Thalassemia: The Consequences of
`Unbalanced Hemoglobin Synthesis*
`
`DAVID G. NATHAN, M.D. and ROBERT B. GUNN, M.D.
`Boston, Massachusetts
`
`T HALASSEMIA major or Cooley's anemia is a
`
`severe, usually fatal form of inherited ane(cid:173)
`mia. Beginning approximately
`two to four
`months after birth, the disease is associated
`with profound anemia, jaundice, splenomegaly,
`expanded marrow space, siderosis and cardio(cid:173)
`megaly. The peripheral smear is characterized
`by extreme poikilocytosis, anisocytosis and aniso(cid:173)
`chromia. Target cells and nucleated erythroid
`precursors abound. Transfusion therapy is man(cid:173)
`datory in most cases. Splenectomy may be
`necessary if marked splenomegaly causes de(cid:173)
`str uction of platelets, white cells or transfused
`red cells, but the basic anemic process is usually
`unchanged by the procedure.
`the
`The devastating clinical effects and
`fascinating genetic and pathophysiologic aspects
`of this severe disease and the various "thalas(cid:173)
`[7] have led to multip le
`semia syndromes"
`probes of these disorders by geneticists, molec(cid:173)
`ular biologists and physicians. The following
`discussion of thalassemia has as its chief purpose
`a re-emphasis of the morphologic, erythro(cid:173)
`metabolic artd erythrokinetic abnormalities
`which attend the disease. Severe thalasscmia is
`less a disorder of depressed hemoglobin synthesis
`and more a disorder of unbalanced hemoglobin
`synthesis. I ndeed, it is our view that the un(cid:173)
`toward sequellae of thalassemia are less due to
`underproduction of normal hemoglobin than to
`overproduction of aberrant hemoglobin.
`To support these remarks it is necessary to
`present a brief review of the genetic basis of the
`common thalassemia disorders.
`
`The amino acid composition of these subunits
`has been established. T o each subunit a heme
`ring is attached. Three other hemoglobins are
`present in smaller amounts in normal hemoly(cid:173)
`sates. Hemoglobin F comprises less than 1 per
`cent of normal adult hemolysates and is com(cid:173)
`posed of two alpha and two gamma subunits.
`Hemoglobin A2 comprises less than 3 per cent of
`the hemoglobin of a normal hemolysate and is
`formed by two alpha and two delta chains.
`Hemoglobin A 3 (approximately 10 per cent of
`the total hemoglobin) has the same subunit
`composition as hemoglobin A and is probably a
`slightly denatured derivative of hemoglobin A.
`In addition, glutathione is bound in disulfide
`linkage probably at the site of the thiol groups of
`the Aa beta chain. A 3 is found in aged red cells
`and is considered part of the A fraction in any
`clinical or genetic analysis of hemoglobin hetero(cid:173)
`geneity. (For detailed reviews, see [7- 3].)
`In the past decade it has been recognized that
`there are four homologous pairs ofloci concerned
`with the production of the subunits of globin
`in normal human erythroblasts. These loci,
`termed alpha, beta, delta and gam1na sites may
`be jointly or severally affected by a thalassemia
`lesion which leads to reduction of the net output
`of a subunit polypeptide. For simplicity we
`refer to the term beta thalassemia to define
`suppression of beta subunit production. Thalas(cid:173)
`semia major or Cooley's anemia, for example,
`is now thought to be due to heritable reduction
`of the output of both loci governing the synthesis
`of the beta chains of hemoglobin A. Thalassemia
`minor, or Cooley's trait, is due to reduction of
`GENETICS OF HEMOGLOBIN
`the output of only one beta locus. These concepts
`SYNTHESrs AND THALASSEMIA
`have been widely accepted and provide a work(cid:173)
`ing basis for the explanation of many of the
`The major component of a normal hemolysate
`phenomena observed in thalassemia.
`(hemoglobin A) is comprised of two pairs of
`T he exact nature of the suppression of the
`homologous subunits, alpha and beta chains.
`• From the Hematology Research Laboratories of the Peter Bent Brigham Hospital and the Children's Hospital
`Medical Center, Boston, Massachusetts. This study was supported by U.S. Public Health Service Grant AM-00965,
`and the John A. Hartford Foundation, Inc.
`
`VOL. 41, NOVEMBER 1966
`
`815
`
`
`1 of 16
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1015
`
`

