HCS

Tfb�piltusisS

HnthSatasac

Ho

paigi

BC

9H

Hn

1

ereditary Spherocytosis—Defects in Proteins Thatonnect the Membrane Skeleton to the Lipid Bilayer

tefan Eber and Samuel E. Lux

he molecular causes of hereditary spherocytosis (HS) have been unraveled in the past decade. No frequent defect isound, and nearly every family has a unique mutation. In dominant HS, nonsense and frameshift mutations of ankyrin,and 3, and �-spectrin predominate. Recessive HS is most often due to compound heterozygosity of defects in ankyrin,-spectrin, or protein 4.2. Common combinations include a defect in the promoter or 5�-untranslated region of ankyrinaired with a missense mutation, a low expression allele of �-spectrin plus a missense mutation, and various mutationsn the gene for protein 4.2. In most patients’ red cells, no abnormal protein is present. Only rare missense mutations,ike ankyrin Walsrode (V463I) or �-spectrin Kissimmee (W202R), have given any insight into the functional domains ofhe respective proteins. Although the eminent role of the spleen in the premature hemolysis of red cells in HS isnquestioned, the molecular events that cause splenic conditioning of spherocytes are unclear. Electron micrographshow that small membrane vesicles are shed during the formation of spherocytes. Animal models give further insightnto the pathogenetic consequences of membrane protein defects as well as the causes of the variability of diseaseeverity.emin Hematol 41:118-141. © 2004 Elsevier Inc. All rights reserved.

Amhst

mnamaitmcstv

Atntdtwbbhwtpp

EREDITARY spherocytosis (HS) is by far themost common congenital hemolytic anemia in

orthern European descendants. The hallmarks ofhe disease are anemia, intermittent jaundice (fromemolysis or biliary obstruction), and splenomegaly.pherocytes are devoid of the normal surface surplusnd rigid. They are trapped during their passagehrough the metabolically unfavorable splenic pulpnd selectively destroyed. The disease was first de-cribed in 1871 by two Belgian physicians, Vanlairnd Masius,1 as microcythemia, referring to the de-reased diameter of spherocytes in the blood smear.

Prevalence and GeneticsS occurs in all ethnic groups. The highest frequencyf 1: 5,000 is found in Northern European countries.

De novo mutations of ankyrin or other membranerotein genes are a frequent cause of sporadic HS. Inbout two thirds of patients, the disease is inheritedn a dominant pattern and can be followed fromeneration to generation, mostly with the same sever-ty. In the remaining cases, both parents are normal.

From the Division of Hematology/Oncology, Children’s Hospitaloston, and the Department of Pediatric Oncology, Dana-Farberancer Institute, Harvard Medical School, Boston, MA.

S.E. is supported by the Deutsche Forschungsgemeinschaft (DFG9/6-1�2). S.E.L. is supported by Grants No. R01 DK34083 and P01L32262 from the National Institutes of Health.

Address correspondence to Professor Dr Stefan Eber, Childrensospital of the Technical University, Koelner Place 1, D80804 Mu-ich, Germany. E-mail: stefan-eber@t.online.de.© 2004 Elsevier Inc. All rights reserved.0037-1963/04/4102-0002$30.00/0

sdoi:10.1053/j.seminhematol.2004.01.002

Seminars in Hematology, Vol 41, N18

bout half of these sporadic cases are due to de novoutations of the type associated with dominant in-eritance2; the others are assumed to be due to reces-ive genes. If this assumption is correct, about 1% ofhe population carries recessive HS genes.

One asymptomatic parent may carry a germ-lineosaicism for an ankyrin mutation or �-spectrin. De

ovo mutations of ankyrin3 or �-spectrin4 genes canrise in one of the parental germ lines and be trans-itted dominantly. Parental mosaicism also occurs

nd must be considered in genetic counseling. Thenheritance of HS is truly recessive in only about 10%o 15% of families. In these cases, both parents haveinor signs of the disease—usually only an in-

reased osmotic fragility in incubated blood or alight reticulocytosis.5,6 Patients with recessive HSend to more profound anemia,7,8 but clinical severityaries greatly.

Homozygosity for dominant HS is (nearly) lethal.s will be described later, dominant HS is mostly due

o null (nonsense or frameshift) mutations: virtuallyo abnormal protein is present. Homozygosity forhese mutations is presumably lethal during fetalevelopment, as homozygosity for dominant inheri-ance has only been described in two cases, neither ofhom was a complete null mutation. A Portugueseaby with severe hydrops fetalis was homozygous forand 3 Coimbra (Y488M),9 and an Italian baby wasomozygous for band 3 Neapolis (16�2 T3C), inhich a splicing defect impedes translation initia-

ion.10 A small amount of abnormal protein may beroduced from both mutations. The parents of bothatients had only mild HS, whereas both children had

evere, life-threatening anemia.

o 2 (April), 2004: pp 118-141

lsslpoco�

T

IatoscdbHac

mts(

cenpaai

iah

foelst

C

Dfss

Fmscs�eadfmPW

Hereditary Spherocytosis 119

HS is due to mutations at different chromosomeoci. Two genetic loci for HS have been identified bytudy of balanced chromosomal translocations ormall deletions, and by linkage analysis. One of theoci, an interstitial deletion of chromosome 8p11.1-21.2,11 eliminates the gene for ankyrin.12 The sec-nd locus is the gene for �- spectrin, which resides onhromosome 14q23-q24.2.13 As will be described,ther loci involve the genes for band 3, protein 4.2,-spectrin, and possibly �-adducin and dematin.

Clinical Presentation

ypical HS

n HS patients the family history is often positive fornemia, gallstones, or splenomegaly. In most caseshe severity of HS is very similar in affected membersf a family. Typically, there are increased number ofpherocytes in the peripheral blood smear and in-reased osmotic fragility of the red blood cells. Otheristinctive morphologic red cell alterations haveeen found in specific membrane defects14 (Fig 1).emolysis (as evidenced by hyperbilirubinemia and

haptoglobinemia) and reticulocytosis (a marker ofompensatory erythropoiesis) are present.

HS typically presents in infancy or childhood, butay first manifest at any age.15 In children, anemia is

he most frequent complaint (50%), followed byplenomegaly, jaundice, and a positive family historyall 10% to 15%).

The majority of patients (60% to 75%) have in-ompletely compensated hemolysis and mild to mod-rate anemia. It is often difficult for parents to recog-ize the general symptoms of anemia (fatigue, mildallor, or nonspecific findings such as “crabbiness”),nd usually the true extent of reduced physical abilitynd school deficits only becomes apparent when pos-tive behavioral changes following splenectomy.

Jaundice occurs in about half of patients, usuallyn association with viral infections. When present it ischoluric, characterized by unconjugated indirectyperbilirubinemia and the absence of bilirubinuria.

The incidence of palpable splenomegaly variesrom about 50% in young children to 75% to 95% inlder children and adults.16 The spleen is modestlynlarged (2 to 6 cm) but may be massive. Althoughikely given the pathophysiology and the response toplenectomy, there is no established correlation be-ween spleen size and the severity of HS.

lassification According to Disease Severity

isease severity of HS can be classified according to aew common clinical laboratory parameters. We clas-ify HS into mild, moderate, moderately severe, and

evere categories based on the hemoglobin and bili-

igure 1. Abnormal red cell morphology in HS due to differentembrane defects. (A) Ankyrin defects: in most blood smearspherocytes and anisocytosis are the typical morphologichange. (B) Band 3 defects: a small number of mushroomhaped (or “pincered”) red cells (arrows) are usually seen. (C)-Spectrin defects: a moderate proportion of acanthocytes andchinocytes are present. (D) �-Spectrin defects: these severelyffected patients have numerous spherocytes, contracted,ense cells, and bizarre poikilocytes. (Reprinted with permissionrom Walensky LD, et al: Disorders of the red blood cellembrane, in Handin RI et al (eds): Blood: Principles andractice of Hematology (ed 2). � 2003 Lippincott Williams &ilkins.)

r(gtat(t

sm8bpthacPawhwitmoihc

slHswwp

fit

pmgcefo8d

aisembsctmaIad

lsmhPguWp

HRBO

TS

120 Eber and Lux

ubin concentrations and the reticulocyte count5

Table 1). Asymptomatic carriers of a recessive HSene represent a separate group. The semiquantita-ive evaluation of the osmotic fragility test in freshnd incubated blood as well as quantitation of spec-rin by specific enzyme-linked immunsorbent assayELISA) or radioimmunoassay (RIA) also can con-ribute to this classification.

Mild HS. Patients with mild HS have compen-ated hemolysis. About one third of patients have aild form of HS, with slight reticulocytosis (3% to

%). Red blood cell production and destruction arealanced or nearly equivalent,17 and the erythrocyteroduction index18 is increased by no more thanhree- to fourfold to achieve a state of “compensated”emolysis. Patients are not anemic or barely anemicnd are usually asymptomatic. The drive for in-reased erythropoiesis is still not fully understood.ossibly the dehydrated, rigid spherocytes do notdequately perfuse the juxtaglomerular renal vessels,here erythropoietin is produced, even when theemoglobin is normal. This hypothesis is consistentith recent observation that serum erythropoietin is

nappropriately high in patients with mild HS, main-aining bone marrow hyperplasia.19 The diagnosisay be difficult because spherocytes are obvious in

nly two thirds of cases. Osmotic fragility is oftenncreased only after the blood is preincubated for 24ours (the incubated osmotic fragility test). The con-entration of spectrin is at least 80% of normal.

Parvovirus infection, pregnancy, exercise, andplenic enlargement may exacerbate mild HS. Hemo-ysis may become more severe in patients with mildS who develop illnesses that cause splenomegaly,

uch as mononucleosis, or during pregnancy20 orith endurance sports. Some patients are diagnosedhen they develop aplastic crises, typically caused by

Table 1. Classification of Spheroc

Mild HS

emoglobin (g/dL) 11-15eticulocytes (%) 3-8ilirubin (mg/dL) 1-2smotic fragilityFresh blood Normal or slightly 1Incubated bloodransfusions† 0-1plenectomy§ Generally not necessary

*Patients depend on regular transfusions.†One transfusion during an aplastic crisis and neonatal exchan‡Some patients need 1 or 2 transfusions during infancy.§May be a total or subtotal splenectomy.

arvovirus B19. Symptomatic gallstones may be the u

rst manifestation of mild HS in an otherwise asymp-omatic patient.

Moderate HS. This is the largest group of HSatients, comprising about 60% to 70% of the total;ost have typical HS as described above. The hemo-

lobin level is between 8 and 11 g/dL and reticulo-ytes are nearly always above 8%. The diagnosis isasy if there is spherocytosis and increased osmoticragility. Family history is positive in about two thirdsf cases. The amount of spectrin is usually below0%. Ankyrin mutations are frequent causes of theisease (see below).Moderately severe HS. A small group of patients,

bout 10%, have a low hemoglobin level beyondnfancy (6 to 8 g/dL) and require occasional transfu-ions.21,22 In some cases the symptoms are morevident during infancy and early childhood anduch improve at school ages. Physical activities may

e normal or impaired. Careful evaluation can showlightly retarded psychomotor development, espe-ially motor coordination (Eber S, personal observa-ion). Moderately severe HS is distinguished fromoderate HS mostly by the low hemoglobin levels

nd the patient’s intermittent need for transfusions.n addition, reticulocytosis is greater (often �15%)nd bilirubinemia more pronounced (often �3 mg/L).Severe HS. Only 3% to 4% of HS patients have

ife-threatening anemia and require regular transfu-ions to maintain a hemoglobin level above 6 g/dL. Inost cases inheritance is autosomal recessive (but weave observed a few dominant cases with severe HS).atients develop hemosiderosis that may lead to or-an failure. Continuous iron chelation therapy issually necessary, starting around 4 years of age.ithout regular transfusions or splenectomy, the

atients may suffer growth retardation, delayed sex-

and Indications for Splenectomy

Moderate HSModeratelySevere HS Severe HS*

5 6-8 �6�10 �10�2-3 �3

ctly 1 Distinctly 1 Distinctly 1clty 1 Distinctly 1 Markedly 1

�3 Regularlity is decreased Necessary

(at �5 yr)Necessary(at � 3 yr)

nsfusions are not counted.

ytosis

8-11.�8�2

DistinDistin0-2‡If vita

ge tra

al maturation, or thalassemia-like facies.

aasemrclttlhvu

i5tsd0c

shwtbTtofp

bpSocih

fbdughpabtt

lc

ouobnnocsmdthwuDfwhpaa

ematstoasnttrmesmtaeiad

bhtpcmee

Hereditary Spherocytosis 121

Silent carrier state. Heterozygous parents or rel-tives of patients with recessive HS are carriers of ansymptomatic trait. They do not have anemia,plenomegaly, or hyperbilirubinemia.5,23 Some carri-rs can only be detected by scrupulous testing forinor laboratory signs of HS. Some have a slight

eticulocytosis (1.5% to 3%) or signs of slightly in-reased hemolysis such as a decreased haptoglobinevel. The incubated osmotic fragility test is probablyhe most sensitive method for detecting carriers, par-icularly the 100% lysis point; the acidified glycerolysis test (AGLT) may also be useful. In carriers, thealftime for hemolysis in the test may lie between thealues for patients with overt HS (AGLT50 � 5 min-tes) and normal controls (�30 minutes).From the estimated prevalence of recessive HS (1

n 40,000 or about 12.5% of all HS, which is 1 in,000 in the United States), about 1% of the popula-ion can be estimated to be silent carriers. Indeed,creens of normal Norwegian24 or German25 bloodonors with osmotic fragility or AGLT tests show a.9% to 1.1% incidence of previously undetected HSarriers.

