THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 267, No . 13, Issue of May 5, pp. 9347-9353,1992 Printed In U. S. A.

Type I Human Complement C2 Deficiency A 28-BASE PAIR GENE DELETION CAUSES SKIPPING OF EXON 6 DURING RNA SPLICING*

(Received for publication, December 9, 1991)

Charles A. Johnson$, Peter DensenQ, Robert K. Hurford, Jr.$, Harvey R. ColtenSlI, and Rick A. Wetsel$ll 11 From the $Edward Mallinckrodt Department of Pediatrics and llDepartment of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 631 10 and the §Department of Internal Medicine, Veterans Administration Medical Center and University of Iowa College of Medicine, Iowa City, Iowa 52242

Two variants of a genetic deficiency of complement protein C2 (C2D) have been previously identified. No C2 protein translation is detected in type I deficiency, while type I1 deficiency is characterized by a selective block in C2 secretion. Type I C2 deficiency was de- scribed in a family in which the C2 null allele (C2QO) is associated with the major histocompatibility haplo- type/complotype HLA-AZS,B18,CZQO,BfS,C4A4, C4B2,DrwZ; this extended haplotype occurs in over 90% of C2-deficient individuals (common complotype/ haplotype). To determine the molecular basis of type I C2 deficiency, the C2 gene and cDNA were character- ized from a homozygous type I C2-deficient individual with the common associated haplotype/complotype. We found a 28-base pair deletion in the type I C2QO gene, beginning 9 base pairs upstream of the 3’-end of exon 6, that generates a C2 transcript with a complete dele- tion of exon 6 (134 base pair) and a premature termi- nation codon. In studies of eight kindred, the 28-base pair deletion was observed in all C2QO alleles associ- ated with the common type I deficient complotype/ haplotype; this deletion was not present in normal C2 nor in type I1 C2-deficient genes. These data demon- strate that: 1) type I human complement C2 deficiency is caused by a 28-base pair genomic deletion that causes skipping of exon 6 during RNA splicing, resulting in. generation of a premature termination codon, 2) the 28-base pair deletion in the type I C2QO gene is strongly associated with the HLA haplotype/complo- type A ~ S , B ~ ~ , C ~ Q O , B ~ S , C ~ A ~ , C ~ B ~ , D ~ W ~ , suggest- ing that all C2-deficient individuals with this haplo- type/complotype will harbor the 28-base pair C2 gene deletion, and 3) type 11 C2 deficiency is caused by a different, as yet uncharacterized, molecular genetic defect.

* This work was supported by United States Public Health Service Grants AI25011 (to R. A. W.), A124836 (to H. R. C.), A124739 (to H. R. C.), and HD17461 (to H. R. C.), a merit review award from the Department of Veterans Affairs (to P. D.), and a grant-in-aid from the American Heart Association (to P. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequencefs) reported in this paper hos been submitted

M86920. to the GenBankTM/EMBL Data Bank with accession numberfs)

11 Recipient of Research Career Development Award AI00919 from the National Institutes of Health. To whom correspondence should be addressed Dept. of Pediatrics, Box 8116, Washington University School of Medicine, 400 S. Kingshighway Blvd., St. Louis, MO 63110.

The complement system is a set of plasma proteins that serves as an effector of several biologic functions associated with inflammation, immunoregulation, and cytotoxicity (1). The second component of human complement (C2)’ is a 110,000 M, single-chain glycoprotein and is the serine esterase component of the classical pathway C3 cleaving enzyme com- plex. C2 is encoded by a 20-kb gene comprised of 18 exons (2, 3) that is located on the short arm of chromosome 6 between the HLA-D and HLA-B loci of the major histocompatibility complex (MHC) (4). The C2 gene is part of a tightly linked class 111 complement gene cluster that includes the homolo- gous gene encoding complement protein factor B and the two C4 loci, C4A and C4B (4).

Deficiency of C2 is the most frequently occurring inherited defect of the complement system in individuals of western European descent. In this population, approximately 1 person in 10,000 is homozygous C2-deficient (5, 6). C2 deficiency exhibits very strong linkage disequilibrium with certain HLA haplotypes and complement polymorphisms. For example, the haplotype/complotype most characteristic of C2 deficiency is HLA-A~~,B~~,C~QO,B~S,C~A~,C~B~,DIWZ (7-9). More than half of C2-deficient individuals have rheumatological disor- ders such as systemic lupus erythematosus (lo), Henoch- Schonlein purpura, and polymyositis (5). In other kindreds, association of C2 deficiency and recurrent pyogenic infection has been observed, and in some cases C2-deficient individuals are asymptomatic (11).

