Four different mutations in codon 28 of alpha spectrin are associated with structurally and functionally abnormal spectrin alpha I/74 in hereditary elliptocytosis

Four different mutations in codon 28 of alpha

spectrin are associated with structurally and

functionally abnormal spectrin alpha I/74 in

hereditary elliptocytosis.

T L Coetzer, … , J Manaster, J Palek

J Clin Invest.

1991;

88(3)

:743-749.

https://doi.org/10.1172/JCI115371

.

Hereditary elliptocytosis (HE) Sp alpha I/74 is a disorder associated with defective spectrin

(Sp) heterodimer self-association and an abnormal tryptic cleavage of the 80-kD alpha I

domain of Sp resulting in increased amounts of a 74-kD peptide. The molecular basis of this

disorder is heterogeneous and mutations in codons 28, 46, 48, and 49 (codons 22, 40, 42,

and 43 in the previous nomenclature which did not include the six NH2-terminal amino

acids) have been reported. In this study we present data on seven unrelated HE Sp alpha

I/74 kindred from diverse racial backgrounds in whom we identified four different mutations

all occurring in exon 2 of alpha Sp at codon 28. Utilizing the polymerase chain reaction we

established a CGT----CTT; Arg----Leu 28 mutation in one kindred of Arab/Druze origin. In

two unrelated white kindred of English/European origin the substitution is CGT----AGT;

Arg----Ser 28 and in two apparently unrelated white kindred from New Zealand, the mutation is

CGT----TGT; Arg----Cys 28. Finally, in one American black kindred and in a black kindred

from Ghana the mutation involves CGT----CAT; Arg----His 28. Allele specific oligonucleotide

hybridization confirmed that the probands are heterozygous for the respective mutant

alleles. All four point mutations abolished an Aha II restriction enzyme site which allowed

verification of linkage of the mutation with HE Sp alpha I/74. Our results […]

Research Article

Find the latest version:

Four Different

Mutations

in

Codon 28 of

a

Spectrin

Are

Associated

with

Structurally

and

Functionally Abnormal Spectrin

al/74

in

Hereditary

Elliptocytosis

Theresa L. Coetzer,** Ken Sahr,* JosefPrchal,1 Hilary Blacklock,11 LoAnnPeterson,' Robert Koler,** JohnDoyle,**J. Manaster,f and Jiri Palek*

*DepartmentofBiomedical Research, DivisionofHematology/Oncology,St. Elizabeth's Hospital ofBoston, Tufts University School of

Medicine, Boston, Massachusetts 02135; tDepartment ofHaematology, South African Institutefor Medical Research, University ofthe

Witwatersrand, Johannesburg, South Africa;

lUniversity

ofAlabama, Birmingham, Alabama 35294; 1lMiddlemoreHospital, Auckland, New Zealand; 'Hennepin County Medical Center, Minneapolis, Minnesota 55415; **University of Oregon Health Science Center, Portland, Oregon 97201; :The Hospitalfor Sick Children, Toronto,Canada;and

fNahariya

Hospital, Nahariya, Israel

Abstract Introduction

Hereditary elliptocytosis (HE)Sp

aoP4

is adisorder associated

withdefective spectrin (Sp) heterodimer self-association and an abnormal tryptic cleavageofthe80-kD aI domain ofSp

resulting in increased amounts of a 74-kD peptide. The molecu-lar basis of this disorder is heterogeneous and mutations in codons 28, 46, 48,and 49 (codons 22, 40, 42, and 43in the

previousnomenclaturewhichdid notincludethesix

NH2-termi-nalamino acids) have been reported. In this study we present data onseven unrelated HESp

all4

kindredfrom diverse racial backgroundsin whom weidentified fourdifferentmutations all

occurringin exon 2ofaSpatcodon 28. Utilizingthe polymer-asechain reactionweestablishedaCGT 7CTT;Arg-- _Leu

28mutation in one kindredofArab/Druze origin. In two unre-lated whitekindredofEnglish/Europeanorigin the substitu-tion is CGT --

_AGT;

_Arg -- _{Ser 28 and in two}

apparently

unrelated white kindred from New Zealand, the mutation is CGT--

_{TGT; Arg}

-*

_Cys

_28.

_Finally,

_inoneAmerican black

kindred and in a black kindred from Ghana the mutation in-volvesCGT--

CAT; Arg

-- His 28. Allele

specific

oligonucleo-tide hybridization confirmed thatthe probands are heterozy-gous for therespectivemutantalleles. All four point mutations

abolishedanAha IIrestrictionenzyme sitewhichallowed

verifi-cation of linkage of the mutation with HE Sp

all4.

