Download Mutations of the Red Blood Cell Membrane Proteins

REVIEW ARTICLE
Mutations of the Red Blood Cell Membrane Proteins: From Clinical Evaluation
to Detection of the Underlying Genetic Defect
By Jiri Palek and Kenneth E. Sahr
S
E V E W RECENT publications have reviewed the
principal structural features of the membrane proteins, the organization of their genes, and the recently
described membrane protein
The objective of
this review is to apply this rapidly expanding knowledge to
the understanding of the pathobiology of red blood cell
(RBC) lesions and the detection of the underlying membrane protein defects in inherited disorders of the RBC
membrane proteins.
MACROMOLECULAR ORGANIZATION OF THE MEMBRANE
SKELETON: IMPLICATIONS FOR MEMBRANE STABILITY,
DEFORMABILITY, AND CELL SHAPE
Electron microscopic images of uniformly extended membrane skeletal preparations show a relatively uniform network of hexagons with occasional pentagons and heptagons
(Fig 1). The hexagon corners contain oligomeric actin
together with proteins 4.1 and adducin, which bind to both
spectrin and actin, thereby facilitating contact between the
two proteins, and protein 4.9.1° The hexagon arms are
composed of fibers of spectrin tetramers (SpT) and, occasionally, double-stranded tetramers or higher oligomers.
Near the center of the SpT, where the two a.p spectrin
heterodimers (SpD) assemble head to head to form SpT,
the SpT are decorated by small globular structures representing ankyrin, the principal attachment site of the skeleton to the major transmembrane protein band 3, the anion
exchange protein."
Destabilization of the lipid bilayer, microvesiculatwn, and
spherocytosk. In the intact erythrocyte membrane, the
skeletal network is in a "condensed" configuration with the
spectrin subunits randomly folded in a close apposition12
(Fig 2). The skeletal proteins cover approximately 60% of
the inner membrane surface and bind weakly to the lipids of
the inner leaflet of the bilayer.14-18 Consequently, the
skeleton confers stability to the fluid lipid bilayer. A
decreased density of the skeletal network, such as seen in
spectrin-deficient RBCs in hereditary spherocytosis (HS),
is thought to facilitate uncoupling of parts of the lipid
bilayer from the underlying skeleton (Fig 2). An enhanced
lipid bilayer-skeleton uncoupling in HS RBCs is readily
demonstrable under a variety of conditions in vitro.' It is
From the Department of Biomedical Research and the Diviswn of
HematologylOncology, St Elizabeth's Hospital of Boston, Tufs University School of Medicine, Boston, MA.
Submitted July 9,1991; accepted April 7,1992.
Supported by National Institutes of Health Grants No. HL-37462
and HL-27215.
Address reprint requests to Jiri Palek, MD, Department of Biomedical Research, St Elizabeth's Hospital of Boston, 736 Cambridge St,
Boston, MA 02135.
0 1992 by The American Society of Hematology.
0006-4971l92/8002-OO31$3.00l0
308
also likely to take place in vivo, thus providing a plausible
explanation for the surface area deficiency observed in
hereditary spherocytosis.'
Membrane fragmentation. The condensed configuration
of the skeleton in intact cells also underlies the high degree
of cell membrane deformability by permitting a considerable extension of the skeletal network followed by elastic
recovery of the biconcave shape after the deforming force
has been discontinued (Fig 3). An example is the transformation of RBCs into extended ellipsoids induced by a shear
~ t r e s s . ' ~Such
J ~ elongated ellipsoids resume a biconcave
shape after the shear stress has ceased. They fragment only
if the length of the deformed ellipsoids exceeds the length
of the maximally unidirectionally extended membrane skeleton. In subjects with hereditary elliptocytosis (HE) carrying a. spectrin or p spectrin mutations that weaken the
SpD-SpD contact, the hexagonal skeletal lattice is disrupted (Fig 4), leading to RBC fragmentation and poikilocytosis.1,20In severely affected subjects, such as homozygotes or double heterozygotes for one or two spectrin
mutations, or patients with hereditary pyropoikilocytosis
(HPP; see below), fragmentation is readily seen during
examination of the peripheral blood film.' In mildly affected subjects (ie, simple heterozygotes carrying a mildly
dysfunctional spectrin) marked RBC fragmentation is seen
only in conjunction with another factor such as a mechanical insult to the cells, exemplified by disseminated intravascular coagulation,2l or another unrelated skeletal defect.
For example, neonates carrying elliptocytogenic spectrin
mutations have a considerably more severe hemolysis,
fragmentation, and poikilocytosisthan a d ~ l t sbecause
~ ~ , ~of~
the additive effect of a weakened spectrin-protein 4.1
contact induced by increased concentrations of free 2,3diphosphoglycerate; the elevation of this compound is due
to its weak binding to fetal h e m o g l ~ b i n ? ~ - ~ ~
Plastic deformation and formation of elliptocytes. When
normal RBCs are deformed for prolonged periods of time,
the proteins of the deformed skeleton undergo an active
rearrangement involvinga disconnection of existing proteinprotein contacts and a formation of new associations that
permanently stabilize the cells in the deformed shapeZ7sz
(Fig 3). It is possible that the elliptical shape of HE RBCs is
a consequence of such permanent plastic deformation.
Cells that flow through microcirculation or cells subjected
to shear stress have either a parachute-like or elliptical
~ h a p e ~ in
~ .HE,
~ ~ ;such shape may become permanent
because the weakened protein-protein contacts (such as the
weakened SpD-SpD association) facilitate skeletal reorganization.28 Indeed, HE-nucleated RBC precursors are
round' and the elliptical deformation progressively increases with increase in cell density and, presumably, cell
age.31
Blood, VOI 80, NO2 (July 15). 1992: pp 308-330
RED BLOOD CELL MEMBRANE PROTEIN MUTATIONS
309
-
. ...
i
A
Fig 1. RBC membrane skeleton prepared by removing membrane lipids and integral proteins by
extraction with Triton-X 100. The skeleton has lmen
artificially extended so that the individual structural
components can be seen. Low magnification (a)
shows a highly ordered, relatively uniform structure.
High magnification images (b through e) show that
this network is assembled into hexagons (b and c) or
heptagons and pentagons (d and e). The arms of each
polygon are formed by spectrin tetramers (Sp4) and,
occasionally, double tetramers (2Sp4) or hexamers
(Sp6). as highlighted In (b) and (c). The tetramer arms
are interconnected by junctional complexes containing actin and protein 4.1. In the middle of the SpT are
globular structures representing ankyrin (b and c).
This composite figure is compiled in part from previously published work’with permission.
BIOGENESIS OF THE RBC MEMBRANE SKELETON:
RELEVANCE TO CLINICAL EXPRESSION OF MEMBRANE
PROTEIN MUTATIONS
Whereas the synthesis of both spectrin and ankyrin can
be readily detected at the earliest stages of erythroid
differentiation,3*-” only a small fraction of the newly
synthesized spectrin and ankyrin is assembled on the RBC
membrane, and the membrane-associated spectrin and
ankyrin turn over rapidly. At the proerythroblast stage,
band 3 synthesis is initiatcd and, together with the synthesis
of the protein 4.1, increases up to the late erythroblast
stage?2.3s.MDuring this time, the messenger RNA (mRNA)
C
levels and the synthesis of spectrin and ankyrin decline. In a
striking contrast, the fraction of the newly assembled
spcctrin and ankyrin on the membrane and their net
amounts progressively
and the turnover of these
proteins on the membrane decline^.^"^.^ This paradoxical
finding is related to a progressive increase in the synthesis
of band 3 and 4.1 proteins that serve as the principle
membrane anchors of the s k e l e t ~ n . ~ ~ . ~ ~ , ~
An important feature of membrane skeletal biogenesis,
common to both mammalian and avian RBCs. is a seemingly wasteful synthesis of both spectrin and ankyrin and, to
a lesser extent, the 4.1 protein, with a relatively small
310
PALEK AND SAHR
Fig 2 Ulhmuctun of the
membrane skeleton in intact normal RBC ghosts (a) and in ghosts
from a patient with severe HS
associated with a deficiency of
spectrin (b). Hemoglobin-free
ghosts were sonicated t o open a
"window" that allows the visualization of the intact skeleton at
the inner membrane surface. The
ghosts were fired and negatively
stained. The skeleton is preferentially stained under these conditions. In ghosts from normal
RBCs (a), the individual skeletal
proteins are in close apposition
t o each other, forming a monomolecular layer. In contrast, the
HS skeleton (b) is considerably
less dense with the individual
proteins separated by skeletonfree areas. (c) Separation of the
lipid bilayer from the underlying
skeleton in normal RBC ghosts.
