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and Systemic Lupus ErythematosusC4B Deficiencies in Human Kidney Diseases Complete Complement Components C4A and

Friedrich Neumair and C. Yung YuYan Yang, Karl Lhotta, Erwin K. Chung, Paula Eder,

http://www.jimmunol.org/content/173/4/2803doi: 10.4049/jimmunol.173.4.2803

2004; 173:2803-2814; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Complete Complement Components C4A and C4B Deficienciesin Human Kidney Diseases and Systemic LupusErythematosus1

Yan Yang,*† Karl Lhotta,§ Erwin K. Chung,*† Paula Eder,¶ Friedrich Neumair,� andC. Yung Yu2*†‡

Although a heterozygous deficiency of either complement component C4A or C4B is common, and each has a frequency of �20%in a Caucasian population, complete deficiencies of both C4A and C4B proteins are extremely rare. In this paper the clinicalcourses for seven complete C4 deficiency patients are described in detail, and the molecular defects for complete C4 deficienciesare elucidated. Three patients with homozygous HLA A24 Cw7 B38 DR13 had systemic lupus erythematosus, mesangial glomer-ulonephritis, and severe skin lesions or membranous nephropathy. Immunofixation, genomic restriction fragment length poly-morphisms, and pulsed field gel electrophoresis experiments revealed the presence of monomodular RP-C4-CYP21-TNX (RCCX)modules, each containing a solitary, long C4A mutant gene. Sequencing of the mutant C4A genes revealed a 2-bp, GT deletion inexon 13 that leads to protein truncation. The other four patients with homozygous HLA A30 B18 DR7 had SLE, severe kidneydisorders including mesangial or membranoproliferative glomerulonephritis, and/or Henoch Schoenlein purpura. Molecular ge-netic analyses revealed an unusual RCCX structure with two short C4B mutant genes, each followed by an intact gene for steroid21-hydroxylase. Nine identical, intronic mutations were found in each mutant C4B. In particular, the 8127 g3a mutation presentat the donor site of intron 28 may cause an RNA splice defect. Analyses of 12 complete C4 deficiency patients revealed two hotspots of deleterious mutations: one is located at exon 13, the others within a 2.6-kb genomic region spanning exons 20–29.Screening of these mutations may facilitate epidemiologic studies of C4 in infectious, autoimmune, and kidney diseases. TheJournal of Immunology, 2004, 173: 2803–2814.

I n the past 30 years, complete complement components C4Aand C4B deficiencies have been identified and studied clin-ically in 13 males and 13 females from 18 families of dif-

ferent racial backgrounds (1, 2). Although 15 HLA haplotypeswere present in these patients, nearly three-quarters of them werehomozygous in HLA alleles. All but one of the complete C4-de-ficient subjects experienced symptoms related to immune complexclearance disorders such as systemic lupus erythematosus (SLE),3

a lupus-like disease, or glomerulonephritis. Many patients had re-current microbial infections. At least four patients died at youngages from severe infections or organ failure during lupus flares.

The human complement C4 genes are located at the class IIIregion of the MHC on the short arm of chromosome 6. The humanC4 locus is remarkably complex, which is featured by 1) segmentalduplication with mono-, bi-, tri-, and quadrimodular RP-C4-CYP21-TNX (RCCX) in the MHC; 2) a gene size dichotomy withlong and short genes; and 3) two classes of polymorphic proteinproducts for C4A and C4B (3). The outcome is a sophisticatedgenetic diversity with multiple length variants of RCCX moduleswith long or short C4 genes coding for polymorphic C4A and/orC4B proteins (3–6). Among different individuals in a population,two to seven (possibly eight) C4 genes may be present in a diploidgenome, leading to a 3- to 5-fold variation in plasma C4 proteinconcentrations and the presence of multiple allotypes (5). C4A ischaracterized by higher binding affinity to peptide Ags and com-plement receptor CR1, a longer half-life of the activated molecule,a relevant role in immunoclearance, and possibly a link betweeninnate and adaptive responses. The activated C4B has a faster re-action rate toward the hydroxyl group containing Ags and a shorthalf-life and is important in propagating the complement activationpathways in a temporally and spatially specific manner. The dif-ferential chemical reactivities between C4A and C4B are attribut-able to the C4A/C4B isotypic residues in modulating the covalentbinding activities of the thioester carbonyl group to its substrates

*Center for Molecular and Human Genetics, Columbus Children’s Research Institute;and Departments of †Molecular Virology, Immunology, and Medical Genetics and‡Pediatrics, Ohio State University, Columbus, OH 43205; §Clinical Division of Ne-phrology, Innsbruck Medical University, Innsbruck, Austria; ¶Bruneck Hospital,Bruneck, Italy; and �Brixen Hospital, Brixen, Italy

Received for publication March 31, 2004. Accepted for publication June 1, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants 1R01AR050078from the National Institute of Arthritis and Musculoskeletal and Skin Diseases,1P01DK55546 from the National Institute of Diabetes and Digestive and KidneyDiseases, and Pittsburgh Supercomputing Center through National Institutes of HealthCenter for Research Resources Cooperative Agreement Grant 1P41 RR06009.2 Address correspondence and reprint requests to Dr. C. Yung Yu, Room W402,Center for Molecular and Human Genetics, Columbus Children’s Research Institute,700 Children’s Drive, Columbus, OH 43205; E-mail address: cyu@chi.osu.edu3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; HERV-K(C4), endogenous retrovirus located in intron 9 of a long C4 gene; PFGE, pulsedfield gel electrophoresis; RCCX, RP-C4-CYP21-TNX, a four-gene duplication moduleconsisting of genes for serine/threonine nuclear protein kinase RP, complement com-ponent C4, steroid 21-hydroxylase CYP21, and extracellular matrix protein tenascin;RFLP, restriction fragment length polymorphism; SSP-PCR, sequence-specificprimer PCR. Gene symbols are italicized throughout the text. For DNA sequences,exon sequences are in uppercase, and intron sequences are in lowercase. For RCCXmodular variants: mono-L, monomodular RCCX with a single long C4 gene; LL,bimodular RCCX with two long C4 genes; LS, bimodular RCCX structure with one

long C4 gene and one short C4 gene; SS, bimodular RCCX structure with two shortC4 genes. RP2 and TNXA are linked, partially duplicated gene segments present in allbimodular, trimodular, or quadrimodular RCCX structures. CYP21A is a nonfunc-tional mutant gene.

