High-voltage Agarose Gel Electrophoresis Research Articles

BF*F allotype of the alternative pathway of complement: A marker of protection against the development of antiphospholipid antibodies in patients with systemic lupus erythematosus.

B factor (BF) from the alternative complement pathway seems to participate in the pathophysiology of systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). To study the allotypic variability of BF in SLE and their associations with clinical and autoantibodies profile. BF allotypes were determined by high-voltage agarose gel electrophoresis, under constant cooling, followed by immunofixation with anti-human BF antibody, in 188 SLE patients and 103 controls. Clinical and serological data were obtained from medical examination and records. No significant differences of BF variants between patients and controls were found, neither in relation to epidemiologic or clinical manifestations. Associations of phenotype BF SS07 and allotype BF*S07 were found with anticardiolipin IgM (aCl-IgM) antibodies (p = 0.014 and p = 0.009 respectively), but not with aCl-IgG, lupus anticoagulant (LA), anti β2GPI or clinical APS. A significant decrease in BF*F allotype (p = 0.043) and BF SF phenotype (p = 0.018) was detected in patients with anti-phospholipid antibodies as a whole (aCl-IgG, aCl-IgM, LA and anti β2GPI). There is a link between phenotype BF SS07 and allotype BF*S07 with aCl-IgM in SLE patients; BF*F allotype could be considered a marker of protection against the development of antiphospholipid antibodies in these patients.

Association of complement factor B allotypes and serum biomarkers inrheumatoid arthritis patients and their relatives.

The aim of the study was to investigate the allotypic variability of complement factor B (BF) in patients and relatives with rheumatoid arthritis (RA) and its association with serological biomarkers and clinical features of the disease. BF allotypes were determined by high-voltage agarose gel electrophoresis in serum samples of 180 patients with RA, 198 relatives and 98 controls from Southern Brazil. Anticyclic citrullinated peptide (anti-CCP), antimutated citrullinated vimentin (anti-MCV) and IgA-rheumatoid factor (RF) were determined by ELISA and IgM-RF by latex agglutination in all samples. No significant differences were found in the allotypic variants of BF between patients with RA, relatives and controls, nor associations with gender and age of RA onset. BF*S07 allotype was significantly associated with extra-articular manifestations (EAMs; Secondary Sjögren Syndrome, pneumonitis, rheumatoid nodules) in patients with RA (P=0.02; OR=6.62). Patients with phenotype BF F had lower positivity for anti-MCV biomarker (P=0.02; OR=0.22) and those with allotype BF*S had higher prevalence of this autoantibody (P=0.02; OR=3.77). An increased frequency of RF-IgA was detected in relatives of patients with RA with BF FS07 phenotype (P=0.02; OR=7.78). Complement BF variability did not influence the development of RA in the studied patients, but BF variants may act as markers of disease prognosis, such as development of EAMs, corroborating with the role of the alternative pathway in the pathogenesis of RA.

Complement factor B allotypes in the susceptibility and severity of coeliac disease in patients and relatives

The alternative pathway of complement plays an important role in the pathogenesis of coeliac disease (CD), where factor B (BF) is central to its activation. CD is a gluten-sensitive enteropathy that results from a complex interplay between genetic, immunologic, and environmental factors. In this study we evaluated the association of BF allotypes with the susceptibility and severity of CD, and with the presence of autoantibodies. Seventy-six non-related patients (56 female; 20 male; 2-77 years) and 150 first-degree relatives (87 female, 63 male; 2-75 years) were investigated. As controls, 97 healthy individuals were included (67 female;, 30 male; 1-71 years). The BF allotypes were determined by high-voltage agarose gel electrophoresis, followed by specific immunofixation. Disease severity was evaluated by anti-endomisial antibody (IgA-EmA) titres and histological findings of intestinal mucosa, which showed a high correlation (r = 0.8; P < 0.00001) in samples collected simultaneously. IgA-EmA was detected in all CD patients ingesting gluten, and in 13.3% of the relatives. The IgA-EmA, smooth muscle, mitochondrial, liver-kidney microsomal, nuclear, gastric parietal cells, and thyroid microsome antibodies were tested by indirect immunofluorescence. A significant decrease in BF S (P = 0.026) and an increasing tendency in BF SF allotype (P = 0.06) were observed in CD patients when compared to their relatives. On the other hand, BF S frequency was increased (P = 0.001 RR = 2.32) and BF SF (P = 0.002) decreased in the relatives when compared to the controls. No differences were observed in the distribution of BF phenotypes amongst the CD patients and the control group, and no association was found with CD severity or with the presence of autoantibodies. These results suggest BF SF as a CD susceptibility marker, and BF S as a protection marker of the disease amongst CD families in the Brazilian population.

