globin chain synthesis in the alpha thalassemia syndromes

Globin Chain Synthesis in the

Alpha Thalassemia Syndromes

YUEr WmKAN, ELIAS ScHwARrz, and DAVID G. NATHAN

From the Hematology Research Laboratory of the Department of Medicine,Children's Hospital Medical Center and the Department of Pediatrics,Harvard Medical School, Boston, Massachusetts 02115

A B ST RAC T Whole blood samples of patientswith various forms of alpha thalassemia includinghemoglobin H disease, alpha thalassemia trait, andthe "silent carrier" state were incubated withleucine-14C for definition of relative rates of pro-duction of alpha and beta chains in these dis-orders. The chains were separated by carboxy-methyl cellulose chromatography in the presenceof 8 M urea and dithiothreitol. Their absorptionsat 280 mu were determined and their radioactivi-ties measured in a liquid scintillation spectrometer.After correction for differences in extinction coef-ficients, the specific activities of the widely sepa-rated alpha and beta peaks were determined. In11 nonthalassemic individuals, the alpha/beta spe-cific activity ratios were found to be 1.02 + 0.07;in nine patients with alpha thalassemia trait, 0.77± 0.05; in six patients with hemoglobin H disease,0.41 + 0.11; and in four "silent carriers," 0.88with a range of 0.82-0.95. The results show thatin peripheral blood, alpha chain production relativeto beta chain production is indeed limited in thealpha thalassemia syndromes. Hemoglobin H dis-ease results from doubly heterozygous inheritanceof a gene resulting in moderate depression ofalpha chain production (alpha thalassemia trait)and a gene resulting in very mild depression ofalpha chain production (the "silent carrier" syn-drome."

Dr. Schwartz's present address is Cardeza Foundationfor Hematologic Research, Department of Pediatrics,Jefferson Medical College, Philadelphia, Pa.

This work was presented in part at the Plenary Ses-sion of the Annual Meeting of the Society for PediatricResearch, 3 May 1968, Atlantic City, N. J.

Received for publication 20 May 1968.

INTRODUCTION

Alpha thalassemia is a complex disorder with atleast four different clinical manifestations (1).The first, alpha thalassemia trait, the hetero-zygous state, is a mild disease characterized mainlyby microcytosis and little if any anemia. Thesecond, hydrops fetalis as a result of homozygousalpha thalassemia, is a lethal disorder in whichdeath occurs in utero or at birth (2). In this dis-ease, there is total absence of hemoglobin contain-ing alpha chains and complete replacement of thenewborn hemoglobin mixture by hemoglobinBart's (y.) (3, 4). The third syndrome, hemo-globin H disease, is an hemolytic hypochromicanemia with intraerythrocyte inclusion formation.In such erythrocytes, hemoglobin H (p4) maycomprise up to 40% of the total hemoglobin (5).Such patients are usually the offspring of oneparent with alpha thalassemia trait and one withthe fourth alpha thalassemia syndrome, the so-called "silent carrier" state. Several differentterms have been employed to describe the lattersyndrome (6-9). The "silent carrier" is heredefined as the hematologically normal parent ofan individual with hemoglobin H disease. Theo-retically he may also be the offspring of an indi-vidual with hemoglobin H disease.

On the basis of gross deficiency of alpha chains,both hydrops fetalis and hemoglobin H diseasemay be ascribed to reduced alpha chain pro-duction. Indeed, evidence for low alpha chainproduction relative to beta chain production inhemoglobin H disease has been provided byWeatherall, Clegg, and Naughton (10) and, bysimilar techniques, low beta chain production rela-

The Journal of Clinical Investigation Volume 47 1968 2515

tive to alpha chain production has been detectedin various beta thalassemia syndromes (10-12).Defective alpha chain production in alpha thalas-semia trait is suggested on the basis of familystudies and red cell morphology. The "silent car-rier" state has been poorly understood and isthought to be a genetic condition which wheninherited in association with a gene for alphathalassemia trait leads to further depression ofalpha chain production (7). In this report, wepresent firm evidence of reduced alpha chain pro-duction in three alpha thalassemia syndromes in-cluding the "silent carrier" state. This evidencewas derived by measurement of the rate of incor-poration of leucine-l"C into the alpha and betachains of the peripheral blood hemoglobin of nor-mal individuals, patients with hemoglobin H dis-ease and alpha thalassemia trait, and of individualsconsidered to be "silent carriers."