The Journal of Immunology

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(7–10). Considering the relevant roles of C4A and C4B in immu-noclearance, memory, and effector functions of the humoral im-mune response, it is not unexpected that a deficiency of C4A orC4B is frequently associated with infectious and/or autoimmunediseases.

An elucidation of the molecular basis of complete C4A and C4Bdeficiencies may help in designing a comprehensive screeningstrategy to determine the prevalence of C4A and C4B mutations inautoimmune, infectious, and kidney diseases. To date, the molec-ular defects of complete C4A and C4B deficiencies have beenelucidated in only five subjects from three families residing inScandinavia and the U.S. (11–13). These patients had single C4Amutant genes in monomodular long (mono-L) RCCX haplotypesor one mutant C4A and one mutant C4B genes from bimodularlong-short (LS) haplotypes. Among the molecular defects found, a2-bp TC insertion into codon 1213 has been detected in C4A mu-tants from several families as well as a C4B mutant from HLA A2Cw7 B39 DR15 (12). In addition, a C nucleotide deletion at codon811 was discovered in a long C4A mutant gene of HLA A30 B18DR3 (11), and another C nucleotide deletion at codon 522 wasdetected in a short C4B mutant gene from HLA A2 B12 DR6 (13).In this study the clinical histories of seven patients with completeC4A and C4B deficiencies from four European families are de-scribed together with detailed molecular genetic analyses to deter-mine the RCCX modular variation and defects in the C4A and C4Bmutant genes.

Materials and MethodsHuman subjects and peripheral blood samples

Seven complete C4-deficient patients from four unrelated families residingin the alpine region close to the border of Austria and northern Italy wererecruited for this study after giving their informed consent. The parentsfrom patient families 1 and 3 were also included. Peripheral blood sampleswere used to isolate genomic DNA and EDTA plasma following estab-lished procedures (14). The protocol of this study was approved by theinstitutional review boards of Columbus Children’s Research Institute inthe U.S. and Innsbruck Medical University in Austria.

Allotyping of human complement components C4A and C4Bproteins

EDTA plasma samples were digested with neuraminidase and carboxyl-peptidase B, resolved by high voltage agarose gel electrophoresis, andfixed by goat antiserum against human C4 (DiaSorin, Stillwater, MN) (15).The C4A and C4B electrophoretic variants were stained by Simply Blue(Invitrogen Life Technologies, Carlsbad, CA). Complement C3 proteinpolymorphisms in EDTA plasma were determined by the immunofixationtechnique similar to that used for C4, with goat antisera against human C3(DiaSorin).

Genomic restriction fragment length polymorphisms (RFLPs)

Three RFLP strategies were used to elucidate the number and size of C4Aand C4B genes in each human subject. TaqI genomic Southern blot anal-ysis was applied to elucidate the RP-C4-CYP21-TNX modular length vari-ants, especially the presence of long and short C4 genes linked to RP1 orRP2 and the relative quantities of CYP21A and CYP21B and of TNXA andTNXB. PshAI/PvuII genomic RFLP analysis was applied to determine therelative dosages of C4A and C4B genes, using a C4d probe spanning exons22–25 for hybridization. Finally, a long-range mapping technique was ap-plied to give independent and confirmative information of the RCCX mod-ules. PmeI-digested genomic DNA trapped in agarose plugs was resolvedby pulsed field gel electrophoresis on a CHEF Mapper (Bio-Rad, Hercules,CA) and was subjected to Southern blot analysis using a C4d-specificprobe (16).

Specific oligonucleotide primers used in this study

For long-range PCR of DNA fragments, the following primers were used:C4E1.5, 5�-TCC AAG AGA GGT TAG ATC CG-3�; C4E9.3, 5�-CTGGAG ACT AAT GAT GGC T-3�; YYE10, 5�-GGA GGC AGA GCT CACATC CTG GTA-3�; Y303IN, 5�-CAG GAA GAA GTG CTG CGG GGA-3�; C4E29.52, 5�-GCT CTT CTC CCT GCC TTC CT-3�; C4E41.3, 5�-

TGG TCC CAG GCT GTG TTC AT-3�; C4E25.3, 5�-CAG GTG CTGCTG TCC CGT GA-3�; and Y23IN, 5�-CTC TGA CAC AGA GTC CTCAGA CC-3�.

For mutation detection using sequence-specific PCR, the followingprimers were used: C4E13D5, 5�-ATC CCG AGG GCA GAT CGT TC-3�;C4E14.3, 5�-CTT GCC CAT GTT GAG GGG CT-3�; C412F, 5�-CTACTA CCC TAT TCG CAC CT-3�; C4E13D3, 5�-GGG CTC TCG ATTCAT GAA CGA-3�; I27F, 5�-CAA GAC CCT CCT CCC GTT TTC-3�;and MBO-28R, 5�-GCC AGA GCC CCT CAC CCC TGA-3�.

For genetic polymorphisms in the two short C4B mutant genes, thefollowing primers were used: E95, 5�-CCC TGG AGA AGC TGA ATATGG-3�; and C4I113, 5�-CCA TGG ATC CTT GGG ACC CCA-3�.

PCR amplification, cloning, and sequencing of the long C4Amutant gene from HLA A24 B38 DR13

The long C4A mutant gene was amplified in three fragments using PCR.The first fragment was 2.3 kb in size and spanned exons 1–9. It was gen-erated using primer sets C4E1.5 and C4E9.3. The PCR conditions were onecycle at 94°C for 2 min; 33 cycles at 94°C for 45 s, 58°C for 45 s, and 72°Cfor 3 min; and one cycle at 72°C for 10 min. The second fragment was 6.6kb in size and covered exons 10–30. It was amplified by primer setsYYE10 and Y303IN. The PCR conditions were one cycle at 98°C for 2min; 40 cycles at 98°C for 45 s, 66°C for 60 s, and 72°C for 9 min; and onecycle at 72°C for 10 min. The third fragment was 5.7 kb in size, corre-sponding to exons 29–41, and was produced using primers C4E29.52 andC4E41.3. The PCR conditions were one cycle at 94°C for 3 min; eightcycles at 94°C for 45 s, 64360°C (�0.5°C/cycle) for 60 s, and 72°C for9 min; 30 cycles at 94°C for 45 s, 59°C for 60 s, and 72°C for 9 min; andone cycle at 72°C for 15 min. All PCR were performed using the FailsafePCR amplification kit (Epicentre Technologies, Madison, WI). The 2.3-kbexon 1–9 DNA fragment was purified using a PCR purification kit (Qiagen,Valencia, CA) and was directly sequenced. The 6.6-kb exons 10–30 frag-ment and the 5.7-kb exons 29–41 fragment were purified by gel filtration,cloned into the pCR4-TOPO vector (Invitrogen Life Technologies), andthen sequenced. Sequencing reactions were performed using the version 3BigDye kit (Applied Biosystems, Foster City, CA). The sequencing prod-ucts were purified by a spinning device (EDGE Biosystems, Gaithersburg,MD), vacuum-dried and processed by a 16-channel capillary sequencingmachine (Applied Biosystems) operated by the Sequencing Core Facilityof the Columbus Children’s Research Institute. DNA sequences were as-sembled and analyzed using GCG software (Genetics Computer Group,Madison, WI). The polymorphisms and mutations discovered in plasmidclones were further confirmed by direct sequencing of the original genomicPCR products.

