Polymorphism of the human complement C4 and steroid 21 hydroxylase genes Restriction fragment length polymorphisms revealing structural deletions, homoduplications, and size variants

Polymorphism of the human complement C4

and steroid 21-hydroxylase genes. Restriction

fragment length polymorphisms revealing

structural deletions, homoduplications, and size

variants.

P M Schneider, … , E J Yunis, H R Colten

J Clin Invest.

1986;78(3):650-657. https://doi.org/10.1172/JCI112623.

Several autoimmune disorders as well as congenital adrenal hyperplasia (CAH) are either

associated or closely linked with genetic variants of the fourth component of complement

(C4A and C4B) and the enzyme steroid 21-hydroxylase (21-OH). These proteins are

encoded by genes that are located downstream from the genes for complement proteins, C2

and factor B (BF) between HLA-B and -DR in the major histocompatibility complex (MHC).

Previous studies of variants and null alleles were based on electrophoretic mobility of C4

protein and linkage with disease phenotypes. These data did not permit analysis of the

basis for the observed null alleles and duplicated variants. We studied this region of the

MHC in 126 haplotypes for a structural analysis of the four adjacent loci, C4A, 21-OHA,

C4B, and 21-OHB. About half of the C4 genes typed as C4 null are deleted and several

unrecognized homoduplicated C4 alleles were detected. Hence the frequencies of different

C4 structural variants must be recalculated based on a direct analysis of the genes.

Analysis of the C4/21-OH genes of patients with the classical (salt-wasting) form of CAH

showed that some involve a deletion of the C4B and 21-OHB genes; whereas for two only

the 21-OHB gene is deleted, i.e., the C4B gene is present. Together, these data provide a

better understanding of the mechanisms generating and importance of […]

Research Article

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Polymorphism

of

the Human Complement

C4 and

Steroid 21-Hydroxylase Genes

Restriction Fragment Length Polymorphisms Revealing Structural

Deletions,

Homoduplications,

and

Size Variants

Peter M. Schneider, Michael C. Carroll, Chester A. Alper, Christian Rittner, Alexander S. Whitehead, Edmond J. Yunis, and Harvey R. Colten

Divisionsof Cell BiologyandImmunology, Children's Hospital, Center for Blood Research, Dana Farber Cancer Institute, Departments

ofPediatrics andPathology,HarvardMedical School, Boston, Massachusetts 02115; Institutfur Rechtsmedizin, UniversitatMainz, D-6500 Mainz, Federal Republic ofGermany

Abstract

Several autoimmune disordersas well ascongenitaladrenal

hy-perplasia (CAH) are either associated or closely linked with

genetic

variants of the fourth component of complement (C4A

andC4B) and theenzymesteroid 21-hydroxylase (21-OH).These

proteinsareencodedbygenes that are locateddownstreamfrom

thegenesfor complement proteins, C2 and factorB(BF)between

HLA-B and -DR in the major histocompatibility complex

(MHC). Previous

studies

of variantsand null alleles were based onelectrophoretic

mobility

of C4 proteinand linkagewithdisease

phenotypes.These datadidnotpermit analysisof the basisfor the observed null alleles and duplicated variants. We studied this region of theMHCin126 haplotypes forastructuralanalysis ofthefour adjacent loci, C4A, 21-OHA, C4B, and 21-OHB.

About half of the C4 genes typedas C4 nullare deleted and

several

unrecognized

homoduplicated C4alleles weredetected.

Hencethefrequencies of different C4 structural variantsmust

be recalculated basedon adirectanalysis of thegenes.Analysis of the

C4/21-OH

genesof

patients

with theclassical

(salt-wast-ing) form of CAH showed thatsomeinvolve adeletionofthe

C4B and 21-OHBgenes;whereas fortwoonly the 21-OHBgene

is deleted, i.e., theC4B geneis present. Together, thesedata provide a better

understanding

of the mechanisms

generating

andimportance of deleted C4 and 21-OH null allelesinhuman

disease.

Introduction

The shortarm of human chromosome 6 carries the genesfor factor B

(BF),'

for the second and the fourth component of

human

complement (C2, C4A,

and

C4B),

and for the

cyto-chrome P-450

steroid

21-hydroxylase (21-OH) (1-4).

C2 and BF are about 0.5 kilobase

pairs (kb)

apart and are

separated

from the C4genes

by

30 kilobases

(kb).

The C4 genesare 10 kb apart,each witha

21-hydroxylase

geneatthe 3' endat adistance AddressreprintrequeststoDr.Colten, DepartmentofPediatrics, Wash-ington University Medical School, 400 S.KingshighwayBlvd., St.Louis, MO65110.

Receivedfor publication5 March 1986.

1.Abbreviationsusedinthispaper: BF, factor B;CAH, congenitaladrenal hyperplasia; MHC, major histocompatibility complex; 17-OHP, 17-hy-droxyprogesterone; RFLP, restrictionfragmentlengthpolymorphism;

SSC,standard salinecitrate;SDS-PAGE,sodiumdodecyl sulfate-poly-acrylamidegelelectrophoresis.

of - 1.5 kb. The orderofthe genes in thedirectionof

transcrip-tion is C2, BF, C4A, 21-OHA, C4B,21-OHB (5-7), andthey have beenmapped between HLA-13 andHLA-DR (8), which areseparated by -0.7cM(9). Because ofthetight linkage,these genes are usually inheritedas a single "complotype" (10). In some haplotypes, linkage disequilibrium includesthe HLA-B

and-DRregion

forming

"extended haplotypes"(I 1).