Our results

implythatcodon 28 of a Sp is a "hot spot" for mutations and alsoindicatethat Arg28is criticalfortheconformational

stabil-ityand functional selfassociationof Spheterodimers.(J.Clin. Invest. 1991. 88:743-749.) Key words: aspectrin gene* "hot

spot" mutations *hereditary pyropoikilocytosis- polymerase

chain reaction * clinical heterogeneity

Portions of thiswork werepresentedat the 32nd Annual Meeting ofthe

American Society of Hematology, November 1990, and have been

publishedas anabstract (1990. Blood.76:37a).

Addressreprintrequests toJiriPalek, M.D., St.Elizabeth's

Hospi-talof Boston,TuftsUniversitySchool of Medicine, Dept. of

Biomedi-calResearch, Division of Hematology/Oncology,736 Cambridge St.,

Boston,MA02135.

Receivedforpublication5February 1991 and in revisedform I May 1991.

Hereditary

elliptocytosis (HE)'

is heterogeneous in terms of

clinicalpresentation, inheritance,andtheunderlying molecu-lar defect (1, 2). The most common abnormalityinvolves a

defective spectrin (Sp) selfassociation

(3-6).

Sp,themajor com-ponentoftheredcellmembraneskeleton,consistsof two

elon-gated chains which arearrangedin anantiparallel fashion to

form

a(3

heterodimers(SpD); the

SpD,

in turn, interact in the

head region ofthe two chains formingtetramers(SpT) and

oligomers (3, 7). Limited

tryptic digestion

ofSphas allowed the

identification of five a Sp (aI-aV) and four

fl

Sp

((3I-fIV)

structural domains

(8).

Theselfassociation of

SpD

into

SpT

involves the

NH2-terminal

alandthe COOH-terminal 3I do-mains (7). a and

f3Sp

arecomposed of multiple homologous

repeatunits,eachconsisting of - 106amino acids (9),except

fortheNH2- and COOH-terminal residuesof bothaand ,B

Sp,

aswellassegment10ofaSp, whichare

atypical

andunrelated

to theconservedrepeatmotif

(10,

11).

Seven distinct structural defects ofthe al domain,

asso-ciated with

impaired SpD-SpD interaction,

havebeendefined

onthebasis of abnormal limited

tryptic digest

maps inwhich

thereisadecreaseinthenormal 80-kDaldomainanda

con-comitant increase inone ormorelowermolecularweight

pep-tides(2). Thesedefects of Spare

designated according

tothe molecularweight oftheabnormal

peptide

(1).HE

subjects

who carry one or moreoftheseabnormal

spectrins exhibit

marked variation in clinical severityas well as the degreeof functional impairment ofSpDself association (12). Recentstudieshave

indicatedthatSp a1174is the most severedefect,whereasSpa165

results in only mild dysfunction (13). Partial amino acid

se-quencing

oftheNH2-terminal portion oftheabnormal

pep-tideshas revealedaminoacidchanges in close

proximity

to the cleavagesite in subjects withSp

aI/65,

Spa146,and Sp

aI/S5b

(14). Knowledgeof the genomic exon/intronarrangementofthe aI

domain of Sp

(15)

has allowed theverification ofthesechanges

atthe DNA level and has alsofacilitatedthestudyof defects

locatedeitherupstream orfurtherawayfromthecleavagesite. Sp

a1/65

appears to be a homogeneous defectdue to a single mutationbecause all cases thusfar investigatedinvolve a dupli-cation oftheleucinecodon (TTG) at position 154(formerly

1. Abbreviations used in this paper: ASO,allele-specific oligonucleo-tide;HE,hereditaryelliptocytosis;HPP,hereditarypyropoikilocytosis;

PCR, polymerase chain reaction; RFLP, restrictionfragment length polymorphism; Sp, spectrin; SpD, spectrin heterodimer; SpT,

spectrin

tetramer.

Codon 28 Mutations inSpectrin //74 Elliptocytosis 743

J.Clin.Invest.

© The American Society forClinicalInvestigation,Inc.

148, see abstract and below) of a Sp

(16-18).

In contrast, recent

studies on Sp

a1174

have revealed thatthis defect is heteroge-neousin origin and that the primary mutation may occur in either thea Sp or#Sp genes

(18-20).

Thus far, four a Sp and oneASppoint mutationshave been described in Spa1/74

sub-jects involvingchangesofaSp amino acids 22 (21), 40 (22), 42 (23), and 43 (22)andaminoacid 2053 inA Sp (20). In these reports, theaminoacidsofaSp have been numbered accord-ingtotheaminoacid sequence of limited tryptic digest

pep-tidesofaSp (9).However, thefull-lengthcDNA sequence ofa

Sp predictsan additional six

NH2-terminal

amino acids (10). Therefore, by agreement with other researchers in the field (Al-loisio,N., J. Delauney, D. Dhermy, and B. Forget), the number-ing usedinthis report has now been modified to include the additional six residues. The previously reported mutations (20-23) are thus located in codons 28, 46, 48, and 47.

