Molecular Bases for Inherited Human Complement Component C6 ...

Molecular Bases for Inherited Human Complement Component C6 Deficiency in Two Unrelated Individuals' Hiroaki Nishizaka," Takahiko Horiuchi,* Zeng-Bian Zhu,t Yasuo Fukumori,* Kohei Nagasawa,* Kenshi Hayashi,§ Richard Krumdieck,t C. Glenn Cobbs,' Masanori Higuchi,* Shin'ichiro Yasunaga," Yoshiyuki Niho,* and John E. Volanakis2t Deficiency of the sixth componentof complement (C6D) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis.We here report the molecular bases of C6D intwo unrelated subjects, one African American (case 1) and the other Japanese(case 2). Screening all 17 exons of the C6 gene and their boundaries by exon-specific PCR/single strand conformation polymorphism demonstrated aberrant single stranded DNA fragments in exon 12 of case 1 and exon 2 of case 2. Nucleotide sequencing of the amplified DNA fragments revealed a homozygous single-base deletion (C,936) in exon 12 of case 1 and a heterozygous single basedeletion ( C ~ g , / C z g z / C ~ g ~ / Cin~exon g ~ ) 2 of case 2. Both mutations resulted in frame shifts and premature termination of the C6 polypeptide. Sequence-specific oligonucleotide probe hybridization and direct sequencing of exon 12 amplified from genomic DNA further supported the homozygosity of the mutation in case 1. Case 2 is apparently compound heterozygote, but the putative mutation in the other allele of the C6 gene remains unknown. Both case 1 and case 2 were homozygous for theC6A allotype. These data indicate that at least three distinct mutational events can cause C6D, single nucleotide deletionsin exons 2 and 12, and a mutation as yet unidentified.Thus, similar to other complement protein deficiencies, the pathogenesis of C6D appears to be heterogeneous. The Journal of Immunology, 1996, 156: 2309231 5.

A

ctivation of either the classical or the alternative pathway of the complement system results in the formation of a large protein-protein complex termed membrane attack complex (MAC),3which consists of the terminal complement components C5b, C6, C7, C8, and C9. Assembly of the MAC on a cell surface results in its gradual insertion into the lipid bilayer and the eventual formation of a transmembrane channel, which can elicit cellular functions or lead to killing of susceptible cells (1,2). Inherited deficiencies have been reported for all proteins participating in the formation of the MACand arefrequently associated with recurrent systemic infections caused by Neisseria meningitidis or Neisseria gonorrhoeae, including meningococcal meningitis, meningococcemia, and disseminated gonococcal infection (3-5). The molecular genetic bases of inherited deficiencies of C5 (6) and C8b (7), and also of subtotal C6 deficiency (C6SD) (S),

~~~~

~

~

* First Department of internal Medicine,

Faculty of Medicine, Kyushu University, Fukuoka, Japan; 'Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294; 'Department of Research, Osaka Red CrossBlood Center,Osaka,Japan; §Institute of Genetic Information, Kyushu University, Fukuoka, Japan; and ¶Veterans Affairs Medical Center, Birmingham, AL 35233 Received for publication September 28, 1995. Accepted for publication January 11, 1996. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported in part by a Grant-in-Aid from the Tokyo Biochemical Research Foundation, by the Kaibara MorikazuMedical Science Promotion Foundation, and by the United States Public Health Service Grants A121067and AR03555. J.E.V. is the Anna Lois Waters Chair of Medicine in Rheumatology.