HS in pregnancy. Transfusion is rarely neces-ary. Unsplenectomized pregnant patients with HSave no significant complications except for anemia,hich is aggravated by the plasma volume expansion

hat occurs normally in pregnancy,26 and sometimesy increased hemolysis19 or megaloblastic crises.ransfusions are rarely necessary but should be insti-

uted if the hemoglobin drops to less than 8 g/dL inrder to guarantee optimal oxygen supply of theetus. Folic acid at 1 mg daily should be provided torevent vitamin deficiency and megaloblastic crises.HS in the neonate. Increased hemolysis should

e suspected when a neonate develops precocious orrolonged icterus, in Western Europe and the Unitedtates not infrequently due to HS. Approximately halff infants with HS develop severe neonatal jaundice,ompared with 8% of normal newborns, and 91% ofnfants discovered to have HS in the first week of lifeave hyperbilirubinemia (bilirubin � 10 mg/dL).Newborns with the combination of HS and the trait

or Gilbert’s syndrome frequently have hyperbiliru-inemia. The presence of the trait for Gilbert’s syn-rome, a TATA box polymorphism in the bilirubinridine diphosphate (UDP) glucuronyltransferaseene (UGT1A1), raises the frequency and severity ofyperbilirubinemia in newborns with glucose-6-hosphate dehydrogenase deficiency27 and probablylso with HS. Kernicterus is a risk if hyperbiliru-inemia is not controlled. In most patients photo-herapy is sufficient, but occasionally an exchangeransfusion is required.

The natural history of HS during the first year ofife was recently re-evaluated.28 The hemoglobin

oncentration was normal at birth (�15 g/dL) in 57% n

f HS patients, consistent with the absence of intra-terine hemolysis. The normal intrauterine survivalf spherocytes is not completely understood but maye due to the functional hyposplenia in neonates. Theumber of pocked red blood cells is increased inormal neonates up to the level of splenectomizedlder children, indicating impaired splenic phago-ytic function at birth. Within the first weeks of life,plenic function improves. The infrequency of ane-ia in neonates with HS contributed to the low

etection rate of HS at birth (approximately twohirds of affected neonates are undiagnosed). Theemoglobin value sharply decreased during the first 3eeks of life, often necessitating transfusion,28 andnderscoring the desirability of early detection of HS.ue to current practice of early discharge of neonates

rom the nursery, physicians, midwives, and parentsith a family history of HS must be alert for a drop ofemoglobin, especially if hyperbilirubinemia wasresent; it is not uncommon for infants with HS to bedmitted at the age of 4 to 6 weeks with severenemia.

Many neonates with HS have transient sluggishrythropoiesis. Many HS infants with only mild tooderate anemia at birth develop a transiently severe

nemia (hemoglobin nadir of �6 g/dL) at the age of 4o 8 weeks, probably due to increased hemolysis asplenic function improves after birth, combined withhe “physiological” reduction of erythropoiesis in thexygen-rich environment outside the uterus. Inbout 10% to 15% of cases, the erythropoietic re-ponse remains sluggish and reticulocyte counts doot rise appropriately for the degree of anemia duringhe first year. A moderate to severe anemia persistshroughout infancy in these patients, who may needepeated red blood cell transfusions up to the age of 9onths. In a recent series,28 only 24% of HS infants

scaped transfusion: 34% needed a single transfu-ion, usually before 2 months of age; 24% requiredultiple transfusions for up to 9 months to reach

ransfusion independence; and 18% had severe HSnd required chronic transfusions. In our combinedxperience of about 1,000 HS patients, the number ofnfants who required transfusions was much lower,nd less than 3% of infants remained transfusion-ependent after the first year of life.If a child is otherwise well, we allow the hemoglo-

in to fall to about 5 to 6 g/dL before we transfuse, toelp to stimulate erythropoiesis. In addition, we onlyransfuse to a hemoglobin of 9 g/dL to avoid sup-ressing the desired marrow response. Regular sub-utaneous administration of erythropoietin is recom-ended by some for infants with HS and sluggish

rythropoiesis to avoid blood transfusions.29 How-ver, considering that subcutaneous injections may

eed to be administered for months and that antibod-

itw

IrmdmlDgea

C

abjtIotwRh

atobltsfisieLrgcdsh

enacfivwi

asawcp

dwcnaempWw

fasmwttic

ssteodmccimwscglttblb

utaa

122 Eber and Lux

es to erythropoietin may develop, further study ofhe use of this hormone is needed in anemic infantsith HS before routine use can be recommendedClose observation of infants with HS is necessary.

t is important to monitor hemoglobin levels andeticulocyte counts in infants with HS at leastonthly during the first 6 months of life in order to

etect and treat late anemia. The observation intervalay be prolonged to 6 to 8 weeks after 6 months of

ife, and to 3 to 4 months in the second year of life.uring childhood, each patient should have hemo-lobin, reticulocyte, and bilirubin levels checked ev-ry 6 to 12 months up to the age of 5 years, andpproximately yearly thereafter.

omplications of HS

Anemic crises. Most patients with typical moder-te HS suffer a few hemolytic crises, often triggeredy viral infections, and characterized by increasedaundice and anemia. Abdominal pain, vomiting, andender splenic swelling are other typical features.ncreased hemolysis is probably due to enlargementf the spleen during infections as well as activation ofhe reticuloendothelial system. For most patientsith hemolytic crises, just supportive care is needed.ed blood cell transfusions are only required if theemoglobin level falls below 5 to 6 g/dL.The characteristic rash of parvovirus infection is

bsent in patients with hemolytic anemias and aplas-ic crises. With rare exceptions, severe aplastic crisesccur only once in life. Most aplastic crises are causedy infection with parvovirus B19,30 which confersife-long immunity. The virus infects and kills ery-hroid precursors, leading to a 10- to 14-day suppres-ion of erythropoiesis.31 The characteristic laboratorynding is a low number of reticulocytes (�2%) de-pite severe anemia. The earliest laboratory sign is anncrease in the serum iron level due to the loss ofrythroblasts and decreased hemoglobin synthesis.oss of erythropoiesis in a patient with hemolysis canapidly lead to a severe drop of hemoglobin (�3/dL), and may be fatal. The authors know of severalhildren who died during an aplastic crisis, probablyue to cardiac failure. Children with aplastic criseshould be carefully observed and transfused if theiremoglobin decreases below 5 to 6 g/dL.Parents should be advised to watch for pallor,

xtreme lassitude, and white conjunctivae after mildonspecific signs of infection such as fever, vomiting,nd abdominal pain. Children with HS should avoidontact with children with erythema infectiosum orfth disease. There are anecdotal reports of the ad-antage of using intravenous immunoglobulin,hich contains anti-parvovirus antibodies, early dur-

32

ng the infection. However, in most patients the t

plastic crisis is discovered at the start of clinicalymptoms, when the patient is producing his ownntibodies to parvovirus; hence, the clinical benefitould be only marginal. Immunoglobulin therapy

annot be recommended before clinical efficacy isroven by a formal study.Even though megaloblastic crises due to folate

eficiency are very rare in developed countries,here nutrition and prenatal care are good, this

omplication can occur in patients who are mal-ourished or pregnant. Folic acid intake may be in-dequate to minimize plasma homocysteine levelsven in normal individuals.33 Due to the higher de-and for folic acid to support increased erythro-

oiesis, the risk of folate deficiency is increased in HS.e recommend 1 mg/d of folic acid for all patientsith HS.Gallstones. Bilirubin (pigment) gallstones are

ound in at least 5% of children less than 10 years ofge with HS; the frequency reaches 40% to 50% in theecond to fifth decade, with the increased incidenceostly during the second and third decades. Patientsho inherit both HS and homozygosity for the muta-

ion in the promoter of the UGT1A gene that charac-erizes Gilbert’s syndrome34 have a four- to fivefoldncreased rate of gallstone formation compared toases with normal bilirubin clearance.35

The best technique to detect gallstones is ultra-onography. We recommend an abdominal ultra-ound every third to fifth year and before splenec-omy. About 40% to 50% of patients with gallstonesventually develop symptoms of gallbladder diseaser biliary obstruction. The treatment of gallbladderisease in HS is debatable, especially in patients withild HS or asymptomatic gallstones. Surgery is

learly necessary if there are recurrent episodes ofholecystitis. Isolated laparoscopic cholecystectomys recommended mostly for children with mild to

oderate HS. Children with more severe disease,ho will need a splenectomy later, may profit from

imultaneous subtotal splenectomy (see below) andholecystectomy. A recent Markov analysis sug-ested that splenectomy (total or subtotal) and cho-ecystectomy should be done in adults who are lesshan 39 years old and have no gallbladder symp-oms.36 If the patient has occasional biliary colic,oth operations are recommended up to age 52. Cho-ecystectomy alone is preferred in older patients withiliary colic.Other complications. Leg ulcers and extramed-

llary hemopoietic tumors are rare complicationshat occur mostly in adults. Spinocerebellar degener-tion and myocardiopathy have been described inssociation with HS, but the relationship, if any, to

he hemolytic anemia is unclear.

FladhwdHp

D

Rirdiccsbcidsdntischs

tp�slnfai

mtTadorwoms

DTw

caosmifeAp

ttcti

man

FvcrartcslwhtpraW

Hereditary Spherocytosis 123

Diagnosis and Laboratory Features

or patients with typical HS, the diagnosis is estab-ished by increased red blood cell osmotic fragilitynd spherocytosis on the blood smear, and if theirect Coombs test is negative. The combination of aigh mean cellular hemoglobin content (MCHC), aidened red cell distribution width, and shifts inistribution curves are often sufficient to suggestS.37 The MCHC is greater than 36 g/L in half ofatients.

iagnostic Findings

equired diagnostic criteria are at least one sign ofncreased hemolysis: reticulocytosis, increased indi-ect bilirubin, increased lactate dehydrogenase, orecreased haptoglobin; increased spherocytes, subtle

n mild cases, and anisocytosis; increased red bloodell osmotic fragility, especially after 24-hour prein-ubation of the blood and increased MCHC or a righthift in the MCHC histogram. All required signs muste present, with the exception that two optional signsan substitute for one of the required signs other thanncreased hemolysis and osmotic fragility. Optionaliagnostic findings include a positive family history;pleen enlargement (a slight increase may escapeetection); anemia (about one third of HS patients areot anemic); and decreased concentrations of spec-rin, or spectrin and ankyrin, or band 3, or protein 4.2n red blood cell membranes. Except for the mostevere variants, anemia and hemolysis are virtuallyured in HS patients by splenectomy. If significantemolysis persists after splenectomy, a faulty diagno-is must be considered.

Detection of hyperdense red cells is a new diagnos-ic tool. Patients with HS consistently have a sub-opulation of hyperdense red blood cells (MCHC40 g/dL) in MCHC histograms. However, the mea-

urement can only be made with modern-generationaser-based blood counters (such as the Bayer Tech-icon/Advia).38 Similar information can be obtained

rom data generated by aperture impedance (Coulter)nalysis.37 These tests are appropriate to screen fam-ly members.

Osmotic gradient ektacytometry is a very sensitiveethod for diagnosing HS: it detects the characteris-

ic decrease of membrane surface area in all cases.39

he deformability index of the red cell is measured asfunction of the osmolality of the suspending me-

ium, which is continuously varied (Fig 2). Thesmolality at which the red cell deformability indexeaches a minimum is the same as the osmolalityhere 50% of the red blood cells hemolyze in ansmotic fragility test.40,41 Unfortunately, the equip-ent needed for this technique is only available in a

mall number of laboratories. t

ifferential Diagnosis of HShere are only a few diseases that can be confusedith HS.As noted earlier, the diagnosis of HS can be diffi-

ult in newborns. Only 35% of affected neonates havereticulocyte count greater than 10% (normal: �8%n the first day of life) and 33% of neonates do nothow distinct spherocytosis.42 In addition, the os-otic fragility of fetal/neonatal red cells is dimin-

shed if adult control values are used; control valuesor neonates have been determined and should bemployed for neonatal osmotic fragility tests.42 TheGLT,43 with modifications,25 offers an easier ap-roach to the diagnosis of HS in neonates.

ABO incompatibility is important in the differen-ial diagnosis of HS in neonates because it is fourimes more often the cause of hyperbilirubinemia; itan be difficult to distinguish from HS, especially inhe rare cases of Coombs-negative ABO incompatibil-ty with spherocytosis.

