Predictors for a dementia gene mutation based on gene-panel next-generation sequencing of a large dementia referral series.

Next-generation genetic sequencing (NGS) technologies facilitate the screening of multiple genes linked to neurodegenerative dementia, but there are few reports about their use in clinical practice. Which patients would most profit from testing, and information on the likelihood of discovery of a causal variant in a clinical syndrome, are conspicuously absent from the literature, mostly for a lack of large-scale studies. We applied a validated NGS dementia panel to 3241 patients with dementia and healthy aged controls; 13,152 variants were classified by likelihood of pathogenicity. We identified 354 deleterious variants (DV, 12.6% of patients); 39 were novel DVs. Age at clinical onset, clinical syndrome and family history each strongly predict the likelihood of finding a DV, but healthcare setting and gender did not. DVs were frequently found in genes not usually associated with the clinical syndrome. Patients recruited from primary referral centres were compared with those seen at higher-level research centres and a national clinical neurogenetic laboratory; rates of discovery were comparable, making selection bias unlikely and the results generalisable to clinical practice. We estimated penetrance of DVs using large-scale online genomic population databases and found 71 with evidence of reduced penetrance. Two DVs in the same patient were found more frequently than expected. These data should provide a basis for more informed counselling and clinical decision making.

Complement components and their autoantibodies.

The purpose of the immune system is to defend the host from constantly changing microbial pathogens. Autoimmune diseases develop as a consequence of the production of antibodies and/or cells that react with self-antigens, and may recruit other effector mechanisms that result in tissue damage. Thus, in this context, autoimmunity represents an immune response to self-antigens that is sufficient to cause disease. This article is specifically devoted to autoantibodies directed against complement components.

Immune complex processing in C1q-deficient mice.

Complement and Fcgamma receptors are known to mediate the processing of immune complexes (IC), and abnormalities in these mechanisms may predispose to the development of lupus. We explored the processing of IC in mice deficient in complement component C1q. 125I-labelled IC comprising Hepatitis B surface antigen (HBsAg)/human anti-HBsAg (HBsAg/Ab) were injected intravenously and the sites of IC clearance determined by direct counting of organ uptake at various time points. The liver and spleen were the main sites of IC uptake in all mice. The splenic uptake of IC was significantly reduced in the C1q-deficient mice compared with the control mice. C1q-deficient mice also exhibited an initial accelerated hepatic uptake of IC similar to that seen in human subjects with hypocomplementaemia. The hepatic localization of IC at later time points was similar in both groups of mice. These data in mice are consistent with previous observations in humans that confirm that the classical pathway of complement plays an important role in the appropriate processing of IC in vivo.

Significance of anticardiolipin and anti-beta(2)-glycoprotein I antibodies in lupus nephritis.

OBJECTIVE: To investigate whether anticardiolipin (aCL) and anti-beta(2)-glycoprotein I (anti-beta(2)GPI) antibodies are associated with lupus nephritis (group II patients), and whether there are differences in the prevalence of these two autoantibodies between group II patients and patients with non-nephritis SLE (group I) and primary antiphospholipid syndrome (PAPS) patients (group III). METHODS: IgG and IgM aCL were measured in 31 patients and anti-beta(2)GPI in 30 patients with systemic lupus erythematosus (SLE) nephritis and 25 without SLE nephritis and in 36 PAPS patients by validated enzyme immunoassays. Relationships of anti-double-stranded DNA (anti-dsDNA) antibodies and antibodies to the collagenous region of C1q (anti-C1q) with SLE nephritis were also examined. RESULTS: The prevalence and levels were higher for aCL, but not for anti-beta(2)GPI, antibodies in group II than in group I patients. Absolute values of aCL and anti-beta(2)GPI in all three patient groups correlated with each other. The prevalences of aCL, anti-dsDNA and anti-C1q antibodies were significantly higher in group II than in group I and group III patients. CONCLUSION: The observations in this paper suggest that raised levels of aCL antibodies are associated with lupus nephritis. We were not able to demonstrate an association between anti-beta(2)GPI antibodies and kidney disease either in patients with lupus or in patients with primary antiphospholipid syndrome. In SLE, we demonstrated that the presence of anticardiolipin antibodies in conjunction with elevated levels of anti-dsDNA and anti-C1q antibodies is highly specific for glomerulonephritis in patients with lupus.

