Letteratura scientifica selezionata sul tema "C4 protein deficiency"

Abstract The complement protein C4, encoded by two genes (C4A and C4B) on chromosome 6p, is the most polymorphic among the MHC III gene products. We investigated the molecular basis of C4 deficiency in a Finnish woman with systemic lupus erythematosus. C4-specific mRNA was present at low concentrations in C4-deficient (C4D) patient fibroblasts, but no pro-C4 protein was detected. This defect in C4 expression was specific in that synthesis of two other complement proteins was normal. Analysis of genomic DNA showed that the proposita had both deleted and nonexpressed C4 genes. Each of her nonexpressed genes, a C4A null gene inherited from the mother, a C4A null gene, and a C4B null gene inherited from the father, all contained an identical 2-bp insertion (TC) after nucleotide 5880 in exon 29, providing the first confirmatory proof of the C4B pseudogene. This mutation has been previously found only in C4A null genes. Although the exon 29/30 junction is spliced accurately, this frameshift mutation generates a premature stop at codon 3 in exon 30. These truncated C4A and C4B gene products were confirmed through RT-PCR and sequence analysis. Among the possible genetic mechanisms that produce identical mutations in both genes, the most likely is a mutation in C4A followed by a gene conversion to generate the mutated C4B allele.

Abstract Human complement C4 is complex with multiple layers of diversity. This study aims to elucidate the copy-number variations (CNVs) of C4A and C4B in disease risk of SLE, and compare the basis of race-specific C4A-deficiency in East-Asians (EA) and Europeans. Our study-populations included (a) 999 EA-SLE patients and 1,347 healthy subjects; and (b) 232 European SLE patients and 500 healthy subjects. Variations in gene copy-numbers (GCNs) for total C4, C4A, C4B, long and short genes were determined and validated by independent genotyping technologies. Genomic regions with C4B96 were investigated to determine the basis of the most basic C4B protein that is concurrent with C4A-deficiency. In EA, strong protective effects for high GCNs of total C4 and C4A against SLE were notable; low and medium GCNs of total C4 and C4A, and the absence of short genes were risk factors for SLE. Homozygous C4A-deficiency was infrequent but had an odds-ratio (OR) of 12.4 (p=0.0015) in SLE. Low serum complement levels were strongly associated with low GCNs of total C4 (OR=3.27, p=7.0×10−7) and C4B (OR=2.55, p=2.5×10−5). Patients with low complement had high frequencies of anti-dsDNA (OR=4.96, p=9.7×10−17), hemolytic anemia (OR=3.89, p=3.6×10−10) and renal disease (OR=2.18, p=8.5×10−6). The monomodular-short haplotype with C4A-deficiency and in linkage-disequilibrium with HLA-DRB1*0301 prevalent in Europeans was scarce in EA. Instead, most EA-subjects with C4A-deficiency shared a recombinant haplotype with bimodular-LS encoding C4B1 and C4B96, which was linked to HLA-DRB1*1501. DNA-sequencing revealed the E920K polymorphism for C4B96. In conclusion, C4 CNVs and C4A-deficiency are important in the risk and manifestations of East-Asian and European SLE.

ObjectiveSystemic lupus erythematosus (SLE) features high frequency of cardiovascular disease (CVD) and fluctuating complement levels. The clinical trial Atherosclerosis Prevention in Pediatric Lupus Erythematosus (APPLE) aimed to evaluate whether atorvastatin treatment reduced the progression of atherosclerosis in 221 patients with childhood-onset SLE (cSLE), using carotid intima media thickness (CIMT) as surrogates. We leveraged APPLE biorepository and trial data to investigate the relationship between complement and CVD in cSLE.MethodsGene copy numbers (GCNs) for total C4, C4A and C4B were measured by TaqMan-based real-time PCR and Southern blotting, and analysed with laboratory and clinical parameters through Student’s t-test and χ2 analyses. Effects of total C4, C4A and C4B GCNs on the response to placebo or atorvastatin treatment and progression of CIMT were examined by regression analyses.ResultsAt baseline, C4 protein levels strongly correlated with GCNs of total C4 (p=1.8×10−6). Each copy of C4 gene increased mean serum C4 by 3.28 mg/dL. Compared with those without hypertension (N=142), individuals with hypertension demonstrated significantly elevated serum levels for C4 and C3 at baseline and serially (C4: P=5.0×10−25; C3: P=5.84×10−20). Individuals with ≥2 C4B genes had 2.5 times the odds of having hypertension (p=0.016) and higher diastolic blood pressure (p=0.015) compared with those with C4B deficiency. At the study end, subjects with ≥2 C4B and atorvastatin treatment had significantly slower increase in CIMT compared with those treated with placebo (p=0.018).ConclusionscSLE with hypertension had elevated serum levels of C4 and C3 and higher GCN of C4B; cSLE with ≥2 C4B genes would benefit from statins therapy to prevent atherosclerosis.

