- Email: [email protected]
Heterozygosity for the IVS-I-5(G→C) mutation with a G→A change at codon 18 (Val→Met; Hb Baden) in cis and a T→G mutation at codon 126 (Val→Gly; Hb Djonburi) in trans resulting in a thalassemia intermedia
Biochimica et Biophysica Acta, 1180(1992) 173-179
173
© 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00
BBAD1S 61210
Heterozygosity for the IVS-I-5 (G ~ C) mutation with a G ~ A change at codon 18 (Val ~ Met; Hb Baden) in cis and a T ~ G mutation at codon 126 (Val ~ Gly; Hb Dhonburi) in trans resulting in a thalassemia intermedia Vladimir Divoky a Emmanuel Biss6 b Jerry B. Wilson a Li-Hao Gu a, Heinrich Wieland b, Irene Heinrichs c, John F. Prior d and Titus H.J. Huisman a a Department of Biochemistry and Molecular Biology, Laboratory of Protein Chemistry, Medical College of Georgia, Augusta, (;.4 (USA), b Department of Clinical Chemistry, University Hospital, Freiburg (Germany), ' Division of Pediatrics, University Hospital, Halle (Germany) and d Hematology Department, The Queen Elizabeth 11 Medical Centre, Sir Charles Gairdner Hospital, Nedlands, Western Australia (Australia)
(Received 30 June 1992)
Key words: fl-Thalassemia; fl-Mutation in cis; fl-Mutation in trans; High-performanceliquid chromatography;DNA sequence We have analyzed the hemoglobins of a young German patient with/3-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the fl-globin genes through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the G_TG --* GGG mutation at codon 126 leading to the synthesis of Hb Dhonburi or a2/32126(H4)Val --* Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G ~ C) mutation that leads to a rather severe /3+-thalassemia and the GTG ~ ATG mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered fl-chain variant, named Hb Baden, was present for only 2-3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G ~ C) thalassemic mutation. The chromosome with the codon 18 (GTG ~ ATG) and the IVS-I-5 (G ~ C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G -~ C) mutation living in different countries failed to detect the codon 18 (G_TG ~ A TG) change.
Introduction /j-Thalassemia (/j-thai) is a most heterogeneous genetic condition that is characterized by the absence or decreased production of normal /J-chains (for review, see Ref. 1). At least 120 mutations and frameshifts have been detected in the /j-globin gene causing this defective /j-chain production, while a few larger deletions will also lead to a /j°-thal (for review and references, see Ref. 2). The phenotypic expression resulting from these numerous changes varies greatly from nearly undetectable to a distinct anemia with severe microcytosis and hypochromia in the heterozygote. Clinical observations in compound heterozygotes also vary considerably being dependent upon the types of changes in
Correspondence: T.H.J. Huisman, Department of Biochemistry and Molecular Biology,Medical College of Georgia, Augusta, GA 309122100, USA.
the /j-globin gene that are present on each of the two chromosomes of such an individual. Recently, we studied a young child from the former German Democratic Republic and of German descent who exhibited the typical clinical features of a /j-thai intermedia. Detailed structural studies of his hemoglobin (Hb) and sequence analysis of the/j-globin genes identified two r a r e / j - c h a i n abnormal Hbs and a common but clinically rather severe /j+-thal determinant; one of the structural mutations occurred in cis to the /j-thal mutation. Here, we provide details of that study which includes comparable analyses of some members of the patient's immediate family. Materials and Methods Blood samples were collected in vacutainers with E D T A as anticoagulant, and studied at the local institution and in Augusta, GA; shipment was by fast air mail service. Informed consent was given.
174 Hematological data were obtained with automated cell counters and with standard laboratory procedures. Hbs in red cell lysates were studied by cellulose-acetate and citrate-agar electrophoresis [3], by isoelectrofocusing (IEF) [4], and by different stability tests, such as isopropanol and heat stability at pH 7.0 and at 60°C [3]. The Hbs were quantified by cation exchange high performance liquid chromatography (HPLC) [5,6] and by reverse-phase HPLC (7,8); both methods played an important part in elucidating the structures of the Hb variants. Separation of a- and 3-chains was by carboxymethyl cellulose (CMC) chromatography [9] or by preparative reverse-phase HPLC [10]. Structural analyses used methodology described before; separation of tryptic peptides of isolated 3X-chains was by reversephase HPLC [11] and amino acid analyses of individual peptides were made with an automated amino-acid analyzer (Pico-Tag, Waters, Milford, MA), while the sequences of a few selected peptides were determined with the method described by Chang e t a [ . [12]. Isolation of a larger quantity of Hb Baden required a preparative HPLC procedure with a 21 x 25(t mm PolyCAT A, 5 ~m, 1000 A column (PolyLC, Columbia, MD). Some 20 to 30 mg were applied and the chromatogram was developed as described before [10] with a flow-rate of 5 ml/min. The isolated fractions were concentrated in a Diaflo YM-10 filter (43 mm) and next applied to a 10× 250 mm Vydac C 4 column, large-pore 5 /xm (The Separations Group, Hesperia, CA). The chromatogram was developed over a period of 70 min at a flow-rate of 2 m l / m i n and with a developing system described elsewhere [10]. The isolated 3X-chains were concentrated by freeze-drying and analyzed by methodology referred to abovc. DNA was isolated from white cells with the method of Poncz et al. [13]. Amplification of 3-globin genes and sequencing of the amplified DNA were done as described before [14,15]. Certain mutations were confirmed by dot-blot analysis that included hybridization of amplified DNA samples with 32p-labeled oligo-
nucleotide probes [16,17]; the same procedure was used to evaluate the possible presence of the Hb Baden mutation in DNA samples of several patients with a specific mutation. Results
The families Family S, consisting of parents with two children and paternal grandparents, originates from the eastern part of Germany. The propositus participated in a screening program for hemoglobinopathies in anemic children. He was found to have 'a rare Hb variant associated with /3-thai' on the basis of the results obtained in the laboratory of one of the authors (H.W.). Family R is also of German origin; only the propositus, a 65-year-old man born in Essen who emigrated to the United States and next to Australia, was available. Hematological data are presented in Table I. The propositus (Ph.S.) had a Hb level of about 9 g / d l with a striking microcytosis and hypochromia, an elevated Hb-A 2 of 6% and a minor increase in Hb-F level; the child was not transfused. The sister (S.S.) had a moderate microcytosis and hypochromia and a slightly elevated level of Hb-A2. Their mother had the characteristics of a /3-thai trait with mild anemia, microcytosis and hypochromia and elevated Hb-A 2. The father had nearly normal values except for a mild microcytosis and hypochromia (MCV 81.3 fl; MCH 26.5 pg). Similar observations were made in the grandfather (J.W.) and also in G.R., the propositus of the second family. The reticulocyte counts in these persons were slightly elevated to about 2-3%.
Hb analysis Electrophoretic examination failed to reveal the presence of a Hb variant; only a slight increase in Hb-F was detectable for the propositus Ph.S. IEF also gave negative data, except for the presence of Hb-F and an elevated level of Hb-A2; however, reexamination of
TABLE 1
Hematological and Hb composition data Subject
SexAg e
Relationship
Hb (g/dl)
PCV (1/1)
RBC (1(1~2/1)
MCV (fl)
MCH (pg)
MCHC (g/dl)
A z~ (%)
F ~ (%)
/3 x b (54")
3 Ab (%)
J.W. E.W. W.M.S. M.S. Ph.S. S.S.
M-73 F-64 M-39 F-30 M-6 F-4
Grandfather Grandmother Father Mother Propositus Sister
14.3 14.9 15.5 1(I.9 9.2 12.5
0.447 0.453 (I.475 0.347 [).302 0.385
5.37 4.85 5.84 5.17 5.43 5.12
83.3 93.5 81.3 67.2 55.6 75.2
26.6 30.7 26.5 21.1 16.9 24.4
32.0 32.9 32.6 31.4 30.5 32.5
2.9 2.1 3.1 5.1 6.(I 3.5
< 1.0 < 1.0 < 1.(1 1.2 ~ 4.l ~ < 1.0
34.7 0 36.0 I1 98.0 32.5
65.3
G.R.