RBC ghosts were suspended in
hypotonic sodium phosphate
buffer, and, after restoring isotonicity, w e n examined after 10
minutes of incubation. Arrows at
the neck region of the spicules
show the separation of the membrane lipidsfrom the filamentous
skeleton. Arrowheads indicate
the vesicles released from the
echinocytic ghosts. (c) deplctl
this process in normal cells; the
release of such vesicles is enhanced in HS.'Z Compiled from
previously published work',"
with permission.
fraction of thc ncwly synthcsizcd a spcctrin, f3 spcctrin, and
ankyrin asscmblcd into thc stablc nctwork.3L3H-'"Particularly striking arc thc diffcrcnccs in thc synthcsis of a
spcctrin and p spcctrin subunits,"' with thc synthesis of a
spcctrin bcing about four timcsgrcater than thc synthesisof
p spcctrin. Howcvcr. thc two chains assemble on thc
mcmbranc in stoichiomctric amounts as stablc hctcrodimcrs via a high-affinity binding of p spectrin to
ankyrin. Thus, thc amount of spcctrin asscmblcd on thc
membrane is principally dctcrmincd by the amounts of
ncwly synthcsizcd p spcctrin and thc numbcr of availablc
ankyrin binding sitcs. Band 3 protcin, thc prcdominant
RBC transmembrane protein, appears to determine the
ovcrall stoichiometry of the components of the membrane
skeleton by providing a high-affinity binding site for
a n k ~ r i n . ~Thc
. ~ ' intcraction behvcen ankyrin and band 3
proteins also appears to be the key determinant of the
pcriphcral localization of the membrane skeIet0n.~3~
The
cxccssivc a spcctrin synthesis may explain why a spectrin
dcfccts that lcad to a reduced synthesis of a spectrin are
clinically apparcnt only when prcscnt in a homozygous or
doubly heterozygous form, such as in recessively inherited
HS associatcd with a severe spectrin defi~iency.~.~'
In a
simplc hetcrozygous carricr of such a defect, thc amount of
RED BLOOD CELL MEMBRANE PROTEIN MUTATIONS
311
In situ
Extended
( lipid-free membrane skeleton)
(intact membrane)
A
Flg 3. Uniaxialelastlc deformation without distention. (A) A schematic diagram of an isolated membrane skeleton that can be uniformly extended t o
cover a surface about seven times larger than the
surface of the normal RBC membrane. In the intact
membrane, the hydrophobicity of the lipid bilayer
precludes an Increase in membrane surface area
(~3%
t o 4’/0) without rupture. However, the membrane can undergo a large deformation under a
constant area because of the large exstensibility of
the membrane skeleton. (6) During uniaxial extension, the skeleton undergoes an isometric uniaxial
stretching from a square t o a long rectangle. After a
cessation of an external force, a square surface area
is resumed because the elastic skeletal network
remains intact, retaining a memory. (C) Prolonged
uniaxial extension leads t o a skeletal protein rearrangement, resulting in a formation of new bonds.
This leads t o a permanent plastic deformation. A
weakening of protein-protein contacts (particularly
the SpD-SpD contacts), such as found in common HE,
facilitates skeletal protein rearrangement, thus permanently stabilizing the cells in elliptical shape,
which is similar t o a shape of RBCs deformed by a
shear force.
-
Uniaxial skeletal extension
y= 1
y = 0.5
B
x = l
x = 2
Extension is no lonaer evident.
Skeletal protein reahangement
produced a permanent (plastic)
deformation.
y=l
C
x = l
x = 2
(3) abnormalities involving defective protein-protein intcractions. This last group includes abnormal spectrin-protein
4.1 interaction in HS,XZ.x3a rcduccd binding of ankyrin to
the ankyrin stripped inside-out membrane
a
OVERVIEW OF MOLECULAR DEFECTS OF THE RBC
defect that was later found in association with and, possibly,
MEMBRANE PROTEINS
secondary to spectrin a1/6s
mutationx5(see below), and
defects Characterized by reduced assembly of SpD to SpT
In analogy to other inherited defects, the number of
(Table 1).
detected membrane protein mutations does not coincide
The latter group of mutations, involving defective selfwith their natural prevalence.4? The detection of these
association of SpD to SpT, represent the most common
mutations is facilitated by their hematologic presentation
group of elliptocytogcnicmutations. This functional defect
and the underlying abnormality of membrane protein
is readily dctcctcd by estimating the relative amounts of
composition and function. Consequently, three groups of
SpD and SpT in spectrin extracts by nondenaturing gel
defects are readily detected either by polyacrylamide gel
electrophoresis.”.”.~ In the majority of patients, these
electrophoresis (PAGE) of membrane proteins solubilized
abnormal spectrins can be further characterized by limited
is sodium dodecyl sulfate (SDS) or by protein association
tryptic digestion of spectrin: some of the tryptic peptides, as
studies (Fig 5): (1) in HS moderate to severe deficiencies of
by two dimcnsional electrophoresis, corrcspond to
the proteins spectrin,u4 ankyrir1,4~anion e x c h a n g ~ r , ~ . ~resolved
~
distinct structural and functional domains of sp~ctrin!~.~
protein 4.2,sh56 and, in HE, protein 4.1 defi~iency~’-~;
(2)
Among these peptides, the 80-Kd a1 domain peptide,
defects characterized by an abnormal protein migration,
such as faster migrating a spectrins6’.“ and f3 ~pectrins~~7-~’
representing the SpD self-association site of a spectrin, is
(most ofwhich are associated with HE and exhibit defective
the most prominent peptide. Abnormal digestion of this
self-association of SpD to SpT), a fast migrating ankyrin in
peptide, resulting either from increased susceptibility of the
HS,74 and a fast or a slow migrating 4.1 protein in HE62-7s’x1; existing cleavage sites to proteolysis or a formation of new
the newly synthesized normal a spectrin, produced by the
single normal a spectrin allele, may still exceed the total
amount of the newly synthesized f3 spectrin.
312
PALEK AND SAHR
Fig 4. Representative electron micrographs of uniformlyspread membrane skeletons from normal RBCs and cells from patients with HS, HE,
and HPP. (a) Normal membrane skeleton showing a primarily hexagonal lattice. (b) Skeleton from HS RBCs with a mild rpechin deficiency. The
membrane skeletal network appears to be only slightly less intact than in normal cells. (c) Membrane skeleton from a patient with mild
heterozygous HE Sp d'", containing dysfunctional spectrin but having normal spectrin content. The integrity of the skeleton is moderately
disrupted because of the reduced ability of SpD to self-associate into SpT. (d) Membrane skeletonfrom a patient with HPP. HPP RBCs are not only
spectrin-deficient but they also contain dysfunctional spectrin, causing a defective self-association of spectrin dimers into tetramers. Note a
striking disruption of the skeleton with a complete loss of skeletal lattice. The magnification of all micrographs is identical. (Adapted from a
previous publication for the author's laboratorymwith permission.)
cleavage sites, has been detected in the majority of patients
with weakened SpD-SpT self-association.'.'2.2~ Several underlying DNA and primary structure defects have been
subsequently detected in the vicinity of the abnormal
cleavage site (Table 1). These mutations are designated
according to size of the largest peptide derived from the
abnormal a1
Below, we summarize the major
structural, functional, and clinical features of the welldefined mutations of the membrane proteins.
Specrrin
mururions. This group of elliptocytogenic
spectrin mutations is associated with the formation of a
74-Kd peptide resulting from cleavage of arginine or lysine
at positions 45 and 48, respectively'3.w.y1
(see Table 1 for
the numbering of amino acids, as based on the cDNA
sequence). The Lys 48 cleavage site is present in normal
spcctrin?' The normal 80-Kd peptide strongly inhibits SpD
self-association; the 74-Kd peptide derived from normal
spectrin by a more extensivc proteolysis has lost this
function,Rgimplying that the critical site involved in SpDSpD self-association is within the first 48 amino acids,
largely residing in the first helix of a spectrin. This helical
segment has bcen proposed to be a part of a combined a,p
spcctrin triple helical unit representing the SpD-SpD contact site4? the first two helices are contributed by the
C-terminus of f3 spectrin, while the helix three is the first
helical segment of a spectrin (Fig 5). The Sp
mutations
are of considerable interest because they appear to be the
most common membrane protein mutations reported to
date, occurring in people of diverse ethnic backgrounds,
including whites, blacks, Arabs, and Melanesian~.'.~~
Therefore, it is not surprising that many different mutations are
associated with this phenotype (Table 1).
Two of these mutations are of particular interest: (1) the
mutations of codon 28, representing the most common
group of mutations detected to date, involve a substitution
of arginine 28 by either histidine, cysteine, leucine, or
serincw.y3.w;
(2) the mutations of p spectrin that lead to a
destabilization of the 48 lysine cleavage site of a spectrin
that gives rise to the 74-Kd p e p t i d ~ . 4 ~
Detection
. ~ ~ - ~ ~of one
of these mutations provided a basis for the above noted
model of spectrin self-association in which the last repeat
unit of p spectrin lies in direct apposition to the first helix of
a spectrin.4' Consequently, mutations within this region
lead to a destabilization of the lysine 48 cleavage site (giving
rise to the 74-Kd peptide) and a marked weakening of the
SpD-SpD self-association.
