaI digest, and 14.8 kb in BglII digest. The 23- and 11kb bands (Fig. 2K, L) present in the proposita, correspond to the region containing the r gene on the (-c~37) chromosome. These bands are present in the normal control as well. Based on previous observations, this band smearing indicates that the deletion extends into the telomere; the smeared bands represent terminal telomeric sequences that are heterogeneous in length (Wilkie et al. 1990). The 2-kb size difference between the smeared bands in Fig. 2K and 2 L corresponds exact-
Fig. 2. Southern blot analysis of the region upstream to the ct-globin gene cluster. The first line represents the distances from the 5' end of the a-globin cluster. Position 0 is defined as the transcription initiation site of the C,-globin gene. The numbers reflect kilobases 5' ( - ) or 3' (+) of this position. The map of the cluster is shown below the ruler (MAP). The positions of the genes (each labeled) are shown asfilled rectangles, the region between the two r genes which is hypervariable in structure is shown as a zig-zag (inter-r the position of relevant restriction sites are noted by X, XbaI; E, EcoRI; Bg, BglII, and the polymorphic XbaI site is represented by ( + / - ) . The telomere of chromosome 16p is shown at the extreme left of the map (Tel). The positions of the DNaseI hypersensitive sites (HS) are shown by the vertical arrows above the first line with the size of the arrow proportional to the degree of sensitivity (Higgs et al. 1990). The line labeled PROBE shows the position of each of the probes used, which are described in Materials and methods, Below each probe is a representative Southern blot analysis of genomic DNA digested with the enzyme indicated below each autoradiograph (labelled A-M) and hybridized with the indicated probe. The size of each of the bands is noted (in kb). N1, N2 are normal controls; IdF, the proposita. Below the autoradiographs is a diagram of the (c~t)Idvchromosome with a dashed line representing the deleted region and brackets indicating the potential borders of the breakpoint
327 ly to the distance between the BglII restriction site at coordinate 11 kb and the XbaI restriction site at coordinate 9 kb (Fig. 2). Comparison of the sizes of the smeared bands from these two digests indicates that the deletion present in the (aa) T chromosome is due to a truncation that occurred between coordinate + 3 . 5 k b (where the intact EcoRI restriction site is located, see Fig. 2) and + 1.0 kb in this cluster (Fig. 2).
Discussion In this report we describe the biochemical and molecular genetic analysis of an unusual patient with hemoglobin H disease. Despite the physical presence of three structurally normal a-globin genes ( - a / a a ) , the total a-globin synthesis programmed by the patient's reticulocytes (in vivo and in vitro) suggested that only a single locus was being expressed. Despite normal structure and function in vitro, the two a-globin genes on the (aa) x cluster were found to be silent in vivo. The silencing of these otherwise normal a-globin genes appears to result from a deletion extending from the cluster in a 5' direction. The Southern mapping data demonstrate that this deletion in the (aa) T chromosome extends from a position between coordinates +1 and + 3 . 5 k b (Fig. 2) within the cluster to the 16p telomere. This deletion removes the region 33-55 kb upstream of the a-globin gene cluster that contains positive regulatory sequences for the transcriptional activation of the a-globin genes. A similar deletion resuiting from a terminal truncation of the 16p arm has been described ((aaTI); Wilkie et al. 1990). However, that deletion can be clearly distinguished from the present case in that it has a 3' terminus significantly more remote from the cluster (coordinate, - 3 0 . 1 ; Wilkie et al. 1990). Whether a common mechanism underlies these two 16p truncations is not known. Comparison of the presently described deletion to the previously described deletions of the a-globin gene cluster L C R - ( { ~ ( l ) R A (Hatton et al. 1990), (ctct)TI (Wilkie et al. 1990), (aa) rJ (Liebhaber et al. 1990), and (act) MM (Romao et al. 1991) - shows that the present deletion is the largest yet described. The similar hematologic phenotypes of all of these patients with various deletions 5' to the ct-globin gene cluster suggest that their region of common overlap, extending from coordinates - 3 0 to - 5 2 kb is important. This region overlies the region defined to contain a set of DNaseI hypersensitive sites in the chromatin of erythroid tissue (Higgs et al. 1990), and a sequence element that fulfills the criteria for an L C R (Jarman et al. 1991). The lack of phenotypic differences between these five patients suggests that regions outside of the common overlap segment do not contain any additional sequences capable of independently influencing the ct-globin gene expression. These observations suggest that all the important regulatory sequences in the L C R for the expression of the a-globin genes are localized on the common overlap region 30-50 kb upstream of the a-globin gene cluster. The importance of the L C R deletions in a-thalassemic individuals (Hatton et al. 1990; Liebhaber et al.
1990; Wilkie et al. 1990; R o m a o et al. 1991), as well as in [3-thalassemic individuals (Kioussis et al. 1983; Curtin et al. 1985; Driscoll et al. 1989) is now clear. The present study expands this newly recognized group of a- and 13thalassemias in which the genes are structurally normal and yet functionally silent. The recognition of this group of globin gene defects should facilitate population screening, prenatal diagnosis, and genetic counseling for thalassemia. In addition, the possibility that similar defects of LCR-like sequences may constitute the molecular defects in other gene systems becomes more likely. The definition of such controlling elements and the delineation of related genetic diseases may be facilitated by the work in this set of thalassemias.
Acknowledgements. This work was supported in part by Assessorato Igiene e Sanita - Regione Sardegna, Legge Regionale no. 11, 30 Aprile 1990. The authors wish to thank Dr.D.Higgs for generously providing probes for this study. S.L. is an Investigator in the Howard Hughes Medical Institute.