Inthisstudy, we describe four mutations in codon 28 (new nomenclature; this corresponds to codon 22 of previous no-menclature) ofaSp in seven unrelated kindred with HE from diverse racialbackgrounds. Allele specific oligonucleotide hy-bridization confirmedthat the probands were heterozygous for the defects. All four point mutations abolished an AhaII restric-tionenzymesiteand this allowed screening of family members toverify linkage of the mutation with HE.

Methods

Subjects. The pedigrees of the seven unrelated Sp a"'74 kindred are shown in Fig. 1. Family 6 is one of the two original black American kindred in whom the Spa1174defect was first detected (24) and clinical and protein data on family 4 have been reported previously (25). Data on the remaining five kindred have not been published. Clinical and

Family1 _Arg-Cys28

1 2

Family 2

1

12

114

Family3 Arg -Ser28 _Family4

1 2

II

E-1KC

ti

SW6

6

6§Th

Family5 Arg-His28 Family6 I K

II

_IKFb

_M

_IC

E

1 2 3 1 2 3

Family7

Arg- Lu 28

II A

1 2

X HPPSpa

Normal

8

notstudied 1/74 Aasymptomatic

11 K"~~~carrier

Figure1. _{Pedigreesof the seven unrelated HE Sp}

a174kindredwith

four differentcodon28 mutations. Families 1-4areCaucasian,

fam-ilies 5 and6 are Black,andfamily 7is ofArab/Druzeorigin.

biochemical dataonaffectedindividualsaregivenin Table I. Criteria for thediagnosisof the clinicalphenotypehave beenoutlined(2)and

arebasedmainlyon(a)reviewof theperipheralbloodfilm, (b)

pres-ence orabsenceof_hemolysis,and(c)theinheritancepattern.

Inallsevenkindred,theclinical severityvariesmarkedlybetween

familymembers and ranges fromasymptomaticcarrierto HPP(Fig.1 andTable

I).

Families 1 and 2areapparentlyunrelated white kindred from New Zealand and families 3 and4 arewhite kindred ofEnglish/

European origin. Family 5 isablackkindred from Ghana, whereas

family7is ofArab/Druzeorigin.

Inall theseindividuals,there isanincrease in the percentage SpD

extracted from the membraneat4VCcomparedtocontrol values of 5.0±3.6% (n=_35).

_Asymptomatic

_{carriers have 25-31%SpD;HE}

sub-jectsrange from 36-55%SpDandHPPindividualshave 60-70%SpD

in their membraneextracts.Thisfunctional Sp self association defect is duetothe presence of abnormalSp

al174

in allcases asevidencedby limitedtryptic digestion ofSp (datanot shown). The membrane Sp content,expressed as aSp/b3 ratio, is decreased in all HPPsubjects

(normalSp/b3 1.0±0.1, n= _{60), as well as in some of the HE}

individ-ualswithseverehemolysis (Table

I).

TheremainingHE subjectsand asymptomaticcarriers haveanormalSpcontent.

Erythrocyte membraneprotein analysis. Themethods used have been previously referredto ordescribed in detail (24, 26, 27). They includeerythrocytemembraneprotein preparation(26,28) and analy-sisbySDS-PAGE(3.5-17%acrylamidegradient gels) (26,29);spectrin

extraction (26)and analysisofSpD and SpTby nondenaturinggel electrophoresis (4); limitedtryptic digestion ofSp (24) followed _by SDS-PAGE

(10%

acrylamide)(30)andimmunoblottingwitha

polyclo-nal anti-a I Sp antibody (26). On some probands, two-dimensional isoelectric-focusing/SDS-PAGE of limited tryptic digests of Sp was per-formed(24).Amino acid sequenceanalysis (31)ofthe abnormal74-kD

aIpeptidewascarriedoutin thelaboratoryof Dr. P. Matsudairaat

MassachusettsInstitute ofTechnology,Cambridge, MA.

Restrictionfragment length polymorphism (RFLP) analysis. Geno-micDNA wasisolated fromperipheralleukocytes of ACD

anticoagu-lated blood (32) and 10 ug DNA were digested with the restriction endonucleases Xba

I,

Msp

I,

Pvu

II,

and TaqI(BoehringerMannheim GmbH, Mannheim, FRG). DNA fragments were fractionated ona

0.7% agarosegel (33)andtransferredto anylonmembrane(Zetaprobe,

Bio-RadLaboratories,Inc.,Richmond, CA)by alkaline Southern blot-ting (34)in 0.4 M NaOH.A 13-kbgenomicaISp probe (clone3021 El) (35) and a 738 bp, Sp cDNA probe (36) were labeledwith 32p dCTP (New England Nuclear, Boston, MA) by random priming (37) andhybridized tothe filtersasdescribed bytheZetaprobe

manufac-turer.