`

`816
`
`Thalassemia-Nathan, Gunn
`
`locus is unknown. Several
`net output of a
`possibilities exist. There may be loss of the
`section of the chromosome which bears all or
`part of the locus which is responsible for the
`encodement of the messenger RNA for one of the
`subunit polypeptides. The loss may be due to
`deletion or to crossing over. In support of the
`latter mechanism are the findings regarding the
`Lepore trait [4,5} and the hemoglobin Pylos
`syndrome [6J which are forms of beta thalas(cid:173)
`semia trait (one beta locus involved) with an
`accompanying abnormal hemoglobin. The ab(cid:173)
`normal hemoglobin is comprised of a normal
`alpha but a distinctly abnormal "beta" chain.
`The beta chain has been found to be an amal(cid:173)
`gam of pieces of beta and delta chains. A
`reasonable cause of this phenomenon would be
`crossing over with insertion of a piece of delta
`site into the midst of a beta site. On the other
`hand, were crossing over to be· the most common
`cause ofloss of beta chain production, more cases
`of the nature of Lepore trait and Pylos syndrome
`might be expected. In the past few years the
`Jacob-Monod model [7J has been invoked to
`provide a more satisfactory explanation for
`depressed beta subunit accumulation. On the
`basis of this model it is proposed that an altered
`repressor of the operator which controls the
`output of the structural beta subunit locus
`provides the basis of the disorder. Proof of such
`a model in human erythroblasts has not as yet
`been obtained. Another theory explores the
`possibility that the relatively stable messenger
`RNA [BJ of human reticulocytes might become
`unstable. A marked instability of beta chain
`messenger due to the inherited production of an
`as yet undetermined substance could be respon(cid:173)
`sible for deficient beta chain production. ltano
`[2] and Ingram and his associates [9] have
`suggested that thalassemia may be due to a
`single base substitution within a given triplet of
`the beta locus. Epstein [70] has recently exam(cid:173)
`ined the concept of a single base substitution in
`mutations, a review to which the reader is
`referred. Such a substitution would produce a
`template or messenger RNA which in turn
`would encode for the same amino acid found in
`the normal beta chain but be necessarily at(cid:173)
`tended by a transfer RNA which might be
`pr-0duced in much smaller quantity than is the
`transfer RNA necessary for the usual triplet
`sequence. This new
`template RNA . would
`oocupy space on the ribosomes and block the
`ribosomal adhesion of normal RNA. This theory
`
`is extremely attractive since it would permit
`many different sites of delay in the assembly of
`beta chains and would therefore partially ac(cid:173)
`count for
`the variable severity of Cooley's
`anemia It is of interest that normal beta chain
`production is associated with a delay point near
`the ninetieth amino acid residue whereas no
`delay points are associated with alpha subunit
`assembly [71]. On the other hand, the theory
`does not by itself account very well for the rise in
`fetal and A2 hemoglobin observed in Cooley's
`anemia. Another possible genetic lesion which
`might occur in thalassemia is specific ribosomal
`injury. Such an injury might also induce a single
`base change in messenger RNA of the type en(cid:173)
`visaged by Ingram and his associates. Strepto(cid:173)
`mycin has been shown to injure cell ribosomes
`in a fashion which alters single base sequence
`enough to lead to a "mis-sense" code and amino
`acid substitution within polypeptides [72]. The
`studies of Bank and Marks [ 13J provide no sup(cid:173)
`port for such ribosomal damage in thalassemia.
`A fundamental and fortunate accompaniment
`of the decreased beta chain production in
`Cooley's anemia is an associated rise in fetal
`hemoglobin. Several theories have been pro(cid:173)
`posed for the persistence of gamma chain pro(cid:173)
`duction in Cooley's anemia, but no satisfactory
`reason for this fortunate circumstance has been
`established. The concentration of fetal hemo(cid:173)
`globin may range from 10 to over 90 per cent of
`the total hemoglobin present in the peripheral
`blood. It is important to realize that the cellular
`distribution of this fetal hemoglobin in Cooley's
`anemia is different from that observed in the
`relatively harmless anomaly, "hereditary per(cid:173)
`sistence of fetal hemoglobin." In the former the
`fetal hemoglobin is distributed heterogeneously,
`whereas in the latter each cell has very nearly the
`same content of fetal hemoglobin.
`
`CLINICAL VARIETIES OF BE·rA THALASSEMIA
`Full expression of clinical. severity of thalas(cid:173)
`semia (thalassemia major) requires that the
`patient inherit an abnormal allele from each
`parent. In thalassemia minor only one abnormal
`allele is inherited and the disease much less
`severe. The heterozygous or trait forms of
`thalassemia are usually accompanied by moder(cid:173)
`ate hypochromia and anisocytosis. In the trait
`form of "classic" (A2) thalassemia a distinct rise
`in A2 and a small increase in F hemoglobin is
`usually observed [74]. In F thalassemia trait,
`hemoglobin F is increased and hemoglobin
`
`AMF.l<ICAN JOURNAL OP MEDICINE
`
`
`2 of 16
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1015
`
`