Sequence-specific primer PCR (SSP-PCR) for the 2-bp deletionat exon 13

The presence of the 2-bp deletion at exon 13 of the C4 gene was detectedin SSP-PCR using primers C4E13D5 and C4E14.3 or using primers C412Fand C4E13D3. PCRs were performed using the Failsafe PCR amplificationkit (Epicentre Technologies). The PCR conditions were one cycle at 94°Cfor 3 min; 30 cycles at 94°C for 30 s, 60°C for 45 s, and 72°C for 1 min;and one cycle at 72°C for 10 min.

PCR amplification, cloning, and sequencing of two short C4Bmutant genes from HLA A30 B18 DR7

The short C4B mutant genes were amplified using two PCR and cloned intothe pCR4-TOPO vector. The first fragment of 7.3 kb spanning exons 1–25was amplified with primers C4E1.5 and C4E25.3, and the second fragmentof 7.4 kb spanning exons 23–41 was amplified with primers Y23IN andC4E41.3. Both PCR were performed using the Failsafe enzyme. The PCRconditions were one cycle at 98°C for 2 min; eight cycles at 94°C for 45 s,64360°C (�0.5°C/cycle) for 60 s, and 72°C for 9 min; 30 cycles at 94°Cfor 45 s, 59°C for 60 s, and 72°C for 9 min with a increase of 10 s/cycle;and one cycle at 72°C for 15 min. The isolated plasmids from both PCRproducts were purified and sequenced to completion. To separate these twomutant C4B genes, multiple clones from each group were sequenced. Thepolymorphisms and mutations were further confirmed by direct sequencingof PCR products.

SSP-PCR-MboI RFLP to detect mutation at the 5� splice site ofintron 28

SSP-PCR was used to determine the g3a mutation in intron 28 of the C4Bgene using primers I27F and MBO-28R. The PCR was performed using theFailsafe PCR amplification kit. The PCR conditions were one cycle at 96°C

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for 3 min; 33 cycles at 96°C for 45 s, 62°C for 45 s, and 72°C for 2 min;and one cycle at 72°C for 5 min. The PCR products were digested over-night with restriction enzyme MboI at 37°C and resolved by electrophore-sis with a 1.5–2% agarose gel.

PCR for exons 9–11 to segregate two mutant C4B genes

To detect the t/c polymorphism in intron 9, DNA fragments spanning exons9–11 from patients with two mutant C4B genes were amplified using prim-ers E95 and C4I113. The PCR conditions were one cycle at 94°C for 2 min;35 cycles at 94°C for 45 s, 58°C for 60 s, and 72°C for 2 min; and one cycleat 72°C for 7 min. The PCR products were digested with HinPI at 37°C andresolved by agarose gel electrophoresis.

ResultsClinical histories of the patients

Seven complete C4 deficiency individuals from four independentfamilies residing in Austria or Sudtirol, an alpine area in northernItaly close to Austria, were recruited for the current study. Theclinical histories of these patients are listed in Table I and de-scribed as follows.

Family 1: 1P. The 42-year-old male patient suffered from severeHenoch Schoenlein purpura at the age of 17 years, with involve-ment of the skin, intestines, and kidneys (17–19). Six years later hedeveloped macrohematuria and nephrotic syndrome. A renal bi-opsy showed mesangial glomerulonephritis with fibrous crescentsand tubular atrophy. At the age of 23 years, hemodialysis had to bestarted. One year later he received a renal allograft. After 2 years,hematuria and proteinuria were noted, and a biopsy of the trans-planted kidney showed recurrence of mild mesangial glomerulo-nephritis and chronic allograft nephropathy. Five years after trans-plantation dialysis had to be resumed. After 8 years ofhemodialysis the patient received a second renal allograft. Sixyears later his serum creatinine is 130 �mol/l, and urinalysis isnormal without signs of recurrent disease. Recent treatment regi-men includes tacrolimus, azathioprine, and prednisolone.

The 37-year-old brother of the patient also has complete C4deficiency. He was unavailable for clinical investigation, but wasreported to be healthy.

Family 2: 2P. The now 20-year-old male presented at the age of10 years with recurrent attacks of fever, vomiting, and macro-

hematuria. A renal biopsy showed mild mesengioproliferative glo-merulonephritis with immune deposits in the mesangium. He wastreated with low dose steroids and amoxicillin (20, 21). At the ageof 15 years, after a wound infection, he developed a nephroticsyndrome with proteinuria of 10 g/day. Renal histology revealed amembranous-type glomerulonephritis with large epimembranousimmune deposits. He responded well to treatment with i.v. Ig (1g/kg body weight monthly for 10 mo) with reduction of proteinexcretion to �1 g. However, after that treatment was stopped,proteinuria recurred and the patient remained unresponsive to Iginfusion. Treatment with mycophenolate mofetil was initiated, anda partial response with reduction of proteinuria to 2.5 g/day wasachieved. Renal function remains normal.

Family 3. The three children in family 3 suffered primarily fromproliferative glomerulonephritis. Two of them developed end-stage renal failure. The third sibling had a life-threatening cerebralinvolvement.

3P1: This 33-year-old female patient developed a lupus-like dis-ease at age 6 years. She had hypertension and erythema of the face,hands, and arms. Urinalysis showed microhematuria and protein-uria of 3 g/day. A renal biopsy showed a membranoproliferative-like glomerulonephritis with immune complex deposition predom-inantly in the mesangium. She received azathioprine and steroids.Despite the treatment, she developed slowly progressive, chronicrenal failure. Hemodialysis had to be started when she was 26years of age. After 5 years of hemodialysis she received a renalallograft. Two years later she has normal transplant function andno signs of recurrence of glomerulonephritis in the allograft. Hercurrent immunosuppressive regimen consists of cyclosporine A,mycophenolate mofetil, and prednisolone (22).

