Characterization of β-globin mRNA in the β0 thalassemias

Cell. Vol. 14. 289-298,

June

1978.

Copyright

0 1978 by MIT

Characterization of @Globin the PO Thalassemias J. M. Old, N. J. Proudfoot,* W. G. Wood, J. I. Longley, J. B. Clegg and D. J. Weatherall Nuffield Department of Clincial Medicine University of Oxford *MRC Laboratory of Molecular Biology Cambridge, England

Summary A number of cases of p” thalassemia have been examined for the presence or absence of /3-globin mRNA. Total RNA extracted from peripheral blood was hybridized to purified complementary DNA specific for P-globin mRNA, and to @cDNA probes specific for the 5’ and 3’ noncoding regions of p-globin mRNA. Three clear-cut categories of j3” thalassemia were identified. The first type had no detectable @globin mRNA. A second type had @globin mRNA sequences which hybridized incompletely to the cDNA probes and probably represented mRNAs with grossly altered structures. A third type appeared to have essentially intact, though untranslatable, P-globin mRNA. Depurination products from 5’ and 3’ flcDNAs synthesized from this latter mRNA were identical to those from normal /3-globin mRNA, but the relative yields were different, suggesting a possible defect near the initiation codon.

The p thalassemias are a heterogeneous group of hereditary anemias characterized by defective synthesis of the p chains of human adult hemoglobin (Weatherall and Clegg, 1972). They can be divided into two main types-P+ thalassemia and Do thalassemia. In p+ thalassemia, some p chain synthesis occurs but at a markedly reduced rate, and there is a correspondingly reduced amount of p-globin messenger RNA sequences present in reticulocytes (Housman et al., 1973; Kacian et al., 1973) but probably not in nuclear RNA, suggesting a possible defect in mRNA processing (Nienhuis, Turner and Benz, 1977). The molecular basis has not been defined further. No p chains are synthesized in p” thalassemia, but studies of globin messenger RNA in this condition have given inconsistent results. Using cDNA/ mRNA hybridization, no detectable p-globin mRNA was found in four cases of homozygous p” thalassemia (Forget et al., 1974; Tolstoshev et al., 1976; Godet et al., 1977). In three of these cases, cDNA/ DNA hybridization data showed that the P-globin genes were present, indicating that the molecular defect must be at the level of DNA transcription or

mRNA in

in the mechanism of mRNA processing. Hybridizable p-globin mRNA, however, has been found in four p” thalassemia homozygotes (Kan et al., 1975b; Ramirez et al., 1976), and evidence for the presence of p-globin messenger RNA has also been found in a fifth patient by fingerprint analysis of a RNAase Tl digest of total RNA (Temple, Chang and Kan, 1977). Forget et al. (1976a) presented preliminary data on seven unrelated individuals with p” thalassemia and concluded that variable but always low levels of p-globin mRNA were present in blood or marrow cells in each case. The mRNA isolated from cells of most p” thalassemics fails to direct the synthesis of p-globin in cell-free protein synthesis systems, suggesting that the hybridizable p-globin mRNA may be structurally abnormal. This is in contrast to the type of p” thalassemia found in Ferrara, in which it appears that it is possible to induce p chain synthesis in vitro in a reticulocyte cell-free system and in vivo by blood transfusion (Conconi et al., 1972, 1975), implying that functional p-globin mRNA is present in this form of p” thalassemia. Hybridization data supporting this concept have been presented by Ottolenghi et al. (1977), although Ramirez et al. (1976) reported that mRNA isolated from patients with the Ferrara form of p” thalassemia hybridizes incompletely with cDNA specific for p-globin mRNA, suggesting that it is structurally abnormal. It therefore appears that p” thalassemia is heterogeneous at the molecular level even within members of the same racial group. Because of this and the conflicting reports regarding the properties of P-globin mRNA in this condition, we have examined a series of p” thalassemia homozygotes from different racial groups who were available for repeated isolation of mRNA from their peripheral blood and for whom complete family studies could be obtained. We have also reexamined mRNA from the p” thalassemia homozygote described by Kan et al. (1975b). In addition to analyzing total RNA from peripheral blood of these patients by hybridization to highly purified cDNA probes specific for the o(- and p-globin mRNAs, we have characterized further any inactive p-globin mRNA by hybridization to cDNA probes specific for the 5’ and 3’ noncoding regions of P-mRNA. These studies have confirmed the heterogeneity of the p” thalassemias and provide an indication of the probable molecular defects in at least some of them. Results Hematological Findings and Hemoglobin Analysis The results of hematological studies and hemoglobin analysis for six of the p” thalassemia homozy-