`

`Thalassemia-Nathan, Gunn
`
`817
`
`A2 is normal. In a few cases both are distinctly
`elevated. Splenomegaly is either mild or ab(cid:173)
`sent, and the disease is well tolerated. The red
`cell life span is normal or nearly normal (75].
`Jaundice is rare and when present [76] may
`mainly be due to ineffective erythropoiesis.
`The blood smear resembles that of iron defi(cid:173)
`ciency except that a greater degree of poikilocy(cid:173)
`tosis usually exists at a given hemoglobin level.
`Since thalassemia trait leads to suppression of
`one of the two sites of beta subunit production,
`doubly heterozygous subjects with combined
`thalassemia minor and a beta-chain hemoglobin(cid:173)
`opathy trait present a predictable picture. The
`production of normal beta subunits is markedly
`curtailed by two different abnormalities affect(cid:173)
`ing the two beta production sites. Therefore,
`little normal hemoglobin is produced. Ac(cid:173)
`cordingly, a higher proportion of the hemoglobin
`that is produced is of the beta hemoglobinopathic
`variety.
`Most cases of t halassemia major and thalas(cid:173)
`semia minor may be easily distinguished from
`each other but there is a small group of patients
`with disease of intermediate severity. For exam(cid:173)
`ple, the union of two patients with thalassemia
`minor, one with increased A2 production and
`the other with i ncreased F production, may
`produce normal children, children with either
`A2 or F
`thalassemia trait or children with
`moderate anemia, jaundice and splenomegaly
`and over 90 per cent fetal hemoglobin [77]. The
`latter group of patients seem to be double
`heterozygotes for two different beta thalassemic
`lesions. These patients often live moderately
`comfortably into adult life. In some cases parents
`with thalassemia minor of the high A2 variety
`may produce children with all the stigmata of
`thalassemia major including jaundice, sple(cid:173)
`nomegaly and moderately severe anemia. But
`for some unknown reason these children seem to
`weather the storm of growth and adolescence
`and, although
`they emerge with clear-cut
`thalassemia major, they have stable hemoglobin
`values as high as 9 to 11 gm. per cent and may
`have highly productive lives. Therefore, it
`behooves the physician to be optimistic in his
`care, with the hope that a given affected child
`will be one of the-se so-called " thalassemia inter(cid:173)
`medias." Just as the homozygous forms are
`sometimes subclassified as thalassemia major and
`intermedia, the heterozygous forms may be sub(cid:173)
`classified as thalassemia minor and minima. The
`terms are useful and self-explanatory.
`
`V O L . 41, NOVEMBER 1966
`
`DELTA AND GAMMA THALASSEMIA
`Although the terms Cooley's anemia and
`Cooley's trait have been applied principally to
`the beta thalassemia syndromes it may be con(cid:173)
`sidered that the existence of alpha and delta
`subunits of hemoglobin represents a physiologic
`form of thalassemia in man since the product of
`the delta subunit gene is formed at only one(cid:173)
`thirtieth the rate of the beta chain. On rare
`occasions complete absence of delta subunit
`production may be noted [18]. Obviously there
`are no clinical sequellae of this abnormality.
`The gradual suppression of the output of gamma
`chains observed during the maturation of the
`normal infant might be considered a form of
`gamma thalassemia.
`
`ALPHA THALASSEMIA
`Three important clinical syndromes result
`from depression of production of alpha chains.
`Alpha thalassemia trait, presumed depression of
`the net output of one of two alpha subunit sites,
`is similar in certain respects to beta thalassemia
`trait. Mild microcytosis, erythrocytosis, poikilo(cid:173)
`cytosis and hypochromia are present, with
`microcytosis usually being the most prominent
`finding. There is no enhanced production at the
`gamma or the delta site. Consequently, no in(cid:173)
`crease in A2 or F hemoglobin is detected in
`adult hemolysates. In fact the total amount of
`A2 hemoglobin may be reduced. The most im(cid:173)
`portant disorder to be differentiated from alpha
`thalassemia trait is that of iron deficiency. The
`latter is ruled out most definitively by exam(cid:173)
`ination of appropriate stains of marrow. Italians,
`Greeks, Chinese and Negroes are the most
`commonly affected groups. When combined
`with a beta hemoglobinopathy such as sickle
`hemoglobin, alpha thalassemia trait does not
`lead to an increased percentage of the abnormal
`hemoglobin in the hemolysates. In fact, the
`percentage of abnormal hemoglobin may be
`somewhat lower than that usually observed. In
`the circumstance in which it is combined with a
`hemoglobinopathy affecting the trans-alpha site
`(hemoglobin I) an increased percentage of the
`abnormal hemoglobin does occur.
`When matings of two afflicted subjects occur,
`the result of such a union may be a patient with
`homozygous. alpha thalassemia. In this fatal
`disease alpha subunit production is either mark(cid:173)
`edly curtailed or totally absent. Gamma chain
`production does occur and the resulting soluble
`hemoglobin is Bart's hemoglobin, a tetramer of
`
`
`3 of 16
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1015
`
`

