Type I Human Complement C2 Deficiency - The Journal of Biological ...

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Charles A. Johnson$, Peter DensenQ, Robert K. Hurford, Jr.$, Harvey R. ColtenSlI, and. Rick A. Wetsel$ll 11. From the $Edward Mallinckrodt Department of ...
THEJOURNALOF

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

BIOLOGICAL CHEMISTRY

Type I Human Complement C2 Deficiency A 28-BASE PAIR GENEDELETION

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$ll11 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 The complement system is a set of plasma proteins that protein C2 (C2D) have been previously identified. No serves as an effector of several biologic functions associated C2 protein translationis detected in type I deficiency, with inflammation, immunoregulation, and cytotoxicity (1). while typeI1 deficiency is characterized by a selective The second component of human complement (C2)’ is a block in C2 secretion. Type I C2 deficiency was de- 110,000 M,single-chain glycoprotein and is the serine esterase scribed in a family in which the C2 null allele (C2QO) component of the classical pathway C3 cleaving enzyme comis associated with the major histocompatibility haplo- plex. C2 is encoded by a 20-kb gene comprised of 18 exons (2, type/complotype HLA-AZS,B18,CZQO,BfS,C4A4, 3) that is located on the short arm of chromosome 6 between C4B2,DrwZ; this extended haplotype occurs in over the HLA-D and HLA-B loci of the major histocompatibility 90%of C2-deficient individuals (common complotype/ complex (MHC) (4). The C2 gene is part of a tightly linked haplotype). To determine themolecular basis of type I class 111 complement gene cluster that includes the homoloC2 deficiency, the C2 gene and cDNA were charactergous gene encoding complement protein factor B and the two ized from a homozygous type I C2-deficient individual with thecommon associated haplotype/complotype. We C4 loci, C4A and C4B (4). Deficiency of C2 is the most frequently occurring inherited found a 28-base pair deletion in the typeI C2QO gene, beginning 9 base pairs upstream of the 3’-end of exon defect of the complement system in individuals of western 6, that generates a C2 transcript with a complete dele- European descent. In thispopulation, approximately 1 person tion of exon 6 (134base pair) anda premature termi- in 10,000 is homozygous C2-deficient (5, 6). C2 deficiency nation codon. In studies of eight kindred, the 28-base exhibits very strong linkage disequilibrium with certain HLA pair deletion was observed in all C2QO alleles associ- haplotypes and complement polymorphisms. For example, the ated with the common type I deficient complotype/ haplotype/complotype most characteristic of C2 deficiency is haplotype; this deletion was not present in normal C2 H L A - A ~ ~ , B ~ ~ , C ~ Q O , B ~ S , C ~ (7-9). A ~ , CMore ~ B ~than ,DIWZ nor in type I1 C2-deficient genes. These data demon- half of C2-deficient individuals have rheumatological disorstrate that: 1)type I human complement C2 deficiency ders such as systemic lupus erythematosus (lo), Henochis caused by a 28-base pair genomic deletion that causes Schonlein purpura, and polymyositis (5). In other kindreds, skipping of exon 6 during RNA splicing, resulting in. association of C2 deficiencyand recurrent pyogenic infection generation of a premature termination codon, 2) the has been observed, and in some cases C2-deficient individuals 28-basepair deletion inthetype I C2QO gene is are asymptomatic (11). strongly associated with the HLA haplotype/complorecently demonstrated heterogeneity among indi, C ~ B ~ viduals , We D ~have Wwith ~, type A ~ S , B ~ ~ , C ~ Q O , B ~ S , C ~ A ~suggestCZ deficiency (12). In type Ideficiency, in which ing that all C2-deficient individuals with this haploC2QO is associated with the common haplotype/complotype, type/complotype will harbor the 28-base pairC2 gene deletion, and 3) type 11 C2 deficiency is caused by a there is no detectable translation of C2-specific mRNA. In different, as yet uncharacterized, molecular genetic contrast, in type I1 deficiency, in which C2QO is associated with two uncommon haplotypes/complotypes, HLA-A2,B5, defect. 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 deficiency have not been defined. Accordingly, we undertook the * This work was supported by United States Public Health Service present study that defines the molecular basis of type I Grants AI25011 (to R. A. W.), A124836 (to H. R. C.), A124739 (to H. deficiency; a 28-bp deletion occurs in theC2 genethat causes R. C.), and HD17461 (to H. R. C.), a merit review award from the aberrant RNA splicing resulting in deletion of exon 6 (134 Department of Veterans Affairs (to P. D.), and a grant-in-aid from bp) from the mature C2 message. The identical gene deletion the American Heart Association (to P. D.). The costs of publication is also present in eight unrelated C2-deficient kindred that of this article were defrayed in part by the payment of page charges. share the common haplotype/complotype, but in some cases This article must therefore be hereby marked “aduertisement” in have different clinical manifestations. These findings suggest accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequencefs) reported in this paper hos been submitted that: 1) all C2-deficient individuals with the common haploto the GenBankTM/EMBLDataBankwith accession numberfs) M86920. 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 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, kilobase(s); MHC, major histocompatibility complex; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate.