In older children and adults an autoimmune he-olytic anemia (AIHA), especially with IgG (warm)

ntibodies or the biphasic IgG antibodies of the Do-ath-Landsteiner type, must be excluded by a nega-

igure 2. Osmotic gradient ektacytometry of red blood cells witharying degrees of spectrin deficiency. In the spectrin-deficientells, the minimum deformability index observed in the hypotonicegion (thin arrow) is shifted to the right of the control (shadedrea), indicating a decrease in the cell surface area-to-volumeatio. The maximum deformability index (DImax) associated withhe spectrin-deficient cells (thick arrow) is less than that of controlells, implying reduced surface area. The more pronounced thepectrin deficiency, the greater is the loss of surface area and theower is the DImax. The osmolality in the hyperosmolar region athich the DI reaches half its maximum value is a measure of theydration state of the red cells. It is decreased in the patient withhe lowest spectrin content, indicating cellular dehydration. (Re-rinted with permission from Walensky LD, et al: Disorders of theed blood cell membrane, in Handin RI et al (eds): Blood: Principlesnd Practice of Hematology (ed 2). � 2003 Lippincott Williams &ilkins.)

ive Coombs test. The demonstration of a subnormal

sdn

SG

Mbstbcr1pfopbmlftau

oasbpm

pdlfcpstmmlct2ntu(

Fotos2

124 Eber and Lux

pectrin concentration in the red cell membrane canistinguish HS from AIHA, in which it is generallyormal.21

pecific Tests of Membrane Proteins andenesMeasuring membrane protein composition.easuring the amount of spectrin (and/or ankyrin,

and 3, or protein 4.2) in the red cell membrane canupport the diagnosis of HS in atypical cases (whenhere is concomitant presence of red blood cell anti-odies). However the measurements are difficult be-ause small variations from normal must be accu-ately measured; some patients with HS have only a0% to 15% decrement in the affected membranerotein. The simplest method is sodium dodecyl sul-ate polyacrylamide gel electrophoresis (SDS-PAGE)f red cell membranes, but to achieve the requiredrecision multiple samples must be tested. Becauseand 3 is used as an internal standard to quantitateembrane proteins on SDS-PAGE and some band 3 is

ost when membrane surface is shed and spherocytesorm, small decreases in the other membrane pro-eins may be hard to measure in mild cases. Themount of spectrin and ankyrin is best determined by

7,44 45

igure 3. Reduced expression of one cDNA allele in an ankyrin nof a patient with a nonsense mutation (stop codon in exon 28 of ahe patient is heterozygous in the genomic DNA, one of the two allf one cDNA allele. (Modified with permission from Ozcan R, et acreening is an efficient strategy for detecting ankyrin-1 mutatio003.)

se of RIA or ELISA. Unfortunately these meth- o

ds have not been developed to a robust routine levelnd they are not presently available. SDS-PAGE mea-urements are done in a few specialized laboratoriesut, in general, investigation of specific membraneroteins plays no practical part in the diagnosis oranagement of HS.Defining specific molecular defects. Several ap-

roaches have been used to define specific molecularefects. In families with multiple affected members,ack of linkage between HS and polymorphic markersor �- or �-spectrin, ankyrin, band 3, or protein 4.2an be used to genetically exclude specific membraneroteins from consideration. Frameshift and non-ense mutations are common in HS (see later sec-ion); they are often accompanied by absence of theutant mRNA as well as the protein due to nonsense-ediated mRNA decay,46 which can be detected by

oss of heterozygosity of a polymorphic reticulocyteDNA marker for one of the four HS genes comparedo the same marker in genomic DNA (Fig 3 and Table).47,48 This method also has been used to detect deovo dominant HS patients who lack a family his-ory.49 Finally, suspect proteins can be sequencedsing genomic DNA and polymerase chain reactionPCR) primers to amplify the exons.50 Because most

se mutation. SSCP analysis of genomic DNA and mRNA (as cDNA)n) on a chromosome with a polymorphic ankyrin marker.48 Whiles (nearly) missing in the cDNA, demonstrating reduced expressionultaneous (AC)n microsatellite polymorphism analysis and SSCPdominant hereditary spherocytosis. Br J Hematol 122:669-677,

nsennkyrieles il: Simns in

f the proteins that cause HS are large and contain

mlupcbpdiss

S

cSsmOtgp

cd1trylt

lntto

asaatiHfctnttasot

irnsasstmcMafationvtar

spdftScw

A

B

�

p

a

Hereditary Spherocytosis 125

any exons, this approach is arduous. Hence, a mo-ecular diagnosis of HS is of scientific interest, fornclear heritage in patients with severe disease, or inatients with atypical HS associated with other clini-al features (such as cardiomyopathy or spinocere-ellar degeneration) that suggest the mutation mayrovide some new insight into the function of theefective protein. A rational procedure in these cases

s to combine the use of polymorphic markers with aingle-strand conformation polymorphism (SSCP)creen,48 as will be discussed later.

Therapy

plenectomyPros and cons of splenectomy. HS is the most

ommon indication for splenectomy in childhood.plenectomy cures almost all patients with HS. Thepherocytes remain but the hemoglobin rises to nor-al and the reticulocyte count falls to 3% or less.nly in very rare patients with extremely severe HS is

he response incomplete, and even they experiencereat improvement following splenectomy7,23 (Lux S,ersonal communication).However, the indications for splenectomy must be

arefully weighed, as between 0.05 and 0.3 patientsie of fulminant postsplenectomy sepsis for every00 person-years of follow-up.51-55 A recent, long-erm 30-year evaluation up of more than 200 patientseported a frequency of 0.073 deaths per 100 person-ears.56 The actual incidence is lower because the risk ofethal sepsis declines with age and time postsplenec-

51,56

Table 2. Heterozygote Frequency of the CommonPolymorphisms of Candidate Genes for Spherocytosis

Gene Polymorphisms of cDNA*†

Proportion ofHeterozygous

Middle Europeans(%)

nkyrin-1 Exon 4 (cd 105 AAC3AAT)/Exon26 (cd 971 CTT3CTG)

39

Exon 18 (cd 691 GGC3GGT)/Exon21 (cd 783 ACC3ACT)

36

Exon 39 (cd 1755 GTG3GTA) 33VNDR of 3�- UTR (AC-repeat)47 54

and 3 Exon 11 (cd 417 CTG3TTG) 13Exon 12 (cd 441 CTG3CTA) 11

-Spectrin Exon 11 (N439S) 50Exon 14 (C3A at nucleotide 2249) 41Exon 16 (D1151N) 45

Abbreviation: cd, codon.*With the exception of exon 11 and 14 in �-spectrin all otherolymorphisms are silent.†�-Spectrin polymorphisms are according to Hassoun et al.101;ll others from Eber et al.50

omy—although it never disappears completely. m

The spleen is important in controlling parasitesike babesia and malaria, and patients who are sple-ectomized have an increased risk of fulminant infec-ions from these organisms, an increasingly impor-ant consideration today, when travel to distant partsf the world is common.Finally, the incidence of coronary heart disease

nd cerebral stroke is increased at least sixfold inplenectomized HS patients older than 40 years ofge.57 However the risk of coronary heart diseasefter splenectomy in patients is only slightly higherhan in the normal population.57,58 Much of thencreased risk of atherosclerosis in splenectomizedS patients seems to due to the loss of protective

actors (low hemoglobin and blood viscosity, lowholesterol level) associated with anemia ratherhan caused by the splenectomy.58 The risk inonsplenectomized (presumably anemic) HS pa-ients appears to be much lower than among con-rols. These results and the increased risk of sepsisrgue against splenectomy in all HS patients. Whohould be splenectomized, when and what kind ofperation should be performed, and how shouldhe patient be treated postoperatively?

Who should have a splenectomy?. Splenectomys indicated in moderate to severe or severe HS ifecurrent transfusions are necessary beyond the neo-atal period. Growth retardation (in severe transfu-ion-dependent HS) or significantly reduced physicalbility due to anemia (in moderate or moderatelyevere HS) probably also justify the operation. Occa-ionally, marked, persistent visible jaundice, due tohe combination of mild to moderate HS and ho-ozygosity for the Gilbert’s polymorphism, leads to

onsideration of a splenectomy for cosmetic reasons.ore controversial justifications include fatigue with

n attention deficit disorder or frequent absencesrom school plus consistently poor scholasticchievement. There is no proven relationship be-ween severity of HS and school performance, but themprovement in energy and behavior that is oftenbserved following splenectomy is suggestive. Sple-ectomy simply to remove a large spleen is not ad-ised because there is very little risk of splenic rup-ure in HS. A transfusion-dependent aplastic crisislso should not require a splenectomy because theisk of a second aplastic crisis is low.

Patients with severe HS and frequent transfusionshould have splenectomy around the age of 5. Werefer an earlier splenectomy rather than to initiateaily subcutaneous iron chelation with deferoxamineor transfusional iron overload, but we never under-ake splenectomy before the patient is 3 years of age.plenectomy is not recommended for mild HS duringhildhood and adolescence; exceptions may be adultsith mild HS undergoing cholecystectomy or with

arked jaundice, as noted earlier.

rstmtcalvatmdm

sccfts

d(e1gH

ofpRpts

anitt

FpsseB incott

126 Eber and Lux

Subtotal (partial) splenectomy. Because of theisk of postsplenectomy sepsis, subtotal (or partial)plenectomy has been advocated as an alterna-ive.59-63 Subtotal splenectomy has been done inore than 100 HS patients, who showed stable long-

erm improvement in hemoglobin and reticulocyteounts (Fig 4). Tchernia and coworkers59 and Rice etl62 remove about 80% to 90% of the enlarged spleen,eaving behind a remnant with about 25% of theolume of a normal spleen (�30 mL). Eber et al61 usemore radical approach, featuring near total splenec-

omy, and the remnant has a volume of only about 10L. Both approaches seem to ensure prolonged re-

uction, although not complete elimination, of he-olysis (Table 3).In the studies of Tchernia et al and Eber et al,

plenic phagocytosis, evaluated by postoperative nu-lear scintigraphic scans and by counts of pitted redells, showed that the splenic remnants retainedunction. It is not known whether subtotal splenec-omy prevents postsplenectomy sepsis, although pre-

igure 4. Long-term follow-up of subtotal splenectomy in heredrocedure in 40 patients (1 to 25 years).59 (A, B) Change in hemplenectomy (arrow). Mean and standard deviation are shown. Bustained for more than a decade. (C) The size of the spleen durspecially early. (Reprinted with permission from Walensky LD, etlood: Principles and Practice of Hematology (ed 2). � 2003 Lipp

umably the risk is decreased. s

Rapid regrowth of the splenic remnant was noteduring the first 2 years after surgery by both groupsFig 4). After this initial spurt, the remnant spleennlarged more slowly; regrowth has not ceased after2 years of follow-up. Thus, it is possible that re-rowth of the splenic remnant will eventually causeS to recur.In most patients the quality of life improves (92%

f patients) and there is a gain in physical growthollowing subtotal splenectomy. Children may alsorofit by improved school performance (Eber SW,iekhof J, unpublished data). In the French study,artial splenectomy did not fully prevent the forma-ion of gallstones, particularly in patients with moreevere disease.59

Indications for subtotal splenectomy. The majordvantages of subtotal splenectomy are long-termormalization of hemoglobin value, improved phys-

cal ability and school performance, comparable tohat obtained after full splenectomy, and avoidance ofransfusions. The procedure appears to reduce the

HS. The data are the result of 12 years of experience with thein and reticulocyte count with time before and after the subtotalthe rise in hemoglobin and decline in reticulocytess have beenhe same period; there is some regrowth of the splenic remnant,isorders of the red blood cell membrane, in Handin RI et al (eds):Williams & Wilkins.)

itaryoglobothing tal: D

everity of HS by about one grade and thus can be

rbetsshbeiHffrot

bsctargfn

pr

mmN

toamleciaatoplnwzUsss

no

O

NOP

P

P

P

S

L

s ved.

Hereditary Spherocytosis 127

ecommended for mild to moderate disease (see Ta-le 1) but not for moderately severe or severe dis-ase—these patients should have a total splenec-omy. Patients with mild disease tend to have aupernormal hemoglobin concentration after a fullplenectomy, which may predispose to coronaryeart disease. Also, a second operation likely will note necessary in patients with mild or moderate dis-ase. Due to the increased risk of postsplenectomynfection, children between 3 and 5 years with severeS who require a splenectomy should be considered

or subtotal splenectomy, with the realization that aull splenectomy may be needed later. We do notecommend any kind of splenectomy before 3 yearsf age or prophylactic removal of gallbladder at theime of subtotal splenectomy.

Subtotal splenectomy is usually an open operation,ut a laparoscopic procedure has been reported. Theubtotal operation is more time-consuming thanomplete splenectomy and recovery is longer, sinceotal splenectomy is frequently laparoscopic.64 Theuthors are aware only of one local bleeding episodeequiring conversion to a total splenectomy. Sur-eons are strongly advised to seek the advice andollow the guidelines of one of the experienced inter-ational groups.Prophylaxis after splenectomy. Virtually every

atient without a spleen has a significantly increased

Table 3. Success of Subtotal Spl

Bader-Meunier et al,59

2001de Bu

perative technique Subtotal (80-90%;ie, about 30 mLresidual volume)

Partithevo

o. of patients (age) 40 (1-15 yr) 5 (1-bservation period 14 yr 6 yrostoperative hemoglobin(g/dL)

12.7 � 1.2 8.3-1

ostoperative reticulocytes �50-66% of preoplevel

NA

ostoperately transfusedpatients

5 (aplastic crisis) 2

ostoperative splenicregrowth

�2� normal size Distin

econdary totalsplenectomy (years aftersurgery)

3 (3-6 yr) 2 (3-

oss of splenic remnant None None

Abbreviation: NA, data not available.*In no case was postsplenectomy sepsis observed.†Data of Stohr G, Sobh H, Eber SW 2003 (unpublished).‡In one patient the splenic remnant was lost due to an inappropplenectomy; after changing procedure, no further loss was obser

isk of severe infection, mostly Streptococcus pneu- a

oniae. Therefore, immunizations against S pneu-oniae, Haemophilus influenzae, and, in cetain cases,eisseria meningitidis are recommended.65

Penicillin should be administered after splenec-omy but there is controversy regarding the durationf prophylaxis. There is no scientific justification forny specific regimen. One of us (S.W.E.) recom-ends treatment for at least 3 years followed by

ifelong administration of broad-spectrum antibioticsarly in any unclear infection or high fever.65 Inhildren with subtotal splenectomy, continuous pen-cillin prophylaxis may be stopped as early as 1 yearfter surgery if phagocytic function is normal, asssessed by the proportions of pocked red cells or byhe uptake of radioactive colloid by the spleen. Thether author (S.E.L.) prefers lifelong penicillin pro-hylaxis. The main argument for this approach is thatate episodes of postsplenectomy sepsis, which areot uncommon,51 almost always occur in individualsho have a poor response to pneumococcal immuni-

ation,51 and who are not taking daily penicillin.56

nless pneumococcal antibody titers are be mea-ured periodically following immunization andhown to be satisfactory, this author believes it isafest to continue penicillin prophylaxis.

