Proc. Nati. Acad. Sci. USAVol. 81, pp. 2821-2825, May 1984Genetics

fl-Thalassemia in Chinese: Use of in vivo RNA analysis andoligonucleotide hybridization in systematic characterizationof molecular defects

(RNA splicing/DNA polymorphism haplotypes/transient gene expression)

TU-CHEN CHENG*, STUART H. ORKINt, STYLIANOS E. ANTONARAKIS*, MICHAEL J. POTTER*,JULIANNE P. SEXTONt, ALEXANDER F. MARKHAMt, PATRICIA J. V. GIARDINA§, ANITA LI¶, ANDHAIG H. KAZAZIAN, JR.**Department of Pediatrics, Genetics Unit, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; tDepartment of Pediatrics,Division of Hematology-Oncology, Children's Hospital and the Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115;WDivision of Pediatric Hematology-Oncology, New York Hospital and Cornell Medical Center, New York, NY 10021; ¶Department of Pediatrics,Queen Mary Hospital, University of Hong Kong, Hong Kong; and tImperial Chemical Industries Pharmaceuticals Division, Mereside, Alderly Park,Macclesfield, Cheshire 5K104TG, United Kingdom

Communicated by Victor A. McKusick, January 3, 1984

ABSTRACT To perform a systematic analysis of (-thalas-semia genes among Chinese, we have determined the DNA ha-plotype in the 13-globin gene region of 37 Chinese -thalasse-mia chromosomes. Only four haplotypes were found. Blot hy-bridization analysis of erythroid RNA from patients ho-mozygous for haplotypes 1, 2, and 3 demonstrated differentpatterns, suggesting that a different mutation was associatedwith each haplotype. The mutation associated with haplotype 1was a C--T substitution at IVS-2, position 654. This mutationproduces a new donor splice site and leads to formation of a 13-globin RNA with an insertion of 73 nucleotides. The mutationassociated with haplotype 2 was a nucleotide insertion of Abetween codons 71 and 72, which results in a frameshift andpremature termination of tglobin synthesis. Haplotype anal-ysis suggests that these two mutations may account for up to85% of f3-thalassemia genes in this ethnic group. The haplo-type 3 gene contained a transcriptional "TATA" box mutationthat has been previously reported. Oligonucleotide hybridiza-tion demonstrated that the mutation associated with haplotype4 was the same IVS-1 position 5 substitution commonly ob-served among j3-thalassemia genes in Asian Indians. Sincehaplotype 4 of Chinese differs at polymorphic sites on eitherside of the IVS-1 position 5 mutation from the haplotype asso-ciated with this mutation in Indians, the mutation presumablyarose independently in these two populations.

The ,B-thalassemias are a heterogeneous group of inheritedanemias that result from mutations within and 5' to the /3-globin gene and affect P3-globin production (1, 2). Whenthese mutations lead to complete lack of /3-globin, they arecalled /3O. When they merely reduce P-globin synthesis, theyare termed ,B. In recent years a number of DNA polymor-phisms have been identified in the ,3-globin gene cluster (3-5). Analysis of these polymorphisms has revealed severaldifferent haplotypes (patterns ofDNA polymorphisms in the/3-globin gene region) in both normal and thalassemia chro-mosomes. In general, specific /3-thalassemia mutations arestrongly associated with particular haplotypes in all ethnicgroups examined (6-8). This observation, along with thefinding that different ethnic groups do not share the same 1-thalassemia mutations (8), has led to an efficient strategy forthe molecular characterization of this disease. Use of thisstrategy-i.e., haplotype analysis of f3-thalassemia chromo-somes in each ethnic group followed by isolation of one ,Bgene from each haplotype-has led to the discovery of more

than 20 different point mutations (see ref. 9 for review).About 2% of Chinese are heterozygous for 0-thalassemia