References Albitar M, Peschle C, Liebhaber SA (1989) Theta, Zeta, and EpsiIon globin messenger RNAs are expressed in adults. Blood 74 : 629-637 Alter B (1979) The G7 :A7 composition of fetal hemoglobin in fetuses and newborns. Blood 54:1158-1163 Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81 : 1191-1995 Curtin P, Pirastu M, Kan YW, Gobert-Jones JA, Stephens AD, Lehmann H (1985) A distant gene deletion affects [3-globin gene function in an atypical 813-thalassemia. J Clin Invest 76: 1554-1558 Deisseroth A, Nienhuis A, Turner P, Velez R, Anderson WF, Ruddle F, Lawrence J, Creagan R, Kucherlapati R (1977) Localization of human n-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay. Cell 12: 205-218 Driscoll MC, Dobkin CS, Alter BP (1989) ~13-Thalassemiadue to a de novo mutation deleting the 5' 13-globingene activation-region hypersensitive sites. Proc Natl Acad Sci USA 86:74707474 Embury SH, Lebo RV, Dozy AM, Kan YW (1979) Organization of the a-globin genes in the Chinese ct-thalassemia syndromes. J Clin Invest 63 : 1307-1310 Embury SH, Miller JA, Dozy AM, Kan YW, Chan V, Todd D (1980) Two different molecular organizations account for the singe a-globin gene of the ct-thalassemia-2 genotype. J Clin Invest 66 : 1319-1325 Goodbourn SEY, Higgs DR, Clegg JB, Weatherall DJ (1983) Molecular basis of length polymorphism in the human ct-globin gene complex. Proc Natl Acad Sci USA 80 : 5022-5026 Grosveld F, Van AG, Greaves DR, Kollias G (1987) Position-independent high-level expression of the human A-, 13-,and hybrid a/13-globingenes in transgenic mice. Cell 51:975-985 Hatton CRS, Wilkie AOM, Drysdale HC, Wood WG, Vickers MA, Sharpe J, Ayyub H, Pretorius IM, Buckle VJ, Higgs DR (1990) ct-Thalassemiacaused by a large 62 kb deletion upstream of the human a-globin gene cluster. Blood 76 : 221-227 Higgs DR, Pressley L, Old JM, Hunt DM, Clegg JB, Weatherall D J, Serjeant GR (1979) Negro ct-thalassemia is caused by deletion of a single c~-globingene. Lancet II : 272-276 Higgs DR, Goodbourn SEY, Wainscoat JS, Clegg JB, Weatherall KJ (1981) Highly variable regions of DNA flank the human aglobin genes. Nucleic Acids Res 9 : 4213-4224
328 Higgs DR, Vickers MA, Wilkie A O M , Pretorius I-M, Jarman AP, Weatherall DJ (1989) A review of the molecular genetics of the human c~-globin gene cluster. Blood 73 : 1081-1104 Higgs DR, Wood WG, Jarman AP, Sarpe J, Lida J, Pretorius IM, Ayyub H (1990) A major positive regulatory region located far upstream of the human o.-globin gene locus. Genes Dev 4: 1588-1601 Jarman AP, Higgs D R (1988) A new hypervariable marker for the human c~-globin gene cluster. Am J Hum Genet 43 : 249-256 Jarman AP, Wood WG, Shapre JA, Gourdon G, Ayyub H, Higgs D R (1991) Characterization of the major regulatory element upstream of the human c~-globin gene cluster. Mol Cell Biol 11 : 4679-4689 Kattamis C, Kanavakis E, Metaxotou-Mavromati A, Tzotzos S, Synodinos J (1988) Correlation of clinical phenotype to genotype in haemoglobin H disease. Lancet I : 442-444 Kioussis D, Vanin E, DeLange T, Flavele RA, Grosveld FG (1983) ]3-thalassaemia. Nature 306:662-666 Liebhaber SA (1989) Alpha thalassemia: a review. Hemoglobin 13 : 685-731 Liebhaber SA, Cash FE (1985) Locus assignment of a-globin structural mutations by hybrid-selected translation. J Clin Invest 75 : 64-70 Liebhaber SA, Kan YW (1981) Differentiation of the m R N A transcripts originating from the al- and c~2-globin loci in normal and a-thalassemics. J Clin Invest 68:439-446 Liebhaber SA, Kan YW (1983) ~-Thalassemia caused by an unstable c~-globin mutant. J Clin Invest 71:461-466 Liebhaber SA, Goossens M, Kan YW (1981) Homology and concerted evolution at the o.1 and ~.2 loci of human c~-globin. Nature 290 : 26-29 Liebhaber SA, Cash FE, Shakin SH (1984) Translationally associated helix-destabilizing activity in rabbit reticulocyte lysate. J Biol Chem 259:15597-15602 Liebhaber SA, Cash FE. Ballas SK (1986) Human ~-globin gene expression: the dominant role of the c~2-1ocus in m R N A and protein synthesis. J Biol Chem 261 : 15327-15333 Liebhaber SA, Griese EU, Weiss I, Cash FE, Ayyub H, Higgs DR, Horst J (1990) Inactivation of human ~t-globin gene expression by a de novo deletion located upstream of the c~-globin gene cluster. Proc Natl Acad Sci USA 87:9431-9435 Moi P, Cash FE, Liebhaber SA, Cao A, Pirastu M (1987) An initiation codon mutation AUG---~GUG of the human ~l-globin gene: structural characterization and evidence for a mild thalassemic phenotype. J Clin Invest 80:I417-1421 Nicholls RD, Higgs DR, Clegg JB, Weatherall DJ (1985) u.~ Thalassemia due to recombination between the a-globin gene and an Alu repeat. Blood 65 : 1434-1438 Nicholls R, Jonasson JA, McGee JOD, Patil S, Ionasescu VV, Weatherall D J, Higgs D (1987a) High resolution gene mapping of the human ~-globin locus. J Med Genet 24 : 39-46 Nicholls RD, Fischel-Ghodsian N, Higgs D R (1987b) Recombination at the human c~-globin gene cluster: sequence features and topological constraints. Cell 49 : 369-378 Orkin SH, Michelson AM (1980) Partial deletion of the a-globin structural gene in human c~-thalassemia. Nature 286 : 538-540 Orkin SH, Goff SC, Hechtman RL (1981) Mutation in an intervening sequence splice junction in man. Proc Natl Acad Sci USA 78 : 5041-5045 Phillips J A III, Scott AF, Smith KD, Young KE, Lightbody KL, Jiji RM, Kazazian H H Jr (1979) A molecular basis for hemoglobin H disease in American blacks. Blood 54 : 1439-1445
Phillips JA III, Vik TA, Scott AF, Young KE, Kazazian HH, Smith KD, Fairbanks VF, Koenig HM (1980) Unequal crossing-over: a common basis of single ~-globin genes in Asians and American blacks with hemoglobin H disease. Blood 55: 1066-1069 Pirastu M, Saglio G, Chang JC, Cao A, Kan YW (1984) Initiation codon mutation as a cause of c~-thalassemia. J Biol Chem 259 : 12315-12317 Pressley L, Higgs DR, Aldridge B, Metaxatou-Mavromati A, Clegg JB, Weatherall DJ (1980a) Characterization of new ~thalassemia-I defect due to a partial deletion of the ~-globin gene complex. Nucleic Acids Res 8 : 4889-4898 Pressley L, Higgs DR, Clegg JB, Perrine RP, Pembrey ME, Weatherall DJ (1980b) A new genetic basis for hemoglobin-H disease. N Engl J Med 303 : 1383-1386 Proudfoot N J, Gil A, Maniatis T (1982) The structure of the human zeta-globin gene and a closely linked, nearly identical pseudogene. Cell 31 : 553-563 Reed KC, Mann DA (1985) Rapid transfer of D N A from agarose gels to