We have recently demonstrated heterogeneity among indi- viduals with CZ deficiency (12). In type I deficiency, in which C2QO is associated with the common haplotype/complotype, there is no detectable translation of C2-specific mRNA. In contrast, in type I1 deficiency, in which C2QO is associated with two uncommon haplotypes/complotypes, HLA-A2,B5, BfS,C4A3,C4Bl,Dw4 and HLA-A11,B35,BfS,C4AO,C4Bl, Dwl, there is a selective block in C2 protein secretion. The exact molecular mutations causing the two types of C2 defi- ciency have not been defined. Accordingly, we undertook the present study that defines the molecular basis of type I deficiency; a 28-bp deletion occurs in the C2 gene that causes aberrant RNA splicing resulting in deletion of exon 6 (134 bp) from the mature C2 message. The identical gene deletion is also present in eight unrelated C2-deficient kindred that share the common haplotype/complotype, but in some cases have different clinical manifestations. These findings suggest that: 1) all C2-deficient individuals with the common haplo-

The abbreviations used are: C2 and C4, the second and fourth complement components, respectively; Sf, factor B; bp, base pair(s); kb, kilobase pair(s); C2D, C2-deficient; C2S, C2-sufficient; kb, kilo- base(s); MHC, major histocompatibility complex; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate.

9347

9348 Type I Human C2 Deficiency

type/complotype are of the type I variant, which is due to a specific 28-bp gene deletion, and 2) the diseases associated with C2 deficiency do not correlate with specific molecular genetic mutations.

EXPERIMENTAL PROCEDURES

C2-deficient Families-Five of the eight C2-deficient kindred stud- ied in this paper have been described in the literature (references in Table I). Family 4 was found to be C2-deficient after clinical and laboratory evaluation of a child with recurrent infection. C2 func- tional assays and HLA and complotyping were performed using standard procedures (13).

RNA Isolation-Fibroblast cell lines were established from normal and C2-deficient members of family 1 as described previously (12). Prior to RNA isolation, the fibroblast cultures were incubated for 24 h with 100 units/ml of y-interferon, conditions previously established to increase C2 gene expression for the mutant and normal alleles. Twice-selected poly(A)+ RNA was prepared from confluent fibroblast cultures by the guanidium isothiocyanate method (14) and oligo(dT) column fractionation (15).

DNA Isolation-Human genomic DNA was isolated from either established fibroblast cell cultures or peripheral blood leukocytes. Approximately lo7 cells were incubated with gentle mixing at 45 “C for 16 h in a 20 ml solution containing 80 pg/ml proteinase K (Boehringer Mannheim), 75 mM NaCI, 25 mM EDTA, 0.1% SDS, 10 mM Tris, pH 8.0. This suspension was extracted twice by gentle rocking for 1.5 h at room temperature with 250 mM NaC104, 0.15% SDS, 25% water-saturated phenol, 24% chloroform, 1% isoamyl al- cohol in a glass 100-ml cylinder. The solution containing genomic DNA was dialyzed for 16 h at room temperature against 4 liters of 10 mM NaCI, 10 mM EDTA, 50 mM Tris, pH. 8.0, followed by treatment with 100 pg/ml RNase A (Boehringer Mannheim) at 37 “C for 3 h. The DNA solution was extracted twice, first with 25% water-satu- rated phenol, 24% chloroform, 1% isoamyl alcohol and then with 48% chloroform, 2% isoamyl alcohol. The purified human genomic DNA was dialyzed, quantitated by absorbance at 260 nm, and stored at 4 “C in the dialysis buffer.

