Genetic basis of human complement C4A deficiency Detection of a point mutation leading to nonexpression

Genetic basis of human complement C4A

deficiency. Detection of a point mutation

leading to nonexpression.

G Barba, … , C Rittner, P M Schneider

J Clin Invest.

1993;

91(4)

:1681-1686.

https://doi.org/10.1172/JCI116377

.

The fourth component of the human complement system (C4) is coded for by two genes,

C4A and C4B, located within the MHC. Null alleles of C4 (C4Q0) are defined by the

absence of C4 protein in plasma. These null alleles are due either to large gene deletions

or to nonexpression of the respective genes. In a previous study, evidence was obtained for

nonexpressed defective genes at the C4A locus, and for gene conversion at the C4B locus.

To further characterize the molecular basis of these non-expressed C4A genes, we selected

nine pairs of PCR primers from flanking genomic intron sequences to amplify all 41 exons

from individuals with a defective C4A gene. The amplified products were subjected to

single-stranded conformation polymorphism (SSCP) analysis to detect possible mutations.

PCR products exhibiting a variation in the SSCP pattern were sequenced directly. In 10 of

12 individuals studied, we detected a 2-bp insertion in exon 29 leading to nonexpression

due to the creation of a termination codon, which was observed in linkage to the haplotype

HLA-B60-DR6 in seven cases. In one of the other two individuals without this mutation,

evidence was obtained for gene conversion to the C4B isotype. The genetic basis of C4A

nonexpression in the second individual is not yet known and will be subject to further

analysis.

Research Article

Genetic Basis

of Human

Complement

C4A

Deficiency

Detection of a Point Mutation Leading to Nonexpression

Giovanna Barba,ChristianRittner, and Peter M. Schneider

InstituteforLegal Medicine, Johannes Gutenberg University,D-6500Mainz, Germany

Abstract

The fourth component of the human complement system (C4) is coded for by two genes, C4A and C4B, located within the MHC. Null allelesof C4 (C4QO)aredefinedbythe absenceof

C4protein in plasma. These null alleles are due either to large gene deletions or tononexpressionof therespective genes. In a

previousstudy,evidencewasobtained for nonexpressed defec-tive genes at theC4Alocus, andforgeneconversionat theC4B

locus. To further characterize the molecular basis of these

non-expressed C4A genes, we selected ninepairsof PCR primers

fromflankinggenomicintronsequences to amplify all 41 exons fromindividualswith adefective C4Agene. Theamplified prod-ucts weresubjected tosingle-stranded conformation

polymor-phism(SSCP) analysistodetectpossiblemutations. PCR

prod-uctsexhibitingavariationintheSSCP patternweresequenced directly.In 10 of12 individuals studied, we detected a 2-bp insertionin exon 29leading tononexpressiondue tothe cre-ationofaterminationcodon, whichwasobserved inlinkageto

the haplotype HLA-B60-DR6 in seven cases. In one ofthe other two individualswithoutthis mutation,evidencewas

ob-tained forgeneconversiontotheC4B

isotype.

The

genetic

basis of C4Anonexpressionin thesecond individual isnotyet known and will be

subject

tofurther

analysis.

(J.

Clin. Invest. 1993.

91:1681-1686.) Key words: genetic

deficiency

- complement

system * insertional mutation *single-stranded conformation

polymorphism-major histocompatibility complex

Introduction

The fourth component ofthe human complement system

(C4)'

is coded for bytwogenes,C4AandC4B, located within

theMHCclass IIIregionon the short armof chromosome 6

(1).The twoC4 isotypes differ in their amino acidsequences

by < 1%. Thetwo C4genes are tandemly arranged together withtwogenesforthecytochrome

_{P4_0}

steroid21-hydroxylase

(CYP2

IA andB), each located 3'tothe C4A and C4B genes, respectively (2, 3). The C4A gene isusually - 22 kb long,

Addresscorrespondenceto Dr.PeterM.Schneider,Institutftir

Rechts-medizin,AmPulverturm3,D-6500Mainz,FRG.

Receivedfor publication 13 August 1992 and inrevisedform30 November 1992.