METHODS

SubjectsPATIENTS EITHER OF CHINESE OR ITALIAN EXTRACTION

Hemoglobin H disease. Six patients with this dis-order were studied. The hematologic diagnosis was con-firmed by demonstration of hemoglobin H with starch-gel electrophoresis (13).

Alpha thalassemia trait. 9 individuals with microcy-tosis and (or) mild hypochromia with normal serum ironconcentration and normal hemoglobin electrophoresis wereincluded in this group. They were either parents of an hy-dropic baby with 100% hemoglobin Bart's or parents oroffspring of patients with hemoglobin H disease.

"Silent carriers." Three individuals whose spouseshad alpha thalassemia trait and who had at least one off-spring with hemoglobin H disease were included in thisgroup. They had no anemia, microcytosis, or hypo-chromia. Rare poikilocytes were observed in their stainedblood smears. One child of a patient with hemoglobin Hdisease was also included in this group.

NONTHALASSEMICCONTROLS

Eight normal individals, including one of Chinese andtwo of Italian extraction, and three patients with hypo-chromic anemia as a result of iron deficiency were se-lected as controls. Two of the latter patients were stud-ied while they were undergoing mild reticulocytosis dur-ing iron therapy.

MethodsHematologic values were determined by standardmethods (14).

INCUBATION

5 ml of heparinized peripheral blood to which dextrose(2 mg/ml) was added were preincubated in a 25 mlErlenmeyer flask at 370C for 10 min in a Dubnoff meta-bolic shaker. 15 Ac of uniformly labeled L-leucine-4C 1was then added. Incubation was continued for 2 additionalhr. The red blood cells were then thrice washed withcold isotonic sodium chloride. The packed red cells werehemolyzed by the method of Lingrel and Borsook (15).The hemolysate was then centrifuged at 20,000 g for 20min to remove stroma.

PREPARATIONOF GLOBIN 2

Hemolysate containing 100 mg of hemoglobin wasadded to 80 ml of 0.05 M barbital buffer, pH 8.6, whichalso contained 0.015 M 2-mercaptoethanol. The hemo-globin was precipitated by the addition of 4 ml of 0.4 Mzinc acetate and was centrifuged at 2000 g for 10 min.It was solubilized in approximately 10 ml of cold acidacetone (1 part 12 M HC1 to 90 parts acetone) to whichwas added 2-mercaptoethanol in a final concentration of0.015 mole/liter. The globin was then precipitated quan-titatively when an additional 80 ml of the acid acetonewas added. It was centrifuged at 2000 g for 10 min andthen thrice washed with cold acetone.

SEPARATIONOF ALPHA AND BETA CHAINS AND DETERMI-NATION OF THEIR SPECIFIC RADIOACTIVITIES

The method was that of Clegg, Naughton, and Weather-all (16) with minor modifications. The starting buffercontained 8 M urea,3 which had been deionized by pass-ing through a column of mixed bed resins,4 2.6 X 10 IMNa2HPO4 and 6 X 10-' dithiothreitol,5 the pH of thebuffer adjusted to 7.2 with 10% phosphoric acid. Globinwas dissolved in 2-3 ml of this buffer and then dialyzedagainst five changes of the same buffer for 2N hr. 75 mgwas then applied to a 1 X 20 cm carboxymethyl cellulosecolumn 6 previously equilibrated with the same buffer.It was eluted with a linear gradient system deliveredfrom a gradient mixing apparatus.7 The first chambercontained 150 ml of the starting buffer and the secondchamber held 150 ml of a buffer containing urea and

1 Supplied by Tracerlab Div., Laboratory For Elec-tronics, Inc., Waltham, Mass., with a specific activity of200 mc/mmole.