Amplification of cDNA and Genomic DNA-Two micrograms of poly(A)’ RNA isolated from fibroblasts of normal and C2-deficient individuals of family 1 were incubated with 10 units of reverse transcriptase at 42 “C for 1 h using the buffers and dNTPs supplied in a cDNA synthesis kit (Invitrogen, San Diego, CA). An oligonucle- otide (Fig. 1, F) anti-sense to the normal C2 cDNA sequence was used as a primer in the first strand synthesis. The cDNA was subsequently amplified by the polymerase chain reaction (16) using the first strand cDNA as template and the oligonucleotide pairs diagramed in Fig. 1. These oligonucleotides were constructed with either BamHI or HindIII restriction sites near the 5’-ends to facilitate subcloning. The first strand cDNA was initially denatured at 95 “C for 3 min with 1 pg of each oligonucleotide in a 100-pl solution containing 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCI2, 0.1% gelatin, 200 p~ dNTPs, and 2.5 units of Taq polymerase (Perkin- Elmer Cetus). Following the initial denaturation, the cDNA was amplified by melting at 95 “C for 2 min, annealing at 60 “C for 2 min, and polymerizing at 72 “C for 5 min. Fifty cycles of amplification were performed using a Tempcycler (Coy Laboratory Products, Ann Arbor, MI) followed by a final elongation at 72 “C for 7 min. The amplified cDNA was digested with BamHI and HindIII, purified by low melt agarose extraction using NuSieve GTG-agarose (FMC Bio- products, Rockland, ME), and subcloned into pBluescript I1 (Stra- tagene, La Jolla, CA). Competent Sure cells (Stratagene) were trans- formed with the ligations, and plasmid DNA was isolated from the recombinants using the alkaline lysis procedure (17). The C2 cDNA was sequenced using double-stranded templates as outlined below.

Genomic DNA was amplified by the polymerase chain reaction as described above. A 5’ oligonucleotide corresponding to the first 25 bp of exon 6 (5’-AAAGCTTGGGCCGTAAAATCCAGCG-3‘) and a 3’ oligonucleotide comprising 25 bp of intron 6 (5”GAGCACAGGA- AGGCCTCTCTGCAGG-3’) were used in the amplification reactions. The amplified DNA products (180 and 150 bp) were separated on a 2.5% agarose gel and visualized by ethidium bromide staining.

Construction of Genomic Cosmid Libraries, Isolation of C2 Genomic Clones, and Southern Blot Analysis-As described above, high molec- ular weight DNA was prepared from peripheral blood leukocytes isolated from a homozygous C2-normal individual and a C2-deficient individual from family 1 that exhibited type I C2 deficiency. This

DNA was partially digested with Sau3Al and used to prepare a normal cosmid library and a C2-deficient type I cosmid library using the methodology described previously (18). Approximately one mil- lion recombinants were plated and screened in duplicate for clones containing the C2 gene by using a nick-translated (19) C2 cDNA as a probe (20). Colony purified clones containing the entire C2 gene were identified subsequently by positive hybridization with ”P-la- beled oliaonucleotides that correwonded to exons 1 and 18 of the human C> gene.

DNA Seauence Analysis-All DNA sequencing was performed using double-stranded templates (21). Two-micrograms of template were denatured in 0.2 M NaOH, 0.2 mM EDTA, neutralized, annealed with specific C2 oligonucleotides (20-mers), and sequenced employing the dideoxy chain termination method (22) and the modified bacte- riophage T7 DNA polymerase (23). Both strands were sequenced at least once.

RESULTS

Characterization of the C2D Type I cDNA-Poly(A+) mRNA was isolated from fibroblast cultures established from

k b 1 2 3 4 5 6 4.4 - 2.3 - 2.0 -

0.6 -

bP

- 1353 - 1078 - 872 - 603

- 310

1 .-.) c- ” “ A B C D E F

FIG. 1. Characterization of amplified cDNA fragments gen- erated from C2-sufficient and type I C2-deficient fibroblast mRNA. The top panel of this figure shows an ethidium-stained 1% agarose gel of PCR-amplified cDNA fragments subjected to electro- phoresis using TAE buffer. The cDNA fragments were generated from normal and type I C2-deficient fibroblast mRNA as described under “Experimental Procedures.” Each pair of lanes consists of normal and type I C2-deficient mRNA samples amplified by the oligonucleotides, the positions of which in the C2S gene are diagramed below. That is, lanes 1, 3, and 5 are cDNA-amplified products from normal fibroblast mRNA; lanes 2, 4, and 6 are cDNA-amplified products from homozygous type I C2D fibroblast mRNA. At the bottom of the gel are shown the oligonucleotide pairs used in the amplification reactions. The diagram shown at the bottom of this figure represents a portion of the normal C2 cDNA structure with the Cls cleavage site, signal peptide, and 5”untranslated sequence indicated by the vertical arrow, dark hatching, and solid line, respec- tively. The small arrows below the C2 structure illustrate the positions of the oligonucleotides employed in the PCR amplification reactions. The sequences of the oligonucleotides used were as follows: A, 5’- GGGGGATCTATTGACCCTATAGATATATTA-3’; B, 5”GACA- AGCTTCGCTATCGCTGCTCCTCGAAT-3’; C, 5”GGATCCA- GAAACAGCTGTGTGTGATAATGG-3’ D, 5”ACCGGATCCTGG- ACTGTTCGCAGAGTG-3’; E, 5”TCATGATGAACAACCAAA- TGCGGATCCTCG-3’; F, 5”GAACATATGCTGGATCCCTCC- AAGCTCACA-3’.