Abstract Mannose-binding lectin (MBL) is a serum protein that has been demonstrated to activate the classical complement pathway and to function directly as an opsonin. Although MBL deficiency is associated with a common opsonic defect and a predisposition to infection, the role of the protein in bacterial infection remains unclear. We have investigated MBL binding to Neisseria meningitidis serogroup B1940 and three isogenic mutants, and the subsequent activation of the two major isoforms of C4 (C4A and C4B) by an associated serine protease, MASP. The mutants lacked expression of the capsular polysaccharide (siaD−), the lipo-oligosaccharide (LOS) outer core that prevented LOS sialylation (cpsD−), or both capsule and LOS outer core (cps−). Using flow cytometry, it was possible to detect strong MBL binding to the cps− and cpsD− mutants over a wide range of concentrations. In contrast, minimal or no MBL binding was detected on the parent organism, with binding to siaD− only at higher MBL concentrations. C4 was activated and bound by mutants that had previously bound MBL/MASP, but there was no significant difference in the amounts of C4A and C4B bound. When sialic acid residues were removed from the parent organism by neuraminidase treatment, the binding of both MBL and C4 increased significantly. Our results suggest that MBL may bind to and activate complement on these encapsulated organisms, and the major determinants of these effects are the LOS structure and sialylation.

Abstract Medium- and short-chain L-3-hydroxyacyl-coenzyme A dehydrogenase (M/SCHAD, SCHAD) deficiency is a mitochondrial fatty acid oxidation disorder (FAOD). This enzyme catalyzes the penultimate step in fatty acid oxidation, the NAD+ dependent conversion of L-3-hydroxyacyl-CoA to 3-ketoacyl-CoA for medium- and short-chain acyl-CoA intermediates (C4-C12). The clinical presentations of most patients are recurrent hypoglycemia associated with hyperinsulinism. We presented four infants with C4 acyl-carnitine elevation identified by newborn screening that also showed an unusual phenotype of congenital hypotonia and gross developmental delay. Enzymatic studies confirmed the disease. Sequencing analysis of all the HADH coding exons on the four patients revealed a homozygous variant of a novel change (c.908G>T, p.Gly303Val). Western blot analysis subsequently confirmed the lack of the SCHAD protein. In addition, there is another previously reported benign variant (c.257T>C) identified in three infants. Therefore, we postulate that the HADH variant (c.908G>T) is indeed pathogenic and associated with a severe phenotype as evidenced by the cases described herein. Population screening for the c.908G>T mutation suggests this mutation to be common among Puerto Ricans. We recommend that SCHAD deficiency is included as part of the differential diagnosis of all infants with congenital hypotonia.

Protein kinases C (PKCs) are a family of serine/threonine kinases that are critical for signal transduction pathways involved in growth, differentiation and cell death. All PKC isoforms have four conserved domains, C1–C4. The C1 domain contains cysteine-rich finger-like motifs, which bind two zinc atoms. The zinc-finger motifs modulate diacylglycerol binding; thus, intracellular zinc concentrations could influence the activity and localization of PKC family members. 3T3 cells were cultured in zinc-deficient or zinc-supplemented medium for up to 32 h. Cells cultured in zinc-deficient medium had decreased zinc content, lowered cytosolic classical PKC activity, increased caspase-3 processing and activity, and reduced cell number. Zinc-deficient cytosols had decreased activity and expression levels of PKC-α, whereas PKC-α phosphorylation was not altered. Inhibition of PKC-α with Gö6976 had no effect on cell number in the zinc-deficient group. Proteolysis of the novel PKC family member, PKC-δ, to its 40-kDa catalytic fragment occurred in cells cultured in the zinc-deficient medium. Occurrence of the PKC-δ fragment in mitochondria was co-incident with caspase-3 activation. Addition of the PKC-δ inhibitor, rottlerin, or zinc to deficient medium reduced or eliminated proteolysis of PKC-δ, activated caspase-3 and restored cell number. Inhibition of caspase-3 processing by Z-DQMD-FMK (Z-Asp-Gln-Met-Asp-fluoromethylketone) did not restore cell number in the zinc-deficient group, but resulted in processing of full-length PKC-δ to a 56-kDa fragment. These results support the concept that intracellular zinc concentrations influence PKC activity and processing, and that zinc-deficiency-induced apoptosis occurs in part through PKC-dependent pathways.