The standard regimens of prophylaxis after sple-ectomy with proven efficiency are penicillin V dosesf 200,000 IU (125 mg) twice per day until 5 years of

omy in Hereditary Spherocytosis

essingh et al,60

002* Rice et al,62 2003*

Eber et al,61 2001� data by Stohr et

al, 2003*†

mnant 1/4 ofrged splenic

Subtotal (80-97%) Near total: 10mL residualvolume

16 (4-15 yr) 29 (3-21 yr)6 yr 9 yr11.5-14.7 12.2-15.9

1.4-11.5% 2.6-13.3%

1 None

Stable at 15-30%of original sizefor 2 yr

To normal size

0 0

None 2 (early period)‡

surgical procedure (cauterization) in the first year after near total

enect

ys Ro2

al; reenla

lume7 yr)

2.8

ct

4 yr)

riate

ge and 400,000 IU (250 mg) twice per day after 5

yc

MsmpEwBudsw(ew

NAH

cpJ

pgedm

btbsil

aaisott8sp

mc

A

B

�

�

P

S

128 Eber and Lux

ears. In case of penicillin allergy an oral macrolide orephalosporin may be given.

Molecular Defects and Etiology of HS

eticulous work of several research groups hashown that HS is caused by defects in the red cellembrane proteins ankyrin, spectrin, band 3, and

rotein 4.2. There is no single frequent defect inuropean populations except in the rare patientsith HS caused by a defect in �-spectrin (see below).ecause most mutations are unique to a family, it issually not worth determining the specific molecularefect. Molecular analysis should be reserved forevere cases needing prenatal diagnosis or for HSith a unique phenotype of special scientific interest.

For a full list of all defects, the reader is referred toxcellent summaries by Walensky et al66 and Ya-ata.67)

ature of Defectssummary of the various molecular defects causingS is given in Table 4 and Fig 5.Ankyrin. Ankyrin defects are estimated to ac-

ount for 30% to 60% of HS in northern Europeanopulations,50,68,69 but only 5% to 10% of cases in

70

Table 4. Membrane Protein a

AffectedProtein

Frequency(% of HS) Heredity Prevailing Mutations

nkyrin US, Europe:30-60%

AD, AR AD: null mutations

Japan: 5-10% AR: missense; �promotermutation (often�108T3C)

and 3 20-30% AD All rare,functionally nullmutations

-Spectrin �5% AR �-Spectrin LEPRA(low expression,splicing defect)� nullmutations

-Spectrin 15-30% AD Null mutations

rotein 4.2 US, Europe:�5% Japan:45-50%

AR Missense (esp. 4.2Nippon)

Abbreviations: AD, autosomal dominant; AR, autosomal recesstates; EU, Europe.

apan. Patients with HS and ankyrin defects have m

rominent spherocytosis without other morpholo-ies. Hemolysis and anemia vary from mild to mod-rately severe.50,71,72 Ankyrin mutations cause bothominant and recessive HS and range from clinicallyild to severe.Because of its double linkage to �-spectrin and

and 3, ankyrin plays a pivotal role in the stabiliza-ion of the membrane. Ankyrin is the high affinityinding site for spectrin heterodimers, which aretable only when bound to the membrane.73 Since its present in limiting amounts, deficiency of ankyrineads to loss of both proteins.

Many spherocytes are deficient in both spectrinnd ankyrin. A combined deficiency of spectrin andnkyrin was first described in two patients with atyp-cal severe HS.22 Later, two independent groups mea-ured ankyrin and spectrin contents either by RIA44

r ELISA45 in 65 unrelated patients with typical HS:heir combined data (Fig 6) show that about two-hirds have a diminution of both proteins to 40% to0% of normal. The deficiency of one protein istrictly correlated with that of the other and is pro-ortional to clinical severity.Null mutations (frameshift, splicing, nonsenseutations) are frequent in dominant HS. Based on a

omprehensive genomic screen of 46 unrelated Ger-

ene Defects in Spherocytosis

rotein Reduction Hemolytic Anemia Abnormal Morphology

ctrin � ankyrin;5-50%

Mild tomoderatelysevere

Mostly typicalspherocytes

d 3 � 4.2: 15-0%

Mild to moderate Spherocytes, occasionalmushroom-shaped orpincered cells

ctrin: 50-75% Severe,transfusiondependent

Spherocytes, contractedcells and otherpoikilocytes,

ctrin: 15-40% Mild to moderate Spherocytes and 5-10%acanthocytes andechinocytes, orSpherocyticelliptocytes

tein 4.2: 95-00%

Mild to moderate Spherocytes,acanthocytes,ovalostomatocytes,

, dominant spherocytosis; R, recessive spherocytosis; US, United

nd G

P

Spe1

Ban4

Spe

Spe

Pro1

ive; D

an kindreds analyzed by SSCP and exon sequenc-

ifsfdqhiumstnsstoiaervn

fSt

mttddt

hln�craw(faB

(criam

ction

Hereditary Spherocytosis 129

ng,50 we concluded that ankyrin mutations accountor 45% to 65% of HS in European populations; aimilar high frequency of ankyrin mutations wasound by others.47,48 De novo mutations leading toecreased expression of one ankyrin allele are fre-uent in patients with HS who lack a positive familyistory49; frameshift or nonsense mutations prevail

n dominant HS. These null mutations result in eithernstable ankyrin transcripts or truncated peptides. Inost cases the mutant mRNA is destroyed by non-

ense-mediated mRNA decay46 and no abnormal pro-ein is detectable. Occasionally, if a truncation occursear the C-terminus, the mutant mRNA is not de-troyed and a stable, usually shortened protein re-ults. Ankyrin Rakovnik, a nonsense mutation withinhe C-terminal domain, leads to selective deficiencyf the major ankyrin isoform, band 2.1, but the minorsoform, band 2.2 is preserved.74 Ankyrin mutationsre located throughout the molecule, and nearly ev-ry family has its own mutation. The amount ofesidual “normal” ankyrin content is diminished inarious degrees due to differing compensation by theormal allele.Promoter defects and compound heterozygosity

or ankyrin defects are common in recessive HS.everal ankyrin defects have been identified in pa-

50

Figure 5. Membrane defects in HS affect the “vertical” intera

ients with recessive HS, mostly missense or pro- r

oter mutations. Mutations in the promoter disruptsranscription factor binding sites or insulator func-ion; examples include �108T3C, �153G3A, andel �72/73. In transgenic mice, these mutations re-uce the expression of a reporter gene, confirmingheir relevance to the pathogenesis of HS.75,76

In a few families with recessive HS, patients havead a defect in the ankyrin promoter or 5�-untrans-

ated region on one ankyrin allele and a missense or aull mutation in the other (Fig 7).50 The mutation108T3C50 in the ankyrin promoter is particularly

ommon and was found in four of eight families withecessive HS of different clinical severity (50%;llele frequency 0.29), in none of 29 patientsith dominant HS, and in 4% of 93 normal controls

allele frequency 0.02). In two of those families aurther missense mutation was found on the otherllele (ankyrin Walsrode [V463I] and ankyrinocholt [5619�16C3T]) (Fig 7).50

Ankyrin Walsrode contains a missense mutationV463I) in the band 3 binding domain and has de-reased affinity for band 377; the affected patient hased blood cells that are more deficient in band 3 thann spectrin or ankyrin, opposite of the trend in othernkyrin defects. Ankyrin Bocholt bears a missenseutation in a rare alternate splice product, that may

s connecting the membrane skeleton and the lipid bilayer.

esult in aberrant splicing. In each family one clini-

c(a

ihCtRTi8toppdrohdiqtn3m

ipaiihbamsb

p1ic(bstcnW3

tpkHf(ai(Kd3uabItwaareakttbb

bi

Fdalwf

130 Eber and Lux

ally unaffected parent carried the promoter defectFig 7). The heterozygous carriers of ankyrin Walsrodend ankyrin Bocholt are healthy.

Band 3 (AE1). Fifty-five band 3 gene mutations,ncluding 27 missense and 23 frameshift mutations,ave been described; all are rare private mutations.onserved arginine residues are frequent sites of mu-

ations; examples include arginines R490C, R518C,760Q, R808C, R808H, and R870W50,78 (Fig 8).hese highly conserved sites are positioned at the

nternal boundaries of transmembrane segments (Fig), and substitution probably interferes with co-ranslational insertion of band 3 into the membranesf the endoplasmic reticulum during synthesis of therotein. In one case, mRNAs for both alleles wereresent but the mutant band 3 was not detected,emonstrating either a functional defect in incorpo-ation of the protein into the membrane or instabilityf the mutant protein.79 The effect of the mutationas been studied in band 3 Prague, a 10 nucleotideuplication near the C-terminus80 that leads to a shiftn the reading frame and an altered C-terminal se-uence after amino acid 821. This mutation affectshe last transmembrane helix and probably elimi-ates the carbonic anhydrase II binding site on band81; it may also impair insertion of band 3 into theembrane and abolish anion transport function.80

igure 6. Spectrin and ankyrin diminution in HS. Combinedata44,45 show a good relationship between deficiency of ankyrinnd spectrin in most patients with HS. Many of the patients wereater proven to have specific ankyrin mutations.50 Some patientsith near normal concentrations of spectrin and ankyrin sufferrom HS due to band 3 defects.

Mutations in the cytoplasmic domain of band 3 can O

nterfere with its binding to other membrane skeletonroteins, resulting in a functional defect. An aminocid substitution (G130A) in the cytoplasmic domainn band 3 Fukuoka possibly affects protein 4.2 bind-ng.82 However, all patients with band 3 deficiencyave a proportional decrease in protein 4.2 (as inand 3 Montefiore and Tuscaloosa) because band 3pparently is needed for 4.2 stability or delivery to theembrane.83 A mild decrement in protein 4.2 may

imply reflect loss of band 3 rather than damage to 4.2inding sites.Band 3 deficiency causes HS in about 30% of Euro-

ean patients.69,70,78,84 The protein is decreased by5% to 40% in HS red cells. HS due to band 3 isnherited dominantly and is generally milder than HSaused by ankyrin or spectrin mutations. Most80%) band 3 deficient patients have a small num-er (1% to 2%) of mushroom-shaped cells in bloodmears, sometimes called “pincered” cells, as thoughhey had been pinched by a tweezers. These peculiarells seem to occur only in band 3 mutants (there areo mushroom-shaped red blood cells in ankyrinalsrode, a mutant ankyrin with predominant banddeficiency).Band 3 mutations sometimes also cause distal renal

ubular acidosis. The red cell form of band 3 is alsoresent in the acid-secreting intercalated cells of theidney cortical collecting ducts where it serves as theCO3-Cl� exchanger. Patients who are homozygous

or band 3 Coimbra (V488M)9 and band 3 Pribram1431�1G3A) have incomplete distal renal tubularcidosis.85 The patient with homozygous band 3 Co-mbra has chronic hyperchloremic metabolic acidosisplasma HCO3 15 mEq/L), a borderline low serum� (3.5 mEq/L), nephrocalcinosis, and evidence of aistal urinary acidification defect.9 In HS due to bandCampinas (694�1G3T), there is increased basal

rinary bicarbonate excretion but efficient urinarycidification.86 However, most patients with HS andand 3 deficiency do not have metabolic acidosis.87

n contrast, band 3 missense mutations other thanhose identified in HS have been found in patientsith dominant distal renal tubular acidosis without

n accompanying hemolytic anemia.87-90 Mutationsffecting Arg 589 are particularly common in distalenal tubule acidosis (eight of 11 families). Appar-ntly mutations in HS and distal renal tubule acidosisffect two different functional sites of band 3, and theidney lesion is not caused by heterozygous loss ofhe anion transport activity of band 3.87-89 Instead,he phenotype may result from faulty targeting ofand 3 to the apical rather than the basolateral mem-rane of collecting tubule type A intercalated cells.Protein 4.2. This form of HS is common in Japan

ut is rare in other populations (Table 4). Protein 4.2s either completely or almost completely absent.91

nly three mutations have been reported outside

Jpfitaehstlvapoctabdmde

ccbmi

sc

mmvtitosns

rdtHtd

pmdnmc3

Ft(t arent

Hereditary Spherocytosis 131

apan (protein 4.2 Tozeur, protein 4.2 Lisboa, androtein 4.2 Nancy). Protein 4.2 Nippon is the mostrequent mutation (A142T)92: it affects the process-ng of 4.2 mRNA, so that red blood cells contain onlyraces of the 72/74-kd isoforms instead of the usuallybundant 72-kd species. Most Japanese patients areither homozygous for 4.2 Nippon or compoundeterozygous for 4.2 Nippon and another missense orplice mutation, such as 4.2 Fukuoka (W119X), No-ame (a splice variant), or Shiga (R317C). The hemo-ytic anemia is moderate and red cell morphology isariable. Also the MCHC (34.8% � 0.1) is not as highs in other HS patients.91 In patients who totally lackrotein 4.2, the red blood cells may be a mixture ofvalocytes and stomatocytes with only a few sphero-ytes. This confusing phenomenon raises the ques-ion whether protein 4.2 Nippon should be classifieds HS.91 In addition, splenectomy markedly improvesut does not abrogate hemolysis in patients with HSue to 4.2 deficiency, also resembling hereditary sto-atocytosis (see article by Delaunay in this issue). The

isease is inherited as an autosomal recessive trait. Het-rozygous parents are asymptomatic.