Genetic polymorphism of the fourth component of human complement: population study and proposal for a revised nomenclature based on genomic PCR typing of Rodgers and Chido determinants.

The fourth component of human complement (C4) is coded for by two homologous genes, C4A and C4B, located in the class III region of the major histocompatibility complex (MHC). Genetic typing of C4A and B alleles is routinely carried out by high-voltage agarose gel electrophoresis. The electrophoretic C4 polymorphism can be further subdivided by the Rodgers (Rg) and Chido (Ch) blood groups, which are antigenic determinants of the C4A and B alpha-chains, respectively. We have used a recently described direct PCR typing method using sequence-specific primers (PCR-SSP) in combination with electrophoretic C4 typing as well as genomic RFLP analysis to determine the frequency of C4 allotypes, Rg/Ch subtypes and C4A-B haplotypes in a family study of the German population. As the current C4 allele designation does not provide any information about the presence or absence of Rodgers and Chido antigens, we have developed an extension to the existing C4 nomenclature. This revised allele designation combines the existing numerical allotypes defined by electrophoretic mobility with eight subtypes (01-08) based on Rg/Ch PCR genotyping results. Using this approach, most electrophoretic allotypes could be subdivided. Among the C4A allotypes, the most common allele was A*0301 (59.9%), and the most common subtype among all electrophoretic allotypes was 01 (85.1%; = Rg1,2-positive, Ch-negative). For C4B, the most common allele was B*0101 (64.3%), and the most common subtype was 01 (79.6%; = Ch1,2,3,4,5,6-positive, Rg-negative). The subtypes 03, 04, 07 and 08 of the C4A allotypes, and the subtypes 03, 07 and 08 of the C4B allotypes, were not detected in this study. The analysis of duplicated C4 alleles revealed considerable heterogeneity of their subtypes. The results demonstrate that all known C4 allotypes can now be assigned unambiguously, which facilitates the identification of MHC haplotypes relevant for transplantation and disease association studies.

No association found between C3 alleles and scar hypertrophy

Serum C3 was examined for structural variants using high-voltage agarose gel electrophoresis at both pH 6.2 and 8.4, thermal stability and stability to attack by hydroxyl radicals, in 450 Chinese people in Hong Kong. The C3 allele frequencies for electrophoretic variants were found to be 0.9911 for C3 ∗S and 0.0089 for all other rare alleles. No variants were found in terms of stability at 54°C in the absence of Ca 2+ or at 62°C in the presence of Ca 2+, and in terms of stability to attack by hydroxyl radicals. Based on a recently proposed single gene hypothesis for scar hypertrophy, statistical analyses were carried out to compare the relevant frequency data of scar hypertrophy with C3 phenotype frequencies, both for the local Chinese population and a European Caucasian population. There is no evidence for any association between C3 alleles or phenotypes and the formation of hypertrophie scars.

Identification of the Heterozygotes for Deficiency of the β-Subunit of the Eighth Component of Complement by Reduced Levels of C8β and Increased Amounts of Free C8α-γ

Sera from obligate heterozygotes for deficiency of the C8 beta subunit of the eighth component of human complement (C8) were analyzed for the molecular composition of C8. The C8 alpha-gamma and C8 beta subunits were separated by SDS-PAGE, visualized by immunoblotting, and the resulting bands were quantitated by laser densitometry. The laser densitometric absorption data were set to 100 arbitrary units (AU) for both subunits of pooled normal human sera. The AU values of individual normal sera ranged from 45 to 150 AU for C8 alpha-gamma (median 99 AU) and from 45 to 140 AU for C8 beta (median 101), whereas the C8 alpha-gamma/C8 beta-ratio varied from 0.7 to 1.4. Sera from C8 beta-deficient heterozygotes differed, as expected, from the normal sera for the markedly reduced levels of C8 beta (20 to 90 AU, median 55 AU) and for the higher C8 alpha-gamma/C8 beta-ratio (1.3 to 3.5). High voltage agarose gel electrophoresis was used to separate free and C8 beta-bound C8 alpha-gamma. The migration of free and C8 beta-bound C8 alpha-gamma subunit was checked by hemolytic overlay gels and by second dimension SDS-PAGE and immunoblotting. Immunochemical evaluation of C8 alpha-gamma using this system revealed about 5-14% free C8 alpha-gamma in sera with normal C8 and higher levels, from 33-71%, in the C8 beta D heterozygous sera. Functional analysis confirmed the substantial increase of free C8 alpha-gamma in the heterozygous group. We conclude that the C8 in C8 beta D heterozygous sera is characterized by increased amounts of free C8 alpha-gamma due to reduced concentrations of the C8 beta subunit. This finding may help to identify individuals heterozygous for C8 beta deficiency.