M-65
Propositus
14.4
0.430
5.54
78.(/
26.0
33.6
2.7
(1.2
21.2
78.S
" By cation-exchange HPLC [5,6]. h By reverse-phase HPLC [7,8]. ' The ¢;y- and Ay-levels in isolated Hb-F were 18.7 and 81.3~ for M.S. and 33.7 and 66.3% for Ph.S., respectively.
64.0 2.(17 67.5
175 Father S
Propositus S
Mother I
I ~-Dhonburi I E HE
~-Dhonburi HEME, I
;-A
HEME
~-A
c
o
,-.-,2", 1'0 2'0 3'0 4'0
50
1'0 2'0 3'0 4'0 5'0 Time in Minutes
1'0 20
3'0 40 50
Fig. 1. Detection of the a-Dhonburi chain in the propositus and his father by reverse-phase HPLC [7,8].
/3A+ /3 x) with trypsin, separation of the tryptic peptides by reverse-phase HPLC, rechromatography of selected peptides and finally the determination of the amino-acid compositions of the individual peptides. Details of this study will not be presented, except to indicate that an abnormal/3T-13 peptide eluted slightly faster than normally observed. When analyzed, it was found that its only valine residue was replaced by a glycine residue. This observation identifies the variant as Hb Dhonburi [18] or Hb Neapolis [19] in which /3126-Val is replaced by Gly.
the gels after the variants were identified suggested that the major Hb of the propositus focused slightly differently from that of his mother. H e a t and isopropanol stability tests were positive for the propositus, his father, grandfather, and sister, and for the propositus of the second family; heat-precipitable Hb was estimated at 25%. Analyses of the Hbs by reverse-phase H P L C revealed the presence of a fast-eluting abnormal/3-chain in the propositus and his father but not in his mother (Fig. 1). Normal /3A-chains were not detectable in the blood of the propositus. The father was an apparent /3 x heterozygote, as were the grandfather and the younger sister and also G.R. of the second family. The relative amount of /3 x (as % of / 3 x + / 3 A) in the heterozygote was 32.5-36.0% in Family S; the lower level of 21.2% in G.R. remains unexplained. Structural analysis of the Hb variant in the propositus (and in G.R. of the second family) followed methodology routinely used in our laboratory which included separation o f / 3 x (/3 x +/3 A for subject G.R.) and a by CMC chromatography, digestion of /3 x (or £, ~..,~,~/J . , ~ •"* ~ AA CODON AR(G) 30 GLY 29 CTG LEU 28 GCC ALA 27 GAG GLU 26 GGT GLY 25 GGT GLY 24 GTT VAL 23 i== ~ GAA GLU 22 ,..., BAT ASP 21 GTG VAL 20 a,o,=,e AAC ASN 19 ~ ~ GTG VAL 18 (ATG;Met) AAG LYS 17 A C G T _ .-
DNA analysis Sequence analysis of amplified D N A samples from the propositus (Ph.S.) and his parents were used to identify the/3-thai allele in Ph.S and his mother. Fig. 2 shows photographs of parts of the sequencing gels of Ph.S. T h r e e mutations were detected, namely a G ~ C change at position 5 of the IVS-I ( G T T G G ~ G T T G C ) , a G ~ A mutation in codon 18 ( G T G for Val to A T G for Met), and a T ~ G mutation in codon 126 ( G T G for Val to G G G for Gly). The first is a /3+-thal muta-
T
G
C
..... ~
A
,~,;~
,,,, ~ . , . ~ ; •
CA G TAT GCC G CT CAG GTG
CCA
.....
CCA ACC TTC
AA Gin Tyr Ala Ala Gin Val Pro Pro
CODON 131 130 129 128 127 126 (GGG;Gly)
125 124
Thr 123 Phi. 122
Fig. 2. Detection of three mutations in the amplified DNA sample from the propositus. Left: The G ~ A mutation at codon 18, indicating a Val ~ Met replacement and a G ~ C mutation at the fifth position of the first intron. Right: The T ---,G mutation at codon 126, indicating a Val --, Gly replacement.
176 'FABLE II
Mutation,~ obsen,ed by .vequenci/zg of amplified DNA from the propositus, ]'ather and mother q]" fi*mily S Mutation
Propositus
Father
Mother
Codon 2 (CA(7 ~ CAT) Codon 18 ( G T G ~ A T G ) ( = 18 Val --+ Met) IVS-I-5 (G ~ C) Codon 126 (G_TG --+ G G G ) ( = 126 Val ~ Gly)
CAC/CAC GTG/ATG
CAC/CAC GTG/GTG G/G GGG/GTG
('AT_/CAC GTG/ATG (3/C GTG/GTG
G/C GGG/GTG
tion (for references, see Ref. 2); homozygotes and compound heterozygotes for this /3-thal allele and a /3 chain variant produce small quantities ( 2 - 8 % ) of normal Hb A [20]. The second mutation ( G T G - ~ ATG) results in a Vaf ~ Met replacement and, thus, in the synthesis of an abnormal /3-chain. This amino-acid replacement has never been observed before and the variant has been named Hb Baden or ce2o~218(A15) Val ~ Met. The third mutation (codon 126, G T G - - , G G G ) confirms the presence of Hb Dhonburi. Table II summarizes the differences in sequences of the DNA samples of three members of Family S. These results indicate that the propositus inherited one chromosome with G T G ~ G G G at codon 126 from his father and a second chromosome with G T G -+ A T G at codon 18 and with G ~ C at position 5 of the first intron from his mother. A well-known polymorphism at codon 2 (CAC ~ CAT) was present in the mother but not in her child, indicating that it occurred on her normal chromosome.
The search for Hb baden ([318(A15)Val --+Met) A study of the Hbs from propositus Ph.S. and his parents by cation-exchange HPLC provided the chromatograms shown in Fig. 3. No major abnormal Hb could be detected; the increase in the level of Hb-A 2 was readily observed, as was the higher level of Hb-F in the propositus. Of special interest is the presence of a small Hb-zone that separated only partially from the
Propositus S
g
10 20 30 40 5'o
ao
4'o
'_2
s'o
8
1'o 2'o Jo & s'o ~
7'o do ~ 4o do ,~o 11o 14o 40 4o 1~o
Time in Minutes (5 ml/min) Fig. 4. Isolation of Hb Baden and Hb Dhonburi by semi-preparative cation-exchange HPLC. The isolated Hb zones were further fractionated into their a and /3 chains by reverse-phase HPLC; see insets [10].
major Hb fraction; it was present in the propositus and the mother in an amount of about 2 - 3 % and was absent in the father. Preparative cation-exchange HPLC was used to isolate a larger quantity of this fraction (labeled Hb Baden); several such separations, as shown in Fig. 4 (bottom), provided a few mg of this material. Isolation
Father S
2'0 3'0 4b 5'0 6'0 7b
io
/Hb Dhonbun
Mother S
Hb-A+ Hb-Dhonburi
Hb-A
Hb-A2
(3.1'/0)
Hb-F Hb-Baden Hb-A2 (1.2%) (21%) I ( 5 1 % ) Hb-AIc|I , . A
" I
LJL__ 1'0
;o
Time in Minutes (2 ml/min)
Hb-A2 Hb-Baden (6O%) (2.5%) Hb-F ',
Q
J-Dhonburi
Ohonburi;
t.t3 8
t~.8aden
~j3
Hb-Dhonburi
E_
HEM
E
;o
2'o a'0 4'0
so
6'0 z'o
lo
2o ao
4o
5o
6b
r'o
Time in Minutes Fig. 3. Separation of the Hb components in red cell lysates from the propositus and his parents by cation-exchange H P L C [5,6]. The detection of a minor Hb-component (labeled Hb Baden) in the lysates of the propositus and his mother was the first indication for the presence of another abnormal Hb.