A deeper understanding of the mechanism of complement activation may help to elucidate the pathogenesis of IgA nephropathy (IgAN). Traditionally, the activation of an alternative pathway (AP) has been recognized as an enhancer mechanism of glomerular damage. This paper documents contemporary information concerning the possible pathological mechanisms of the lectin pathway (LP) in the circulation and in the glomerulus. The circulating initiator of LP activation is not fully understood. However, ligands for mannose-binding lectin (MBL) which are among the starter molecules of the LP are aberrant glycosylated molecules-containing immune complex. Recent reports have focused onN-glycans on secretory IgA as a candidate ligand. Mesangial deposits of MBL are seen in 25% of patients with IgAN. Mesangial deposits of MBL and C4 and/or C4 breakdown products are implicated as markers for disease progression of IgAN. On the other hand, patients with MBL deficiency tend to show better clinical presentation and lower levels of urinary protein and serum creatinine than MBL-sufficient patients. It is now recognized that involvement of AP and LP constitutes an additional mechanism for explaining the progression of IgAN.

Because tandem mass spectrometry- (MS/MS-) based newborn screening identifies many suspicious cases of fatty acid oxidation and carnitine cycle disorders, a simple, noninvasive test is required to confirm the diagnosis. We have developed a novel method to evaluate the metabolic defects in peripheral blood mononuclear cells loaded with deuterium-labeled fatty acids directly using the ratios of acylcarnitines determined by flow injection MS/MS. We have identified diagnostic indices for the disorders as follows: decreased ratios of d27-C14-acylcarnitine/d31-C16-acylcarnitine and d23-C12-acylcarnitine/d31-C16-acylcarnitine for carnitine palmitoyltransferase-II (CPT-II) deficiency, decreased ratios of d23-C12-acylcarnitine/d27-C14-acylcarnitine for very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, and increased ratios of d29-C16-OH-acylcarnitine/d31-C16-acylcarnitine for trifunctional protein (TFP) deficiency, together with increased ratios of d7-C4-acylcarnitine/d31-C16-acylcarnitine for carnitine palmitoyltransferase-I deficiency. The decreased ratios of d1-acetylcarnitine/d31-C16-acylcarnitine could be indicative of β-oxidation ability in patients with CPT-II, VLCAD, and TFP deficiencies. Overall, our data showed that the present method was valuable for establishing a rapid diagnosis of fatty acid oxidation disorders and carnitine cycle disorders and for complementing gene analysis because our diagnostic indices may overcome the weaknesses of conventional enzyme activity measurements using fibroblasts or mononuclear cells with assumedly uncertain viability.

This report describes the clinical, biochemical, and pathological findings in three infants with hepatic short-chain l-3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) deficiency, a recently recognized disorder of the mitochondrial oxidation of straight-chain fatty acids. Candidate subjects were identified from an ongoing study of infant deaths. SCHAD analysis was performed on previously frozen liver and skeletal muscle on subjects with a characteristic urine organic acid profile. Autopsy findings were correlated with the biochemical abnormalities. Enzyme analysis in liver revealed marked deficiency in SCHAD with residual activities of 3–11%. All subjects had normal activity in skeletal muscle. However, Western blot analysis of SCHAD revealed an identical truncated protein in both liver and muscle from one patient, suggesting that SCHAD is similar in liver and muscle and that the normal activity in muscle may be due to other enzymes with C4 activity. Autopsy findings revealed marked steatosis and a muscle pattern consistent with spinal muscular atrophy in one patient. Lipid storage was less pronounced in one patient and not detected in the third patient who had a well-documented history of recurrent hypoglycemia. This is the initial pathological characterization of this enzyme defect, and our observations suggest that SCHAD deficiency is a very severe disorder contributing to early infant death.