As noted above, mild red cell protein 4.2 deficien-ies are associated with primary loss of band 3, whichontains its binding site.93 Protein 4.2 probably alsoinds to ankyrin. Protein 4.2 Nippon–deficientembranes lose 70% of their ankyrin during low

igure 7. Compound heterozygosity for ankyrin defects in recesshe ankyrin promoter (�108T3C) on one allele and a missense m[619�16C3T]) on the other allele.50 Note the greater reduction oo the weakened binding of ankyrin Walsrode to band 3.76 Both p

onic strength extraction, where ankyrin is usually p

table,70,92 and the ankyrin loss is blocked by prein-ubation of the membranes with purified protein 4.2.

Spectrin. Spectrin deficiency is the most com-on protein alteration in HS. Red blood cells withutations in the ankyrin or spectrin genes show

arious degrees of spectrin deficiency,5,7,21,23 the ex-ent of which is related to red cell spheroidic-ty,21,23,40 the severity of hemolysis,21,23 and the resis-ance of red cell membranes to shear stress.40,94 Theverall architecture of the membrane skeleton is pre-erved in spectrin-deficient red blood cells, but theumber of junctional complexes interconnectingpectrin, actin, and protein 4.1 is reduced.

In general, HS caused by �-spectrin defects is aecessive trait and that due to �-spectrin mutations isominant, because �-spectrin chains are produced inhree- to fourfold excess compared with �-spectrin.95

ence, a moderate reduction of �-spectrin produc-ion, as would be seen in a heterozygote, would notecrease formation of the spectrin ��/�� dimer.Patients with �-spectrin defects are often com-

ound heterozygous for missense and low expressionutations. They have a marked reduction of spectrin

imer content (25% to 50% of normal). In many butot all of these families, one allele contains a missenseutation in the �-II domain (�-spectrin Bughill) that

hanges an alanine to an aspartic acid at residue09 (A970D).96 Peptide analysis of the �-spectrin

S. In two families with recessive HS, the patients had a defect inion (ankyrin Walsrode [V463I]) or a splice defect (ankyrin Bocholtd 3 than ankyrin in the patient with ankyrin Walsrode—this is dues arere unaffected carriers of one of the two mutations.

ive Hutatf ban

eptides from the affected patients shows only

�vwpddtpotsaT(laHa

ct

sscu

qsminftctne

tPmb2al

scnt�aCctpTswet

PIP

Idpfmaoniro

FpamaF(citAti

132 Eber and Lux

-spectrin Bughill, but genomic DNA analysis re-eals both an allele with �-spectrin Bughill and oneithout, indicating that the second �-spectrin allelerobably contains a null mutation.96 A candidateefect was discovered in a family with severe, non-ominant HS.8 One of the alleles, designated �-spec-rin Prague (5187-2A3G), had a mutation in theenultimate position of intron 36, leading to skippingf exon 37 and premature termination of the �-spec-rin peptide. The other �-spectrin allele had a partialplicing abnormality in intron 30 and produced onlybout one sixth of the normal amount of �-spectrin.his low-expression allele, named �-spectrinLEPRA

low-expression allele Prague) (4339-99C3A), wasinked to the �-spectrin Bughill variant in this patientnd several others with severe, nondominant HS.97

omozygosity for �-spectrinLEPRA alone does notppear to cause disease.

Patients with recessive HS and �-spectrin defi-iency are rare. Clinically, only homozygous �-spec-

6-8,21

igure 8. Defects in the transmembrane region of band 3 inatients with HS. The structure of band 3 is shown schematicallynd is not to scale or correctly folded. Missense mutations areostly substitutions of critical arginine residues (closed triangles)t the internal boundaries of transmembrane segments of band 3.rameshift mutations (open doughnuts) and nonsense mutationsclosed rectangles) are also shown. Homozygous band 3 Coimbraauses severe hydrops fetalis. Band 3 Coimbra is associated withncomplete renal tubular acidosis. Band 3 Prague is a 10 nucleo-ide duplication near the C-terminal end. The deletion in South Eastsian ovalocytosis (SAO) just at the transition from the cytoplasmico the transmembrane portion of band 3 is marked because of itsmportance, although SAO is not a form of HS.

rin deficiency causes hemolytic anemia. Blood m

mears contain numerous spherocytes and micro-pherocytes. Patients with very severe spectrin defi-iency may also have misshapened spherocytes, spic-lated red blood cells, and bizarre poikilocytes.21,23

Monoallelic expression of �-spectrin occurs fre-uently in HS patients with spectrin deficiency,2,6,98

uggesting that null mutations of �-spectrin are com-on. About 15 null mutations have been described,

ncluding initiation codon disruption, frameshift andonsense mutations, gene deletions and splicing de-

ects. �-spectrin Kissimmee, from a missense muta-ion (W202R),99 is both unstable and defective in itsapacity to bind protein 4.1100 (Fig 9). Patients withhis spectrin–4.1 binding defect have only 80% oformal red blood cell spectrin, which may be the realxplanation of their HS.

Overall, �-spectrin defects account for about 15%o 30% of HS in northern European populations.atients with �-spectrin deficiency typically haveild to moderately severe HS. Where described, their

lood smears contain moderate numbers (8% to0%) of spiculated red blood cells (echinocytes andcanthocytes)4,100 as well as spherocytes, a fairly re-iable differentiating feature.

Spectrin defects that affect the self-association ofpectrin �/�-heterodimers lead to hereditary ellipto-ytosis (HE) or hereditary pyropoikilocytosis (HPP),ot HS (see article by Gallagher in this issue). Most ofhese defects involve the N-terminal end of the-spectrin chain but some are missense mutationst the C-terminus of the �-chain (as in �-spectrinsagliari, Providence, and Buffalo). C-terminal trun-ations of the �-spectrin chain that damage the spec-rin self-association site and remove the more distalhosphorylation region (as in �-spectrins Prague,andil, Nice, Campinas, and Gottingen) causepherocytic elliptocytosis—rounded elliptocytesith an increased osmotic fragility. The molecular

xplanation of this interesting intermediate pheno-ype is unknown.

rocedure for Molecular Screening Usingnnocent Polymorphisms of Membranerotein Genes

n addition to pathogenetically relevant molecularefects in HS, a wide variety of silent or innocentolymorphisms have been detected during screeningor ankyrin, spectrin, and band 3 genes. These poly-

orphisms offer a rational approach to the study ofnkyrin and other membrane proteins.48 Frameshiftr nonsense mutations are most common in domi-ant HS in most cases, and no mutant RNA or protein

s present in reticulocytes and mature erythrocytes,espectively, reflecting either reduced transcriptionf one of the ankyrin alleles or instability of its

RNA. In either case, finding that one ankyrin,

�pcadt

tsagr(aa3odrbba

LTit

tsuiststb

tvttaapsst8tHiisrs

F�btddb Prac

Hereditary Spherocytosis 133

-spectrin, or band 3 gene is not expressed, by com-aring frequent polymorphisms in genomic DNA andDNA (mRNA), is proof that a null mutation exists,nd is an efficient procedure for identifying candi-ates for frameshift or nonsense mutations in one ofhe three genes.

Limited characterization of the underlying muta-ion may be useful in families with unclear heritage orevere or atypical disease. Because ankyrin mutationre most common, one option is to first compareenomic DNA and reticulocyte mRNA (as cDNA) foreduced expression of one cDNA allele using theAC)n repeat of ankyrin47-49 or other commonnkyrin (ANK1) polymorphisms (Table 2).50 If neg-tive, reduced expression of one allele of band50,84or �-spectrin2,101 can be sought. SSCP screeningf all the exons in the candidate gene can then beone in patients who show absence of one allele ineticulocyte RNA. Alternatively, candidate genes maye identified by SDS-PAGE analysis of red cell mem-rane proteins and then characterized by SSCPnd/or DNA sequencing.

Pathophysiology

oss of Membrane Surface by Vesiculationhe primary membrane lesions described above all

nvolve the “vertical interactions” between the skele-

igure 9. Defect in the binding site for protein 4.1 in �-spectrin-spectrin Kissimmee was passed over a column containing immobound and could be eluted under conditions unfavorable for specthe column and, in a separate assay, this fraction (Sp Kissimmee)efect to �-spectrin (W202R).99 The mutation is in a conserved reomain. The region is a likely location for the 4.1 binding site. (Relood cell membrane, in Handin RI et al (eds): Blood: Principles and

on and the bilayer, consistent with the prevailing a

heory that HS is caused by local disconnection of thekeleton and bilayer, followed by vesiculation of thensupported surface components. These processes,

n turn, lead to progressive reduction in membraneurface area and to a “spherocyte,” actually a shapehat ranges between a thickened discocyte and apherostomatocyte. The phospholipid and choles-erol contents of isolated spherocytes are decreasedy 15% to 20% due to the loss of surface area.102

Since budding red cells are rarely observed inypical blood smears of patients with HS, microscopicesicles are probably lost; loss may occur preferen-ially in bywaters of the circulation, such as the re-iculoendothelial system. When membrane vesiclesre induced in normal red blood cells, they originatet the tips of spicules, where the lipid bilayer uncou-les from the underlying skeleton.103 The vesicles aremall, about 100 nm, and devoid of hemoglobin andkeletal proteins, so that they are invisible on conven-ional examination of stained blood films. Tiny 50- to0-nm bumps that may be vesicles have been de-ected in HS red cells on atomic force microscopy.104

owever, the hypothesis that spherocytes in HS orig-nate by membrane vesiculation during shear stressn the circulation or in the metabolically inhospitableplenic cord and reticuloendothelial system has beenecently questioned by Costa et al,105 who found aimilar decrease in the surface area of mature red cells

mmee. Spectrin from a normal individual or from a patient withnormal protein 4.1. All normal spectrin and 61% of the HS spectrin1 interactions. However, 39% of the HS spectrin failed to bind tod the ability to bind protein 4.1. Subsequent studies localized thein the N-terminal end of �-spectrin, just beyond the actin bindinged with permission from Walensky LD, et al: Disorders of the redtice of Hematology (ed 2). � 2003 Lippincott Williams & Wilkins.)

Kissiilizedrin-4.lackegionprint

nd reticulocytes in HS. Their findings suggest in-

sd

ISITdep1acda

saEmlmli

irbmmctivdpd

aotma3crmTrmtad

LHrtiblcts

StFpswtw

cfcgtt

Fb“dtbiiloicwWH(

134 Eber and Lux

tead that surface area loss occurs in the bone marrowuring the genesis of HS red blood cells.105

s HS Caused by Disconnection of thekeleton or a Lack of Band 3-Lipidnteractions?he observation that spectrin or spectrin-ankyrineficiencies are common in HS has led to the hypoth-sis that interactions of spectrin with bilayer lipids orroteins are required to stabilize the membrane (Fig0, Hypothesis 1). Budding off of spectrin-deficientreas would lead to HS. Unexplained is how sphero-ytes develop in patients whose red blood cells areeficient in band 3 or protein 4.2 but have normalmounts of spectrin.83,92

The alternate hypothesis argues that the bilayer istabilized by interactions between lipids and thebundant band 3 molecules (Fig10, Hypothesis 2).ach band 3 contains about 14 hydrophobic trans-embrane helices, many of which must interact with

ipids. In deficient red cells the area between band 3olecules would increase, on average, and the stabi-

izing effect would diminish. Transient fluctuations

igure 10. Two hypotheses concerning the mechanism of mem-rane loss in hereditary HS. Hypothesis 1 assumes that themembrane” (the lipid bilayer and integral membrane proteins) isirectly stabilized by interactions with spectrin or other elements ofhe membrane skeleton. Spectrin-deficient areas, lacking support,ud off, leading to HS. Hypothesis 2 assumes that the membranes stabilized by interactions of band 3 with neighboring lipids. Thenfluence of band 3 extends into the lipid milieu because the firstayer of immobilized lipids slows the lipids in the next layer and son. In band 3–deficient cells the area between lipid moleculesncreases and unsupported lipids are lost. Spectrin-ankyrin defi-iency allows band 3 molecules to diffuse and transiently cluster,ith the same consequences. (Reprinted with permission fromalensky LD, et al: Disorders of the red blood cell membrane, inandin RI et al (eds): Blood: Principles and Practice of Hematologyed 2). � 2003 Lippincott Williams & Wilkins.)

n the local density of band 3 could aggravate this

nstability and allow unsupported lipids to be lost,esulting in HS. Spectrin- and ankyrin-deficient redlood cells could become spherocytic by a similarechanism. Since spectrin filaments corral band 3olecules and limit their lateral movement,106 a de-

rease in spectrin would allow band 3s to diffuse andransiently cluster, fostering vesiculation. However,t is more likely that both mechanisms operate toariable degrees in different diseases: hypothesis 1ominating in spectrin and ankyrin defects, and hy-othesis 2 controlling in band 3 and protein 4.2isorders.