Type I Human C2 Deficiency 9349

kb

23.1 - 9.4 - 6.6 - 4.4 -

2.3 - 2.0 -

0.6 -

cis 1

C2D

cis

T K jE S L G R ’ Protein } c 2 s

A C A A A G G j A A A G C C T G G G C C G T CDNA

I I I ! ‘ I I I I nucleotide position 709 712 715; 718 721 724 727

j 852 855 858 861 I I I I

A C A A A G G I A T C T T C A G C T T T G A C2D

T K D L Q L * Protein

FIG. 2. Schematic comparison of the C2-sufficient and type I C2-deficient cDNA sequences. The cDNA structures are diagramatically represented by the stick figures. The Cls cleavage site is indicated by the vertical arrows; the 134-bp deletion is indicated as an open box in the C2S sequence. The stop codons are indicated by asterisks. The cDNA and deduced protein sequences at the B’-end of the 134-bp deletion are shown below the diagrams. The nucleotides are numbered starting from the translation initiation site for the C2-sufficient and C2- deficient cDNAs.

c2s C2D

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 FIG. 3. Southern blot of C2-sufficient and type I C2-defi-

cient cosmid clones probed with an exon 6-specific oligonu- cleotide. Two micrograms of cosmid DNA, isolated from clones containing the complete C2-sufficient gene (C2S) or the complete type I C2-deficient gene (CZD), were digested with seven different restriction enzymes, subjected to 1% agarose gel electrophoresis, Southern blotted, and probed with oligonucleotide D of Fig. 1 (cor- responding to exon 6 of the C2 gene) as described under “Experimen- tal Procedures.’’ Lane 1 contains X DNA digested with HindIII that was radiolabeled with ’*P and used as a size maker. Lane 9 contains a human C2 restriction fragment containing exon 6 that was used as a positive hybridization control. Lanes 2-8 contain cosmid DNA digested to completion with the following restriction enzymes: lanes 2, BglII; lanes 3, EcoRI; lanes 4, BamHI; lanes 5, HindIII; lanes 6, PstI; lanes 7, PuuII; lanes 8, SphI. The EcoRI restriction fragment length polymorphism (lanes 3) detected in the C2S (30 kb) and C2D (6.0 kb) clones is not a marker for type I C2 deficiency, because it is detected in a population of normal C2 genes (data not shown).

a normal and a C2D type I individual. The cDNA was syn- thesized from this RNA using C2-specific oligonucleotide primers and was amplified by the polymerase chain reaction (PCR) as described under “Experimental Procedures.” In a series of overlapping fragments, ranging in size from 960 to 566 bp, the entire coding, 3’-untranslated, and 220 bp of the 5”untranslated sequences were amplified and analyzed by

agarose gel electrophoresis. An obvious difference was ob- served in only one area in comparison of the normal and C2D cDNA (Fig. 1). This difference suggested a deletion in the C2D cDNA corresponding to the region encoding the Cls cleavage site. That is, a smaller C2D cDNA fragment (lune 4 ) was generated compared with the normal C2 cDNA (lune 3) when oligonucleotides spanning the Cls cleavage site were used. In addition, no C2D cDNA was generated when an oligonucleotide near the Cls cleavage site was used in the PCR reaction ( l a n e 6 ) . Fragments covering the entire C2D cDNA were then amplified, subcloned, and sequenced. The nucleotide sequences of the C2D and normal (20, 24) cDNA were identical, except for 134 bp that were missing in the C2D cDNA inclusive of the region encoding the Cls cleavage site. This 134-bp deletion shifts the reading frame of the C2D mRNA such that a termination codon UGA is encountered 12 bp downstream from the deletion (Fig. 2). This mutation, therefore, should generate a truncated primary translation product of 212 amino acids which would have an unglycosyl- ated molecular weight of 23,000.