177 TABLE III The amino-acid compositions of a few selected tryptic peptides of the fl-chains of Hb Dhonburi, Baden and Olympia a
Amino-acid
/3-Dhonburi T-3
Aspartic acid Glutamic acid Glycine Arginine Threonine Alanine Proline Tyrosine Valine Methionine Leucine Phenylalanine Lysine
/3-Baden b T-3 x
T-13 x
1.95 (2) 2.10 (2) 3.18 (3) 1.00 (1)
1.95 (2) 1.98 (2) 3.15 (3) 0.99 (1)
3.00 (3) 1.02 (0) 0.96 (1) 2.05 (2) 2.14 (2) 0.78 (1) 0 (1)
1.03 (1) 3.03 (3)
1.09 (1) 1.94 (3) 0.87 (0) 0.97 (1)
0.91 (1) 0.94 (1) 13
3.00 (3) 0.93 (1) 1.93 (2)
1.89 (2) 1.96 (2) 2.95 (3) 1.16 (1) 0.95 (1)
1.92 (2)
1.03 (1)
Total
T-13
/3-Olympia c T-3 x
0.89 (1) 1.00 (1)
2.09 (3) 0.53 (0) 0.96 (1)
0.97 (1) 1.00 (1)
12
13
12
13
In mol/mol of peptide. b Amino-terminal sequence: Met-Asn-Val---. c Amino-terminal sequence: Val-Asn-Met--- (by microsequencing with the method of Chang et al. [12]); sequencing of amplified DNA confirmed the expected G ~ A mutation at codon 20 (Val --* Met). "
of the/3X-chains was by semi-preparative reverse-phase H P L C . T h e c h r o m a t o g r a m of Fig. 4 (top at left) shows that the major /3-chain of the H b B a d e n fraction has the mobility of /3 A, while a notable quantity of the /3-Dhonburi chain is also present. T h e H b D h o n b u r i fraction was nearly pure and contained a- and /3D h o n b u r i chains (Fig. 4, top at right). A b o u t 0.5-1.0 mg of the /3-Baden and /3-Dhonburi chains were di-
[
I[/ [3-Ohonburi([~126 Val~Gly)
/T-oT-7,,-,,T-1, i
T-13(Abn)
,-,
1
~ _ _ l T13
i
i
T-14,15
J
II
i
~ I 1/13-Baden(!318 Val~Met)
i silT-4 T-2
h-13
"
I-9 ,
,
I/
,
i
[11
T-3(Abn) I T-13A
i
J
,
v
,--
l
-
----:
,
7
Time in Minutes (1.5 ml/min)
Fig. 5. Separation of peptides in tryptic digests of three abnormal /3 chains by reverse-phase HPLC [11]. See text for details. Hb Olympia (a2/3220(B2)Val ~ Met) is a high-oxygen-affinity Hb variant that was observed in a Caucasian adult from Kentucky (unpublished data); the sample was kindly provided by Dr. Marie M. Keeling (Louisville, KY).
gested with trypsin and the tryptic peptides were separated by reverse-phase H P L C [11]. Fig. 5 illustrates the two c h r o m a t o g r a m s , as well as that for the /3-chain of H b Olympia that is characterized by a Val ~ Met m u t a t i o n at position /320 [21]. Most peptides eluted in the expected positions and had normal amino-acid compositions. Exceptions were t h e / 3 T - 1 3 and /3T-13A peptides in t h e / 3 - D h o n b u r i digest, and the /3T-3 peptide in the /3-Baden and /3-Olympia digests. The amino-acid compositions of the /3T-3 and /3T-13 peptides are listed in Table III; the data confirm the Val ~ Gly r e p l a c e m e n t in /3T-13 of the /3-Dhonburi chain and the Val ~ Met replacement in the /3T-3 peptides of the /3-Baden and /3-Olympia chains. T h e latter two peptides differ in the location of the Val Met substitution; that in fl-Baden is in the aminoterminal position (or position 18 of the intact /3-chain) and that in /3-Olympia is in the third position of the fiT-3 peptide (or position 20 of t h e / 3 chain). Discussion
T h e condition of patient Ph.S. can be designated as thalassemia intermedia; the child has a m o d e r a t e anemia with severe microcytosis and hypochromia, an elevated H b - A 2 level and a m o d e s t increase in Hb-F. N o blood transfusions are required. H e inherited from his father a /3-chain variant with a Val ~ Gly substitution at position 126. This rare variant, that has b e e n n a m e d H b D h o n b u r i [18] and H b Neapolis [19], is mildly unstable, and in the heterozygote is associated with minor thalassemic features such as a slight decrease in M C V and M C H values without an increase in the level
178 of H b - A e ( T a b l e 1) a n d with biosynthetic a l t e r a t i o n s c o n s i s t e n t with a mild /3-thalassemic p h e n o t y p e lit)]. His s e c o n d c h r o m o s o m e carries a G--+ C m u t a t i o n at the fifth p o s i t i o n of IVS-I. This well-known /3-thai allele is o b s e r v e d mainly a m o n g A s i a n Indians, M e l a n e s i a n s a n d C h i n e s e [22-24]; the greatly dec r e a s e d /3-chain p r o d u c t i o n results from an u n d e r u t i lization of the IVS-I d o n o r splice-site a n d the activation of o n e o r m o r e cryptic splice-sites. C o m p o u n d h e t e r o z y g o s i t y for this IVS-I-5 ( G - + C) m u t a t i o n a n d the c o d o n 126 ( G T G - - + G G G ) m u t a t i o n a p p a r e n t l y leads to a d i s t i n c t / 3 - t h a i i n t e r m e d i a , as is e v i d e n t from the clinical c o n d i t i o n o f the p r o p o s i t u s o f the S family. T h e s u r p r i s e in this study was the finding of a G T G -+ A T G m u t a t i o n in c o d o n 18 that results in the synthesis of an a b n o r m a l ,8-chain with a V a l - + M e t r e p l a c e m e n t at that location. This /3-chain a b n o r m a l H b m u s t be a stable variant, b e c a u s e the s a m e r e p l a c e m e n t is p r e s e n t in the n o r m a l e-chain a n d e m b r y o n i c H b s c o n t a i n i n g this chain are stable p r o t e i n s [25,26]. T h e two m u t a t i o n s (at c o d o n 18 ( G --+ A ) a n d at IVS-I-5 ( G --+ C)) m u s t occur on the s a m e c h r o m o s o m e b e c a u s e the c o d o n 18 c h a n g e was only o b s e r v e d in the p r o p o s i tus a n d his m o t h e r w h o also carries the IVS-I-5 (G -+ C) m u t a t i o n . T h e synthesis of t h e /3-Baden chain is low and c o m p a r a b l e to that of H b A in u n t r a n s f u s e d patients with H b E a n d the IVS-I-5 ( G -+ C) t h a l a s s e m i a allele [20,27]. T h e u n u s u a l c h r o m o s o m e in this family is the s e c o n d e x a m p l e of a c h r o m o s o m e in which a / 3 - t h a i m u t a t i o n occurs in cis to that resulting in the synthesis of an a b n o r m a l /3-chain; the o t h e r was r e p o r t e d by B a k l o u t i et al. [28] w h o o b s e r v e d a c o m m o n p r o m o t e r m u t a t i o n (C--+ T at p o s i t i o n - 8 8 ) in cis to the G A G ---) G T G m u t a t i o n at c o d o n 6 (i.e., the /3 s m u t a t i o n ) . W e have t e s t e d D N A s a m p l e s from m o r e than 200 /3-thai p a t i e n t s for t h e G T G --+ A T G m u t a t i o n at c o d o n 18 by d o t - b l o t h y b r i d i z a t i o n with 32p-labeled specific p r o b e s a n d w e r e u n a b l e to d e t e c t this change; a m o n g the 208 p a t i e n t s w e r e 49 subjects with a h e t e r o z y g o s i t y for the IVS-I-5 ( G - - ) C) m u t a t i o n ( t h r e e f r o m C z e c h o s l o v a k i a , o n e f r o m Italy, 13 f r o m Malaysia, 32 from I n d i a ) a n d two M a l a y s i a n p a t i e n t s with a homozygosity for this allele. It a p p e a r s most likely that the H b B a d e n m u t a t i o n o c c u r r e d as an i n d e p e n d e n t event on a c h r o m o s o m e t h a t c a r r i e d the IVS-I-5 ( G -+ C) m u t a tion. T h e two m u t a t i o n s p r e s e n t on the /3-thai c h r o m o s o m e o c c u r in the s e g m e n t of D N A w h e r e the d i f f e r e n t cryptic splice sites a n d t h e 5' d o n o r splice-site of the IVS-I a r e l o c a t e d ( r e v i e w e d in Ref. 1). T h e cryptic splice-site at c o d o n s 17-18-19 (AAG,I, G T G - A A C ) is a b o l i s h e d by t h e G --+ A m u t a t i o n at c o d o n 18, i.e., the H b B a d e n m u t a t i o n ( A A G . A T G . A A C ) . T h e G--+ C m u t a t i o n at IVS-I-5 has a n e g a t i v e effect on the 5' d o n o r splice site of the I V S - I ( C A G S G T T G G T - - + C A G J, G T T G C T ) . Thus, t h e s e two m u t a t i o n s e l i m i n a t e
one cryptic splice-site a n d nearly c o m p l e t e l y d c a c t i v a t e the d o n o r splice-site, l e a d i n g to the activation of (one of) thc two r e m a i n i n g cryptic splicc-sites, namcly G T G { G T G A G at c o d o n s 24-25-26 and / \ A G 1 (fVT A C at positions 9 t h r o u g h 16 of the IVS-I: which one of these two sites will bc most active c a n n o t be pred i c t e d without f u r t h e r analyses.
