Analysis of fas ligand gene mutation in patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the possible association of a Fas ligand (FasL) gene mutation(s) or polymorphism(s) with systemic lupus erythematosus (SLE). METHODS: For amplification of the introns of the FasL gene, long polymerase chain reaction (PCR) using exon-based primers was utilized, followed by partial sequencing to construct exon-specific oligonucleotide primers for the analyses of FasL genomic DNA in SLE patients. Structural defects were studied by use of a composite analysis of reverse transcriptase-PCR/single-strand conformational polymorphism (SSCP) analysis of messenger RNA (mRNA) transcripts of the FasL gene in 35 SLE patients and PCR/SSCP analysis of FasL genomic DNA in 143 SLE patients. RESULTS: The sizes of the introns were approximately 0.6 kb for intron 1, 4.3 kb for intron 2, and 1.3 kb for intron 3. By SSCP analysis, we did not identify any mutations or polymorphisms in the FasL mRNA transcripts or in any of the 4 exons or areas of the introns adjacent to the exons. CONCLUSION: Using the same methods used in the present studies (PCR/SSCP), one group of investigators identified a structural defect of the FasL molecule in 1 of 75 SLE patients evaluated. Among the 143 SLE patients in the present study, however, we did not identify any mutations or polymorphisms of the FasL gene. Our results suggest that a FasL defect is not the major contributing factor in the pathogenesis of SLE.

An NcoI polymorphism in the human complement component 7 (C7) gene.

A novel polymorphic site has been found in the 3' untranslated region (UTR) of the human complement component 7 (C7) gene. The polymorphic site at 14-bp down-stream from the TAG stop codon was either C or A (Nco I-digested), with allele frequencies of 0.660 and 0.340. This NcoI polymorphism would be useful to perform a DNA marker haplotype study in patients with deficiencies of the complement genes, such as C6, C7, C9, which are located closely on chromosome 5p13.

Association of Fas/APO-1 gene polymorphism with systemic lupus erythematosus in Japanese.

OBJECTIVES: This study was undertaken to investigate the possible association of Fas gene mutation(s) or polymorphism(s) with systemic lupus erythematosus (SLE) in Japanese. METHODS: Screening for structural defects of the Fas gene was performed by using reverse transcriptase-polymerase chain reaction (RT-PCR)/single-strand conformation polymorphism (SSCP) analysis in 57 patients with SLE, followed by direct sequencing for the aberrantly migrating bands. The frequency of Fas polymorphism was determined by sequence-specific oligonucleotide probe (SSOP) hybridization in 82 SLE patients and 132 ethnically matched healthy individuals. RESULTS: We found a novel polymorphism at nucleotide 297 (T297C), which was linked to Fas polymorphism at nucleotide 416 (A416G). The 297C/416G genotype was present in four of the 132 (3.0%) healthy controls, none of whom was homozygous for the genotype. The allele frequency for 297C/416G in the controls was 1.5%. In contrast, 10 of the 82 (12.2%) SLE patients carried the 297C/416G allele, including one patient homozygous for the genotype. The allele frequency in SLE patients was 6.7%. The 297C/416G allele was significantly frequent in SLE patients (P = 0.01, chi2) with a relative risk of 5.00. CONCLUSION: As the polymorphism 297C/416G is silent at the amino acid level, it may affect the expression of Fas itself or be linked to a neighbouring genetic abnormality that is responsible for the pathogenesis of SLE.

Analysis of CD40 ligand gene mutations in patients with primary biliary cirrhosis.

An elevated immunoglobulin (Ig)M concentration in serum is a common and distinctive feature of primary biliary cirrhosis (PBC). Little is known, however, about the mechanism of hyper-IgM in PBC. CD40 ligand (CD40L) has a crucial role in immunoglobulin class switching in B cells. Mutations in the gene encoding CD40L are known to induce X-linked hyper-IgM syndrome. To identify mutations in the gene for CD40L in PBC patients, we analyzed CD40L gene mutations, using reverse transcription (RT)-PCR single-strand conformation polymorphism (SSCP) analysis. No mutations were detected in cDNA from any of 24 PBC patients by the RT-PCR-SSCP technique. These data suggest that other, unidentified mechanisms are involved in hyper-IgM in PBC patients.