Overrepresentation of the Fcgamma receptor type IIA R131/R131 genotype in caucasoid systemic lupus erythematosus patients with autoantibodies to C1q and glomerulonephritis.

OBJECTIVE: To test the hypothesis that there is an association between the Fcgamma receptor type IIA (FcgammaRIIA)-H/R131 polymorphism and autoantibodies to the collagenous region (CLR) of C1q in patients with systemic lupus erythematosus (SLE). METHODS: One hundred ninety-five Caucasoid lupus patients were studied. Anti-C1q(CLR) antibodies in serum were measured by enzyme-linked immunosorbent assay (ELISA) and FcgammaRIIA genotype analysis was performed by polymerase chain reaction. Immunoglobulin subclass of the autoantibodies was measured by ELISA. RESULTS: Fifty-six patients were anti-C1q antibody positive, and Ig subclass analysis indicated a predominance of IgG2 anti-C1q antibodies. Analysis of the SLE population as a whole revealed no significant difference in the allele frequencies of R131 and H131 compared with controls. There was, however, a significantly increased frequency of the R131 allele both in the anti-C1q-positive subgroup of patients (chi2 = 7.66, P<0.01) and in the 71 patients with nephritis (chi2 = 7.76, P< 0.01), compared with controls. CONCLUSION: FcgammaRIIA-R131 constitutes a heritable susceptibility factor for the development of SLE with manifestations in the kidney in Caucasoid patients. The close associations demonstrated between this FcgammaRII variant, antibodies to C1q(CLR), and glomerulonephritis may be due to a failure of clearance of the potentially pathogenic IgG2 autoantibody.

Cloning of the mouse homolog of the 126-kDa human C1q/MBL/SP-A receptor, C1qR(p).

Binding of C1q to cell surfaces has been shown to mediate a number of biological activities including enhancement of phagocytosis and stimulation of superoxide production. Several C1q binding proteins have been proposed as candidate receptors for these functions. The 126-kDa human C1q membrane receptor, termed C1qR(p), has recently been cloned. This molecule is believed to play a role in the enhancement of phagocytosis in monocytes and macrophages, and its expression has been shown to be restricted to cells of the myeloid lineage, endothelial cells, and platelets. Here we report the isolation and genomic characterization of the murine homolog of C1qR(p). Degenerate oligonucleotide primers based on the published human sequence were used to amplify a region of the murine homolog spanning from the carbohydrate recognition domain to the fourth epidermal growth factor (EGF) domain. This fragment was used as a probe to isolate the murine gene from a 129/Sv genomic lambda library. The predicted primary protein sequence displayed 68.1% identity with the human homolog. All the major structural domains were conserved between the two molecules. The coding sequence of the murine gene was contained within two exons separated by a small intron of approximately 250 bp. The structure of the human gene was found to be similar, with the position of the intron conserved. Cloning of the murine C1qR(p) will facilitate further investigation of the physiological function of this molecule.

A high-resolution radiation hybrid map of the proximal region of rat chromosome 4.

Radiation hybrid (RH) mapping has been used to produce genome maps in the human and mouse, but as yet the technique has been applied little to other species. We describe the use of RH mapping in the rat, using a newly available rat/hamster RH panel, to construct an RH map of the proximal part of rat Chromosome (Chr) 4. This region is of interest because quantitative trait loci (QTLs) for defective insulin and catecholamine action, hypertension, and dyslipidemia map to this region. The RH map includes 23 rat genes or microsatellites previously mapped to this part of Chr 4, one rat gene not previously mapped in the rat, and markers for four new genes, homologs of which map to the syntenic region of the mouse genome. The RH map integrates genetic markers previously mapped on several rat crosses, increases the resolution of existing maps, and may provide a suitable basis for physical map construction and gene identification in this chromosomal region. Our results demonstrate the utility of RH mapping in the rat genome and show that RH mapping can be used to localize, in the rat genome, the homologs of genes from other species such as the mouse. This will facilitate identification of candidate genes underlying QTLs on this chromosomal segment.

Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats.

The human insulin-resistance syndromes, type 2 diabetes, obesity, combined hyperlipidaemia and essential hypertension, are complex disorders whose genetic basis is unknown. The spontaneously hypertensive rat (SHR) is insulin resistant and a model of these human syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridaemia and hypertension map to a single locus on rat chromosome 4. Here we combine use of cDNA microarrays, congenic mapping and radiation hybrid (RH) mapping to identify a defective SHR gene, Cd36 (also known as Fat, as it encodes fatty acid translocase), at the peak of linkage to these QTLs. SHR Cd36 cDNA contains multiple sequence variants, caused by unequal genomic recombination of a duplicated ancestral gene. The encoded protein product is undetectable in SHR adipocyte plasma membrane. Transgenic mice overexpressing Cd36 have reduced blood lipids. We conclude that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridaemia in SHR and may be important in the pathogenesis of human insulin-resistance syndromes.

Molecular basis of hereditary C1q deficiency associated with SLE and IgA nephropathy in a Turkish family.

Two siblings (case 1 and case 2) with homozygous C1q deficiency are described. Both presented with a photosensitive rash, and during follow-up case one developed SLE with nephrotic range proteinuria. Case 2 had microscopic hematuria with a past history of macroscopic hematuria. Renal biopsies revealed mesangioproliferative glomerulonephritis in case 1 and IgA nephropathy in case 2, a new finding in association with C1q deficiency. Since the classical pathway of complement plays a role in the development of antibody responses, the family was also evaluated for the immune response to hepatitis B vaccine. Antibody response to hepatitis B vaccine was normal in both affected members and the rest of the family. The A-, B- and C- chain genes of C1q were amplified by PCR and directly sequenced. A homozygous C to T point mutation was identified in genomic DNA isolated from the patients at codon 186 in the A chain that resulted in a premature stop codon. This mutation was present in both parents and both unaffected sibs in the heterozygous state. This mutation was identical to that previously described in a Slovakian family with C1q deficiency. Because of this finding, a series of 92 genomic DNA samples was screened from ethnically distinct patient groups with SLE to test the hypothesis that this mutation of C1q may be a widespread disease susceptibility gene. No further examples of this mutation were found.

IFN-gamma up-regulates expression of the complement components C3 and C4 by stabilization of mRNA.

We investigated the mechanisms underlying the regulation of complement genes C3 and C4 by IFN-gamma. IFN-gamma (500 U/ml, 24 h incubation) increased steady state mRNA levels for both C3 and C4 in three different cell types (Hep G2, U937, and primary fibroblasts). The response to IFN-gamma in Hep G2 cells was time and dose dependent. At all doses of IFN-gamma and at all incubation times, the transcription rate for these two genes, determined by nuclear run-on assays, was reduced (0.3 +/- 0.1; unstimulated rate = 1.00). The t1/2 of mRNA for C3 and C4 in unstimulated cells was 1.8 +/- 0.3 and 2.2 +/- 0.2 h, respectively. After high-dose IFN-gamma stimulation, both C3 and C4 mRNA levels remained at 100% with respect to baseline at 5 h, but after 12 h, levels fell to 13 +/- 2% (C3) and 8 +/- 3% (C4) of baseline values, giving a half-life for these mRNA species of between 5 and 12 h. IFN-gamma stimulation increased C3 and C4 protein synthesis measured at 24 h. We suggest that it is the increase in mRNA stability that is the major effector mechanism by which IFN-gamma regulates C3 and C4 gene expression.

Autoantibodies to the collagenous region of C1q occur in three strains of lupus-prone mice.