have been determined. However, molecular defects leading to complete C6 deficiency (C6D) have not been reported. The single polypeptide chain of C6 is composed of 913 amino acids and is,homologous to the other terminal complement proteins C7, CSa, C8b, andC9 (9, 10). All of these polypeptide chains have a mosaic structure consisting of protein modules apparently derived from diverse protein families. The genes for C6, C7, and C9 are located on chromosome 5p13(1 1).In humans the genes for C6 and C7 are about 160 kb apart and are arranged in a tail-to-tail orientation relative to transcription, whereas the C9 gene is located more than 2.5 megabases away (12, 13). The C6 gene spans about 70 kb of DNA and consists of 17 exons (14). The nucleotide sequences of all exons and of the exonlintron boundaries have been reported (14). Here we report a simple and sensitive strategy for studying the entire coding region of C6 by using exon-specific PCWsingle strand conformation polymorphism (SSCP) (15, 16). By using this methodology we have determined the molecular bases for two cases of complete C6D, one inan African American male and the other in a Japanese male. The deficiency of the former individual is apparently due toa homozygous deletion of nucleotide G,,,, in exon 12, which causes a shift in the reading frame and generates a stop codon 9 nucleotides downstream. In the Japanese subject, a heterozygous deletion of nucleotide C291/C2921C2931C294 in exon 2 also results in frame shift and a termination codon 129 nucleotides downstream. Since this individual also has complete C6D, hemust be considered as compound heterozygote with an as yet unidentified defect in the other allele of the C6 gene.

*

Methods

Abbreviations used in this paper: MAC, membrane attack complex; C6D, C6 deficiency; C6SD, subtotal C6D; CSF, cerebrospinal fluid; SSCP, single strand conformation polymorphism; SSOP, sequence-specific oligonucleotide probe.

Two unrelated individuals were included in this study. Case 1 was a 45-yr-old African American man who wasadmitted to the Veterans Affairs Medical Center of Birmingham with fever and disorientation. The patient

Address correspondence and reprint requests to Dr. John E. Volanakis, Division of Clinical Immunology and Rheumatology, THT 437, University of Alabama at Birmingham, Birmingham, AL 35294-0006.

Copyright 0 1996 by The American Association of Immunologists

C6D subjects

0022-1 767/96/$02.00

231 0

MOLECULAR BASES

FOR HUMAN COMPLEMENT C6 DEFICIENCY

Table I. Oligonucleotides for amplification of human complement C6 exons Exon 1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

5’ Oligonucleotide Sequence (5’ to 3 ’ )

3’ Oligonucleotide Sequence (5’to 3 ’ )

C T G A C T C A G G A T GT AC CA T TC GC T GT AG A G T C C T T C C A G TATGGAGCAGGT AC TAG TAG TC GC GC TT GCTATTGTGATT TTGACCCTGCCA TG CT AGG AGG TA TC ATGAACATTTCAC AGACTTGCCTTCAACTTTACC TTGTTCTGATACCTGTTCTCC G T T C T A C A TT AC GT CG TT TT AG TA AA AT TT CC AC C T G A C G G T A G A T A T C C A C A GA TG AT TC TG AC GT AC AC GC T C A T A C T T G G T A C T G C T A G G T A C T GT TC A TA TC GC CT AT T GC C T A T C A T A C C A G GCACCATGTTCCTCTTTGAAT ATGGTCTGTAAATGACAGCCA G G A G C T A C A T A TC GT GG GT CA TC GC CA TT AC G A G A A C T A C T T TCAGAGAACTGGGCAGTAATG GTACAAGGTGGAGGTTGCTAA T T C A G C T G C A C TC CA T G TA GT TC TC GA GT C A T A G G T A A A G T GTATGCAATGTA GC TC AA CAA CAG TT GG TAGGTTTAGGAT GCCTTAAAGAGTTTAGAGAGT ATATTGGGCACATTCATTCTG A T C A C C T C TT GT GT CG TG AT TT AG GC AA GC GA CA T A T A C T A C A C T T A G A A T C C AA TA GA CG TC GT TC GA CG AC T C T T C C A T C A A A G T G A A C A C A C GT TA AC CA T AT G CG TC GT A G A T G A A G G T T T A G A T A A A G C C A A TT GC GC TC AT AA AA TT CG TT G T T C A T T G T G C T

had a history of meningococcal meningitis, with episodes in 1969 and in 1982. The father of the patient died at age 52 of silicosis, The mother had six additional offspring by a different father. There is no history of meningococcal infection in the family. On admission, the white cell count was 19,000 per ml with a differential of 93% neutrophils, 6% lymphocytes, and 1% monocytes. The cerebrospinal fluid (CSF) showed a white cell count of 12,900 per ml with a differential of 100% neutrophils and a red blood cell count of 240 per ml. The CSFglucose concentration was less than 10 mg/dl with a plasma glucose of 138 rng/dl, and the protein concentration was 650 mg/dl. Microscopic examination of the CSF showed Gram-negative diplococci within leukocytes, and cultures of the blood and CSF grew N . ntmingiridis. The patient was treated with intravenous cefotaxime for 10 days with complete resolution of his fever and mental status changes. Case 2 was a 37-yr-old Japanese man who was found to he C6 deficient during a large scale screening for inherited deficiencies of late-acting complement components among healthy blood donors in Osaka, Japan (case 6-1 in Ref. 17). This individual gave no history of systemic neisserial infections.