nylon membranes. Nucleic Acids Res 13:7207-7221 Romao L, Osorio-Almeida L, Higgs DR, Lavinha J, Liebhaber SA (1991) u-Thalassemia resulting from deletion of regulatory sequences far upstream of the a-globin structural genes. Blood 78:1589-1595 Rovera G, Magarian C, Borun TW (1978) Resolution of hemoglobin subunits by electrophoresis in acid urea polyacrylamide gels containing Triton X-100. Anal Biochem 85:506-518 Sanger F, Nicklen S, Coulson A R (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 4 : 54635467 Sophocleus T, Higgs DR, Aldridge B. Trent R J, Pressley L, Clegg JB, Weatherall DJ (1981) The molecular basis for the hemoglobin Bart's hydrops fetalis syndrome in Cyprus. Br J Haematol 47:153-156 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98 : 503517 Surrey S, Chambers JS, Muni D, Schwartz E (1978) Restriction endonuclease analysis of human globin genes in cellular DNA. Biochem Biophys Res Commun 83:1125-1131 Weiss I, Cash FE, Coleman MB, Pressley A. Adams JG, Sanguansermsri R, Liebhaber SA (1990) Molecular basis for a-thalassemia associated with the structural mutant hemoglobin SuanDok (Ct2109LEU~ARO).Blood 76 : 2630-2636 Wilkie A O M , Lamb J, Harris PC, Finney RD, Higgs D R (1990) A truncated human chromosome i6 associated with ~-thalassemia is stabilized by addition of telomeric repeat (TTAGGG)n. Nature 346 : 868-871 Wilkie AOM, Higgs DR, Rack KA, Buckle VJ, Spurt NK, FischelGhodsian N, Ceccherini I, Brown W R A , Harris PC (1991) Stable length polymorphism of up to 260kb at the tip of the short arm of human chromosome 16. Cell 64 : 595-606 Wilson JT, Wilson LB, De Rial JK, Villa-Komaroff L, Efstratiadis A, Forget BG, Weissman SM (1978) Insertion of synthetic copies of human globin genes into bacterial plasmid. Nucleic Acids Res 5 : 563-581 Yamada M, Hudson S, Tournay O, Bittenbender S, Shane SS, Lang B, Tsiyirmoto Y, Caton AJ, Rovera G (1989) Detection of minimal disease in hematopoietic malignancies of the B-cell lineage by using third complementary-determining region (CRD-III)-specific probes. Proc Natl Acad Sci USA 86:51235127
9 Springer-Verlag1992
Human a-globin gene expression is silenced by terminal truncation of chromosome 16p beginning immediately 3' of the -globin gene Luisa Romao 1'2, Faith Cash 1, Ingrid Weiss I, Stephen Liebhaber 1, Mario Pirastu 3, Renzo Galanello 4, Angela Loi 3, Elisabetta Paglietti 4, Panos loannou 5, and Antonio Cao 4 1Howard Hughes Medical Institute and Departments of Genetics and Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 2Laboratorio de Genetica Humana, Instituto Nacional de Saude, Lisboa, Portugal 3Istituto di Ricerca sulle Talassemie ed Anemia Mediterranee, CNR, Cagliari, Italy 4Instituto di Clinica e Biologia dell'Eta Evoluttiva, Universit~tdegli Studi, Cagliari, Italy 5Thalassemia Centre, Nicosia, Cyprus Received October 7, 1991 / Revised December 30, 1991
Summary. The high level expression of the human aglobin genes in erythroid tissue appears to require a set of DNaseI hypersensitive sites located upstream of the human a-globin gene cluster. These sequences, termed the locus control region (LCR), include two erythroid specific and a number of less restricted DNaseI hypersensitive sites. In this report we describe an individual with a-thalassemia associated with a truncation of the short arm of chromosome 16 that removes the LCR region and inactivates the adjacent intact a-globin genes. This genetic study supports the critical role of the L C R in the transcriptional activation of the human a-globin gene cluster and substantiates the importance of L C R deletions in the etiology of ~-thalassemia.
Introduction The human a-globin gene cluster, located subtelomeric on the short arm of chromosome 16, contains four functional genes: a single embryonic ~-globin, two fetal/ adult a-globin genes (al and ct2), and one poorly expressed fetal/adult 0-globin gene (Deisseroth et al. 1977; Nicholls et al. 1987a; Albitar et al. 1989). The two a-globin genes are coexpressed in the adult (Liebhaber et al. 1986). Synthesis of the normal hemoglobin tetramer, ct2132, depends on balanced expression of the ~- and [3globin gene clusters. The transcriptional activation of both of these clusters depends on the presence and activity of their respective locus control regions (LCRs) located 5' to each cluster ([~: Grosveld et al. 1987; a: Higgs et al. 1990; Jarman et al. 1991). The a-globin L C R consists of a set of two major erythroid specific, and a number of less restricted, DNaseI hypersensitive sites loCorrespondence to: S.A.Liebhaber, Room 437A, Clinical Re-
search Building, 422 Curie Boulevard, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104, USA
cated between 33kb and 55kb 5' to the a-globin gene cluster. These sites, which support high levels of position-independent expression of linked genes when expressed in stably transfected mouse erythroleukemia cells and when integrated in the genome of transgenic mice (Higgs et al. 1990), are critical to the expression of the cluster in vivo (Hatton et al. 1990; Liebhaber et al. 1990; Wilkie et al. 1990; Romao et al. 1991). Hemoglobin H disease (Hb H), the most severe form of ct-thalassemia compatible with postnatal viability, usually reflects the loss of function of three of the four ctglobin genes (Higgs et al. 1989; Liebhaber 1989). The most common molecular basis of this disease state is the deletion of both adjacent a-globin loci on one chromosome and the deletion of a single a-globin gene in trans (genotype: - - / - a ; Phillips et al. 1979, 1980; Embury et al. 1979; Orkin and Michelson 1980; Pressley et al. 1980a; Sophocleus et al. 1981; Nicholls et al. 1985). Less frequently, Hb H disease can also be due to coinheritance of deletion and non-deletion defects (genotype: - - / c t a z ; Moi et al. 1987), homozygosity for nondeletion defects (~taz/aar; Kattamis et al. 1988; Pressley et al. 1980b) or coinheritance of an a-cluster containing a deletion of a single a-globin gene on one chromosome and a deletion of the L C R in trans (Hatton et al. 1990; Liebhaber et al. 1990; Wilkie et al. 1990; Romao et al. 1991). In this report, we describe an Italian patient with Hb H disease, in whom both intact ct-globin genes are silenced by deletion in cis of the LCR. These results further substantiate the functional role of the a-globin cluster L C R and the importance of its loss in the etiology of a-thalassemia.