We have developed an ELISA to measure murine autoantibodies to the collagenous region (CLR) of C1q, using the whole human C1q molecule as the solid-phase ligand, in the presence of 1 M NaCl. The assay was validated by testing positive sera from 20 mice using purified mouse C1q, and from 10 mice using purified human C1q-CLR, as the solid-phase ligands. There were highly significant correlations between results obtained with human C1q (whole molecule) and: (i) mouse C1q (rsp = 0.73, P less than 0.001), and (ii) human Clq-CLR alone (rsp = 0.86, P = 0.001). Antibodies to Clq were measured in 53 MRL/lpr, 17 BXSB and 25 NZB/W lupus-prone mice. Median (range) anti-C1q (CLR) antibody levels in MRL/lpr, BXSB, and NZB/W autoimmune mice aged 3 months were 22 (16-66), 21 (17-39) and 19 (15-27) EU, respectively. The median anti-Clq antibody level in MRL/lpr mice aged 5 months was 76 (35-142) EU, significantly higher than that at 3 months (U = 558, P less than 0.0005). Median anti-C1q antibody level in NZB/W mice at 8 months was 37 (13-74) EU and in BXSB mice at 11 months was 62 (31-231) EU, significantly higher than corresponding values at 3 months (U = 326, and U = 4, P less than 0.001, respectively). This is the first demonstration of anti-C1q (CLR) antibodies in NZB/W and BXSB mice. The pathologic significance and the potential utility of these antibodies for monitoring disease in lupus-prone mice are under evaluation.

Homozygous hereditary C1q deficiency and systemic lupus erythematosus. A new family and the molecular basis of C1q deficiency in three families.

OBJECTIVE: To describe a new kindred with Clq deficiency and to identify the molecular lesions responsible for complete functional C1q deficiency in this and 2 other previously described kindreds. METHODS: The A-, B-, and C-chain genes of C1q were amplified by polymerase chain reaction, cloned, and sequenced. The DNA sequence was checked for mutations. RESULT: Patient 1 had a homozygous G-to-A change at codon 6 of the C chain, causing an amino acid change from Gly to Arg. Patient 2 had a homozygous deletion of a C nucleotide at codon 43 of the C-chain, causing a frame shift, leading to a premature stop codon at codon 108. Patient 3 had a homozygous C-to-T mutation at amino acid position 41 of the C chain, resulting in a premature stop codon. CONCLUSION: In the homozygous state, the mutations are sufficient to cause complete deficiency of Clq. The mutation in patient 1 has been previously reported in a patient of different ethnic origin. A survey of a series of 158 DNA samples from patients with systemic lupus erythematosus showed no other examples of this mutant allele.

Clearance pathways of soluble immune complexes in the pig. Insights into the adaptive nature of antigen clearance in humans.

Efficient delivery of immune complexes (ICs) to the mononuclear phagocytic system, and subsequent IC processing, may prevent their potentially harmful effects in other tissues and may also be important in the development of humoral immune responses. In mice, rabbits, and primates, the liver and spleen are the main sites of IC clearance. It has been demonstrated previously that the pulmonary capillaries in the pig are lined with macrophages and that certain particulates, including bacteria, localize to this organ. In this study, we used gamma scintigraphy to explore the sites and kinetics of clearance of soluble IC comprising 123I-labeled hepatitis B surface Ag (HBsAg):porcine anti-HBsAg in the Large White pig. At t = 10 min after i.v. injection, 43 +/- 5% (mean +/- SE) IC localized in the lungs, and 36 +/- 6% counts in the liver. At t = 85 min, values were: lungs, 15 +/- 4% and liver, 29 +/- 2%. Findings were similar following intraarterial injection. Complement depletion resulted in more rapid initial IC clearance (t1/2 = 5 min), reduced lung uptake (23 +/- 3% at 10 min), and impaired IC catabolism. In normal animals, 5 to 7% injected IC bound to PBMCs, but no E binding was seen. A fall in PBMC numbers (46 to 59% of baseline), was observed following IC injection. These findings contrast with our previous observations using analogous IC in humans, in which we did not observe any change in peripheral blood leukocyte counts consequent upon complex processing, suggesting that in humans, Es may function as a buffering system for complement-bearing IC in the circulation, preventing their interaction with leukocytes bearing complement and FcR, and the potential activation of these cells.

