Genome-wide comparative analysis of atopic dermatitis and psoriasis gives insight into opposing genetic mechanisms.

Atopic dermatitis and psoriasis are the two most common immune-mediated inflammatory disorders affecting the skin. Genome-wide studies demonstrate a high degree of genetic overlap, but these diseases have mutually exclusive clinical phenotypes and opposing immune mechanisms. Despite their prevalence, atopic dermatitis and psoriasis very rarely co-occur within one individual. By utilizing genome-wide association study and ImmunoChip data from >19,000 individuals and methodologies developed from meta-analysis, we have identified opposing risk alleles at shared loci as well as independent disease-specific loci within the epidermal differentiation complex (chromosome 1q21.3), the Th2 locus control region (chromosome 5q31.1), and the major histocompatibility complex (chromosome 6p21-22). We further identified previously unreported pleiotropic alleles with opposing effects on atopic dermatitis and psoriasis risk in PRKRA and ANXA6/TNIP1. In contrast, there was no evidence for shared loci with effects operating in the same direction on both diseases. Our results show that atopic dermatitis and psoriasis have distinct genetic mechanisms with opposing effects in shared pathways influencing epidermal differentiation and immune response. The statistical analysis methods developed in the conduct of this study have produced additional insight from previously published data sets. The approach is likely to be applicable to the investigation of the genetic basis of other complex traits with overlapping and distinct clinical features.

Maternal filaggrin mutations increase the risk of atopic dermatitis in children: an effect independent of mutation inheritance.

Epidemiological studies suggest that allergy risk is preferentially transmitted through mothers. This can be due to genomic imprinting, where the phenotype effect of an allele depends on its parental origin, or due to maternal effects reflecting the maternal genome's influence on the child during prenatal development. Loss-of-function mutations in the filaggrin gene (FLG) cause skin barrier deficiency and strongly predispose to atopic dermatitis (AD). We investigated the 4 most prevalent European FLG mutations (c.2282del4, p.R501X, p.R2447X, and p.S3247X) in two samples including 759 and 450 AD families. We used the multinomial and maximum-likelihood approach implemented in the PREMIM/EMIM tool to model parent-of-origin effects. Beyond the known role of FLG inheritance in AD (R1meta-analysis = 2.4, P = 1.0 x 10-36), we observed a strong maternal FLG genotype effect that was consistent in both independent family sets and for all 4 mutations analysed. Overall, children of FLG-carrier mothers had a 1.5-fold increased AD risk (S1 = 1.50, Pmeta-analysis = 8.4 x 10-8). Our data point to two independent and additive effects of FLG mutations: i) carrying a mutation and ii) having a mutation carrier mother. The maternal genotype effect was independent of mutation inheritance and can be seen as a non-genetic transmission of a genetic effect. The FLG maternal effect was observed only when mothers had allergic sensitization (elevated allergen-specific IgE antibody plasma levels), suggesting that FLG mutation-induced systemic immune responses in the mother may influence AD risk in the child. Notably, the maternal effect reported here was stronger than most common genetic risk factors for AD recently identified through genome-wide association studies (GWAS). Our study highlights the power of family-based studies in the identification of new etiological mechanisms and reveals, for the first time, a direct influence of the maternal genotype on the offspring's susceptibility to a common human disease.

DNA methylation within melatonin receptor 1A (MTNR1A) mediates paternally transmitted genetic variant effect on asthma plus rhinitis.