Thehigh degreeof polymorphism in C4 has led to

specu-lation that

susceptibility

to someautoimmune diseases might beduetodifferences in the interaction ofC4variants withother

polymorphic

complement

proteins,

antigens,andantibodies

(12,

13).

The

frequency of null alleles attheC4loci (0.10-0.15) is relativelyhigh (14), and somepossible associations ofC4 null

alleles(C4 QO) with diseases oftheimmunesystem have been

suggested,e.g.,

systemic

lupus erythematosus (SLE), scleroderma, and subacute

sclerosing panencephalitis

(SSPE)

(15-17).

Adefect ofthefunctional21-OH gene

results

in congenital adrenal

hyperplasia (CAH)

andone

mutation

shows linkage

dis-equilibrium

witha rare MHC

haplotype (18,

19). Ithasbeen

shown that 2 1-OHB is the functionalgeneand thata

deletion

of the 2 1-OHA gene doesnot affect steroid metabolism (20). Since

deletions of

the 21-OHgenes arecorrelated with null alleles

attheadjacent C4

loci,

ithasbeenpostulated that these C4 QO

genesrepresent deletionsaswell (6). Several CAH haplotypes have therefore been included in this study.

Restriction fragment length polymorphisms (RFLPs)have

proved useful in the investigation ofthe basis of C4 genetic vari-ants(21) andcansubdividecommonC4allotypes

(22). Analysis

of

genomic

andcomplementary

(cDNA)

sequencesofC4Aand

C4B hasshown that only eight nucleotide differences

resulting

in aregional change in six amino acids accountfor the class differences between the C4

isotypes.

Even fewerexchangeswere

detected in

allotypic

variantsateach locus

(23, 24).

Avariation

insizeof the C4B gene (the short form is 16 kb,whereas the long form is identical in sizetothe C4Ageneat22

kb)

isaresult ofthepresenceorlackofa6.8-kb intron in the 5'

region

of the C4Bgene(25,

26).

Somebut notall C4A and C4B null allelesaredueto

large

deletionsthat includeadeletionofa

flanking

21-OH gene

(25).

Molecularmapsof

genomic

DNAshowedadeletion ofthe C4A and21-OHA genesorthe21-OHAand C4B genes.Three

hap-lotypeswith nondeleted C4null alleleswerealso

identified (25).

Here we present the use of a restriction

fragment length

polymorphism

locatedatthe 5' end of bothC4 genesto inves-tigatethemolecular basis ofC4

QO

alleles and

duplications

as

well as the frequencyofthe long and the shortform oftheC4B gene.

Applied

together witha

probe (7)

to detecta

fragment

size difference ofthe 21-OH genes(20), this

polymorphism

provides

informationabout the structureofthe

C4/2

1-OHgene

complex,

which can beusedtofurtheranalyzethe

genetic

basis ofdisorders associatedwithdeficiencies ofthe C4 and21-OH loci.

J.Clin.Invest.

©TheAmerican Societyfor ClinicalInvestigation,Inc.

0021-9738/86/09/0650/08 $1.00

Methods

Selectionofindividuals.Thisstudyis based on the DNAtyping patterns of 92 individuals from 32 Caucasianfamilies, including8patients with CAH, 5patients with celiac disease, and4patients withmultiplesclerosis. ARl other subjects were healthy and were selected for the presence of haplotypes carrying null alleles at one or both of the C4 loci.

Complement and HLA typing. Blood forcomplement typingwas collected intoImg/mlEDTA,centrifuged,and the serum was stored at -80'C. BF typing was carried out by agarose gel electrophoresis followed byimmunofixation with goat anti-human BF antibody (Atlantic Anti-bodies, Scarborough, ME)(27). ForC2 typing, plasma sampleswere

subjectedtoisoelectric focusing in polyacrylamide gels (28). C4 typing wascarried out by high voltage agarose electrophoresis inadiscontinuous buffer system after desialation of the samples withneuraminidase, fol-lowed byimmunofixationwith goat anti-human C4antibody(Atlantic Antibodies) (29). The alpha-chains of C4A and C4Bwereanalyzed by sodium dodecyl sulfate(SDS) polyacrylamidegelelectrophoresis (PAGE) (30). Blood for HLAtypingwascollectedintoheparinat afinal

con-centration of 10 U/mi. HLA-A, -B, -C, and-DRantigenswereassigned by themicrolymphocytotoxicityassay(31,32).

Complotypesaregiven inarbitraryorderasBF,C2,C4A,and C4B types in abbreviated form. Thus, "SCOI" represents BF*S, C2*C, C4A*QO, C4B*1.