The anti-lipid A monoclonal antibody E5 binds to rough gram-negative bacteria, fixes C3, and facilitates binding of bacterial immune complexes to both erythrocytes and monocytes.

Treatment of patients with septic shock using monoclonal antibodies (mAbs) to endotoxin is still controversial. Clinical trials of E5, one of the mAbs directed against the lipid A moiety of lipopolysaccharide (LPS), are currently in progress. The mechanisms of action of this, and other antibodies under clinical evaluation, are, however, poorly understood. In this study we examined in vitro the ways in which E5 interacted with Gram-negative bacteria, complement, erythrocytes and monocytes. By fluorescence-activated cell sorter (FACS) analysis we showed direct, dose-dependent binding of E5 to Escherichia coli (E. coli) and Salmonella minnesota (S. minnesota). Antibody binding to S. minnesota was enhanced by treatment with the beta-lactam antibiotic amoxycillin, but not by treatment with the aminoglycoside gentamicin. Immune complexes formed between E5 and both species of Gram-negative bacteria activated both classical and alternative complement pathways, but only in the case of S. minnesota did this facilitate binding to erythrocyte CR1 and monocyte CR3. Bacterial C3b and iC3b fixation by E5 was quantified using specific mAbs. These observations suggest that E5 may enhance bacterial clearance in several ways: (1) by facilitating direct complement fixation; (2) by facilitating the binding of opsonized bacteria to cells of the mononuclear phagocyte system; (3) by enabling bacteria to bind to erythrocyte CR1 (CD35), allowing safe carriage in the circulation to the fixed macrophages of the liver and spleen; (4) by acting synergistically with beta-lactam antibiotics.

Hereditary C1q deficiency and systemic lupus erythematosus.

We describe a 27-year-old women with systemic lupus erythematosus, C1q deficiency and cytomegalovirus retinitis. She suffered from severe SLE, with cutaneous and CNS involvement, and died of CNS disease aged 28. Review of 29 other published cases of C1q deficiency shows that SLE in these patients is often severe (five with CNS disease, ten with glomerulonephritis). The results of autoantibody studies in this and another patient with C1q deficiency and SLE are presented--both patients had autoantibodies to the extractable nuclear antigens, Sm, RNP and Ro, and one patient had high titres of antibodies to dsDNA. One of the patients had previously been treated with fresh frozen plasma, and antibodies to C1q were present in his serum. Homozygous C1q deficiency is associated with a very high prevalence of severe SLE with the full panoply of autoantibodies characteristic of this disease.

The anti-lipid A antibody HA-1A binds to rough gram-negative bacteria, fixes complement and facilitates binding to erythrocyte CR1 (CD35).

MoAbs to bacterial cell wall lipopolysaccharide are currently under evaluation for the treatment of Gram-negative sepsis. The mode of action of these reagents remains poorly understood. In this study we examined the ability of radiolabelled HA-1A (an IgM anti-lipid A MoAb) to bind in vitro to Salmonella minnesota (Re 595), Escherichia coli, and Streptococcus pyogenes. HA-1A was able to bind specifically to the 'rough' mutant Salm. minnesota, but not to a 'smooth' E. coli, or Strep. pyogenes. Binding to Salm. minnesota led to complement fixation which resulted in bacterial adherence to erythrocyte CR1, suggesting a possible mechanism whereby the antibody might enhance clearance of bacteria by facilitating delivery to the fixed mononuclear phagocytic system. We were not able to demonstrate the formation of immune complexes between free lipopolysaccharide and HA-1A in the presence of serum, nor the enhancement of complement-mediated binding of HA-1A:Salm. minnesota immune complexes to erythrocytes by antibiotic treatment. Binding of HA-1A to small bacterial fragments was, however, demonstrable after in vitro treatment with a beta-lactam antibiotic, which disrupts the bacterial cell wall, but not with gentamicin, an aminoglycoside antibiotic which blocks protein synthesis.