Materials and methods Clinical data on the patient
Diagnostic procedures including complete blood counts, red cell indices, hemoglobin electrophoresis at pH 8.6 and 7.0, demonstra-
324 tion of HbH precipitates by brilliant cresyl blue staining of peripheral blood, and determination of relative a- and 13-globin chain synthetic ratios were perfomed according to standard laboratory procedures.
DNA analysis" and mapping Isolation of total genomic DNA from peripheral blood leukocytes, Southern blotting, and hybridization were carried out as described (Southern 1975; Reed and Mann 1985; Church and Gilbert 1984). The probes used to map the ct-globin gene cluster itself were an aglobin gene cDNA cloned in the plasmid JW101 (Wilson et al. 1978) and a Hinfl fragment of ,~-globin gene clone, pBR~ (Proudfoot et al. 1982). The insert in the later clone represents the entire 4globin gene excluding the 5' part of the first exon. To screen for the presence of previously defined nondeletion defects in the ctland a2-globin genes, DNA from the patient was digested with Ncol to detect the AUG initiation codon mutations and Hphl to identify the IVS-I splice junction mutation (Pirastu et al. 1984; Moi et al. 1987: Orkin et al. 1981). The region 5' to the cluster was mapped with the following probes: ~,-globin: pBR, inter-~-HVR: pSG21 (Goodbourn et al. 1983), LI: pBam6.8 (Nicbolls et al. 1987b), RA0.6:pRNot2 and RA2.2: pRA5' (Nicholls et al. 1987b), and NFG400 (Wilkie et al. 1991).
Gene cloning and sequencing Total genomic DNA from the patient (IdF) was digested to completion with BamHI and size fractionated on a continuous sucrose density gradient. The fraction containing 14- to 15-kb fragments was purified and ligated to EMBL3 in the BamHI site, packaged in vitro, and propagated in Escherichia coli. The recombinant phage with the 14.5-kb fragment encompassing the two adjacent o,-globin genes was identified by hybridization to the a-globin gene probe. It was plaque purified and its structure was confirmed by restriction analysis. The fragments containing c,1- and ~.2-globin genes were subcloned from the recombinant phage in the PstI site of the Bluescript plasmid and sequenced by the dideoxy-chain termination method (Sanger et al. 1977).
Expression studies Assessment of ct-globin gene function was carried out in mouse erythroleukemia (MEL) cells by transient expression of the cloned c~2-globin gene from the transcriptionally silent chromosome. MEL cells were maintained in suspension culture in minimal essential media with 9% fetal bovine serum, 20 mM glutamine, 100 lag/ml streptomycin, and 100 units penicillin. The 1.5-kb PstI fragment containing the c~2-globin gene from the patient's silent cluster was subcloned into the EcoRI site of the expression vector pSV2Aneo as previously detailed (Weiss et al. 1990). MEL cells were co-electroporated at 800 laF with 50 lag of this plasmid and an equal quantity of pSV2Aneo.Hc~2/4. The Hc~2/4 gene contains an c~2-globin gene with a functionally silent mutation in the 3' nontranslated region that serves as a hybridization marker (Liebhaber et al. 1990). 2h after electroporation live cells were isolated on a Percoll gradient and placed in culture (Weiss et al. 1990). The total cellular RNA was isolated 48 h after transfection and analyzed by a quantitative reverse transcription/polymerase chain reaction assay as described (Weiss et al. 1990; Yamada et al. 1989).
RNA analysis Total reticulocyte RNA was isolated from peripheral blood by phenol extraction of acid precipitated polysomes (Liebhaber and Kan 1983). In vitro translations, using 1 lag of total reticulocyte RNA, were carried out in a micrococcal nuclease treated rabbit reticulocyte lysate, in a 15-lal reaction mix, in the presence of (35S) methionine (L-(35S) methionine 1400ci/mmol, Amersham), ana-
lyzed on a Triton X-100/acid/urea gel (Rovera et aI. 1978: Alter 1979) and quantitated by soft-laser densitometry, as described (Liebhaber et al. 1984; Liebhaber and Cash 1985). Analysis of the relative c~2-and c~l-globin mRNA levels was carried out by primer extension of a (32p) 5'-end-labeled primer followed by HaeIiI digestion (Moi et al. 1987; Liebhaber and Kan 1981). The digested products were analyzed on a 8% acrylamide/SM urea gel, and the band intensities on autoradiographs were quantitated as previously described (Liebhaber and Kan 1981).