`

`818
`
`Thalassemia- Nathan, Gunn
`
`gamma chains. Bart's hemoglobin has virtually
`no Bohr effect and has the additional unhappy
`ability to bind oxygen nearly irreversibly at the
`oxygen tensions of the tissues [1g], hence con(cid:173)
`ducts the function of oxygen transport extremely
`poorly. The result is that such infants are usually
`born dead or dying at approximately the thirty(cid:173)
`second week of pregnancy bearing all the evi(cid:173)
`dence of severe hydrops fetalis whether anemia
`is severe or moderate. Extreme macrocytosis,
`anisocytosis and anisochromia, with marked
`erythroblastemia, are present. The liver is huge,
`the spleen remarkably small. It should be
`pointed out that Bart's hemoglobin may be
`quite easily detected in the hemolysates of in(cid:173)
`fants with the alpha thalassemia trait, but it
`disappears when normal gamma chain pro(cid:173)
`duction virtually ceases and is not routinely
`detectable in the hemolysates of adults with
`pure alpha thalassemia trait. When highly sen(cid:173)
`sitive technics are used, Bart's hemoglobin may
`be detected in a large proportion of the hernoly(cid:173)
`sates of premature and newborn infants who
`have no familial evidence of alpha thalassemia
`[ZO]. This implies that gamma chain production
`normally outstrips alpha chain production
`during fetal life, and that the small gamma
`chain excess forms Bart's hemoglobin.
`Another form of alpha thalassemia syndrome
`is hemoglobin H disease. This disorder also
`occurs in the family setting of alpha thalassemia
`trait, but probably cannot be regarded either as
`a severe form of alpha thalassemia trait or a
`mild form of homozygous alpha thalassemia.
`The disease resembles thalassemia intermedia.
`All the signs of hemolysis are present together
`with evidence of a disorder of hemoglobin
`production. When hypochromic hemoglobin H
`blood samples are exposed to a redox agent such
`as brilliant cresyl blue, characteristic robin's
`egg blue fine precipitates appear within nearly
`all the cells. The larger cells appear to have
`more precipitates .. Starch block electrophoresis
`of hemolysates at pH 7 reveal that the hemoly(cid:173)
`sates contain an abnormally rapid fraction which
`may include from 5 to 25 per cent of the total
`and which has been shown to be comprised of
`beta subunit tetramers. This so-called hemo(cid:173)
`globin H has several unusual properties. Like
`hemoglobin Bart's, it is a useless respiratory
`pigment. Its lack of Bohr effect and its high
`affinity for oxygen inhibits its capacity to
`deliver oxygen to the tissues at physiologic pH or
`oxygen tension [27 ]. Therefore, as noted by
`
`[22], hemoglobin H
`traverses the
`Gabuzda
`circulation "forever in unhappy union" with
`oxygen. H emoglobin H is an unstable tetramer
`and is readily oxidized within the red cell. It
`therefore precipitates during the life span of the
`cell. As it precipitates, it apparently binds
`glutathione to its thiol groups in a final embrace
`du mort [22]. The results of the intracellu lar
`precipitation of such a substantial quantity of
`hemoglobin are large intracellular Heinz bodies
`which may be most easily observed in the pe(cid:173)
`ripheral blood of splenectomized patients with
`hemoglobin H disease. The relationship of
`Heinz body formation to hemolytic anemia and
`hemoglobin H disease will be discussed sub(cid:173)
`sequently.
`The mode of inheritance of hemoglobin H
`disease is not at all clear. One of the parents of
`the patient usually has alpha thalassemia trait
`and the other appears normal. The apparently
`normal parent seems to carry a defect which
`permits the expression of hemoglobin H in a
`child who otherwise might have only alpha
`thalassemia trait. The defect may be one which
`depresses the output of the trans-alpha site, or
`increases the output of the unaffected beta sites.
`Hemoglohin H disea.<;t>, tht>n, is a form of alpha
`thalassemia in which the imbalance between
`alpha and beta subunit production is greater
`than that observed in ordinary alpha thalassemia
`trait. Hemoglobin H may be detected after
`exposure to brilliant cresyl blue in a small
`percentage of the erythrocytes of patients with
`ordinary alpha thalassemia trait. In fact, the
`detection of these cells constitutes a major clue
`in establishment of the diagnosis of alpha
`. thalassemia trait. Presumably these rare cells are
`the progeny of erythroblasts which were so
`seriously affected by a gross imbalance of beta
`and alpha production that the excessive beta
`chains form the tetramer hemoglobin H.
`The forms of alpha thalassemia serve as a
`convenient and constructive example of a con(cid:173)
`cept of the pa tho physiology of thalassemia which
`this review intends to illustrate. The basis of this
`concept is the central role of unbalanced subunit
`production in the pathogenesis of these dis(cid:173)
`orders; unbalanced production in which the
`subunit synthesized in excess is as important if
`not more important in the pathogenesis of
`disease than is the underproduced subunit. To
`refine this illustration we must return to a
`morphologic and erythrokinetic evaluation of
`Cooley's anemia.
`
`AMERICAN JOURNAL OF MEDIC I NE
`
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`4 of 16
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1015
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`