Preparationofhuman genomicDNAand Southernblotting.For DNA

preparation, individual blood samples of 30 ml eachwerecollected into

asyringecontaining2 ml of 100mMEDTA. The DNAwasextracted from white blood cellspurified bysucrosesedimentationaccordingto

standard procedures(33).IndividualDNAsamplesof 10Mgweredigested with restriction enzymesat aconcentration of 5 U/ugaccordingtothe manufacturer'sinstructions (New EnglandBiolabs, Beverly,MA). The

DNAfragmentswereseparatedbyelectrophoresisina0.8% agarose-gel (ultrapure agarose, BRLLaboratories, Bethesda, MD)andblottedonto

nitrocellulose(BA85, Schleicher &Schuell,Keene, NH)(34,35).

Pre-hybridization and Pre-hybridization werecarriedoutinamixturecontaining

4 Xstandardsaline citrate (SSC), 50mMphosphate buffer pH6.8, 1 mM EDTA, 0.1% SDS, 5% dextransulfate,5XDenhardt'ssolution,0.1 mg/ml yeast RNA, and 50pg/mlsheared denatured salmon spermDNA.

Ashybridization probe, 1MgofpurifiedcDNAinsertwaslabeled with 100MCi

_{alpha-[32P]dCTP}

(deoxycytidine triphosphate, New England Nuclear,Boston,MA) inanick translation reaction(36). Hybridization wasperformed for 15 h at650C,followed bytwo tothreesubsequent washes of 30 min each in 0.2XSSC/0.1% SDSatthesametemperature. Thehybridization patterns on the blotswerevisualizedby autoradiog-raphy.

C4and 21-OH

specific

DNAprobes. The cDNAprobe used to

gen-eratethe 5' C4 polymorphism was derived from thefulllength C4 cDNA clone pAT-A (23). The 500-bp probe specific for the 5' ends of both C4 genes wasobtainedafter Bam HI/KpnIdouble digest, separated from theotherfragmentsby agarose electrophoresis and purified by electro-elution intoadialysis bag (37). The probe specific for the 21-OH genes wasderivedfrom the cosmid clone cos I E3 and represents a 900-bp Bgl

Ifragmentof the 2 1-OHA region (7). The 300-bp insert of cDNA clone pALU-7 is specific for the C4d region of both C4 genes (38). These inserts were purified as described above.

Interpretation ofrestrictionfragmentpatterns. The5'C4 fragments were mapped relative to the 12- and 3.5-kb Kpn Ifragmentscharacteristic for the 5' ends of the C4A and the C4B genes (5). Thecorrelationof thesefiagmentswith the two forms of the C4B genes has been established byhybridizationof KpnIdigestedDNAwith thecDNAprobe pALU-7,which yields a 7.5-kb band for the long C4 genes and an 8.5-kb band for the short C4B gene (26, 39).

Several precautionswereobserved to obtain accurate and reproducible results. The evaluation of relative intensities of DNA

fragments

on Southern blotsasevidence for the number ofgenes was only carried out within the same digest on the blot. Intensities of C4 and 21-OHbands within the same digest were not compared, as they depend on the size

andspecific activityof the radiolabeledprobe.Assignmentofadeletion orduplicationwasestablished infamilystudiestoconfiri the_findings

bysegregationanalysis.

Results

Pattern

of

the

Taq

I

polymorphism.

After

digestion

ofgenomic DNA with the restriction enzymeTaqIandhybridizationwith the labeled 5' C4

probe,

four differentfragmentswerevisualized.

Fig.

1 shows the

position

ofthese

firagments

within theC4/2 1-OH

region

and the genotypes in which they occur. Southern blot

analysis

of DNA from

homozygous

individuals shows that the

longest fragment (7.0 kb)

isspecificfor the C4A gene

locus,

andthe other three represent different alleles oftheC4B gene. One of them (6.4 kb) isamarker foradeletion of the C4A gene in thecomplotype SCOI (BF*S, C2*C, C4A*QO,C4B* 1). This

complotype

isusuallypart of the extendedhaplotype

HLA-B8,

DR3, SCOJ.

The 6.4-kbfragmentis locatedatthe 5' end of the remaining shortC4B gene in this haplotype. The 6.0-kb

(C4B

long)and 5.4-kb(C4B short)bands represent thetwoforms of C4B in normalhaplotypeswith two C4 loci. The 21-OH genes areidentifiedbythe 3.2-kb(21-OHA)and3.7-kb (2 1-OHB)

Taq

Ibands.

Two different forms of C4B deletions were detected in

family

EM.(Fig. 2a). Father A. hasnoC4Bbands,but both the 21-OHA and the 2 1-OHB bandare _{visible. This pattern suggests} that both C4B genesaredeleted. Ononechromosome, the dele-tion includes a21-OHA gene, andon the other the 2 1-OHB gene. Mother R. has a stronger C4A than C4B band, and a

stronger 2 1-OHB than 2 1-OHA band. A possible deletion could include the 21 -OHA and C4B genes on one chromosome,

whereas the other carries all four genes. This assumedparental

deletion pattern is confirmedbythe analysis ofsegregating chro-mosomes in the children. DaughterP. has inherited both

21-OHA/C4Bdeleted haplotypes; i.e.,no21-OHAorC4B

fiagments

canbedetected. Son L. hasadeletion ofthe C4B/21-OHB genes on onechromosome, inherited from A.; i.e., the C4A and the 21-OHA bands are stronger than the C4B/2 1-OHB bands.