1.Abbreviations used in this paper: C2 andC4, second and fourth components ofcomplement;C4A andC4B, isotypesAandBof the fourth component ofcomplement;C4QO,C4 nullallele; SSCP,

single-stranded conformationpolymorphism.

whereas the C4B gene ispolymorphicinsize with either22 or 16 kb.This sizevariation is due to the presence ofa 7-kb intron located - 2.5kbfromthe 5' endof the C4genes(4, 5).

At theprotein level C4 is highly polymorphic, with >40

variants including null alleles (C4QO) at both loci (6). Null

allelesaredefined bythe absenceof C4protein inplasma and arepresentinthe normalpopulationatfrequenciesof0.1-0.3

(7). Complete deficiency of C4 isa rare event andiscorrelated with severeimmunecomplex disease (8). Partial deficiency of oneof thetwoisotypes (C4AorC4B) ismore common and is

alsoassociated withanincreasedsusceptibilitytoautoimmune

or immune complex disease (e.g., in patients with systemic

lupus erythematosus [9, 10], subacute sclerosing panencepha-litis

[11

], orprimarybiliarycirrhosis[ 12 ] ).

Genetic variation is also evidentatthegenomic level, as

demonstrated bythedetection ofgenedeletionsand duplica-tions. C4null alleles are dueeithertolargedeletions compris-inganentireC4 gene and theflanking21-hydroxylase gene, or to

nonexpression

of therespectivegene (13, 14). In aprevious studyonthe molecularbasis of C4nullalleles, evidencewas

obtained for defectivegenes at the C4A locus, andforgene

conversionattheC4B locusasdemonstratedby the presenceof

C4A-specific

sequences(15).

Tofurther characterizethemolecularbasis ofthedefective C4Agenes,wedevelopedastrategy based onPCR

amplifica-tion and

genomic

DNA sequence

analysis.

We selectednine

pairs

of

primers

fromthe

published

C4A

genomic

sequence(5)

to

amplify

all 41 exons from individuals homozygous for C4AQO. All individuals studied carried a nonexpressedC4A gene on onehaplotypeand aC4Agenedeletionon the other

haplotype. The

amplified

DNA fragments were subjected to

single-stranded

conformation

polymorphism (SSCP) analysis

todetect

possible

mutations.

Finally,

directDNA

sequencing

ofthePCRproduct

exhibiting

avariation intheSSCPpattern wasusedto define thetype of mutation. Using this approach

wedetecteda2-bp insertionin exon29leadingto

nonexpres-sion. However, this mutationwas

only

foundin 10of12

haplo-typesstudied.

Methods

Selectionofindividuals and genetic typing. 12 individuals with

homo-zygous C4A deficiencywereselected.Allindividuals had been haplo-typed for MHCclassI,II,and IIIgene productsinfamily studies by our laboratoryaswell as by other collaborating laboratories (6, 15, 16)

according tothe following methods: HLA-A, -B, and -C, and-DR

antigenswereassigned by the microlymphocytotoxicity assay ( 17); C4

typingwascarriedoutby high voltage agarose gel electrophoresisin a

discontinuous buffer system after desialation of the samples with

neur-aminidase, followed by immunofixation orhemolytic overlay(18); C4QO alleleswereconfirmed by C4 a-chaintyping usingSDS-PAGE

(19);restriction fragmentlengthpolymorphism analysiswascarried

outbySouthern blot withTaqI-digested genomicDNAand

hybridiza-tiontoC4- and CYP2 1-specificprobesasdescribed (14).

J.Clin.Invest.

C) The AmericanSocietyfor ClinicalInvestigation,Inc.