A very slow BF variant (S085) detected in a Japanese population.

Using high-voltage agarose gel electrophoresis and immunofixation a very slow BF variant was detected in a Japanese person living in Yamanashi district. The family analysis suggested the hereditary occurrence of a new allele, BF*S085.

Allelic variants for complement factors C3, C4, and B in acute necrotizing ulcerative gingivitis.

Impaired immune defense mechanisms and genetic factors appear to play a role in susceptibility to acute necrotizing ulcerative gingivitis (ANUG). Therefore, possible etiological mechanisms might involve genes at the Major Histocompatibility Complex, which include the complement factor loci. We have tested for a possible association between certain complement factor alleles and ANUG using a case-control study design. Specific alleles at complement factors C3 and C4, and properdin factor B (Bf) loci were determined indirectly by high voltage agarose gel electrophoresis in 58 subjects with a history of ANUG and in 58 age-sex-matched healthy controls. The highest relative risk of ANUG, as obtained by conditional logistic regression, for alleles at the C3 locus was 1.9 (90% confidence limits 0.8 to 4.8; p = 0.229) for C3*F-positive individuals. The highest relative risk for alleles at the C4 locus was 2.6 (0.5 to 14.9; p = 0.358) for C4A*3-positive individuals. There was no evidence for an association between Bf allotype and risk of ANUG, with a relative risk of 1.2 for Bf*F- and relative risk of 1.0 for B*S-positive individuals. None of our estimates was statistically significant. We conclude, therefore, that it is unlikely that there is any association between complement factor gene haplotype and susceptibility to ANUG.

Genetic polymorphism of properdin factor B (BF) in a Chinese population: existence of two rare variants.

Genetic polymorphism of properdin factor B (BF) was investigated in sera from 259 healthy individuals living in Guangzhou, southern China, by means of high voltage agarose gel electrophoresis followed by immunofixation. Besides the common phenotypes, SS, FS, FF and SS07, two rare heterozygous phenotypes tentatively named as SSG1 and SFG2 were observed. The allele frequencies estimated forBF*S, BF*F, BF*S07, BF*SG1 andBF*FG2 were 0.8668, 0.1197, 0.0077, 0.0019 and 0.0039, respectively. Family data indicated an autosomal, codominant inheritance for theBF*FG2 allele.

Genetic polymorphism of properdin factor B (BF) in Chinese patients with two types of diabetes mellitus.

The distribution of phenotypes for properdin factor B (BF) has been investigated in 50 IDDM patients and 52 NIDDM patients living in Guangzhou, southern China, using high voltage agarose gel electrophoresis followed by immunofixation. A significant decrease in the frequency of BF F positive patients (p<0.01) and an increase in the gene frequency ofBF*S (p<0.005) were found in IDDM as compared to 259 normal controls. The relative risk for the BF F positive patients in IDDM was 0.15. On the other hand, no significant association was observed between BF types and NIDDM.

Placental alkaline phosphatase types and subtypes determined by agarose gel electrophoresis and separator isoelectric focusing.

Two techniques for phenotyping the human placental alkaline phosphatase system were developed: high-voltage agarose-gel electrophoresis and thin-layer separator isoelectric focusing on agarose. These methods enabled a more rapid and sensitive phenotyping of all common phenotypes than the traditionally employed starch-gel electrophoresis. An extended polymorphism of placental alkaline phosphatase was revealed by isoelectric focusing. The existence of two suballeles of Pl1 allele and two suballeles of Pl2 allele was postulated.

C3 variants in Japanese.

By high-voltage agarose gel electrophoresis, seven phenotypes of C3 were found in Japanese. The allele frequencies for C3*S, C3*S025, C3*S02, C3*F, C3*F06, C3*F065, and C3*F08 were 0.9943, 0.0003, 0.0003, 0.0006, 0.0003, 0.0021, and 0.0021, respectively. CH50, C3/C3c protein concentrations, and C3 hemolytic activities in fresh sera with variant C3 phenotypes were within the normal ranges.

