A variant hemoglobin assicated with α-Thalassemia-2

Btochlmtca et Btophyswa Acta, 747 (1983) 65-70

65

Elsevier BBA 31686

H E M O G L O B I N EVANSTON: a14(A12) Trp --, Arg A VARIANT H E M O G L O B I N A S S O C I A T E D W I T H a - T H A L A S S E M I A - 2 WINSTON F MOO-PENN, ROSALIE M BAINE, DANNY L JUE, MARY H JOHNSON, JAMES E McGUFFEY and JANE M BENSON

Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA 30333 ( U S A ) (Received March 23rd, 1983)

Key words Hemoglobm variant, a-cham, a-Thalassemta, (Human)

Hb Evanston (a14 Trp --, Arg) was detected on cellulose acetate at pH 8.4 as a band with an electrophoretic mobility similar to that of Hb S. In addition, a band migrating cathodic to Hb A 2 suggested the presence of a variant Hb A 2 with a substitution in the a-chain, a fact that was later confirmed by structural analysis. An unusual feature of Hb Evanston is its low percentage; less than l(Wo occurs in the hemolysate. Studies indicate that the variant is not unstable, but there appears to be a defect in globin-chain synthesis. Gene mapping also shows that it is associated with the et-thalassemia-2 gene. The variant has high oxygen affinity with normal cooperativity and a normal Bohr effect. The combination of l i b Evanston with a-thalassemia-2 produced anemia in this black family.

Introduction

Materials and Methods

Screening for abnormal hemoglobins is becoming more routine in m a n y hospitals m the U m t e d States as part of the normal hematologic work-up of pauents. Because of tins practice m a n y new variants have been characterized and our understanding of this unique molecule continues to grow. In this report we describe a new variant, a14 Trp---, Arg, that was found in a patient from Tuscaloosa, AL. Since our mlUal observation, an abstract [1] reporting on the same variant has appeared in wtuch the variant is referred to as H b Evanston. In keeping with the priority nanung of variant hemoglobins, we will refer to the a14 Trp Arg variant as H b Evanston in our report.

Standard hematological methods were used in thas investigation [2]. Electrophoresls of samples on cellulose acetate and citrate agar, and globmchain analysis procedures have been described [3]. Alkali-resistant hemoglobin was deternuned by the Singer test [4], and the heat and lsopropanol tests [5,6] were used to detect the presence of unstable hemoglobins. The abnormal hemoglobin was isolated at 4°C on DEAE-Sephadex by the method of H m s m a n and Dozy [7], and oxygen equilibrium measurements on the sample were done by the tonometrlc techmque of Raggs and Wolbach [8]. A 60 # M hemoglobin solution In 0.05 M Blstrls or Trls buffers at" 20°C, in both the absence and the presence of the allostenc effectors 2,3-dlphosphoglycerate and inosltol hexaphosphate, was used for these studies. Control experiments with H b A

Abbreviations Hb, hemoglobin, Blstrls, 2-[bls(2hydroxyethyl)armno]-2-(hydroxymethyl)propane-1,3-dlol 0167-4838/83/$03 00 © 1983 Elsevier Science Pubhshers B V

66

isolated from the same column were run simultaneously Globln chains for structural analysis were prepared according to the method of Clegg et al. [9], reduced with dlthaoerythrltol, modified with ethylenmune, and digested with trypsin [10] The peptlde digest was analyzed by peptlde fingerprinting [ 11] and by hagh-performance hquld chromatography (HPLC) [12]. Amino a o d compositions were deternuned on a Beckman analyzer [13] after the peptldes had been hydrolyzed at l l0°C for 24 h m vacuo. Sequence data were obtained from a Beckman 890C sequencer using the program (121078) provided by the manufacturer. Polybrene was added to the spmmng cup to enhance retentxon of peptldes, and repetmve yields were increased by m sltu purification of quadrol [14]. The phenylthlohydantom-denvatlves were identified and quantltated by HPLC [15]. Globln-cham synthesxs stu&es were done as prevaously descnbed [16]. [3H]Leucme was used as a label, and the cells were incubated for 2 h at 37°C. Globm was prepared by precxpltatlon m acid acetone at - 2 0 ° C . The globm chains were separated [9], and total counts m each chain were calculated by summation of the counts m each peak after subtractmg background counts To calculate specific radloactlvmes, protein concentratlons were determined by the method of Lowry [ 17]. For gene mapping, DNA was extracted from peripheral blood leukocytes [18] and digested with the restncUon endonucleases EcoRI, BamHI, BglII, HmdIII, and XbaI according to manufacturer's directions (Bethesda Research Laborato-