3P2: This male patient is currently 26 years old. At the age of5 years he developed lupus-like skin lesions, microhematuria, andproteinuria of 2 g/day. When he was 9 years old, a renal biopsywas performed, which showed severe membranoproliferative-likeglomerulonephritis with mesangial proliferation. All glomerularcapillaries showed proliferative changes of variable severity.Despite immunosuppressive treatment with azathioprine and

Table I. A summary of clinical histories in seven complement C4 deficiency patientsa

PatientNo.

Sex and Age(years; diseaseonset/current) Disease and Organ Involvement Medical Procedures Recent Medication

HLA A24 Cw7 B38 DR132P M; 10/20 Kidney (mesangial GN;

membranous GN)Mycophenolate mofetil (1.5 g),

prednisolone (5 mg)4P1 M; 5/29 SLE, skin, kidney (mesangial GN) Hydroxychloroquine (200 mg),

prednisolone4P2 F; 2/40 SLE, skin, kidney (mesangial GN) Skin autotransplantation (chin) Hydroxychloroquine (200 mg),

azathioprine (100 mg),prednisolone (8 mg)

HLA A30 B18 DR71P M; 17/42 Schoenlein Henoch purpura, kidney

(mesangial GN), skin, gutTwo kidney transplantations Tacrolimus (6 mg),

azathioprine (50 mg),prednisolone (5 mg)

3P1 F; 6/33 SLE, kidney (MPGN) Kidney transplantation Cyclosporin A (150 mg),mycophenolate mofetil (2 g)

3P2 M; 5/26 SLE, kidney (MPGN), skin Kidney transplantation Hemodialysis, sevelamer (4.8g), furosemide (500 mg),mycophenolate mofetil (1 g)prednisolone (4 mg)

3P3 F; 5/23 SLE, kidney (MPGN), skin,cerebral vasculitis

a GN, glomerulonephritis; MPGN, membranoproliferative GN.

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prednisolone, his renal functions deteriorated. Cyclophospha-mide bolus therapy stabilized renal disease for some time, butat the age of 16 years the patient was started on hemodialysis.After 2 years a cadaveric renal transplantation was performed.The patient was treated with tacrolimus, azathioprine, andsteroids. Because of proteinuria and increasing serum creatininelevels 5 years after transplantation, a graft biopsy was per-formed. The biopsy showed no signs of recurrence of lupusnephritis, but indicated chronic allograft nephropathy. Six yearsafter transplantation hemodialysis again became necessary. Af-ter 3 mo of hemodialysis the patient suffered from Aspergillusfumigatus meningitis. He was treated with liposomal amphoter-icin B and made a full recovery. Currently, patient 3P2 isreceiving hemodialysis and is on the waiting list for a secondrenal transplantation (22–24).

3P3: This female patient is 23 years of age. At 5 years of age shewas noted to have hematuria and proteinuria of 4 g/day. A renalbiopsy showed mesangial and focal endocapillary proliferativeglomerulonephritis, and the patient was diagnosed with SLE.Treatment with azathioprine and prednisolone was started and re-sulted in marked improvement of proteinuria. The patient was welluntil the age of 22 years, when she developed a febrile illness witha facial maculopapular rash. A skin biopsy revealed vasculitis withimmune complex deposits. The patient’s mental status deterioratedrapidly, and magnetic resonance imaging showed severe cerebralvasculitis. The patient was unresponsive to high dose steroids,plasma infusion and exchange, and i.v. Igs. However, she re-sponded to immunoadsorption treatment and mycophenolatemofetil. An almost complete recovery of cerebral function wasachieved. At present she is maintained on mycophenolate mofetiland low dose steroids without signs of glomerulonephritis or vas-culitis (25).

Family 4. The prominent clinical presentation in the patients offamily 4 is lupus-like skin disease. The two patients also sufferedfrom mild glomerular disease.

4P1: This male patient is now 29 years old. At the age of 5years he suffered from acute oliguric renal failure. A renalbiopsy revealed mild mesangial glomerulonephritis. Steroidtreatments led to a complete resolution and normalization ofrenal functions. Serum creatinine and urine analyses werenormal at the time of this report. The patient developed skinmanifestations of SLE primarily on the face. For that conditionhe was treated with hydroxychloroquine (200 mg daily) and lowdose prednisolone (22, 23, 26).

4P2: The female patient is 40 years of age. When she was 2years old she developed recurrent attacks of fever; rashes on theface, trunk, and extremities; and oral ulcers. She was treated withsystemic and later topical steroids. Skin lesions became atrophicand scarring. At the age of 24 years a renal biopsy was obtainedbecause of microscopic hematuria. Histologic studies showed mes-angial glomerulonephritis with an increase in mesangial matrixand extensive mesangial immune complex depositions. At presentrenal function is normal, and there is no hematuria or proteinuria.However, the patient continues to suffer from skin involvement.She required plastic surgery with autologous skin transplantationto her chin at age 29 years. Her current medications are azathio-prine (100 mg), hydroxychloroquine (200 mg), and low dose ste-roids to control skin disease (22, 23).

A brother of patients 4P1 and 4P2 died at the age of 3 years fromcerebral vasculitis and sepsis caused by staphylococci and strep-tococci infections (23).

Additional immunologic and hematologic observations of thecomplete C4 deficiency patients

The seven patients tested positive for antinuclear Abs, but sub-types, including anti-dsDNA, were negative. Patients 3P3 and 4P1had leucopenia and thrombocytopenia, but these were not constantfeatures. Interestingly, IgG and IgA deficiencies were observed inpatients 2P and 3P3, which probably reflected a defect in Ig classswitching.

The patients were vaccinated against various pathogens withoutcomplication. They also appeared to have normal responses to tet-anus toxin vaccination. However, repeated vaccinations of patient1P against hepatitis B surface Ag were not successful, because thepatient did not develop a specific Ab response. Other patients weresuccessfully vaccinated against hepatitis B surface Ag.

RCCX modules and C4A and C4B mutant genes in the sevencomplete C4 deficiency patients

HLA typing revealed that two common haplotypes were presentamong the seven patients described above. Patients 2P, 4P1, and4P2 were homozygous with HLA A30 B18 DR7; patients 1P, 3P1,3P2, and 3P3 were homozygous with HLA A24 B38 DR13 (18, 22,27). Immunofixation experiments of EDTA plasma confirmed thecomplete absence of complement C4A and C4B proteins in theseseven patients (Fig. 1A, upper panel). The results also showed thatthe mother of 1P expressed C4A3 and C4B5, and both parents ofpatients 3P1, 3P2, and 3P3 expressed C4A3 and C4B1. Comple-ment C3 proteins were detectable in all patient samples and theirrelatives using the same EDTA plasma samples and immunofix-ation technique (Fig. 1A, lower panel), suggesting that the absenceof C4 proteins was not likely to have been caused by proteindegradation.