Results The subject of this study was a 5-year-old girl (IdF) from Sardinia, who was referred for evaluation of anemia. Hematological studies were fully diagnostic of H b H athalassemia; H b of 8.6g/dl, M C V 62fl, M C H 18.5pg, H b A 2 1.62%, a/13 synthetic ratio 0.32, and a H b H level of 5.5% of total hemoglobin. Brilliant cresyl blue stain of her peripheral blood d e m o n s t r a t e d inclusion bodies in the majority of her red cells consistent with the high levels of H b H. T h e r e was no evidence by starch gel electrophoresis or globin chain c h r o m a t o g r a p h y of a b n o r m a l hemoglobins k n o w n to p r o d u c e a-thalassemia (data not shown). As an initial step in the molecular analysis of the H b H disease, S o u t h e r n blot analysis of the c~-globin genes was carried out. T h e most frequent molecular etiology of H b H disease is deletion of three of the four c~-globin genes ( - - / - c t ; Higgs et al. 1989; L i e b h a b e r 1989). Surprisingly, the m a p p i n g studies on this patient d e m o n s t r a t e d that she was h e t e r o z y g o u s for a c o m m o n 3.7-kb deletion that r e m o v e s a single c~-globin gene (-a3v/ao~; Surrey et al. 1978; Higgs et al. 1979; E m b u r y et al. 1980). With the exception of this deletion, mapping of the entire cluster with BamHI, BglII, and HindIII was entirely n o r m a l using both a- and ~,-globin probes. NcoI and HphI digestions excluded the presence of two previously described nondeletion defects in the c~-globin gene: loss of the A U G translation initiation codon (Pirastu et al. 1984; Moi et al. 1987) or the pentanucleotide deletion at the splice acceptor site of intron 2 of the ~2-globin gene (Orkin et al. 1981). Since the H b H p h e n o t y p e usually reflects the presence of only a single functional c~globin gene, this single deletion cannot fully account for her disease state. T h e substantial loss of c~-globin gene expression usually seen in H b H disease was confirmed by analysis of the patient's reticulocyte R N A . In vitro translations d e m o n s t r a t e d a severely depressed ct: [3 synthetic ratio. The translation of normal reticulocyte m R N A resulted in an ~ : [3 globin synthesis ratio of 1.77 while in the proposita the synthetic ratio was 0.22 (Fig. 1A). The loss of (t-globin gene expression was further explored by determining the relative levels of the (,1- and ~2-globin m R N A s . This was d e t e r m i n e d by a primer extension analysis (Liebhaber and Kan 1981) on reticulocyte R N A (Fig. 1B). In normal individuals, peripheral reticulocytes contain about 2.6-fold m o r e c~2- than al-globin m R N A (range: 2.6 + 0.6; L i e b h a b e r and Kan 1981: L i e b h a b e r et al. 1984, 1986). In this patient, the results d e m o n s t r a t e a complete lack of c~2-globin m R N A . This absence of c~2-globin m R N A was confirmed on extreme o v e r e x p o s u r e of
325
Fig. 1A, B. Analysis of reticulocyte mRNA. A In vitro translation of reticulocyte m R N A from a normal control (lane 2), and the proposita (lane 3). Lane 1 contains the translation of a water control. 1 gg total reticulocyte R N A was added to a translational lysate in the presence of (35S) methionine. Translation products were analyzed by Triton X-lOO/acid/urea polyacrylamide gel and autoradiographed. The positions of the 13- and ct-globins are indicated to the left of the autoradiograph. The ratios of c~: 13globin synthesis as detemined by densitometric scanning of the gel autoradiograph are shown below each of the respective lanes. B Primer extension analysis of the ct2:ctl m R N A ratio in the reticulocyte R N A of a water control (lane 1), a normal control (lane 2), and the proposita (lane 3). 1 Bg reticulocyte R N A was reverse transcribed using a (32p) Y-end-labeled primer hybridizing to the 3' terminus of the ct2- and a l - m R N A s . After synthesis, c D N A was digested with HaeIII and the end-labeled digestion products, specific for each of the two ct-globin m R N A species, were resolved on a 8 M urea/8% acrylamide gel and autoradiographed. The position of the al- and et2-globin mRNA-specific fragments are indicated to the left of the autoradiograph and the ratios of the a2-globin to ctl-globin m R N A specific bands are shown below each respective lane
the autoradiograph. The very low level of total et-globin mRNA as determined by the et: [3 globin synthesis in intact reticulocytes and in the in vitro translation, and the total absence of et2-globin mRNA as determined by the primer extension, suggests that both et-globin genes in the intact cluster (etet) are inactive. Based on this data the genotype of the propositus was tentatively described as _ et/(etet)T.
To characterize the presumed thalassemic defect(s) on the (etet)T chromosome, both of the et-globin genes were structurally analyzed. Both the etl- and et2-globin genes in the (etet)T cluster were cloned, and the two 1.5kb PstI fragments, extending from 570 base pairs (bp) 5' to the cap site to 84bp 3' to the poly A addition site were sequenced in their entirety. Both genes had a normal structure when compared to previously reported sequences (data not shown; Orkin and Micholson 1980; Liebhaber et al. 1981; Higgs et al. 1981). To confirm the normal structure and function of the a-globin gene on the (eta) T chromosome, we assessed the function of the et2-globin gene in transfected cells. The 1.5-kb PstI genomic fragment containing this gene was electroporated into mouse erythroleukemia (MEL) cells along with an equal quantity of the normal a2-globin gene containing a functionally silent" hybridization marker (Liebhaber et al. 1990). The relative levels of mRNA from the normal et2- and et2T-globin gene as determined by a quantitative reverse transcriptase/polymerase chain reaction assay (Weiss et al. 1990) yielded a