Acknowledgements T h e a u t h o r s greatly a p p r e c i a t e the c o o p e r a t i o n of the m e m b e r s of Family S. in this study. This r e s e a r c h was s u p p o r t e d in p a r t by the Ministry o f Science and Arts, B a d e n - W i i r t t e m b e r g , G e r m a n y (to H . W . ) and by the U n i t e d S t a t e s Public H e a l t h Service r e s e a r c h grants HLB-05168 a n d HLB-41544 (to T.H.J.H.).
References 1 Bunn, H.F. and Forget, B.G. (19861 Hemoglobin: Molecular, Genetic and Clinical Aspects. W.B. Saunders, Philadelphia. 2 Baysal, E. (1992) Hemoglobin 16, 237-258. 3 Huisman, T.H.J. and Jonxis, J.H.P. (19771 The Hemoglobin+ opathies Techniques of Identification. Clinical and Biochemical Analysis, Vol. 6, Marcel Dekker, New York. 4 Righetti, P.G., Gianazza, E., Bianchi-Bosisio, A. and Cossu, G. (1986) in The Hemoglobinopathies, Huisman, T.HJ. (ed.) Methods in Hematology, Vol. 15, pp. 47-70, Churchill Livingstone, Edinburgh. 5 Biss~, E. and Wieland, H. (1988) J. Chromatog. 434, 95-11(1. 6 Kutlar, A., Kutlar, F., Gu, L-G., Mayson, S.M. and Huisman, T.H.J. (19901 Hum. Genet. 85, 106-110. 7 Shelton, J.B., Shelton, J.R. and Schroedcr, W.A. (19841 J. l,iq. Chromatogr. 7, 1969-1977. 8 Kutlar, F., Kutlar, A. and Huisman, T.HJ. (1986)J. Chromatogr. 357, 147-153. 9 Clegg, J.B., Naughton, M.A. and Weatherall, D.J. ( 10661 J. Mot. Biol. 19, 91-108. 10 Huisman, T.H.J. (19871 J. Chromatogr. 418, 277 3114. 1l Wilson, J.B., Lam, H., Pravatmuang, P. and Huisman, T.HJ. (1979) J. Chromatogr. 179, 271-290. 12 Chang, J.Y., Brauer, D. and Winmann-Liebold, B. (19781 FEBS Len. 93, 205-214. 13 Poncz, M., Solowiejczyk, D., Harpel, B., Mory, Y., Schwartz, E. and Surrey, S. (1982) Hemoglobin 6, 27-36. 14 Gonzalez-Redondo, J.M., Kattamis, C. and Huisman, T.It.J. (1989) Hemoglobin 13, 377-392. 15 Gonzalez-Redondo, J.M., Stoming, T.A., Kutlar, A., Kutlar, F.. Lanclos, K.D., Howard, E.F., Fei, Y.J., Aksoy, M., Altay, (~., Gurgey, A., Basak, A.N., Efremov, G.D., Petkov, G and ttuis man, T.H.J. (1989) Blood 73, 1705-17l 1. 16 Diaz-Chico, J.C., Yang, K.G., Stoming, T.A., Efremov, D.(J.. Kutlar, A., Kutlar, F., Aksoy, M., Altay, ~., Gurgey, A.. Kilinq, Y. and Huisman, T.H.J. (1988) Blood 71,248-251. 17 Gonzalez-Redondo, J.M., Stoming, T.A., Lanclos, K.D., Gu, Y.C.. Kutlar, A., Kutlar, F., Nakatsuji, T., Deng, B., ltan, 1.S., McKic, V.C. and Huisman, T.HJ. (1988) Blood 72, 101)7-1(/14. 18 Bardakdjian-Michau, J., Fucharoen, S., Delanoe-Garin, J., Kister, J., Lacombe, C., Winichagoon, P., Blouquit, Y., Riou, J., Wasi, P. and Galacteros, F. (1990) Am. J. Hematol. 35, 96-09. 19 Pagano, L., Lacerra, G., Camardella, L., De Angioletti, M.. Fioreni, G., Maglione, G., De Bonis, C.. Guarino. E.. Viola, A..
179
20
21
22 23
Cutolo, R., De Rosa, L. and Carestia, C. (1991) Blood 78, 3070-3075. Yang, K.G., Kutlar, F., George, E., Wilson, J.B., Kutlar, A., Stoming, T.A., Gonzalez-Redondo, J.M. and Huisman, T.H.J. (1989) Br. J. Haematol. 72, 73-80. Stamatoyannopoulos, G., Nute, P.E., Adamson, J.W., Bellingham, A.J., Funk, D. and Hornung, S. (1973) J. Clin. Invest. 52, 342-349. Treisman, R., Orkin, S.H. and Maniatis, T. (1983) Nature 302, 591-596. Kazazian, H.H., Jr., Orkin, S.H., Antonarakis, S.E., Sexton, J.P., Boehm, C.D., Goff, S.C. and Waber, P.G. (1984) EMBO J. 3, 593-596.
24 Cheng, T-C., Orkin, S.H., Antonarakis, S.E., Sexton, J.P., Giardina, P.J.V., Li, A. and Kazazian, H.H., Jr. (1984) Proc. Natl. Acad. Sci. USA 81, 2821-2825. 25 Kleinschmidt, T. and Sgouros, J.G. (1987) Biol. Chem HoppeSeyler 368, 579-615. 26 Bashford, D., Chothia, C. and Lesk, A.M. (1987) J. Mol. Biol. 196, 199-216. 27 George, E., Li, H-J., Fei, Y-J., Reese, A.L., Baysal, E., Cepreganova, B., Wilson, J.B., Gu, L-H., Nechtman, J.F., Stoming, T.A., Liu, J-C., Codrington, J.F. and Huisman, T.H.J. (1992) Hemoglobin 16, 51-66. 28 Baklouti, F., Ouazana, R., Gonnet, C., Lapillone, A., Delaunay, J. and Godet, J. (1989) Blood 74, 1817-1822.