It is also of interest that in the majority, but not in all of
these mutations, the self-association of spectrin is severely
impaired and the patients have a symptomatic hemolytic
anemia. This is particularly the case in the f3 spectrin
mutations leading to the Sp
p h e n ~ t y p e ~and,
~ . ~ "to a
lesser extent, mutations involving a spectrin codon 28.9'
However, additional presently unknown genetic factors
may modify clinical expression of these mutations: in the
case of the 28 Arg to His mutation, a simple heterozygous
state for this mutation may present either as a dominantly
inherited elliptocytic hemolytic anemia or as an asymptomatic carrier state, an offspring of which has HPP.9'
RED BLOOD CELL MEMBRANE PROTEiN MUTATiONS
IC
313
t
NH2 ,COOH
B
N
Fig 5. A schematic diagram illustrating molecular assembly of membrane skeleton and the approximate location of the molecular defects in
HS, HE, and HPP. Known skeletal protein deficiencies in HS and HE include those of spectrin, ankyrin (both in HS), protein 4.1, and glycophorin C
(both in HE). The principalskeletal protein dysfunctions (mutations)include (1) defects of the head region of either a or p spectrin, which lead to a
defective self-association of SpD into tetramers (arrows, all in HE and/or HPP); (2) defective p spectrin, which binds poorly to protein 4.1 (in HS);
and (3) abnormal protein 4.1, which binds weakly to spectrin, thereby destabilizing spectrin4.1-actin interaction (in HE). (A) A schematic
illustration of the skeletal protein constituentsand their assembly. Spectrin is composed of two chains, a and p, that are twisted along each other
into heterodimers (SpD). At the head region (arrows), SpD are assembled into tetramers (SpT). At their distal end, SpT binds to the junctional
complexes of oligomeric actin with the aid of protein 4.1 and adducin. Spectrin is attached to transmembrane proteins by linkage of p spectrin to
ankyrin (band 2.1). which binds to the cytoplasmic domain of the anion transport protein. Protein4.2 is likely to be located at this site as well. (B)
Schematic illustration of the protein constituentsof the junctional complex. Each SpT end is attached to oligomeric actin by the spectrin binding
protein 4.1 and by adducin that binds to two SpT ends. Actin oligomers are stabilized by tropomyosin that lies in the groove of F-actin. Other
actin-associated proteins include dematin (an actin-binding protein) and myosin (not shown). The 4.1 protein also binds to glycophorinC. (C) The
model of spectrin structure. The u and p chains of spectrin are arranged in an antiparallel fashion with the N-terminus of u chain and the
C-terminus of the p chain forming the SpD-SpD contact site. Both spectrin chains are composed of multiple repeat units. The open blocks are
highly homologous; the repeat 10 (stippled)of a spectrin is nonhomologous. The repeat units' structure breaks down at the distal end of spectrin
tetramers, sharing homology with analogous segments of a actinin and dystrophin (solid rectangles).mal ID) Model of the contact site between
the u and p spectrin chains of the opposite heterodimer. Each repeat unit is composed of three u helical segments, connected by short nonhelical
regions. The proposed contact site is a combined triple helical unit containing2 u helical segments from repeat 17 of the C-terminus of p spectrin
(hatched area) and the first u helical segment of u spectrin near the N4erminus.e Mutations of 0 and p spectrin involving a defective assembly of
SpD to SpT are the most common membrane protein defects reported to date (Table 1). The abnormal cleavage sites giving rise to the most
commonly seen abnormal tryptic peptides in these mutationsare shown by solid lines; the sites of the mutations, most of which are in the vicinity
of the cleavage sites, are designated. Mutations of p spectrin giving rise to a truncated protein are also depicted in this diagram. They are
spectrins LePuy.'23 Gottingen.126 Rouen.'z4 Tandil," and Nice.'a (Adapted and reprinted with permission.l2u3)
The mutations involving a spectrin codons 46 and 49
appear to be associated with a milder clinical expression,91,99although the percentage of unassembled SpD in
the crude spectrin extract is increased from approximately
6% (control value) to values exceeding 50% of the SpD +
SpT p 0 0 1 ? ~ ,The
~ reason for this discrepancy between the
apparently severe spectrin dysfunction and the relatively
mild clinical presentation is not known. The high percentage of SpD could be an artifact of extractions8; in contrast
to the normal SpD self-association, which is kinetically
immobilized at a temperature of near 0°C (the temperature
at which spectrin is extracted from the membrane),lOOJO1
the mutant spectrin self-association may not be kinetically
frozen at this temperature, thus permitting some of the
mutant SpT to dissociate into SpD during the low ionic
strength extraction.
Spectrin a1/78
mutations. Although rare, these mutations
are of interest because they highlight the complexity of
SpD-SpD
contact
Lower molecular weight
and reduced phosphorylation
p Sp Gottingen SpD-SpD
p Sp Rouen
Lower molecular weight
and absent phosphorylation
SpD-SpD
contact
p Sp Nice
contact
Lower molecular weight
and absent phosphorylation
SpD-SpD
contact
Lower molecular weight
and absent phosphorylation
Four abnormal spots (31.
26,21.18 Kd) and a reciprocal decrease of four
normal a11 peptides)
78-Kd peptide cleaved at al
16 Lys
$ Sp LePuy
p Spectrin
Sp all
Not defined
SpD-SpD
contact and another unknown defect
sp a11131
(Sp Jendouba)
SpD-SpD
Sp all*
Truncated C terminus due to
absence of sequences of
exon Y and a premature
chain termination
GAT
G to T substitution (+3) in the 5’ splice site
causing skipping of exon Y and aframeshift with a premature chain termination
T to A substitution in the 5’ splice site following exon X leading to deletion of
exon X cDNA sequences and a frameshift leading to premature termination of
translation
AG duplication at codons 2045/2046 in
axon X leading to frameshift and premature chain termination
A to G mutation in +4 position of the 5’
donor consensus splice site of the intron
following exon X, leading to deletion of
exon X cDNA sequences and a frame
shift leading to premature termination of
translation
GCT
(Continuedon following page)
Truncated C terminus due to
absence of sequences encoded by exon X and a premature chain termination
Truncated C terminus due to
premature chain termination
Truncated C terminus due to
absence of sequences encoded by exon X and a premature chain termination
all Ala + Arg in repeat a9
all 748 Asp + Glu in repeat a7
GAC + GAA
45 AGG + AGT in exon 2 of a Sp
a145 Arg + Ser in repeat a1
41 CGG + TGG in exon 2 of a Sp
-
46 GGT + G l T in exon 2 of a Sp
49 (37- I T in exon 2 of a Sp
48 AAG + AGG in exon 2 of a Sp
28 CGT TGT in exon 2 of a Sp
28 CGT + C l T in exon 2 of a Sp
28 CGT + AGT in exon 2 of a Sp
28 CGT + CAT in exon 2 of a Sp
2053 GCT CCT in exon X of p Sp
a1 28 Arg Cys in repeat a1
a1 28 Arg Leu in repeat a1
aI 28 Arg Ser in repeat a1
a 128 Arg -* His in repeat al
pl2053 Ala + Pro in repeat
1317
a1 41 Arg Trp in repeat a1
a1 46 Gly
Val in repeat a1
aI 49 Leu -* Phe in repeat a1
aI 48 Lys Arg in repeat al
---
74-Kd peptide cleaved at aI
48 Lys andlor aI 45 Arg
SpD-SpD
contact
a1 471 Glu + Pro in repeat a5
a1469 His + Arg in repeat a5
50-Kd peptide cleaved at a1
468 Arg or 470 Arg
SpD-SpD
contact
Sp a1/-b
Sp
471 CAG -* CCG in exon 11 of a Sp
469 CAT -* CGT in exon 11 of a Sp
a1 260 Leu + Pro in repeat a3
a1 261 Ser + Pro in repeat a3
aI 207 Leu +Pro in repeat a2
50-Kd peptide cleaved at aI
256 Arg o r a l 258 Lys
SpD-SpD
contact
Sp allm
-
260 CTG + CCG in exon 6 of a Sp
261 T C + CCC in exon 6 of a Sp
CTG CCG in exon 5 of a Sp
aI 154 Leu duplication in repeat a2
154 T G duplication in exon 4 of a Sp
DNA Defect
65-Kd peptide cleaved at al
137 Arg
Primary
Structure.
SpD-SpD
contact
Function
Sp
a Spectrin
Provisional
Designation
Protein Defect
Limited Proteolysis
or Other Study
Mild to moderately
severe common
HE
Asymptomatic
carrier
Mild common HE
Mild HE
Autosomal recessive HS
HPP
A rare group of p
spectrin mutations
Probably rare
A common group of
a Sp mutations,
heterogeneousat
the primary structure and DNA levels, found in
whites, blacks, arabs, and melanesians
124
123-126
41,119
120
102,103
90.91.93.94
104,107,112
Apparently rare
Asymptomatic HPP
carrier or mild to
moderately severe
common HE
104,107,115
Relativelycommon in
blacks, heterogeneous at the primary structure1
DNA levels
HPP
Asymptomatic HPP
carrier or mild,
common HE
References
Relatively common in 98,104-107
blacks, homogeneous, at protein
and DNA levels
Prevalence
and Comments
HE of moderate
severity
Heterozygote
Double
Heterozygote
or Homozygote
Clinical Expression
Mild HE
Table 1. Membrane Protein Mutations in HS, HE, and HPP
W
$
x
Fm
0
P
2
Incompletely studied
Incompletely
studied
Membrane instability
resulting from
weakened specwin-actin interaction
Truncated C terminus due to
7-bp insertion causing a
frameshift and premature
termination
Primary
Structure*
-
Absence of glycophorin C
Partial deficiency of the 4.1
band
Deletion of exons 3 and 4 resulting in
frameshift and premature stop codon
TGGCCG +lTGCG in codons 44.45 resulting in frame shift and a stop codon (nucleotides 166-168)
Mutations in codons 44 and
45
Asymptomatic
Mild HE
Mild common HE
Deletion of three exons (including the
erythroid initiation codon) precluding
mRNA translation
Probably rare
About 10% of HE
cases in North Africa the molecular
basis of the defect
may be heterogeneous, probably
rare
Probably rare
Mild common HE
240 nucleotide deletion spanning the
codons for Lys 407-Gly 486 due to deletion of two exons encoding the spectrin
binding
Rare
Probablyabout 10%
of kindred with autosomal dominant
HS
Prevalence
and Comments
Probably rare
HE with severe hemolysis
Double
Heterozygote
or Homozygote
Mild common HE
HS of moderate severity
Dominantly inherited
HS
Heterozygote
369 nucleotide duplication of codons for
Lys 407-Glu 529 due to a duplication of
three exons encoding the spectrin binding domain
Deletion of one copy of ankyrin gene due
to heterozygousdeletion of chromosome8[de1(8) ( p l l - p Z l . l ) ]
TGG + CGG
7-bp deletion in exon X leading to a frameshift and premature chain termination
DNA Defect
Deletion of exon 3 and 4
15-Kd insertion adjacentto
New 44-Kd and 60-Kd chymotryptic peptides with
the binding domain
the same isoelectric point
as the normal 34-and
50-Kd peptides
New -40-Kd chymotryptic Lack of the entire spectrin
binding domain
peptides presumably resulting from fusion of
peptides adjacent to the
C and N terminus of the
deleted sequence
About 40% decrease in
ankyrin and spectrin content
Abnormal digestion pattern Trp + Arg substitution in POsition 202 of p Sp
of p Sp N-terminus
Lower molecular weight
and absent phosphorylation
Limited Proteolysis
or Other Study
Clinical Expression
161
154-156
80.81
80.81
139
130
73
References
Putative mutations, which are yet to be characterized at the DNA level, are not included. They are tabulated in Palek and Lambert.' Asymptomatic variants are not included in this table.