Since the initial discovery of the association between C4 protein deficiency and autoimmune disease, much effort has been devoted to the characterisation of C4 protein and its molecular composition (Dawkins et al., 1983). From the outset, researchers have found the C4 protein difficult to work with as it is unstable and degrades rapidly. However, with the advent of molecular techniques, studies of the C4 gene have accelerated, uncovering a very complicated and large duplicated gene region. This thesis reviews the literature on complement component C4, and reports on three interrelated research areas involving protein and DNA analysis of complement C4. Contributions to the molecular characterisation of the C4 complement gene region and population genetics are discussed in relation to current knowledge, and direction for further research is provided. This study set out to establish whether indigenous people of PNG and Aboriginal Australians have shared a common gene pool. By analysing the distribution of polymorphic C4 alleles these studies found the Aboriginal Australian and PNG populations to be distinctly different. Analysis of the distribution of C4 alleles in the two ethnic groups indicated that the two Aboriginal populations showed significant similarities but the three geographically distinct PNG populations were significantly different. Studies here suggested these ethnic populations are more reliably distinguished from each other by differences in the frequencies of the most frequently occurring C4 alleles, than by the incidence of rare alleles. The high frequency of C4 null alleles in northern located Darwin Aboriginal's reported by Ranford et al. (1987), is not an isolated incident as this study also found the highest frequencies of C4 null alleles in northern located Aboriginal Australian's compared to any ethnic group studied. This could be associated with the high occurrence of the autoimmune disease Systemic Lupus Erythematosus (SLE) in Aboriginal Australians, and strong susceptibility of Aboriginal Australians to this disease (Anstey et al., 1993). Accurate identification of C4 alleles including C4 null alleles is essential both for investigating gene-linked disease associations, as well as for advancing future population genetics studies. Studies here demonstrate that C4 protein allotyping does not accurately identify the C4 genotype of an individual. This was highlighted by the lack of Hardy-Weinberg equilibrium with respect to C4 allele frequencies of the East Cape York Aboriginal population as well as the identification of a number of alleles by molecular analysis which could not be identified by protein typing. The difficulties associated with determining C4 allotypes by C4 protein gel electrophoresis include instability of the C4 protein, high degree of technical expertise for interpretation of allotypes, and limitations of this technique in distinguishing overlapping alleles. It was established in these studies that the polymorphic C4d region of C4 genes may be used by molecular means to identify and type C4 alleles. With the advent of molecular biological techniques such as protein and DNA sequencing, and RFLP analysis of the C4 gene region, information required for development of a molecular-based typing protocol has become available. Some of the previously described C4A and C4B alleles have been found to have unique combinations of polymorphic amino acid residues distributed within a short stretch of DNA, otherwise known as the C4d region. This region of the gene is highly polymorphic encoding amino acid residues known to be involved in immune-complex binding, and also encodes antigenic determinants. Therefore the C4d region was considered an ideal candidate for the development of a molecularbased allotyping protocol. The molecular-based C4 typing technique developed here is based on PCR-RFLP analysis of the C4d region. Protein and DNA sequence information, C4 protein allotyping data, and computer analysis which estimates the predicted isoelectric value of a protein were used to develop this protocol. Application of the molecular C4 typing protocol enabled the identification of C4 alleles which co-migrate by C4 protein gel electrophoresis as well as a number of rare and novel C4 alleles. The molecular basis of C4 null alleles has been extensively studied in Caucasians, however, very little is known about the genetic basis of C4 null alleles in other ethnic populations. C4 null alleles from Aboriginal Australians were investigated here in order to identify the genetic basis of these alleles compared to those in Caucasians. Many Caucasian C4 null alleles result from large deletions. However, this study found that the C4B null allele in Aboriginal Australians results from either duplication of C4A genes on a single haplotype, or possession of a hybrid C4A/C4B type of novel allele, which was denoted as C4A *CAN1. These findings have considerable significance in relation to the high frequency of C4 null alleles found in Aboriginal Australian populations. Many of these "null alleles" may indeed be functional alleles which have been misinterpreted by C 4 protein typing. The molecular basis of C4A null alleles remains unresolved, however, this study indicates that the defect is likely to be due to post-transcriptional processing of the allele. This thesis describes a detailed investigation into the molecular basis of C4 alleles including rare and null alleles. This information together with the vast amount of molecular data available on C 4 enabled the development of a molecular-based C4 typing protocol. This protocol is considered to be a more reliable and accurate typing method than the traditional protein typing method, and can be easily adopted in any laboratory for routine allotyping.