Hemolytic assays Total serum hemolytic activity was measured as described (18). C6 hemolytic activity was measured by using C6-depleted serum (Advanced Research Technologies. San Diego, CAI. Briefly, 100 pl of Ab-coated sheep erythrocytes ( I .5 X I Ox/ml), 100 pI of C6-depleted serum diluted 1/20, and 100 pl of test serum dilutions were incubated in half-strength Veronalbuffered saline containing 2.5% dextrose. 0.1% gelatin, 0.15 mM CaCl,, and 0.5 mM MgClz for 60 minat 37°C. The reaction was stopped by of ice-cold Veronal-buffered saline containing 0.01 M adding 1.2ml EDTA, and the C6 activity was calculated in CH,,, units from the OD,,, of the supernatants.

PCR/SSCP analysis Primers for exon-specific PCR for all 17 exons of the C6 gene were synthesized on the hasis of the fanking intronic sequences (14) and are listed in Table 1. Genomic DNA was isolated from blood cells as described previously (19). PCR was performed i n a thermal cycler PJ2000 (PerkinElmer Cetus, Norwalk, CT) by using 50 ng of genomic DNA as template, 0.2 pM concentrations of each primer, 25 pM dNTP including 2 pCi [a-”PP]dCTP (ICN Radiochemicals, Irvine, CA), and 0.125 U Taq polymerase in a 5-p1 total reaction volume (20). Reactions were conducted for 30 cycles consisting of I min at 95°C and 2 min at 60°C. The PCR products were subjected to electrophoresis at 25°C on 5% nondenaturing acrylamide gels containing 5% glycerol or at 4°C without glycerol, using 45 mM Tris-borate, I mM EDTA buffer, pH 8.3 and 13 V/cm. PCR products of exons 4,6. 7, 9, 10, 11, 14, and 16 that were longer than 300 hp were digested with HueIII, Sucl, BgIII, Sucl, TnqI, HueII1, RsuI, and DmI, respectively, before SSCP analysis. Two DNA fragments were derived from each restricted PCR product and all fragments were smaller than 250 bp. DNA fragments were visualized by exposing the gels to Kodak XAR film and Fuji imaging plate (Fuji Photo Film Co., Ltd., Kanagawa, Japan).

Nucleotide sequencing DNA fragments of interest were excised from PCWSSCP acrylamide gels, purified on Suprec-01 (Takara Shuzo Co. Ltd., Otsu, Japan), and reampli-

Fragment Sire (hp) 290 271 223 301 234 381 354 258 341 414 345 237 256 350 226 383 286

fied by PCR for 20 cycles consisting of I min at 95°C and 2 min at 60°C by using 2 pM of each primer, 200 pM dNTP, and 0.625 U Taq polymerase in a 25.~1total reaction volume. Alternatively, DNA fragments of interest were amplified directly from genomic DNA. In either case, reaction products were purified by Microcon 100 (Amicon, Beverly, MA) and directly sequenced by using the ATaq Cycle sequencing kit (USB, Cleveland, OH) and radiolabeled primers according to the manufacturer’s instructions. Primers were labeled by using T4 polynucleotide kinase (New England Biolabs, Inc., Beverly, MA) and [y”P]ATP at 37°C for 20 min.