Analysis of Genomic DNA in Type I C2 Deficiency-To determine the molecular genetic mutation that causes the 134-bp deletion in the C2D message, genomic cosmid clones containing the C2 gene were isolated from libraries prepared from normal and type I C2D DNA as described under “Ex- perimental Procedures.’’ Normal and C2D cosmid clones, containing the entire C2 gene, were digested with seven dif- ferent restriction enzymes, Southern-blotted, and probed with an oligonucleotide (30-mer) contained within the 134-bp dele- tion to ascertain whether this deletion was represented in the C2 gene. Restriction fragments from both the normal and C2D clones hybridized to the 30-mer (Fig. 3). The normal and C2D BumHI restriction fragments (3.5 kb) that hybridized to the oligonucleotide probe were preparatively isolated, sub- cloned into pSP72 (Promega), and partially sequenced (Fig. 4). From the genomic sequence data, it was determined that the 134 bp deleted from the C2D message was encoded by a single, complete exon (exon 6) in the normal C2 gene. In the C2D gene, 28 bp were missing that included 9 bp of the 3‘- end of exon 6 and the donor splice site of the adjacent intron

9350 Type I Human C2 Deficiency EXON 5 ' mlmn 5

A C A T G C T T G G G G C C A C C A A T C C C A C C C A G A A G A C A A A G G G T G A G T G T T T G A G G T G G G G T T T C T G G T T G A G A C G G G T G C l G G A T C T G G G C C G G A G A A C G G G M s I L e ~ G I y A I a T n r A r n P l a T h r G , n L y r T h i L y r G

677 715 G

A G G A T G C A A C C T T C C T G G A G G C C A G G A G C C T ~ G G T G G C T C A G C C A C T G A A A G G G A G G G A G G C A G A G A A G C T G G A C C T G C T T G G C G A G A G C G ~ A ~ G A A ~ ~ A

lntmn 5 ' EXON 6 I u S B r L B u G l y A l Q L y B l l e G l n i l e G l n A ~ Q S e r G l y ~ ~ s L e u A r n L e u T y r L e u L e u L e u

G G T G G G G A ~ C C T C C C C T T C C A C A T T T C T C C A G A A A G C C T G G G C C G T A A A A T C C A A A T C C A G C G C T C T ~ G T C A T C T G A A ~ ~ T ~ ~ A ~ ~ ~ ~ ~ T ~ ~ ~ ~

716

A I p C y B S e r G I n S ~ ~ V a l S e r G l u A l n A S p P h e L e u I l e P h e L y s G I u S e r A l a S e r i e u M e f Y s i A s p A r p G A C T G T T C G C A G A G T G T G T C G G A A A A T G A C T T T C T C A T C T T C A A G G A G A G C G C C T C C C T C A T G G l G G A C A G G G l C A G G A A T C A G G A G T C T G C C T G C A G C A

EXON 6 , mllm 6

849

G A G G C C T T C C T G T G C T C A C T A T C T C T C T C T G T C T C C T T C C C C T C C T C A G A A C C C C A C T C A C A G C C C A C C T C C T C C A A G A A G T C T T C T C A G A T T A T A C T C A

T G C C A T G T A G G A ~ T C A T G A A T T C A A T T T A T A C A T C A T A A T l T T T A T T C C A C A G C A C T G T T G G G A C A C T G T G C T G G G C T G G C G A C A C G A A G A T G G A A A G G C

T G A G T C T T A C T C A G A T C A T C A T C T A G A C A G T G T C A G A A G T A G T A G A T A C C A C A G A T A C G A G A A T C T G T C T G A T A T A T C A G T A T A T T A T A T T A T A T T C T T A

A A C T T C A A C A T T G T G C G A G C T T A A A T G T G T G T G A T A G A C T G G C A T G G T G C T A G T G C C T G T A A T C C C A A A C A T T T G G G A G G C C G A G G C A G G T G G A T C A C T T

G A G G T C A G G A G T T T G A G A C C A G C C T G A C C A A C A T G A T G A A A C C C T G T C . ( 9 C O b p t - C T A G C T C T G G T A G C T G G T A T A G T G C T G C A C A T A C A G C T A A T

T T T G T A T T T T A G T A G A G A T G G G T T T T G C A T G T T G G T C A G G C T G G T C T C G A A C T C C T G A C C T C A G G T G A T C C A C C T G C C T A A G T G T T G G G A T T C A G G C A T G