(10). Although the Chinese are the most populous ethnicgroup in the world, f3-thalassemia mutations have not beenstudied adequately in this population. Even so, the first (toour knowledge) 03-thalassemia mutation characterized, anonsense mutation in codon 17, was found in a Chinese pa-tient (11). In addition, a deletion of four nucleotides in co-dons 41 and 42 (12) and an A->G nucleotide substitutionwithin the "TATA" box that affects 83-globin gene transcrip-tion (13) have been found in this group.To perform a systematic molecular analysis of the /3-thal-

assemia genes among Chinese, we have determined the ha-plotype of 37 f3-thalassemia chromosomes. Only four haplo-types were found, and one /3-thalassemia gene representingeach haplotype was analyzed. We previously reported themutation associated with one of the four haplotypes (haplo-type 3) (13). In this communication we describe mutationsassociated with the remaining three haplotypes.

EXPERIMENTAL PROCEDURESSubjects. Our subjects were (i) families of unselected Chi-

nese patients with S3-thalassemia from Hong Kong and (ii)unselected Chinese-American families with the disease fromNew York City. Both groups originated mainly from Kwang-tung province of China.

Analysis of DNA Polymorphisms. High molecular weightDNA was prepared as described (14). Restriction site poly-morphisms in the j3-globin gene cluster were analyzed (5).The particular haplotype of the /3-globin gene cluster was de-termined by analysis of these sites in various members ofeach family. The DNA fragments used as probes for theseanalyses have been described elsewhere (5, 15, 16).RNA Blot Hybridization Analysis. Whole cells of peripher-

al blood samples were lysed in a buffer containing 7 M ureaand 2% sodium dodecyl sulfate (17). Total RNA was thenprepared as described (18). Formaldehyde-treated RNAsamples were electrophoresed in vertical 1.1% agarose/2.2M formaldehyde slab gels (19), blotted, and hybridized sepa-rately with a-globin cDNA and /3-IVS-2 (BamHI/EcoRIfragment) probes. 83-Globin RNA levels in different sampleswere compared by normalizing them against a-globin RNAlevels.Gene Cloning and DNA Sequencing. The /3-thalassemia

genes were cloned in bacteriophage X Charon 28 as a 7.5-kilobase (kb) HindIII fragment and then subcloned as a 3.7-

Abbreviations: IVS, intervening sequence; kb, kilobase(s); nt, nu-cleotide(s).

2821

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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kb Bgl II/Pst I fragment in the expression plasmid frSVplac(20, 21). DNA sequence analysis was by either the method ofMaxam and Gilbert (22) or the M13 dideoxy method of Sang-er et al. (23).

Transient Expression in HeLa Cells. Normal and mutant f-globin genes subcloned as 3.7-kb Bgl II/Pst I fragments inirSVplac were introduced into HeLa cells by calcium phos-phate precipitation (20, 21). The transfected cells were har-vested 48 hr later, and RNA was prepared (24). S1 nucleasemapping of the cellular RNA was performed with variousprobes (21, 25, 26). Protected DNA fragments were electro-phoresed in urea/acrylamide gels and the gels were exposeddirectly to x-ray film with an intensifying screen. Primer-ex-tension analysis of RNA from infected HeLa cells was per-formed exactly as described (20).

Oligonucleotide Analysis. The IVS-I nucleotide 5 mutationwas detected by oligonucleotide blot analysis using a syn-thetic oligodeoxynucleotide (19-mer) of the following se-quence 5'-G-C-A-G-G-T-T-G-C-T-A-T-C-A-A-G-G-T-T-3'.After filters were hybridized at 470C overnight (27), theywere washed with 0.9 M NaCl/0.09 M sodium citrate/0.05%sodium pyrophosphate: (i) twice at 250C, 5 min each, (ii)twice at 47TC, 45 min each, and (iii) once at 540C for 2 min.