The organization of the MHC complement gene cluster and thecharacteristic RFLP patterns of the RCCX modular variants aredepicted in Fig. 1B (5, 16). The RCCX structures of the patientsand relatives were determined by TaqI RFLP (Fig. 1C) and furtherconfirmed by PmeI-PFGE (Fig. 1D) of genomic DNA samples.The dosages of C4A and C4B genes were determined by PshAI-PvuII RFLP (Fig. 1E).

The three patients with homozygous HLA A24 B38 DR13 (2P,4P1, and 4P2) had the identical TaqI restriction patterns that arecharacteristic of the mono-L RCCX structures. As shown in Fig.1C, left panel, each patient had RP1 linked to a long C4 gene (7.0kb), followed by a CYP21B gene (3.7 kb) and a TNXB gene (2.5kb) in the RCCX modules. PmeI PFGE revealed the presence of a113-kb fragment, confirming the presence of homozygous (L/L)RCCX structures (Fig. 1D, left panel). PshAI-PvuII RFLP using aC4d probe further revealed that the mutant C4 gene present in 2P,4P1, and 4P2 is C4A, because only the 1.7-kb C4A-specific re-striction fragments were detectable (Fig. 1E, left panel). In es-sence, there is a solitary, long C4A mutant gene (C4AQ0) presentin the monomodular RCCX structure with RP1-C4AQ0 (L)-CYP21B-TNXB between HLA A24 -B38 and HLA DR13.

For the four patients with homozygous HLA A30 B18 DR7 hap-lotypes from families 1 and 3, TaqI RFLP showed the presence ofbimodular RCCX structures. These structures were characterizedby RP1 linked to a short C4 gene (6.4 kb), followed by CYP21B(3.7 kb) and TNXA (2.4 kb) in the first module, and then RP2linked to another short C4 gene (5.4 kb), followed by CYP21B (3.7kb) and TNXB (2.5 kb) in the second module. The presence of sucha bimodular RCCX structure with two short C4 genes was furtherconfirmed by the 139-kb PmeI fragment in the PFGE (Fig. 1D,right panel).

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FIGURE 1. Phenotypic and genotypic analyses of the complete complement C4 deficiency patients. Patients 2P, 4P1, and 4P2 are homozygous withHLA A24 Cw7 B38 DR13. Patients 1P, 3P1, 3P2, and 3P3 are homozygous with HLA A30 B18 DR7. 1M is the mother of patient 1P; 3M and 3F are parentsof patients 3P1, 3P2, and 3P3. A, Immunofixation of EDTA plasma complement proteins. EDTA plasma samples treated with neuraminidase and car-boxypeptidase B were resolved by high voltage agarose gel electrophoresis, fixed with goat serum with Abs against human C4 (upper panel) or Abs againsthuman C3 (lower panel), and stained. B, A map of the MHC complement gene cluster with selected RCCX modular variants and their defining restrictionenzyme fragment sizes. Horizontal arrows represent gene transcriptional orientations; inverted arrows represent locations of DNA probes hybridized in aSouthern blot analysis. The white box in the shaded C4 genes represents the endogenous retrovirus HERV-K(C4). 21A, CYP21A; 21B, steroid 21-hydroxylase CYP21B. An asterisk on bimodular SS highlights the presence of two CYP21B genes. C, TaqI RFLP elucidates the number and length variantsof RCCX modules. Genomic DNA samples digested with TaqI restriction enzyme were hybridized to three probes in a Southern blot analysis thatdistinguished RP1 linked to a long or a short C4 gene, RP2 linked to a long or a short C4 gene, CYP21A or CYP21B, and TNXA and TNXB. D, PmeI pulsedfield gel electrophoresis (PFGE) to determine the RCCX haplotypes. Intact genomic DNA from peripheral blood lymphocytes in agarose plugs was digestedwith PmeI and resolved by PFGE, processed by Southern blotting, and hybridized to C4d (exons 28–31) genomic DNA probe. RCCX haplotypes deducedfrom the autoradiographs were labeled on top of the lanes. E, PshAI-PvuII genomic RFLP to determine the relative gene dosages of C4A and C4B. GenomicDNAs digested with PshAI and PvuII were hybridized to a C4d genomic probe (exons 22–25). The DNA sequence for the C4A isotypic residues containsa PshAI restriction site and is therefore used to define the presence of C4A and C4B genes in the RFLP.

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PshAI-PvuII RFLP of the C4d region revealed that the C4 genesin 1P, 3P1, 3P2, and 3P3 were all C4B, because only the 2.2-kbC4B-specific restriction fragments were detectable (Fig. 1E, rightpanel). Therefore, the four patients with homozygous HLA A24B38 DR13 haplotypes contained two short C4B mutant genes(C4BQ0) and no C4A genes.

TaqI RFLPs showed that CYP21A was present in none of theseven complete C4 deficiency patients. This is unusual because abimodular RCCX structure often contains a CYP21A pseudogenetogether with TNXA-RP2 gene fragments between the two C4genes. As shown in Fig. 1C, no CYP21A-specific TaqI restrictionfragments were detectable in any of the complement C4A- andC4B-deficient patients engaged in this study. The relative bandintensities of CYP21B:CYP21A in the mother of family 1 and theparents of family 3 both showed a ratio of 3:1, which suggested aconfiguration of CYP21B-CYP21B in the HLA A30 B18 DR7 hap-lotype. To further confirm the absence of CYP21A, BsaI RFLP wasperformed. The 8-bp deletion in exon 3 of the CYP21A pseudogenecreates a novel BsaI restriction site (28). As shown in Fig. 2, noCYP21A-specific BsaI fragments were detectable in the completeC4 deficiency patients. Based on these genotypic and phenotypicanalyses, the RCCX modules in HLA A30 B18 DR7 are interpretedas bimodular short-short (SS) with the following configuration:RP1-C4BQO (S)-CYP21B-TNXA-RP2-C4BQ0 (S)-CYP21B-TNXB.

Genotype analysis of the mother from patient 1P revealed LS/SSheterozygous RCCX structures (1M; Fig. 1, C and D). In additionto the SS structure with two mutant C4B genes, her other two C4genes from the LS structure (Fig. 1, C and E) coded for C4A3 andC4B5 proteins (Fig. 1A). The parents of patients 3P1, 3P2, and 3P3were also heterozygous in RCCX structures. The mother of family3 (3M) had long-long (LL)/SS haplotypes, and the father (3F) hadLS/SS haplotypes (Fig. 1, C and D). Both of the LL haplotypefrom 3M and the LS haplotype from 3F expressed C4A3 and C4B1(Fig. 1A).