ratio of et2T to et2 normal of 1.1 (data not shown). This demonstrates that the et2T gene, although transcriptionally silent in vivo, is structurally normal and encodes normal levels of mRNA when expressed as the isolated gene. The above results demonstrate that the Hb H phenotype in the proposita results from coinheritance of a _et3.7 deletion and a nonfunctional (etet):r gene cluster that contains structurally and functionally (in vitro) normal et-globin genes. The possibility of a deletion in the region upstream to the cluster was next investigated. The hypervariable locus 5'HVR (Nicholls et al. 1987b; Rao.6, Fig. 2) 90 kb upstream of the et-globin genes was first analyzed. As expected, based upon the highly polymorphic nature of this site (Jarman and Higgs 1988), both of the normal controls had two distinct alleles. The proposita has only a single allele, which is also present as one of two distinct alleles in both normal controls (N1 and N2, Fig. 2D,E). The equivalent intensity of the single allele present in the patient to the same size allele in the control lanes (DNA loading is balanced - see [3globin control; Fig. 2F, G) indicates that this locus is present in only one copy in the patient. This would be consistent with deletion of the region encompassing this locus in the (eta) a" chromosome. To further define this deletion, an XbaI digest of the patient's DNA was hybridized with L1 probe (Nicholls et al. 1987b; coordinate, -10.5 kb; Fig. 2). The single band detected had an intensity one-half that of the corresponding band in the normal controls (N1 and N2: Fig. 2I) when normalized for the intensity of the [3-globin gene signal (Fig. 2G). This indicated that the patient was hemizygous for the region encompassing this locus as well. Further analysis by an EcoRI digestion hybridized to the same probe (Fig. 2H), again demonstrated a band that was half the intensity of that in the normal controls when normalized to [3-globin gene hybridization on the same filter (Fig. 2F). A similar half intensity band was detected in an XbaI digest analyzed with the RA 2.2 probe (coordinate, -87; Nicholls et al. 1987b; Fig. 2C). These results document a deletion extending from at least - 8 7 to - 1 0 kb 5' to the cluster. Such a deletion would include the et-globin cluster LCR (Higgs et al. 1990). To localize the 5' breakpoint of the deletion, we used the most upstream available probe: NFG400 (Wilkie et al. 1991). This probe recognizes a set of loci, one of which is located in the subtelomeric region of chromosome 16 (coordinate, -112kb; Fig. 2). Hybridization of this probe to an XbaI digest results in the identification of five hybridizing fragments in the normal control and only four in the patient (IdF; Fig. 2A). Similarly, the EcoRI analysis (Fig. 2B) shows the loss of a single allele in the patient compared to the control. The isolated loss of a single band in each of these digest hybridizations and the identical intensity of the remaining bands between the patient and controls indicates that the deletion includes this subtelomeric locus located 112kb 5' to the cluster. To determine more accurately the position of the 3' breakpoint, we determined whether the ~-globin gene located at the 5' terminus of the cluster was present or
326 deleted. This was done by hybridizing an E c o R I digest of the patient's D N A with a ~,-globin gene probe (Proudfoot et al. 1982). The 23-kb band containing the pseudo-~, gene is intact and of normal intensity (data not shown) indicating that the deletion does not extend 5' of the E c o R I site at +3.5 kb. The 4.5-kb allele in Fig. 2J represents the region containing the ~-globin gene. In the proposita (IdF, Fig. 2J), this allele is half the intensity of the 13-globin gene hybridization on the same filter (Fig. 2F). These data indicate that the 3' terminus of the deletion is positioned between the E c o R I site at coordinate +3.5 kb and the ~,-globin gene. To identify the b r e a k p o i n t in this region, we hybridized X b a I , BgIII, and E c o R I digests (Fig. 2K, L, M) with the I ~,HVR probe (Goodbourn et al. 1983), which detects a hypervariable locus between the ~,-globin gene and the pseudo-r locus ( G o o d b o u r n et al. 1983). The E c o R I digest revealed two alleles in the proposita: an 18.0-kb fragment containing the c~2- and a l globin genes in the -c~37 chromosome, and a 23-kb allele containing the corresponding region on the (cm) T chromosome. The hybridization to a normally sized E c o R I fragment from the (ac0 T chromosome with this probe confirms that the deletion begins 5' to the E c o R I site at coordinate + 3 . 5 k b (Fig. 2). Surprisingly, however, hybridization with the same probe to XbaI and BglII digests failed to generate discrete junction fragments; instead they generated smeared bands beginning at 13.2kb in XbaI digest, and 14.8 kb in BglII digest. The 23- and 11kb bands (Fig. 2K, L) present in the proposita, correspond to the region containing the r gene on the (-c~37) chromosome. These bands are present in the normal control as well. Based on previous observations, this band smearing indicates that the deletion extends into the telomere; the smeared bands represent terminal telomeric sequences that are heterogeneous in length (Wilkie et al. 1990). The 2-kb size difference between the smeared bands in Fig. 2K and 2 L corresponds exact-
Fig. 2. Southern blot analysis of the region upstream to the ct-globin gene cluster. The first line represents the distances from the 5' end of the a-globin cluster. Position 0 is defined as the transcription initiation site of the C,-globin gene. The numbers reflect kilobases 5' ( - ) or 3' (+) of this position. The map of the cluster is shown below the ruler (MAP). The positions of the genes (each labeled) are shown asfilled rectangles, the region between the two r genes which is hypervariable in structure is shown as a zig-zag (inter-r the position of relevant restriction sites are noted by X, XbaI; E, EcoRI; Bg, BglII, and the polymorphic XbaI site is represented by ( + / - ) . The telomere of chromosome 16p is shown at the extreme left of the map (Tel). The positions of the DNaseI hypersensitive sites (HS) are shown by the vertical arrows above the first line with the size of the arrow proportional to the degree of sensitivity (Higgs et al. 1990). The line labeled PROBE shows the position of each of the probes used, which are described in Materials and methods, Below each probe is a representative Southern blot analysis of genomic DNA digested with the enzyme indicated below each autoradiograph (labelled A-M) and hybridized with the indicated probe. The size of each of the bands is noted (in kb). N1, N2 are normal controls; IdF, the proposita. Below the autoradiographs is a diagram of the (c~t)Idvchromosome with a dashed line representing the deleted region and brackets indicating the potential borders of the breakpoint
327 ly to the distance between the BglII restriction site at coordinate 11 kb and the XbaI restriction site at coordinate 9 kb (Fig. 2). Comparison of the sizes of the smeared bands from these two digests indicates that the deletion present in the (aa) T chromosome is due to a truncation that occurred between coordinate + 3 . 5 k b (where the intact EcoRI restriction site is located, see Fig. 2) and + 1.0 kb in this cluster (Fig. 2).