RESULTSAnalysis of DNA Polymorphic Sites in f3-Globin Gene Clus-

ters of Chinese. Analysis ofDNA polymorphisms associatedwith 31 pA-bearing chromosomes in Chinese revealed eightdifferent haplotypes (Fig. 1). However, among 37 pthal-bear-ing chromosomes only four haplotypes were detected, withfrequencies of 0.38 (14/37), 0.47 (18/37), 0.10 (4/37), and0.03 (1/37) (Fig. 1). Haplotypes 1 and 2 account for 85% ofthe P-thalassemia genes, suggesting that the mutations asso-ciated with these two haplotypes may be responsible for themajority of ,-thalassemia in these Chinese patients. Thesehaplotypes also account for nearly 60% (17/31) of the pA_bearing chromosomes. We have recently reported an A--Gsubstitution at position -28 in the TATA box in a Chinese ,8-thalassemia gene (13). This gene was associated with haplo-type 3. We now report thalassemia alleles associated withhaplotypes 1, 2, and 4.

Analysis of P-Globin RNA of Erythroid Cells. When totalerythroid RNA of four Chinese P-thalassemia patients wasanalyzed by blot hybridization assay (Fig. 2), we observedabnormal f3-globin RNA intermediates in two patients. One

patient was homozygous for haplotype 1 (Fig. 2 A and B,lane 3), while the other was heterozygous for haplotypes 1and 2 (data not shown). The predominant RNA intermediatewas about 75 nucleotides (nt) larger than mature mRNA (700nt), while the larger RNA intermediate was about 200 ntsmaller than unspliced 8-globin RNA precursor (1680 nt).These data suggest that the mutation associated with haplo-type 1 results in production of abnormal RNA intermediates.In addition, normal-sized B-globin RNA was absent from theerythroid RNA of these two patients.The RNA intermediates in these patients appeared similar

to those detected in a Greek patient known to be homozy-gous for the IVS-2 position 1 mutation (Fig. 2A, lane 6).However, in contrast to the RNA pattern observed in thisMediterranean subject, the apparent abundance of the small-er intermediate in the Chinese patients was greater than thatof the larger. Furthermore, the presence of the IVS-2 posi-tion 1 defect in the Chinese patients was excluded by restric-tion mapping with Hph 1 (28), an enzyme that recognizes thenormal but not the mutated IVS-2 junction.When the erythroid RNA of a patient homozygous for ha-

plotype 2 was analyzed, no detectable ,B-globin RNA wasobserved (Fig. 2A, lane 4). The concentration of a-globinRNA was comparable to that found in the erythroid RNA ofa normal control (Fig. 2B, lanes 4 and 5).A third 3-globin RNA pattern was observed in a patient

homozygous for haplotype 3 and the TATA box mutation(Fig. 2 A and B, lane 2). One-tenth of the normal amount off3-globin RNA was detected by densitometric analysis of theautoradiogram. Thus, different RNA patterns are detectedby blot hybridization analysis of erythroid RNA from pa-tients homozygous for haplotypes 1, 2, and 3, suggesting thata different mutation is associated with each of these haplo-types.

P-Thalassemia Gene Associated with Haplotype 1 and ItsExpression in Heterologous Cells. The cloned 83-globin genederived from a haplotype 1 chromosome contained a novelnucleotide substitution (C-T) at position 654 of IVS-2 (Fig.3A). Common sequence polymorphisms found in other 13-globin genes (6) were absent, indicating that this mutant genewas of the framework 1 varietyll (6, 16). Inspection of thenucleotide sequence of IVS-2 revealed that the C-*T substi-

'Three common types of normal ,B-globin gene sequences, termedframeworks, have been observed in various populations by DNApolymorphism and nucleotide sequence analysis.

,r ^r7 p I t A_7A _f3 _ _

t t tt ttt t2 3 4 5 678 910

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+++ + 6+ - + 11+_+ + 2+__ + 1+__ + 2+++ _ 7

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+3] 1 FIG. 1. Haplotypes of pA and pthal-bearing chro-mosomes in Chinese. Polymorphic sites, designated

Me s 1 to 10, were identified by the following restrictionendonucleases: 1, 4, and 5, HincII; 2, 3, and 9, Hin-

+++ - + 14 dIII; 6, Hinfl; 7, HgiAI; 8, Ava II; and 10, BamHI. A+-- - + 18 + indicates cleavage and a - indicates absence of+-- - + 4 cleavage at a site. The bracket [ ] indicates that this+ + + + + 1 haplotype has not yet been conclusively ascertained,

3 7 but it does differ from haplotypes 1-7.