Al-Probe: 5'C4

H ToqIFragments: 7.0

21-OH 50C4

H H 21-OHH 3.2 5.4 3.7

U~j

0. CIL

H H H H

70 3.2 6.0 3.7

b.I~.CA IC

b. _{{ s [' 3}

H H

6.4 3.7

C. A c4

'B

1OKb

I

a b c d

C4.04' -_6

Iw 60

21-OH WS104 -372

L4 0-.

Figure1.Mapof the human C4 and 21-hydroxylase gene region illus-tratingtheTaqIrestrictionfragmentpattern. (a) 7.0/5.4-kb

a. b.

A. R J. M

P L. E.

a c b a a c b

Kb Kb

70-

wVW

_i70

60- - ^

-64

37-

_-37

32- -32

U 21-OHA/C4B * C4A/21-OHA+Iong C4B

De/eflicn

of

0

C4B/21-OHB

ED

C4A/21-OHA+short

C4B

Figure2.TaqIrestrictionfragmentpatterns ofhaplotypes carrying deletionsof the C4 and 2

1-hydroxylase

genes.(a)Familytreeand Southern blotshowingthesegregationof

21-OHA/C4B

andC4B/2 1-OHB deletions infamilyEM. The

haplotypes

indicated above the blot

are:a,HLA-A3,Cw6, Bw47, DR7, FC91,0,deletion of C4B and

21-OHB; b,

HLA-AlJ,

Cw4, B35, DR], SC30,deletion of21-OHAand

C4B;c,

HLA-Al,

C'4,

B35, DRw6,

SC3J,

nodeletion; d,HLA-A2,

Cw4,

B35,

DR], SC30, deletionof21-OHAandC4B.(b)Familytree andSouthern blot

showing

thesegregationofC4A/21-OHAdeletions in

family

AD.Thehaplotypesindicated above the blotare:a,

HLA-Cw5,

B44,

DR3,

SCOl,

deletion of C4A and

21-OHA

in combination with short C4B gene;b,

HLA-A2,

B7, DR2,

SC3J,

nodeletion;c,

HLA-CwS,

B44, DR4,

SCO),

deletionofC4A and 21-OHA in combi-nation withlongC4B gene;d,

HLA-AJ,

B8,

DR3, SCO1,

deletionof C4A and 21-OHA incombinationwith short C4B gene.

though

the C4

fragment

patternsof R.and L.are

identical,

the reversed intensities of the 21-OH bands indicate thepresence

of

different

deletions.

Arare

_complotype

withaC4Adeletionwasfoundin

_family

AD

(Fig.

2

_b).

Mother

M. hasno detectable C4A

_protein

as

assesse

_by

agarose

electrophoresis

and immunofixation of

neuraminidase-treated

plasma. However,

a7.0-kb5'C4 bandis

clearly

visible

together

witha6.4-kb bandfromtheother

chro-mosome,

butnosecond5' C4B

fragment

isapparent.Basedon

the lackofaC4Agene

product

according

tothe

protein

typing,

onecouldassumethat the

7.0-kb

bandrepresents in thiscase

the 5'endofa

long

C4Bgeneincombination withaC4A dele-tion.The 21-OHAgenesonboth chromosomesaredeletedas well.This deletion is

inherited

with

HLA-B44,

DR4.

Incontrast,

thedeleted

AQO

haplotype

fromfatherJ. segregateswith

HLA-B44,

DR3 andshows the usual 6.4-kb band. This indicatesthe presenceofashort C4Bgene,asit isfound inthemost common

haplotype

withaC4Adeletion andashort C4Bgene,the

ex-tended

haplotype B8, DR3,

SCOJ.

The

paternal

chromosome

thus

_might

have been

generated

by

anancestral

crossing-over

eventbetween the HLA-B44 locus and the

complement

genes

of the extended

haplotypej

whereas theC4A deletion withthe

long

C4Bgene on the maternal chromosome has

apparently

originated independently.

Analysis

of

the

21-hydroxylase

genesinCAH

patients.

Eight

patientswith theclassical

(salt-wasting)

form ofCAHwere

stud-ied together with their families. Intwo

patients,

the21-OHB gene alonewas

deleted,

andtheC4B genewasintact

(Fig.

3

a).

Five

patients

had atleast one21-OHBgene

(Fig.

3 cand

d),

whereas the other threeindividuals lacked the 3.7-kb TaqI

frag-ment thatrepresents the 21-OHB gene

(Fig.

3 a and

b).

One

haplotype

hadadeletion of the

21-OHA/C4B

genes

(Fig.

3

e),

andeight haplotypes carriedaC4B/21-OHB deletion. Of

these,

five occurred in the CAH-linkedextended

haplotype

Bw47,DR7,

FC91,0.

This haplotype was

originally typed

as

FC0,31,

and renamedonthe basisof structural data

(see

discussion)

and pro-tein

typing

in

comparison

tothe knownC4

allotypes

A

3,

A

1,

A91,A92,and B3(datanot

shown).