2 Gerald, P. S. Personal communication.3 Supplied by J. T. Baker Chemical Co., Phillipsburg,

N. J., and by Mallinckrodt Chemical Works, St. Louis,Mo.

4 Rexyn I-300, supplied by Fisher Scientific Company,Pittsburgh, Pa.

5 Supplied by Nutritional Biochemicals Corporation,Cleveland, Ohio. /

6 Whatman CM52, supplied by Reeves Angel & Co.,Clifton, N. J.

7 Contigrad, manufactured by Metaloglass, Inc., Boston,Mass.

2516 Y. W. Kan, E. Schwartz, and D. G. Nathan

dithiothreitol, at the same concentrations but with 2.8 X102 M Na2HPO4, the pH adjusted to 7.2 with H3PO4.The flow rate was regulated at approximately 0.4 ml/minwith a Technicon proportioning pump, and 6 ml fractionswere collected. The optical density at 280 mu of each ofthe collected fractions was determined in a Hitachi-Perkin-Elmer spectrophotometer. In later experiments,improved resolutions of the protein peaks were obtainedby a somewhat different elution system. Four chambersof the gradient mixer were each filled with 100 ml ofbuffer. Each contained urea and dithiothreitol as above.In the first chamber, the concentration of Na2HPO4was2.6 X 10' mole/liter, in the second and third 1.5-X 10'mole/liter and in the fourth 3.2 X 102 mole/liter. All ofthe buffers were adjusted to pH 6.7 with 10% HPO4.

In two experiments the absorptions at 280 m;L of iso-lated alpha and beta chains were compared by dissolvinga weighed amount of each chain in buffer derived fromthat fraction of the ionic strength gradient in which therespective chain emerged. The absorption at 280 mi//mgof each chain was then determined. It was found that theabsorption of beta chains exceeded that of alpha chainsby a value of 1.52, both at pH 7.2 and at pH 6.7.

Measurement of radioactivity. 1-ml aliquots of eachfraction were added to 15 ml of a counting solution ofthe following composition: toluene, methanol, ethyleneglycol monomethyl ether, and ethanolamine, 3: 3:2: 1.This contained 2,5-diphenyloxazole (PPO), 0.3 g andp-bis [2-(5-phenyloxazolyl) ]benzene (POPOP), 0.025 g/100 ml. This constituted a single phase liquid system.There was some precipitation of phosphate in the laterfractions, without effect on the counting efficiency. Theradioactivity was determined in a Packard model 3375liquid scintillation counter with 30% gain and windowsettings of 50-500 divisions. The efficiency for 14C in thissystem was 50% at 10'C. Automatic external standardcounting did not reveal variable quenching in the frac-tions. The counting error (2 SD of the count rate andbackground divided by the net count rate) was no greaterthan 5% and was usually 3%.

Specific activity was computed as counts per minuteper absorbance units (cpm/OD). The calculation wasperformed only on homogeneous fractions which immedi-ately surrounded and included the maximum optical den-sities of the separated chains. The cpm/OD of eachfraction from the same peak agreed with one anotherwithin ±5%o.

The relative rates of synthesis of the two chains werespecific activity of alpha chain

expressed as the ratiosspecific activity of beta chain

after correction for the 280 mA absorption differences ofthe two chains.

RESULTSThe hematologic values of the patients affectedwith alpha thalassemia are summarized in TableI. The patients with hemoglobin H disease weremoderately anemic; their red cells were hypo-

chromic and microcytic. The predominant altera-tion in alpha thalassemia trait was microcytosis.None of these patients was anemic, and erythro-cytosis was common. The hematologic values forthe "silent carriers" were normal. A few irregularelongated erythrocytes were apparent in stainedsmears of their peripheral blood.