Cdl 290

gotes and their parents are summarized in Table 1. In each case, family studies were compatible with the diagnosis of the homozygous state for p” thalassemia. In vitro globin chain synthesis studies were carried out in all the homozygous individuals and many of their heterozygous parents. The homozygotes showed a total absence of p chain synthesis and marked imbalance of (Y and y chain production, and the findings in the parents were compatible with their being heterozygotes for p thalassemia.

ence of fetal hemoglobin (HPFH). The two latter samples have been shown to have no p-globin mRNA and to be derived from red cell precursors in which the p- and possibly the Gglobin genes are deleted (Ottolenghi et al., 1976). The three RNAs lacking p-globin hybridized to about 12% SI nuclease resistance (Figures le-lg). The hybridization curves obtained with the RNA from the Spa thalassemia and HPFH homozygotes reached a plateau at this level, while that from the p”lSp” thalassemia behaved similarly but then continued to hybridize with the cDNA probe at higher RNA inputs but at a much lower rate. This is almost certainly due to the low amounts of b-globin mRNA in the peripheral blood of the p”/Spo thalassemia, as previously described (Ottolenghi et al., 1975). These findings suggest that the cDNA, probe used in these experiments was contaminated with approximately 12% cDNA,. The cDNA, and cDNA, probes were then used to determine whether there was p-globin mRNA in the cells of seven p” thalassemia homozygotes (Figure 1). In one case (Figure la), the cDNA, probe hybridized to the level of that obtained with normal mRNA but at a slower rate, indicating that the thalassemic p-globin mRNA was present in a lower amount than in normal reticulocytes. From the ratio of the 50% hybridization values, an (Y/P ratio of 4.511 was obtained, compared with the 1.41 1 obtained with normal mRNA (unpublished obser-

Hybridization of Purified cDN& and cDNA, Complementary DNAs specific for normal (Y- and @globin mRNA sequences were prepared by hybridization of cDNA,, to cytoplasmic RNA lacking P-mRNA sequences prepared from the peripheral blood reticulocytes of a compound p”/Sp” thalassemia heterozygote (Ottolenghi et al., 1975). The cDNA, probe was protected to a maximum level of 90% Sl nuclease resistance, and the cDNA, probe to a maximum level of 80% Sl nuclease resistance when hybridized with normal @-mRNA. The amount of cDNA, contamination of the cDNA, probe was determined by hybridizing to three RNA samples which were known to be lacking p-globin mRNA. These were obtained from the peripheral blood of the compound p”lSp” thalassemia heterozygote, a Spa thalassemia homozygote and a homozygote for the Negro form of hereditary persistTable

1. Hematological

and Hemoglobin

Origin

Initials and Age of Propositus (Years)

Greek

EP5

English

SM 21

Saudi Arabian

AW8

Chinese

DC 14

Pakistani

Pakistani

NM2

BM4

Analytical

Data on Propositi

and Immediate

Relatives Globin Chain Synthesis

Family Member

Hb (cdl)

MCH (Pg)

MCV (fl)

Hb F W)

(%)

a/P

UJY

Propositus Mother Father

9.8 11.2 12.9

27 21 20

70 63 61

T 0.8 -

T 4.2 4.5

1.89 -

1.84 -

Propositus Mother Father

8.4 10.8 12.2

26 22 21

77 71 67

98.7 3.9 4.4

1.3 6.4 5.9

1.64 2.27

3.8 -

Propositus Mother Father

8.3 10.5 13.1

28 19 22

89 68 65

97.2 2.4 3.2

2.8 8.8 7.1

3.5 2.1

1.98 -

Propositus Mother Father

7.3 11.1 12.3

28 20 21

82 65 66

1.4 1.1

T 6.4 7.0

Propositus Mother Father

6.8 11.5 15.9

20 18 21

61 54 62

96.1 0.7 0.3

3.9 5.0 6.9

1.5 1.6

2.7 -

Propositus Mother Father

10.2 10.8 13.9

21 19 20

65 59 64

97.8 2.7 1.2

2.2 5.3 5.6

1.9 1.2

3.0 -

Globin chain synthesis studies on the peripheral blood of each propositus and BM had not received blood during the period of study: the remainder cell hemoglobin; (MCV) mean cell volume.

showed complete absence were on regular high-level

Hb A2

of 8 chain transfusion

-

3.5 -

production. SM, AW, NM regimes (T). (MCH) mean

a

100

b

r

loo,

1

RNA/

cDNA

2

(xIO-~I

3

L

RNA I cONA

s

20

5

6

6

.

.

.F+-----•

7

1~10-~1

/ 0

5

IO

15

RNA I cDNA e

100

20 1xW3

25

30

l

4 1

1

2 RNA I CDNA

100

4

(x10-31

r

RNA I cDNA g

3

r .A

.