Spectrin and ankyrin deficiency lead to secondarylterations of band 3, such as increased mobility,ligomerization, and aggregation, which could con-ribute to the loss of spectrin-free vesicles from theembrane. Red blood cells from HS patients with

nkyrin mutations exhibit a marked increase in bandrotational diffusion.107 The magnitude of the in-

rease correlates inversely with the ankyrin/band 3atio and with the fraction of band 3 retained in theembrane skeleton following detergent extraction.hese data suggest that ankyrin deficiency relaxesotational constraints on the population of band 3olecules. Increases in band 3 rotation could be due

o release of band 3 from low-affinity binding sites onnkyrin, and there is evidence for increased band 3ensity and aggregation in HS.108

oss of Cellular Deformabilityereditary spherocytes hemolyze because of the

heologic consequences of their decreased surface-o-volume ratio. The red cell membrane is very flex-ble but can only expand its surface area 2% to 3%efore rupturing. Thus, the cell becomes increasinglyess deformable as surface area is lost. For red bloodells in HS, poor deformability is a hindrance only inhe spleen, since the cells have a nearly normal life-pan following splenectomy.

equestration of Hereditary Spherocytes inhe Spleenigure 11 illustrates our current understanding of theathophysiology of HS. The dominant role of thepleen is unquestioned, but the exact mechanism byhich HS red cells are further damaged (“condi-

ioned”) and ultimately removed in the spleen is notell understood.Spherocytes are selectively sequestered at the

ordal-sinus junction. As a consequence, spleensrom patients with HS have massively congestedords and relatively empty sinuses. In electron micro-raphs, few spherocytes are seen in transit throughhe sinus wall, in contrast to normal spleens whereransiting cells are readily found.109

Spherocytes are “conditioned” in the metaboli-

cddctmtnt

itdtcov(tavbhp

CcmhChr

ewsbnp

S

Rampms

FdmptaooW

Hereditary Spherocytosis 135

ally inhospitable splenic cords. There is also abun-ant evidence that red blood cells in HS suffer duringetention in the spleen. The mechanism of this pro-ess, called “splenic conditioning,” is less certain dueo the lack of information about the cordal environ-ent.110 The average residence time of HS red cells in

he splenic cords is 30- to 300-fold longer than that oformal red blood cells; still, this delay is far short ofhe time required for metabolic depletion.

Consequences of splenic trapping. The follow-ng consecutive damage in the spleen should lead tohe premature hemolysis of spherocytes. (1) Oxi-ants may exacerbate membrane and water loss. Po-assium loss and membrane instability may be in-reased by the high concentrations of acids andxidants that must exist in a spleen filled with acti-ated macrophages ingesting trapped HS red cells.2) Splenic residence may activate membrane pro-eases. Oxidatively damaged membrane proteins arelso subject to proteolysis in vitro111; proteolysis inivo would contribute to skeletal weakness and mem-rane loss. (3) Macrophages may directly conditionereditary spherocytes. The involvement of macro-

igure 11. Pathophysiology of the splenic conditioning andistinctly with functionally similar defects (such as frameshiftodifying factors must exist that regulate the clinical expressirotein 4.2. The primary defect leads to a weak “vertical” interahe plane of the lipid bilayer (such as band 3). Secondary eventsnchoring of the lipids and may contribute to the loss of spectrinf “splenic conditioning” that ultimately lead to premature hemf the red blood cell membrane, in Handin RI et al (eds): Blooilliams & Wilkins.).

hages is supported by the historic observations of e

oleman and Finch,112 who found that large doses ofortisone markedly ameliorated HS in nonsplenecto-ized patients. Similar doses of corticosteroids in-ibit splenic processing and destruction of IgG-or3b-coated red blood cells in patients with immuno-emolytic anemias, probably by suppressing mac-ophage-induced red cell sphering and phagocytosis.

Binding of naturally occurring band 3 antibodiesnhances the premature clearance of spherocytesith band 3 deficiency from the circulation.113 In

plenectomized patients with band 3 deficiency, redlood cell deformability inversely correlates with theumber of red cell–bound IgG molecules (up to 140er cell).

ummary of Pathophysiology

ed cells in HS are selectively detained by the spleennd this custody is detrimental, leading to a loss ofembrane surface that fosters further splenic trap-

ing and eventual destruction (Fig 11). The primaryembrane defects involve deficiencies or defects of

pectrin, ankyrin, protein 4.2, or band 3, but the

uction of hereditary spherocytes. The clinical symptoms varyonsense mutations with no abnormal protein present). Thusprimary defects in the genes for ankyrin, band 3, spectrin, orbetween the membrane skeleton and the integral proteins in

a disturbed structure and function of band 3 that decreases thelipid vesicles. The circle at the right represent the many factors. (Modified with permission from Walensky LD, et al: Disordersinciples and Practice of Hematology (ed 2). � 2003 Lippincott

destror n

on ofctionare-freeolysisd: Pr

tiologic relationship of these defects to surface loss

ibqlitacdbblcm

Tboccdssbtaa

STc(

atslcHlasnoo

cafe

ANo

sdcaptn1crhblrad

maclbn

CIo

amfipiHni

smptccstarssbfTibl

136 Eber and Lux

s less clear. Current speculation is that the mem-rane skeleton (including band 3) may not ade-uately support all regions of the lipid bilayer in HS,eading to loss of small areas of untethered lipids andntegral membrane proteins. Uncertain is whetherhe effect is directly due to deficiency of spectrin andnkyrin, or spectrin-ankyrin deficiency indirectly in-reases the lateral mobility of band 3 molecules andecreases their stabilization of the lipid bilayer, oroth (Fig 11). In addition, the loss of band 3 due toand 3 or protein 4.2 defects may directly diminishipid anchoring in HS. The mechanisms of spleniconditioning and red blood cell destruction also re-ain uncertain.

Animal and Fish Models of HS

he availability of well-characterized mouse and ze-rafish models has contributed to our understandingf the pathophysiology of HS. Four types of sphero-ytic hemolytic anemia have been identified in theommon house mouse, Mus musculus114: ja/ja (jaun-ice); sph/sph (spherocytosis) and its alleles [sph1J/ph1J (hemolytic anemia), sph2J/sph2J (now lost),ph2BC/sph2BC, and sphDem/sphDem]; nb/nb (normo-lastosis); and wan/wan. The nomenclature indicateshat anemia is observed only in the homozygous statend that the mutants represent four loci: ja, sph, nb,nd wan. All of the mutants have severe hemolysis.

pectrin Mutantshe ja/ja mutant has no detectable spectrin. The micearry a nonsense mutation in the �-spectrin geneR1160X).

The sph/sph variants lack �-spectrin but have smallmounts of �-spectrin; they have defects in �-spec-rin synthesis, function, and/or stability. The sph andph2BC alleles are frameshift mutations and null al-eles. These mice have both spherocytes and ellipto-ytes, and some poikilocytes, and are a cross betweenS and hereditary pyropoikilocytosis. The sph1J al-

ele, previously called ha and sphha, lacks the last 13mino acids of �-spectrin and exhibits markedpherocytosis.115 The mutant protein is produced inearly normal amount, proving that the C-terminusf �-spectrin has some critical although still mysteri-us function in supporting the lipid bilayer.

Cardiac thrombi, fibrotic lesions and renal hemo-hromatosis are found in ja/ja and sph/sph mice indulthood.116 Transplantation of hematopoietic cellsrom sph/sph mice are sufficient to induce thromboticvents in the recipients.117

nkyrin Mutantsb/nb mice have 50% to 70% of the normal quantity

f spectrin and no normal ankyrin. They have normal t

pectrin synthesis118 but are moderately spectrin-eficient because their ankyrin is very unstable (inontrast with human ankyrin deficiency wherenkyrin and spectrin levels are comparably de-ressed). The nb mutation causes premature termina-ion of the ankyrin protein in exon 36 at the begin-ing of the C-terminal domain. A small amount of the57-kd remnant is found in mature nb/nb red bloodells and, along with some expression of an Ank2-elated peptide, may explain why fetal nb/nb miceave normal reticulocyte counts and no anemia atirth.119 Humans may also be protected in utero, ateast partially, since hydrops fetalis has not beeneported in patients with ankyrin defects or probablenkyrin defects (such as combined spectrin-ankyrineficiency).

The nb/nb mice develop ataxia when they reachaturity, due to loss of cerebellar Purkinje cells120;

nk-1 protein is markedly reduced in the Purkinjeells, which may explain their fragility. Spinocerebel-ar degeneration and related syndromes have alsoeen reported in a few adults with HS, although it isot yet known whether ankyrin is affected.

linical Variability of Band 3 Mutationsndicates the Presence of Genetic Modifiersf Disease Severity

Complete absence of band 3 was first described inrecessive form of HS in cattle , due to a nonsenseutation at codon 646.89 The cattle, like band 3–de-

cient mice and humans, have deficient anion trans-ort, lack protein 4.2, and have a reduced number ofntramembranous particles by electron microscopy.owever, they have a relatively mild hemolytic phe-otype compared to other band 3–deficient organ-

sms.New mouse mutants with defects in membrane

keleton proteins have been generated by targetedutagenesis in embryonic stem cells. Mice com-

letely deficient in band 3 survive gestation but tendo die in the neonatal period,83 often from thromboticomplications.121 Survivors have a profound sphero-ytic hemolytic anemia, closely resembling the mostevere forms of HS in humans. The mice have unde-ectable protein 4.2 and glycophorin A but normalmounts of spectrin, actin, and protein 4.1 in theired cell membrane skeletons, and normal membranekeleton architecture by electron microscopy. De-pite their normal skeletons, red blood cells lackingand 3 shed astonishing amounts of membrane sur-ace in small vesicles and long tubules (Fig 12).83

hese observations indicate that band 3 is, surpris-ngly, not required for membrane skeleton assemblyut has a critical function in stabilizing membraneipids. Loss of this function may be fundamental to

he pathogenesis of HS.

Cg(wms

vcgb(mttm

cgireDTabbtvnka

ibssdpja3ttTpwr

Tds

FShNMOfr(tPW

Hereditary Spherocytosis 137

A newly discovered mouse mutation (wan), in a3H/HeJ background, is a null defect in the band 3ene. wan/wan mice also have a very severe anemia100% lethal in the neonatal period). But when wan/an mice are crossed to wild-type Mus castaneusice, the F2 generation shows a wide variation in

igure 12. Marked membrane fragility of mice lacking band 3.canning electron micrographs of red cells from wild-type (A),eterozygous (B), and homozygous band 3–deficient (C-E) mice.83.ote the markedly spherocytic shape of the band 3 (-/-) red cells.any are shedding multiple tiny membrane vesicles (arrowheads).thers extrude rod-like membrane extensions (arrows), which arerequently detached (D, arrow). The membrane extensions ofteneach considerable length and are highly coiled (E). Bar, 1 �m.Reprinted with permission from Walensky LD, et al: Disorders ofhe red blood cell membrane, in Handin RI et al (eds): Blood:rinciples and Practice of Hematology (ed 2). � 2003 Lippincottilliams & Wilkins.)

everity, from lethal anemia to normal hematologic

alues (hemoglobin, hematocrit, and mean corpus-ular volume). These observations show that a strongenetic modifier is segregating in the M castaneusackground. A genome-wide scan for the modifiertermed a “quantitative trait locus” or QTL), usingean corpuscular volume as the variable trait, iden-

ified a single QTL on chromosome 12, centered overhe �-spectrin gene,122 an obvious candidate for aodifier gene.Band 3–wan/wan and band 3 knockout mice also

ontain excess binucleate erythroblasts, which sug-ests that absence of band 3 is associated with a defectn cytokinesis. Indeed, in homozygous retsina (ret/et) zebrafish with HS the mitotic spindles of laterythroblasts are grossly disrupted and the masses ofNA separate poorly and interfere with cell cleavage.his behavior explains the high frequency of binucle-te cells in zebrafish and mouse erythroblasts lackingand 3, but not in other forms of severe HS.123 Sinceand 3 is only expressed in erythroblasts and one cellype in the kidney collecting duct, the data imply thatertebrates have developed a special kind of cytoki-esis for the last one or two cell divisions, a cyto-inesis that depends on band 3—perhaps to helpttach the mitotic spindle to the poles of the cell.