0021-9738/93/04/1681/06 $2.00

TableI.SequenceofPrimerPairs Used

for Exon-specificPCR

Amplification

Primer C4exons

pairs Sequence (5'-3') amplified Size

bp

5' CCACAACTCTGGGCCTGAGGCCA 1-4 1,323

2 CACAGGCAGAGGTCACATCAGTG

3 GACCTCTGCCTGTGACCTACTTC 5-9 1,228

4 GTTAGCTCAGAGGTCAGAGGCAA

5 TCCCTCTCTTCTTGGGCTCTGTC 10-13 1,129

6 TCGGCACAAGTGCCATCTCTCCT

7 GAAGCTCTCCCACTCTGACCGCC 14-17 1,324

8 CAAGCGCCGCCACCTGTGCCCTA

9 GCGCTTGGAAAGGCAGAACGGTC 18-22 1,386

10 ACTTAGGAAACCAATGGCTGGAG

11 ACAGCAGGACAGGCTGCCAACTT 23-28 1,569

12 CCTCAGGCTCAGTGCCAGAGCGC

13 CTTCCTGTTTACTTGTGGTCTCC 29-31 1,021

14 ACCCTCTCCTCCACCAGTGCCTG

15 CCTCACATGTCCCACGTCCTCTC 32-35 827

16 ATCGAGTCCCTTCCTGCCTCATC

17 AACAGGCCAAGGAAACCCAGTAC 36-41 1,735

18 GCAACACACACTGACACTTCGCT

PCR

amplifcation.

Nine pairs of oligonucleotide primers were

chosenaccordingtothepublishedC4Agenomicsequence from flank-ing intron sequences (5) as shown in TableIandFig. 1.With these primers all41 exonsof both C4A and C4B genescanbeamplified. The PCRwasperformed inathermocycler (Perkin-Elmer Corp., Norwalk, CT) with 100 ng ofgenomicDNA, 25pmol of eachprimer, 200jM

dNTP, and2UTaqpolymerase(Boehringer, Mannheim,FRG)using

a"touchdown"protocol(20). The first 10 cycleswerecarriedout at

decreasing annealing temperatures in 1 °C steps fortwocycles each from 69to64°C, followed by 20 cycles using the following conditions: 1 min 94°C;2 min64°C; 5 min 72°C. For primers 3/4, a different touchdown programwascarried out beginning with 12 cycles from 64

to58°C.Isotype-specific PCRwasperformed to amplify only the C4A alleleusingaC4A-specific primer located in the C4d region and includ-ing the isotype-specific Chido-4 sequence as well as a second primer closetothepossible mutation site: A-down, 5'-GACCCCTGTCCA-GTGTTAGAC-3'; 13N, 5'-TTTCAATCCACAGGGCTGGGGCC-3' usingatouchdown protocol from 67to62°C.

SSCPanalysis. PCR products were purified by adsorption to glass

milk(Geneclean; BIO 101, LaJolla, CA) and digested with appro-priate restriction enzymes to obtain small fragments with a size range

between 700and 50 bp. Thesamples were denatured with formamide

5'

13

C4d

,a I

5 ₂₁₃ 4 5 ₆₇ 89 1011 1213 14 1516 17

1.3 1.2 lkb 1.1I1.3 1.3 1.5 160.8 1.

1 5 10 14 18 23 29 32 36

byheatingat80'Cfor 3 min and keptonice untilloading. The mixture was separatedeither in a 0.75-mm-thick MDE gel in 0.6X TBE buffer (ATBiochem, Malvern, PA) or in a standard polyacrylamide gel (5% total acrylamide, 1% crosslinker, 5% glycerol) polymerized on Gel-BondPAG film(Pharmacia LKB, Freiburg, FRG)atroom tempera-ture, applying 150 Vand 5 mAfor 2.5-20h (21, 22). Using these conditions, single-strandedDNA aswellasheteroduplexfragments can beanalyzed. TheDNAwasvisualized by silverstainingasdescribed withminor modifications (23).

Detection of C4

isotype-specifc

sequences. Restriction fragment analysis of PCR productswascarriedout aspublished by Braun etal.

(15).Briefly, the amplified DNA using PCR primers L3 / L4 was puri-fied byabsorption to glass milk (see above)anddigested with 2 U Nla

IV(NewEnglandBiolabs, Schwalbach, FRG). The digestedDNAwas electrophoresed ina 1.5% agarosegel and visualized by ethidiumbro-midestaining.