Sequence-specific oligonucleotide probe hybridization A sequence-specific oligonucleotide probe (SSOP) hybridization assay was used to confirm homozygosity of C6D in case 1, Two I &mer SSOP were synthesized. The first included the identified nucleotide deletion of exon 12 (C6-Ex 12-SSOPm: S’GTATCAATGATATGAAGA-3’).while the second corresponded to the normal sequence (C6-Ex 12-SSOPn5-GTATCAAT GATGATGAAG-3’). The procedure was described previously (2 1 ). Briefly, genomic DNA was used as template to amplify by PCR exon 12 of the C6 gene, by using primers listed in Table 1. Fifty nanograms of amplified DNA in 2 ml were dot-spotted onto nylon membrane filters, alkaline-denatured with 0.4 N NaOH for 5 min, and incubated at 54°C for 1 h with hybridization buffer (50 mM Tris-HCI, pH 8.0, 3 M tetrdmethylammonium chloride, 2 mM EDTA, 5X Denhardt’s solution, 0.1% SDS, 100 mg/ml of heat-denatured herring sperm DNA) containing 5 to I O pmol of 32P-end-labeled SSOP. After rinsing with 2X SSPE (0.3 M NaCI, 0.02 M sodium phosphate, 2 mM EDTA, pH 7.4) containing 0.1% SDS atroom temperature, filters were washed in TMAC solution (50 mM Tris-HC1, pH 8.0, 3.0 M tetramethylammonium chloride, 2 mM EDTA, 0.1% SDS) at room temperature for 10 min, followed by washing in TMAC solution at 58°C for 10 min twice. The filters were then subjected to autoradiography. TheSSOP were end labeled by using T4 polynucleotide kinase and [ y-”P]ATP at 30°C for 20 min.

Results Definition of C6D

C6 protein was undetectable in serum from case I by double immunodiffusion analysis using a goat anti-human C6 serum (Cappel, Durham, NC). Total and C6 hemolytic activities were also undetectable (less than 10 CH,,Jml each). Addition of purified C6 (Advanced Research Technologies, San Diego, CA) to case I serum to a final concentration of 20 mglml restored total hemolytic activity to 188 CH,,Jml (control value 192 CH,,Jml). The patient’s mother had total and C6 hemolytic activities of 277 and 69,290 CH,,,/ml, respectively, both of which are within normal limits. Results of complement determinations of case 2 have been reported before (17). Briefly, this individual had undetectable serum C6 protein by single radial immunodiffusion and also undetectable total and C6 hemolytic activities in serum. Hemolytic activities of all other complement components were within normal limits. Total

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The Journal of Immunology

231 1

a.

a. Case 1 / Exon 12 1

2

3

4

I

Glycerol (-). 4°C

1

2

3

b.

4

Fragment 1,4 (; .A

-1 c'

i

Glycerol (+), 25°C

b. Case 2 / Exon 2 1

21 32 43

Glycerol (-), 4°C

4

C. Normal allele

I#J :I>

I GGACAACCATGTATCAATGATGATGAAGAAATGAAA Glycerol (-). 4°C

Glycerol (+). 25°C

PCWSSCP analysis of C6Dindividuals. a , Exon-specific PCWSSCPforexon 1 2 using genomic DNAfrom case 1 (lane 2 ) , his mother (lane 3 ) , andtwoC6-sufficient controls (lanes I and 4 ) . b, Exon-specific PCR for exon 2 using genomic DNA from case 2 ( h n e 3 ) and 3 C6-sufficient controls (lanes 7, 2, and 4 ) .