Fig. 1 indicates typical separations into alphaand beta chains on carboxymethyl cellulose col-umns of the globin derived from a control subject,a patient with alpha thalassemia trait and a pa-tient with hemoglobin H disease. The identities ofthe alpha and beta chains have been confirmed byClegg and his coworkers (16) and urea starch-gelelectrophoresis 2 performed on our first few col-umns confirmed their data. Both absorbance andradioactivity are shown in these figures. Thelower specific absorption of alpha chains at 280m,u is revealed by the data derived from the nor-mal subject. This is in agreement with the dataof Clegg and coworkers (16) who also found sucha difference in absorbance. Fantoni, Bank, andMarks also confirmed the finding in globin ob-tained from mice (17), but Bank and Marks didnot find this difference in human hemoglobin ( 11 ).We believe that the difference in absorbance isbest explained by the presence of two tryptophansin the beta chain compared to one in the alphachain in human hemoglobin (18). Tryptophan hasthe highest molar extinction coefficient in theultraviolet range (19). At pH 7.2 a small peakwhich emerged just before the beta chains con-tained neither alpha nor beta chains at least asmeasured with urea starch-gel electrophoresis.The radioactivity associated with this peak wasclose to the beta chain peaks and occasionally"spilt over" to the front portion of the beta chainpeak. For uniformity in calculation, only the tubesat the peak and around the descending border ofthe beta chain peak were used for calculation ofbeta chain specific activity. The validity of thismethod of calculation was supported by the simi-larity of our results in the normal control to thosepreviously reported (10, 11).

Table I and Fig. 2 summarize the results of allof our studies of the alpha/beta specific activityratios in the control subjects and in patients withthe alpha thalassemia syndromes. In the controlgroup, the ratio was 1.02 + 0.07 with no differ-

Globin Synthesis in Alpha Thalassemia 2517

TABLE IHematologic Values and Ratios of Specific Activity

Identification Red blood cell values Subunit synthesis

Meancorpus-

Mean cularMean corpus- hemo-

Red corpus- cular globinFamily blood Hemo- Hemat- Reticu- cular hemo- concen- Ratio of specificname Subject Race* cells globin ocrit locyte volume globin ti ation activity a/,8

10'6/mm. g/100 ml % % Asa ppg/cell g/100 mlHemoglobin H disease

J. E.L. U. II 3D. A. II 4D. A. II 5D. A. 11 6M. A. II 1

Alpha thalassemia traitL.U. I IL. U. III2L. U. III3D. A. III1M.A. I IG.E. I IG.E. I 2Y.E. I 1Y.E. 1 2

C 6.28I 4.88I 5.02I 6.58I 6.08I 5.26

I 6.06I 4.98I 4.87I 5.68I 6.83C 6.35C 5.59C 6.85C 5.60

10.08.88.8

11.611.29.3

13.711.211.811.815.514.212.214.513.3

40.532.538.043.040.036.0

43.037.038.539.046.544.039.543.038.5

7.26.66.03.84.6

16.4

1.21.61.61.60.41.10.81.01.5

64.566.675.765.365.868.4

70.974.379.168.768.162.370.762.868.7

15.918.017.517.618.417.7

22.622.524.220.522.722.421.821.223.7

24.627.023.126.928.025.8

31.830.230.630.233.332.230.833.734.5

0.310.620.340.300.490.42

0.41 ( 4 0.11)t

0.700.810.780.820.690.750.770.810.80

0.77 (4 0.05)t

Silent carriersL. U. 1 2 I 5.07M. A. I 2 I 4.36D. A. I 1 I 4.45L. U. III 1 I 4.49

14.311.312.615.0

44.0 1.6 86.8 28.2 32.536.5 0.6 83.0 26.0 31.042.0 1.2 94.4 28.3 30.042.5 0.5 94.7 33.4 35.2