Discussion In this report we describe the biochemical and molecular genetic analysis of an unusual patient with hemoglobin H disease. Despite the physical presence of three structurally normal a-globin genes ( - a / a a ) , the total a-globin synthesis programmed by the patient's reticulocytes (in vivo and in vitro) suggested that only a single locus was being expressed. Despite normal structure and function in vitro, the two a-globin genes on the (aa) x cluster were found to be silent in vivo. The silencing of these otherwise normal a-globin genes appears to result from a deletion extending from the cluster in a 5' direction. The Southern mapping data demonstrate that this deletion in the (aa) T chromosome extends from a position between coordinates +1 and + 3 . 5 k b (Fig. 2) within the cluster to the 16p telomere. This deletion removes the region 33-55 kb upstream of the a-globin gene cluster that contains positive regulatory sequences for the transcriptional activation of the a-globin genes. A similar deletion resuiting from a terminal truncation of the 16p arm has been described ((aaTI); Wilkie et al. 1990). However, that deletion can be clearly distinguished from the present case in that it has a 3' terminus significantly more remote from the cluster (coordinate, - 3 0 . 1 ; Wilkie et al. 1990). Whether a common mechanism underlies these two 16p truncations is not known. Comparison of the presently described deletion to the previously described deletions of the a-globin gene cluster L C R - ( { ~ ( l ) R A (Hatton et al. 1990), (ctct)TI (Wilkie et al. 1990), (aa) rJ (Liebhaber et al. 1990), and (act) MM (Romao et al. 1991) - shows that the present deletion is the largest yet described. The similar hematologic phenotypes of all of these patients with various deletions 5' to the ct-globin gene cluster suggest that their region of common overlap, extending from coordinates - 3 0 to - 5 2 kb is important. This region overlies the region defined to contain a set of DNaseI hypersensitive sites in the chromatin of erythroid tissue (Higgs et al. 1990), and a sequence element that fulfills the criteria for an L C R (Jarman et al. 1991). The lack of phenotypic differences between these five patients suggests that regions outside of the common overlap segment do not contain any additional sequences capable of independently influencing the ct-globin gene expression. These observations suggest that all the important regulatory sequences in the L C R for the expression of the a-globin genes are localized on the common overlap region 30-50 kb upstream of the a-globin gene cluster. The importance of the L C R deletions in a-thalassemic individuals (Hatton et al. 1990; Liebhaber et al.
1990; Wilkie et al. 1990; R o m a o et al. 1991), as well as in [3-thalassemic individuals (Kioussis et al. 1983; Curtin et al. 1985; Driscoll et al. 1989) is now clear. The present study expands this newly recognized group of a- and 13thalassemias in which the genes are structurally normal and yet functionally silent. The recognition of this group of globin gene defects should facilitate population screening, prenatal diagnosis, and genetic counseling for thalassemia. In addition, the possibility that similar defects of LCR-like sequences may constitute the molecular defects in other gene systems becomes more likely. The definition of such controlling elements and the delineation of related genetic diseases may be facilitated by the work in this set of thalassemias.
Acknowledgements. This work was supported in part by Assessorato Igiene e Sanita - Regione Sardegna, Legge Regionale no. 11, 30 Aprile 1990. The authors wish to thank Dr.D.Higgs for generously providing probes for this study. S.L. is an Investigator in the Howard Hughes Medical Institute.
References Albitar M, Peschle C, Liebhaber SA (1989) Theta, Zeta, and EpsiIon globin messenger RNAs are expressed in adults. Blood 74 : 629-637 Alter B (1979) The G7 :A7 composition of fetal hemoglobin in fetuses and newborns. Blood 54:1158-1163 Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81 : 1191-1995 Curtin P, Pirastu M, Kan YW, Gobert-Jones JA, Stephens AD, Lehmann H (1985) A distant gene deletion affects [3-globin gene function in an atypical 813-thalassemia. J Clin Invest 76: 1554-1558 Deisseroth A, Nienhuis A, Turner P, Velez R, Anderson WF, Ruddle F, Lawrence J, Creagan R, Kucherlapati R (1977) Localization of human n-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay. Cell 12: 205-218 Driscoll MC, Dobkin CS, Alter BP (1989) ~13-Thalassemiadue to a de novo mutation deleting the 5' 13-globingene activation-region hypersensitive sites. Proc Natl Acad Sci USA 86:74707474 Embury SH, Lebo RV, Dozy AM, Kan YW (1979) Organization of the a-globin genes in the Chinese ct-thalassemia syndromes. J Clin Invest 63 : 1307-1310 Embury SH, Miller JA, Dozy AM, Kan YW, Chan V, Todd D (1980) Two different molecular organizations account for the singe a-globin gene of the ct-thalassemia-2 genotype. J Clin Invest 66 : 1319-1325 Goodbourn SEY, Higgs DR, Clegg JB, Weatherall DJ (1983) Molecular basis of length polymorphism in the human ct-globin gene complex. Proc Natl Acad Sci USA 80 : 5022-5026 Grosveld F, Van AG, Greaves DR, Kollias G (1987) Position-independent high-level expression of the human A-, 13-,and hybrid a/13-globingenes in transgenic mice. Cell 51:975-985 Hatton CRS, Wilkie AOM, Drysdale HC, Wood WG, Vickers MA, Sharpe J, Ayyub H, Pretorius IM, Buckle VJ, Higgs DR (1990) ct-Thalassemiacaused by a large 62 kb deletion upstream of the human a-globin gene cluster. Blood 76 : 221-227 Higgs DR, Pressley L, Old JM, Hunt DM, Clegg JB, Weatherall D J, Serjeant GR (1979) Negro ct-thalassemia is caused by deletion of a single c~-globingene. Lancet II : 272-276 Higgs DR, Goodbourn SEY, Wainscoat JS, Clegg JB, Weatherall KJ (1981) Highly variable regions of DNA flank the human aglobin genes. Nucleic Acids Res 9 : 4213-4224