GenomicDNAamplificationandsequencing. Exons1 and 2 ofaSp

wereamplified by the polymerase chain reaction (PCR) (38)utilizing oligonucleotide primers complementary to the flanking intron

se-quences (Fig. 2). P1

(5'GAATTCGACTGGACAGTTCCAT3')

and P2(5qTAGGGTCTGCTCTGAGGCAAT3) amplified a ±1,500-bp

fragmentcontaining both exons, whereas P2 and P3 (5'CACATATA-AGCGGGGCAACAT3)amplifieda348-bpfragment containing only

exon2. 1 Mg ofgenomicDNA wasamplified for 30cyclesusing Taq polymerase (Perkin Elmer Corp., Norwalk, CT) in a Perkin Elmer Cetus DNA thermal cycler. Eachcycleconsisted of1 min denaturation

at

94°C,

1

min

annealingat

55°C

and 2 min extensionat72°C.

Subcloning

and sequencingofamplifiedDNA. ThePCR reaction mixeswereanalyzed on1% agarosegels and theamplifiedDNAwas

purified by elution from the gelusingGeneclean (Bio 101,LaJolla,

CA). The PCR productswereblunt-endligatedinto Hinc IIdigested

pGEM 3Z vector(PromegaBiotec, Madison, WI) (33). Positive sub-clones were sequenced on both strands bydideoxy sequencing(39)

using T7 DNA polymerase (Sequenase; United States, Biochemical Corp., Cleveland,OH)andplasmid

Sp6

andT7promoterprimers.

Direct sequencingofamplifiedDNA.Genomic DNA wasamplified

asaboveusing primersP2 and P3. 5 Ml of the PCR reaction mixwere

reamplified

asdescribed,usingonly P2. This resulted in accumulation ofsingle stranded DNA which was purified either on Centricon 30

TableLClinical and Biochemical Data on HE Sp

a"17"

Kindred

Subject Splenectomy Hct Retics %SpD* Sp/b3* Clinicalphenotype Mutation

Family 1

1.2 Yes 30 2.1 52 0.74 HPP Arg Cys28

11.2' No 41 1.5 49 0.91 MildHE Arg Cys28

111.1 Yes 34 6.3 68 0.67 HPP Arg- Cys 28

Family 2

1.2 Yes 34 2 53 0.86 SeverehemolyticHE Arg Cys28 II.2 Yes 36 1.5 53 0.83 Severe hemolyticHE Arg- Cys 28 I1.3 Yes 32 1.8 55 0.81 Severe hemolytic HE Arg- Cys 28

III.2 No 36 1.9 48 0.90 MildHE Arg Cys 28

III.4 Yes 15 17$ 60 0.71 HPP Arg Cys28

Family 3

1.1 No 45 2 36 1.00 MildHE Arg Ser 28

11.3 No 12-18 5-14 50 0.77 Severe hemolyticHE Arg - Ser 28 Family4

II.2 Yes 35 4-5 61 0.69 HPP Arg Ser 28

III.1 No 35 1.9 44 0.96 Mild HE Arg- Ser28

Family5

1.1 No NA N 26 0.99 Asymptomatic carrier Arg- His28

11.1 No NA t 48 0.97 HemolyticHE Arg - His 28

II.2 No NA t 51 0.96 HemolyticHE Arg- His28

11.3 No NA t Transfused HemolyticHE Arg - His28

Family 6

1.2 No 40 0.9 25 1.02 Asymptomaticcarrier Arg - His 28 1.3 No 47 1.8 31 0.97 Asymptomaticcarrier Arg - His 28

11.2 No 21-25 8-16 76 0.80 HPP Arg His 28

11.3 No 34.5 5.2 32 0.98 MildHE Arg - His 28

Family7

1.2 No NA 1.4 39 0.91 MildHE Arg Leu28

11.1 No NA 12 73 1.00 SeverehemolyticHE Arg - Leu28

111.2 No NA 20 66 0.82 HPP Arg - Leu 28

NA, not available.

*_{Normal values for%of SpD,}

_5.0±3.6%;

_Sp/b3,

(Amersham,UK) ormodel 30,000filters(Millipor MA). The supernatantwasconcentratedbyethanol sequenced usingT7 DNApolymeraseandP3asseq

Allele-specificoligonucleotide hybridization. Noi allele-specific oligonucleotides (ASO)weresynthesize Biosystemsoligonucleotide synthesizer (AppliedBim

terCity, CA)andpurified.Thenormal ASO sequenc GAGGCGTCAGGAAGTG3'(codon28 isunderlir

tantASO sequenceswereidentical except for the re] substitutionatcodon 28.