.TO

. i’-

0

“.

.

.

I

./’

l

RNA I cDNA

txW3

1

291

(~10-~1

Figure 1. Hybridization of Purified cDNA, and cDNA,, to RNA Prepared from the p0 Thalassemia Homozygotes Listed in Table 1, to RNA from a Chinese p0 Thalassemic Patient Reported by Kan et al. (1975b) and to Three Control RNA Preparations Lacking &Globin mRNA Each cDNA probe (100 pg) was hybridized with increasing amounts of RNA as described in Experimental Procedures. (a) Hybridization of cDN& (O-0) and cDNA, (+--0) to RNA from the p” thalassemic patient SM, together with the hybridization of cDN& (6w) and cDNA, (w-0) to RNA from normal reticulocytes. (b) Hybridization of cDN% (C--O) and cDNA, (O--O) to RNA from the $ thalassemic patient EP. (c) Hybridization of cDNA, (& 0) and cDNA, (60) to RNA from the p” thalassemic patient DC. (d) Hybridization of CON& (O0) and cDNA, (GO) to RNA from the p” thalassemic patient BM. (e) Hybridization of cDN& (6 0) and cDNA, (O---O) to RNA from the p” thalassemic patient AW, together with the hybridization of cDNA, (C-m) and cDNA, (-Cl) to p”/Sp” thalassemic RNA. (f) Hybridization of cDN& (C-0) and cDNA, (e-0) to RNA from the p” thalassemic patient NM, together with the hybridization of cDN& (LB) and cDNA, (L-0) to homozygous 8a” thalassemic RNA. (g) Hybridization of cDN& (O0) and cDNA, (60) to RNA from the Chinese p” thalassemia patient reported by Kan et al. (1975), together with the hybridization of cDN& (+m) and cDNA, ([r--O) to homozygous HPFH RNA.

Cell 292

vations). RNA prepared from three patients, EP (Figure lb), DC (Figure lc) and BM (Figure Id), all hybridized to the cDNA, probe in the same manner as the p”/Spo thalassemia RNA, indicating that no p-globin mRNA was present in these mRNA preparations. In the three remaining cases, AW (Figure le), NM (Figure If) and the Chinese patient reported by Kan et al. (1975b) (Figure lg), the cDNA, probe hybridized to an initial level of 2540% Sl nuclease resistance, followed by the slower increase at higher RNA concentrations. Repeat hybridizations from a second blood sample gave identical hybridization curves, indicating that the red cells of these three patients contain p-mRNA sequences which hybridize incompletely to the cDNA, probe. Synthesis of Specific 5’ and 3’ &cDNA One serious limitation of the above data related to the 3H-cDNA probe used. The cDNA, was heterogeneous in length with a mean size of about 250 nucleotides (complementary to the entire 3’ noncoding region and a substantial part of the coding region). To obtain a more precise characterization of the p thalassemic mRNAs, cDNA probes specific for the 5’ and 3’ noncoding regions of p-globin mRNA were synthesized. Using the hexanucleotide d(G-C-A-C-C-A) as a primer, reverse transcriptase will synthesize a cDNA transcript of the 5’ noncoding region of p-globin mRNA (Baralle, 1977). Similarly, using the primer d(TIo-G-C) and omitting dCTP from the reaction, reverse transcriptase will synthesize cDNA complementary to the 3’ terminal 29 nucleotides adjacent to the poly(A) of p-globin mRNA. a-cDNA is not synthesized under these conditions since its 3’ terminal sequence is rich in dC residues (Proudfoot and Longley, 1976). The 29 residue 3’ terminal cDNA, probe was found to be too short to allow efficient hybridization under stringent conditions. Longer 3’ noncoding region cDNA, probes were therefore obtained by adding limiting concentrations (1 PM) of dCTP to the reverse transcriptase reaction mixture (Proudfoot, 1977). Figure 2a shows a gel fractionation of these 5’ and 3’ noncoding region p-cDNAs. Slot 1 shows the cDNA products synthesized using the hexanucleotide primer. As previously described (Baralle, 1977), two predominant products are obtained (labeled (Y and p) which have been demonstrated by sequence analysis to be the (Y and /3 5’ noncoding region cDNAs. The larger cDNA, band (56 nucleotides long) was eluted and used as the 5’ cDNA, probe. Slot 2 shows the cDNA products synthesized using TIO-G-C as primer and omitting dCTP from the reaction. Two major products were obtained (pC1 and pC2) and, as previously described