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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 studied 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 functional 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 a t 45 “C for 16 h in a 20ml solution containing 80 pg/ml proteinase K (Boehringer Mannheim), 75 mM NaCI, 25mM 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 alcohol in a glass 100-ml cylinder. The solution containing genomic DNA was dialyzed for 16 h a t room temperature against 4 litersof 10 mM NaCI, 10 mM EDTA, 50 mM Tris, pH. 8.0, followed bytreatment with 100 pg/ml RNase A (Boehringer Mannheim) a t 37 “C for 3 h. The DNA solution was extracted twice, first with 25% water-saturated phenol, 24% chloroform, 1%isoamyl alcohol and thenwith 48% chloroform, 2% isoamyl alcohol. The purified human genomic DNA was dialyzed, quantitated by absorbance at 260 nm, and stored a t 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 a t 42 “C for 1 h using the buffers and dNTPssupplied in a cDNA synthesis kit (Invitrogen, San Diego, CA). An oligonucleotide (Fig. 1, F) anti-sense to the normal C2 cDNA sequence was used asa 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 nearthe 5’-ends to facilitate subcloning. The first strand cDNA was initially denatured a t 95 “C for 3 min with 1 pgof each oligonucleotide ina 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 (PerkinElmer Cetus). Following the initialdenaturation, the cDNA was amplified by melting a t 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 a t 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 Bioproducts, Rockland, ME), and subcloned into pBluescript I1 (Stratagene, La Jolla, CA). Competent Sure cells (Stratagene) were transformed 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’ oligonucleotidecorresponding to thefirst 25 bp of exon 6 (5’-AAAGCTTGGGCCGTAAAATCCAGCG-3‘) and a 3’ oligonucleotide comprising 25 bp of intron 6 (5”GAGCACAGGAAGGCCTCTCTGCAGG-3’) wereused 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 CosmidLibraries, Isolation of C2 Genomic Clones, and Southern Blot Analysis-As described above, high molecular weightDNAwas 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

DNAwas 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 million 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-labeled oliaonucleotides that correwonded to exons 1 and 18 of the human C> gene. DNA Seauence Analysis-AllDNA 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 bacteriophage 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

1

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FIG. 1. Characterization of amplified cDNA fragments generated 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 electrophoresis 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 geneare 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 bottomof the gel are shown the oligonucleotide pairs used in the amplification reactions. The diagram shown at the bottomof 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, respectively. The small arrows below the C2 structure illustratethe 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”GACAAGCTTCGCTATCGCTGCTCCTCGAAT-3’; C, 5”GGATCCAGAAACAGCTGTGTGTGATAATGG-3’ D, 5”ACCGGATCCTGGACTGTTCGCAGAGTG-3’; E, 5”TCATGATGAACAACCAAATGCGGATCCTCG-3’; F, 5”GAACATATGCTGGATCCCTCCAAGCTCACA-3’.

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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 anopen box in the C2S sequence. The stop codons are indicated by asterisks. The cDNA and deduced protein sequences at theB’-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 C2deficient cDNAs. c2s

kb

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1 2 3 4 5 6 7 81 92 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 oligonucleotide. 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 (corresponding to exon 6 of the C2 gene) as described under “Experimental 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 notshown).

a normal and a C2D type I individual. The cDNA was synthesized 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 wasobserved 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 Clscleavage 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 unglycosylated molecular weight of 23,000. Analysis of GenomicDNA 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 “Experimental Procedures.’’ Normal and C2Dcosmid clones, containing the entire C2 gene, were digested with seven different restriction enzymes, Southern-blotted, and probed with an oligonucleotide (30-mer) contained within the 134-bp deletion toascertain whether this deletion was represented in the C2gene. Restriction fragments from both the normal and C2D clones hybridized to the30-mer (Fig. 3). The normal and C2D BumHI restriction fragments (3.5 kb) that hybridized to the oligonucleotide probe were preparatively isolated, subcloned 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 thedonor splice site of the adjacent intron

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EXON 5 ' MsILe~GIyAIaTnrArnPlaThrG,nLyrThiLyrG