A G C C A C C G C G C C C A G C C C C T A G C T T C T T C C T A A C A G C C A T T T C C T A G T G T C T C C C C T G G T C C T T G C C T C T G T C G G T C T C A C T C C A G T T T C T C T G C C T C C T

8 n m n 6 EXON 7

C C A G G G C C C T T G T T T G C T C T C T T A C C A T C T C C C C T T T G G C T T C A G G G C C ~ C T G C C T C T C A C T T G C C C C G C A C A G A T C T T C A G C T T T G ~ G A T C A A I I ~ P ~ ~ S ~ ~ P ~ ~ G I U I I ~ A S

850

FIG. 4. Nucleotide sequence and exon/intron characterization of the C2-sufficient and type I C2- deficient genes (exons 5-7). The sequence of the C2-sufficient gene is shown. The single nucleotide difference in the type I C2-deficient gene (intron 5) is shown above the corresponding nucleotide in the sufficient sequence. The 28-bp deletion in the type I C2-deficient gene is indicated by the shaded box. Putative branch sites (38) are denoted by black boxes. The sequence of intron 6 was partially determined; the entire intron is approximately 1.7 kb in length as determined by PCR and Southern blot experiments (data not shown).

Genomic Sequence TCATGbTGGACAGGGTCAGGAATCAGGAGTCTGbCTG ""_"""""""""""""""""""""""""""

Genomic Structure cis

FIG. 5. Schematic representation of the 28-bp deletion in the type I C2-deficient gene. The 28-bp deletion from the C2QO gene in type I C2 deficiency is represented by the dashed box. The wedge indicates the exon/intron boundary. The relative position of the deletion is shown on the genomic structure. The asterisk indicates the guanine substitution in intron 5 of the C2-deficient type I gene.

(intron 6) (Fig. 5). All other nucleotide sequences between exons 5 and 6 were identical in the normal and C2D genes, except for an adenine to guanine substitution that was up- stream of the putative branch site of intron 5. These results indicate that the 28-bp deletion at the 5'-splice site of intron 6 causes skipping of the preceding exon (exon 6) during type I C2D RNA processing.

Screening of Other C2-deficient Kindred for t h 28-bp Gene Deletion-To determine if the 28-bp gene deletion is present in C2D individuals from other families, genomic DNA span- ning the deletion was amplified in individuals from eight different kindred as described under "Experimental Proce- dures." Amplified DNA from normal and homozygous C2D individuals with the 28-bp deletion yielded fragments of 180 and 152 bp, respectively. Amplified DNA from heterozygous C2D individuals with the 28-bp deletion yielded both frag- ments. An ethidium bromide-stained agarose gel from a rep- resentative set of samples is shown in Fig. 6, and the results from all eight kindred are presented in Table I. In all individ- uals examined, the C2D gene contained the 28-bp genomic deletion when linked to the common C2D complotype/hap- lotype; however, this deletion was not present in the C2D genes linked to the uncommon complotype/haplotypes; i.e. the haplotype associated with type I1 C2 deficiency (kindred 2, B I1 8 and B I11 9). In addition, the association of the common complotype/haplotype with the 28-bp gene deletion apparently is not associated with a specific clinical problem,

because some of these homozygous type I C2D individuals were asymptomatic (kindred 3, brother) and others had re- current pyogenic infections (kindred 1, A I11 1 and A I11 3; kindred, 3, 88; kindred 4, son and daughter; kindred 5, male) or systemic lupus erythematosis (kindred 7, 1 I1 2; kindred 8, 2 I1 2) (Table I).

DISCUSSION

We have previously identified two types of human C2 deficiency (12). In type I deficiency, no C2 protein synthesis is detected in fibroblast cultures from homozygous deficient individuals in a kindred with the characteristic MHC haplo- type/complotype (HLA-A~~,B~~,C~QO,B~S,C~A~,C~B~,D~W~) that is associated with the C2 mutant allele. We now present data that establish the molecular genetic defect responsible for type 1 C2 deficiency is a 28-bp deletion in the C2 gene that removes 9 bp of the 3'-end of exon 6 and 19 bp of the adjacent intron, inclusive of the donor splice site. This dele- tion causes aberrant RNA splicing (exon skipping) resulting in deletion of exon 6 from the mature C2 message. Analysis of eight C2-deficient kindred with the common haplotype/ complotype revealed that the C2QO gene in each of these families contains the 28-bp deletion. This finding, together with the fact that no crossover events have been detected among the class I11 complement genes (7), strongly suggests that all homozygous C2-deficient individuals with the com- mon haplotype/complotype will have the 28-bp C2 gene deletion.