173
© 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00
BBAD1S 61210
Heterozygosity for the IVS-I-5 (G ~ C) mutation with a G ~ A change at codon 18 (Val ~ Met; Hb Baden) in cis and a T ~ G mutation at codon 126 (Val ~ Gly; Hb Dhonburi) in trans resulting in a thalassemia intermedia Vladimir Divoky a Emmanuel Biss6 b Jerry B. Wilson a Li-Hao Gu a, Heinrich Wieland b, Irene Heinrichs c, John F. Prior d and Titus H.J. Huisman a a Department of Biochemistry and Molecular Biology, Laboratory of Protein Chemistry, Medical College of Georgia, Augusta, (;.4 (USA), b Department of Clinical Chemistry, University Hospital, Freiburg (Germany), ' Division of Pediatrics, University Hospital, Halle (Germany) and d Hematology Department, The Queen Elizabeth 11 Medical Centre, Sir Charles Gairdner Hospital, Nedlands, Western Australia (Australia)
(Received 30 June 1992)
Key words: fl-Thalassemia; fl-Mutation in cis; fl-Mutation in trans; High-performanceliquid chromatography;DNA sequence We have analyzed the hemoglobins of a young German patient with/3-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the fl-globin genes through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the G_TG --* GGG mutation at codon 126 leading to the synthesis of Hb Dhonburi or a2/32126(H4)Val --* Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G ~ C) mutation that leads to a rather severe /3+-thalassemia and the GTG ~ ATG mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered fl-chain variant, named Hb Baden, was present for only 2-3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G ~ C) thalassemic mutation. The chromosome with the codon 18 (GTG ~ ATG) and the IVS-I-5 (G ~ C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G -~ C) mutation living in different countries failed to detect the codon 18 (G_TG ~ A TG) change.
Introduction /j-Thalassemia (/j-thai) is a most heterogeneous genetic condition that is characterized by the absence or decreased production of normal /J-chains (for review, see Ref. 1). At least 120 mutations and frameshifts have been detected in the /j-globin gene causing this defective /j-chain production, while a few larger deletions will also lead to a /j°-thal (for review and references, see Ref. 2). The phenotypic expression resulting from these numerous changes varies greatly from nearly undetectable to a distinct anemia with severe microcytosis and hypochromia in the heterozygote. Clinical observations in compound heterozygotes also vary considerably being dependent upon the types of changes in
Correspondence: T.H.J. Huisman, Department of Biochemistry and Molecular Biology,Medical College of Georgia, Augusta, GA 309122100, USA.
the /j-globin gene that are present on each of the two chromosomes of such an individual. Recently, we studied a young child from the former German Democratic Republic and of German descent who exhibited the typical clinical features of a /j-thai intermedia. Detailed structural studies of his hemoglobin (Hb) and sequence analysis of the/j-globin genes identified two r a r e / j - c h a i n abnormal Hbs and a common but clinically rather severe /j+-thal determinant; one of the structural mutations occurred in cis to the /j-thal mutation. Here, we provide details of that study which includes comparable analyses of some members of the patient's immediate family. Materials and Methods Blood samples were collected in vacutainers with E D T A as anticoagulant, and studied at the local institution and in Augusta, GA; shipment was by fast air mail service. Informed consent was given.
174 Hematological data were obtained with automated cell counters and with standard laboratory procedures. Hbs in red cell lysates were studied by cellulose-acetate and citrate-agar electrophoresis [3], by isoelectrofocusing (IEF) [4], and by different stability tests, such as isopropanol and heat stability at pH 7.0 and at 60°C [3]. The Hbs were quantified by cation exchange high performance liquid chromatography (HPLC) [5,6] and by reverse-phase HPLC (7,8); both methods played an important part in elucidating the structures of the Hb variants. Separation of a- and 3-chains was by carboxymethyl cellulose (CMC) chromatography [9] or by preparative reverse-phase HPLC [10]. Structural analyses used methodology described before; separation of tryptic peptides of isolated 3X-chains was by reversephase HPLC [11] and amino acid analyses of individual peptides were made with an automated amino-acid analyzer (Pico-Tag, Waters, Milford, MA), while the sequences of a few selected peptides were determined with the method described by Chang e t a [ . [12]. Isolation of a larger quantity of Hb Baden required a preparative HPLC procedure with a 21 x 25(t mm PolyCAT A, 5 ~m, 1000 A column (PolyLC, Columbia, MD). Some 20 to 30 mg were applied and the chromatogram was developed as described before [10] with a flow-rate of 5 ml/min. The isolated fractions were concentrated in a Diaflo YM-10 filter (43 mm) and next applied to a 10× 250 mm Vydac C 4 column, large-pore 5 /xm (The Separations Group, Hesperia, CA). The chromatogram was developed over a period of 70 min at a flow-rate of 2 m l / m i n and with a developing system described elsewhere [10]. The isolated 3X-chains were concentrated by freeze-drying and analyzed by methodology referred to abovc. DNA was isolated from white cells with the method of Poncz et al. [13]. Amplification of 3-globin genes and sequencing of the amplified DNA were done as described before [14,15]. Certain mutations were confirmed by dot-blot analysis that included hybridization of amplified DNA samples with 32p-labeled oligo-
nucleotide probes [16,17]; the same procedure was used to evaluate the possible presence of the Hb Baden mutation in DNA samples of several patients with a specific mutation. Results
The families Family S, consisting of parents with two children and paternal grandparents, originates from the eastern part of Germany. The propositus participated in a screening program for hemoglobinopathies in anemic children. He was found to have 'a rare Hb variant associated with /3-thai' on the basis of the results obtained in the laboratory of one of the authors (H.W.). Family R is also of German origin; only the propositus, a 65-year-old man born in Essen who emigrated to the United States and next to Australia, was available. Hematological data are presented in Table I. The propositus (Ph.S.) had a Hb level of about 9 g / d l with a striking microcytosis and hypochromia, an elevated Hb-A 2 of 6% and a minor increase in Hb-F level; the child was not transfused. The sister (S.S.) had a moderate microcytosis and hypochromia and a slightly elevated level of Hb-A2. Their mother had the characteristics of a /3-thai trait with mild anemia, microcytosis and hypochromia and elevated Hb-A 2. The father had nearly normal values except for a mild microcytosis and hypochromia (MCV 81.3 fl; MCH 26.5 pg). Similar observations were made in the grandfather (J.W.) and also in G.R., the propositus of the second family. The reticulocyte counts in these persons were slightly elevated to about 2-3%.
Hb analysis Electrophoretic examination failed to reveal the presence of a Hb variant; only a slight increase in Hb-F was detectable for the propositus Ph.S. IEF also gave negative data, except for the presence of Hb-F and an elevated level of Hb-A2; however, reexamination of
TABLE 1
Hematological and Hb composition data Subject
SexAg e
Relationship
Hb (g/dl)
PCV (1/1)
RBC (1(1~2/1)
MCV (fl)
MCH (pg)
MCHC (g/dl)
A z~ (%)
F ~ (%)
/3 x b (54")
3 Ab (%)
J.W. E.W. W.M.S. M.S. Ph.S. S.S.
M-73 F-64 M-39 F-30 M-6 F-4
Grandfather Grandmother Father Mother Propositus Sister
14.3 14.9 15.5 1(I.9 9.2 12.5
0.447 0.453 (I.475 0.347 [).302 0.385
5.37 4.85 5.84 5.17 5.43 5.12
83.3 93.5 81.3 67.2 55.6 75.2
26.6 30.7 26.5 21.1 16.9 24.4
32.0 32.9 32.6 31.4 30.5 32.5
2.9 2.1 3.1 5.1 6.(I 3.5
< 1.0 < 1.0 < 1.(1 1.2 ~ 4.l ~ < 1.0
34.7 0 36.0 I1 98.0 32.5
65.3
G.R.