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A. , probe

2 3 4 5

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-3 pre RNA 3 pre RNA

-/ mRNA .RN.

6 7

io -G-pre RNA

-/3 mRNA

B. a probe

2 3 4 5

FIG. 2. Blot hybridization analysis of total ery-throid RNA from various subjects. In A the probe is aP-IVS-2 fragment and in B the probe is a-cDNA.Lanes: 1, 5, and 7, RNA of a normal control; 2, RNAof a patient homozygous for haplotype 3 (13); 3, RNAof a patient homozygous for haplotype 1; 4, RNA of apatient homozygous for haplotype 2; 6, RNA of aMediterranean patient homozygous for the IVS-2 posi-tion 1 mutation (28). Bands due to precursor RNAs areindicated.

tution at position 654 generated a new G-T dinucleotide that,within the context of the surrounding nucleotides, forms anapparent donor-like splice sequence (A-A-G-G-T-A-A-A). Inview of the RNA analysis shown in Fig. 2, it seemed likelythat this substitution adversely affects RNA processing, per-haps in a manner similar to that previously described for twoother IVS-2 mutations (positions 745 and 705) that also gen-erate new internal donor-like sequences (6, 21, 29).To investigate the influence of the IVS-2 position 654 sub-

stitution on RNA processing, the cloned mutant gene wasintroduced in HeLa cells by transfection on a simian virus40-based expression plasmid (20, 21). Total cellular RNAwas analyzed by S1 nuclease mapping (25, 26) and by primerextension assay (20, 21). S1 nuclease mapping demonstrated

A

5'

5'

31IVS-2

.....AAGGCAATA4T654

3'_v_ I\/S-2

70 71 72 73/Ala Phe Ser Asp. GCCTTTAGTGAT .

GCC TTT AAGTGAT

FIG. 3. B-Thalassemia mutations. (A) A C--T nucleotide substi-tution is located at position 654 of IVS-2 in the 83-thalassemia geneassociated with haplotype 1. (B) An insertion of A between the co-

dons for amino acids 71 and 72 is present in the ,3-thalassemia geneassociated with haplotype 2.

that the normal donor and acceptor sites of both IVS-I andIVS-2 were utilized (not shown). Primer extension analysisusing a 47-nucleotide fragment of exon 3 as primer, however,revealed that the f-globin RNA was abnormally long, 491rather than 418 nt (Fig. 4). Particularly striking, and in con-trast with the splicing pattern of the IVS-2 position 745 mu-tant (21), was the absence of detectable normal-sized RNA(see the right of Fig. 4). These results demonstrated the pres-ence of an insertion within the abnormal RNA species. Di-rect sequence analysis of the primed cDNA showed that thenew donor-like site generated by the position 654 substitu-tion was spliced to the normal IVS-2 acceptor, and the previ-ously characterized cryptic acceptor site within IVS-2 at po-sition 579 (21, 29) was spliced to the normal IVS-2 donor site(not shown). Thus, the processed RNA from this mutantgene contains exons 1, 2, and 3 as well as nucleotides 580-652 of IVS-2 (illustrated at the bottom of Fig. 4). This HeLacell transcript corresponds precisely to the major abnormalspecies identified by blot hybridization analysis of erythroidRNA of the affected patients (Fig. 2).

,6-Thalassemia Gene Associated with Haplotype 2. Thecloned gene derived from a haplotype 2 chromosome con-tained a frameshift mutation due to the insertion of an A nu-cleotide between the codons for amino acids 71 and 72 (Fig.3B). This insertion results in a shift in the reading frame suchthat premature termination of translation would occur at thenew codon 90 of the p-gene coding sequence. The apparentabsence of 8-globin RNA in erythroid cells of a patient ho-mozygous for this defect is similar to the low level ofmRNAseen in association with other nonsense and frameshift muta-tions (11, 30-32). This mutant gene contained the sequencepolymorphisms typical of framework 3 Asian genes (6, 16).