EXON

P1 I

E XON

P3

51

44bp 240bp

1.0±0.1. *_{Presplenectomy values.}

e Corp., Bedford, Genomic DNAwas

_amplified

asdescribed and 10 ul of the PCR

precipitationand reaction mixweretransferredtoanylonmembrane

(Zetaprobe) using

luencing

primer. a_{slot blot apparatus}

_(40).

Normal andmutantASO

probes

wereend

rmal and mutant labeled with gamma 32PATP

using

T4

polynucleotide

kinase

(33).

The

edon anApplied slotblotswere

_hybridized

with eithermutantornormal ASO

probes

for systemsInc.,Fos- 2 hat 620CinSX SSPE, 5X Denhardts

solution,

0.5% SDS.

Mem-:ewas5'CAGGA- braneswerewashed twice for 15 minat room_temperaturein 2X

SSPE,

ied) and themu- followedbytwotothree washes for 5-10 minat620Cin 2x

SSPE,

levant nucleotide 0.1% SDS andsubjectedto_{autoradiography}at-70'Cusing

intensify-ingscreens(NewEngland Nuclear).

Restriction endonucleaseanalysis of

amplified

DNA. DNA from normal and affectedfamilymemberswasamplifiedasdescribedusing

P2andP3. Theamplified productwas

purified

byphenolchloroform

3' DNA extraction and ethanol

precipitation; digested

with Aha II

(New

En-- 3 glandBiolabs, Beverly, MA)andanalyzedon2% agarosegels.

Results

8AA

I-8

80 AA

9-88

Figure 2. Exon/intronarrangement of theNH2-terminal portionof aSp.Pl-P3areoligonucleotide primersusedfor PCRamplification.

AAdenotesaminoacids.

Coinheritance

ofmutant Sp

a'74

withHE

and

HPP. In allseven

kindred,

the

clinically

affected individuals exhibiteda

striking

increasein theamount of

SpD

in thered cell membrane ex-tracts(Table

I), ranging

from 25-76%SpDoutofthetotalSpD and

SpT pool

as

compared

withcontrol valuesof 5.0±3.6%

(n

= 35). Limited trypticdigestionofSp revealed thepresenceof mutant Sp a1/74 as evidenced by a marked increase in the amount ofthe 74-kD a Ipeptideanda concomitantdecreasein the normal 80-kD a I domain ofSp (data not shown). The clinical presentation was variable, includingasymptomatic car-rier state, mild HE, HE with hemolysis and HPP. The latter wascharacterizedbymicrospherocytosisand micropoikilocy-tosis with onlyoccasional elliptocytes,which correlated with a

decreasedmembranespectrincontent as well as a more severe

clinicalandbiochemicalpresentation (TableI).In

hematologi-cally normal family members, SpD selfassociation and limited

tryptic digests,aswell asSpcontent, were all normal.

Assignmentofthe defect toaSp.The primary defect in Sp a1/74canreside either on theaSp orthe (3 Sp chain as indicated

by RFLPstudies (18) orlimited tryptic digestion ofa ( Sp

mixed hybrids (19).In the present study, either RFLP analysis orlimited tryptic digestion of isolateda and

_#

chains was

em-ployed insome kindred inanattempttoassign thedefect to oneof the two Spchains. For RFLPstudies, DNA from

af-fected andnonaffected individualswere analyzedwitha

geno-micaISp probewhichispolymorphicforthe restriction en-zymes Xba I, Pvu II, and Msp I (41). The Xba Ipolymorphism, for example,

distinguishes

twoalleles corresponding to

restric-tionfragment lengths of 4.05and2.20+ 1.85 kb,respectively.

Inthreepedigrees,oneofthe4.05-kb allelessegregatedwiththe

inheritance ofHE Sp a174 which would be compatible with

linkageto a Spindicatingthat the primary defect could be in theaIdomainofSp(data notshown). RFLPstudies usinga Taq Ipolymorphismfor(3 Sp were notinformative.

Elucidation

of the a Sp gene mutation. Amino acid se-quencingof the 74-kD al peptide in four probandsrevealed

that the cleavage site was at Lys 48 (residue 42 in previous nomenclature) comparedwith thenormalcleavage siteatArg 45(previously residue 39) (42).Wethereforeanalyzed exons 1 and 2 ofa Sp, which code for the first 88 amino acids (six NH2-terminalamino acidsaspredictedbytheDNA sequence [15] plus 82 amino acids accordingtoSpeicher's nomenclature [42]). Genomic DNA was amplified by PCR andsequenced either

directly

or after

subcloning.

DNA

sequencing

of

sub-cloned DNAfrom probandsJF

(family

1) andBF

(family

2) revealed a

single

base change of C -* T in exon 2 of both probandsasexemplifiedin

Fig.