328 Higgs DR, Vickers MA, Wilkie A O M , Pretorius I-M, Jarman AP, Weatherall DJ (1989) A review of the molecular genetics of the human c~-globin gene cluster. Blood 73 : 1081-1104 Higgs DR, Wood WG, Jarman AP, Sarpe J, Lida J, Pretorius IM, Ayyub H (1990) A major positive regulatory region located far upstream of the human o.-globin gene locus. Genes Dev 4: 1588-1601 Jarman AP, Higgs D R (1988) A new hypervariable marker for the human c~-globin gene cluster. Am J Hum Genet 43 : 249-256 Jarman AP, Wood WG, Shapre JA, Gourdon G, Ayyub H, Higgs D R (1991) Characterization of the major regulatory element upstream of the human c~-globin gene cluster. Mol Cell Biol 11 : 4679-4689 Kattamis C, Kanavakis E, Metaxotou-Mavromati A, Tzotzos S, Synodinos J (1988) Correlation of clinical phenotype to genotype in haemoglobin H disease. Lancet I : 442-444 Kioussis D, Vanin E, DeLange T, Flavele RA, Grosveld FG (1983) ]3-thalassaemia. Nature 306:662-666 Liebhaber SA (1989) Alpha thalassemia: a review. Hemoglobin 13 : 685-731 Liebhaber SA, Cash FE (1985) Locus assignment of a-globin structural mutations by hybrid-selected translation. J Clin Invest 75 : 64-70 Liebhaber SA, Kan YW (1981) Differentiation of the m R N A transcripts originating from the al- and c~2-globin loci in normal and a-thalassemics. J Clin Invest 68:439-446 Liebhaber SA, Kan YW (1983) ~-Thalassemia caused by an unstable c~-globin mutant. J Clin Invest 71:461-466 Liebhaber SA, Goossens M, Kan YW (1981) Homology and concerted evolution at the o.1 and ~.2 loci of human c~-globin. Nature 290 : 26-29 Liebhaber SA, Cash FE, Shakin SH (1984) Translationally associated helix-destabilizing activity in rabbit reticulocyte lysate. J Biol Chem 259:15597-15602 Liebhaber SA, Cash FE. Ballas SK (1986) Human ~-globin gene expression: the dominant role of the c~2-1ocus in m R N A and protein synthesis. J Biol Chem 261 : 15327-15333 Liebhaber SA, Griese EU, Weiss I, Cash FE, Ayyub H, Higgs DR, Horst J (1990) Inactivation of human ~t-globin gene expression by a de novo deletion located upstream of the c~-globin gene cluster. Proc Natl Acad Sci USA 87:9431-9435 Moi P, Cash FE, Liebhaber SA, Cao A, Pirastu M (1987) An initiation codon mutation AUG---~GUG of the human ~l-globin gene: structural characterization and evidence for a mild thalassemic phenotype. J Clin Invest 80:I417-1421 Nicholls RD, Higgs DR, Clegg JB, Weatherall DJ (1985) u.~ Thalassemia due to recombination between the a-globin gene and an Alu repeat. Blood 65 : 1434-1438 Nicholls R, Jonasson JA, McGee JOD, Patil S, Ionasescu VV, Weatherall D J, Higgs D (1987a) High resolution gene mapping of the human ~-globin locus. J Med Genet 24 : 39-46 Nicholls RD, Fischel-Ghodsian N, Higgs D R (1987b) Recombination at the human c~-globin gene cluster: sequence features and topological constraints. Cell 49 : 369-378 Orkin SH, Michelson AM (1980) Partial deletion of the a-globin structural gene in human c~-thalassemia. Nature 286 : 538-540 Orkin SH, Goff SC, Hechtman RL (1981) Mutation in an intervening sequence splice junction in man. Proc Natl Acad Sci USA 78 : 5041-5045 Phillips J A III, Scott AF, Smith KD, Young KE, Lightbody KL, Jiji RM, Kazazian H H Jr (1979) A molecular basis for hemoglobin H disease in American blacks. Blood 54 : 1439-1445
Phillips JA III, Vik TA, Scott AF, Young KE, Kazazian HH, Smith KD, Fairbanks VF, Koenig HM (1980) Unequal crossing-over: a common basis of single ~-globin genes in Asians and American blacks with hemoglobin H disease. Blood 55: 1066-1069 Pirastu M, Saglio G, Chang JC, Cao A, Kan YW (1984) Initiation codon mutation as a cause of c~-thalassemia. J Biol Chem 259 : 12315-12317 Pressley L, Higgs DR, Aldridge B, Metaxatou-Mavromati A, Clegg JB, Weatherall DJ (1980a) Characterization of new ~thalassemia-I defect due to a partial deletion of the ~-globin gene complex. Nucleic Acids Res 8 : 4889-4898 Pressley L, Higgs DR, Clegg JB, Perrine RP, Pembrey ME, Weatherall DJ (1980b) A new genetic basis for hemoglobin-H disease. N Engl J Med 303 : 1383-1386 Proudfoot N J, Gil A, Maniatis T (1982) The structure of the human zeta-globin gene and a closely linked, nearly identical pseudogene. Cell 31 : 553-563 Reed KC, Mann DA (1985) Rapid transfer of D N A from agarose gels to nylon membranes. Nucleic Acids Res 13:7207-7221 Romao L, Osorio-Almeida L, Higgs DR, Lavinha J, Liebhaber SA (1991) u-Thalassemia resulting from deletion of regulatory sequences far upstream of the a-globin structural genes. Blood 78:1589-1595 Rovera G, Magarian C, Borun TW (1978) Resolution of hemoglobin subunits by electrophoresis in acid urea polyacrylamide gels containing Triton X-100. Anal Biochem 85:506-518 Sanger F, Nicklen S, Coulson A R (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 4 : 54635467 Sophocleus T, Higgs DR, Aldridge B. Trent R J, Pressley L, Clegg JB, Weatherall DJ (1981) The molecular basis for the hemoglobin Bart's hydrops fetalis syndrome in Cyprus. Br J Haematol 47:153-156 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98 : 503517 Surrey S, Chambers JS, Muni D, Schwartz E (1978) Restriction endonuclease analysis of human globin genes in cellular DNA. Biochem Biophys Res Commun 83:1125-1131 Weiss I, Cash FE, Coleman MB, Pressley A. Adams JG, Sanguansermsri R, Liebhaber SA (1990) Molecular basis for a-thalassemia associated with the structural mutant hemoglobin SuanDok (Ct2109LEU~ARO).Blood 76 : 2630-2636 Wilkie A O M , Lamb J, Harris PC, Finney RD, Higgs D R (1990) A truncated human chromosome i6 associated with ~-thalassemia is stabilized by addition of telomeric repeat (TTAGGG)n. Nature 346 : 868-871 Wilkie AOM, Higgs DR, Rack KA, Buckle VJ, Spurt NK, FischelGhodsian N, Ceccherini I, Brown W R A , Harris PC (1991) Stable length polymorphism of up to 260kb at the tip of the short arm of human chromosome 16. Cell 64 : 595-606 Wilson JT, Wilson LB, De Rial JK, Villa-Komaroff L, Efstratiadis A, Forget BG, Weissman SM (1978) Insertion of synthetic copies of human globin genes into bacterial plasmid. Nucleic Acids Res 5 : 563-581 Yamada M, Hudson S, Tournay O, Bittenbender S, Shane SS, Lang B, Tsiyirmoto Y, Caton AJ, Rovera G (1989) Detection of minimal disease in hematopoietic malignancies of the B-cell lineage by using third complementary-determining region (CRD-III)-specific probes. Proc Natl Acad Sci USA 86:51235127