raes, Galthersburg, MD). The DNA fragments were separated by electrophoresls on 0.7% agarose gels and transferred to mtrocellulose filters by a modification of Southern's method [19]. The filters were hybridized to a rock-translated 32p-labelled probe derived from the recombmant plasmad JW101 which carries the human a-globin DNA insert [20]. After extensive washang, filters were autoradlographed for 4 - 7 days Results Hemoglobin Evanston was detected in a black male as a variant with an electrophoreUc mobdlty hke that of Hb S on cellulose acetate (pH 8.4), but it has the same mobdxty as Hb A on citrate agar (pH 6.2). The presence of a variant Hb A 2 nugratlng between Hb A 2 and carbonic anhydrase suggested that it was an a-chain variant. The abnormal globm chain could not be distinguished from the normal chains under acid condmons (pH 6.0), but m alkahne buffer it resolves between a A and a ~ Phdadelphaa (pH 8.6) Although the test for inclusion bodies wxth bnlhant cresyl blue was posltxve, the heat and lsopropanol tests for unstable hemoglobins gave negatxve results The hematologic data m Table I show that the proposltus and has brother are anemic The normal levels of iron, iron binding capaoty, and ferrmn indicate that the anemm ~s not due to iron defioency There ~s marked rmcrocytos~s and hypochrorma, and target cell and spherocytes are present Blood smears show amso- and potlolocytosls. The retlculocyte counts are elevated: 2.8% m the proposltus, and 1.6% m his brother These hemato-

TABLE I H E M A T O L O G I C DAT A RBC, red blood cell count, Hct, hematocnt, MCV, mean corpuscular volume, MCH, mean corpuscular hemoglobin, MCHC, mean corpuscular hemoglobin concentratton

Proposltus Sister Sister Brother Daughter

Age (years)

RBC (1012/1)

Hb (g/dl)

Hot (%)

MCV (fl)

MCH (pg)

MCHC (g/dl)

HbA (%)

Varlant-Hb (%)

HbA 2 Vanant-HbA 2 HbF (%) (%) (%)

HbS (%)

47 54 59 62 27

627 487 472 500 572

109 148 138 115 95

375 430 390 370 314

60 88 83 74 544

173 304 292 230 166

288 3 44 3 54 310 305

908 978 979 942 761

71 --31 --

13 22 21 26 28

----21 1

08 --01 --

08 07 07 05 06

67

variants contribute approximately 25% to the total hemoglobin present. Informatton on the substitution m Hb Evanston was obtained from peptlde fingerprints The fingerprmt showed that aTp-3 was rmssing, and two new peptlde spots were evtdent, one directly below aTp-7, and the other approximately equidistant between aTp-7 and aTp-l,2 [21] Amino acid compositions mdlcated that tryptophan at posttlon 14 had been replaced by ar~nme, and the two new peptides observed on fingerprinting had resulted from cleavage by trypsin at this new site (Table II) These peptldes were also resolved by HPLC chromatography and the results are shown m Fig. 1. Normal aTp-3 ts absent, and new peptldes representing aTp-3a and a-3b are present Sequence analysis of the intact abnormal chain confirmed this substitution. The oSygen-affimty results (Fig. 2) show that Hb Evanston has a marginally increased affinity for oxygen. When the allosterlc effector 2,3-dlphosphoglycerate IS added, the oxygen-affinity curve is shifted to the left compared to HbA. However, the magnitude of the slufts is similar for Hb Evanston and HbA in the absence and presence of the allostenc effector Inosttol hexaphosphate appears to have a slightly greater effect in

TABLE II A M I N O ACID ANALYSIS OF PEPTIDES FROM EVANSTON

Hb

Values are expressed as molar ratios HPLC peptides were obtamed after rechromatography of the first peak from Ftg l on a Vanan MicroPak AX-10 column [24] This column resolved peptides Tp-3a, -3b and -7,8 Amino acid

Trp Lys Arg Gly Ala

Expected

1 1

Found Fingerprinting

HPLC

Tp-3a

Tp-3a

.

Tp3b

.

.

-09 -18

1 2

Tp-3b

.

10

10

- -

09 -21

- -

10 - -

- -

10 --

logic findings are also observed in his daughter and suggest that a thalassemla condition may exist in these individuals. In addmon, his daughter ts tron-defictent Quantttation of the abnormal hemoglobin by column chromatography agrees with the electrophoretic data, which reveal very low percentages of Hb Evanston. Tlus low percentage is a strflang feature, and m this respect resembles Hb Fort Worth, another a-chain vartant that makes up less than 10% of the hemolysate. Most other a-chain

20

, 100

II II II I I II ii

18-~

16" 14"

I-- - -

I

"¢

B

• 90

"80

12-

I"~

10-

=

I

1/

08"

ii ~

• 70

m~

"60

--

"50

I o~

-4e

T"

-30

/

z

06-

I

L

04-

!