Genetic basis of human complement C8 alpha-gamma deficiency.

Deficiency of the alpha-gamma subunit of the eighth component of complement (C8alpha-gammaD) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis. We here report the molecular basis of C8alpha-gammaD in two unrelated Japanese subjects. Screening all 11 exons of the C8alpha gene and all 7 exons of the C8gamma gene and their boundaries by exon-specific PCR/single-strand conformation polymorphism demonstrated aberrant single-stranded DNA fragments in exon 2 of C8alpha gene in case 1 and in exons 2 and 9 of C8alpha gene in case 2. Nucleotide sequencing of the amplified DNA fragments in case 1 revealed a homozygous single-point mutation at the second exon-intron boundary, inactivating the universally conserved 5' splice site consensus sequence of the second intron (IVS2+1G-->T). Case 2 was a compound heterozygote for the splice junction mutation, IVS2+1G-->T, and a nonsense mutation at Arg394 (R394X). R394X was caused by a C to T transition at nucleotide 1407, the first nucleotide of the codon CGA for Arg394, leading to a stop codon TGA. No mutations were detected in the C8gamma gene by our method. Our results indicate that the pathogenesis of C8alpha-gammaD might be caused by heterogeneous molecular defects in the C8alpha gene.

A non-sense mutation at Arg95 is predominant in complement 9 deficiency in Japanese.

Deficiency of the ninth component of complement (C9D) is one of the most common genetic abnormalities in Japan, with an incidence of one homozygote in 1000. Although C9D individuals are usually healthy, it has been shown that they have an significantly increased risk of developing meningococcal meningitis. In the present study we report the molecular bases for C9D in 10 unrelated Japanese subjects. As a screening step for mutations, exons 2 to 11 of the C9 gene were analyzed using exon-specific PCR/single-strand conformation polymorphism analysis, which demonstrated aberrantly migrating DNA bands in exon 4 in all the C9D subjects. Subsequent direct sequencing of exon 4 of the C9D subjects revealed that eight of the 10 C9D subjects were homozygous for a C to T transition at nucleotide 343, the first nucleotide of the codon CGA for Arg95, leading to a TGA stop codon (R95X). R95X is a novel mutation different from those recently identified in a Swiss family with C9D. Cases 6 and 7 were heterozygous for the R95X mutation. Family study in case 10 confirmed the genetic nature of the defect. In case 6, the second mutation for C9D of the C9 gene was identified to be the substitution of Cys to Tyr at amino acid residue 507 (C507Y), while the genetic defect(s) in the other allele in case 7 remains unknown. Our results indicate that a novel mutation, R95X, is present in most cases of C9D in Japan.

C5a induces tissue factor activity on endothelial cells.

Tissue factor (TF) is an integral membrane glycoprotein that serves as a cofactor for blood coagulation factor VIIa. The induction of TF on the surface of endothelial cells is initiated by various stimuli including lipopolysaccharide, interleukin-1 beta, and tumor necrosis factor alpha. We have demonstrated that recombinant human C5a induces TF activity in a dose-dependent fashion in human umbilical vein endothelial cells (HUVEC). Peak activity (4.9-fold increase) was obtained 3-6 h after treatment with 10 microM C5a. TF mRNA as assessed by RT-PCR method was also significantly increased (3.75-fold) after 3 h incubation with C5a, suggesting that C5a induces TF activity on HUVEC, at least in part, by enhancing the level of TF mRNA. The increase in TF activity by C5a was inhibited by methylprednisolone. The induction of TF on endothelial cells by C5a may represent one of many potential interrelationships between the inflammatory and coagulation schemes.