GenomicDNAsequencing. For directsequencingofPCR-amplified

products, theamplifiedDNAfragmentswerepurified byadsorptionto

glass milk (see above). Thesequencingreactionwascarriedout on

double-stranded DNAusingtheT7SequencingKit(Pharmacia LK.B)

and a-[35S]dATP (DuPont NEN, Dreiech, FRG). Isotype-specific

PCRfragmentsweresequenced by cycle sequencingwith TaqDNA

polymerase using the "fmol" DNA sequencing system (Promega Corp.,Madison, WI).

Results

5 ofthe 12individualswithhomozygousC4Adeficiencyand

onenonexpressed C4A gene selected for this studyhadbeen examined inaprevious studyforthe presenceof defective C4A

genesusingPCRamplificationandsubsequentrestriction

frag-mentanalysis (15).The othersevenindividualswereselected

from aC4 reference typing workshop (6)and from a recent

disease associationstudy ( 16). Inthesetwostudies,C4typing

had beencarriedout attheproteinand DNA level in addition

toHLAtypingtoestablish complete MHC haplotypes.These

sevensamplesweretestedasdescribed above for the presence

of nonexpressed C4Agenesusing restriction

analysis

withthe enzyme NlaIV,which detects theisotype-specificChido-4/

+4site oftheC4dregion(amino acids 1101-1 106 located in

exon 26) (24, 25; seealso Fig. 1). Insix of these samples, a

defective C4Agenewasdetected (not shown),whereas inone

sample (typed C4AQO,QO,B 1,2), only the C4B-specific

se-quence(i.e.,Chido+4)wasfound,asindicatedby the absence

ofthe Nla IV restriction site(Fig. 2). Thus,the C4Adeficiency

canbeexplainedfor this genebyconversion from C4AtoC4B. TocharacterizetheC4AQOnonexpressed alleles in the

re-maining 11individuals,weselected 18oligonucleotideprimers

from thepublished genomicC4A sequence that allow the

am-plification ofallC4exons asshownin Fig. 1.Thenine

ampli-fiedgenomic fragmentsrange insize from 0.8 to 1.7 kb, and

contain three to six exons each (Table I). The resulting PCR productsfromanormal individualwithout the C4 null allele (A) andan individual withanonexpressed C4A gene on one

haplotypeandaC4Adeletiononthe other haplotype (B) are

3'

Figure1.Mapof the C4 gene structure and location of PCR primers for exon amplification. (a) C4 protein subunit organization and location of the

isotype-spe-78

(b) cific C4d region; (b)location of PCR primer pairs;

JI.r

(c) sizes ofamplifiedfragments(inkilobases); (d)

'l*numbers of the firstexonof each amplified fragment.

7 (C) The asteriskindicates the location of the

S 1 2 3 4

S

Figure 2.

NlaIV

restric-tionfragment analysis ofPCR-amplified DNA

for theidentification of the C4 genotype. The amplified 926-bp

frag-(bP) ment coverstheC4

iso-(bp) _ type-specificsequences:

467 - after

NlaIV

digestion,a

single 467-bp fragment

216

- is characteristic of the

191 - C4Bisotype(Chido +4)

191- andthe276-andl9l-bp fragmentsare

character-istic of the C4A isotype (Chido -4). The

geno-typesstudiedare asfollows: lane 1, C4A genotype control C4A3,2 BQO,QO with C4BtoC4Ageneconversion; lane2, C4AQO,QOBl,2

withonenonexpressedC4A gene and one C4A gene deletion; lane

3,C4AQO,QO B2,lwithoneC4Agenedeletion anda geneconversion from C4AtoC4B; lane 4, C4Bgenotypecontrol C4AQO,QOBl,1 withbothC4Agenesdeleted;S, (DX 174-HaeIII-digestedDNA assize

marker.

represented in Fig. 3. Since both the C4A and C4B genes are

amplifiedsimultaneouslyinthisexperiment,any structural de-viation in the defective gene should be visible as anadditional fragmentinindividualB. Noobvious difference in the size of the amplifiedexons can be seen, providing further evidence

that thenondeletedC4A null geneis structurallyintact.