*Note that the numbering of amino acids and the respective codons of a spectrin are based on the cDNA sequence.220 thus including the first six amino acids that were not detected by protein
sequence.92 The repeat numbering of (Y and B spectrin proteins are based on the protein sequences derived from cDNA sequencing.220~227
Glycophorin C
deficiency
Glycophorin C
4.1 deficiency
Low molecular Membrane instability
weight 4.1
resulting from
(4.168'ffi)
weakened spectrin-actin interaction
High molecular weight
4.1 (4.1")
4.1 protein
Ankyrin deficiency
Reduced binding of
spectrin to the
membrane leading
to a secondary deficiency of spectrin
Sp-4.1 contact, formation of Sp-4.1actin complex
p Sp-4.1
Ankyrin
SpD-SpD
contact
Function
p Sp Tandil
Provisional
Designation
Protein Defect
Table 1. Membrane Protein Mutations in HS, HE, and HPP (Cont'd)
4z
0
3
-u
m
g
m
2
8
Rr
r
5
U
m
n
316
PALEK AND SAHR
regulation of the SpD-SpD self-association as well as the
an A to G substitution in position 3 of codon 362, creating a
susceptibility of the spectrin a1 domain to proteolysis. The
5' splice site that produces an inframe splicing out of
cleavage site giving rise to the 78-Kd peptide involves a
codons 363 to 371. As in the majority of other spectrin
mutations, the change involves helix 3. Several other
spectrin lysine 16 in helix 3 of the combined cup spectrin
defects of spectrin, such as the spectrin a1/61
repeat unit.102J03 However, the mutations are 25 and 29
117and spectrin
amino acids downstream at positions 41 or 45, the latter
a1/43118
have been reported, but the underlying primary
being next to one of the amino acid mutations that give rise
structure and DNA defects remain to be defined. The
spectrin a1/61,first detected in a doubly heterozygous
to the Sp a1/74
phenotype?l
Spectrin a1165 mutation. This HE mutation is unique
individual for this particular mutation, and spectrin ~ $
among the spectrin mutations in that it is homogeneous
illustrate some of the technical limitations of two-dimenboth at the protein and DNA levels, being confined to the
sional tryptic peptide mapping. Because the amount of the
black race?8J04-107This feature is of interest in light of
abnormal 61-Kd peptide is very small, the defect is difficult
to discern on Coumassie blue-stained gels and it can be
recent reports of the relatively high prevalence (0.6%) of
HE in equatorial Africa.lo8 Furthermore, RBCs containing
detected only on Western blots with polyclonal antibodies
mutant spectrin are somewhat resistant to invasion by
directed against the a1 domain.
malaria and exhibit a reduced parasite growthl@J'lO;
thus, it
Mutations of the spectrin dZ domain. These rare mutais possible that this mutant spectrin and, possibly, other
tions have been detected because of an abnormal migration
similar spectrin mutations provide some degree of protecof the a11 domain peptides as shown by two-dimensional
tion to malaria parasite invasion. The Sp a1/65
mutation
tryptic peptide mapping.41J19-122In contrast to the a1
appears to be the mildest among all a1 domain mutations:
domain mutations, in which the disease pathobiology can
the spectrin SpD self-association is only mildly impaired
be explained by the disruption of the SpD-SpD selfand the clinical expression is very mild; even homozygous
association, the link between the a11 mutations and disease
subjects carrying this mutation have a relatively mild
pathobiology is not clear. For example, the mutation
hemolytic anemia that does not require s p l e n e ~ t o m y . ~ ~ J ~ ~involving helix 3 of the repeat 7 of a spectrin is expressed as
Spectrin
mutations. The cleavage sites leading to
a mild dominantly inherited HE with a mild increase in SpD
the generation of the 50-Kd tryptic peptide involves either
in the crude 0°C spectrin extract," while a replacement of
256 arginine or 258 lysine due to a1260 Leu + Pro or a1261
alanine (GCT) codon by arginine (GAT) in the ninth
Ser + Pro substitutions, r e s p e c t i ~ e l y . ~This
~ J ~relatively
~
repeat unit of spectrin is asymptomatic in a simple heterozycommon mutation should be distinguished from a rare
gous state; in a homozygous form it is associated with a
mutation that is a consequence of either 471 Glu --f Pro
severe recessively inherited ~pherocytosis.4~J~~
In another
substitution or 469 His + Arg substitution in the fourth
defect, the abnormality was located in the C-terminus of the
repeat unit (the Sp a1/50b mutation).104J07J12
The group of
a11 domain.lZ1The underlying genetic defect involves an
Sp a1/50a
mutations appears identical to defects designated
inframe skipping of exon 18, due to G to A substitution in
as Sp a1I6 by another l a b ~ r a t o r y , the
~ ~ differences
~J~~
in the
the acceptor splice site of intron 17 (ag/GA + aa/GA).
size of the peptides reflecting the difference in estimation of
The ensuing protein is shortened by 41 amino acids,
the apparent molecular mass. Although apparently conproducing an abnormal tryptic cleavage site after Arg 890 in
fined to the black population, the Sp a1/46defects are
helix 3 of repeat a9 (J. Delauney et al, personal communicaheterogeneou~.~~
OfJ ~particular
~ J ~ ~ interest is a recently
tion, 1992). The phenotype is that of recessively inherited
reported Leu + Pro mutation,l15 the site of which is at a
severe hemolytic anemia with microspherocytes, poikiloconsiderable distance from the cleavage site, residing 51
cytes, thermal instability of RBCs, and a mild defect in SpD
amino acid to the N-terminus in helix 2 of the second repeat
self-association. Although a11 domain defects may have
unit, thereby destabilizing the cleavage site located in the
some influence on the SpD-SpD self-association, it is
apposed helix 3.
apparent that the underlying pathobiology may be related
It is of interest that in the spectrin mutants discussed
to other mechanisms that are yet to be defined.
above, most of the amino acid substitutions are located in
Mutations involving the C-terminus of p spectrin. With
the exception of the p spectrin defects leading to increased
helix 3 of a given repeat unit, near the connecting segment
among the individual repeats. Thus, this region of the
susceptibility of the lysine 48 of a spectrin to proteolysis
mutations, see above), all known p spectrin
spectrin molecule is important in regulating spectrin func(the Sp
tion.
mutations have been identified on the basis of their increased mobility on SDS-PAGE.67-73
Some of the shortened
Other d domain mutations. While the abnormal spectrins giving rise to abnormal a1peptides of 74,65, and 50 Kd
p spectrins also enhance the susceptibility to proteolysis of
(the Sp a1/74,
Sp
and Sp (YI/~O, respectively) appear to
the 48 lysine of a spectrin, thus causing increased formation
represent the most common group of spectrin mutations,
of the 74-Kd peptide derived from a ~ p e c t r i n In
. ~ ~most of
several other defects have been reported, all leading to a
the reported truncated p spectrins, the underlying defect
defect of SpD-SpD self-association and all creating new
has now been defined. In three of them, it involves skipping
tryptic cleavage sites. In spectrin Sfax (Sp
the first
of an exon located near the 3' end of the p spectrin gene
reported mutation of repeat 014, the abnormal 36-Kd
(designated exon X or exon Y), due to mutations of the 5'
fragment starts at Ala 357 and contains a deletion involving
donor consensus splice site sequence of the intron following
amino acid 363 to 371.116 This deletion is a consequence of
the particular exon; the resulting mRNA lacking sequences
1
~
~
RED BLOOD CELL MEMBRANE PROTEIN MUTATIONS
from exon X or Y contains a frameshift and premature stop
~ o d o n . In~ two
~ ~other
- ~ ~reports, the truncated p spectrin
is a result of a premature chain termination because of a
change in the reading frame due either to a 2-bp insertion
in codon 2O46lz6or a 7-bp deletion following codon 2041.73
These mutations resulting in a truncated p spectrin
protein also result in a decreased phosphorylation of the
C-terminus of p spectrin. Indeed, in spectrin LEPuY,'~~
spectrin Gottigen,lZ and spectrin
phosphorylation
is absent. This group of mutations is also characterized by
defective SpD self-association. Spectrin Rouen is of special
interest in this regard because it lacks only the very
C-terminal64 residues of p spectrin that are not part of the
combined ap triple helical structure, suggesting that this
sequence is also involved in SpD self-association.