(Proudfoot and Longley, 1976), derive from “pileups” before the first and second dC in the 3’ terminal p-cDNA sequence. The small products are due to limiting concentrations of the input label, a-32P-dATP. In the presence of 1 PM dCTP (slot 3), longer cDNA bands were obtained, and the longest (pC5) was eluted and characterized by depurination analysis (Ling, 1972; Proudfoot and Longley, 1976). Figure 2b shows a two-dimensional fingerprint of the pC5 depurination products. Proudfoot (1977) has described the complete 3’ noncoding region sequence of human P-globin mRNA. The complete set of depurination products labeled by &*P-dATP can be predicted from these data. pC5 contains all of these products up to 73 nucleotides from the poly(A) (represented by T3 A). However, the next characteristic depurination product, T,-CA, 84 nucleotides from the poly(A) is absent. PC5 may therefore be accurately sized as between 83 and 93 nucleotides in length, including the primer. Furthermore, virtually no contaminating depurination products were observed, proving that it is pure p-globin cDNA. Hybridization of Specific 5’ and 3’ cDNA, to Reticulocyte mRNAs Table 2 contains the hybridization data obtained upon annealing the 5’ and 3’ cDNA, probes with normal reticulocyte RNA and with the various p” thalassemia reticulocyte RNAs described earlier. The numbers indicate the percentage Sl nuciease resistance of the 32P-labeled cDNA probes after hybridization for 7 days. The 5’ cDNA probe hybridized to values approaching 80% Sl nuclease resistance with all the RNA preparations that had previously been shown to contain detectable p-globin mRNA. Many of these values were obtained in duplicate. Similarly, normal reticulocyte RNA gave an 80% hybridization value. The data strongly argue that these particular thalassemic patients have p-globin mRNA with essentially normal and complete 5’ noncoding regions, although single-point mutations cannot be ruled out. The RNA preparations that had no detectable p-globin mRNA sequences when using the 250 nucleotide 3H-cDNA, as described above gave considerably lower levels of Si nuclease resistance. Reticulocyte RNA prepared from both the 86” thalassemia homozygote and from an individual homozygous for the Negro form of hereditary persistence of fetal hemoglobin (HPFH) gave essentially zero levels of hybridization, consistent with the previously described deletions of the p (? and 6) globin genes in these conditions. The compound p”lSpo thalassemia RNA and that from DC gave low, although significant, levels of Sl nuclease resistance, possibly representing hybridization to &globin mRNA pres-

@-Globin 293

mRNA

1

Figure

2. Synthesis

in p” Thalassemias

23

and Characterization

of 5’ and 3’ Noncoding

Region

Human

&Globin

Complementary

DNA

(a) Gel electrophoresis (12% polyacrylamide, 7 M urea) (Erownlee and Cartwright, 1977) of d(G-C-A-C-C-A)-primed globin cDNA (slot l), of d(T,,-G-C)-primed globin cDNA minus dCTP (slot 2) and of d(T,,-G-C)-primed globin cDNA with 1 PM dCTP (slot 3). (0) denotes origin; see text and Saralle (1974), Proudfoot and Longley (1976) and Proudfoot (1977). Bands were located by autoradiography and eluted from the gel using a procedure described by Maxam and Gilbert (1977). (b) Two-dimensional fractionation of the depurination products of pC5. (1) denotes pH 3.5 electrophoresis; (2) denotes homochromatography (Ling. 1972; Proudfoot and Longley. 1976). (M) denotes molar yield of product; brackets denote nucleotide predicted from nearestneighbor considerations. (Pi), (B) and (Y) denote inorganic phosphate, bromophenol blue marker and xylene cyanol FF marker, respectively.

ent at somewhat elevated levels in these two patients (see above). EP and BM were not investigated further due to lack of material. The 3’ pC5 cDNA probe gave lower values of Sl nuclease resistance even with the control normal RNA sample. Furthermore, the values were somewhat irreproducible. Two explanations may account for these observations. First, the 3’ pC5

cDNA is extremely AT-rich (about 70%), and second, the cDNA can form stable hairpin loops. Annealing the 3’ cDNA, in the absence of complementary RNA sequence gave an Sl nuclease resistance level of 20%. The p-globin mRNA sequences in the RNA samples will therefore have to compete with the intramolecular structure of the cDNA probe, and once hybridized to the cDNA, will exist

Cell 294

Table

2. Percentage

Hybridization

of Specific Amount

Source

RNAlcDNA Ratio x IV

Region

5’ P-cDNA

of RNA

Ml) 1 .o

50

79

pa SM

1 .o

50

79

Normal British

5’ and 3’ Noncoding

Saudi Arabian

6”AW

3.0

150

2.0

100

Chinese

p”