A 2-bp deletion in exon 13 of the long C4A mutant gene fromHLA A24 Cw7 B38 DR13

Sequencing determination and analyses were first initiated on pa-tient 2P at the polymorphic C4d region that spanned 2.3 kb. It wasfound that the mutant gene contained sequences characteristic ofC4A, which included the sequences coding for D1054, PCPVLD1101–1106, and VDLL 1188–1191. In addition, the mutant genehad the typical indels in introns 28 and 29 that are present in longC4A genes. However, no deleterious mutations were detected inthe C4d genomic region of the mutant gene.

To identify the nucleotide mutations contributing to the nonex-pression of complement protein, the long C4A mutant gene frompatient 2P was amplified by PCR in three fragments. These frag-

ments corresponded to exons 1–9 (2.3 kb), exons 10–31 (6.6 kb),and exons 29–41 (5.7 kb). The 6.4-kb endogenous retrovirusHERV-K(C4) located in intron 9 was not included. The three am-plified fragments were cloned and sequenced using 60 primers thatallowed sequence determination of all 41 exons and their inter-vening introns in both orientations (Fig. 3A) (3, 29, 30).

Compared with the annotated sequence for C4A and C4B (3,30), 10 nucleotide substitutions or indels present in the C4A mutantgene that deserve special attention are listed in Table II. The mostconspicuous change is a deleterious 2-bp deletion at the sequencefor codon 497 from exon 13 (Fig. 3B). At nt 9968 –9969, thesequence from patient 2P was missing the GT dinucleotide.Such a deletion would change the protein reading frame andgenerate terminations at codons 607 and 613 from exon 15 (Fig.3C). Two other novel nucleotide changes were C9775T (fromexon 12), which was a synonymous mutation at the sequencefor codon 476, and the c3t substitution at nucleotide 14,701(from intron 28).

Subsequently, genomic fragments at exon 13 from patients2P, 4P1, and 4P2 were independently amplified by PCR andsubjected to sequencing. The identical mutation was found ingenomic DNA samples from all three patients with HLA A24Cw7 B38 DR13.

To facilitate screening of the 2-bp deletion at exon 13 fromgenomic DNA samples, specific forward (E13D5) and reverse(E13D3) PCR primers were designed for two independent exper-iments. The application of E13D5 and E14.3 yielded a 492-bpfragment from patients 2P, 4P1, and 4P2 (Fig. 5A). The applicationof 12f and E13D3 yielded 391-bp fragments from the same sub-jects. In each set of experiments, the positive control was a 757-bpfragment amplified by 21A5 and 21A3.

Identical mutations in the short C4B mutant genes from HLAA30 B18 DR7

Genomic DNA fragments corresponding to the two short C4Bgenes from patient 1P were amplified together by PCR in twoindependent experiments as shown in Fig. 4B. Each of these DNAfragments was sequenced to completion. Variant sequences wereidentified by comparison with C4B sequences in public databasesand are listed in Table III.

Nine novel nucleotide changes were detected in the mutant C4Bgenes. However, none of these changes was located in the codingsequences (Table III). Peculiarly, five novel single-nucleotide mu-tations were clustered in intron 19. The remaining four mutationswere present in introns 20, 28, 30, and 31, respectively. Remark-ably, the g3a substitution at intron 28 was present at the introndonor site (position 8127; Fig. 4D). Such a substitution (i.e.,gt3at) would abrogate the correct splicing of C4 RNA transcripts.A new potential splice junction is present in seven nucleotidesdownstream of the original donor site. If the C4 heteronuclearRNA were spliced according to this cryptic donor site, a new ter-mination codon, TAA, would be generated nine nucleotides down-stream of Gly1206.

The homogeneity of the genomic DNA sequences at the 3� re-gion of exon 28 from patient 1P (Fig. 4C, right panel) suggestedthat both short C4B genes in the PCR product had the samemutation. In contrast, direct sequencing of the correspondinggenomic region amplified for subject 1M (the mother of patient1P) yielded both g and a sequences. The latter was expectedbecause subject 1M had two functional C4 genes coding for C4A3and C4B5 in addition to the two mutant C4B genes (Fig. 1A).

Sequencing of the mutant C4B genes from patients 3P1, 3P2,and 3P3 also revealed homogeneous and identical sequences with

FIGURE 2. BsaI RFLP to determine the presence of CYP21A andCYP21B. An 8-bp deletion in exon 3 of the CYP21A pseudogene generatesa novel restriction site for BsaI. Genomic Southern blot analysis of BsaI-digested DNAs was hybridized to a CYP21 genomic DNA probe. The0.37-kb fragment is common to both CYP21A and CYP21B.

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the g3a substitution at nt 8127, the 5� splice junction or the donorsite of intron 28.

Screening of mutation g8127a

To facilitate screenings of genomic DNA samples for the g3a mu-tation at the donor site of intron 28 (nt 8127), a new PCR strategy wascreated that used a reverse primer (primer MBO-28R) with one mu-

tagenized nucleotide to create a novel MboI restriction site after am-plification across the intron 28 donor site (Fig. 4D). As shown in Fig.5B, the DNA fragments corresponding to intron 27-intron 28 from 1P,3P1, 3P2, and 3P3 were homogeneous with the presence of theg8127a mutation. As expected, samples from subjects 1M and 3Fwere heterozygous, and the normal control gave rise to the uncleavedfragment only.

FIGURE 3. RCCX structure, PCR amplification, cloning, and sequence determination of the monomodular long C4A mutant gene from patient 2P withthe HLA A24 Cw7 B38 DR13 haplotype. A, PCR strategy to amplify the mutant C4A gene. Arrows above exon-intron structures represent transcriptionorientations of RP, C4, and endogenous retrovirus HERV-K(C4). Horizontal arrows below exon-intron structures represent PCR primer sets to amplify thegenomic DNA fragments. B, The 2-bp (GT) deletion in exon 13. Left panel, Exon 13 sequence from a wide-type C4A gene. Middle and right panels,Sequences of patient 2P. Both forward and reverse sequences reveal the GT dinucleotide deletion. C, The 2-bp deletion leads to frameshift mutations andstop codons (marked by red asterisks) at codons 608 and 614 from exon 15.