M-65
Propositus
14.4
0.430
5.54
78.(/
26.0
33.6
2.7
(1.2
21.2
78.S
" By cation-exchange HPLC [5,6]. h By reverse-phase HPLC [7,8]. ' The ¢;y- and Ay-levels in isolated Hb-F were 18.7 and 81.3~ for M.S. and 33.7 and 66.3% for Ph.S., respectively.
64.0 2.(17 67.5
175 Father S
Propositus S
Mother I
I ~-Dhonburi I E HE
~-Dhonburi HEME, I
;-A
HEME
~-A
c
o
,-.-,2", 1'0 2'0 3'0 4'0
50
1'0 2'0 3'0 4'0 5'0 Time in Minutes
1'0 20
3'0 40 50
Fig. 1. Detection of the a-Dhonburi chain in the propositus and his father by reverse-phase HPLC [7,8].
/3A+ /3 x) with trypsin, separation of the tryptic peptides by reverse-phase HPLC, rechromatography of selected peptides and finally the determination of the amino-acid compositions of the individual peptides. Details of this study will not be presented, except to indicate that an abnormal/3T-13 peptide eluted slightly faster than normally observed. When analyzed, it was found that its only valine residue was replaced by a glycine residue. This observation identifies the variant as Hb Dhonburi [18] or Hb Neapolis [19] in which /3126-Val is replaced by Gly.
the gels after the variants were identified suggested that the major Hb of the propositus focused slightly differently from that of his mother. H e a t and isopropanol stability tests were positive for the propositus, his father, grandfather, and sister, and for the propositus of the second family; heat-precipitable Hb was estimated at 25%. Analyses of the Hbs by reverse-phase H P L C revealed the presence of a fast-eluting abnormal/3-chain in the propositus and his father but not in his mother (Fig. 1). Normal /3A-chains were not detectable in the blood of the propositus. The father was an apparent /3 x heterozygote, as were the grandfather and the younger sister and also G.R. of the second family. The relative amount of /3 x (as % of / 3 x + / 3 A) in the heterozygote was 32.5-36.0% in Family S; the lower level of 21.2% in G.R. remains unexplained. Structural analysis of the Hb variant in the propositus (and in G.R. of the second family) followed methodology routinely used in our laboratory which included separation o f / 3 x (/3 x +/3 A for subject G.R.) and a by CMC chromatography, digestion of /3 x (or £, ~..,~,~/J . , ~ •"* ~ AA CODON AR(G) 30 GLY 29 CTG LEU 28 GCC ALA 27 GAG GLU 26 GGT GLY 25 GGT GLY 24 GTT VAL 23 i== ~ GAA GLU 22 ,..., BAT ASP 21 GTG VAL 20 a,o,=,e AAC ASN 19 ~ ~ GTG VAL 18 (ATG;Met) AAG LYS 17 A C G T _ .-
DNA analysis Sequence analysis of amplified D N A samples from the propositus (Ph.S.) and his parents were used to identify the/3-thai allele in Ph.S and his mother. Fig. 2 shows photographs of parts of the sequencing gels of Ph.S. T h r e e mutations were detected, namely a G ~ C change at position 5 of the IVS-I ( G T T G G ~ G T T G C ) , a G ~ A mutation in codon 18 ( G T G for Val to A T G for Met), and a T ~ G mutation in codon 126 ( G T G for Val to G G G for Gly). The first is a /3+-thal muta-
T
G
C
..... ~
A
,~,;~
,,,, ~ . , . ~ ; •
CA G TAT GCC G CT CAG GTG
CCA
.....
CCA ACC TTC
AA Gin Tyr Ala Ala Gin Val Pro Pro
CODON 131 130 129 128 127 126 (GGG;Gly)
125 124
Thr 123 Phi. 122
Fig. 2. Detection of three mutations in the amplified DNA sample from the propositus. Left: The G ~ A mutation at codon 18, indicating a Val ~ Met replacement and a G ~ C mutation at the fifth position of the first intron. Right: The T ---,G mutation at codon 126, indicating a Val --, Gly replacement.
176 'FABLE II
Mutation,~ obsen,ed by .vequenci/zg of amplified DNA from the propositus, ]'ather and mother q]" fi*mily S Mutation
Propositus
Father
Mother
Codon 2 (CA(7 ~ CAT) Codon 18 ( G T G ~ A T G ) ( = 18 Val --+ Met) IVS-I-5 (G ~ C) Codon 126 (G_TG --+ G G G ) ( = 126 Val ~ Gly)
CAC/CAC GTG/ATG
CAC/CAC GTG/GTG G/G GGG/GTG
('AT_/CAC GTG/ATG (3/C GTG/GTG
G/C GGG/GTG
tion (for references, see Ref. 2); homozygotes and compound heterozygotes for this /3-thal allele and a /3 chain variant produce small quantities ( 2 - 8 % ) of normal Hb A [20]. The second mutation ( G T G - ~ ATG) results in a Vaf ~ Met replacement and, thus, in the synthesis of an abnormal /3-chain. This amino-acid replacement has never been observed before and the variant has been named Hb Baden or ce2o~218(A15) Val ~ Met. The third mutation (codon 126, G T G - - , G G G ) confirms the presence of Hb Dhonburi. Table II summarizes the differences in sequences of the DNA samples of three members of Family S. These results indicate that the propositus inherited one chromosome with G T G ~ G G G at codon 126 from his father and a second chromosome with G T G -+ A T G at codon 18 and with G ~ C at position 5 of the first intron from his mother. A well-known polymorphism at codon 2 (CAC ~ CAT) was present in the mother but not in her child, indicating that it occurred on her normal chromosome.
The search for Hb baden ([318(A15)Val --+Met) A study of the Hbs from propositus Ph.S. and his parents by cation-exchange HPLC provided the chromatograms shown in Fig. 3. No major abnormal Hb could be detected; the increase in the level of Hb-A 2 was readily observed, as was the higher level of Hb-F in the propositus. Of special interest is the presence of a small Hb-zone that separated only partially from the
Propositus S
g
10 20 30 40 5'o
ao
4'o
'_2
s'o
8
1'o 2'o Jo & s'o ~
7'o do ~ 4o do ,~o 11o 14o 40 4o 1~o
Time in Minutes (5 ml/min) Fig. 4. Isolation of Hb Baden and Hb Dhonburi by semi-preparative cation-exchange HPLC. The isolated Hb zones were further fractionated into their a and /3 chains by reverse-phase HPLC; see insets [10].
major Hb fraction; it was present in the propositus and the mother in an amount of about 2 - 3 % and was absent in the father. Preparative cation-exchange HPLC was used to isolate a larger quantity of this fraction (labeled Hb Baden); several such separations, as shown in Fig. 4 (bottom), provided a few mg of this material. Isolation
Father S
2'0 3'0 4b 5'0 6'0 7b
io
/Hb Dhonbun
Mother S
Hb-A+ Hb-Dhonburi
Hb-A
Hb-A2
(3.1'/0)
Hb-F Hb-Baden Hb-A2 (1.2%) (21%) I ( 5 1 % ) Hb-AIc|I , . A
" I
LJL__ 1'0
;o
Time in Minutes (2 ml/min)
Hb-A2 Hb-Baden (6O%) (2.5%) Hb-F ',
Q
J-Dhonburi
Ohonburi;
t.t3 8
t~.8aden
~j3
Hb-Dhonburi
E_
HEM
E
;o
2'o a'0 4'0
so
6'0 z'o
lo
2o ao
4o
5o
6b
r'o
Time in Minutes Fig. 3. Separation of the Hb components in red cell lysates from the propositus and his parents by cation-exchange H P L C [5,6]. The detection of a minor Hb-component (labeled Hb Baden) in the lysates of the propositus and his mother was the first indication for the presence of another abnormal Hb.