1 .o

50

Chinese

p0 DC

3.0

150

Pakistani

p” NM

(2) (2)

p-cDNA

Probes Average

3’ 6-cDNA 56

69

51

51

57

56

51

53

55

45

26

49

32

36

66 (2)

47

30

38

-

41

79(l)

47

-

-

-

47

34 (1)

25

-

15

20

17 (2)

-

13

18

77 (2)

57

Italian

g”lSpo

1 .o

50

-

24

Sicilian

66”

0.5

25

6 (2)

21

20

26

1 .o

50

80)

-

18

-

-

18

4 (1)

21

17

-

-

19

HPFH No mRNA

-

The number of hybridizations performed with the 5’ 6-cDNA probe on the same patient hybridized with the above RNA preparations as described in Experimental Procedures.

in duplexes of relatively low stability. Nevertheless, significant data were obtained with this probe by repeating the different hybridizations as many as 4 times in several cases. The normal RNA sample and the RNA from SM both gave average levels of hybridization of about 55%. This result provides strong evidence that the 3’ terminal 80 nucleotides adjacent to the poly(A) of p-globin mRNA are intact in the RNA from SM. Four of the RNA samples that had previously been shown to contain no p-globin mRNA with the 3H-labeled 250 nucleotide cDNA, all gave values of 20% Sl nuclease resistance, indicating essentially no detectable 3’ p-globin mRNA. Of particular interest was the observation that the three remaining p” RNA samples [AW, NM and the Chinese patient of Kan et al. (1975b)] all gave significant levels of hybridization (about 40%), but in each case, lower levels than those observed with normal mRNA. This result is entirely consistent with the lower levels of hybridization obtained with these samples with the purified 3H-labeled cDNA, probe (Figure 1). Furthermore, this strongly suggests that these different RNAs have either different 3’ noncoding region sequences or, more probably, a deletion or insertion in the 3’ noncoding region. Sequence Studies on mRNA from SM The results outlined above suggest that SM has intact p-globin mRNA with both 5’ and 3’ noncoding region sequences apparently normal. To investigate this particular case further, larger amounts of total cytoplasmic RNA were prepared, and poly(A)-containing RNA was purified from this using oligo(dT)-cellulose chromatography (Aviv and Leder, 1972). The preparation obtained was tested for mRNA activity by in vitro translation in wheat germ and ribonuclease-treated reticulocyte lysate cell-free systems (Roberts and Patterson,

is in parentheses.

9

Each P-cDNA

19

probe

(20 pg) was

1973; Pelham and Jackson, 1976; Pritchard et al., 1976). Both systems gave OL- and yglobin translation products but no p-globin (data not shown), confirming that this patient was a true p” thalassemic and not simply lacking some essential translational control factor as suggested for Ferrara 6” thalassemia (Conconi et al., 1972, 1975). SM’s globin mRNA was then tested as a template for 5’ and 3’ noncoding region P-cDNA synthesis. Slots 1 and 3 in Figure 3a show control 5’ and 3’ cDNA synthesized using the hexanucleotide a(G-CA-C-C-A) and dodecanucleotide a(T,,-G-C), respectively, with normal human (Y- and p-globin mRNA (see previous section). Slots 2 and 4 show the cDNA products synthesized under the same conditions using the mRNA preparation from SM. Apparently normal 3’ P-cDNA products are visible (slot 2), but the 5’ P-cDNA (slot 4) was either absent or in low yield when compared with the 5’ a-cDNA. The higher molecular weight band (R) present in slot 4 derives from ribosomal RNA contaminating the PO-mRNA preparation (unpublished observations). To confirm these observations based solely on comparisons with the control cDNA products, the putative 5’ cu-cDNA, the possible trace amounts of 5’ p-cDNA and the 3’ 6-cDNA @Cl) from SM were subjected to depurination analysis (Figures 3b-3d). As expected, the 5’ a-cDNA gave a normal depurination fingerprint. More surprisingly, however, the traces of 5’ fi-cDNA also gave the complete set of correct depurination products, although other contaminating products were visible in almost equivalent yields (Baralle, 1977). The 3’ P-cDNA product, pCl, was also shown to be correctly identified, as judged from its depurination fingerprint giving all the correct products (Proudfoot and Longley, 1976). These data therefore provide direct sequence evidence confirming previous

p-Globin 295

mRNA

in p” Thalassemias

(a) 1234

R

Figure

3. Synthesis

and Characterization

of 5’ and 3’ Noncoding

Region

Globin

Complementary

DNA from

SM’s mRNA

(a) Gel electrophoresis (12% polyacrylamide, 7 M urea) (Brownlee and Cartwright, 1977) of d(T,,-G-C)-primed cDNA using normal globin mRNA (slot 1) and SM’s globin mRNA (slot 2). and of d(G-C-A-C-C-A)-primed cDNA using normal globin mRNA (slot 3) and SM’s globin mRNA (slot 4). (0) denotes origin: see text for explanation of other lettering. p-cDNA and 3’ pC1, respectively (Ling. 1972; (b-d) Two-dimensional fractionations of the depurination products of SM’s 5’ a-cDNA, Proudfoot and Longley, 1976). See legend to Figure 2b for details.