`

`Thalassemia-Nathan, Gunn
`
`819
`
`MORPHOLOGY AND ERYTHROKINETICS
`TN COOLEY'S ANEMIA
`
`The appearance of the peripheral smear in
`Cooley's anemia depends upon the presence or
`absence of the spleen. When the spleen is intact,
`there are numerous tear-drop forms. (Fig. 1 and
`2.) The red cells are heterogeneous with respect
`to hemoglobin content and size. Large pale
`cells and small dense cells may be observed
`together with intermediate forms. Nucleated
`red cells are usually present in small numbers.
`In wet preparations viewed with Nomarski
`optics, the tear-drop cells may be very striking,
`and contrast sharply with the cells observed
`following splencctomy. (Fig. 2.) The acid elution
`technics of Betke and Kleihauer [23], which
`define the cellular content of fetal hemoglobin,
`are somewhat confusing in cases of Cooley's
`anemia . Just as the peripheral smear stained with
`Romanowsky dyes shows marked heterogeneity
`of hemoglobin distribution so does the Betke
`preparation. (Fig. 3.) As a result, it may be sur(cid:173)
`mised that some cells contain more fetal hemo(cid:173)
`globin tha n others. Incubation of the Cooley's
`anemia erythrocytes of patients with intact
`spleens in 1 per cent methyl violet at 37°c for
`10 minutes reveals few cells containing Heinz
`bodies.
`Measurements of the autologous life span of
`Cooley's anemia erythrocytes in patients whose
`spleens are intact usually reveal biphasic curves.
`C~1 survival studies may be more impressive
`than C14-glycine technics in revealing the fact
`
`Fie. 1. Peripheral smear of a pa tient with Cooley's
`anemia a nd an intact spleen. Tear-drop forms are evident
`as well as anisochromia and target cells. Note the tear(cid:173)
`d rop form with a vacuole a t its t ip. (See text .)
`
`that there is a population of cells which is
`rapidly removed from
`the circulation and
`another with a much longer life span [24]. The
`glycine-2-C14 studies do reveal, however, that
`the survival of hemoglobin A is considerably
`shorter than that of hemoglobin F [25]. There(cid:173)
`fore, the heterogeneous distribution of hemo(cid:173)
`globin F in thalassemic cells has a functional
`significance, and the cells rich in hemoglobin
`enjoy a more favorable survival.
`In splenectomized pa tients the morphology of
`the peripheral blood in Cooley's anemia
`is
`markedly different from that observed in non(cid:173)
`splcnectomized patients. Tear-drop formation
`is not as impressive. Instead, there is an increase
`
`F10. 2. Nomarski optics view of the cells of two patients with Cooley's anemia; one with an intact spleen (lejt) and
`the other splencctomized (right ). Note the tear -dr op formation in the pa tient with an intact spleen and the crater al
`distortions in the splenectomized patient.
`
`VOL . 41, NOVEMBER 1966
`
`
`5 of 16
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1015
`
`

`

`820
`
`Thalassemia- Nathan, Gunn
`
`(Fig. 5.) The Heinz bodies are more irregularly
`shaped and larger than those observed following
`incubation of normal or glucose-6-phosphatase
`dchydrogenase (G6PD)-deficient cells in phenyl(cid:173)
`hydrazine. Examination of wet preparations of
`the cells with Nomarski optics (27) shows that
`the cells have large crateral distortions which