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716 EXON 6 , m m l 6 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 GACTGTTCGCAGAGTGTGTCGGAAAATGACTTTCTCATCTTCAAGGAGAGCGCCTCCCTCATGGlGGACAGGGlCAGGAATCAGGAGTCTGCCTGCAGCA 849 GAGGCCTTCCTGTGCTCACTATCTCTCTCTGTCTCCTTCCCCTCCTCAGAACCCCACTCACAGCCCACCTCCTCCAAGAAGTCTTCTCAGATTATACTCA TGCCATGTAGGA~TCATGAATTCAATTTATACATCATAATlTTTATTCCACAGCACTGTTGGGACACTGTGCTGGGCTGGCGACACGAAGATGGAAAGGC TGAGTCTTACTCAGATCATCATCTAGACAGTGTCAGAAGTAGTAGATACCACAGATACGAGAATCTGTCTGATATATCAGTATATTATATTATATTCTTA AACTTCAACATTGTGCGAGCTTAAATGTGTGTGATAGACTGGCATGGTGCTAGTGCCTGTAATCCCAAACATTTGGGAGGCCGAGGCAGGTGGATCACTT GAGGTCAGGAGTTTGAGACCAGCCTGACCAACATGATGAAACCCTGTC.(

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TTTGTATTTTAGTAGAGATGGGTTTTGCATGTTGGTCAGGCTGGTCTCGAACTCCTGACCTCAGGTGATCCACCTGCCTAAGTGTTGGGATTCAGGCATG

AGCCACCGCGCCCAGCCCCTAGCTTCTTCCTAACAGCCATTTCCTAGTGTCTCCCCTGGTCCTTGCCTCTGTCGGTCTCACTCCAGTTTCTCTGCCTCCT

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FIG.4. Nucleotide sequence and exon/intron characterization of the C2-sufficient and type I C2deficient 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).

GenomicSequence

"_""""""""" 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 shownon the genomicstructure.The asterisk indicates the guanine substitution in intron5 of the C2-deficient typeI 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 upstream of the putative branch site of intron 5. These results indicate that the28-bp deletion at the5'-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 spanning the deletion was amplified in individuals from eight different kindred as described under "Experimental Procedures." 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 fragments. An ethidium bromide-stained agarose gel from a representative set of samples is shown in Fig. 6, and the results from all eight kindred are presented in Table I. In all individuals examined, the C2D gene contained the 28-bp genomic deletion when linked to the common C2D complotype/haplotype; 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 recurrent 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 haplotype/complotype ( H L A - A ~ ~ , B ~ ~ , C ~ Q O , B ~ S , C ~ A ~ , C 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 deletion 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 common haplotype/complotype will have the 28-bp C2 gene deletion.

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TABLE I PCR analysis of 28-bp genomicdeletion in 8 C2-deficient kindred Kindred

Individual”

Clinical presentation

Haplotype/ 28-bp complotype’ deletion‘

Well A/N +/Well A/N +/Infections A/A +/+ Well N/N -1Infections A/A +/+ Well B/C -/Well A/N +/Infections A/B +/Infections A/A +/+ Well A/A +/+ Well A/N +/Well A/N +/Well A/N +/Well A/N +/Infections A/A +/+ Infections A/A +/+ Infections AIA +I+ Well A/N +/Systemic lupus er+I+ A/A ythematosus Systemic lupus er8 (Ref. 10) 2 I1 2 A/A +I+ ythematosus ‘The individuals are identified as indicated in the corresponding references when possible. bThe capital letters indicate the following haplotypes: A = A25, B18, C2Q0, BfS, C4A4, C4B2, Drw2; B = A2, B5, C2Q0, BfS,C4A3, 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.

A I1 1 A I1 2 A I11 1 A I11 2 A I11 3 2 (Ref. 12) B I1 8 B I1 9 B 111 9 3 (Ref. 11) 88 Brother” Father“ Mother” 4 Fathere Mothere Sone Daughter‘ 5 Male’ 6 Female’ 7 (Ref. 10) 1 I1 2 1 (Ref. 12)

180 bpz 152 bp

1 2 3 4 5 6 7 8

0

QTB

-eo

FIG. 6. Screening of C2-deficient individuals for the 28-bp C2 gene deletion. GenomicDNA fragments spanning the 28-bp deletion were amplified starting from 1 pgof 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 electrophoresis through a 2.5% agarose gel and visualized by ethidium bromide staining. Lane 1, homozygous C2S daughter (A I11 2) kindred 1; lane 2, homozygousC2D 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 C2Dfemale (1I1 2) kindred 7; lane 8, homozygous C2Dfemale (2 I1 2) kindred 8.