finding that SM has p-globin mRNA with apparently intact 5’ and 3’ noncoding regions. An estimate of the relative yields of 5’p- and 3’pcDNAs synthesized with SM’s mRNA may be made by comparison with the control cDNA products. The same amount of mRNA was used to generate the two cDNA products in both the control and p” thalassemia mRNA experiments. Thus the PC1 and pC2 products synthesized with SM’s mRNA are 6.7 and 6.1%, respectively, of the control products. The 5’ ,&cDNA synthesized by the SM mRNA, however, was only 1.7% of the control. Furthermore, as shown in Figure 3d, the PC1 depurination fingerprint contained no detectable contaminants, while the 5’ /3-cDNA (Figure 3c) was seriously contaminated. From these considerations, we conclude that the hexanucleotide primer is up to 6 times less efficient than the dodecanucleotide primer on the p-globin mRNA of this particular p thalassemia mRNA preparation. This finding represents the only structural difference detectable so far between SM’s p-globin mRNA and normal pglobin mRNA, and its possible significance is discussed below.

Discussion The experiments described in this paper provide convincing evidence for the heterogeneity of 6” thalassemia at the molecular level with the hybridization data falling into three clear-cut categories. First, several RNA preparations contained no detectable p-globin mRNA. Second, one RNA preparation appeared to have an essentially intact pglobin mRNA. Finally, three RNA preparations contained detectable p-globin mRNA sequences, although with probably grossly abnormal structures. The patterns of hybridization of total reticulocyte RNA to cDNA, were consistent using several different preparations from the same patient, findings which do not support the suggestion that variability in cDNA/RNA hybridization in 6” thalassemia is due to degradation of untranslated @globin mRNA or to artifacts produced by blood transfusion or other factors (Forget et al., 1976a). Because of the increasing heterogeneity of the p” thalassemias, even in members of the same race, we suggest that these forms of p” thalassemia be classified provisionally as types 1, 2 and 3,

Cell 296

respectively. If the type of p thalassemia characteristic of the Ferrara region of Northern Italy is eventually encountered in other races, it might be better to designate it as type 4 p” thalassemia. EP, DC and BM fit into the category of type 1 /3” thalassemias. DNA from these patients has not yet been examined for the presence of p-globin genes, so it is possible that some of them may be caused by a gene deletion, as has been demonstrated in Spa thalassemia and in the Negro form of hereditary persistence of fetal hemoglobin (Ottolenghi et al., 1975, 1976; Kan et al., 1975a; Forget et al., 1976b). Because, however, all previously reported cases of p” thalassemia with no detectable P-globin mRNA have been shown to have intact p-globin genes, it seems probable that these three cases are similar to those reported previously (Forget et al., 1974; Tolstoshev et al., 1976; Godet et al., 1977). The possibility remains, of course, that the cells of these patients contain very small amounts of pglobin mRNA which cannot be detected by current techniques. A total abolition of p-globin mRNA production in the presence of intact p-globin genes might result from a transcriptional or processing defect. The latter can be examined by looking for p-globin mRNA sequences in nuclear RNA from bone marrow cells by cDNA hybridization techniques. Comi et al. (1977) have reported a case of p” thalassemia with p-globin RNA sequences present in nuclear RNA but none detectable in cytoplasmic RNA using such techniques. A defect in transcription could be due to a deletion or mutation of DNA sequences in control regions adjacent to the p-globin gene, and it may soon be possible to detect such mutations by the techniques of gene mapping using restriction enzymes currently being developed. Of the types of p” thalassemia with detectable p-globin mRNA sequences, only that from SM appeared to have intact p-globin mRNA-that is, type 2, although we do not yet have evidence that this is of normal length. Other previously reported cases of p thalassemia which may come into this category are the one reported by Temple et al. (1977) and the Ferrara cases (Conconi et al., 1975). Unlike the Ferrara form of p” thalassemia, however, mRNA from SM failed to synthesize any p-globin chains in vitro, even though the 5’ and 3’ noncoding regions of the P-globin mRNA were shown to be normal from an analysis of the depurination products of 5’ and 3’ /3-cDNA synthesized from SM’s RNA. The only detectable difference between SM’s and normal p-globin mRNA was in the relative amounts of 5’ and 3’ /3-cDNA synthesized with the primers. The low synthesis of 5’ pcDNA compared with 3’ /I-cDNA could result from poor hybridization of the hexanucleotide primer d(G-C-A-C-C-A), while the primer d(TIo-G-C) hybrid-