C3 polymorphism in patients with chronic uremia.

The C3 polymorphism phenotypes were determined by high-voltage agarose gel electrophoresis in 83 patients with chronic uremia on maintenance hemodialysis. In 45 patients with chronic pyelonephritis, the frequency of C3F and C3FS was significantly lower than in 38 patients with chronic glomerulonephritis and in a large control group.

GLO Polymorphism in Iceland

The phenotypes of red cell glyoxalase I (GLO) were determined in two Icelandic population samples using starch-gel electrophoresis and high-voltage agarose-gel electrophoresis. The gene frequencies of 178 individuals were 0.46 for GLO1 and 0.54 for GLO2. In a group of Icelandic insulin-dependent diabetics the gene frequencies were found to be very similar. The evaluation of 30 mother-child pairs is also shown.

The C4 system: formal and population genetics.

The present study shows that the C4 system as investigated by high voltage agarose gel electrophoresis is highly polymorphic. In a series of unrelated Norwegian adults, where C4 types have been ascertained through segregation in families, six different haplotypes have been found to occur with a frequency exceeding 1%. The genotype frequencies in the population fit expected Hardy-Weinberg distribution. In family material comprising 89 matings with 327 children the distribution of offspring is as expected according to autosomal codominant inheritance of haplotypes.

The Structural C6 Polymorphism: Nonlinkage Between C6 and Chromosome 6 Marker Loci

Abstract The complement factor 6 polymorphism has been examined in a Norwegian family material by two techniques: either high voltage agarose gel electrophoresis or isoelectric focusing in polyacrylamide gel slabs. Proteins with C6 activity were visualized by a specific, hemolytic assay. Agarose electrophoresis gave good separation of common allele products and easy discrimination between the common C6 types: C6A, C6AB, and C6B. Prolonged isoelectrofocusing gave better band separation, but the discrimination between the common C6 types is not noticeably better than with the agarose technique. For detection of rare alleles isoelectrofocusing is presumably the method of choice. In a Norwegian population sample, C6 gene frequencies were: C6A: 0.61 and C6B: 0.39. The data on the linkage relationship between the HLA region loci and C6 show that C6 complement locus is not within measurable distance to the HLA region. Linkage studies also indicate nonlinkage between C6 and another chromosome 6 marker locus, PGM3. C6 has therefore been excluded from a substantial portion of the short arm of chromosome 6.

Investigations on the polymorphism of glyoxalase I (EC 4.4.1.5) in the population of Hessen, Germany

The phenotypes of glyoxalase I (GLO) were determined in a random population from Hessen (Germany) by high-voltage agarose gel electrophoresis. The gene frequencies in 1150 unrelated individuals were 0.4391 for GLO1 and 0.5609 fro GLO2. Rare phenotypes were not observed. The segregation of phenotypes in 50 families and 32 mother-child combinations supports the assumed autosomal codominant inheritance. The possibility of a simultaneous typing for GLO, esterase D (EsD), and carbonic anhydrase2 (CA2) on one gel is discussed.

Nonlinkage between C6 and chromosome 6 markers.

C6 typing was performed in a family material by two different techniques: serum or plasma samples were subjected either to high-voltage agarose gel electrophoresis or to isoelectric focusing in polyacrylamide gel slabs. Proteins with C6 activity were then visualized by a specific, hemolytic assay. In 81 unrelated adults within the family material the following allele frequencies were found: C6A:0.61 and C6B:0.39. Linkage studies exclude linkage between C6 and HLA region marker loci, and also between C6 and another chromosome 6 marker locus PGM3.

The distribution of the third complement component and other polymorphic traits in rheumatoid arthritis.

The distribution of C'3 phenotypes in 61 seropositive rheumatoid arthritis patients and 464 normal blood donors in Tokyo was examined by high-voltage agarose gel electrophoresis. The allele of the common European variant, C3(F), is absent in Japanese, but another F allels, C3(Frare), is present with a frequency of 0.025 and 0.013 in RA patients and blood donors, respectively; the difference is not statistically significant. The distribution of phenotypes for several other enzyme systems was also determined in an attempt to detect association with RA Again, no statistically significant differences were found.

Transferrin variants designated by their relative mobilities in high-voltage agarose gel electrophoresis.

A unified nomenclature for the designation of transferrin variants, based on their relative mobilities in a standardized high-voltage agarose gel electrophoresis, is proposed. This is aimed at improving terminology of Tf variant typing results with reference to the relatively common variant Tf B2.