Zebrafish with spherocytosis and null mutationsn �-spectrin (reisling) and protein 4.1 (merlot/cha-lis) also have been identified.124,125 These fishhould prove useful for in vivo structure-functiontudies, since it is relatively easy to test whether aerivative of the missing protein can rescue the HShenotype. For example, normal band 3 cDNA in-ected into a one- or two-cell embryo rescues thenemia of band 3–deficient ret/ret zebrafish, but bandlacking both 4.1 binding sites is unable to rescue

hese deficient animals124; band 3 with only one ofhe two 4.1 sites has intermediate rescue function.he 4.1-band 3 interaction appears functionally im-ortant, at least in fish, and likely band 3–skeleton asell as band 3–lipid interactions have pathogenic

oles in HS.

Acknowledgmenthe authors thank K. Zurbriggen and B. Siegfried for re-actional work and S. Staubli for drawing (all from Univer-itakts-Kinderklinik Zurich).

References1. Vanlair CF, Masius JB: De la microcythemie. Bull R Acad

Med Belg 5:515-613, 18712. Miraglia del Giudice E, Lombardi C, Francese M, et al:

Frequent de novo monoallelic expression of �-spectrin gene(SPTB) in children with hereditary spherocytosis and iso-lated spectrin deficiency. Br J Haematol 101:251-254, 1998

3. Ozcan R, Kugler W, Feuring-Buske M, et al: Parental mosa-icism for ankyrin-1 mutations in two families with heredi-

tary spherocytosis. Blood 90:4a, 1997 (suppl, abstr)

138 Eber and Lux

4. Basseres DS, Duarte AS, Hassoun H, et al: �-Spectrin S(ta)Barbara: A novel frameshift mutation in hereditary sphero-cytosis associated with detectable levels of mRNA and agerm cell line mosaicism. Br J Haematol 115:347-353, 2001

5. Eber SW, Armbrust R, Schroter W: Variable clinical severityof hereditary spherocytosis: Relation to erythrocytic spec-trin concentration, osmotic fragility, and autohemolysis.J Pediatr 117:409-416, 1990

6. Miraglia del Giudice E, Nobili B, Francese M, et al: Clinicaland molecular evaluation of non-dominant hereditaryspherocytosis. Br J Haematol 112:42-47, 2001

7. Agre P, Orringer EP, Bennett V: Deficient red-cell spectrin insevere, recessively inherited spherocytosis. N Engl J Med306:1155-1161, 1982

8. Wichterle H, Hanspal M, Palek J, et al: Combination of twomutant �-spectrin alleles underlies a severe spherocytichemolytic anemia. J Clin Invest 98:2300-2307, 1996

9. Ribeiro ML, Alloisio N, Almeida H, et al: Severe hereditaryspherocytosis and distal renal tubular acidosis associatedwith the total absence of band 3. Blood 96:1602-1604, 2000

10. Perrotta S, Nigro V, Iolascon A, et al: Dominant hereditaryspherocytosis due to band 3 Neapolis produces a life-threat-ening anemia at the homozygous state. Blood 92:9, 1998(abstr)

11. Chilcote RR, Le Beau MM, Dampier C, et al: Association ofred cell spherocytosis with deletion of the short arm ofchromosome 8. Blood 69:156-159, 1987

12. Lux S, Tse W, Menninger J, et al: Hereditary spherocytosisassociated with deletion of the human erythrocyte ankyringene on chromosome 8. Nature 345:736-739, 1990

13. Fukushima Y, Byers MG, Watkins PC, et al: Assignment ofthe gene for � spectrin (SPTB) to chromosome 14q23-q24.2by situhybridization. Cytogenet Cell Genet 53:232-233,1990

14. Palek J, Lambert S: Genetics of the red cell membraneskeleton. Semin Hematol 27:290-332, 1990

15. Friedman EW, Williams JC, Van Hook L: Hereditary sphero-cytosis in the elderly. Am J Med 84:513-516, 1988

16. Krueger HC, Burgert EOJ: Hereditary spherocytosis in 100children. Mayo Clin Proc 41:554-567, 1966

17. Jensson O, Jonasson JL, Magnusson S: Studies on hereditaryspherocytosis in Iceland. Acta Med Scand 201:187-195,1977

18. Koepke JF, Koepke JA: Reticulocytes. Clin Lab Haematol8:169-179, 1986

19. Guarnone R, Centenara E, Zappa M, et al: Erythropoietinproduction and erythropoiesis in compensated and anaemicstates of hereditary spherocytosis. Br J Haematol 92:150-154, 1996

20. Pajor A, Lehoczky D, Szakacs Z: Pregnancy and hereditaryspherocytosis. Report of 8 patients and a review. Arch Gy-necol Obstet 253:37-42, 1993

21. Agre P, Casella JF, Zinkham WH, et al: Partial deficiency oferythrocyte spectrin in hereditary spherocytosis. Nature314:308-383, 1985

22. Coetzer TL, Lawler J, Liu SC, et al: Partial ankyrin andspectrin deficiency in severe, atypical hereditary spherocy-tosis. N Engl J Med 318:230-234, 1988

23. Agre P, Asimos A, Casella JF, et al: Inheritance pattern andclinical response to splenectomy as a reflection of erythro-cyte spectrin deficiency in hereditary spherocytosis. N EnglJ Med 315:1579-1583, 1986

24. Godal HC, Heist H: High prevalence of increased osmoticfragility of red blood cells among Norwegian donors. ScandJ Haematol 27:30-34, 1981

25. Eber SW, Pekrun A, Neufeldt A, et al: Prevalence of in-

creased osmotic fragility of erythrocytes in German blooddonors: Screening using a modified glycerol lysis test. AnnHematol 64:88-92, 1992

26. Maberry MC, Mason RA, Cunningham FG, et al: Pregnancycomplicated by hereditary spherocytosis. Obstet Gynecol79:735-738, 1992

27. Iolascon A, Faienza MF, Moretti A, et al: UGT1 promoterpolymorphism accounts for increased neonatal appearanceof hereditary spherocytosis. Blood 91:1093, 1998 (letter)

28. Delhommeau F, Cynober T, Schischmanoff PO, et al: Natu-ral history of hereditary spherocytosis during the first year oflife. Blood 95:393-397, 2000

29. Tchernia G, Delhommeau F, Perrotta S, et al: Recombinanterythropoietin therapy as an alternative to blood transfu-sions in infants with hereditary spherocytosis. BJH 1:146-152, 2000

30. Young N: Hematologic and hematopoietic consequences ofB19 parvovirus infection. Semin Hematol 25:159-172, 1988

31. Pekrun A, Eiffert H, Eber SW, et al: Aplastische Krisen beihereditarer Spharozytose. Monatsschr Kinderheilkd 136:173-175, 1988

32. Peter G: Parvovirus B19, in Red Book: Report of the Com-mittee on Infectious Diseases (ed 24). Elk Grove Village, IL,American Academy of Pediatrics, 1997, p 383

33. Malinow M, Duell PB, Hess DL, et al: Reduction of plasmahomocyst(e)ine levels by breakfast cereal fortified with folicacid in patients with coronary heart disease. N Engl J Med338:1009-1015, 1998

34. Bosma PJ, Chowdhury JR, Bakker C, et al: The genetic basisof the reduced expression of bilirubin UDP-glucuronosyl-transferase 1 in Gilbert’s syndrome. N Engl J Med 333:1171-1175, 1995

35. Miraglia del Giudice E, Perrotta S, Nobili B, et al: Co-inheritance of Gilbert syndrome increases the risk for devel-oping gallstones in patients with hereditary spherocytosis.Blood 92:470a, 1998 (suppl, abstr)

36. Marchetti M, Quaglini S, Barosi G: Prophylactic splenec-tomy and cholecystectomy in mild hereditary spherocytosis:Analyzing the decision in different clinical scenarios. J In-tern Med 244:217-226, 1998

37. Michaels LA, Cohen AR, Zhao H, et al: Screening for hered-itary spherocytosis by use of automated erythrocyte indexes.J Pediatr 130:957-960, 1997

38. Mohandas N, Kim YR, Tycko DH, et al: Accurate and inde-pendent measurement of volume and hemoglobin concen-tration of individual red cells by laser light scattering. Blood68:506-513, 1986

39. Cynober T, Mohandas N, Tchernia G: Red cell abnormalitiesin hereditary spherocytosis: relevance to diagnosis and un-derstanding of the variable expression of clinical severity.J Lab Clin Med 128:259-269, 1996

40. Chasis JA, Agre P, Mohandas N: Decreased membrane me-chanical stability and in vivo loss of surface area reflectspectrin deficiencies in hereditary spherocytosis. J Clin In-vest 82:617-623, 1988

41. Clark MR, Mohandas N, Shohet SB: Osmotic gradient ekta-cytometry: Comprehensive characterization of red cell vol-ume and surface maintenance. Blood 61:899-910, 1983

42. Schroter W, Kahsnitz E: Diagnosis of hereditary spherocy-tosis in newborn infants. J Pediatr 103:460-463, 1983

43. Zanella A, Izzo C, Rebulla P, et al: Acidified glycerol lysistest: A screening test for spherocytosis. Br J Haematol 45:481-486, 1980

44. Savvides P, Shalev O, John KM, et al: Combined spectrin andankyrin deficiency is common in autosomal dominant he-

reditary spherocytosis. Blood 82:2953-2960, 1993

Hereditary Spherocytosis 139

45. Pekrun A, Eber SW, Kuhlmey A, et al: Combined amkyrinand spectrin deficiency in hereditary spherocytosis. 67:89-93, 1993

46. Byers PH: Killing the messenger: New insights into non-sense-mediated mRNA decay. J Clin Invest 109:3-6, 2002

47. Jarolim P, Rubin HL, Brabec V, et al: Comparison of theankyrin (AC)n microsatellites in genomic DNA and mRNAreveals absence of one ankyrin mRNA allele in 20% ofpatients with hereditary spherocytosis. Blood 85:3278-3282, 1995

48. Ozcan R, Jarolim P, Lux SE, et al: Simultaneous (AC)nmicrosatellite polymorphism analysis and SSCP screening isan efficient strategy for detecting ankyrin-1 mutations indominant hereditary spherocytosis. Br J Haematol 122:669-677, 2003

49. Miraglia del Giudice E, Francese M, Nobili B, et al: Highfrequency of de novo mutations in ankyrin gene (ANK1) inchildren with hereditary spherocytosis. J Pediatr 132:117-120, 1998

50. Eber SW, Gonzalez JM, Lux ML, et al: Ankyrin-1 mutationsare a major cause of dominant and recessive hereditaryspherocytosis. Nature Genet 13:214-218, 1996

51. Eber SW, Langendorfer CM, Ditzig M, et al: Frequency ofvery late fatal sepsis after splenectomy for hereditary sphero-cytosis: Impact of insufficient antibody response to pneumo-coccal infection. Ann Hematol 78:524-528, 1999

52. Green JB, Shackford SR, Sise MJ, et al: Late septic complica-tions in adults following splenectomy for trauma: A pro-spective analysis in 144 patients. J Trauma 26:999-1004,1986

53. Hansen K, Singer DB: Asplenic-hyposplenic overwhelmingsepsis: Postsplenectomy sepsis revisited. Pediatr Dev Pathol4:105-121, 2001

54. Holdsworth RJ, Irving AD, Cuschieri A: Postsplenectomysepsis and its mortality rate: Actual versus perceived risks.Br J Surg 78:1031-1038, 1991

55. Schwartz PE, Sterioff S, Mucha P, et al: Postsplenectomysepsis and mortality in adults. JAMA 248:2279-2283, 1982

56. Schilling RF: Estimating the risk for sepsis after splenectomyin hereditary spherocytosis. Ann Intern Med 122:187-188,1995

57. Schilling RF: Spherocytosis, splenectomy, strokes, and heatattacks. Lancet 350:1677-1678, 1997

58. Robinette CD, Fraumeni JFJ: Splenectomy and subsequentmortality in veterans of the 1939-45 war. Lancet 2:127, 1977

59. Bader-Meunier B, Gauthier F, Archambaud F, et al: Long-term evaluation of the beneficial effect of subtotal splenec-tomy for management of hereditary spherocytosis. Blood97:399-403, 2001

60. de Buys Roessingh AS, de Lagausie P, Rohrlich P, et al:Follow-up of partial splenectomy in children with heredi-tary spherocytosis. J Pediatr Surg 37:1459-1463, 2002

61. Eber SW, Bolkenius M, Heidemann P, et al: Subtotale Sple-nektomie bei hereditarer Spharozytose im Kindesalter. ChirGastroenterol 17:12-17, 2001 (suppl)

62. Rice HE, Oldham KT, Hillery CA, et al: Clinical and hema-tologic benefits of partial splenectomy for congenital hemo-lytic anemias in children. Ann Surg 237:281-288, 2003

63. Tchernia G, Gauthier F, Mielot F, et al: Initial assessment ofthe beneficial effect of partial splenectomy in hereditaryspherocytosis. Blood 81:2014-2020, 1993

64. Katkhouda N, Hurwitz MB, Rivera RT, et al: Laparoscopicsplenectomy: Outcome and efficacy in 103 consecutive pa-tients. Ann Surg 228:568-578, 1998

65. Eber SW, Belohradsky BH, Weiss M: Infektionsprophylaxe

bei Asplenie. Klin Padiatrie 213 Sonderheft 1:1-4, 2001

66. Walensky LD, Narla M, Lux SE: Disorders of the red bloodcell membrane, in Handin RI, Lux SE, Stossel TP (eds):Blood: Principles and Practice of Hematology (ed 2). Phila-delphia, PA, Lippincott Williams & Wilkins, 2003, pp 1709-1858