ized normally. A point mutation at the hexanucleotide primer binding site could impair the hybridization of the primer sufficiently to reduce the activity of the hybrid as a substrate for the reverse transcriptase. If the point mutation occurred at the first or second nucleotide of the primer binding site (the U or G of the AUG codon), it might also explain the failure of the mRNA to be translated both in vivo and in vitro. Alternatively, a deletion or insertion of a sequence close to the hexanucleotide primer binding site might alter the three-dimensional structure of the mRNA in such a way that the primer binds less efficiently to the mRNA. Such a deletion or insertion might similarly prevent translation of the mRNA. In this context, it is interesting to note that all of SM’s cytoplasmic p-globin mRNA is associated with ribosomes (data not shown), suggesting that it is capable of forming an initiation complex. Further experiments are in progress to determine the nucleotide sequence of this p-globin mRNA. The type 3 category consists of a group of p” thalassemias in which the red cells contain pglobin mRNA which hybridizes incompletely to the cDNA, probe. Several previously reported cases may fit into this category. Kan et al. (1975b) reported the presence of nonfunctional P-globin mRNA in two Chinese patients which hybridized with a cDNA, probe to a slightly lesser extent than normal mRNA. The RNA from one of the patients described here originated from one of these two patients, and our results therefore confirm those of Kan et al. (1975). RNA from an Italian p” thalassemia homozygote (Ramirez et al., 1976) has also been reported to hybridize similarly, although the hybridization was not taken to completion. (Conceivably, some “homozygous” individuals in this group may actually be doubly heterozygous for types 1 and 3.) The incomplete hybridization of the cDNA, probe observed in Figure 1 could be due to a high level of &globin mRNA, but the hematological data and the kinetics of hybridization suggest only the presence of the usual small amounts. Hybridization of P-cDNA to 6-globin mRNA would produce a hybrid with a lower thermal stability than a P-globin RNAcDNA hybrid (Comi et al., 1977). Although thermal stability curves have not been determined for the P-cDNA hybrids of any of the type 3 patients, Kan et al. (1975b) showed that the thermal stability curve of the RNA-@DNA hybrid of their patient was similar to that of normal RNA, indicating a perfectly matched hybrid. This suggests that the incomplete hybridization observed in Figure 1 is due to only part of the /3-cDNA hybridizing perfectly with the p” thalassemic RNA to give a hybrid with a melting temperature similar to that of a complete p-globin RNA-&DNA hybrid, and not due to the P-cDNA imperfectly hybridizing to &RNA. It seems more

fl-Globin 297

mRNA

m p” Thalassemias

probable, therefore, that the observed hybridization is due to a structurally abnormal @globin mRNA with possibly a deletion or insertion of sequences at the 3’ end of the mRNA. The results from the hybridization of the specific 5’ and 3’ pcDNAs confirmed that each 3’ noncoding region contained less than the normal amount of hybridizable P-globin sequences. The hybridization analysis gives no information about the precise nature and position of any deleted or inserted sequences. Each ,B”-mRNA hybridized to ~50% of the cDNA ,!3 probe (calculated from the curves in Figure l), suggesting that the deleted or inserted sequences are between loo-150 nucleotides in length. The 3’ p-cDNA probe hybridized to a level of 75% of that with normal mRNA (calculated from the results in Table 2), suggesting that at least 50 nucleotides of the extreme 3’ noncoding region were present. Thus the affected sequence could possibly be internal, starting in the coding region and extending into the 3’ noncoding region, thereby making chain termination impossible. Determination of the size of p” thalassemia P-globin RNAs by gel electrophoresis would yield useful information about the molecular defect, but this has not been attempted at present because of the limited amount of these RNAs available. More precise information on the defect in these p” thalassemic p-globin mRNAs can only come from a comparison of the nucleotide sequences with that of normal human p-globin mRNA. Experiments to determine the sequence of these nonfunctional mRNAs are in progress, and these may provide an insight into how a defect in the 3’ noncoding region prevents translation of the mRNA. Experimental

