Complete deficiencies of complement C4A and C4B including 2-bp insertion in codon 1213 are genetic risk factors of systemic lupus erythematosus in Thai populations

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Abstract

The complement component C4 is encoded by two genes: C4A and C4B on human chromosome 6p in the major histocompatibility complex (MHC). Most studies have linked the deficiencies in C4 with systemic lupus erythematosus (SLE) in Angio-Irish, North American, Black American, Mexican American, Australian and Japanese populations. Null alleles at either locus (C4AQ0 or C4BQ0) are relatively common in Americans occurring at the C4A and C4B loci in approximately 10% and 16% of normal individuals, respectively.

In the present study, we extensively examined the possible association between homozygous C4Q0 and SLE in a large cohort of Thai populations diagnosed as SLE and further attempted to identify the genetic basis of C4Q0. One hundred and eighteen cases of SLE patients and 145 matched controls were genotyped by touchdown PCR. The results confirmed the previous studies that 5.93% (7/118) of C4 null genes: 2.54% (3/118) of C4AQ0 and 3.39% (4/118) of C4BQ0 were found in SLE patients. In contrast to other studies, we found no cases of C4 null genes in normal control (0 from 145 samples). To further investigate the genetic basis of C4 deficiency, all genomic DNAs were also analyzed for 2-bp (TC) insertion at codon 1213 in exon 29 which is a common mutation in many C4A null genes and a novel 1-bp deletion (C) at codon 522 in exon 13 that is common in most C4B null genes. Both mutation results in a flame-shift mutation and premature stop codon using sequence specific primers PCR (SSP-PCR) and direct sequencing.

The results showed that there was 2-bp insertion in exon 29 of mutant C4B gene in one SLE patient carrying C4AQ0. There was no 2-bp insertion in exon 29 of both C4A and C4B genes in normal individual and the rest of SLE patients. All patients with C4AQ0 exhibited more than 5 ACR criteria including malar rash, oral ulcers, renal disorder, immunological disorder, anti-nuclear antibody, without hematological disorder. In contrast, all of C4BQ0 SLE patients showed 5 or 6 ACR criteria including hematological disorder, malar rash, oral ulcers, renal disorder, immunological disorder and anti-nuclear antibody. A patient who possesses C4AQ0 and 2-bp insertion in exon 29 of mutant C4B showed 9 ACR criteria but no discoid rash and hematological disorder.

In conclusion, both C4AQ0 and C4BQ0 are the strong predisposing factors for SLE in Thais. It was supported by the absence of either C4A or C4B deletion in healthy control. We suggested that the different racial and genetic backgrounds could alter the thresholds for requirement of C4A or C4B protein levels in immune tolerance and regulation.

Introduction

The fourth component of human complement system (C4) is coded by two genes: C4A and C4B located within the MHC class III region on the short arm of chromosome 6. The two C4 isotypes differ in their amino acid sequences by <1%. The two C4 genes are tandemly arrayed together with two genes for the cytochrome P450 and steroid 21-hydroxylase (CYP21A and B), each located 3′ to the C4A and C4B genes, respectively. These genes produce single chain precursors that give rise to native C4-a three-chain glycoprotein of about 185 kDa. The C4A and C4B genes specify functional distinct C4 proteins, C4A is a hundred times more reactive with targets containing free amino group and 10 times less reactive with hydroxyl group than C4B [1], [2]. The functional differences in these C4 proteins are due to four amino acid variations between residues 1101 and 1106; PCPVLD for C4A gene, and PCPVIH for C4B gene [3], [4].

The genetics of human complement C4 are complex. There is a frequent variation in the number and size of the C4 genes presenting in an individual [5]. The majority of the population has two C4 loci coding for C4A and C4B proteins, respectively. The C4 genes are either 21 or 14.6 Kb in size due to the presence of an endogenous retrovirus HERV-K (C4) in intron 9 of the long C4 genes [6], [7], [8], [9]. Deletion or duplication of the C4 genes is always accompanied by neighboring genes RP at the 5′ region, steroid hydroxylase gene CYP21, and extracellular matrix protein gene tenascin TNX at the 3′ region. Along with C4, these genes make up a genetic module known as the RCCX [10], [11], [12]. There can be one, two, or three RCCX (RP-C4-CYP21-TNX) modules on one copy in the HLA class III region. Variation in RCCX module numbers between chromosomes may favor unequal crossing-over events resulting in deletions and homoduplications of C4 and its neighboring genes. Indeed, such haplotypes have been recognized with a reasonably high frequency [13], [14], accounting for a major fraction (∼60%) of known C4 null (Q0) alleles, although other molecular mechanisms that can generate C4A or C4B null alleles have been identified [15].