Type I Human C2 Deficiency 9351

180 bpz 152 bp

1 2 3 4 5 6 7 8 0 QTB - e o

FIG. 6. Screening of C2-deficient individuals for the 28-bp C2 gene deletion. Genomic DNA fragments spanning the 28-bp deletion were amplified starting from 1 pg of DNA isolated from individuals of four unrelated C2-deficient kindreds as described under “Experimental Procedures.” The C2-deficient families are listed in Table I. The amplified DNA fragments were separated by electropho- resis through a 2.5% agarose gel and visualized by ethidium bromide staining. Lane 1, homozygous C2S daughter (A I11 2) kindred 1; lane 2, homozygous C2D son (88) kindred 4; lane 3, heterozygous C2D mother kindred 4; lane 4, heterozygous C2D father kindred 4; lane 5, homozygous C2D daughter kindred 4; lane 6, no DNA added during amplification; lane 7, homozygous C2D female (1 I1 2) kindred 7; lane 8, homozygous C2D female (2 I1 2) kindred 8.

The splicing of mRNA precursors requires at least two conserved sequences at the ends of introns: the 5’ donor splice site consensus sequence 5’-CAG/GTAAGT-3’, which is com- plementary to the 5”terminal region of U1 small nuclear RNA (snRNA), and the 3‘ acceptor splice site consensus sequence 5’-CAG/(25, 26). In addition, other less well char- acterized sequence elements in mRNA precursors are involved in correct splicing of mRNA precursors.

The molecular genetic basis of several inherited diseases has been elucidated in recent years (27). Some of these dis- eases occur as a result of abnormal splicing of mRNA precur- sors caused by gene mutations at 5’ and 3’ splice site se- quences. Mutations in these consensus splice sequences usu- ally abrogate or reduce the frequency of correct splicing of the affected intron. A few examples have been reported where mutations at the 5’-splice site cause skipping of the preceding exon. In most of these genes (phenylalanine hydroxaylase (28), &globin (29, 30), pro-a2 collagen (31, 32), and murine c-kit (33)), the preceding exon is abberantly removed as the result of a single base pair substitution at position 1 (G) of the intron 5”splice site consensus sequence. In addition, a G to A mutation in the last position of exon 12 in the porphobi- linogen deaminase gene (34), and a 7-base pair deletion start- ing at position 5 of the 9th intron of the rat albumin gene (35) cause deletions of the mutated or preceding exons, re- spectively. Recently, an intra-exon deletion directly upstream of the 5”splice site consensus sequence in exon 19 of the dystrophin gene was reported in a patient with Duchenne

TABLE I PCR analysis of 28-bp genomic deletion in 8 C2-deficient kindred

Kindred Individual” presentation complotype’ deletion‘ Clinical Haplotype/ 28-bp

1 (Ref. 12) A I1 1 Well A/N +/- A I1 2 Well A/N +/- A I11 1 Infections A/A +/+ A I11 2 Well N/N -1- A I11 3 Infections A/A +/+

2 (Ref. 12) B I1 8 Well B/C B I1 9 Well A/N

-/- +/-

B 111 9 Infections A/B +/- 3 (Ref. 11) 88 Infections A/A +/+

Brother” Well A/A +/+ Father“ Well A/N Mother” Well A/N

+/- +/-

4 Fathere Well A/N +/- Mothere Well A/N +/- Sone Infections A/A +/+ Daughter‘ Infections A/A +/+

6 Female’ Well A/N +/- 7 (Ref. 10) 1 I1 2 Systemic lupus er- A/A +I+

8 (Ref. 10) 2 I1 2 Systemic lupus er- A/A +I+

5 Male’ Infections AIA +I+

ythematosus

ythematosus ‘The individuals are identified as indicated in the corresponding

references when possible.

B18, C2Q0, BfS, C4A4, C4B2, Drw2; B = A2, B5, C2Q0, BfS, C4A3, bThe capital letters indicate the following haplotypes: A = A25,

C4B1, Drw4; C = All , B35, C2Q0, BfS, C4A0, C4B1, Drwl; N = C2 haplotype not associated with C2 deficiency.