The splicing of mRNA precursors requires at least two conserved sequences at theends of introns: the 5’ donor splice site consensus sequence 5’-CAG/GTAAGT-3’, whichis complementary 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 characterized 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 diseases occur as a result of abnormal splicing of mRNA precursors caused by gene mutations at 5’ and 3’ splice site sequences. Mutations in these consensus splice sequences usually abrogate or reduce the frequency of correct splicing of the affected intron. A few examples have been reported where mutations at the5’-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 lastposition of exon 12 in the porphobilinogen deaminase gene (34), and a 7-base pair deletion starting at position 5 of the 9th intron of the rat albumin gene (35) cause deletions of the mutated or preceding exons, respectively. 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



muscular dystrophy (36). Althoughthis deletion did not occur precisely at the5’-splice site, the mutated exon 19was 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 thiscase, 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 notbeen 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 protein has been detected by immunochemical methods in sera or within fibroblast cells fromtype I C2-deficient individuals; therefore, it seems likely that the truncated C2D protein is either rapidly degradedor 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

C2D

GO

AA

AA

TG cc

1 7 p

1

Step

1

1

Step 2

Step 2

(

-

2.0 kb) intron 6

FIG. 7. Proposed model illustrating exon skipping mechanism in type I C 2 deficiency. Proposed processing of the C2-sufficient and type I C2-deficient primary transcripts areshown 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 the5”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. REFERENCES with the majority (-93%) ofC2 deficiency (C2QO) genes occurring on the haplotype HLA-A25,B18,BfS,C4A4,C4B2, 1. Kinoshita, T. (1991) Zmmunol. Today 12,291-295 Drw2, and almost all remaining C2QO genes occurring in the 2. Ishikawa, N., Nonaka, M., Wetsel, R. A., and Colten, H. R. (1990) J. Biol. Chem. 265, 19040-19046 context of parts of this haplotype. This tight HLA association 3. Ishii, Y., Zhu, Z. B., Macon, K. J., and Volanakis, J. E. (1991) has led to the suggestion that C2 deficiency associated with Compl. Znflamm. 8, 167 these MHC markers originated 600-1300 years ago with the 4. Carroll, M. C., Campbell, R. D., Bentley, D. R., and Porter, R. R. complete haplotype and that nearly all current C2 null genes (1984) Nature 307,237-241 are descendants of this original mutation (7). Our data 5. Glass, D., Raum, D., Gibson, D., Stillman, J. S., and Schur, P. (1976) J. Clin. Znuest. 58,853-861 strongly support thishypothesis in thatall type I C2-deficient 6. Rynes, R. I., Britten, A. F., and Pickering, R. J. (1982) Ann. genes that we examined associated with these common hapRheumatic Dis. 41,93-96 lotype/complotype markers contain the 28-bp gene deletion. 7. Alper, C.A. (1987) Zmmunol. Lett. 14, 175-181 Of interest, however, is the fact that although these C2QO 8. Fu, S. M., Kunkel, H. G., Brusman, H. P., Allen, F. H., and genes harbor the same mutationandare associated with Fotino, M. (1974) J. Exp. Med. 140, 1108-1111 common extended haplotypes/complotypes, they do not ex9. Awdeh, Z. L., Raum, D. D., Glass, D., Agnello, V., Schur, P. H., Johnston, R. B., Gelfand, E. W., Ballow, M., Yunis, E., and hibit identical clinical manifestations. This finding suggests Alper, C.A. (1981) J. Clin. Znuest. 67,581-583 that thediseases associated with C2 deficiency are notsimply the result of a particular molecular genetic mutation in com- 10. Zitnan, D., Starsia, Z., Mozolova, D., Bosak, V., Cebecauer, L., Lukac, J., Lulovicova, M., Ondrasik, M., Buc, M., Niks, M., bination with expression of certain cellular HLA antigens. Stefanovic, J., Bircak, J. (1988) Rheumatologia 2, 15-22 Instead, the clinical manifestations associated with C2 defi- 11. Figueroa, J. E., and Densen, P. (1991) Clin. Microbiol. Reu. 4, ciency are likely to be the result of more complexphenomena 359-395 that will be difficult to ascertain in human studiesbecause of 12. Johnson, C.A., Densen, P., Wetsel, R.A., Cole, F. S., Goeken, N. E., and Colten, H. R. (1992) N. Engl. J. Med., in press the limited patient pool. With the advent of gene targeting methods(37), C2-deficient murine model systems can be 13.Amos, D.B. (1980) in NZAZD Manual of TissueTechniques, Public Health Service Publication NZH 80545 (Ray, J. G., ed) established in avariety of different genetic backgrounds. Such pp. 42-45, Government Printing Office, Washington, D. C. strains of mice should prove useful in identifying relevant 14. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, genes that when expressed in combination with C2 deficiency W. J. (1979) Biochemistry 18,5294-5299 result in increased susceptibility to infection and/or develop- 15. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 197-198, Cold Spring Harment 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 MartinMohren for assistance in isolating cosmid clones and Barb Pellerito for preparation of the manuscript.

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