3 forJF.To

verify

the

muta-Normal

G A T C

dw

am

do T

_ ,_ -G

UL

CGT Arg 22

Mutant

tion,

several subclones from separate PCR reactionswere

se-quenced

to ensurethat both alleleswereanalyzed. In

subject

JF, fouroutof 10subclonesof PCR 1containedthemutation,

aswellasthreeoutof four subclones of PCR 2. Insubject

BF,

the mutationwaspresent in fouroutof 12 subclones of PCR 1

and,

two outof 11 subclones of PCR 2

(Fig.

3).This

changed

the normal CGT codon to TGT

resulting

in an

_Arg

-.

Cys

substitutionatamino acid 28 (previouslyresidue 22). All

re-mainingcodonsofexon2,as well as exon 1, werenormal. Intheremaining five kindred, direct sequencing of PCR-amplified DNAwascarriedoutand threedifferent mutations

atcodon 28wereidentified(Fig. 4).In this type ofsequencing

both normal andmutantallelesaresequenced

simultaneously

resultinginthepresence ofthenormal and mutantnucleotides

atthe same position in the sequencing ladder. In twounrelated white families (kindred 3 and 4), the point mutation was CGT -* AGT which resulted in an

Arg

-- _{Ser substitution}

(Fig. 4, left). In the Arab Druze kindred (family 7) (Fig. 4,

center),

the

point

mutationwasCGT-- CTT

causing

substitu-tion ofArgbyLeuand intwounrelated black kindred(families

5and

6),

aCGT-- _CATmutation

_{changed Arg}

_toHis

_{(Fig. 4,}

right).

Allele-specific

oligonucleotide hybridization. Toverifythe

various different nucleotide substitutions, PCR-amplified

DNA oftheprobandswerehybridized with either a normal ora mutantASO probe and the results are shown in Figs. 5 and 6. CE(II.2, family 6)and KF(II. 1, family 5)areheterozygous for theCAT(His 28)defect because their DNA hybridized to both the normal and the mutant probe (Fig. 5).Likewise,SW(I1.2,

family4)andKC (11.3, family 3) areheterozygousfor the AGT

(Ser 28)mutation and AP(II.1,family 7) is a heterozygote for theCTT(Leu 28) abnormality (Fig. 6).

Linkage of the codon 28 mutations to HE

Sp a74.

The

normal DNA sequence has an Aha II restriction cleavage site at codon28 which is eliminated by the base changes and therefore restriction enzyme analysis could be used to test for the pres-enceof the mutations. Exon 2 ofaSp wasamplifiedin affected and normal individuals from all kindred and afterdigestion withAha II,thesamples wereanalyzedon 2% agarosegels.An example of one kindred(family 1) is shown inFig. 7. In the normal individual from thiskindred,both normal alleles con-tain the restriction site and are therefore cleaved intotwo frag-ments of 222 and 126 bp, respectively. In contrast, the HE individuals from this family areheterozygousforSp

aI/74

and contain a normal allele which isdigested byAha II anda

mu-G A T C

-

_

-_. ,.... f...

._

^!-i

-T

-G

-_T

TGT Cys 22

Figure 3. Normal and mutant DNA sequence of part of theaSpgene in proband11.2(JF)offamily 1. Genomic DNA was amplified by PCR,subcloned,and sequenced.Apoint mutation in codon 28 that changes the normal subclone sequence of CGT (Arg)toTGT (Cys) in the mutant subclones was detected.

G A T C

.~t'

4--_

Arg -Ser 28

G1 A T C

I= .. I,,w,"