G

Q

P

C

I

N

D

D

Detection of C6 gene mutations by PCWSSCP analysis

PCWSSCP analysis revealed aberrant bands in exon 12 of case I and exon 2 of case 2. As shown in Figure Itr, the exon 12-specific PCR product of case I ( / m e 2 ) displayed two bands migrating differently than those of the two C6-sufficient controls run in parallel (lnrles I and 4 ) . Distinct migration of the patient's bands was evident whether electrophoresis was performed at 25°C in glycerol-containing gels or at 4°C in the absence of glycerol. Genomic DNA from the mother of case I ( h e 3 ) displayed four bands at 4°C. two of which corresponded to those of her son's DNA and the other two to those of the controls' DNA. At 25°C the single band produced by the mother's DNA migrated with a mobility intermediate between those produced by control DNA and case I DNA. The combined results suggested homozygosity of an exon 12 dehis fect for case l and heterozygosity of the samedefectfor mother. In case 2 (Fig. I h, l117e 3 ) the exon 2-specific PCR product contained four bands. two migrating with the same mobility as those present in C6-sufficient controls (krrzes I , 2, and 4 ) and two migrating either faster or slower under both conditions of electrophoretic analysis. This result suggested heterozygosity of an exon 2 defect for case 2. No other aberrant bands were detected in any other exons in either C6D case. Nucleotide sequencing of exon 12 of case 1

The two distinctly migrating bands (2 and 3 ) detected by PCRI SSCP analysis of exon 12 in case I were isolated from the gel shown in Figure 2a. amplified by PCR, and sequenced in their entirety in parallel with the two control bands ( I and 4 ) . The nucleotide sequence obtained from hrrnds 2 and 3 was identical to the reported sequence of exon I2 of the normal C6 gene (9. 14). except that nucleotide cDNA sequence numbering (9) was deleted

E

M

K

I 1,l I X

Mutant allele GGACAACCATGTATCAATGATATGAAGAAATGA G Q P C I N D M I hlM,

hemolytic activity could be restored to normal values by addition of purified C6.

E

I 0 1 Ih

FIGURE 1.

K

KSTOP I

(>I 15

FIGURE 2. Definition of exon 12 mutation in case 1. a, PCWSSCP analysis of exon 1 2 using genomic DNA from case 1, his mother, and 2 C6-sufficient controls. The single stranded DNA fragments are designated as I to 4. Each fragment was purified separately from the gel, amplified byPCR, and subjected to nucleotide sequencing. b, Partial nucleotide sequences of the SSCP bands. The sequences of fragments 1 and 4 are identical to the corresponding sequence of the normal C6 gene. Fragments 2 and 3 display deletion of nucleotide C,,,,. c, Nucleotide sequence and deduced amino acid sequence (one letter code) around the 1936delC mutation. The C6 protein encoded by the C6D gene is truncated at amino acid residue608 with the last three residues being different from those of the native protein.

(Fig. 2h). This deletion causes a shift in the reading frame, generating a premature termination codon 9 nucleotides downstream (Fig. 2c). To confirm the homozygosity of the defect we subjected exon 12-specific PCR products of case I , his mother, and a C6sufficient control to SSOP hybridization. As shown in Figure 3, the patient's PCR product hybridized only with the mutant oligonucleotide(SSOPm), while that of his mother hybridized equally well with both SSOPm and the oligonucleotide based on the normal gene sequence (SSOPn), and that of the C6-sufficient control hybridized only with SSOPn. Further confirmation of homozygosity was obtained by amplifying exon 12 directly from genomic DNA andsequencingthe product in its entirety. Figure 4 showsthenucleotidesequence around nucleotide Gly3,,of exon 12 of case 1, his mother, and a C6-sufficient control. As shown, G,y30is deleted from the gene of case I , generating three codonsfor aminoacids different from those of the normal gene, followed by a premature termination codon. A single sequencing ladder is present above the deletion; thus the 1936delG mutation is homozygous. The patient's mother has two sequencing ladders above nucleotide1936, one corresponding to the normal sequence and the other to the sequence of case I . indicating heterozygosity of the 1936delG mutation.

MOLECULAR BASES FOR HUMAN COMPLEMENT C6 DEFICIENCY

231 2

a.

SSOPmSSOPn

b. r e 4

*)

b i 5 N Z Case 1

EE 2

Mother

0 0 0 0

Fragment 1,3 Fragment 2,4

-

O O r n O

Control

,

12-

Sequence-specificoligonucleotide probe (SSOP) hybridization. Dotblotsofexon 12-specific PCR products of case 1, the mother of case 1, and a C6-sufficient control are shown. T h e mutant oligonucleotide (SSOPm) hybridized withdot-blotted PCR products of exon 1 2 of case 1 and his mother, while the oligonucleotide corresponding to the normal gene (SSOPn) hybridizedwi th the dots of the mother and the control. FIGURE 3.