Mutations of the pIVdomain of spectrin. This domain of
p spectrin is involved in interaction with protein 4.1.loJz7A
recently reported mutation underlies a markedly weakened
binding of spectrin to protein 4.1. It is associated with a
clinical phenotype of dominantly inherited HS with some
acanthocytes on the peripheral blood film.82J28The same
functional defect of p spectrin was previously reported by
another
but the underlying mutation has not been
yet defined. The pathophysiologic mechanism whereby this
mutation leads to the HS phenotype is unclear. For unexplained reasons, these patients are also partially deficient in
spectrin as are nearly all other patients with HS.lZyAs
discussed above in the section on the macromolecular
assembly of the skeleton, the spectrin deficiency is likely
responsible for the HS phenotype in these subjects. HS cells
carrying this defect are less mechanically stable than cells
from other HS patients30; this is possibly related to the
weakened p spectrin-4.1 interaction. In contrast, a disruption of the spectrin-protein 4.1 interaction due to a reciprocal defect involving the spectrin binding domain of the 4.1
protein leads to a phenotype of mild to moderately severe
elliptocytosis (see below). Because the abnormal function
of the above p spectrin mutant is normalized by reducing
agents in vitro,130it is possible that in circulating RBCs the
p Sp-protein 4.1 binding is near normal owing to the
relatively high concentrations of reducing compounds in
the cells.
Spectrin polymorphisms. Tryptic peptide mapping has
shown several asymptomatic variants involving the aII, aIII,
and a V domains of a spectrin, as well as the pIV domain of
p ~ p e c t r i n . ' , ~InJ ~the
~ common a11 domain polymorphism,
characterized by four distinct phenotypes of different size
and isolectric point,132the underlying genetic basis is four
haplotypes involving substitutions of Arg 701 to His, Ile 809
to Val, and Thr 853 to Arg in exons 6, 17, and 18,
re~pective1y.l~~
Additional reported polymorphisms should
be useful in linkage studies attempting to compare such
polymorphism with the disease ~ h e n o t y p e . ' ~ ~ - ' ~ ~
Deficiency of ankyrin. In contrast to the rapidly increasing list of the mutations of spectrin, our knowledge of the
molecular defects of human ankyrin is incomplete. The
observation that the deficiency of ankyrin may represent
the primary defect underlying spectrin deficiency in inherited spherocytosis was first made in spherocytic mice
317
carrying the nb m ~ t a t i 0 n . lThe
~ ~ description of ankyrin
deficiency in humans followed.46Subsequent studies of one
of the originally reported subjects showed a profound
decrease of ankyrin synthesis due to an as yet unclear defect
leading to a marked decrease in ankyrin mRNA expression.
Consequently, assembly of spectrin on the membrane is
reduced despite normal spectrin synthesis.138Furthermore,
a complete deletion of the ankyrin gene resulting from an
interstitial deletion within a short arm of chromosome 8 is
associated with the phenotype of moderately severe HS,139
thereby explaining the known association between HS and
abnormalities of chromosome 8.140-14 Furthermore, recent
studies of linkage of the inheritance of HS with a restriction
fragment length polymorphism (RFLP) of the ankyrin gene
implicate ankyrin as the primary molecular lesion in a large
HS kindred.145 It is possible, but is yet to be firmly
established, that ankyrin deficiency in dominantly inherited
HS is quite common.146J47
Abnormally migrating ankyrin. Recently, a fast migrating
ankyrin (Ankyrin Prague)74 was detected due to a yet
undefined defect within the regulatory domain of ankyrin; it
is dominantly coinherited with mild HS. Recent cloning of
the ankyrin C D N A , ~ ~progress
* J ~ ~ in characterization of the
detection of a new
spectrin binding site of ankyrin,5J50-152
ankyrin gene polymorphism,153as well as new advances in
the detection of DNA defects are likely to open this
potentially exciting field.
Deficiency of 4.1 protein. Partial deficiency of the 4.1
protein, although rare in the United States, has been
detected more frequently in HE subjects in Southern
France and North A f r i ~ a . ~In~ a, simple
~ ~ , ~heterozygous
~~
state, it is expressed as a mild, common HE; the homozygous state is associated with a severe hemolytic anemia that
is partially corrected by splenectomy.6°The molecular basis
of 4.1 deficiency is heterogeneous. Detailed studies of the
index case showed a deletion of 318 nucleotides that
included the translation initiation site that gives rise to the
major erythroid 4.1
In a recent study (N.D.
Venezia and J. Delaunay, personal communication), a
Spanish patient with homozygous 4.1(-) HE was found to
have a point mutation in the downstream erythroid initiation codon (AUG -+ AGG), resulting in a complete absence of RBC 4.1 protein. Although the protein 4.1 gene is
expressed in erythroid and nonerythroid tissues, protein 4.1
deficiency in these individuals is erythroid specific.lS6This is
related to the fact that the protein 4.1 pre-mRNA is subject
to extensive alternative splicing that affects the function of
the protein157J58and that nonerythroid tissues express high
molecular weight isoforms through the use of an upstream
5' translation initiation site that is not affected by the above
noted mutations. In another kindred, protein 4.1 mRNA
was lacking approximately 2 kb at the 5' end, possibly
reflecting aberrant mRNA sp1i~ing.l~~
Abnormally migrating 4.1 protein. Two other protein 4.1
defects have been reported, each involving the spectrin
binding domain of the protein as shown by enzymatic and
chemical digestion of the 4.1 protein and amplification of
appropriate regions of the CDNA.~O,~~
In an elongated form
of 4.1 protein (95 Kd), the Lys 407 -+ Glu 529 sequence
318
PALEK AND SAHR
spanning the spectrin binding domain is duplicated due to a
Plasmodium falciparum m e r o z o i t e ~ , ~and
~ ~the
J ~ Ge
~ Gerbich blood group phenotype is common in certain parts of
duplication of three consecutive exons of the protein 4.1
malaria infested M e l a n e ~ i a . ' ~ ~ , ' ~ ~
gene?' In a shortened 4.1 protein, the Lys 407 + Glu 486
Protein 4.2 defects. Many case reports of patients having
sequence of the spectrin binding domain is deleted as a
either a reduced or absent 4.2 protein have been reresult of a deletion of two adjacent exons of the 4.1 protein
gene.s1As expected, the patient with the shortened protein
p ~ r t e d ,but
~ ~the
- ~molecular
~
basis of the defect is unknown
and the function of the 4.2 protein is not fully understood.'
4.1 had a more severe hemolytic anemia than the patient
In two reports, protein 4.2 deficiency appeared to result
with a high molecular 4.1 protein, presumably because the
latter protein retains some if not most of its ability to bind p
from a defective binding of the 4.2 protein to the band 3
p r ~ t e i n , ' ~as~evidenced
J~
by a weak binding of the normal
spectrin. A second shortened 4.1 variant (4.1 Presles) has
been described." This defect appears to result from a
4.2 protein to inside out membrane vesicles from the
preferential splicing of protein 4.1 pre-mRNA to generate a
patient. In one of these cases, the underlying genetic defect
relatively abundant RBC transcript lacking motif I1 (S.
was reported to represent substitution of 327 Pro by Arg in
Feddal and J. Delaunay, personal communication, 1992).
a highly conserved region of the band 3 protein.181 In
Other abnormalities of the 4.1 protein, characterized by
another report, a mutation of the 4.2 protein involving
abnormal mobility, have been reported,' but detailed studcodon 142 from GCT (Ala) to ACT (Thr) was detected.ls2
ies of the molecular basis of these defects are not available.
However, it remains to be established whether this mutaDeficiency of glycophorin C. Deficiencies of each of the
tion is coinherited with protein 4.2 deficiency, thus causing
RBC membrane glycoproteins have been reported.' Gethe clinical phenotype of hemolytic anemia with spheroovalnetic abnormalities of glycophorins A and B, the genes of
ocytes.
which are physically linked on the q28-q31 region of
Defects of the anion exchanger. While several functional
chromosome 8, including deficiencies of both glycophorin A
and structural defects of the anion exchanger have been
and B and the formation of fusion proteins, are clinically
reported, only a few have been characterized at the level of
asymptomatic.
primary structure and DNA. Two interesting defects have
Glycophorin C deficiency is clinically expressed as a mild,
been found in association with acanthocytosis. In one,
recessively inherited HE with no hemolysis and mild
associated with hereditary acanthocytosis without obvious
e1liptocytosis.l The molecular basis of the deficiency is
hemolysis, band 3 had a larger molecular mass, a restricted
heterogeneous, including deletions of two exons (exons 3
rotational diffusion, and a decreased number of highand 4) and alterations leading to a frameshift and premaaffinity binding sites for ankyrin.ls3At the structural level,
ture codon termination.161J62
Except for elliptocytosis, these
two-dimensional peptide mapping showed changes in a
subjects are clinically asymptomatic. RBC membrane vis17-Kd anion transport segment consistent with the addition
coelastic properties, as measured by micropipette, are
of tyrosines or tyrosine-containing peptides. A second
normal,31 suggesting that the diminished deformability of
defect, reported in a patient with choreacanthocytosis
glycophorin C-deficient elliptocytes during shear, as desyndrome, involved structural abnormalities of the C-termitected by the ectocytometer, is likely to reflect abnormal
nus of the anion exchange protein without obvious funcflow of elliptocytes in the shear stress field rather than
tional alterations.1s4 However, the linkage of these two
changes of intrinsic membrane mechanical proper tie^.^'
abnormalities with the inheritance acanthocytosis is yet to
Deficiency of glycophorin C is also accompanied by the loss
be established, and the contribution of these abnormalities
of the expression of all antigens of the Gerbich blood group
to the acanthocytic shape is likewise unknown.