0.830.820.910.95

0.88 (4- 0.06)t

Nonthalassemic controls

Normal (8) and iron deficiency (3)

* C, Chinese; I, Italian.$ Mean 4 1 SD.

ences detected between normals and individualswith iron deficiency. In alpha thalassemia trait itwas 0.77 + 0.05, and in hemoglobin H disease itwas 0.41 0.11. No attempt was made to correctthe value observed in hemoglobin H disease forthe large pool of free beta chains which were notpresent in the other types of cells under study.In the four "silent carriers" the average ratio was0.88 with a range of 0.82-0.95. These results are

highly significant. The nonthalassemic controls andthe patients with alpha thalassemia trait andhemoglobin H disease showed no overlap in theirratios and could be separated into three distinctgroups (P < 0.001). The ratios obtained from thefour "silent carriers" lie just below 1 SD of thoseof the normal group and just above 1 SD of thoseof the alpha thalassemia trait group. Althoughindividually they cannot be precisely distinguished


1.02 (-4 0.07)t

a

' a-THALASSEMIATRAIT

HEMOGLOBINHDISEASE

.A3 1~~~I \

k~~~~~~

NIKX

ftJ

C0.s

k-'4.01.5

1.21

1.0!

0.8

0.6

0.4

0.2

1.0 0:.t0kI

1.5

FIGURE 2 Corrected ratios of specific activities alpha/beta in the four groups studied. Mean and 1 SD are

shown by the horizontal lines. The ratios for the foursilent carriers lie just outside 1 SD of the alpha thalas-semia trait and the control groups (dashed line).

who were so studied. These ratios corresponded tothe values which were expected from currentlyheld concepts of the mode of inheritance of thealpha thalassemia syndromes. The presumed "si-lent carrier" in generation III of family L.U. hada synthetic ratio at the low border of normalvalues. This case is discussed more thoroughlybelow.

1.0

0.5

I I5 10 IS 20 25 30 35 40 45

TUsE. NUmsER

FIGURE 1 Typical separation on a carboxymethyl cellu-lose column at pH 7.2 of alpha and beta chains from thehemolysate prepared from control, alpha thalassemia trait,and hemoglobin H patients. (Absorbancy is uncorrected.)

from normal individuals or those with alphathalassemia trait, taken as a whole the ratios ob-tained in the "silent carriers" are significantlydifferent from normal and from alpha thalassemiatrait (P = 0.01-0.001).

Family studies of the patients affected withalpha thalassemia are represented in Fig. 3. Inthis figure, the establishment of the diagnosis ofeach of the different syndromes was based onclinical and genetic criteria, and the alpha/betaspecific activity ratios are noted in those patients

DISCUSSION

The results of this study of globin synthesis inthe peripheral blood firmly support the classifica-tion of the alpha thalassemia syndromes into dis-tinct clinical and genetic entities: alpha thalassemiatrait, hydrops fetalis associated with hemoglobinBart's, hemoglobin H disease, and the "silentcarrier" syndrome. Although it is not known ifglobin chain synthesis in reticulocytes of periph-eral blood is representative of that in the bonemarrow precursors, these results clearly distin-guish the four groups of subjects studied. The lowreticulocyte counts encountered in some of thesesubjects posed a serious technical obstacle to thisstudy. This was overcome by prolonged radioactivecounting of aliquots of the widely separated pro-tein peaks in a highly stable liquid scintillationsystem.

The presence of alpha thalassemia trait is usu-ally recognized on genetic grounds (20). This

Hb H a-THAL SILENT Control-DISEASE TRAIT CARRIER

- A;&e0:

.0

I .1.