Theanalysis ofthenineamplified fragmentsby SSCP gel

electrophoresis did not reveal any differences between the DNAfrom three control individualsand threeunrelated

indi-viduals with nonexpressedC4A nullallelesfor primers 5'/2,

3/4,5/6,7/8,9/10,15/16,and17/18asshownin Fig.4A for primers 15 / 16. Characteristic SSCPand heteroduplex

poly-morphisms were obtained using primers 11 / 12 thatamplify

the centralportionof the C4dregion, sincethese exons 25-28 contain well-definedisotype-specificsequencedifferences

cod-ing forthe Rodgers(C4A)andChido (C4B) blood groupsof theC4 molecule (26) (not shown; manuscriptinpreparation).

572

S A B

3/4

A

B

5/6

A B

Finally, using primers 13/

14 and restrictionenzyme

diges-tion with HinfI,wedetectedanadditional

fragment

of - 250

bp

in twoof the three

samples

with

nonexpressed

C4A null alleles

(Fig.

4

B,

lanes 3 and

4),

whichwasnotfound inanyof the controlindividuals.

Therefore,

this PCR

fragment covering

exons29-31wasselected for detailed DNAsequence

analysis.

Genomic DNA sequence analysis ofthis fragment

using

PCR

primer

13assequencing primerrevealedamixed

C4A/

C4Bsequence

containing

a

2-bp

shift in the

reading

frame

(not

shown).

Sincethe

origin

ofthispossible insertionwas not visi-ble with thissequencing reaction, ithadtobeveryclosetothe

sequencing primer

annealing site. Therefore, we

synthesized

primer 13N,which annealstothe opposite coding strand and extendsthesequencingreactionintothedirection ofthe

possi-bleinsertionsite. Thuswedetecteda2-bp insertionat nucleo-tide

position

5877

(numbers

accordingtoreference 5)inexon 29 asdemonstratedinFig. 5.Thesequenceofthenoncoding

strand ofacontrolindividual typed C4A3B1 (A)isshown in

comparisonto that ofan individual with thedefective C4A

geneand normal C4B1 alleles (B). A mixed DNAsequence pattern canbe observed beginningatthe C inposition 5877 andextendingupstream,resulting inatwo-nucleotidereading frame shift. This observationcould beconfirmed after

isotype-specific amplificationwith theprimer A-down,which only

an-nealstotheC4A-specificDNAsequencecodingfortheCh -4

sequenceinexon26(seeFig. 1), together with primer14, and

by sequencingboth strands in thisregionwithprimers 13 and 13N(not shown).

The genomicDNAsequenceofexon29,intron29,andthe beginningofexon 30, aswellasthe translated amino acid se-quenceisillustratedinFig.6. In the mutationally altered C4A

sequence, thelastcorrectlytranslated codon would be amino acidposition 1214. Beginning with position 1215,a sequence

of 68 amino acidresiduesfollowstothe end ofexon29, which iscompletely different fromthe normal C4sequence.

Assum-ingaregular splicingoftherelativelyshort intron 29,two more

codonsarefound inexon 30.Thenexttriplet in thissequence representsthestopcodonTGA (Zin Fig. 6), which terminates the translation of this C4Atranscript.

Our initial resultsusing SSCP analysis (Fig. 4) provided

7/8

9/10

11/12

13/14

15/16

17/18

A B A B S

A B A B A

B A B

; ,.

(kb)

1.35

1.08

0.87-

0.60-Figure 3.Exon-specificPCRamplificationofDNAfromonecontrolindividual(A, C4A3Bl)andoneindividual withanonexpressedC4A gene

(B, C4AQOB

1).

Therespectiveprimerpairsfor eachfragment (see Fig. 1 andTableI)areindicatedabove;S. (X

_{174-HaeIII-digested}

DNA

assize marker.

l

",*.1;I<.xSw

A12

3

4

5

6

|I ~-..S.b-~s .