Genetic bases of human complement C7 deficiency.

Complement C7 deficiency (C7D) is associated frequently with recurrent bacterial infections, especially meningitis caused by Neisseria meningitidis. We report in this work the molecular bases of C7D in two unrelated Japanese males. We used exon-specific PCR/single-strand conformation polymorphism analysis as a screening step for mutations. Subsequent direct sequencing of the target exons identified homozygous mutations in exon 16 of case 1 and in exon 15 of case 2. The mutation of case 1 was a homozygous T to A transversion at nucleotide 2250, the third nucleotide of the codon TGT for Cys728, leading to a stop codon TGA (C728X). In case 2, a homozygous 2-bp deletion (2137delTG/2138delGT/2139delTG) caused a frameshift, generating a premature termination codon 4 to 6 nucleotides downstream. Family study in case 1 confirmed the genetic nature of the defect. Moreover, we detected a novel polymorphism in intron 11 that presumably is linked to the mutation responsible for C7D in case 1. Our results indicate that the pathogenesis of C7D is heterogeneous like most of the other deficiencies of complement components.

Production of the third and fourth component of complement (C3, C4) by smooth muscle cells.

Production of the third and fourth components of complement (C3, C4) by smooth muscle cells was investigated by using normal human aortic smooth muscle cells (AoSMC), human smooth muscle cell line (G402) and vascular smooth muscle cells obtained from human umbilical cord vein (UVSMC). AoSMC spontaneously produced both C3 and C4 at 15 ng/10(6) cells/72 hr and 22 ng/10(6) cells/72 hr, respectively, and both were enhanced by interferon-gamma (IFN-gamma). Although phorbol 12-myristate 13-acetate (PMA) and tumour necrosis factor-alpha (TNF-alpha) enhanced C3 production, C4 production was reduced by these agents. On the other hand, G402 produced C4 but not C3 in a dose-dependent manner when cultured with IFN-gamma. UVSMC produced only a small amount of C3 and C4 compared with AoSMC or G402. C3 and C4 produced by AoSMC were confirmed to be identical with their human serum counterparts as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and measurement of haemolytic activity. Northern blotting analysis showed that the expression of mRNA of C3 and C4 was enhanced by TNF-alpha and IFN-gamma, respectively, in AoSMC. Our findings suggest the importance of smooth muscle cells as a source of components of complement in vascular diseases including vasculitis.

Molecular bases for inherited human complement component C6 deficiency in two unrelated individuals.

Deficiency of the sixth component of complement (C6D) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis. We here report the molecular bases of C6D in two 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 (G1936) in exon 12 case 1 and a heterozygous single base deletion (C291/C292/C293/C294) in exon 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 the C6A allotype. These data indicate that at least three distinct mutational events can cause C6D, single nucleotide deletions in exons 2 and 12, and a mutation yet unidentified. Thus, similar to other complement protein deficiencies, the pathogenesis of C6D appears to be heterogeneous.

[Two cases of systemic lupus erythematosus associated with fatty liver and Basedow's disease].

We present two cases of systemic lupus erythematosus (SLE) associated with both Basedow's disease and fatty liver. The first case is a 46-year-old Japanese female who was admitted because of high fever and general fatigue. She had been diagnosed as having Basedow's disease and treated with thiamazole for over 4 years. Since thiamazole-induced lupus was unlikely because of high titer anti-nuclear antibody and anti-DNA antibody and low levels of complements, a diagnosis of SLE was made. The upper abdominal ultrasound study and the specimen obtained by liver biopsy performed before initiating steroid therapy demonstrated marked fatty liver. SLE itself is considered as an etiology of fatty liver in this case. The second case was a 25-year-old Japanese female with SLE. She had been treated with prednisolone for 13 years and was complicated with Basedow's disease 10 years later. Fatty liver was also demonstrated in this patient on ultrasonography, and was thought to be resulted from long-term steroid hormone administration.