...

~~~~~~~~~-G

C

Arc

' -_eG2 T

G A T C

imii.k4- r..

GA C

Arq -His 28

Figure 4. Normal andmutantDNA sequence of partoftheaSpgene inproband 11.3 of family 3 (left, CGT-- _{AGT); proband I. 1 of}

fam-ily7(center, CGT -* CT1)andprobandII.1 offamily5 (right, CGT-- _{CAT).Genomic DNAwas}amplified by_{PCR andsequenced}

directlyresultingin the presence of the normal andmutantnucleotide

atthesameposition inthesequencingladder.

Mutant CAT

His 28 12 112 liii 1112 M

*t

1

CE4

a.

4.

Figure 5.Allele specific oligo-nucleotide(ASO)

hybridiza-tion. DNA from various af-fected HESpa1/74and normal

familymemberswere ampli-fied byPCRandhybridizedto

eitheranormal ASOor a

mu-KF~

~ (11.2,

family 6)andKF(11. 1, family 5)arebothheterozygous for the mutation.

tant allele which lacks the cleavage site and remains intact, resulting in thepresenceof threefragmentsonthe gel (348 bp

and 222+ 126bp). The codon 28 mutation is therefore linked

tothe inheritance of HESpaT'74inthis kindred. AhaIIdigests

ofamplifiedDNAfrom theremaining six kindredgave

identi-cal results(datanotshown).

Discussion

In this study, we have described four different mutationsin codon 28(previously referredtoascodon22) ofaSpinseven

unrelatedHESp

a1174

kindred from diverse racialbackgrounds. Thepoint mutations resultedinthe substitution of Arg 28by eitherCys,Ser,Leu,orHis. The latter mutation is identicalto

thatpreviously describedinonewhite French kindred(21).In

anindependentsimultaneousstudytheArg Leu mutation

was also found in one black individual and the Arg -> Ser

substitutioninonewhitesubject (23).

Normal CGT

Arg 28

Mutant AGT

Ser 28

Mutant CTT

Leu 28

APAUM

SW Avo

Figure6. Allelespecific oligonucleotide (ASO) hybridization. DNA

from variousaffected HE Sp a1/74 and normal family memberswere

amplified byPCR andhybridizedtoeitheranormalASOor amutant

ASO. Note thatSW(11.2, family 4),KC(11.3, family 3)and AP(11.1

family 7)areheterozygousfor therespectivemutations.

bp

348-222s.

126\

Figure7.Aha IIrestriction enzymeanalysis. Genomic DNAfrom family 1 was amplified by PCR produc-ing a 348-bp fragment which was digested with AhaIIandanalyzed on 2% agarosegels.AffectedHE/ HPPSp a/14individuals are heterozygousfor the

mu-tantallelewhich has lost the Aha IIsite and hence remains intact (348-bp fragment), and the normal allele which is cleaved by Aha 11 (222- and 126-bp fragments). The normal familymember doesnot

contain themutantallele.

Mdenotessize markers.

The Arg 28 mutations only occurred in individuals whose red cells contained the structurally and functionally defective Spa1/74andwereabsent innormal family members and control

individuals. Furthermore, because Arg 28 is locatednearthe

74-kD tryptic cleavage site and ispart of the self-association site, these data imply that the codon 28 mutationsareinvolved

in the observed functional and structural Spa174

abnormali-ties. Inthetriple-helical model proposed for Sp (9), Arg 28 is located inrepeat IofaSp,attheNH2-terminal end of helix 3, adjacenttotheturnregion. Helix 3 thusappearstobecritical in maintaining the structural and functional integrity of the Sp molecule. The importance of helix 3 is further evidenced by the

presenceof mutations in this helix in various otherrepeatunits ofaSp. In thesecasesthemutations occurredatthe COOH-terminal end of thehelix, adjacenttotheconnecting region: in Sp aI/65 (repeat 2), Spa1/46(repeat 3), Sp a1I/S' (repeat 5), Sp Lyon (repeat 1), and Sp Culoz (repeat 1) (14-16, 22).

Ourstudies further indicate that Arg28 isavital residue for

thenormal function andconformationalstability ofaSp.

Sub-stitutionof Arg 28 withanaminoacid residue of different size

and/or charge alters the conformation of the head region ofa

Sp which inturninfluences thefunction and structural integ-rity of the molecule. This is manifested bya decreased SpD

self-association interaction and anenhancedsusceptibility of

the a1/74tryptic cleavage site atArg 45 or Lys 48(previously

residues 39 and 42), resulting inanincreased production ofthe

74-kD peptide after digestion with trypsin. Interestingly, the typeof amino acid substitutionatposition 28 doesnotappear

toaffect thestability of the molecule inadifferentialmanner,

becauseall four mutations (Arg 28-* Cys/Ser/His/Leu)

pro-duceasimilarrangeoffunctional(%SpD) and structural

(74-kDpeptide) abnormalities (Table I).

Within each familyinvestigated, there isa marked varia-tioninthe clinicalexpression ranging fromanasymptomatic

carrierstate to HPP. This heterogeneity has previously been showntocorrelate with theamountsof bothSpD and the 74-kDpeptide,aswellaswith theabsoluteSpcontentofthe

mem-brane(12). Becauseall affectedindividuals from onekindred areheterozygotes for the identical mutation, which ispresent inonlyoneof thetwoalleles, it is clear that additional factors modulate the expression of the defectivegeneand the

abnor-malprotein product. Thesemaybecis-acting factors suchas a