25 consecutive patients (15M, 10F ; mean age 30 years) with nephrotic syndrome (NS) of different grade were studied. Control group consisted in 18 healthy adult volunteers. Total protein S antigen (TPS:Ag) and free protein S antigen (FPS:Ag) after precipitation of C4-BP-bound protein S by PEG 3-5 % final concentration were measured by Laurell's technique. PS:Ag was also searched in concentrated urine of 9 patients by ELISA method, more sensitive than the Laurell's technique. In the same plasma samples we measured C4-BP, Protein C Ag and AT III biological activity (all reagents from D.Stago). Serum albumin level, proteinuria, proteinuria selectivity index, triglycerides, cholesterol levels were recorded. TPS:Ag was found elevated in NS (1.30±0.3 U/ml) in comparison with control group (1.09±0.32 U/ml) and the difference was statistically significant (p<0.05). The mean values of FPS:Ag observed in patients and controls were not statistically different, but if we consider 95 % confidence limits (0.99-1-35 U/ml), 16 pts had normal or elevated FPS:Ag level, whereas 9 had decreased FPS:Ag level. A positive correlation was found between TPS:Ag and FPS:Ag in control group (r=0.66 ; p< 0.001) and in patients with NS (r=0.4l, p<0.05). C4-BP was significantly (p<0.01) increased in nephrotic patients ( 1.37 ± 0.36 U/ml) in comparison with control group (1.04±0.27 U/ml). A negative correlation was found between FPS:Ag and C4-BP levels in control group (r = −0.57, P< 0.01) but not in nephrotic patients. A positive correlation was found between FPS:Ag and albumin level and between FPS:Ag and cholesterol level. No correlation was found between TPS:Ag or FPS:Ag and proteinuria, proteinuria selectivity index, AT III and protein C levels. Traces of PS were found in urine (0. to 2.5 U/day) in 9 patients tested. 2/25 pts suffered thromboembolic events : one had a very low level of FPS:Ag in addition to a decreased level of AT III. The other one had normal FPS:Ag and AT III level but a borderline Protein C level. In conclusion. An acquired FPS:Ag deficiency was observed in 9/25 pts with NS despite an increased level of TPS:Ag. In this small series of patients the acquired FPS deficiency does not seem to be related either to an urinary loss of FPS or to an increased binding to C4-BP, as suggested by some authors.

Protein S deficiency has been demonstrated in 5 members from the same family with a history of severe recurrent venous thrombosis over three generations. The propositus, a 16 year old female, had a first spontaneous thrombotic episode at age 15. Phlebography revealed a total obstruction of her left ilio-femoral vein with an extension to the vena cava. She was treated with heparin followed by oral anticoagulant therapy. The four other affected members (mother, aunts and uncle of the propositus) had also presented recurrent thrombosis with onset at a young age. The grandfather, not tested, had died from massive pulmonary embolism at age 54. The immunological assay of protein S was performed in plasma by Laurell, using a monospecific antiserum to human protein S, or by an ELISA, using a kit from Diagnostica Stago (Asserachrom Protein S). In order to separate free protein S, the functionally active form, from protein S complexed with C4-binding protein, plasma was adsorbed with 3.75 % polyethyleneglycol (PEG 6000). Following PEG precipitation, the levels of free protein S antigen remaining in the supernatant were quantitated by the usual immunological methods. In addition, two-dimensional immunoelectrophoresis (DDIE) also provided information on the distribution of both forms. In plasma protein S levels were decreased (40 to 55 % of the normal range) in two untreated patients and lower levels (17 to 20 96) were observed in the three others, including the propositus, who were under dicoumarol therapy. In PEG treated-plasma, protein S was undetectable (less than 5 %) in all patients, indicating a lack of free protein S. This was confirmed by DDIE : whereas protein S migrated as two distinct peaks, corresponding to free and complexed protein S in normal plasma, only a single peak of complexed protein S was observed in all affected patients. These results clearly demonstrate a total lack of free protein S which appears to be responsible for the thromboembolic disorder in this family as there was no deficiency of the other plasma inhibitors (antithrombin III, heparin cofactor II and protein C). According to the classification recently proposed by Comp et al., this family belongs to type I protein S deficiency, with an autosomal dominant mode of inheritance.