(Ge:l, Ge:2 and Ge:3), the so called Leach p h e n ~ t y p e . * ~ ~ J ~A partial deficiency of the band 3 protein has recently
In contrast, the loss of expression of only one or two Ge
been detected in a subset of patients with dominantly
inherited HS.48,49The deficiency is linked with the inheriantigens (Ge-2,+3, the Yus phenotype, and Ge-2,-3, the
tance of HS. The nature of the defect remains unknown,
Gerbich phenotype) is asymptomatic because these RBCs
carry an abnormal glycophorin C-related glycoprotein.164-168 but RFLP linkage studies in one large family assigned the
primary defect to the band 3 gene.185The synthesis of band
The underlying genetic defect involves a deletion of 2 exons
3 protein has been studied in erythroblasts of one patient
(exons 3 and 4) in the Leach phenotype or a deletion of one
and was found normal.ls6 In several patients, separation of
of the duplicated domains encoded by exon 3 (the Gerbich
RBCs on density gradients showed that band 3 deficiency is
phenotype) or exon 2 (the Yus p h e n ~ t y p e ) . ' ~ ~ - ' ~ ~
Several in vitro studies have suggested that both glycomore profound in the high-density
Although the
phorin A and glycophorin C provide an attachment point of
increase in RBC density may not be directly proportional to
increasing RBC age,187 the data, nevertheless, raise a
the skeletal protein 4.1 to the membrane.173J74The experiments of nature described above, however, show that
possibility of a release of the band 3 protein from the
complete deficiency of glycophorin A in vivo has no
membrane of circulating RBCs, possibly resulting from
apparent effects on cell function. Likewise, complete defiweakened binding of the band 3 protein to ankyrin. This
ciency of glycophorin C, although producing a mild elliptolatter defect may reside in the ankyrin binding domain of
cytosis, does not lead to significant alterations of membrane
the cytoplasmic segment of the band 3 protein or, conproperties?l On the other hand, both glycophorin A and
versely, the band 3 binding domain of ankyrin.
glycophorin C may be involved in the attachment of
Several groups have recently detected abnormalities of
RED BLOOD CELL MEMBRANE PROTEIN MUTATIONS
319
ASSESSMENT OF RBC MORPHOLOGY, MEMBRANE
the band 3 protein in RBCs in Southeast Asian ovalocytosis
PROTEIN ANALYSIS, AND DETECTION OF THE
(SAO),188-192a condition highly prevalent in malaria inUNDERLYING GEN ETlC DEFECT
fected regions of Malaysia, the Philippines, and Papua New
Guinea. S A 0 RBCs are rigid, resist invasion by various
When we attempt to define the underlying molecular
strains of malaria parasites, and exhibit reduced agglutindefect in a patient with HE, HPP, or HS, we begin with a
ability to antisera against several blood group a n t i g e n ~ . l ~ ~ - lcareful
~~
assessment of RBC morphology. In a patient
The underlying genetic defect involves an intra-exon delecontaining elliptocytes on the peripheral blood film, it is
tion of codons 400-408 at the boundary of the cytoplasmic
essential that a clinical diagnosis of HE be unequivocally
segment and the first transmembrane segment of the band 3
established (1) by finding of an autosomal dominant pattern
p r ~ t e i n . ~This
~ ~ mutation
, ’ ~ ~ is tightly linked with a substituof inheritance in the family, and (2) by excluding one of the
tion of lysine 56 by glutamic acid.189The latter mutation
common causes of acquired elliptocytosis, including iron
and B12 deficiencies, myelodysplasia, myofibrosis, and other
alters the mobility of the cytoplasmic segment of the band 3
conditions.1~22~23
The percentage of elliptocytes or the deprotein or its proteolytic fragments and was originally
gree of elliptical deformation as shown by elliptocyte length
designated band 3
One group reported
that the ovalocyte band 3 contains an altered N - t e r m i n ~ s . ’ ~ ~to width ratio, previously used as one of the diagnostic
criteria, have no clinical use, as the underlying defect can be
However, this is contrary to the recent sequencing data
detected even in patients with elliptocytes not exceeding
indicating that, except for the 56 Lys + Glu mutation, the
20% (normal range, 0% to 5%).’ The second step is to
cDNA encoding the first 110 amino acids, including 150 bp
categorize
HE into one of the previously described morphoof the 5’ untranslated sequence is n01mal.l~~
Furthermore,
logical
subtypes1J3:
(1) common HE, characterized by
the sequence of the “extended” terminus192is, with the
biconcave elliptocytes, rod-shaped cells, and a varying
exception of one amino acid, homologous to the sequence
number of poikilocytes; (2) HPP, a disorder closely related
of a region of the ring-infected erythrocyte surface antigen
to
HE characterized by a striking micropoikilocytosis and
(RESA),lWsuggesting that the sequence of the “extended
microspherocytosis;
(3) spherocytic HE, a phenotypic hyN terminus” is derived from a protein other than band 3.
brid
of
HE
and
HS;
and
(4),stomatocytic elliptocytosis, also
S A 0 band 3 also exhibits a markedly reduced lateral
called
Southeast
Asian
or
Melanesian ovalocytosis (Fig 6).
mobility in the membrane.’%Ym It is also excessively phosCommon
hereditary
elliptocytosis.
In common HE, the
ph0rylated~~~,~01;
it is presently unknown whether or not this
underlying defect can be detected in about 60% to 70% of
abnormality is responsible for the abnormal function of the
cases by using the following screening approaches (Fig 7).
band 3 protein. The membrane domain of S A 0 band 3 also
The first step involves a standard SDS-PAGE, which in
appears to have an altered structure based on its abnormal
about 10% of cases may show a partial deficiency or an
binding of some anion transport inhibitors, and to have a
absence of the 4.1 protein, or abnormally migrating 4.1, a
reduced capacity for anion transportJo2 The mechanism
spectrin, or p spectrin proteins. The second step involves an
whereby this mutation leads to increased membrane rigidity
electrophoretic separation of SpD and SpT from the crude
and malaria resistance is currently under study.
spectrin extract under nondenaturing conditions at 0°C.
Involvement of nonerythroid tissues in hereditary defects of
This temperature kinetically immobilizes the SpD-T equilibthe RBC membrane skeleton. Although spectrin, ankyrin,
rium so that the fraction of unassembled SpD in the extract
and the band 3, 4.1, and 4.2 proteins are present in many
approximates the fraction of the assembled SpD in the
nonerythroid tissue^,^,^^^ clinically significant symptoms inmembrane in ~ ~ v o . ~ J ~ This
J O ~ fraction does not exceed 10%
volving nonerythroid tissues are rare. One of the possible
of the SpD + SpT pool in healthy subjects. An increase in
explanations of the lack of a multisystem involvement is the
the fraction of unassembled SpD reflects a defective SpD to
fact that nonerythroid isoforms of spectrin and ankyrin are
SpT self-association that is typically due to a mutation of a
encoded by distinct genes.203However, both the erythroid f3
or f3 spectrin involving the spectrin heterodimer contact
spectrin and ankyrin genes are expressed in skeletal muscle
site. The next essential step involves a limited proteolysis of
and some neurons as tissue-specific isoforms by alternative
spectrin, followed by a separation of the resulting peptides
pre-mRNA s p l i ~ i n g . 2This
~ ~ may possibly account for the
by electrophoresis in two dimensions, involving isoelectric
findings of mental retardation in two reports of HS associfocusing in the first dimension and SDS-PAGE in the
ated with ankyrin d e f i ~ i e n c y 4 ~and
J ~ ~an earlier report of a
second dimension.22~u~87~89
Under carefully controlled condispinal cord disease in an HS family in which membrane
tions, the tryptic peptide maps are highly reproducible, and
protein studies are not available.m The importance of
the 80-Kd peptide, corresponding to the a1 domain and the
these clinical observations is highlighted by recent reports
SpD-SpD self-association site of a spectrin, is the most
that distinct isoforms of ankyrin are associated with specific
prominent spot. Mutations of (Y spectrin involving the a1
structures of mouse brain and that the erythroid ankyrin
domain, as well as some of the mutations of f3 spectrin, give
isoforms are missing in ankyrin-deficient (nb/nb) spherorise to abnormally migrating peptides that are reactive with
cytic mice.2o5One of the structures containing erythroid
polyclonal antibodies raised against the normal a1 domain.
ankyrin isoforms are cerebellar Purkinje cells; the progresAt the present time, this step is essential before the
sive loss of these cells in ankyrin deficient (nb/nb) mice
characterization of the defect at the DNA level. In most,
coincides with a development of a psychomotor disorder.2M but not all cases, the site of the mutation is in the vicinity of
320
Fig 8. Abnormalities of RBC shape in inherited disorders of RBC
membrane proteins. (1) Typical HS. Although spherocytes are prominent, many cells retain some degree of biconcave shape. (2) HS with
pincered (mushroom shaped) cells (arrow). Plncered RBCs are found
in about 80% of HS patients with band 3 deficiency, whereas they are
rare in other forms of HS (D. Arnold, P. Jarolim, and J. Palek, recent
unpublished data). (3) HS with acanthocytes, as reported in HS
containing mutant p spectrin that binds poorly t o the 4.1 protein.u.lz*
(4) Severe hemolytic HS associated with a combined deficiency of
spectrin and ankyrin. In addition t o spherocytosis, many ceils have an
irregular contour; some of the cells resemble RBCs in HPP, shown in
(8). 15) Spherocytic elliptocytosis (hemolytic ovalocytosis). Many cells
have an oval shape. These patients often have hemolytic anemia
despite minimal changes in RBC morphology. (6) Common mild HE
associated with Sp d I4 mutation. In addition t o elliptocytes, occasional rod-shaped ceils (arrow) are present. (7) "Homozygous" common HE in a compound heterozygote for Sp dt74and Sp d's. Note
prominent elliptocytosis with numerous fragments and poikilocytes.