Codon 28 Mutations inSpectrina1174Elliptocytosis 747

Normal CGT Arg 28

4.0

a.

m.

a.0

a.M

linked

polymorphism

ormutation occurringelsewhereinthe a

Sp

geneor

alternatively,

trans-acting factors involving linkage with another,asyet unknown,defect.Anexampleof the latter

possibilitywould be the concurrentinheritance ofan aSp co-don 28 mutation

together

witha"thalassemia-like" defect of

Sp synthesis (1),

which wouldcause apartial Sp deficiencyand

result in an altered red cell morphology and a more severe

clinical

presentation,

asseeninsome

individuals

withsevere

hemolytic

HEand HPP.Identical

point mutations,

associated with variable clinical severity, have also been noted in

hemo-philia

A(43).

The occurrence of fourdifferent mutations in one codon allows

interesting speculation

as tothe

origin

of these

muta-tions.

Firstly,

theexistence ofa

positive

selectionpressurefor individuals heterozygous for a mutantallele has been docu-mented inthe caseof hemoglobin S, E, and thalassemic

dis-orders,

whereresistancetomalaria caused byamutationin one of the

globin

genes confersaselective survival advantage(44). In our

study

ofa

Sp mutations,

this would probably be an

unlikely

eventbecause thefamiliesareof different ethnic and

geographic origins

unrelatedtomalarialendemic regions.

Sec-ondly,

Arg28 may beacrucialamino acid intheSp molecule

sothat any

change

in thisresidue wouldalterthefunctionand structuralconformation of

Sp

and hencemanifestas adisease

state, whereas

changes

in the

surrounding

residueswould not haveadeleteriousinfluenceand henceonlyresult inclinically silent

polymorphisms

whichwould notbedetected.This like-wiseseems an

unlikely explanation

because the molecularbasis of

_{Sp a1174}

is

heterogeneous

and mutationsat codons46, 48,

and49

(previously

referredto ascodons

40, 42, 43)

havebeen

reported (22, 23).

Anequally

unlikely possibility

is that theArg

28mutationsaretoleratedbecause the

resulting

structural and functionalalterations couldbeassimilated

by

the red cell

mem-brane,

whereasmutationsin other

possibly

morecritical resi-dues

maybe

potentially

lethal and thus selected

against.

A strongcase

against

this

possibility

isa recent

study

ofa

family

witha

severely

dysfunctional Sp

mutation

(13).

The

homozy-gote

proband

carrying

thismutant

Sp

had a life

threatening

hemolysis

and her mutant

Sp

was

completely

devoid of the

propensity

to form

SpT.

In contrast,

heterozygote family

members hada

relatively

mild

hemolytic

HE

only,

because the adverse consequencesof this mutant

Sp

werediminished

by

the

product

ofthe normal

Sp

allele.

Webelievethatthemost

likely

conclusion is that thecodon

28may representa"hotspot" for mutationsbecauseit is en-coded

by

the nucleotides

CGT,

and CG dinucleotides have

been

implicated

asmutation "hot

spots"

for severalreasons:

(a)

CGdinucleotidesare observedat amuch lowerratethan

expected

inexonsequences,

implying negative

selectionor

re-pression (45); (b) evolutionary

studiesonvertebrate genes

indi-cated a

high

rate of CG substitution

(46); (c)

restriction en-zymes thatcontain CG dinucleotides in their

recognition

se-quence

(e.g.,

Taq

I,

Msp

I)

detect a

high frequency

of

polymorphisms (47);

and

(d)

a

large

numberofrecurrent

point

mutations

involving

CG-- TG and CG-- CAtransitions have

beenfound inhuman

genetic disease,

e.g.,

hemophilia

A(48,

49)

andadenosine deaminase

deficiency

(50).

Themechanism

underlying

this

frequent generation

of

mu-tations is

presumably

dueto

preferred methylation

of C resi-duesinthis

position

in the

heavily methylated

human genome

(51)

which can

subsequently undergo

spontaneous

deamina-tiontoT

(52, 53).

This would result inaC Ttransition or

alternatively in a G -E A transition if 5 methyl-C deamination occurred on theantisense strand. Two of the mutations noted in our study, in four unrelated kindred, involve such transi-tions

_(LGT

TGT;Arg

-*

Cys

andCGT--

CAT;Arg

-*

His)

and thus codon 28 may be regarded as a "hot spot" for a Sp

mutations. The mechanism underlying the recurrent muta-tions

involving

C -- _{AandG - T}transversions

_(Arg

₂₈ -i

Ser/Leu)remainstobe resolved.

Acknowledgments

Wewish to thank Tricia Duquette and Patty Cerulle for expert techni-cal assistance; Joan Joos for the artwork, and Loretta Wencis and Gabriella Maitino for typing the manuscript. We also thank Dr. Ber-nard Forget, YaleUniversity School of Medicine, New Haven, CT, for providing us the information on the DNA sequence and exon-intron boundaries of theaIdomain before the publication of these data in Reference 15.

This study was supported by grants HL3746 and HL27215 from theNational Institutes of Health, Bethesda, MD, and by a grant from theMedical Research Council of SouthAfrica.

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