!

02-

01 10

~

. . 40 . .50

30

60

;o

.

80

.90 . 100 .

.

110

.

120

.

130

. 140 .

.

150

.

160

170

180

1~o

2O0 '

2 0

220

-20 -10

230

TUBE N U M B E R S

Fig 1 HPLC of a tryptlc digest from Hb Evanston The sample (10 rag) was separated on a Waters #Bondapak Cts column (0 4 × 30 cm) Buffer A was 12 5 mM sodmm acetate, pH 5,7, and buffer B was a 1 2 ddutlon of buffer A v~th acetomtnle The gradient of 1-60% B was run for 2 h at a flow rate of 1 5 m l / n u n , and fractions were collected at 30-s intervals

68

lowering the oxygen affinity. The cooperatwlty and the Bohr effect are unchanged both m the absence and presence of effectors Globln-cham synthesis and gene mapping experiments were undertaken in an attempt to determine the molecular basis for the observed hematologtc findings, as well as to provide an explanatmn for the low percentage of the variant Globm-chaln synthes~s studies were performed twice using peripheral retlculocytes from the proposltus. Both samples showed a pronounced deficiency in a-chain synthesis, with total ( o t A + otEvanst°n)/fl A ratios of 0 50 and 0.54. These ratios are lower than we usually see in a-thalassemla trmt (range 0.65-0.80) The proportion of the total a-chain counts Incorporated into the OtEvanst°n chain was similar to the proportion of the variant m peripheral blood: the o~Evanst°n/o/T°tal synthests ratios were 0 06 and 0 09 The specific radnoactwlties of the flA, aA and a Evanst°n chains were 10.4,

xx

15

n"\ x \\

10

\

x

g

\\

,_1

O5

•

• \\ \N

\\ \

x \

O\\

X \\

\\

\\

\\

\\

\

\

\

\

\

\\

\\ \\

-0 5

5

,,~, ....

, ....

, ....

6

r ....

\

~.........

7

~ , , ,",~ . . . .

8

9

05

pH

Fng 2 The effect of pH and orgamc phosphates on the oxygen affimty of Hb Evanston Experimental detaals are in the text *, no addmon, =, + 2,3-d~phosphoglycerate, II, + lnOSltOl hexaphosphate

Eco RI

Bam HI

Bgl II

H,nd III

Kb

m~

QIm~v~

Kb

(-I

9 /8

,,d

14--

16 --15 15

J

105-"

~74

)

45t ¸ 37~

(*) 1

2

3

4

5

1

2

3

4

5

]

1

2

3

4

5

3

4

5

Fig 3 Autoradnograms showing a-gene-specnfic restrtctlon fragments D N A was d~gested wtth restrnct~on endonucleases as indicated, and the sizes of the fragments m kdobase pairs are shown m the margins The snzes were determined from X-DNA &gested w~th HmdIII The numbers at the base of the figure refer to members of tlus family (1) known homozygote for the black form of ot-thalassemla-2, (2) daughter of the proposltus who does not have Hb Evanston, (3) proposntus wtth 7 170 Hb Evanston, (4) brother of the propomus with 3 170 Hb Evanston; and (5) normal control (non-thalassemtc)