`may be either indentations of the surface or
`vacuolar distortions. (Fig. 2.) Electronmicro(cid:173)
`graphs of shadow casts of the cells confirm that
`the surface is indeed irregular and indented
`either by vacuoles into which the membrane
`invaginates, or by actual pits on the surface.
`(Fig. 6.) Sections of the cells (F ig. 7) ,examined
`with the electron microscope also reveal surface
`inclu(cid:173)
`indentations, vacuole formations and
`sions of precipitated hemoglobin and degen(cid:173)
`erated mitochondria. Vacuole formation may
`also be observed in splenectomized patients
`with other forms of hemolytic anemia and
`reticulocytosis [28] .
`Studies of the survival of the hemoglobins in
`a splenectomized thalassemic subject have re(cid:173)
`vealed an extremely rapid turnover of a pool of
`hemoglobin A. In one case, it was shown that
`this pool represented as much as 50 per cent of
`the total circulating pool of hemoglobin A and
`that the turnover of the pool proceeded with a
`half-time of one day. The turnover of hemo(cid:173)
`globin F was considerably slower [25]. Studies
`of total heme turnover with glycine-2-C14 in
`splenectomized patients have also demonstrated
`a component undergoing rapid, early destruc(cid:173)
`tion [29J. Thus, the morphology and erythro(cid:173)
`kinetics of Cooley's anemia suggest an hetero(cid:173)
`geneous distribution of hemoglobins A and F
`and further suggest that the large spleen of
`these patients specifically sequesters those wide,
`pale cells which contain Heinz bodies and
`crateral membrane distortions but which possess
`little hemoglobin F.
`These studies lend further documentation to
`the theory tha t the cells that are poor in hemo(cid:173)
`globin F are more liable to destruction. The
`following data further support this concept.
`
`MORPHOLOGY AND METABOLISM
`OF CENTRIFUGED COOLEY'S
`ANEMIA ERYTHROCYTES
`When the blood of splenectomized thalas(cid:173)
`semic patients is centrifuged at 15,000 r.p.m.
`for 1 hour, the top and bottom 10 per cent of
`erythrocytes are found to be quite different in
`character. The upper layer cells are predomi-
`
`AMERICA N JOURNAL OF MEDICINE
`
`Fm. 3. Betke-Kleihauer fetal hemoglobin stain of the
`blood of a splenectomized patient with Cooley's anemia.
`T he cells rich in feta l hemoglobin arc darkly stained.
`T he tcchnic a lso stains inclusion bodies. 1 t is clear that
`the largest inclusions are found in the cells with the least
`fetal hemoglobin.
`
`in wide, hypochromic erythrocytes together
`with small piscine forms which are little more
`than fragments of stroma. (Fig. 4.) The degree
`of anisochromia is markedly increased, as is the
`number of nucleated red cells. The reticuloc yte
`count is elevated to a lesser extent than in other
`there may be
`hemolytic anemias, whereas
`marked increases in the nucleated red cell count.
`Incu bation of such cells in 1 per cent methyl
`violet for 10 minutes reveals that many of the
`wide pale cells contain large Heinz bodies [26}.
`
`FIG. 4. Peripheral smear of a splcncetomized patient
`with Cooley's anemia. Tear-drop cells are not as evident.
`There is marked adsochromia and a nisocyt~is.
`
`
`6 of 16
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1015
`
`