100

80

60

40

20

t4ik

Z:

'4

a

0uKc0

k

500

400

300

200

100


LU DAI

I

YEI

GE

MA

069 0.82

0.42 1.10

0.95 0.81 0.78 0.82

FIGURE 3 Pedigree of the families. The symbols represent diagnoses made on clinical and geneticgrounds. The numbers are the alpha/beta specific activity ratios obtained in this study.

diagnosis is ascribed to those patients who exhibitchanges in red blood cell morphology similar tomild beta thalassemia trait but who have normalpercentages of hemoglobins A and F and are notiron deficient. The patients are found withinfamilies in which hemoglobin H disease or hydropsfetalis occurs. They may also be recognized eitherin family studies in which interaction fails to occur

in the presence of a beta chain abnormality such as

hemoglobin S (6) or in families in which thalas-semia interacts with an alpha chain abnormality(21, 22). Evidence for deficiency of alpha chainsynthesis in alpha thalassemia trait, has been lack-ing except in the neonatal period when cord bloodconcentrations of hemoglobin Bart's may exceed5%o of the total hemoglobin (20). In the adult,precipitable hemoglobin, stainable with brilliantcresyl blue, can be demonstrated in a tiny propor-

tion of the erythrocytes of some individuals (23).This is believed to represent hemoglobin H (1,23). Despite this minimal evidence of subunit im-balance, our present studies show that a definitereduction of alpha chain synthesis by the reticulo-cytes to a level of approximately 75% of beta chainsynthesis occurs in the heterozygous state. Thoseindividuals in this study who have alpha thalas-

semia trait on clinical and genetic grounds all havesubunit synthetic ratios which confirm this diag-nosis (Fig. 3).

In hemoglobin H disease, the presence of thebeta chain tetramers clearly indicates that alphachains are deficient in the hemoglobin mixture.Our present study confirms the findings of Cleggand Weatherall who showed previously that alphachain specific activity is diminished in this syn-

drome (10, 24). Our results, and those of Cleggand Weatherall, show a wide variation in thealpha/beta specific activity ratios among patientswith hemoglobin H disease. The alpha/beta ratioin this disorder tends to underestimate the relativerates of synthesis of beta chains, since the largepool of preformed beta chains (hemoglobin H)dilutes the radioactive beta chains that are formedduring the relatively short time of incubation withleucine-14C. The observed beta specific activity isaffected by the size of this pool which may varyfrom patient to patient. Little or no influence ofunequal pool size is exerted in alpha thalassemiatrait and the "silent carrier" state, since excess

beta chains are extremely difficult if not impossi-ble to demonstrate in these disorders. Therefore,the alpha/beta specific activity ratios are more


* Hydrops Fetalis FM Silent Carrier

R Hb H Disease M Normal

° a-Thal Trait D Not Examined

L---j MR-i '411_,

exact functions of relative synthetic rates in thelatter conditions.

If hydrops fetalis associated with hemoglobinBart's represents the homozygous state of alphathalassemia, the clinically less severe hemoglobinH disease must represent a disorder intermediatein severity between the homozygous and hetero-zygous state. The concept of the "silent carrier"gene has been proposed to explain this phe-nomenon (6-9). It is allegedly carried by thehematologically normal parent of a patient withhemoglobin H disease, a disease which representsdouble heterozygosity for an alpha thalassemiagene and a "silent carrier" gene. Up to now, noevidence for a deficit in alpha chain synthesis hasbeen demonstrated when the latter gene occursalone in the "silent carrier" state. Our presentstudy of the three "silent carriers," who are thehematologically normal parents of patients affectedwith hemoglobin H disease, clarifies this issuebecause all three had very mild reduction of alphachain synthesis relative to beta chain synthesis intheir reticulocytes. Thus, as originally postulatedby Sturgeon, Jones, Bergren, and Schroeder (7),the gene for the "silent carrier" state is itselfresponsible for a degree of alpha chain suppressionand contributes to the establishment of hemoglobinH disease in an additive fashion. The reduction ofalpha chain specific activity to only 75% of betachain specific activity in alpha thalassemia traitmay result either from incomplete suppression ofthe one gene affected by alpha thalassemia or fromcompensatory increase in production by the nor-mal gene. The total suppression of alpha chainsynthesis in the homozygous state (hydropsfetalis) (2-4) supports the latter alternative. Inhemoglobin H disease, compensation by the genenot affected with alpha thalassemia is limited bythe gene responsible for the "silent carrier" state.