(8) HPP Sp d'". Note prominent microspherocytosis and marked RBC
fragmentation and poikilocytosis. but only occasional elliptocytes.
Some poikilocytes are in the process of "budding" (snow). In addition
t o the presence of spectrin mutation, these cells are also possibly
deficient in s p e ~ t r i n . (9)
~ ~SA0
~ ~ (stometocytic
~.~~
elliptocytosis). The
cells often contain a longitudal slit or a transverse ridge (arrow).
(Reprinted with pewnission.l)
the abnormal cleavage site that gives rise to the abnormally
migrating peptide. In some of the previously studied mutations, the site of cleavage has been mapped by transferring
the tryptic peptides to immobilon membrane, followed by
PALEK AND SAHR
direct N-terminal sequencing of the peptide of interest.*Jm
Occasionally, the mutation can be detected during this
step."J''
More often, the mutation is subsequently found
by analyzing exon or cDNA sequences encoding both the
N-terminal and C-terminal sequences adjacent to the cleavage site (Tablc 1). This can be readily accomplished using
the polymerase chain reaction (PCR) technique2m.mto
amplify genomic DNA or cDNA. In two spectrin mutations,
this step can now be bypassed. The HE Sp u116s
mutation
appears to be homogeneous involving a duplication of the
leucine codon at position 154, thus permitting the use of
appropriate allele-specificoligonucleotide (ASO) probes to
verify or exclude this m u t a t i ~ n . ~In~8-of~ ~
12' patients
~
with
the spectrin u1/74mutation examined so far, the defect
involves codon 28ms99'.w
and eliminates an Aha 11 restriction
site9'; this change can be used as diagnostic of this mutation. However, because several different changes can result
in ablation of a restriction enzyme site, this type of change
must be confirmed by sequence analysis or use of AS0
hybridization probes. As discussed above, in some patients
with the Sp u1174
defect, the mutation may involve p
spectrin. Likewise, in one of the spectrin ailMdefects, the
mutation is not adjacent to the abnormal cleavage site.Ils
The majority of HE patients who are simple heterozygotes for a spectrin mutation are asymptomatic, presenting
either without detectable hemolysis or with a minimal
hemolysis only. However, rare HE patients present with
moderate to severe hemolysis. This clinical picture is
typically accompanied by the presence of poikilocytes and
fragments on the peripheral blood film. In some patients,
this clinical syndrome is dominantly transmitted, and coinherited with a mutant spectrin (such as Sp c P 4 ) that is
grossly dysfunctional, as also reflected by a marked increase
in unassembled dimeric spectrin. reaching 50% of the total
SpD + SpT p ~ o l . ~Other
. ~ . ~rare
~ patients are either
homozygous or doubly heterozygous for one or two spectrin
mutations involving defective SpD self-association?* In
some patients, the augmentation of hemolysis together with
poikilocytosis reflects an interaction of the elliptocytogenic
spectrin mutation with another factor. This other factor can
be either inherited or acquired. An example of an inherited
factor is a silent genetic factor, in trans, ie, linked to the
normal spectrin allele as detected either by analysis of
tryptic peptide maps (the Sp uv/4ipolymorphism)131
or by
studies of linkage with a spectrin RFLP.*I" Examples of
acquired factors are an altered intracellular environment
such as found in neonates whose RBCs contain high
hemoglobin F (neonatal poikilocytosis, see section on
membrane fragmentation), microcirculatory damage to
RBCs during disseminated intravascular coagulation or
thrombotic thrombocytopenia purpura,1u certain infections, or other conditions such as superimposed BIZdeficiency.L3 The presence of a second interacting factor is
suggested by the finding of a relatively mild defect of
spcctrin, as reflected by a mild increase in unassembled
dimeric spectrin in conjunction with a symptomatic hemolytic disorder.'-'
Hereditary pyropoikilqtosis. The diagnosis of HPP is
suggested by the presence of a severe recessively inherited
RED BLOOD CELL MEMBRANE PROTEIN MUTATIONS
321
1A
1
3
2
.band 3’
Fig 7. Evaluation of a membrane proteln defect In
HE and HPP. (A) Identification of f3 speetrin mutant
with abnormal mobility (Spectrin Nice, gel 2) and the
deficiency of protein 4.1 (gel 4) in HE. Membrane
proteins from control RBCs (gels 1and 3) and RBCs of
the two patients (gels 2 and 4) were solubilized in
SDS and electrophoresed in a Fairbanks nonlinear
3.5% t o 17% polyacrylamide gradient gel‘“ (first two
gels) and Laemmli gel’.’
(last two gels). Note the
excellent resolution of a spectrin, f3 spectrin, and
ankyrin bands, as well as the f3 spectrin mutant
(Spectrin Nice) in the modified Fairbanks system. In
the Laemmli system, f3 spectrin and ankyrin bands
overlap. However, the Laemmli system resolves band
4.1 into two bands (4.la and 4.lb). Both bands are
absent in the HE 4.1 (gel 4). (B) Nondenaturing
polyacrylamide gels of crude spectrin extracts. Lane
1 is a control, 2 is one of the two parents who both
have common HE, and 3 is their homozygous HE
offspring. RBCa of the HE parent, who is heterozygous for the Sp
contain both dimeric (SpD) and
tetrameric ( S p n Sp. In the homozygous offspring,
spectrin is almost exclusively in a dimeric state. (C)
Two-dimensional peptide map (isoelectric focusing
followed by SDS-PAGE [IEFISDS-PAGE]) of limited
tryptic digest of spectrin from a control and HE
subject with the Sp d Mdefect. The positions of the
normal d domain and those of the abnormal d
peptides of molecular weight of 65 Kd are indicated
by arrows. (Modified and reprinted with permission.’)
Fairbanks
4
B
1
2
3
1
Laemmli
Nondenaturing
gel
c 6
-1
P
65kd’-
hemolytic anemia, associated with the presence of numerous poikilocytcs, microsphcrocytcs, and fragments on the
periphcral blood film. The fragmcntation often leads to a
decrcase in mcan corpuscular volume and a marked increase in the RBC size distribution width. Incubation of
RBCs at tempcratures of 45 to 47°C shows a gross thcrmal
instability in the majority of cases?’ further supporting the
diagnosis of HPP. It should be pointed out, howevcr, that
varying dcgrccs of thermal instability of RBCs are noted in
the majority of elliptocytogenic spectrin mutations rcgardless of whether thc clinical phenotype is that of HE or HPP.
Although the molccular basis of HPP is heterogcncous,
the common feature of HPP is the presence of one of
several spcctrin mutations involving a defective SpD selfassociation togethcr with a partial dcficicncy of spcctrin.22J’.2”’ Some patients are homozygous for the mutant
spectrin and their parcnts may both have mild HEvn;others
are cithcr doubly hctcrozygous for two spcctrin mutations
(one of which may often present with HE in one of thc
parents) or, more commonly, thcy are compound hetcrozygotcs for a spcctrin mutation charactcrizcd by dcfcctivc
SpD self-association and anothcr unknown genetic defect
that augments the clinical cxprcssion of the mutant spcctrin
and produccs a concomitant spcctrin deficiency, such as a
thalassemia-likc dcfcct in spcctrin synthesis.” Thc prcsencc of such a second genetic defect was recently verified
(1) by findings of rcduccd a spcctrin synthesis in an in vitro
culturc systcm (both in thc HPP offspring carrying the
elliptogenic spcctrin mutation, and her fully asymptomatic
father) and (2) by findings of rcduccd amounts of mRNA
produccd by thc structurally normal spcctrin allclc.21i.212
In
HPP patients who are cithcr homozygous or doubly het-
PALEK AND SAHR
322
erozygous for one or two spectrin mutations involving a
defective SpD contact, the spectrin deficiency results from
an instability of spectrin leading to accelerated spectrin
degradation before assembly on the membrane.w7,213In
RBCs of homozygotes for a severely dysfunctional spectrin
that leads to a marked increase in the fraction of unassembled dimeric spectrin, the spectrin deficiency may be
related to the binding stoichiometry of one copy of ankyrin
per one spectrin tetramer; thus, in patients with a marked
increase of unassembled SpD, the number of ankyrin copies
may not be sufficient to bind all SpD to the RBC membrar~e.',~~
SoutheastAsian ovalocytosis. In contrast to common HE
and HPP that is heterogeneous in molecular terms, SA0
appears h o m o g e n e ~ u s , ~
presumably
~~J~
because its prevalence is related to a selective pressure resulting from a
resistance of ovalocytes to invasion by malaria parasifes.195J96,214
In addition to characteristic RBC morphology, the presence of this defect is suggested by the finding of
resistance of ovalocytes or their ghosts to changes in
~ h a p e , ' ~ ~and
, * l by
~ analysis of limited proteolytic digests of
whole cells or ghosts (showing the "band 3 Memphis"
polymorphism, which is tightly linked with SAO),188or by
PCR amplification of the region of cDNA or genomic DNA
that contains the deletion of eight c o d o n ~ . ' ~ ~ J ~ ~
In our experience, the strategy outlined above detects the
underlying defect in about 70% of patients with HE and in
all cases of HPP and SAO. In a rare patient with a mild
recessively inherited elliptocytosis, staining of membrane
proteins with periodic acid (PAS) may show a lack of
glycophorin C'; the heterozygous carriers of this defect are
asymptomatic.