177 TABLE III The amino-acid compositions of a few selected tryptic peptides of the fl-chains of Hb Dhonburi, Baden and Olympia a
Amino-acid
/3-Dhonburi T-3
Aspartic acid Glutamic acid Glycine Arginine Threonine Alanine Proline Tyrosine Valine Methionine Leucine Phenylalanine Lysine
/3-Baden b T-3 x
T-13 x
1.95 (2) 2.10 (2) 3.18 (3) 1.00 (1)
1.95 (2) 1.98 (2) 3.15 (3) 0.99 (1)
3.00 (3) 1.02 (0) 0.96 (1) 2.05 (2) 2.14 (2) 0.78 (1) 0 (1)
1.03 (1) 3.03 (3)
1.09 (1) 1.94 (3) 0.87 (0) 0.97 (1)
0.91 (1) 0.94 (1) 13
3.00 (3) 0.93 (1) 1.93 (2)
1.89 (2) 1.96 (2) 2.95 (3) 1.16 (1) 0.95 (1)
1.92 (2)
1.03 (1)
Total
T-13
/3-Olympia c T-3 x
0.89 (1) 1.00 (1)
2.09 (3) 0.53 (0) 0.96 (1)
0.97 (1) 1.00 (1)
12
13
12
13
In mol/mol of peptide. b Amino-terminal sequence: Met-Asn-Val---. c Amino-terminal sequence: Val-Asn-Met--- (by microsequencing with the method of Chang et al. [12]); sequencing of amplified DNA confirmed the expected G ~ A mutation at codon 20 (Val --* Met). "
of the/3X-chains was by semi-preparative reverse-phase H P L C . T h e c h r o m a t o g r a m of Fig. 4 (top at left) shows that the major /3-chain of the H b B a d e n fraction has the mobility of /3 A, while a notable quantity of the /3-Dhonburi chain is also present. T h e H b D h o n b u r i fraction was nearly pure and contained a- and /3D h o n b u r i chains (Fig. 4, top at right). A b o u t 0.5-1.0 mg of the /3-Baden and /3-Dhonburi chains were di-
[
I[/ [3-Ohonburi([~126 Val~Gly)
/T-oT-7,,-,,T-1, i
T-13(Abn)
,-,
1
~ _ _ l T13
i
i
T-14,15
J
II
i
~ I 1/13-Baden(!318 Val~Met)
i silT-4 T-2
h-13
"
I-9 ,
,
I/
,
i
[11
T-3(Abn) I T-13A
i
J
,
v
,--
l
-
----:
,
7
Time in Minutes (1.5 ml/min)
Fig. 5. Separation of peptides in tryptic digests of three abnormal /3 chains by reverse-phase HPLC [11]. See text for details. Hb Olympia (a2/3220(B2)Val ~ Met) is a high-oxygen-affinity Hb variant that was observed in a Caucasian adult from Kentucky (unpublished data); the sample was kindly provided by Dr. Marie M. Keeling (Louisville, KY).
gested with trypsin and the tryptic peptides were separated by reverse-phase H P L C [11]. Fig. 5 illustrates the two c h r o m a t o g r a m s , as well as that for the /3-chain of H b Olympia that is characterized by a Val ~ Met m u t a t i o n at position /320 [21]. Most peptides eluted in the expected positions and had normal amino-acid compositions. Exceptions were t h e / 3 T - 1 3 and /3T-13A peptides in t h e / 3 - D h o n b u r i digest, and the /3T-3 peptide in the /3-Baden and /3-Olympia digests. The amino-acid compositions of the /3T-3 and /3T-13 peptides are listed in Table III; the data confirm the Val ~ Gly r e p l a c e m e n t in /3T-13 of the /3-Dhonburi chain and the Val ~ Met replacement in the /3T-3 peptides of the /3-Baden and /3-Olympia chains. T h e latter two peptides differ in the location of the Val Met substitution; that in fl-Baden is in the aminoterminal position (or position 18 of the intact /3-chain) and that in /3-Olympia is in the third position of the fiT-3 peptide (or position 20 of t h e / 3 chain). Discussion
T h e condition of patient Ph.S. can be designated as thalassemia intermedia; the child has a m o d e r a t e anemia with severe microcytosis and hypochromia, an elevated H b - A 2 level and a m o d e s t increase in Hb-F. N o blood transfusions are required. H e inherited from his father a /3-chain variant with a Val ~ Gly substitution at position 126. This rare variant, that has b e e n n a m e d H b D h o n b u r i [18] and H b Neapolis [19], is mildly unstable, and in the heterozygote is associated with minor thalassemic features such as a slight decrease in M C V and M C H values without an increase in the level
178 of H b - A e ( T a b l e 1) a n d with biosynthetic a l t e r a t i o n s c o n s i s t e n t with a mild /3-thalassemic p h e n o t y p e lit)]. His s e c o n d c h r o m o s o m e carries a G--+ C m u t a t i o n at the fifth p o s i t i o n of IVS-I. This well-known /3-thai allele is o b s e r v e d mainly a m o n g A s i a n Indians, M e l a n e s i a n s a n d C h i n e s e [22-24]; the greatly dec r e a s e d /3-chain p r o d u c t i o n results from an u n d e r u t i lization of the IVS-I d o n o r splice-site a n d the activation of o n e o r m o r e cryptic splice-sites. C o m p o u n d h e t e r o z y g o s i t y for this IVS-I-5 ( G - + C) m u t a t i o n a n d the c o d o n 126 ( G T G - - + G G G ) m u t a t i o n a p p a r e n t l y leads to a d i s t i n c t / 3 - t h a i i n t e r m e d i a , as is e v i d e n t from the clinical c o n d i t i o n o f the p r o p o s i t u s o f the S family. T h e s u r p r i s e in this study was the finding of a G T G -+ A T G m u t a t i o n in c o d o n 18 that results in the synthesis of an a b n o r m a l ,8-chain with a V a l - + M e t r e p l a c e m e n t at that location. This /3-chain a b n o r m a l H b m u s t be a stable variant, b e c a u s e the s a m e r e p l a c e m e n t is p r e s e n t in the n o r m a l e-chain a n d e m b r y o n i c H b s c o n t a i n i n g this chain are stable p r o t e i n s [25,26]. T h e two m u t a t i o n s (at c o d o n 18 ( G --+ A ) a n d at IVS-I-5 ( G --+ C)) m u s t occur on the s a m e c h r o m o s o m e b e c a u s e the c o d o n 18 c h a n g e was only o b s e r v e d in the p r o p o s i tus a n d his m o t h e r w h o also carries the IVS-I-5 (G -+ C) m u t a t i o n . T h e synthesis of t h e /3-Baden chain is low and c o m p a r a b l e to that of H b A in u n t r a n s f u s e d patients with H b E a n d the IVS-I-5 ( G -+ C) t h a l a s s e m i a allele [20,27]. T h e u n u s u a l c h r o m o s o m e in this family is the s e c o n d e x a m p l e of a c h r o m o s o m e in which a / 3 - t h a i m u t a t i o n occurs in cis to that resulting in the synthesis of an a b n o r m a l /3-chain; the o t h e r was r e p o r t e d by B a k l o u t i et al. [28] w h o o b s e r v e d a c o m m o n p r o m o t e r m u t a t i o n (C--+ T at p o s i t i o n - 8 8 ) in cis to the G A G ---) G T G m u t a t i o n at c o d o n 6 (i.e., the /3 s m u t a t i o n ) . W e have t e s t e d D N A s a m p l e s from m o r e than 200 /3-thai p a t i e n t s for t h e G T G --+ A T G m u t a t i o n at c o d o n 18 by d o t - b l o t h y b r i d i z a t i o n with 32p-labeled specific p r o b e s a n d w e r e u n a b l e to d e t e c t this change; a m o n g the 208 p a t i e n t s w e r e 49 subjects with a h e t e r o z y g o s i t y for the IVS-I-5 ( G - - ) C) m u t a t i o n ( t h r e e f r o m C z e c h o s l o v a k i a , o n e f r o m Italy, 13 f r o m Malaysia, 32 from I n d i a ) a n d two M a l a y s i a n p a t i e n t s with a homozygosity for this allele. It a p p e a r s most likely that the H b B a d e n m u t a t i o n o c c u r r e d as an i n d e p e n d e n t event on a c h r o m o s o m e t h a t c a r r i e d the IVS-I-5 ( G -+ C) m u t a tion. T h e two m u t a t i o n s p r e s e n t on the /3-thai c h r o m o s o m e o c c u r in the s e g m e n t of D N A w h e r e the d i f f e r e n t cryptic splice sites a n d t h e 5' d o n o r splice-site of the IVS-I a r e l o c a t e d ( r e v i e w e d in Ref. 1). T h e cryptic splice-site at c o d o n s 17-18-19 (AAG,I, G T G - A A C ) is a b o l i s h e d by t h e G --+ A m u t a t i o n at c o d o n 18, i.e., the H b B a d e n m u t a t i o n ( A A G . A T G . A A C ) . T h e G--+ C m u t a t i o n at IVS-I-5 has a n e g a t i v e effect on the 5' d o n o r splice site of the I V S - I ( C A G S G T T G G T - - + C A G J, G T T G C T ) . Thus, t h e s e two m u t a t i o n s e l i m i n a t e
one cryptic splice-site a n d nearly c o m p l e t e l y d c a c t i v a t e the d o n o r splice-site, l e a d i n g to the activation of (one of) thc two r e m a i n i n g cryptic splicc-sites, namcly G T G { G T G A G at c o d o n s 24-25-26 and / \ A G 1 (fVT A C at positions 9 t h r o u g h 16 of the IVS-I: which one of these two sites will bc most active c a n n o t be pred i c t e d without f u r t h e r analyses.