One individual (in generation III of familyL.U.) who was classified within the "silent car-rier" group on genetic grounds requires specialattention. He is the hematologically normal childof a patient affected with hemoglobin H disease.His two siblings both have alpha thalassemia traitsubstantiated by clinical criteria and by theiralpha/beta specific activity ratios. His specificactivity ratio is at the lower limit of normal. It isnot possible in this study to determine absolutelywhether he is normal or a "silent carrier," and this

differentiation would be necessary for completeunderstanding of the question of allelism in thisdisorder. At the present, there is no definite evi-dence to indicate whether or not the "silent car-rier" gene is allelic with the alpha thalassemiagene (8, 25). Indeed in several family studies,hemoglobin H disease has been detected in a parentand a child with the second putative parenthematologically normal (8, 26-28). This would,on the surface, favor nonallelism of the two genes.However, these data do not actually rule outallelism since it has not been possible with hereto-fore available techniques to detect the "silentcarrier" gene in the apparently normal spouse ofan individual with hemoglobin H disease. Thegene frequency for the silent carrier state is un-known. If a large group of hematologically normalchildren of hemoglobin H parents are analyzedwith the technique reported in this study and theresult compared to a large number of known"silent carriers" (viz. the normal parent of a pa-tient with hemoglobin H disease), it will be pos-sible to determine whether the two genes areallelic. If they are allelic, the result of the specificactivity ratio determinations in the two groups willbe similar. If they are not allelic, many of thehematologically normal offspring of hemoglobin Hpatients will fall within the normal range ofalpha/beta synthetic ratios.

ACKNOWLEDGMENTSWe thank Drs. Thomas Necheles, Shirley Driscoll, andJohn Craig for referring several of their patients forour studies, and Rebecca Tiedemann, Domenica Paci, andLynn Esler for technical assistance.

This work was supported by U. S. Public Health Serv-ice Grants HD 02777, HE 5255, and K3 AM35361, andby a grant from the John A. Hartford Foundation.

REFERENCES1. Weatherall, D. J. 1965. The Thalassemia Syndromes.

F. A. Davis Co., Philadelphia.2. Lie-Injo, Luan Eng. 1962. Alpha-chain thalassemia

and hydrops fetalis in Malaya: report of five cases.Blood. 20: 581.

3. Kan, Y. W., A. Allen, and L. Lowenstein. 1967.Hydrops fetalis with alpha thalassemia. New Engl. J.Med. 276: 18.

4. Nathan, D. G., P. S. Gerald, S. Driscoll, and J. M.Craig. 1966. Hydrops fetalis and erythroblastosis inhomozygous alpha thalassemia. Abstract from themeeting of the Society for Pediatric Research, Atlan-tic City, N. J.


5. Rigas, D. A., and R. D. Koler. 1961 Decreased eryth-rocyte survival in hemoglobin H disease as a resultof the abnormal properties of hemoglobin H: thebenefit of splenectomy. Blood. 18: 1.

6. Ingram, V. M., and A. 0. W. Stretton. 1959. Geneticbasis of the thalassemia diseases. Nature. 184: 1903.

7. Sturgeon, P., R. T. Jones, W. R. Bergren, and W. A.Schroeder. 1962. Observations on "Bart's" and the"Fast" hemoglobins of thalassemia-H disease. Proc.Intern. Congr. Haematol. 2:1041.

8. Wasi, P., S. Na-Nakorn, and A. Suingdumrong. 1964.Haemoglobin H in Thailand: a genetic study.Nature. 204: 907.

9. Necheles, T. F., M. Cates, R. G. Sheehan, and H. J.Meyer. 1966. Hemoglobin H disease, a family study.Blood. 28: 501.

10. Weatherall, D. J., J. B. Clegg, and M. A. Naughton.1965. Globin synthesis in thalassemia: an in vitrostudy. Nature. 208: 1061.