B123

4

5

6

h1

-

Ez

Figure4. PCR-SSCP andheteroduplexanalysisof selectedexonsof the C4 genes for the detection of sequence variations.(A)0.8-kb fragment withexons32-35amplifiedwithprimers 15/16and di-gested withPvull. Electrophoresiswascarriedoutina 14-cm-long

0.5xMDEgel for 2.5 hat150 V.(B) 1-kbfragmentwithexons 29-31 amplifiedwithprimers 13/14anddigestedwith HinfI. Electro-phoresiswascarriedoutina14-cm-long 5%polyacrylamidegel for 2.5hat150 V. The samplesstudiedare asfollows:lane 1, C4A ge-notype controlC4A3,2BQO,QOwith C4BtoC4A geneconversion;

lane2, C4 genotype control C4A3B 1; lane 3,C4AQO,QO

Bl,

1 with

onedefective C4A gene andoneC4A gene deletion(witha2-bp in-sertion inexon29);lane4,C4AQO,QOB1,2withonedefective C4A gene andoneC4A gene deletion(witha2-bpinsertion inexon29);

lane 5,C4AQO,QOB1,2 withonedefective C4A gene and one C4A gene deletion(withouta2-bp insertion inexon29); lane 6, C4B ge-notypecontrolC4AQO,QOB

l,

1 with both C4A genes deleted.

A G C T

A N - C

. - A

G

-G

. -AA

t - C

,;HBO,__

B

C G

-A

-A G C T

-i i

II..wo'...&-i

C C

C ₍₅₈₇₇₎

G

A

G

r

c A

G A c C

Figure5.DNA sequence of C4exon29(noncoding strand)obtained

byPCRamplificationwithprimers 13/13N and sequencingprimer

13N.(A)ControlsampleC4A3BI; (B)C4AQO,QO Bl,l withone

defective C4A gene andoneC4A gene deletion: the normal sequence isshownattherightside and themutationallyaltered sequence with the2-bpinsertion is shown at the left side of B (5877 is the nucleotide position of the insertion).

haplotypewasidentifiedthat didnotcarrythe2-bp insertion. 8 ofthe 10 C4A geneswith the insertionwere linkedto

HLA-B60,

and,withasingle exception,toHLA-DR6. Thetwoother

haplotypeshad HLA-B8 and either DR3orDR6. Theadjacent

C4Bgenesofthesehaplotypesexpressed either the BI orthe B2

allotype. AllC4Bgenes wereshort, i.e.,without the 6.5-kb in-tron,asdemonstrated bythedetectionofa5.4-kbTaqI restric-tion fragment. Thehaplotype notcarrying the mutation was

HLA-B35,DR3.

(exon 29)

5867 CTG TAC TGG GGC TCA GTC ACT GGT TCT CAG AGC .. (normal C4

1210 L Y W G S V T G S Q S .. sequence) 2 bp insertion: * *

5867 CTG TAC TGG GGC TCT CAG TCA CTG GTT CTC AGA GCA ATG CCG 1210 L Y W G S Q 8 L V L R A X P

TGT CGC CCA CCC CGG CTC CTC GCA ACC CAT CCG ACC CCA TGC C R P P R L L A T H P T P C CCC AGG CCC CAG CCC TGT GGA TTG AAA CCA CAG CCT ACG CCC

P R P Q P C G L K P Q P T P TGC TGC ACC TCC TGC TTC ACG AGG GCA AAG CAG AGA TGG CAG

C C T 8 C F T R A K Q R W Q ACC AGG CTG CGG CCT GGC TCA CCC GTC AGG GCA GCT TCC AAG

T R L R P G 8 P V R A A 8 K

GGG GAT TCC GCA GTACCC AA GTAGGGGCCGTCCCCGGGCTCTGGGGG

a D 8 A V P K (intron 29 ... 9071 GGGTGGGTAGTCCTCAGACCAAGGGCTTGCTTGAGTCCTGGCTCAACCTCCCTAG

...)

evidence thatthe mutation leading to nonexpression ofthe

defectiveC4A gene is not present in allhaplotypeswith C4A null genes. The individual in lane S of (Fig. 4 B) does not

exhibit the additional 250-bp fragment observed in the two

other

C4A-deficient

individuals inlanes 3 and 4.Therefore,we

studiedatotalof11 individuals carrying haplotypeswith

non-expressed C4Anullallelesusing amplification ofexons 29-31 andsubsequent SSCP electrophoresis forthe presence of this

mutation. The results are summarized inTable II. Only one

(exon 30)