Procedures

Materials Peripheral blood samples were obtained from the p” thalassemia homozygotes listed in Table 1. In addition, a sample of total reticulocyte RNA from a Chinese patient was obtained from Dr. Y. W. Kan (Kan et al., 1975b). Blood was collected into heparin and either processed immediately, or the cells were washed and frozen at -70°C. In one case, blood was transported from Saudi Arabia in cooled containers and processed within 8 hr of collection. Hemoglobin Analysis and Globin Chain Synthesis Hematological studies and hemoglobin analysis wasxarried out as described in Weatherall and Clegg (1972). The relative rates of globin chain synthesis were determined by incubating peripheral blood reticulocytes with 3H-leucine for 1 hr at 3PC, after which the cells were washed, lysed and converted to “globin” without further purification (Weatherall, Clegg and Naughton, 1965). The globin chains were separated by CM-cellulose chromatography as previously described (Clegg, Naughton and Weatherall, 1966). RNA Isolation Total cytoplasmic RNA was prepared from peripheral blood reticulocytes by phenol-chloroform extraction (Pritchard et al., 1976). mRNA was prepared from total RNA by affinity chromatography with oligo (dT)-cellulose (Aviv and Leder, 1972).

cDNA Preperatlon 3H-cDNA,B was prepared with normal reticulocyte mRNA as a template, oligo-d(T,,,,) as primer and RNA-dependent DNA polymerase prepared from avian myeloblastosis virus (a gift from Dr. Beard, Life Sciences Research Laboratories, St. Petersburg. Florida) as previously described (Old et al., 1976). The cDNA was purified by alkaline sucrose gradient centrifugation. and a fraction with a mean molecular weight of 70,000-80,000 daltons was taken for further purification. cDNA, and cDNA, probes were prepared by hybridizing cDN&, to @lSfl” thalassemic mRNA and separating the hybridized component (cDNA.-enriched) from the nonhybridized component (cDNA,-enriched) by hydroxylapatite chromatography (Old et al., 1977). The cDN& probe hybridized lo mRNA., to 90% completion (Figure 1) and contained approximately 10% cDNA, (Old et al., 1977). The cDNA, hybridized lo mRNA,, to 65% completion and contained approximately 12% cDNA, (Figure 1). 52P-cDNA specific for the 5’ noncoding region of ,+mRNA was synthesized with normal or SM’s reticulocyte mRNA as template, d(G-C-A-C-C-A) as primer and RNA-dependent DNA polymerase using &‘P-dGPT as input label (spec. act. 200 Ci/mmole) according to the method described by Baralle (1977). 3ZP-cDNA specific for the 3’ noncoding region of P-mRNA was synthesized with normal or SM’s mRNA as template, d(Tl,,-G-C) as primer and RNA-dependent polymerase with a limiting amount concentration of dCTP (1 PM) and a-32P-dATP as input label (spec. act. 200 Ci/ mmole) as described by Proudfoot (1977). Hybrldizatlon of cDNA 10 RNA A constant amount of cDNA, or cDNA, (100 pg) was mixed with increasing amounts of RNA in 2 ~1 of hybridization buffer (0.5 M NaCI. 25 mM HEPES, 10 mM EDTA, 50% formamide and 500 pg/ ml of E. coli RNA) in a sealed siliconized glass capillary tube and incubated at 43°C for 100 hr. Under these conditions. the hybridization of the cDNA went to completion. Hybridization of the 32PcDNAs was conducted with a fixed amount of cDNA (1000 cpm) and a fixed excess amount of each RNA (to contain enough ,¶mRNA to saturate 100 pg of cDNA,, as calculated from the results in Figure 1) as described above, except that the period of incubation was 160 hr. The samples were diluted with 350 ~1 of Sl nuclease assay buffer [0.03 M sodium acetate (pli 4.5), 2.8 mM ZnSO,, 0.14 M NaCl and 10 pg/ml denatured calf thymus DNA)]; 40 units of Sl nuclease (Sigma Chemical, London) were added, and the mixtures were incubated for 3 hr at 37°C. An aliquot from each sample was used to determine total radioactivity, and the proportion of Sl nuclease-resistant cDNA was determined after precipitation with perchloric acid. Acknowledgments We wish to thank the clinicians who referred cases, particularly Drs. J. Mann, J. Stewart, M. Pembrey. B. Modell. W. Turner, D. Evans and W. Forsythe. We are also most grateful to Dr. H. Drysdale for supplying normal reticulocyte-rich blood; lo Dr. Beard for supplying avian myeloblastosis virus reverse transcrip tase; to Dr. F. Baralle for donating the hexanucleotide primer; and to Drs. S. Gillam and M. Smith for the d(,rG-C) primer. We thank Miss D. Wilde for assistance with the ribonuclease-treated reticulocyte lysate cell-free system, and Mrs. J. Nash, Mr. M. Shepherd and Mrs. Ii. Ayyub for their excellent technical help. The work was supported by the MRC and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

January

17, 1978;

revised

March

13, 1978

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