`

`Thalassemia-Nathan, Gunn
`
`821
`
`F10. 5. Top {left) and bottom (right ) layers of the centrifuged blood of a splenectomized
`patient with Cooley's anemia. The top layer cells ( 1 and 2) are larger and more hypo(cid:173)
`chromic. They also contain larger irregular Heinz bodies and larger erateral indentations
`than the bottom layer cells (3,4).
`
`F10. 6. Platinum carbon shadow cast of the membra ne of the spleneetomized patient (same
`as in Fig. 5). Note the numerous surface indentations.
`
`VOL . 41, NOVEMBl. R. 1 966
`
`...
`
`
`7 of 16
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1015
`
`

`

`822
`
`Thalassemia-Nathan, Gunn
`
`F10. 7. Electron micrograph of a section of glutaraldehyde fixed erythrocytes of a splenec·
`tomized patient with Cooley's anemia. Note surface indentations, degenerated mito(cid:173)
`chondria in vacuoles and Heinz bodies.
`
`nantly newly formed erythrocytes. Reticulocytes
`are present in much higher proportion in the
`upper layer. If Fe69 is administered to the patient
`one week before the centrifugation study, nearly
`all the radioactivity is fou nd in the upper layer.
`In time the radioactivity leaves the upper layer
`
`and is found nearer the lower layer of cells. The
`morphology and hemoglobin composition of the
`upper and lower layers also differ. The volume
`and diameter of the upper layer of cells is much
`greater. Heinz bodies and siderotic granules are
`larger and more frequent. (Fig. 5.) The hemo·
`
`TABLE I
`.RESULTS OF 2 H OUR INCUBATIONS OF 1'H ALASSEMIC, H EMOGLOBIN H , ABNOR MAL AND MYELOPl8ROTIC BLOOD IN AUTOLOOOUS
`PLASMA AT 37 °c. WJTll ADDED GLUCOSE AND AT pH 7.5
`
`Reticulo· H emoglobin Hemoglobin
`H
`F
`cytcs
`(3)
`( %)
`(%)
`
`Glucose
`Consumption
`(mM / L. cells/ hr.)
`
`Potassium Flux
`(mEq.jL. cells/hr.) ATP Content
`l n
`O ut
`( µM / L. cells)
`Net
`
`Erythrocyte
`
`Thalassemia
`Whole blood
`Top layer
`Bottom layer
`Hemoglobin H
`Whole blood
`Top layer
`Bottom layer
`Normal
`M yelofibrosis
`
`10
`3
`
`12
`3
`
`35
`20
`50
`
`12
`25
`6
`
`5 . 5
`7
`3
`
`4 .0
`3
`3.5
`1. 7
`3
`
`4
`7
`3
`
`3
`3
`5
`1. 7
`2.5
`
`5
`9
`3.5
`
`-1
`-2
`-0 .5
`
`-0.5
`3.5
`0
`3
`-1.0
`6
`0
`1. 7
`2 . 3 +0 . 2
`
`946
`1 ,040
`800
`
`1,200
`1 , 400
`900
`1 , 500
`1 , 850
`
`NOTE: The thalassemic and hemoglobin H blood samples were centrifuged at 15,000 r.p.m. for 1 hour and the
`top and b ottom 10 per cent of cells were suspended in a utologous plasma for similar studies. An aliquot was retained
`for starch block or 1 minute alkali denaturation estimates of hemoglobin H or F. The myelofibrotic blood is included
`as a control because this particular specimen of blood was from a splcncctomized patient with many nucleated red
`cells. The p a tient was also iron d eficient and the mean corp uscular volume and mean corpuscular hemoglobin closely
`approximated the blood of the patients with thalassemia and hemoglobin H disease.
`
`AMERI CAN J OURNAL OF MEOICINI!
`
`
`8 of 16
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1015
`
`