Detection

of

heteroandhomoduplicated C4 loci. An indi-vidual

carrying

aheteroduplication of theC4Blocus(two

dif-ferent C4B alleles onthesame chromosome) togetherwith a

C4A null alleleon onechromosome (C4A*QO, B*1,2) anda

common C4 haplotype on the other (C4A*3, B*l) has been analyzed byC4proteinandDNAtyping(Fig.4aandb).Father B.(aandb)hasequallystrong 7.0 kb(C4A) and5.4kb

(C4B

short)bands andaweak6.0-kb(C4B long) band,thus

indicating

the presenceof five C4 alleles (oneofthemanondeleted C4A

nullallele). With the 21-OHprobe, the 3.7 kb (21-OHB)band isslightlystrongerthan the 3.2-kb(21-OHA) band, andsuggests thatthe 21-OHB gene is alsoduplicatedon onechromosome. Chromosome bprobablyhastheduplicated C4B/21-OHBgenes as sonM. (a andd)does not appear to have inherited this pattern. Similar restrictionfragmentpatternswereobserved in other

individuals withduplicated C4 locithat were notdetected

by

C4 protein typing. In family CH., a homoduplication ofthe C4B locus(twoidentical C4B alleles on the same chromosome)was detected on Southern blotanalysis.Thisduplicationsegregates with chromosomebfromfatherS.tohisdaughters (Fig.5,

top).

Thefinding was supported by the C4 typing gels (Fig.5, bottom),

where stronger C4B 1 protein patterns in individualsS.,JI. and D. were observed. A stronger C4B than C4A alpha chainwas also observed after separation of immunoprecipitated and re-ducedC4 from plasma samples bySDS-PAGE(data notshown).

To obtain further evidence for the duplication, DNAsamples

offamily CH. were also digested with the restriction enzymes Bgl IIand Kpn I and hybridized with the C4 and 21-OHprobes

(data not shown). These patterns were used to establish apartial

restricion map ofthe duplicated gene region

(Fig.

6). Inaddition,

another homoduplication ofthe C4B locus in combination with anondeleted C4A null allele in thehaplotype HLA-AII,BJ7, DRw6,FC01,I hasbeen detected(datanotshown).

Screening for deletions of the C4/21-OHgenes. To obtain

data on the occurrence and type of deletions in the C4/21-OH

region, 126 individual haplotypes were analyzed in family studies

usingthe 5' C4TaqIpolymorphism. The families were selected forthepresence of one or more C4 null alleles, based onprotein typing. Also,members of some families are carriers of disorders like CAH, multiple sclerosis, and celiac disease. The data ob-tained thus are notrepresentativeforfiequenciesinthe normal population.

TheSouthern blot analysis of C4 haplotypes carrying a null allele(C4QO)showed thefollowing: (a)Deleted to nondeleted allelesshowedaratio of 3:2.(b)AlldeletedC4A genes occurred with thecomplotypeSCOIandincluded deletions ofthe 21-OH gene.(c) C4B null haplotypes with a deletion included either deletion of a 21-OHA or a21-OHB gene. (d) Deletion of the C4B gene was not observed inSC30andFC30 complotypes in

Name HLA- A C B DR BF/C2/C4A/C4B

TaqI-fragments:

C4A OHA C4B OHB

a. F.C. w24 w24 w2

C.S. 3

2 w2

44

w62 w6

w47 7

51 3

F C 4 QO

F C 3 1

F C 91 QO

S C 3 1

7.0 3.2 -

-7.0 3.2 6.0

-7.0 3.2 -

-7.0 3.2 6.0

--70 -6.0

* -32

b. K.G. 3 w47 7 F C 91 QO

3 w3 40 4 S C 3 QO

c. C.D. 1 8 1 S B 4 QO

3 51 2 F C 3 1

7.0 3.2 -

-7.0 3.2 -

-7.0 3.2 -

-7.0 3.2 6.0 3.7

-70

-3.2

V -70

-60

3 w6 w47 7

1 w4 35 1

2 w5 44 4

11 w47 7

F C 91 QO

S C 3 1

S C 3 QO

F C 91 QO

7.0 3.2 -

-7.0 3.2 6.0 3.7

7.0 3.2 6.0 3.7

7.0 3.2 -

-28 w3 w60 S C Q0 2 7.0 3.2 5.4 3.7

28 w6 w47 7 F C 91 Q0

28 14 4 FS C 3 1

3 w60 1 FS C 3 Q0

7.0 3.2 -

-7.0 3.2 5.4 3.7

7.0 - - 3.7

-70 Figure 3. Haplotypesand Taq I restriction frag--54 mentpatterns ofpatientswith the classical

(salt-wasting) formofCAH.(a) homozygous deletionof

3.7 21-OHB and heterozygous deletion ofC4B;(b) ho-3 2 mozygous deletionofC4B and21-OHB; (c)

het-erozygous deletion of C4B and 21-OHBwith

re-maininglong C4B gene;(d)heterozygous deletion of C4B and 21-OHB withremainingshort C4B

7.0 _gene;heterozygous deletion of 21-OHAand C4B -5 4 with bothremaining21-OHBgenes.(The

South-ernblot figuresin lanes a,d,ande areobtained '

-37 _{fromsequentialhybridizationsof the C4 and}

21--32

OHprobesonthesameblot.)

