PCR-RFLP Genotyping of C1q Mutations and Single Nucleotide Polymorphisms in Malaysian Patients with Systemic Lupus Erythematosus

Abstract

Five types of known mutations within the C1q gene [located at C1qA-Gln186 (C > T), C1qB-Gly15 (G > A), C1qB-Arg150 (C > T), C1qC-Gly6 (G > A), and C1qC-Arg41 (C > T)] and two SNPs located at C1qA-Gly70 (G/A) and C1qC-Pro14 (T/C) were screened in a multiracial Malaysian population. One hundred thirty patients with systemic lupus erythematosus (SLE) and 130 matched healthy control subjects were genotyped using PCR-RFLP methods. We found no occurrence of the five types of mutations in either the homozygous or heterozygous form among the 260 samples studied. Statistical analysis also revealed that there were no significant associations observed in the genotype distributions and allele frequencies among the patients with SLE and healthy control subjects with both C1qA-Gly70 (G/A) and C1qC-Pro14 (T/C) SNPs. Overall, C1q deficiency was not proven as a primary causative genetic predisposition factor for SLE in the Malaysian population.

KEY WORDS: C1Q DEFICIENCY, C1Q GENE, MALAYSIAN POPULATION, MUTATION, PCR-RFLP METHOD, SINGLE NUCLEOTIDE POLYMORPHISM (SNP), SYSTEMIC LUPUS ERYTHEMATOSUS (SLE).

Systemic lupus erythematosus (SLE) is a prototypic, systemic, chronic inflammatory multisystem disease of unknown pathoetiology (Woods 1993; Kotzin 1996). SLE susceptibility is likely to be multifactorial as a result of the combined effects of genetic, environmental, and hormonal factors (Mok and Lau 2003).

Homozygous hereditary deficiency of each of the classical pathway components (C1q, C1r, C1s, C4, C2) is greatly associated with increased severity and susceptibility to SLE (Pickering et al. 2000). Individuals who have a congenital genetic deficiency of the C1q gene showed more than 90% prevalence in development of SLE-like symptoms (Walport et al. 1998; Pickering et al. 2000). C1q deficiency may cause SLE by failing to maintain self-tolerance and impairment in the physiological clearance of dying cells and immune complexes (Walport et al. 1998; Carroll 2004; Manderson et al. 2004; Sontheimer et al. 2005).

C1q is a macromolecular complex of 18 polypeptide chains, composed of six A-, six B-, and six C-polypeptide chains (Sellar et al. 1991). The three chains are 223, 226, and 217 residues long, respectively, and are encoded by three individual genes, C1qA (2.5 kb), C1qB (2.6 kb), and C1qC (3.8 kb) (Sellar et al. 1991; Slingsby et al. 1996). Each gene contains two exons separated by one intron. The C1q gene has been mapped to chromosome 1p34.1-36.3 (Sellar et al. 1991, 1992).

C1q plays versatile roles in immunological processes. A defect in any one of the C1qA, C1qB, and C1qC genes may lead to failure in the synthesis of intact C1q molecules. Today, the molecular basis for hereditary C1q deficiencies is well-defined single-base mutations leading to termination codons, frame shifts, or amino acid exchanges. Currently, three types of nonsense mutations, located at C1qA-Gln186 (C > T) (Petty et al., 1995, 1997a, 1998; Topaloglu et al. 1996; Berkel et al., 2000), C1qB-Arg150 (C > T) (McAdam et al. 1988), and C1qC-Arg41 (C > T) (Slingsby et al. 1996), and two types of missense mutations, located at C1qB-Gly15 (G > A) (Petty et al. 1997b) and C1qC-Gly6 (G > A) (Petry et al. 1995; Slingsby et al. 1996; Suwairi et al. 1997), have been identified in several families of different ethnic backgrounds from various geographic regions.

Recently, the association of the C1qA-Gly70 (G/A) and the C1qC-Pro14 (T/C) ttanslationally silent exonic single nucleotide polymorphisms (SNPs) with predisposition to SLE has been reported (Racila et al. 2003; Petry and Loos 2005). The homozygous C1qA-Gly70 GGA SNP showed a significant association with decreased levels of C1q (Racila et al. 2003). However, the effect of the CqC-Pro14 SNP remains unknown.

In Malaysia the incidence of SLE is estimated to be 43 per 100,000 (Wang et al. 1997). In this study we screened Malaysian samples for possible C1q deficiencies caused by mutations and SNPs.

Materials and Methods

Study Subjects. One hundred thirty blood samples were obtained from patients with SLE attending the Renal Unit of the University of Malaya Medical Center (UMMC), Kuala Lumpur, Malaysia. All patients had fulfilled at least 4 of 11 of the American College of Rheumatology classification criteria for SLE (Tan et al. 1982; Hochberg 1997). In general, the clinical manifestations found in these patients were malar rash, renal disorder with proteinuria (>0.5 g/day), arthritis, and photosensitivity. Besides the patient samples, another 130 blood samples from healthy individuals admitted to the same medical center served as control subjects for this study. Whole blood samples were collected in tubes containing EDTA and stored at -70°C until DNA extraction.

This study was approved by the University of Malaya Ethics Committee (Ethics Approval 380.6), and informed consent was obtained from all subjects.

DNA Extraction. Genomic DNA was extracted from whole blood using a standard proteinase K treatment followed by a phenol:chloroform method (Sambrook and Russell 2001).