34-

-

G A T C G A T C

'*

"=-

1 del C

-7

C.

-

Normal allele

29 I .?94

Control Mother Case 1 5'

C A T CC A T CC A T C

5'

CAAAGATGC Q R C

...

CCC ATCAAC P I N I

GCC A

I

su

1N

GAG CCTCTGGTA E P L V 1 0

Mutant allele 29 1-294 I

CAAAGATGCCCATCAACT Q R C P

...

S

T

AGC CTC TGG TAG 5 L W STOP

I

I

sx

loo

.

Definition of exon 2 mutation in case 2. a. PCfUSSCP analysis of genomic DNAof case 2. The single stranded DNAfragments are designated as I to 4. Each fragment was purified separately from the gel, amplified byPCR, and sequenced. b, PartialDNA sequence of the SSCP bands. The nucleotide sequences of fragments 1 and 3 were identical with the corresponding sequence of the normal C6 gene. Fragments 2 and 4 displayed a single base deletion of one C atposition 291, 292, 293, or 294. c, The nucleotide sequence and deduced amino acid sequence (one letter code) around the mutation 291 delC/292delC/293delC/294delC. The encoded C6 protein is truncated at amino acid residue 100 with 42 amino acid residues being different from those in the native protein. FIGURE 5.

I

3'

3'

FIGURE 4. Partial DNA sequence of exon 1 2 amplified from genomic DNAof case 1, his mother, and a C6-sufficient individual. The arrow indicates the position of the deletion (1936delC), which leads to premature termination of the C6 transcript at amino acid residue 608. As there is a single sequencing ladder above the deletion, the mutation of 1936delC in case 1 is homozygous. The presence of two overlapping sequencing ladders above nucleotide 1936 in the DNA of the patient's mother indicates heterozygosityof the mutation. The single letter code has been used for amino acid residues.

Nucleotide sequencing of exon 2 of case 2

All four single stranded DNA fragments detected by PCWSSCP analysis of exon 2 of case 2 were isolated from the gel shown in Figure 5a and sequenced in their entirety. As shown in Figure Sh, the nucleotide sequences of two of the fragments ( I and 3 ) were identical with the corresponding sequence of the normal C6 gene (9). The other two single stranded DNA fragments ( 2 and 4 ) displayed deletion of one of the four cytidines at positions 29 1-294 (numbering according to Ref. 9). The mutation causes a frame shift generating codons for 42 amino acidsdifferent from those encoded by the normal gene followed by a premature termination codon, corresponding to amino acid residue 101 of the C6 polypeptide. The results indicate that case 2 is heterozygous for the 291delC/ 292deIC/293deIC/294delC mutation. Analysis of exon 3 polymorphism

Two common allotypes of C6, termed C6A and C6B. have been described. They differ from each other in the second position of the codon for residue 98 in exon 3 where a A+C transversion results in an amino acid change from Glu in the A allotype to Ala in the B allotype (22, 23). PCWSSCP analysis, of exon 3 of case I in

comparison to three C6-sufficient controls, is shown in Figure 60. Case 1 and control 1 displayed two bands, each migrating ditferently, thus indicating that these two subjects are homozygous for differing allotypes. Controls 2 and 3 showed four bands, each suggesting heterozygosity. All four bands were isolated from the gel, amplified by PCR using exon 3-specific primers, and subjected to nucleotide sequencing. As shown in Figure 6h the sequence of hcrrzds I and 3 encoded the C6A allele, while hands 2 and 4 encoded the C6B allele. Thus, case I is homozygous for the C6A allotype. Similar analysis of exon 3 of case 2 demonstrated that he was also homozygous for the C6A genotype.