Expression of the frameshift gene in HeLa cells revealed alow level (about 20% of normal) of normally spliced B-globinRNA. This reduced steady-state level of RNA from non-sense and frameshift mutant p-genes is quite typical (refs. 31and 32 and unpublished data) and may be the result of intra-

Proc. Natl. Acad. Sci USA 81 (1984)

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aE0z

0

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2

01

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2-622-527

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-309

*3 418 491

3'

242s -238

_ -217

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S -160

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FIG. 4. Primer extension analysis of cytoplasmic RNA producedby the cloned mutant gene associated with haplotype 1. Normal andmutant genes were transfected separately into HeLa cells. Total cel-lular RNA was prepared from the transfected HeLa cells and hy-bridized to a 47-nt EcoRI/Bst NI fragment labeled at its 5' end. Afterextension of the primer with reverse transcriptase, cDNA productswere fractionated on an 8% polyacrylamide sequencing gel. Markersare indicated in nt. A 491-nt "abnormal" cDNA product was madefrom the mutant RNA (lanes at both sides of the autoradiogram) anddepicted on the left side of the figure by the solid lines. No "normal"cDNA product (418 nt) was detected even after prolonged exposure(shown by the arrow to the right). (Lower) Splicing pattern of theIVS-2 position 654 mutant transcript.

nuclear degradation of abnormal nonfunctional mRNA spe-cies or inherent RNA instability.

Oligonucleotide Detection of the Mutation Associated withHaplotype 4. Upon screening 22 Chinese 3-thalassemiagenes by hybridization with a synthetic oligonucleotide spe-cific for a common Asian Indian mutation (G- C at IVS-Jposition 5) (8, 21), we detected this mutation in one of them.That gene was the sole gene in our panel associated withhaplotype 4 (Figs. 1 and 5). Positive hybridization with theoligonucleotide was also observed in three family memberswho carried the haplotype 4 ,f-thalassemia chromosome. It isparticularly noteworthy that the 8-thalassemia gene in thisChinese family has the normal HgiAI and Ava II restrictionsites [designated 8 gene framework 1 (6)], whereas that ofAsian Indians always lacks these intragenic sites [designatedframework 3 Asian (8, 16)].

FIG. 5. Detection of the IVS-J position 5 mutation in a Chinesef3-thalassemia gene. For each lane 10 jig of DNA was digested withBamHI, electrophoresed, and hybridized with the specific oligonu-cleotide probe. DNA samples: 1, an Asian Indian homozygous forthe IVS-J position 5 mutation; 2, a Mediterranean homozygous forthe nonsense mutation at codon 39 of the f3 gene; 3, an Asian Indianheterozygote who does not have the IVS-J position 5 mutation; 4, 5,and 6, members of a Chinese family, all of whom carry a haplotype 4/B-thalassemia chromosome and are heterozygous for ,3-thalassemia.DNA in lane 4 is from the mother of subjects whose DNA is in lanes5 and 6. Note that in all individuals a roughly 2.0-kb non-globinDNA fragment hybridizes with the probe.

DISCUSSIONWe have systematically analyzed ,-thalassemia mutationsamong Chinese individuals. Four haplotypes were identifiedamong the p-thalassemia chromosomes in our panel andeach was associated with a different mutation. This correla-tion of specific mutations with haplotypes has also been ob-served in Mediterraneans and Asian Indians (6, 8). Excep-tions to such correlations do exist (6) and may reflect theinterplay of DNA recombination as well as multiple originsof these defects (6, 15, 16).The mutant gene associated with haplotype 1 is particular-

ly striking in its pattern of RNA splicing (Fig. 4). From bothin vivo (Fig. 2) and heterologous cell (Fig. 4) results, we con-clude that this mutation abolishes normal RNA splicing. Thepredominant RNA species in vivo is the exclusive heterolo-gous cell product. It contains 73 nt of IVS-2 inserted be-tween exons 2 and 3. The nature of the minor, larger in vivoRNA species evident in the blots is unknown, but it may be aP3-globin RNA precursor from which IVS-J has been re-moved.