11. Bank, A., and P. A. Marks. 1966. Excess alpha chainsynthesis relative to beta chain synthesis in thalas-semia major and minor. Nature. 212: 1198.

12. Heywood, J. D., M. Karon, and S. Weissman. 1966.Studies of the kinetics of alpha and beta hemoglobinchain synthesis. J. Lab. Clin. Med. 67: 246.

13. Gammack, D. B., E. R. Huehns, E. M. Shooter, andP. S. Gerald. 1960. Identification of abnormal poly-peptide chain of haemoglobin G-Ib. J. Mol. Biol. 2:372.

14. Cartwright, G. E. 1964. Diagnostic Laboratory Hema-tology. Grune & Stratton, Inc., New York. 3rd edition.

15. Lingrel, J. B., and H. Borsook. 1963. A comparison ofamino acid incorporation into hemoglobin and ribo-somes of marrow erythroid cells and circulatingreticulocytes of severely anemic rabbits. Biochemistry.2: 309.

16. Clegg, J. B., M. A. Naughton, and D J. Weatherall.1965. An improved method for the characterization ofhuman haemoglobin mutants: identification of a2g2'GLU haemoglobin N (Baltimore). Nature. 207:945.

17. Fantoni, A., A. Bank, and P. A. Marks. Globin com-position and synthesis of hemoglobins in developingfetal mice erythroid cells. Science. 157: 1327.

18. Hill, R. J., W. Konigsberg, G. Guidotti, and L. C.Craig. 1962. The structure of human hemoglobin. I.The separation of the alpha and beta chains and theiramino acid composition. J. Biol. Chem. 237: 1549.

19. Greenstein, J. P., and M. Winitz. 1961. Spectropho-tometry. In Chemistry of the Amino Acids. JohnWiley & Sons, Inc., New York. 2: 1688.

20. Fessas, P. 1965. Forms of thalassemia. In AbnormalHaemoglobins in Africa. A symposium organized bythe Council for International Organizations of Medi-cal Sciences, Ibadan, 1963. Blackwell Scientific Publi-cations Ltd., Oxford. 71.

21. Atwater, J., I. R. Schwartz, A. J. Erslev, T. D. Mont-gomery, and L. M. Tocantins. 1960. Sickling oferythrocytes in a patient with thalassemia-Hemo-globin-I disease. New Engl. J. Med. 263: 1215.

22. Dormandy, K. M., S. P. Lock, and H. Lehmann. 1961.Haemoglobin Q-alpha-thalassemia. Brit. Med. J. 1:1582.

23. Fessas, P. 1959. Observations on a second haemo-globin abnormality in haemoglobin H disease. Proc.Congr. European Soc. Haematol. 1043.

24. Clegg, J. B., and D. J. Weatherall. 1967. Haemoglobinsynthesis in alpha thalassemia (haemoglobin H dis-ease). Nature. 215: 1241.

25. Koler, R. D., and D. A. Rigas. 1961. Genetics of hemo-globin H. Ann. Human Genet. 25: 95.

26. Gouttas, A., P. Fessas, H. Tsevrenis, and E. Xef-teri. 1955. Description d'une nouvelle variete d'anemiahemolytique congenitale. Sang. 26: 911.

27. Minnich, V., S. Na-Nakorn, S. Tuchinda, W. Pravit,and C. V. Moore. 1956. Inclusion body anemia inThailand (hemoglobin H-thalassemia disease). Proc.Intern. Congr. Intern. Soc. Haematol. 743.

28. Ramot, B., C. Sheba, S. Fisher, J. A. M. Ager,and H. Lehmann. 1959. Haemoglobin H disease withpersistent haemoglobin "Bart's" in an oriental Jewessand her daughter. Brit. Med. J. 2: 1228.


globin chain synthesis in the alpha thalassemia syndromes

Documents