Discussion We describe here the molecular bases of C6D in two unrelated subjects, one of African American and the other of Japanese descent. To our knowledge, this is the first description of molecular defects leading to complete C6D. Among complement deficiencies, C6D is relatively common, although its true incidence in the general population can only be estimated from a single study performed in Japan (17). Four C6D individuals were identified among 145,640 consecutive blood donors screened for deficiencies of late complementcomponents. Since the defect is inherited as an autosomal recessive trait. a rate

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1-

2

1

23 1 4-

Glycerol (-), 4%

b

Glycerol (+), 25%

Fragment 1,3 Fragment 2,4

(CW

(CW

I 5'

FIGURE 6. Definition of C6 allotypes. a, PCWSSCP analysis of exon 3 of case 1 and of three C6-sufficient controls.All four fragments were

purified from the gel separately, amplified by PCR, and sequenced. b, Nucleotide sequence of the fragments and the deduced partial amino sequence (one letter code). The nucleotide at position 41 3 was A in fragments 1 and 3, resulting in Clu at amino acid residue 98 (phenotype C6A). In fragments 2 and 4, the nucleotides at position 41 3 were C, resulting in Ala at amino acid residue 98 (phenotype C6B).

of 0.52% can be estimated for the null gene in the general populationof Japan. Interestingly, the three C6D individuals of that study for whom data were available had no history of recurrent infections. This contrasts sharply with a high frequency of systemic neisserial infections ascertained by several other studies of C6D individuals of diverse ethnic backgrounds. A comprehensive review of complement deficiencies published in 1984 identified 33 C6D individuals in 24 kindreds (3). At least eight of these kindreds were African American and at least three European American. Eighteen of the 33 C6D subjects had had one or more episodes ofmeningococcal meningitis and four one or more episodes of disseminated gonococcal infection. A similarly high incidence of recurrent meningococcal infections was found among 27 C6D individuals from I O South African kindreds (24). Most of these individuals were Cape Coloured (mixed Caucasoid, SouthEastAsian,andSouthernAfrican),andtwowereBlack, belonging to the Xhosa tribe. Although the differencein susceptibility to neisserial infectionsbetween Japanese on one hand and all other ethnic groups on the other could be due to genetic factors unrelated to C6D, it seems likely that ascertainment bias a major is contributory factor. This view is supported by the finding that both in the literature review (3) and in the South African study (24) the frequency of meningococcal disease was much lower among nonprobands than among probands.

Regardless of possibleethnicdifferences,theassociationbetween C6D and increased susceptibility to neisserial infections is well documented (3, 24-26). It is generally assumed that Ab-and complement-mediated serum bactericidal activity is a major host defense mechanism against neisseriae (27, 28). It follows that failure of this mechanism due to a terminal complement component deficiency leads to susceptibility to infections by these pathogens. However, Ross and Densen( 3 ) have presented strong argumentsin favor of the existence of additional factors contributing to the observed susceptibility of C6D to neisserial infections. Quantitation of C6 in 27"C6-deficient"serabyusingan ELISA sensitive to I nglml demonstrated thatC6 was undetectable in 17 (63%). Of the remaining IO, 4 sera had less than 60 ng C6/ml and 6 had 0.37 to 4.8 mg/ml, as compared with normal control values of 20 to 80 mg/ml. In four of the latter six sera, C6 was functional, being incorporated into a terminal component complex during complement activation(29). This form of C6 deficiency has been termed subtotal C6 deficiency (C6SD) and has been studied in considerable detail (30). The C6 polypeptide present in the serum of 8 C6SD individuals from four kindreds was found to be about 14% shorter than normal C6. This truncated C6 expressed both hemolytic and bactericidal activity againstN. rneningitidis (8, 29, 30). The molecular defect leading to C6SD appears to be an abnormal 5' splice donor site of intron 15, which would probably prevent splicing and result in a translation product 13.5% smaller than normal C6 (8). Neither of the cases presented here has this molecular defect. To identify the molecular defects causing C6Din the two individualsinvestigatedhere weadapted a two-stepprocedurethat avoids sequencing the entire coding region of the gene. In a first step, all 17 exons of the C6 gene were amplified and the resulting DNA fragments were analyzed by SSCP. This