9126 G ACA CGG TGA ... T R Z (-stop)

Figure 6. DNA and translated amino acid sequences of exon 29 aroundtheinsertion site and location of the stop codon in exon 30

(underlined;Z) incomparisontothe respective sequence of exon 29 ofanormalC4 gene; the changed amino acid sequenceafterthe in-sertion site is printed in boldface. The GT/AG splice signal of intron 29is alsoprintedin boldface. The nucleotide and amino acid posi-tionsareindicatedattheleft,and theinserted nucleotides are marked by asterisks.

Table11. SSCPAnalysis ofC4A Gene Exon 29ofHLAHaplotypes with SilentC4ANullGenes

Taq Ifragment

HLA- Allotypes

CYP CYP Ex. 29

A C B DR C4A C4B C4A 21A C4B 21B Ins. No.

(kb)

2(32) w3 60 6 QO 1 7.0 3.2 5.4 3.7 + (3) 2(24) w3 60 6 QO 2 7.0 3.2 5.4 3.7 + (4) 2 w3 60 3 QO 2 7.0 3.2 5.4 3.7 + (1) 1 w7 8 6 QO 2 7.0 3.2 5.4 3.7 + (1)

2 8 3 QO 1 7.0 3.2 5.4 3.7 + (1)

19 35 3 QO 2 7.0 3.2 5.4 3.7 _ (1)

Discussion

The strategy ofexon-specificPCRamplification using oligonu-cleotide primers from flankingintron sequences,screening for possible mutation sites by SSCP analysis, and subsequent

di-rectgenomic sequencing of specifictargetfragmentsrepresents

arapidandpowerful approach forthedetection ofsequence

variationinwell-defined geneticsystems.Genomic cloning in

lambda orcosmidvectors, as well asextensiveDNA

sequenc-ing, isavoided,and a large numberofsamples can bestudied simultaneously. However,theanalysisof SSCP and

heterodu-plex DNA fragments may not detect all existing mutations.

The resolutiondepends stronglyonthesizesofthefragments

to be analyzed, the composition ofthe gel system, and the

electrophoretic conditions. Accordingtomethodological

stud-iesregardingSSCP and heteroduplexanalysis,thepossibility of detectingapoint mutation decreases with increasing fragment size (21, 27).Theinsertiondetected in thenonexpressed C4A

gene represents a

major

conformational alteration of the

re-spective

fragment

thatfacilitated its detection.

Fromprevious studies itwasknown that only aproportion ofC4A null allelesaredue to genedeletions. Themajority of

these C4A gene deletions were found among the Caucasoid

population

within the extended haplotype HLA-A

l-Cw7-B8-C2C-BfS-C4AQO-BI-DR3 (10, 14). The molecular basis of

C4A null allelesnotduetogenedeletions remained unclear.It could beestablished, however, thatthese genesare

dysfunc-tional,

since theycarry

C4A-specific

DNA sequenceswithout expressingany C4A geneproduct. Conversionofthe C4A gene

toC4Basdescribed forthe geneconversion fromC4BtoC4A

in theHLA-B44-C4A3-BQO haplotype was not detected ina

previousstudy (15). Only a single HLAhaplotype has been

described with a conversion from C4A to C4B in the HLA

haplotypeB13-C4AQ0-B6,1 -DR7(28).Inthis study,we iden-tifiedoneindividual with conversion fromC4AtoC4B,

possi-bly

leading

to

homoduplication

and

-expression

oftheC4B2 allotypeon the haplotype

HLA-A3-Cw4-B35-C4AQO-C4B2-DR4(Fig. 2,Table II).