At the protein level, C4 is the highly polymorphic with >40 variants including null alleles (C4Q0) at both loci [16]. Null alleles are defined by the absence of C4 protein in plasma and are present in the normal population at frequencies of 0.1–0.3 [17]. Complete deficiency of C4 is a rare event and is correlated with severe immune complex disease [18]. Cumulative results from more than 35 different studies revealed that heterozygous and homozygous deficiencies of C4A were present in 40–60% of SLE patients from almost all ethnic groups. These studies included Northern and Central Europeans, Anglo-Saxons, Caucasians in the US, African Americans, Asian Chinese, Koreans and Japanese [19], [20], [21], [22], [23], [24], [25], [26], [27]. In French SLE and control populations showed relatively low frequencies of C4AQ0, but the differences between the patient and control groups were statistically significant [28]. The major causes for C4AQ0 in Caucasian and African SLE patients are the presence of a mono-S RCCX module with a single, short C4B gene and the 2-bp insertion into the sequence for codon 1213 at exon 29 of the mutant C4A gene. These events are absent or extremely rare in the C4AQ0 of Oriental SLE patients. Studies of other racial group [29] suggest that the C4A null alleles are variables, independent of or in addition to the closely linked MHC class II genes in the expression of SLE. Moreover, studies in Spanish, Mexican, British, Australian Aborigine and Taiwan SLE patients had increased frequencies of C4B deficiency instead of C4A deficiency and mono-L C4B gene do exist in the Oriental populations [30], [31], [32], [33]. To date, the frequency of mono-L RCCX structures coding for C4B protein and the molecular basis leading to the non-expression of C4A in Oriental SLE patients has not been elucidated. Therefore, we investigated the association of complement component C4 null genes: C4AQ0 and C4BQ0 and their common mutations with SLE in Thai patients.

Section snippets

Genomic DNA extraction

Genomic DNA was extracted from the individual EDTA-blood leukocytes by standard phenol–chloroform technique from 118 SLE patients and 145 normal individuals. The SLE patients attended the out patient clinic at Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. All patients met at least 4 of the American College of Rheumatology (ACR) revised criteria for the classification of disease [34]. Their clinical manifestations were summarized in Table 1, Table 2. The controls included 145

Genotyping study of C4Q0 genes by touchdown PCR

In our study, genomic DNA of individual EDTA treated-blood leukocytes from 118 patients and 145 non-related healthy controls was genotyped by “touchdown” PCR for C4AQ0 and C4BQ0 using 4 primer pairs: Aup or Bup/L3 and Adown or Bdown/L4, which amplified the different fragments: 377 bp and 578 bp in non-C4 deficiency (Fig. 1). No PCR product from C4AQ0 and C4BQ0 were amplified by these primers. The results revealed that 3 of 118 SLE patients (2.54%) were C4AQ0 indicated by showing only fragments

Discussion

We suggest that both C4AQ0 and C4BQ0 be the risk factors of SLE in Thai population. We found surprisingly no homozygous genetic deficiency of both isotypes in healthy population. C4Q0 was not found in the 145 control samples which was different from most intensive studies in Europeans and American Caucasians that showed the C4AQ0 frequencies in 0.5–2.93% among different normal ethnic groups [31]. However, we confirmed the studies in Europeans, Anglo-Saxons, Caucasians, African Americans,

Acknowledgements

This work was supported by the Research Fund of “Genetic studies in transgenic animal models of SLE” (no. 02011854-0005) to Associate Professor Yindee Kitiyanant and the Postdoctoral Fellow from the Thailand Research Fund (no. TRG46-4680002) to Dr. Wannaporn Ittiprasert. The sponsors of the study had no role in the study design, data analysis or data interpretation. I would like to thank Professor John Mullins and Dr. Linda Mullins, Wellcome Trust Cardiovascular Research Initiatives, Edinburgh,

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