allowed the identification of aberrant bandsin exon 12 of case 1 and exon 2 of case 2. This screening strategy is sensitive, relatively simple, and applicable to the study of other C6D cases. In a second step, the aberrant SSCP bands were sequenced in their entirety. This all as a lowedthe identification ofthemoleculardefectofcase homozygous deletionof the first nucleotide of the codon for Asp606 ( 1 936delG). Direct nucleotide sequencing of exon 12 amplified from genomic DNA and SSOP hybridization assay confirmed the homozygous nature ofthe defect. As expected, the patient's mother was heterozygous for the same mutation. The identified 1936delG mutation causesa shift in the reading frame, giving rise to a premature termination codon. The putative polypeptide chain encoded by this gene has 608 amino acid residues and, therefore, is missing the carboxyl thirdof the C6 molecule (Fig. 7). This part of the molecule contains two short consensus repeats. which have been implicated in binding C5b (31). In addition,thetruncated polypeptide ismissingtwofactor I domains,which also are thought to provide binding sites for C5b (10). Thus, even if secreted, the truncated C6 polypeptide should not be able to participate in the assembly of the MAC. In contrast to case I , the molecular defect identified in case 2, was present in only one of 29 I delC/292delC/293delC/294delC, the two alleles of the C6 gene. This mutation also caused a frame shift and a termination codon 43 codons downstream.The putative C6 polypeptide encodedby this mutant gene would consistof only 1 0 0 amino acid residues, 42 of which would be different from the correspondingresidues of nativeC6. Since this individualhas complete C6D. a mutation must also be present on the other C6 allele, but we have not identified it as yet. There are several possible explanations for our failure to identify the putative second defect.Thesensitivity of thePCR/SSCPprocedure weusedis about 90%. provided the DNA fragments used for SSCP are less

MOLECULAR BASES FOR H U M A N COMPLEMENT C6

231 4

FIGURE 7. Schematic diagram structure of C6 Ref. (adapted from

of themolecular 14). Modules are designated according to the recommendations of a recentworkshop (39) as follows: T1, thrombospondin, type 1; LA, LDR typereceptor, A; EG, epidermal growth factor-like; CP, complement controlprotein; FM, complementfactor I, MAC proteins. Arrows indicatetheapproximatelocations of the molecular defects in case 2 (arrow 2) and case 1 (arrow 1).

Exons

1

Structural

2

3

4

5

7

6

8

9

10

11 12

2

1

J.

J.

1314

16

17

@ m m m m m m rm m m m m m n

Motifs

LA

T1T1

Acid number

DEFICIENCY

0

than 300 bp long (32). This was assured in this study by restriction digesting the larger PCR products. It thus seems possible that PCW SSCP failed to identify the putative mutation within one of the exons. It also seems possible that the defect consists of a mutation within an intron. The primer pairs we used for amplifying the exons were designed to include at least six nucleotides of flanking intronic sequences. However, it still is possible that a mutation away from a splice junction may affect splicing, thus interfering with translation. For example, it has been reported that neurofibromatosis type I is due to the insertion of an Alu sequence in an intron of the NFl gene (33). This insertion results in skipping of a downstream exon during splicing and shifts the reading frame. Finally, it also seems possible that a mutation in the promoter of the C6 gene is responsible for defective gene expression Direct repeats between 2 and 8 bp long have been shown to be located in the immediate vicinity of short gene deletions (34). It has been suggested that tandem repeats cause misalignment during gene replication, generating small (less than 20 bp) deletions (35). In case 1, the deleted G,,,, resides in the middle of three tandem ATG repeats. In case 2, one of four tandem Cs is deleted. Thus, in both cascs “slipped nlispairing” could be responsible for the mutations. In conclusion, our results provide evidence that, like most other complement deficiencies (36), C6D is caused by heterogeneous mutational events. Genetic heterogeneity of C6D was indicated previously (37) by RFLP analysis of genomic DNA from deficient individuals. However, at least among South African individuals, the heterogeneity of C6D was believed to be quite restricted (38). Evaluation of the true extent of genetic heterogeneity underlying C6D would require analysis of additional kindreds of diverse ethnic backgrounds.

Acknowledgment We t h a n k Mrs. Paula Kiley for a s s i s t a n c e with t h e p r e p a r a t i o n of t h e manuscript.

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