BACKGROUND: Asthma and allergic rhinitis (AR) are common allergic comorbidities with a strong genetic component in which epigenetic mechanisms might be involved. OBJECTIVE: We aimed to identify novel risk loci for asthma and AR while accounting for parent-of-origin effect. METHODS: We performed a series of genetic analyses, taking into account the parent-of-origin effect in families ascertained through asthma: (1) genome-wide linkage scan of asthma and AR in 615 European families, (2) association analysis with 1233 single nucleotide polymorphisms (SNPs) covering the significant linkage region in 162 French Epidemiological Study on the Genetics and Environment of Asthma families with replication in 154 Canadian Saguenay-Lac-Saint-Jean asthma study families, and (3) association analysis of disease and significant SNPs with DNA methylation (DNAm) at CpG sites in 40 Saguenay-Lac-Saint-Jean asthma study families. RESULTS: We detected a significant paternal linkage of the 4q35 region to asthma and allergic rhinitis comorbidity (AAR; P = 7.2 × 10(-5)). Association analysis in this region showed strong evidence for the effect of the paternally inherited G allele of rs10009104 on AAR (P = 1.1 × 10(-5), reaching the multiple-testing corrected threshold). This paternally inherited allele was also significantly associated with DNAm levels at the cg02303933 site (P = 1.7 × 10(-4)). Differential DNAm at this site was found to mediate the identified SNP-AAR association. CONCLUSION: By integrating genetic and epigenetic data, we identified that a differentially methylated CpG site within the melatonin receptor 1A (MTNR1A) gene mediates the effect of a paternally transmitted genetic variant on the comorbidity of asthma and AR. This study provides a novel insight into the role of epigenetic mechanisms in patients with allergic respiratory diseases.

A functional IL-6 receptor (IL6R) variant is a risk factor for persistent atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a common inflammatory skin disease. Previous studies have revealed shared genetic determinants among different inflammatory disorders, suggesting that markers associated with immune-related traits might also play a role in AD. OBJECTIVE: We sought to identify novel genetic risk factors for AD. METHODS: We examined the results of all genome-wide association studies from a public repository and selected 318 genetic markers that were significantly associated with any inflammatory trait. These markers were considered candidates and tested for association with AD in a 3-step approach including 7 study populations with 7130 patients with AD and 9253 control subjects. RESULTS: A functional amino acid change in the IL-6 receptor (IL-6R Asp358Ala; rs2228145) was significantly associated with AD (odds ratio [OR], 1.15; P = 5 × 10(-9)). Interestingly, investigation of 2 independent population-based birth cohorts showed that IL-6R 358Ala specifically predisposes to the persistent form of AD (ORpersistent AD = 1.22, P = .0008; ORtransient AD = 1.04, P = .54). This variant determines the balance between the classical membrane-bound versus soluble IL-6R signaling pathways. Carriers of 358Ala had increased serum levels of soluble IL-6R (P = 4 × 10(-14)), with homozygote carriers showing a 2-fold increase. Moreover, we demonstrate that soluble IL-6R levels were higher in patients with AD than in control subjects (46.0 vs 37.8 ng/mL, P = .001). Additional AD risk variants were identified in RAD50, RUNX3, and ERBB3. CONCLUSION: Our study supports the importance of genetic variants influencing inflammation in the etiology of AD. Moreover, we identified a functional genetic variant in IL6R influencing disease prognosis and specifically predisposing to persistent AD.

The eczema risk variant on chromosome 11q13 (rs7927894) in the population-based ALSPAC cohort: a novel susceptibility factor for asthma and hay fever.

In a genome-wide association study, a common variant on chromosome 11q13.5 (rs7927894[T]) has been identified as a susceptibility locus for eczema. We aimed to analyze the effect of this risk variant on asthma and hay fever and to determine its impact on the general population level in over 9300 individuals of the prospectively evaluated Avon Longitudinal Study of Parents and Children birth cohort. We demonstrate an association of rs7927894[T] with atopic asthma and with hay fever. The largest effect sizes were found in patients with the combined phenotype atopic asthma plus eczema [odds ratio (OR) = 1.50; 95% confidence interval (CI) 1.20-1.88; P = 3.7 × 10(-4)] and hay fever plus eczema (OR = 1.37; 95% CI 1.15-1.62; P = 3.8 × 10(-4)). We replicated the effects of rs7927894[T] on eczema-associated asthma and hay fever independently in the German GENUFAD (GEnetic studies in NUclear Families with Atopic Dermatitis) study and show that they are significantly larger than the effect observed in eczema. The estimated population attributable risk fractions for eczema, eczema-associated atopic asthma or hay fever were 9.3, 24.9 and 23.5%, respectively. Finally in eczema, we found a synergistic interaction of rs7927894[T] with filaggrin gene (FLG) mutations, which are a major cause of epidermal barrier dysfunction, and replicated the interaction in the German Multicenter Allergy Study birth cohort. The synergistic effect of rs7927894[T] and FLG mutations on eczema risk as well as the association of both variants with eczema-associated atopic asthma and hay fever point to an involvement of rs7927894[T] in a functional pathway that is linked to the barrier defect.