Hereditary spherocytosis. The principal cellular lesion of
hereditary spherocytosis is a surface area deficiency reflecting a loss of membrane lipids. In most cases, the cause
of the lipid loss is a partial deficiency of spectrin.' As a
result of the deficiency of spectrin, the density of the
submembrane skeletal network in the HS RBC membrane
is reduced, presumably underlying the lipid bilayer destabi-
lization and microvesiculation.' Indeed, the degree of
spectrin deficiency is proportional to the increase of osmotic fragility, a crude indicator of cell surface area
deficiency, and to the loss of membrane material from the
cells in vitro.",216 Consequently, clinical severity is proportional to the amounts of RBC spectrin with either a
life-threatening44 or even fatal disease217in subjects with
severe spectrin deficiency. In the latter report of a severe
spectrin deficiency (26% of normal) with a normal content
of ankyrin and band 3,217 progenitor-derived erythroblasts
in culture exhibited a strikingly reduced synthesis of a
spectrin and a markedly reduced assembly of a and p
spectrin on the membrane. A moderate to severe deficiency
of spectrin can be detected by protein separation by
SDS-PAGE, with densitometric estimation of spectrin
content by measuring a ratio of spectrin and band 3 peaks.
It should be noted, however, that this technique is too
insensitive to detect mild spectrin deficiencies. Furthermore, the loss of membrane lipids from the HS RBC
membrane is accompanied by a loss of the band 3 protein,
producing a mild deficiency of this protein that can spuriously normalize the spectrin to band 3 ratio.' Thus, a direct
quantitation of the number of spectrin copies per cell by
radioimmunoassay represents the definitive method to
measure spectrin content per cell.
Although varying degrees of spectrin deficiency are
detected in the majority of patients with HS, it should be
emphasized that the underlying molecular basis of HS is
likely to be heterogeneous and that the primary molecular
lesion is likely to involve several membrane proteins,
including spectrin, ankynn, band 3, and band 4.2 proteins
(see above). This biochemical heterogeneity of HS is
evident by analysis of membrane protein composition by
SDS-PAGE (Table 2). This approach shows abnormalities
in about 60%of HS and segregates the disorder into at least
four categories: (1) isolated spectrin deficiency; (2) a
combined deficiency of spectrin and ankyrin; (3) a deficiency of the band 3 protein as reflected by a marked
increase in the ratios of spectrin, actin, 4.1, and 4.2 protein
Table 2. Membrane ProteinAbnormalities in HereditarySpherocytosisas Shown by SDS-PAGE
Membrane Protein
Abnormalities
Severe ( - 50%) spectrin deficiency
Mild to moderate (20% to 30%)
spectrin deficiency
Severe ( - 50%) deficiency of both
spectrin and ankyrin
Mild to moderate (20% to 30%) deficiency of spectrin and ankyrin
Deficiency of band 3 protein
Deficiency of protein 4.2
Inheritanceand
Clinical Presentation
Autosomal recessive HS with severe
hemolysis
Autosomal dominant HS with mild to
moderate hemolysis
HS with severe hemolysis, inheritance unknown
Autosomal dominant HS with mild to
moderate hemolysis
Autosomal dominant HS with mild to
moderate hemolysis
Autosomal recessive hemolytic anemia with sphero-ovalocytes
Underlying
Molecular Defects*
Prevalence
References
a spectrin
Probably rare
41.1 19.217
p spectrin
Ankyrin'
Ankyrin
About 10% of patients
Unknown
Probably rare
82.83.1 28
Ankyrin'
20% or more of kindred
219.t
Band 3 protein
About 15% to 20% of kindred 48,49,185,186
Band 4.2 protein*
Band 3 protein*
Relatively common in Japan
47.138
50-56.181
Although less sensitive than direct quantitation of the number of copies of spectrin, ankyrin, or band 3 protein per cell, SDS-PAGE followed by
densitometrictracing and determination of the ratio of spectrin and ankyrinto band 3 shows abnormalitiesin about 70% of patients with HS.
*Presumed primary defect, the DNA defect is likely to be heterogeneous.
tJarolim P, Palek J, unpublished observations, 1992.
RED BLOOD CELL MEMBRANE PROTEIN MUTATIONS
323
to the band 3 protein; and (4) a deficiencyof the 4.2 protein.
verified by a direct immunoassay and the characterization
of this defect is currently in progress.
However,subsequent detailed characterizationof the underThe last group of defects shown by the above approach is
lying molecular defect is difficult, as there is no simple
a partial or complete deficiency of the 4.2 protein. Both
functional or structural test that would provide further
partial and complete deficiencies have been reported, and
clues in regard to the nature of the underlying molecular
the hematologicphenotype is either that of HS and, in some
defect.
patients, ovalo-stomatocytosis.50-55
It remains to be estabIn a patient with a severe spectrin deficiency and a
lished whether the 4.2 deficiency represents the primary
recessively inherited HS, the primary molecular defect has
defect or simply a marker of another yet unknown defect.
been recently assigned to ci spectrin by protein and RFLP
In a striking contrast to HE, HPP, and SAO, the
linkage studies; in a group of recently reported patients, the
underlying
cDNA or genomic DNA defect remains unclear
underlying defect involves a mutation that leads to spectrin
in the majority of patients with HS. In many patients with
deficiencyby a yet undefined molecular me~hanism.4~9~~~
In
HE or HPP, the approximate site of the genetic defect
dominantly inherited HS, isolated spectrin deficiency is
could be deduced from studies of protein self-association
likely to be heterogeneous. As noted above, a subset of
(ie, involving SpD-SpD contact) or from limited proteolytic
patients may have an abnormal p spectrin that binds poorly
peptide
maps of spectrin. In HS, a similar strategy has been
to the 4.1 pr0tein.82~~
It is yet to be determined whether a
useful
in
characterizing one defect involving a weakened p
subset of patients with spectrin deficiency carries a defect
spectrin-protein 4.1 c0ntact.8~,*~
In the majority of HS
involving a mutant ankyrin, which.has a reduced ability to
patients, however, the assignment of the tentative site of
bind spectrin to the membrane. Another possible defect
the mutation to a specific region of the protein has not been
may involve spectrin that binds weakly to ankyrin; one such
made. For several reasons this area of investigation will
defect has been noted in an asymptomatic parent of a
likely open up in the near future. Firstly, the cDNA
patient manifesting with HPP,Z18 but the nature of the
sequences of human a n k ~ r i n , ' ~ciJ ~spectrin,m
~
specdefect remains unclear.
trin,221protein 4.1,2u protein 4.2,2',224 and band 3,2253226
and
In patients with combined spectrin and ankyrin deficienthe gene organization of human ci ~ p e c t r i n land
~ , ~mouse
~~
cies, the defect underlying a deficiency of ankyrin has been
band 3228have been recently described. Secondly, several
detected in several p a t i e n t ~ ! ~ J ~In
~ Jone
~ ~ report, the entire
rapid and sensitive techniques have been developed to
ankyrin gene was found absent due to an interstitial
detect mutations in cDNA and genomic DNA. These
deletion within a short arm of chromosome 8.139In another
scanning techniquesu9 include chemical cleavage of mispatient with severe spectrin and ankyrin deficiency, the
matched base pairs,m RNase protection,B1 denaturing
mRNA levels and the synthesis of ankyrin in reticulocytes
gradient gel electrophoresis (DGGE),U2-u5and singlewere reduced.138Spectrin was synthesized normally but the
stranded conformational polymorphism analysis
assembly of spectrin on the membrane was reduced be(SSCP).u6,u7These methods can be used in conjunction
cause of the underlying defect involving the synthesis of
with the PCR technique to detect small deletions or
ankyrin. However, in the majority of patients with domiinsertions and single base substitutions. If by one of these
nantly inherited HS, the nature of combined spectrin and
methods the amplified DNA appears to be abnormal, the
ankyrin deficiencies remains unknown. The detection of the
specific change can then be determined by subsequent
underlying defects is of considerable interest as this biosequence analysis. Although neither RNase protection,
chemical phenotype is very common, found in up to
DGGE, or SSCP techniques have been applied to the
two-thirds of all patients219(Table 2). It is possible, but
analysis of gene defects in HE or HS, their potential has
presently not proven, that some of these subjects could have
been illustrated in experiments to analyze mutations of the
a thalassemia-like defect of ankyrin synthesis.
type I collagen gene by RNase protection238and mutations
Analysis of membrane skeleton proteins by SDS-PAGE
in p-thalassemia by DGGE.B9
has recently shown a unique subset of patients with domiACKNOWLEDGMENT
nantly inherited HS with high ratios of spectrin, ankyrin,
4.1, and actin to the band 3 protein, implying a partial
The authors are indebted to Gabriella Maitino and Loretta
deficiency of the band 3 pr0tein.4~This deficiency has been
Wencis for typing the manuscript and to Joan Joss for the art work.
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