67. Yawata Y: Hereditary spherocythosis, in Cell Membrane.Weinheim, Germany, Wiley-VCH, 2003, pp 173-212

68. Lanciotti M, Perutelli P, Valetto A, et al: Ankyrin deficiencyis the most common defect in dominant and non dominanthereditary spherocytosis. Haematologica 82:460-462, 1997

69. Saad ST, Costa FF, Vicentim DL, et al: Red cell membraneprotein abnormalities in hereditary spherocytosis in Brazil.Br J Haematol 88:295-299, 1994

70. Yawata Y, Kanzaki A, Yawata A, et al: Characteristic featuresof the genotype and phenotype of hereditary spherocytosisin the Japanese population. Int J Hematol 71:118-135, 2000

71. Miraglia del Giudice EM, Hayette S, Bozon M, et al: AnkyrinNapoli: A de novo deletional frameshift mutation in exon 16of ankyrin gene (ANK1) associated with spherocytosis. Br JHaematol 93:828-834, 1996

72. Randon J, Miraglia del Giudice E, Bozon M, et al: Frequentde novo mutations of the ANK1 gene mimic a recessivemode of transmission in hereditary spherocytosis: Threenew ANK1 variants: Ankyrins Bari, Napoli II and Anzio. Br JHaematol 96:500-506, 1997

73. Woods CM, Lazarides E: Spectrin assembly in avian ery-throid development is determined by competing reactions ofsubunit homo- and hetero-oligomerization. Nature 321:85-89, 1986

74. Jarolim P, Rubin HL, Brabec V, et al: A nonsense mutation1669Glu3Ter within the regulatory domain of human ery-throid ankyrin leads to a selective deficiency of the majorankyrin isoform (band 2.1) and a phenotype of autosomaldominant hereditary spherocytosis. J Clin Invest 95:941-947, 1995

75. Gallagher PG, Nilson DG, Wong C, et al: A two base pairdeletion at position -72/-73 of the ankyrin-1 promoter asso-ciated with hereditary spherocytosis disrupts TFIID com-plex binding and decreases transcription of a linked reportergene. Blood 100:77, 2002 (suppl, abstr)

76. Weisbein JL, Wong C, Cline AP, et al: Mutations at positions�108 and �153 of the erythroid ankyrin promoter decreaseexpression in transgenic mice by disrupting insular func-tion. Blood 100:78, 2002 (suppl, abstr)

77. Eber SW, Pekrun A, Reinhardt D, et al: Hereditary sphero-cytosis with ankyrin Walsrode, a variant ankyrin with de-creased affinity for band 3. Blood 84:362a, 1994 (suppl,abstr)

78. Dhermy D, Galand C, Bournier O, et al: Heterogenous band3 deficiency in hereditary spherocytosis related to differentband 3 gene defects. Br J Haematol 98:32-40, 1997

79. Jarolim P, Rubin HL, Brabec V, et al: Mutations of conservedarginines in the membrane domain of erythroid band 3 leadto a decrease in membrane-associated band 3 and to thephenotype of hereditary spherocytosis. Blood 85:634-640,1995

80. Jarolim P, Rubin HL, Liu SC, et al: Duplication of 10 nucle-otides in the erythroid band 3 (AE1) gene in a kindred withhereditary spherocytosis and band 3 protein deficiency(band 3 PRAGUE). J Clin Invest 93:121-130, 1994

81. Vince JW, Reithmeier RA: Carbonic anhydrase II binds tothe carboxyl terminus of human band 3, the erythrocyteC1-/HCO3- exchanger. J Biol Chem 273:28430-28437, 1998

82. Kanzaki A, Hayette S, Morle L, et al: Total absence of protein4.2 and partial deficiency of band 3 in hereditary spherocy-

tosis. Br J Haematol 99:522-530, 1997

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

140 Eber and Lux

83. Peters LL, Shivdasani RA, Liu SC, et al: Anion exchanger 1(band 3) is required to prevent erythrocyte membrane sur-face loss but not to form the membrane skeleton. Cell86:917-927, 1996

84. Jarolim P, Murray JL, Rubin HL, et al: Characterization of 13novel band 3 gene defects in hereditary spherocytosis withband 3 deficiency. Blood 88:4366-4374, 1996

85. Rysava R, Tesar V, Jirsa MJ, et al: Incomplete distal renaltubular acidosis coinherited with a mutation in the band 3(AE1) gene. Nephrol Dial Transplant 12:1869-1873, 1997

86. Lima PR, Gontijo JA, Lopes de Faria JB, et al: Band 3CampinasA novel splicing mutation in the band 3 gene(AE1) associated with hereditary spherocytosis, hyperactiv-ity of Na�/Li� countertransport and an abnormal renalbicarbonate handling. Blood 90:2810-2818, 1997

87. Jarolim P, Shayakul C, Prabakaran D, et al: Autosomaldominant distal renal tubular acidosis is associated in threefamilies with heterozygosity for the R589H mutation in theAE1 (band 3) Cl-/HCO3- exchanger. J Biol Chem 273:6380,1998

88. Bruce LJ, Cope DL, Jones GK, et al: Familial distal renaltubular acidosis is associated with mutations in the red cellanion exchanger (Band 3, AE1) gene. J Clin Invest 100:1693-1707, 1997

89. Inaba M, Yawata A, Koshino I, et al: Defective anion trans-port and marked spherocytosis with membrane instabilitycaused by hereditary total deficiency of red cell band 3 incattle due to a nonsense mutation. J Clin Invest 97:1804-1817, 1996

90. Karet FE, Gainza FJ, Gyory AZ, et al: Mutations in thechloride-bicarbonate exchanger gene AE1 cause autosomaldominant but not autosomal recessive distal renal tubularacidosis. Proc Natl Acad Sci USA 95:6337-6342, 1998

91. Palek J, Jarolim P: Clinical expression and laboratory detec-tion of red blood cell membrane protein mutations. SeminHematol 30:249-283, 1993

92. Rybicki AC, Heath R, Wolf JL, et al: Deficiency of protein 4.2in erythrocytes from a patient with a Coombs negativehemolytic anemia. Evidence for a role of protein 4.2 instabilizing ankyrin on the membrane. J Clin Invest 81:893-901, 1988

93. Lombardo CR, Willardson BM, Low PS: Localization of theprotein 4.1-binding site on the cytoplasmic domain of eryth-rocyte membrane band 3. J Biol Chem 267:9540-9546, 1992

94. Waugh RE, Agre P: Reductions of erythrocyte membraneviscoelastic coefficients reflect spectrin deficiencies in he-reditary spherocytosis. J Clin Invest 81:133-141, 1988

95. Hanspal M, Yoon SH, Yu H, et al: Molecular basis of spectrinand ankyrin deficiencies in severe hereditary spherocytosis:Evidence implicating a primary defect of ankyrin. Blood77:165-173, 1991

96. Tse WT, Gallagher PG, Jenkins PB, et al: Amino-acid substi-tution in �-spectrin commonly coinherited with nondomi-nant hereditary spherocytosis. Am J Hematol 54:233-241,1997

97. Jarolim P, Wichterle H, Palek J, et al: The low expression�-spectrin LEPRA is frequently associated with autosomalrecessive/nondominant hereditary spherocytosis. Blood 88:4a, 1996 (suppl, abstr)

98. Hassoun H, Vassiliadis JN, Murray J, et al: Molecular basis ofspectrin deficiency in �-spectrin Durham. A deletion within�-spectrin adjacent to the ankyrin-binding site precludesspectrin attachment to the membrane in hereditary sphero-cytosis. J Clin Invest 96:2623-2629, 1995

99. Becker PS, Tse WT, Lux SE, et al: �-Spectrin Kissimmee: A

spectrin variant associated with autosomal dominant hered-

itary spherocytosis and defective binding to protein 4.1.J Clin Invest 92:612-616, 1993

00. Wolfe LC, John KM, Falcone JC, et al: A genetic defect in thebinding of protein 4.1 to spectrin in a kindred with heredi-tary spherocytosis. N Engl J Med 307:1367-1374, 1982

01. Hassoun H, Vassiliadis JN, Murray J, et al: Characterizationof the underlying molecular defect in hereditary spherocy-tosis associated with spectrin deficiency. Blood 90:398-406,1997

02. Cooper RA, Jandl JH: The role of membrane lipids in thesurvival of red cells in hereditary spherocytosis. J Clin Invest48:736-744, 1969

03. Liu SC, Derick LH, Duquette MA, et al: Separation of thelipid bilayer from the membrane skeleton during discocyte-echinocyte transformation of human erythrocyte ghosts.Eur J Cell Biol 49:358-365, 1989

04. Zachee P, Boogaerts MA, Hellamans L, et al: Adverse role ofthe spleen in hereditary spherocytosis: Evidence by the useof the atomic force microscope. Br J Haematol 80:264-265,1992

05. Da Costa L, Mohandas N, Sorette M, et al: Temporal differ-ences in membrane loss lead to distinct reticulocyte featuresin hereditary spherocytosis and in immune hemolytic ane-mia. Blood 98:2894-2899, 2001

06. Corbett JD, Agre P, Palek J, et al: Differential control of band3 lateral and rotational mobility in intact red cells. J ClinInvest 94:683-688, 1994

07. Cho MR, Eber SW, Liu SC, et al: Regulation of band 3rotational mobility by ankyrin in intact human red cells.Biochemistry 37:17828-17835, 1998

08. Reinhardt D, Witt O, Miosge N, et al: Increase in band 3density and aggregation in hereditary spherocytosis. BloodCells Mol Dis 27:399-406, 2001

09. Weiss L: A scanning electron microscopic study of thespleen. Blood 43:665-691, 1974

10. Groom AC: Microcirculation of the spleen: New concepts,new challenges. Microvasc Res 34:269-289, 1987

11. Fujino T, Ishikawa T, Inoue M, et al: Characterization ofmembrane-bound serine protease related to degradation ofoxidatively damaged erythrocyte membrane proteins. Bio-chim Biophys Acta 1374:47-55, 1998

12. Coleman DH, Finch CA: Effect of adrenal steroids in hered-itary spherocytic anemia. J Lab Clin Med 47:602-610, 1956

13. Reliene R, Mariani M, Zanella A, et al: Splenectomy prolongsin vivo survival of erythrocytes differently in spectrin/ankyrin- and band 3-deficient hereditary spherocytosis.Blood 100:2208-2215, 2002

14. Peters LL, Barker JE: Spontaneous and targeted mutations inerythrocyte membrane skeleton genes: Mouse models ofhereditary spherocytosis, in Zon LI (ed): Hematopoiesis, aDevelopmental Approach. Oxford, UK, Oxford UniversityPress, 2001, pp 582-609

15. Wandersee N, Birkenmeier C, Bodine D, et al: Mutationsin the murine erythroid �-spectrin gene alter spectrinmRNA and protein levels and spectrin incorporation intothe red blood cell membrane skeleton. Blood 101:325-330,2003

16. Kaysser TM, Wandersee NJ, Bronson RT, et al: Thrombosisand secondary hemochromatosis play major roles in thepathogenesis of jaundiced and spherocytic mice, murinemodels for hereditary spherocytosis. Blood 90:4610-4619,1997

17. Wandersee NJ, Lee JC, Kaysser TM, et al: Hematopoieticcells from �-spectrin-deficient mice are sufficient to inducethrombotic events in hematopoietically ablated recipients.

Blood 92:4856-4863, 1998

1

1

1

1

1

1

1

1

Hereditary Spherocytosis 141

18. Bodine DM, Birkenmeier CS, Barker JE: Spectrin deficientinherited hemolytic anemias in the mouse: Characterizationby spectrin synthesis and mRNA activity in reticulocytes.Cell 40:959-969, 1984

19. Peters LL, Turtzo LC, Birkenmeier CS, et al: Distinct fetalAnk-1 and Ank-2 related proteins and mRNAs in normaland nb/nb mice. Blood 81:2144-2149, 1993

20. Peters LL, Birkenmeier CS, Bronson RT, et al: Purkinje celldegeneration associated with erythroid ankyrin deficiencyin nb/nb mice. J Cell Biol 114:1233-1241, 1991

21. Hassoun H, Wang Y, Vassiliadis J, et al: Targeted inactiva-tion of murine band 3 (AE1) gene produces a hypercoagu-lable state causing widespread thrombosis in vivo. Blood

92:1785, 1998

22. Peters LL, Andersen SG, Gwynn B, et al: A QTL on mousechromosome 12 modifies the band 3 null phenotype: � spec-trin is a candidate gene. Blood 98:437a, 2001 (suppl, abstr)

23. Paw BH, Zhou Y, Li R, et al: Cloning of the zebrafish retsina bloodmutation: Mutations in erythroid band 3 in dyserythropoiesis andcytokinesis defects. Blood 96:440a, 2000 (suppl, abstr)

24. Liao EC, Paw BH, Peters LL, et al: Hereditary spherocytosis inzebrafish riesling illustrates evolution of erythroid �-spectrinstructure, and function in red cell morphogenesis and mem-brane stability. Development 127:5123-5132, 2000

25. Shafizadeh E, Paw B, Foott H, et al: Characterization ofzebrafish merlot/chablis as non-mammalian vertebratemodels for severe congenital anemia due to protein 4.1

deficiency. Development 129:4359-4370, 2002