The ANO3/MUC15 locus is associated with eczema in families ascertained through asthma.

BACKGROUND: A previous genome-wide linkage scan in 295 families of the French Epidemiological Study on the Genetics and Environment of Asthma (EGEA) reported strong evidence of linkage of 11p14 to eczema. OBJECTIVE: Our purpose was to conduct fine-scale mapping of the 11p14 region to identify the genetic variants associated with eczema. METHODS: Association analyses were first conducted in the family sample from the French EGEA by using 2 methods: the family-based association method and logistic regression. Replication of the EGEA findings was sought in French Canadian and United Kingdom family samples, which, similarly to EGEA samples, were ascertained through asthma. We also tested for association in 2 German samples ascertained through eczema. RESULTS: We found significant association of eczema with 11p14 genetic variants in the vicinity of the linkage peak in EGEA (P = 10(-4) for rs1050153 by using the family-based association method, which reached the multiple testing-corrected threshold of 10(-4); P = .003 with logistic regression). Pooled analysis of the 3 asthma-ascertained samples showed strong improvement in the evidence for association (P = 6 × 10(-6) for rs293974, P = 3 × 10(-5) for rs1050153, and P = 8 × 10(-5) for rs15783). No association was observed in the eczema-ascertained samples. CONCLUSION: The significant single nucleotide polymorphisms are located within the overlapping anoctamin 3 (ANO3) and mucin 15 (MUC15) genes. Several lines of evidence suggest that MUC15 is a strong candidate for eczema. Further investigation is needed to confirm our findings and to better understand the role of the ANO3/MUC15 locus in eczema and its relationship with respect to asthma.

Multi-locus stepwise regression: a haplotype-based algorithm for finding genetic associations applied to atopic dermatitis.

BACKGROUND: Genome-wide association studies (GWAS) provide an increasing number of single nucleotide polymorphisms (SNPs) associated with diseases. Our aim is to exploit those closely spaced SNPs in candidate regions for a deeper analysis of association beyond single SNP analysis, combining the classical stepwise regression approach with haplotype analysis to identify risk haplotypes for complex diseases. METHODS: Our proposed multi-locus stepwise regression starts with an evaluation of all pair-wise SNP combinations and then extends each SNP combination stepwise by one SNP from the region, carrying out haplotype regression in each step. The best associated haplotype patterns are kept for the next step and must be corrected for multiple testing at the end. These haplotypes should also be replicated in an independent data set. We applied the method to a region of 259 SNPs from the epidermal differentiation complex (EDC) on chromosome 1q21 of a German GWAS using a case control set (1,914 individuals) and to 268 families with at least two affected children as replication. RESULTS: A 4-SNP haplotype pattern with high statistical significance in the case control set (p = 4.13 × 10(-7) after Bonferroni correction) could be identified which remained significant in the family set after Bonferroni correction (p = 0.0398). Further analysis revealed that this pattern reflects mainly the effect of the well-known FLG gene; however, a FLG-independent haplotype in case control set (OR = 1.71, 95% CI: 1.32-2.23, p = 5.6 × 10(-5)) and family set (OR = 1.68, 95% CI: 1.18-2.38, p = 2.19 × 10(-3)) could be found in addition. CONCLUSION: Our approach is a useful tool for finding allele combinations associated with diseases beyond single SNP analysis in chromosomal candidate regions.

Immune disease risk variants regulate gene expression dynamics during CD4+ T cell activation.

During activation, T cells undergo extensive gene expression changes that shape the properties of cells to exert their effector function. Understanding the regulation of this process could help explain how genetic variants predispose to immune diseases. Here, we mapped genetic effects on gene expression (expression quantitative trait loci (eQTLs)) using single-cell transcriptomics. We profiled 655,349 CD4+ T cells, capturing transcriptional states of unstimulated cells and three time points of cell activation in 119 healthy individuals. This identified 38 cell clusters, including transient clusters that were only present at individual time points of activation. We found 6,407 genes whose expression was correlated with genetic variation, of which 2,265 (35%) were dynamically regulated during activation. Furthermore, 127 genes were regulated by variants associated with immune-mediated diseases, with significant enrichment for dynamic effects. Our results emphasize the importance of studying context-specific gene expression regulation and provide insights into the mechanisms underlying genetic susceptibility to immune-mediated diseases.