The individual haplotypes with deletednull allelesarelisted

inTableI A. Allbut three C4A QO deletionsarelinkedto HLA-B8, DR3, and all 21 show thecomplotype SCOJ. Inthisgroup,

the 21-OHAgeneis also deleted.Allof the four 2 1-OHA/C4B deletions occurinthe SC30 and FC30 complotypes, but with

various HLA determinants. Six of nine C4B/21-OHB deletions segregate withHLA-Bw47, DR7, FC91,0, ahaplotype linkedto

CAH.The other threehaplotypes withadeletion ofthe 2 1-OHB

gene arealso obtained fromCAHfamilies.

C4AQO haplotypeswherethegeneisnotdeleted showno

linkagetospecific HLAtypes(Table I B).Incontrast, 12of17 nondeleted C4B null allelessegregatewith HLA-B44,SC30, and

sevenofthese withthe extended haplotype B44, DR4, SC30.

Allnondeleted C4B null allelesarefound in thecommonSC30

andFC30 complotypes.

Distribution of the long and short C4Bgenes. Theanalysis

oflongand short C4Bgenesshowed the following: 61 of 13 haplotypes without deletion of the C4B locuscarry along C4B

gene.Only the longC4Bvariantisfound when the C4Bgeneis

presentbutnotexpressed. C4A null alleles thatarenotdeleted

may occurwith both sizes of C4Bgenes. Most C4A null

hap-lotypes withadeletion of the C4Agene(19/21)carrythe short

C4Bgene.

Themostcommoncomplotypesinvestigatedarerepresented

inFig. 7togetherwith thedistribution of thelongandthe short

form ofthe C4Bgene.Most of the SC3J complotypes (34/38)

havethe long C4Bgene,and11ofthesearefound in the extended haplotype HLA-B7, DR2, SC31.In contrast,amajorityof the FC31 complotypes (9/13) have the short C4B gene; three of themarelinkedtoHLA-B44,DR7. With thesingle exception oftheFC32complotype,which segregates withHLA-B51, DR4, all haplotypeswithaC4B2 allelecarrythe short C4Bgene, as

doallHLA-Bw57, SC61 haplotypes.

Discussion

The biochemical analysis ofpolymorphic complement gene

products has alreadyrevealedsignificantfunctional differences betweenisotypicandallotypicC4 variants(40-42).Theanalysis ofthe structural basis of C4 null allelesbyrecombinantDNA

techniquesmayhelptodescribemechanismsleadingto dupli-cations and deletionsaswellasto understandthe effect of these events in regardto possible autoimmune disease associations

(15, 17, 43).InthecaseofCAH,aclosegenetic linkageof this

disease toa limited numberofMHC-haplotypes has been

es-tablished(18, 19).Thislinkageis duetothe locationofthegene forsteroid 21-hydroxylasewithin theC4region.A DNA

frag-mentlength polymorphism generated bythe restrictionenzyme

Taq Iprovidesan effective andconvenientwaytoanalyze si-multaneouslythegenomicstructure of the fouradjacentloci of A.E.

S.P.

d. A.R.

-37 _ -3.2

a.

B 01<

a ca

b d d

b.

I'll

Kb

6.0- , C4A

5.4-C48 3.7-heCWN

3.2- - _

1 2 3 0 C4Bduplication

Figure 4. Heteroduplication of C4B locus as detected by Taq I restric-tionfragmentpattern and C4 protein typing. (a)Familytree and Southern blot revealing duplication of a short C4B gene and 21-OHB infamilyBR. Thehaplotypes indicated above the blot are: a, HLA-A31,Cw2,BSJ,DRw8,SC31;b,HLA-A26,Cw3,Bw60,DR5, SC01,2, duplication of the C4B locus with nondeleted C4A null allele;

c,HLA-Al,Cw6,Bw57,DR5,SC31;d, HLA-A2,B18, DR2,SC3O. (Thefiguresin thefirst two lanes are obtained from sequential hybrid-izations of the C4 and 21-OH probes on the same blot.) (b) C4 protein typing pattern, obtained by agarose electrophoresis of

neuraminidase-treatedplasma andimmunofixation;lane 1, control pattern with two expressed C4A and C4B loci; lane 2, C4 pattern of father B. (4a, lane 1)with duplicated C4B locus and a single expressed C4A locus; lane 3, control pattern with two expressed C4A loci and duplication of the C4B locus.

theC4A,21-OHA, C4B, and 21-OHB genes, sincethe C4 and 21-OHbands are well separated on the blot and easy to identify.

Ourresults on the structureofC4A null allelesconfirm in

family

studiesprevious

findings

thatmappedaC4Adeletionin

combination withashortC4Bgene,obtained by

analyzing

clones

fromagenomic library (25).Inaddition,we wereabletodetect two deleted C4AQO haplotypes withalongC4B gene

(Fig.

2

d,

TableI).The Taq Irestrictionsiteupstreamfrom the C4region appeared preserved

(Fig.