+ indicates deletion is detected, i.e. 152-bp amplified fragment; - indicates deletion is not detected, i.e. 180-bp amplified fragment. “ P. Densen, unpublished data.

e P. Shackelford and C. Johnson, unpublished data. ’C. Alper, unpublished data.

muscular dystrophy (36). Although this deletion did not occur precisely at the 5’-splice site, the mutated exon 19 was skipped during RNA processing.

The 28-bp gene deletion in type I C2 deficiency is the first report of a mutation that completely removes the 5’-splice site consensus sequence. In this case, removal of the 5‘-splice site sequence causes skipping of the preceding exon (exon 6). Deletion of exon 6 during RNA processing appears to occur in all transcripts in type I C2 deficiency, since no other C2 mRNA species were detected.

The mechanism by which exon skipping proceeds during splicing of primary transcripts with mutated or deleted 5’- splice sites has not been studied in depth. We propose here a mechanism whereby exon skipping might occur in type I C2 deficiency (Fig. 7). In this model, intron 6 in the C2-deficient gene is not cleaved because of the deletion at the 5”splice site. Intron 5 is cleaved normally but forms a lariat with a cryptic branch site in intron 6, suggesting that the 5’-end of intron 5 has a strong preference for the cryptic branch site over its normal branch site. Cleavage and removal of the lariat containing exon 6 is followed by ligation of exon 5 to exon 7. The resulting mature type I C2D mRNA contains a 134-bp deletion that would direct the synthesis of a truncatedprimary translation product. In our previous studies (12), no C2 pro- tein has been detected by immunochemical methods in sera or within fibroblast cells from type I C2-deficient individuals; therefore, it seems likely that the truncated C2D protein is either rapidly degraded or not recognized by the anti-C2 antisera used in those studies. In any event, functional C2 protein is not generated from the type I C2-deficient gene (12).

As discussed above, the major histocompatibility complex markers associated with C2 deficiency are highly restricted,

9352 Type I Human C2 Deficiency

c2s A A GO

1 Step

1 Step 2

C2D A A TG

cc 1 7 p

1 Step 2

( - 2.0 kb) intron 6

FIG. 7. Proposed model illustrating exon skipping mechanism in type I C2 deficiency. Proposed processing of the C2-sufficient and type I C2-deficient primary transcripts are shown on the left and right panels, respectively. In Step 1, the 5”splice sites are cleaved and corresponding lariats are formed at specific branch sites. No cleavage occurs at the 5”splice site of intron 6 of the C2-deficient gene, since the 28-bp deletion has removed the required gt recognition sequence. As a consequence, the 5”splice site of intron 5 forms a lariat at the branch site of intron 6. Cleavage occurs a t the 3”splice site in Step 2, resulting in liberation of the lariats and ligation of the exons. Hence, exon 6 is removed as part of the lariat structure in type I C2 deficiency.

with the majority (-93%) of C2 deficiency (C2QO) genes occurring on the haplotype HLA-A25,B18,BfS,C4A4,C4B2, Drw2, and almost all remaining C2QO genes occurring in the context of parts of this haplotype. This tight HLA association has led to the suggestion that C2 deficiency associated with these MHC markers originated 600-1300 years ago with the complete haplotype and that nearly all current C2 null genes are descendants of this original mutation (7). Our data strongly support this hypothesis in that all type I C2-deficient genes that we examined associated with these common hap- lotype/complotype markers contain the 28-bp gene deletion. Of interest, however, is the fact that although these C2QO genes harbor the same mutation and are associated with common extended haplotypes/complotypes, they do not ex- hibit identical clinical manifestations. This finding suggests that the diseases associated with C2 deficiency are not simply the result of a particular molecular genetic mutation in com- bination with expression of certain cellular HLA antigens. Instead, the clinical manifestations associated with C2 defi- ciency are likely to be the result of more complex phenomena that will be difficult to ascertain in human studies because of the limited patient pool. With the advent of gene targeting methods (37), C2-deficient murine model systems can be established in a variety of different genetic backgrounds. Such strains of mice should prove useful in identifying relevant genes that when expressed in combination with C2 deficiency result in increased susceptibility to infection and/or develop- ment of autoimmune diseases.

Acknowledgments-We thank Dr. Chester A. Alper for genomic DNA samples, Dr. Z. Starsia for fibroblast cells, and Dr. John E. Volanakis for sharing unpublished data. We also thank Martin Moh- ren for assistance in isolating cosmid clones and Barb Pellerito for preparation of the manuscript.

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