Jorge Esparza-Gordillo

Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS) is an important cause of acute renal failure in children. Mutations in one or more genes encoding complement-regulatory proteins have been reported in approximately one-third of nondiarrheal, atypical HUS (aHUS) patients, suggesting a defect in the protection of cell surfaces against complement activation in susceptible individuals. Here, we identified a subgroup of aHUS patients showing persistent activation of the complement alternative pathway and found within this subgroup two families with mutations in the gene encoding factor B (BF), a zymogen that carries the catalytic site of the complement alternative pathway convertase (C3bBb). Functional analyses demonstrated that F286L and K323E aHUS-associated BF mutations are gain-of-function mutations that result in enhanced formation of the C3bBb convertase or increased resistance to inactivation by complement regulators. These data expand our understanding of the genetic factors conferring predisposition to aHUS, demonstrate the critical role of the alternative complement pathway in the pathogenesis of aHUS, and provide support for the use of complement-inhibition therapies to prevent or reduce tissue damage caused by dysregulated complement activation.

Genetic and environmental factors influencing the human factor H plasma levels.

Factorxa0H is a plasma protein that plays a critical role in the regulation of complement activation in fluid phase and on cellular surfaces. Over the years numerous reports have illustrated the association of factorxa0H deficiencies with chronic renal and infectious diseases. Plasma levels of factorxa0H show a five-fold range of variation in humans (116–562xa0μg/ml), which may also be relevant to disease susceptibility. To quantify the effects of the genetic and environmental factors responsible for the variation in the factorxa0H plasma levels, we have applied variance-component methods to a family-based sample. Factorxa0H plasma levels show an age-dependent increase (P<0.0001) and are decreased in smokers (P<0.0001). Interestingly, the heritability of the factorxa0H trait is very high (h2=0.62±0.07; P<0.0001), indicating that 62% of the factorxa0H phenotypic variance is due to the additive effects of genes. On this premise, we conducted a genome-wide linkage screen in order to identify genes regulating the factorxa0H trait. Three genomic regions (1q32, 2p21–24 and 15q22–24) provided suggestive evidence of linkage (LOD scores 2.03, 2.15 and 2.00, respectively) with the plasma levels of factorxa0H.

The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine catabolism.

3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of alpha (biotin-containing) and beta subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCC alpha and MCC beta subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted CGA and CGB. We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCC alpha. Two MCCB missense mutations and one splicing defect mutation leading to early MCC beta truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCC beta truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the alpha and beta subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.

Functional analysis of MCCA and MCCB mutations causing methylcrotonylglycinuria

Methylcrotonylglycinuria (MCG; MIM 210200) is an autosomal recessive inherited human disorder caused by the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC, E.C.6.4.1.4), involved in leucine catabolism. This mitochondrial enzyme is one of the four biotin-dependent carboxylases known in humans. MCC is composed of two different types of subunits, alpha and beta, encoded by the nuclear genes MCCA and MCCB, respectively, recently cloned and characterized. Several mutations have been identified, in both genes, the majority are missense mutations along with splicing mutations and small insertions/deletions. We have expressed four missense mutations, two MCCA and two MCCB mapping to highly evolutionarily conserved residues, by transient transfection of SV40-transformed deficient fibroblasts in order to confirm their pathogenic effect. All the missense mutations expressed resulted in null or severely diminished MCC activity providing direct evidence that they are disease-causing ones. The MCCA mutations have been analysed in the context of three-dimensional structural information modelling the changes in the crystallized biotin carboxylase subunit of the Escherichia coli acetyl-CoA carboxylase. The apparent severity of all the MCC mutations contrasts with the variety of the clinical phenotypes suggesting that there are other cellular and metabolic unknown factors that affect the resulting phenotype.