Acknowledgements T h e a u t h o r s greatly a p p r e c i a t e the c o o p e r a t i o n of the m e m b e r s of Family S. in this study. This r e s e a r c h was s u p p o r t e d in p a r t by the Ministry o f Science and Arts, B a d e n - W i i r t t e m b e r g , G e r m a n y (to H . W . ) and by the U n i t e d S t a t e s Public H e a l t h Service r e s e a r c h grants HLB-05168 a n d HLB-41544 (to T.H.J.H.).
References 1 Bunn, H.F. and Forget, B.G. (19861 Hemoglobin: Molecular, Genetic and Clinical Aspects. W.B. Saunders, Philadelphia. 2 Baysal, E. (1992) Hemoglobin 16, 237-258. 3 Huisman, T.H.J. and Jonxis, J.H.P. (19771 The Hemoglobin+ opathies Techniques of Identification. Clinical and Biochemical Analysis, Vol. 6, Marcel Dekker, New York. 4 Righetti, P.G., Gianazza, E., Bianchi-Bosisio, A. and Cossu, G. (1986) in The Hemoglobinopathies, Huisman, T.HJ. (ed.) Methods in Hematology, Vol. 15, pp. 47-70, Churchill Livingstone, Edinburgh. 5 Biss~, E. and Wieland, H. (1988) J. Chromatog. 434, 95-11(1. 6 Kutlar, A., Kutlar, F., Gu, L-G., Mayson, S.M. and Huisman, T.H.J. (19901 Hum. Genet. 85, 106-110. 7 Shelton, J.B., Shelton, J.R. and Schroedcr, W.A. (19841 J. l,iq. Chromatogr. 7, 1969-1977. 8 Kutlar, F., Kutlar, A. and Huisman, T.HJ. (1986)J. Chromatogr. 357, 147-153. 9 Clegg, J.B., Naughton, M.A. and Weatherall, D.J. ( 10661 J. Mot. Biol. 19, 91-108. 10 Huisman, T.H.J. (19871 J. Chromatogr. 418, 277 3114. 1l Wilson, J.B., Lam, H., Pravatmuang, P. and Huisman, T.HJ. (1979) J. Chromatogr. 179, 271-290. 12 Chang, J.Y., Brauer, D. and Winmann-Liebold, B. (19781 FEBS Len. 93, 205-214. 13 Poncz, M., Solowiejczyk, D., Harpel, B., Mory, Y., Schwartz, E. and Surrey, S. (1982) Hemoglobin 6, 27-36. 14 Gonzalez-Redondo, J.M., Kattamis, C. and Huisman, T.It.J. (1989) Hemoglobin 13, 377-392. 15 Gonzalez-Redondo, J.M., Stoming, T.A., Kutlar, A., Kutlar, F.. Lanclos, K.D., Howard, E.F., Fei, Y.J., Aksoy, M., Altay, (~., Gurgey, A., Basak, A.N., Efremov, G.D., Petkov, G and ttuis man, T.H.J. (1989) Blood 73, 1705-17l 1. 16 Diaz-Chico, J.C., Yang, K.G., Stoming, T.A., Efremov, D.(J.. Kutlar, A., Kutlar, F., Aksoy, M., Altay, ~., Gurgey, A.. Kilinq, Y. and Huisman, T.H.J. (1988) Blood 71,248-251. 17 Gonzalez-Redondo, J.M., Stoming, T.A., Lanclos, K.D., Gu, Y.C.. Kutlar, A., Kutlar, F., Nakatsuji, T., Deng, B., ltan, 1.S., McKic, V.C. and Huisman, T.HJ. (1988) Blood 72, 101)7-1(/14. 18 Bardakdjian-Michau, J., Fucharoen, S., Delanoe-Garin, J., Kister, J., Lacombe, C., Winichagoon, P., Blouquit, Y., Riou, J., Wasi, P. and Galacteros, F. (1990) Am. J. Hematol. 35, 96-09. 19 Pagano, L., Lacerra, G., Camardella, L., De Angioletti, M.. Fioreni, G., Maglione, G., De Bonis, C.. Guarino. E.. Viola, A..
179
20
21
22 23
Cutolo, R., De Rosa, L. and Carestia, C. (1991) Blood 78, 3070-3075. Yang, K.G., Kutlar, F., George, E., Wilson, J.B., Kutlar, A., Stoming, T.A., Gonzalez-Redondo, J.M. and Huisman, T.H.J. (1989) Br. J. Haematol. 72, 73-80. Stamatoyannopoulos, G., Nute, P.E., Adamson, J.W., Bellingham, A.J., Funk, D. and Hornung, S. (1973) J. Clin. Invest. 52, 342-349. Treisman, R., Orkin, S.H. and Maniatis, T. (1983) Nature 302, 591-596. Kazazian, H.H., Jr., Orkin, S.H., Antonarakis, S.E., Sexton, J.P., Boehm, C.D., Goff, S.C. and Waber, P.G. (1984) EMBO J. 3, 593-596.
24 Cheng, T-C., Orkin, S.H., Antonarakis, S.E., Sexton, J.P., Giardina, P.J.V., Li, A. and Kazazian, H.H., Jr. (1984) Proc. Natl. Acad. Sci. USA 81, 2821-2825. 25 Kleinschmidt, T. and Sgouros, J.G. (1987) Biol. Chem HoppeSeyler 368, 579-615. 26 Bashford, D., Chothia, C. and Lesk, A.M. (1987) J. Mol. Biol. 196, 199-216. 27 George, E., Li, H-J., Fei, Y-J., Reese, A.L., Baysal, E., Cepreganova, B., Wilson, J.B., Gu, L-H., Nechtman, J.F., Stoming, T.A., Liu, J-C., Codrington, J.F. and Huisman, T.H.J. (1992) Hemoglobin 16, 51-66. 28 Baklouti, F., Ouazana, R., Gonnet, C., Lapillone, A., Delaunay, J. and Godet, J. (1989) Blood 74, 1817-1822.