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Two different short C4B genes in HLA A30 B18 DR7Sequence determination of the amplified DNA fragments showedthat both short C4B genes had virtually identical sequences, except

for a single nucleotide at intron 9. At position 2601, both t and cnucleotides were detectable, suggesting a possible diversion be-tween the two mutant C4B genes. The C4BQ0 gene with 2601c is

FIGURE 4. RCCX structure, PCRamplification, cloning, and sequencedetermination of the bimodular struc-ture of patient 2P with HLA A30 B18DR7. A, Gene organization in the bi-modular SS structure. 21B, CYP21B.B, PCR strategy to amplify the mu-tant C4B genes. C, Mutation at thedonor splice site of intron 28. Leftpanel, Heterozygous sequences withboth G and A nucleotides (marked bya red arrow) from the patient’smother, 1M; right panel, sequence ofthe patient. D, The probable splicedefect in intron 28 leads to a stopcodon (asterisk) in exon 29. The g3amutation in 1P is marked by a redarrow. A cryptic splice signal ispresent seven nucleotides down-stream of the original donor se-quence. A specific reverse primer(MBO-28R) designed to detect theg3a mutation at the splice junctionis shown in purple (see Fig. 5B).

Table II. The sequence changes in the mutant long C4A gene in families 2 and 4

Exon/Intron Positiona Nucleotide Change Amino Acid Change (if any) Remarks

Exon 9 2296 C3A (TCT to TAT) S 328 YExon 12 9775 (3401) C3T (GCC to GCT) 476 A ncExon 13 9968 (3594) �GT (deletion) nc; frameshift and premature stop codonExon 20 12169 (5796) T3C (GTT to GTC) 806 VIntron 20 12299 (5923) g3aExon 21 12526 (6150) A3G (ACC to GCC) T 888 AIntron 28 14510 (8140) g3cIntron 28 14514 (8144) � cIntron 28 14701 (8332) c3t ncIntron 29 14980 (8611) c3g

a Nucleotide numbering is based on the long C4A gene; the nucleotide numbering in a short C4 gene is in parentheses. nc, novel change in sequence. An update and annotatedsequence for a short C4 gene can be found in Ref. 2.

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detectable by restriction enzyme HinPI, which recognizes theDNA sequence gcgc. To confirm the presence of two differentC4BQ0 genes in the HLA A30 B18 DR7 haplotype, a 931-bpgenomic DNA fragment spanning exons 9–11 was amplified byPCR and digested by HinPI. If a HinPI site is present, the restric-tion enzyme-digested products would be 585 and 346 bp in size.Fig. 5C showed the result of such an experiment. With respect tothe presence of 2601c, the control sample (lane C) was homoge-neously positive. In contrast, patient members of families 1 and 3all yielded heterogeneous results, because both 2601c and 2601talleles were present.

By long-range PCR, a genomic DNA fragment spanning be-tween RP1 and C4B exon 11 was amplified. The mutant C4B genefrom the first RCCX module was found by HinPI RFLP and South-ern blot analysis to contain the 2601t allele (data not shown).

DiscussionIn this study we described the clinical histories and the molecularbases of seven patients with complete complement C4 deficiencies.These patients are from four independent families, and they arehomozygous in two different HLA haplotypes. The first group withHLA A24 B38 DR13 contains the monomodular RCCX structures,each with a single long C4A mutant gene. No C4B gene is present.The molecular defect leading to the absence of C4A protein pro-duction is a 2-bp deletion at exon 13. The three patients with thismolecular defect have either SLE with severe skin lesions andfrequent infections, or membranous nephropathy.

The second group with HLA A30 B18 DR7 contains the bi-modular RCCX configurations with two short mutant C4B genes.No C4A gene is present in this haplotype. In each of the two C4Bmutant genes, the molecular defect is probably a point mutation atthe donor site of the intron 28 splice junction. The four patientswith this defect have SLE or Henoch Schoenlein purpura withsevere glomerulonephritis. Three of these patients had end-stagekidney failure and required hemodialysis and kidney transplanta-tions. Two of these grafts were eventually lost due to allograftnephropathy. A successful second renal transplantation was per-formed in one of the patients (1P). The SLE disease in anotherpatient (3P3) progressed to a life-threatening cerebral vasculitis, anillness that claimed the life of another complete C4 deficiencypatient who we had studied previously (13). A complex treatment

regimen, which included the application of mycophenolate mofetilto suppress B cell proliferation and a potential alloimmune re-sponse and immunoadsorption using a column coated with poly-clonal sheep Abs against human IgG, appeared to have reversedthe disease course, and the patient regained much of her CNSfunction (21).

For therapies of complete C4 deficiency patients with severeorgan involvements, such as glomerulonephritis or cerebral vas-culitis, mycophenolate mofetil could be used as a basic treatment.In addition, either i.v. Ig or immunoadsorption procedures could beapplied. Low dose steroids appeared effective toward lesser prob-lems, such as skin involvement. Vigorous treatments of all infec-tions in the C4-deficient patients are essential. As severe glomer-ulonephritis does not recur in a renal allograft, C4 deficiency is nota contraindication for kidney transplantation.

The roles of complement C4A and C4B in immunity, autoim-munity, and kidney physiology remain perplexing. Currentthoughts are that the complete absence of C4 proteins probablyimpaired the clearance of immune complexes and apoptotic ma-terials, which contributes to inflammatory and vasculitic lesions invarious organs, including skin and kidneys. It is postulated that thepresence of complement C4-decorated self-Ags would facilitatethe deletion of autoreactive B cells in the bone marrow in theprocess of central tolerance. It is also suggested that the depositionof activated C4 on foreign Ags facilitates the activation of Ag-specific B cells and enhances the class switching of Igs in theperipheral lymphoid system (31, 32). Thus, it is plausible that thereis a strong association between complete C4 deficiency with sys-temic autoimmune diseases and kidney disorders.

Multiple investigators observed very low levels of complementC4 in patients with lupus nephritis (33–36). Such a phenomenoncould be explained by high consumption rates caused by patho-genic immune complexes, inherited deficiencies (or low gene dos-ages) of C4A or C4B, and possibly lower C4 biosynthesis rates.The surface deposition of C4d, which is a split product of inacti-vated C4 containing the thioester residues, has been found recentlyto be one of the most consistent markers for acute and chronicrenal allograft rejections caused by the humoral immune response(2, 37). These phenomena reflect the complement-mediated tissueinjuries caused by effector functions of C4 in the complement ac-tivation pathways.