`

`Thalassemia-Nathan, Gunn
`
`:
`
`globin content of cells in the upper layer is
`lower than that in the bottom layer and crateral
`distortions viewed with Nomarski optics appear
`much larger a nd more frequent. (Fig. 5.)
`Fetal hemoglobin content is much lower than
`it is in the small dense cells in the bottom layer.
`(Table l.)
`When cells from the upper and lower layers
`are incubated separately in vitro, the membrane
`defect of the cells in the upper layer, those with
`less hemoglobin and increased size and number
`of Heinz bodies, crateral membrane distortions
`and siderotic granules, is very obvious. The flux
`of K 42 across such cells may be extraordinarily
`high. Glycolysis and lactate formation arc
`excessive. ATP content is low and often unstable.
`(Table l)
`The smaller cells of the bottom layer, those
`with more hemoglobin (much of it fetal hemo(cid:173)
`globin), and smaller and fewer Heinz bodies,
`crateral distortions and sidcrocytes, have a much
`more normal Au:x of potassium an~ a lower
`glycolytic rate. ATP levels, however, are lower
`than in the cells of the upper layer. T hese
`data suggest that the cells with an ability to form
`ga mma chains have a higher degree of mem(cid:173)
`brane integrity than do the cells which have
`more limited gamma chain production. I t may
`also be suggested that cells rich in fetal hemo(cid:173)
`globin are smaller than F-poor cells because t he
`former divide more frequently in the marrow
`a nd that the larger volume of the F-poor cells
`is largely comprised of water.
`
`PATHOGENESIS OF HEMOLYSIS IN
`COOLEY'S ANEMIA-AN HYPOTHESIS
`Fessas [26) has postulated that the Heinz
`bodies in Cooley's anemia stem from the un(cid:173)
`balanced production of alpha chains in cells
`lacking sufficient beta chain production to com(cid:173)
`bine with the alpha subunits. Since unpaired
`alpha subunits are extremely unstable, it seems
`reasonable that they might precipitate and
`become the antecedents to the Heinz bodies. A
`logical extension of this argument is that the
`Heinz bo<:lics may contribute to membrane
`damage and hemolysis by some mechanism as
`yet undefined and, furthermore,
`that ceUs
`capable of increased gamma chain production
`sunrive more favorably because they contain
`smaller and fewer Heinz bodies due to the
`gamma chains which provide a combining site
`or trap for excessive alpha chains by forming
`fetal hemoglobin. (Fig. 3.)
`
`V O L. 41 , NOVE M BE R 1 966
`
`_,
`'
`
`823
`
`'
`
`F'IC. 8. Methyl violet prep aration of a bone
`marrow aspirate of a child with Cooley's anemia.
`No Heinz bodies were detectable in the peripheral
`blood. Note the large dark inclusions one of which
`may be seen extruding from an opening in the
`cell.
`
`\¥hen the spleen remains in situ it removes
`Heinz bodies from the cells [30). In fact, some of
`the splenic hypertrophy observed may be due to
`its constant phagocytosisofHeinz bodies (37 ). I n
`such patients Heinz bodies arc seen only rarely
`in peripheral smears, although many of the
`bone marrow erythroblasts and some marrow
`erythrocytes are full of these inclusions. (Fig. 8.)
`The Heinz body-laden erythrocytes which. do
`escape from t he marrow are quickly engulfed by
`the spleen. The splenie littoral cells may seize
`them at a projecting point on the cell surface
`under which lies the Heinz body (32). The
`erythrocyte may be stretched into a tear-drop
`shape while it is firmly ancho