1),andin thecaseofaC4Adeletion

thelength oftheresulting

fragment

dependsonthe

position

of the 3' restriction site insidethe C4Bgene;

i.e.,

6.4 kbwith a short B gene and 7.0kbwitha longB gene. Deletionsofthe

21-OHA/C4Bgenessimilartotheonedescribed (25) in the hap-lotypeHLA-B7, DR2, SC30 weredetected infour other

hap-lotypes (TableI),and allshare thesamecomplotype SC30. Deletions affectingtheC4B/2

l-OHB

genes occurmostly in haplotypes foundinCAHpatients (Table I). With the

exception

ofHLA-Bw47, DR7,thesehaplotypes differ inHLA typeaswell

as incomplotype.Theextendedhaplotype Bw47, DR7,

FC91,0

has afiequency of >20%amongCAHpatients (19).The func-tional21-OHB gene(6) and theC4Bgene are deleted in this haplotype. Southern blotanalysis of

genomic

DNA indicated

that thedeletion extendsfrom the end of the 3.2-kb

Taq

I

fiag-ment inthe 2 1-OHA gene to thehomologousregion 3'tothe

21-OHBgene(44).

While in most cases (eight haplotypes) deletions of the

21-OHBgeneincluded the C4Bgene, in twounrelated CAH

patients

only the 21-OHB gene was deleted. In contrast, five haplotypes in CAH

patients

withno apparentdeletion of the

21-OHBgene were observedas well. Thus, the classical (salt-wasting) form ofCAHiscausedbymajor structural deletions, but isalsoaresult of defects in 21-OH

expression. Therefore,

a

b

b

d

b

c

c

_0

O- _{_V} %

-#

__

C4Ar

C48L

* C4B duplication

[ffR

C4A/21-OH

A

deletion

Figure S.Homoduplication of C4B locusasdetectedby TaqIrestrictionfragment pattern and C4protein

typ-ing.Family tree, Southern blot and C4protein typing patterndemonstratingthe segregationofaC4B homo-duplicationinfamilyCH. Thehaplotypes indicated above the blotare:a, HLA-Al, Cw6, BwS7,SC61;b, HLA-Aw33,B44,DR7,

SC3J,1,

duplicationof the

21-OHAandC4B genes; c, HLA-Al,B8,DR3,SCOJ,

deletionof the C4A and 21-OHA genes;d, HLA-A24, Cw7,B7,DR2,SC3J.

every CAH haplotype notcarryingaC4B null allele must be

analyzed further. On a functional level, the analysis of the

17-hydroxyprogesterone (17-OHP) response to ACTH stimu-lationhasprovidedabiochemical basistoassesstheseverityof

thedisease in heterozygousandhomozygous carriers of CAH haplotypes (45).

Unequal crossing-over mechanismshave beensuggested by

several authorsto account forthe

generation

of deletionsand

duplications (25, 29, 46, 47).The highdegree ofstructural

ho-mologyin theC4/21-OH region might facilitateunequalpairing ofchromatids during meiosis.

Duplication

ofthenearby C2and Factor B genes has not been detected (48, 49), although the genes show asubstantial degree of homology

[39%

identity (50)].

NullallelesforC2 that have beenanalyzed do not appear to be

duetolarge

deletions,

astheDNArestriction fragmentpatterns

offunctionaland nullallelesareidentical (49). Thenearidentity

(>99%)

amongthe

duplicated

C4/21-OHgenesfavors

recom-binantevents.

Unequal crossing-over intheC4region would result inone

chromosome witha

single

andthe otherwith three C4genes. Based on the C4 protein typing data, the

frequencies

ofnull

JE

s

7.0

m

7.0

6.4

1 1

3.2 5.4

m l

L Lii

3.2 5.4

3.7

m

L LJ

3.2 5.4 3.7

3.7

m-3

7.0 3.2 6. 3.7

7n A2) ~Ai

. .v, Figure6.Homoduplicationof C4B locus:partial

restric-b MC4A3 - tionfragmentmapsofsegregating haplotypesoffamily

D/JI b CH. Restrictionfragmentmap of the segregating

haplo-C < > _ types

showing

the location and number of the_TaqI L'~JL~L'fragmentsin theC4/2I-OHregionof both

chromo-LTJ !somesofindividuals S., JE., and D./JI. offamilyCH.

alleles[0.10-0.15 (14)]andduplications [0.0075-0.01 (46)]do alleles weredeleted. This figure includes nine rare CAH hap-notcorrelate. Our finding that not all C4 null alleles are deleted lotypes, so that only about 50% of all C4 null alleles might be changes this ratiosignificantly. Inthisstudy, 34 of 57 C4 null due todeletions in the normal population. Also, wehave

ob-TableLHaplotypesCarrying C4NullAlleleswith andwithoutDeletions

A. Haplotypes with deletions oftheC4and21-hydroxylasegenes:

HLA-A C B DR Bf / C2 / C4A / C4B (total) (CAH)

I. C4A/21-OHA deletions * - 8 3 S C QO 1 (18)

w5 44 3 S C QO 1 (1)

2 w5 44 4 S C Q0 1 (1)

2 w2 51 1 S C Q0 1 (1) (=21)