Table III. Nucleotide changes in the short C4B genes from patient 1P (HLA A30 B18 DR7; RCCX-SS)

Exon/Intron Nucleotide No.a Nucleotide Change (c.f., C4B short) Amino Acid Substitution (if any) Remarks

Exon 9 2296 C3A (TCT to TAT) S 328 YIntron 9 2601 c or t Segregates 2 C4B mutant genesExon 16 4659 C3T (AAC to AAT) N 661Intron 19 5546 t3c ncb

Intron 19 5653 a3g ncIntron 19 5666 g3a ncIntron 19 5753 a3g ncIntron 19 5761 t3c ncExon 20 5793 T3C (GTT to GTC) V 806Intron 20 5985 g3a ncExon 21 6150 A3G (ACC to GCC) T 888 AIntron 21 6307 g3a ncIntron 21 6331 a3gIntron 21 6419 t3cExon 26 7535 C3A (GGC to GGA) G 1076Intron 28 8127 g3a nc; donor splice siteIntron 30 8920 c3aIntron 31 9576 g3a ncIntron 31 10248 t3c

a Nucleotide number is based on the sequence of a short C4B gene.b nc, novel change in sequence.

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To date, six deleterious mutations in the C4A or C4B gene havebeen detected in 12 human subjects with complete C4A and C4Bdeficiencies. All except one of those mutations are 1- or 2-bp in-sertions or deletions (indels) in coding sequences that lead toframeshift and nonsense mutations (Fig. 6). SSP-PCR and SSP-PCR plus RFLP techniques to screen mutations of C4 genes havenow been created, and these would help clarify the roles of C4Aand/or C4B deficiencies in infectious and autoimmune disease pa-tients. The molecular basis of C4 mutations in 10 other HLA hap-lotypes are yet to be elucidated (Table IV). It is worthwhile topoint out that deleterious nonsense mutations tend to be race- orethnic group-specific (38). For example, the presence of the 2-bpinsertion in exon 29 of the C4A genes have been detected inhealthy Caucasians (39) and in Caucasian and black SLE patients(40). Such a mutation has not been detectable in Asians (41). Al-though considerable progress has been made in understanding themolecular basis of C4A and/or C4B deficiencies in European andNorthern American Caucasians, very little or no knowledge isavailable on the basis of C4A or C4B deficiencies in any otherethnic group (in the U.S.).

As established previously, a bimodular RCCX is regularly char-acterized by the presence of an RP1 gene, followed by C4A, the

FIGURE 5. PCR-based mutation detections in C4A and C4B mutantgenes. A, SSP-PCRs to show the 2-bp deletion in exon 13 of the HLA A24B38 DR13 haplotype. Two complementary PCR strategies were applied.The first four lanes show results using primer set E13D5/E143 by whichthe 2-bp deletion is incorporated in the forward primer. The next four lanesshow the results of the primer set 12f/E13D3 by which the deleted se-quence is incorporated in the reverse primer; lane C, normal control. B,SSP-PCR and MboI RFLP to show the mutation in intron 28 donor site.Genomic DNA samples were amplified by primer set I27F/MBO-28R anddigested with restriction enzyme MboI. C, HinPI RFLP to segregate thetwo C4B mutant genes of the bimodular SS structure, based on a singlenucleotide polymorphism (t2601c) in intron 9. Genomic DNAs were am-plified by primer set E95/I113 and digested with HinPI. The C4 gene in thecontrol sample (lane C) has the c allele only. All others contain both 2601 tand c alleles.

FIGURE 6. Deleterious mutations in the long (upper) and short (lower) C4 genes leading to complete complement component C4A and C4B defi-ciencies. Locations of mutations in the mutant genes are marked (�). The indels or point mutations are listed on the right panel, which included the HLAand RCCX haplotypes. The mutant C4A or mutant C4B containing the defect are in bold. The 2-bp insertion in exon 29 (codon 1213) is relatively commonin C4A mutant genes in Caucasians (54).

Table IV. Other known HLA haplotypes with uncharacterized mutantC4A and/or C4B genes in complete C4 deficiency patientsa

HLA RCCX (if known) Ref. No.

A11 Cw5 B18 DR2 n.d.b 43A1 Cw7 B7 DR2

A2 Bw15 Dw8 n.d. 44–47(A2 B12 Dw2)c

A26 B49 DR2 n.d. 48

A11 Cw4 B35 DR1 L 49, 50

A1 B17 LS 50, 51

A2 B15 DR2 n.d. 52A2 B15 DR3

A31 B51 DRw9 n.d. 53A24 B52 DR2

a Modified from Ref. 2.b n.d., not determined.c Mutations in C4AQ0 and C4BQ0 genes of HLA A2 B12 DR6 were determined

(see Fig. 6)

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pseudogene CYP21A, gene fragments TNXA and RP2, C4B, thesteroid 21-hydroxylase CYP21B, and then the extracellular matrixprotein TNXB. Two major bimodular RCCX haplotypes arepresent in Caucasians, LL and LS. With some exceptions, the firstlong gene usually codes for C4A. The second gene may be long orshort; it generally codes for a C4B protein, but sometimes for aC4A protein. The C4 genes and RCCX constituents in the HLAA30 B18 DR7 haplotype contain some distinct features. The first isthe presence of two short C4B genes in a row (i.e., bimodular SS).These two C4B genes share the identical mutations, and only onenucleotide change is detectable between the two genes, of whicheach spans 14.2 kb. The second is the presence of CYP21B-CYP21B instead of CYP21A-CYP21B in the bimodular RCCXstructure. The presence of this unusual configuration could be ex-plained by a recent gene duplication event or by a genetic process,such as a long-range gene conversion that homogenized both C4and CYP21 sequences.

The four complete C4 deficiency patients with HLA A30 B18DR7 each have four intact and probably functional CYP21B genesin a genome. Our ongoing epidemiologic study of the RCCX mod-ular variations in autoimmune diseases reveals that a considerablepercentage of healthy subjects and patients have more than twofunctional CYP21B genes in a diploid genome, particularly sub-jects with trimodular RCCX structures (4, 39). Although the ho-mozygous deficiency of CYP21B leads to congenital adrenal hy-perplasia (42), the possible impact of the presence of high genedosage of functional CYP21 on steroid biosynthesis, including cor-tisols, mineralocorticoids, and sex hormones, deserves furtherstudy.

AcknowledgmentsWe are indebted to the patients and family members who participated inthis study, and we are grateful to Huachun Zhong and Dr. Bob Munson(DNA Sequencing Core Facility, Columbus Children’s Research Institute),and Dr. Dan Birmingham (Ohio State University) for help with DNAsequencing.

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