Genetic determinants of variation in the plasma levels of the C4b-binding protein (C4BP) in Spanish families

The C4b-binding protein (C4BP) is a plasma glycoprotein implicated in the homeostasis of the complement and coagulation systems. It is composed of two polypeptides (α and β), which form three plasma oligomers with different subunit compositions (α7β1, α7β0, and α6β1). The β chain-containing C4BP isoforms (C4BPβ+ isoforms) bind and inactivate protein S (PS), downregulating the activated protein C (APC)-dependent anticoagulatory pathway. Because PS deficiency is associated with recurrent thrombosis, it has been suggested that increased levels of C4BPβ+ isoforms might diminish the free PS plasma level, affecting the risk of developing thromboembolism. Previous work has tested this hypothesis, but no definitive conclusions were reached, mostly because nothing is known about the factors influencing the high variability in C4BP plasma levels in humans. As a part of the GAIT project, using variance component analysis, this work provides the first estimation of the relative contributions of genetic and environmental influences on the plasma levels of total C4BP and C4BPβ+ isoforms. Plasma levels of total C4BP and C4BPβ+ isoforms showed strong evidence of genetic regulation (heritability 37.7% and 42.5%, respectively). They were also affected by age, smoking, and exogenous sex hormones. Our results constitute the first step in localizing and evaluating potential quantitative trait loci that affect the plasma levels of C4BP and C4BPβ+. Furthermore, analysis of phenotypic and genetic correlations between C4BPβ+ plasma levels and the components of the APC anticoagulatory pathway (total PS, free PS, functional PS, and functional PC) suggests a genetic co-regulation of the proteins. These observations might have important implications in the individual susceptibility to thrombotic disease.

Genetic correlation between plasma levels of C4BP isoforms containing beta chains and susceptibility to thrombosis.

Thrombosis is a complex trait with both genetic and environmental components.1 In general, disease status is a qualitative trait where individuals are diagnosed as affected or unaffected, but it is now accepted that underlying the disease there is a continuum trait termed liability, susceptibility, or risk. Liability cannot be measured directly, but it can be modelled and estimated. Disease results when an individual’s liability is above a critical value or threshold, whereas liability values below the threshold correspond to healthy individuals. These threshold models of an underlying continuous scale of risk allow inferences that are compatible with current models of gene action.2nnQuantitative plasma phenotypes, such as those related to haemostasis, are also complex traits, whose regulation depends on multiple genetic and environmental factors.3 Classical statistical methods are unable to quantify or partition the genetic and environmental factors determining the variability in such complex traits. However, variance component methods have been developed which allow the examination of sources of correlation between quantitative physiological measures and disease outcomes.4 These statistical genetic methods also permit the localisation and evaluation of the relative effects of the genes involved.5nnWe have applied these methods to the Genetic Analysis of Idiopathic Thrombophilia Project (GAIT Project) to characterise the genetic determinants responsible for idiopathic thrombophilia. Using a family based approach, we estimated that idiopathic thrombophilia has a heritability of 0.61, indicating that 61% of variation in liability to thrombosis at the population level can be attributed to genetic factors.6 Phenotypic correlations were also evaluated between 27 plasma phenotypes related to haemostasis and thrombosis liability.6 It was found that genetic factors were mainly responsible for these phenotypic correlations,6 indicating that some of the genes that regulate quantitative variation in these plasma phenotypes also affect the risk of thrombosis. These …

Expression of the peptide C4b-binding protein β in the arthritic joint

Background: C4b-binding protein (C4BP) is a plasma oligomeric glycoprotein that participates in the regulation of complement and haemostasis. Complement-regulatory activity depends on the C4BPα-polypeptide, whereas the C4BPβ-polypeptide inactivates protein S, interfering with the anti-coagulatory protein C-dependent pathway. Objective: To investigate the expression of C4BPβ in the rheumatoid joint. Methods: Expression of C4BP was studied in synovial explants from patients with rheumatoid arthritis, osteoarthritis and healthy controls, using immunohistochemistry and in situ hybridisation. C4BP isoforms and free C4BPβ were studied in synovial effusions from patients with rheumatoid arthritis, osteoarthritis and microcrystalline arthritis (MCA) by immunoblotting; total and free protein S levels were studied by enzyme immunoassay. Results: C4BPβ was overexpressed in the synovial membranes of patients with rheumatoid arthritis, in close association with the severity of synovitis and the extension of interstitial fibrin deposits. As many as 85% fluids from patients with rheumatoid arthritis contained free C4BPβ, whereas this unusual polypeptide was present in 50% fluids from patients with MCA and 40% fluids from patients with osteoarthritis. Free protein S at the effusions was pathologically reduced in patients with rheumatoid arthrits and MCA, and remained normal in patients with osteoarthritis. Conclusion: C4BPβ is expressed by the inflamed synovial tissue, where it can participate in processes of tissue remodelling associated with invasive growth.