69 5.3 and 5.9 d p m / m o l , respectively. Both the a A and the a Evanst°n chains had lower specific radioactivities than the flA-chaln, suggesting a form of a-thalassemla. The sirmlar specific radioactlvlUes of the a A and otEvanst°n chains suggests that instabihty is not the primary cause of the low proportion of Hb Evanston in peripheral blood. Gene mapping results are shown in Fig. 3 The patterns derived from digestions of D N A with BamHI and BgllI showed that the proposltus and his daughter are homozygous for a-thalassenna-2. In the BamHI analysis the normal 14 kb band was nnssmg, and only the shorter 10.5 kb band was found. With BgllI, both the normal 12 kb and 7 4 kb bands were nnssing, but a single band of approx. 15.0 kb was found, indicating that both chromosomes have lost the BgllI split point located between the a-chain genes. The brother is heterozygous for a-thalassenna-2 and showed the above patterns as well as the normal bands. Analysis with EcoRI showed that neither the propositus nor ins daughter has the normal 23 kb band. The daughter has only the 19 kb band that is characteristic of homozygous a-thalassenna-2, whereas the propositus has, in addition to tins 19 kb band, another band of approximately 18 kb that is also seen in ins brother. Analysis with H m d l I I showed a pattern typical of homozygous a-thalassenna-2 for the daughter and the proposltus, with bands of 16 kb and 4.5 kb. The normal 3.7 kb 'bridging' D N A between the two a-chain genes is missing. The brother as previously mentioned is heterozygous for et-thalassemta-2 and has a normal 3.7 kb band, but, in ad&tlon, a band of approx. 15 kb was observed. Tins band is also present in the proposltus. The results of gene analysis indicate that the propositus and ins daughter are homozygous for a-thalassemia-2. The loss of a functional a-chain gene plus the D N A that normally connects the two a-cham genes probably arose from n u s p a m n g during genetic recombination. The result Is a single ' h y b r i d ' a-chain gene sinular to the non-et chmn gene of Hb Lepore [22,23]. The propositus and ins brother are also careers of Hb Evanston, and both show an additional 18 kb band in EcoRI digestion as well as a 15 kb band in HmdlII digestion. These two bands must be associated with the Hb Evanston gene cluster and could represent a dele-

txon of approramately 1 kb located in the 5' flanking sequence of the non-functional ~l-gene. Such a deletion between the HmdlII and the BgllI restriction sites to the 5' side of the ~l-gene would be consistent with these bands [25,26]. However, further experiments would be required to verify this suggesuon. In summary, gene analysis of tins family m & cate that the propositus has a genotype of - a A / - - OtEvanst°n, the daughter a genotype of - a A / - a A, and the brother a genotype of aAetA/a Evanst°n The brother's genotype is consistent with the even lower percentage of H b Evanston seen m tins individual. Discussion

One of the most unusual features of H b Evanston is its low percentage in peripheral blood. Since the proposltus has only two a-chain genes, of winch one is H b Evanston, it is surprising that he has less than 10% of the variant. No chermcal or biosynthetic ewdence for mstablhty of the variant was found. It seems most hkely that the low level of synthesis is probably due to some deficiency in the synthesis, processing, transport or stabdlty of the m R N A winch codes for the ctEvanst°n chain. We cannot rule out the possibility that the small deletion 5' to the ~l-gene might affect m R N A synthesis. In one form of y-fl-thalassenna, the deleuon of the 7 and 8 chain genes is associated with complete suppression of fl-chain production from that chromosome although the fl gene itself plus 2.5 kb of its 5' flanking sequence appears to be intact [27]. It should be noted that both m&xaduals with H b Evanston also have athalassenna-2 and the proposltus is homozygous for a-thalassenna-2 as well, which indicates that a Evanst°~ must be hnked to a-thalassenna-2. Were these genes not linked, normal hematologic finding would be expected in lndwlduals heterozygous for H b Evanston, since tins condition would resemble heterozygous Hb Constant Spnng or heterozygous a-thalassemta-2, both of which are bemgn syndromes. Inclusion bodies were found in the presence of bnlhant cresyl blue, and it is surprising that we did not detect H b H in the proposltus, since phenotyplcally we would expect ins condition to resemble H b H disease or H b H disease with Constant Spring

70

The explanation for the increased affimty for oxygen shown by the variant is related to the replacement of the large aromatic tryptophan residue with argmme. Tryptophan is a hydrophoblc residue and is located internally m the molecule. Replacement wtth a charged arglnme residue wdl d~srupt the structural relationships in this area, since arg/nlne will tend to be located on the external surface of the molecule. Increase in oxygen afflmty indicates that this substitution is more favorable to the formation and stabdlty of the R conformation. The effects of the replacement do not extend to the Bohr residues, the 2,3-dlphosphoglycerate binding site, or the critical residues revolved in the cooperatlvity of the subumts, since these properties are unchanged. This discussion of the properties of Hb Evanston suggests that individuals with anemia should be carefully evaluated to determine the correct basis for dmgnosxs

Addendum Further studies w~th XbaI, which splits between the HmdlII and the BgllI sites, show that the deletion assoctated with Hb Evanston is located between the HmdlII and the XbaI sites, rather than between the XbaI and BgllI sites.

Acknowledgements We thank Drs P.L. Walker and W A. Hall of Tuscaloosa, AL, and Dr. J.H. Gentry of Carrolltown, AL, for bringing this farmly to our attenuon and for collecting blood samples. S. Holland and H G Brown of the Hemoglobmopathy Standardization Laboratory, CDC, assisted m screening blood samples. We thank Dr. B. Forget for plasmid JWl01.

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