The observed 2-bp insertion in exon 29 ofthe defective

C4A genes is inlinkagewith HLA-B60for8ofthe 10

haplo-typesstudied. Of these,seven also havethe HLA-DR6 allele. Thetwo remaining haplotypes have theHLA-B8 allele, one

with DR3 and one with DR6. All haplotypes haveashort C4B geneasindicated bythe presenceofa 5.4-kbTaqI

fragment,

butshowavariation

regarding

theC4B

allotype,

whichmay be

eitherB 1 orB2. Thusit isverylikely that all these haplotypes

mighthavearisen fromasingle mutationalevent in an

ances-tralHLA-B60, DR6 haplotype. Since C4B1 and B2 arefound

in almost equal numbers in this study (four and six,

respec-tively), it might bespeculated thatthe C4Ballotypic variation

wasintroduced shortly after the mutational event, either by an independent mutation changing the amino acid sequence of theadjacentC4B gene orby arecombinationbetweenthe

de-fectiveC4A gene and the C4B gene. The sequence differences

explaining the C4B 1/B2 variation resulting in a net charge

difference ofthe gene product with C4B 1 being more basic thanC4B2have not yetbeendefined.The sequencedifferences describedsofaroriginate fromtheC4dregion(exons23-30), but donotaccountforthe observedelectrophoreticmigration difference betweenthe B 1 and B2variants(29, 30).Therefore, subtypes ofB2 may existthat havearisen from independent mutationalevents resulting inanet chargeof the protein

char-acteristic forthe B2 allotype.

Insertional mutagenesisas a cause of genetic defects has

alreadybeenobservedinanumberofgenes(e.g., in alpha-2-antiplasmin deficiency

[31],

beta-thalassemia

[321,

andcystic fibrosis [33

]).

A recentsurvey onthistypeof mutationalevent with insertionssmaller than 10 bp by Cooper and Krawczak

(34) showed thattwo mechanisms seem tobe involved: (a)

slipped mispairing mediated by directrepeatsor runsof identi-calbases,and

(b) misincorporation

of basesduetothe forma-tion of secondarystructureintermediates inducedby palindro-micsequencesorsymmetricelements. Theinsertiondetected in theC4Apseudogenes belongstothefirstcategory,sincethe

original sequence ofthe insertion site CTC is changed to

CTCTCby addition ofaCTor aTCdinucleotide(Figs.5and

6).Asimilar insertionwasobserved in

beta-thalassemia,

where

aTGTGsequence(at residue 148-Leu)waschangedto

TGT-GTG by insertion ofaTGdinucleotide (32). No functional

geneproductcanbeexpected fromthe C4A

mutationally

al-tered sequence,since the

reading

frame shiftcauses acomplete change ofthe amino acidsequence in the lastthirdofthe

a-chain, followed by a termination codon atthe

beginning

of

exon30

(Fig.

6).The

resulting

truncatedC4translation

prod-uctisprobablynotstable andtherefore

degraded rapidly

intra-cellularly. Thusthe2-bpinsertion is sufficientto

explain

the

nonexpression ofthesedefectiveC4A genes.However,we

can-notcompletelyexclude the

possibility

that othermutationsare

also present in these genes that were not detected

by

SSCP

does not have thedescribed2-bp insertion (Table II), investi-gations are currently being carried out to identify the unknown

genetic defect also causingC4A nonexpression.

Similar observations regarding the heterogeneity of the ge-netic basis of deficiency as well as the finding of strong MHC haplotype linkage have been described recently for comple-ment C2deficiency(35). The authorsidentified a 28-bp dele-tion spanning the splice junction of exon/intron 6 of the C2 gene as the cause for deficiency. This mutation is linked to the haplotype HLA-A25-B 1 8-C2QO-B fS-C4A4-C4B2-DR2, whereas C2 deficiency outside thisextended haplotype has a

differentand as yet unknown genetic basis. Our findings dem-onstratethatamajority ofthe nonexpressed C4A genes among theCaucasian populationhave arisen from a single insertional

mutationlinkedto the HLA haplotype B60-DR6, whichwas

then spread by recombination to haplotypes with different

HLA-B and/or DR alleles. However, this mutation was not

foundin allhaplotypes withadefective C4Agenestudied, pro-viding evidence for heterogeneity in the genetic basis of C4A

deficiency.

Acknowledgments

This studywassupported byagrant from the Deutsche Forschungsge-meinschaft (DFG Schn 284/3-1).

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