II. 21-OHA/C4Bdeletions * w4 35 1 S C 3 Q0 (2)

3 w4 35 w6 S C 3 QO (1)

3 w3 w60 1 FS C 3 QO (1) (=4) (1)

III. C4B/21-OHB deletions * [w6] w47 7 F C 91 QO (6) (5)

3 w3 40 4 S C 3 QO (1) (1)

1 8 1 S B 4 QO (1) (1)

w24 44 F C 4 QO (1) (=9) (1)

IV. 21-OHBdeletions w24 w2 w62 w6 F C 3 1 (1) (1)

2 w2 51 3 S C 3 1 (1) (=2) (1)

B. C4QOhaplotypeswithoutdeletions:

HLA-A C B DR Bf / C2 / C4A / C4B (total) (CAH)

I. C4AQ0 haplotypes * - 7 [2] S C QO 1 (2)

2 w2 51 1 S C QO 1 (1)

28 w3 w60 S C QO 2 (1) (1)

1 w4 35 S C QO 5 (1) (=5)

II. C4BQOhaplotypes 3 7 S C 3 QO (1)

2 [w5] 44 4 S C 3 QO (7) (1)

* - 44 * S C 3

QO

(5)

w26 w6 13 3 F C 3 QO (1)

* - 44 w6 F C 3

QO

(2)

28 51 w6 F C 3 QO (1) (17)

*Various determinants; [ Ithis determinantor notdefined;-notdefined.

0

9R

MI

TableII.Extended Haplotypes [1]Recognized Among the SelectedHaplotypes

1[1 (*1 (Extendedhaplotype)

HLA-A C B DR Bf / C2 / C4A / C4B Frequency No.observed Deletion C4B

(normal, 1) inthis study of C4 size

1 w7 8 3 S C QO 1 0.082 18 A Short

3 - 7 2 S C 3 1 0.043 11 Long

2 w5 44 4 S C 3 QO 0.029 7 Long

23 w4 44 7 F C 3 1 0.027 3 Short

1 w6 w57 7 S C 6 1 0.019 1 Short

2 w3 w62 4 S C 3 3 0.016 1 - Long

[1] FrequenciesamongnormalCaucasians (11);[*]PredominantHLA-Aand -C alleles.

tained evidence for an increased number of homoduplicated

haplotypes, whichhave not been identified previously. Therefore,

deletionsand duplicationsmay bemore common than

previ-ously suggestedand only by direct analysis of this region on the DNAlevelcan thefrequenciesbedeterminedaccurately.

Inthefirst analyses ofthe C4 generegion, only shortC4B

genes(16 kb)were detected (5, 7, 25). In the present study, a

majority ofthe C4B genes analyzed were the same size as the

C4A gene (22 kb). This includes all 17 C4B null haplotypes

studied thusfar, in which the C4B gene is present but not

ex-pressed. The C4Bgenesize polymorphism, due to the 6.8-kb

intron,

raises questions about thestructureofthe ancestral C4 genethatgaverisetothe duplicatedhuman C4A and C4B genes

and themouseSlp and C4genes.

Thesixmostcommonextended haplotypesamong normal

Caucasians (1 1)wereincluded intheanalysisof the 5' C4

poly-morphism (Table II). AlthoughC4Bgene lengthpolymorphisms subdividesome groupscomplotyped by protein polymorphisms

(Fig.

7), within specific extendedhaplotypes only asingletype

of C4Bgeneis observed. Thesame homogeneity has been ob-servedamongextendedhaplotypes by using theBamHI/XbaI

polymorphism of

the C4

gamma-chain region

(A. S. Whitehead, unpublished observations). Uniformityof extendedhaplotypes

wasalso demonstrated by comparing the mixed lymphocyte re-sponsematched for the extended

haplotype

even in unrelated individuals (51). This high degree offunctionalandstructural preservation is also supported byouranalysisat the DNAlevel.

B

A

Comp/otypes: SC31 No.Anolyzed: (36)

B

FC31

(11)

C

E

A A

SCot SC30 FC30 (25) (17) (4)

A B

SC61 SB/C42

(3) (8)

Figure7.Distribution of differentC4/21-hydroxylasegenestructures

amongcommoncomplotypes.The TaqIpatternsarelisted in the structural order of the genesC4A/21-OHA/C4B/21-OHB ("-"

indi-cates adeletion): (A) 7.0/3.2/6.0/3.7,C4Blong;(B) 7.0/3.2/5.4/3.7, C4Bshort; (C) -/-/6.4/3.7, C4B short;(D*)-/-/7.0/3.7,C4Blong; (E*)7.0/-/-/3.7;F=_7.0/3.2/-/-;(*distinguishedonthebasisof C4protein typing patterns).

Acknowledaments

This work was supported by grants from the National Institutes ofHealth (HD-17461,AI-21157,HL-29583,'AI-14157,and CA-20531) andfrom theDeutscheForschungsgemeinschaft(Ri 164/16-2). P. M.Schneider isarecipient ofafellowship fromtheGermanAcademicExchnge Service (DAAD), andA.S. Whiteheadisarecipient ofaHelen HayWhitney Foundation Fellowship (F488).

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