It is intriguing that no normal B3-globin RNA is producedfrom this mutant gene. In the instance of a similar type ofmutation (IVS-2 position 745) in which a new internal donor-like sequence was also generated, both normally and abnor-mally processed RNAs were observed in heterologous cellsby the same primer extension assay and by blot hybridiza-tion (21). Whether any normal-sized /-globin RNA was pro-duced from a similar mutant with a substitution at IVS-2 po-sition 705 is not clear (29). In the case of the Chinese genedescribed here, the mutation alters RNA processing so as toeliminate joining of the normal IVS-2 donor to the normalIVS-2 acceptor. The mechanism(s) by which this substitu-tion exerts such a dramatic effect is uncertain. One possibili-ty is that splicing in this segment of IVS-2 is processive in a3'-to-5' direction, such that the first available acceptor (thenormal IVS-2 acceptor site) joins to the first acceptable do-nor (either the normal donor or any newly generated internaldonors in IVS-2). Although evidence has been presented for

2824 Genetics: Cheng et aL

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Proc. Natl. Acad. Sci. USA 81 (1984) 2825

a 5'-to-3' selection of splice sites (33), other data are not con-sistent with a simple scanning model of this kind (34).The identification of a common Asian Indian mutation in

Chinese haplotype 4 is unusual. In general, specific f3-thalas-semia mutations are restricted to particular ethnic groups.Since this mutation in the Chinese is within a 83 gene thatcontains different intragenic polymorphisms than those as-sociated with the mutation in Asian Indians (8, 16), it mostlikely represents an independent origin of the same defect.However, the possibility of a rare gene conversion event hasnot been excluded. Independent origins of P3-globin genestructural variants P3S and pE have been postulated on thebasis of similar findings in different populations (15, 35).We have now assigned a different defect to a gene of each

haplotype. However, we cannot rule out the presence of oth-er different mutations in these haplotypes or other undiscov-ered haplotypes in this ethnic group. Although a nonsense

mutation in codon 17 (11) and a deletion of four nucleotidesin codons 41 and 42 (12) were also found in Chinese P3-thalas-semia, no haplotype analysis was performed in those stud-ies. Taken together, six different mutations leading to ,8-thal-assemia have been defined in Chinese. All differ from thosefound in Mediterraneans (6), while two, the deletion in co-dons 41 and 42 and the substitution at IVS-1 position 5, havealso been observed in Asian Indians (8). In addition, theIVS-2 position 654 mutation has been inferred by RNA anal-ysis in a Vietnamese patient homozygous for haplotype 1(unpublished data).The direct analysis of P3-globin gene RNA in erythroid

cells is a useful adjunct to a systematic strategy for the iden-tification of different P3-thalassemia mutations. The detec-tion of an abnormal RNA species in the erythroid cells ofhaplotype 1 patients facilitated characterization of the IVS-2position 654 defect. Previously, others have reported evi-dence ofRNA abnormalities in vivo as a consequence of cer-tain P3-thalassemia mutations in Mediterraneans and Ameri-can Blacks (36, 37).

Finally, the use of oligonucleotides to screen for specificmutations in genomic DNA will allow quantitative assess-ment of haplotype-mutation correlations. As exemplifiedhere, oligonucleotide assays may reveal unsuspected muta-tions within an ethnic group and suggest a multicentric originof specific mutations.

We thank Emily Pasterfield for help on manuscript preparation.This work was supported by grants from the National Institutes ofHealth to T.-c.C., S.H.O., and H.H.K., a grant from the March ofDimes to H.H.K., a National Institutes of Health Research CareerDevelopment Award to S.H.O., and a National Institutes of HealthNew Investigator Research Award to S.E.A.

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