Trans-ancestry analysis reveals genetic and nongenetic associations with COVID-19 susceptibility and severity.

COVID-19 presents with a wide range of severity, from asymptomatic in some individuals to fatal in others. Based on a study of 1,051,032 23andMe research participants, we report genetic and nongenetic associations with testing positive for SARS-CoV-2, respiratory symptoms and hospitalization. Using trans-ancestry genome-wide association studies, we identified a strong association between blood type and COVID-19 diagnosis, as well as a gene-rich locus on chromosome 3p21.31 that is more strongly associated with outcome severity. Hospitalization risk factors include advancing age, male sex, obesity, lower socioeconomic status, non-European ancestry and preexisting cardiometabolic conditions. While non-European ancestry was a significant risk factor for hospitalization after adjusting for sociodemographics and preexisting health conditions, we did not find evidence that these two primary genetic associations explain risk differences between populations for severe COVID-19 outcomes.

Rare variant analysis in eczema identifies exonic variants in DUSP1, NOTCH4 and SLC9A4.

Previous genome-wide association studies revealed multiple common variants involved in eczema but the role of rare variants remains to be elucidated. Here, we investigate the role of rare variants in eczema susceptibility. We meta-analyze 21 study populations including 20,016 eczema cases and 380,433 controls. Rare variants are imputed with high accuracy using large population-based reference panels. We identify rare exonic variants in DUSP1, NOTCH4, and SLC9A4 to be associated with eczema. In DUSP1 and NOTCH4 missense variants are predicted to impact conserved functional domains. In addition, five novel common variants at SATB1-AS1/KCNH8, TRIB1/LINC00861, ZBTB1, TBX21/OSBPL7, and CSF2RB are discovered. While genes prioritized based on rare variants are significantly up-regulated in the skin, common variants point to immune cell function. Over 20% of the single nucleotide variant-based heritability is attributable to rare and low-frequency variants. The identified rare/low-frequency variants located in functional protein domains point to promising targets for novel therapeutic approaches to eczema.

Variants in a novel epidermal collagen gene (COL29A1) are associated with atopic dermatitis.

Atopic dermatitis (AD) is a common chronic inflammatory skin disorder and a major manifestation of allergic disease. AD typically presents in early childhood often preceding the onset of an allergic airway disease, such as asthma or hay fever. We previously mapped a susceptibility locus for AD on Chromosome 3q21. To identify the underlying disease gene, we used a dense map of microsatellite markers and single nucleotide polymorphisms, and we detected association with AD. In concordance with the linkage results, we found a maternal transmission pattern. Furthermore, we demonstrated that the same families contribute to linkage and association. We replicated the association and the maternal effect in a large independent family cohort. A common haplotype showed strong association with AD (p = 0.000059). The associated region contained a single gene, COL29A1, which encodes a novel epidermal collagen. COL29A1 shows a specific gene expression pattern with the highest transcript levels in skin, lung, and the gastrointestinal tract, which are the major sites of allergic disease manifestation. Lack of COL29A1 expression in the outer epidermis of AD patients points to a role of collagen XXIX in epidermal integrity and function, the breakdown of which is a clinical hallmark of AD.

An interaction between filaggrin mutations and early food sensitization improves the prediction of childhood asthma.

BACKGROUND: Asthma prediction in early infancy is essential for the development of new preventive strategies. Loss-of-function mutations in the filaggrin gene (FLG) were identified as risk factors for eczema and associated asthma. OBJECTIVE: We evaluated the utility of the FLG mutations for the prediction of asthma. METHODS: Eight hundred seventy-one individuals of the prospective German Multicenter Allergy Study cohort were genotyped for 3 FLG mutations. Information on asthma, eczema, and food sensitization was available from birth to 13 years of age. Pulmonary function was measured from 7 to 13 years of age. The predictive value of the FLG mutations and of atopic phenotypes in infancy was assessed for asthma. RESULTS: In infants with eczema and sensitization to food allergens, the FLG mutations predicted childhood asthma with a positive predictive value of 100% (95% CI, 65.5% to 100%). This subgroup was characterized by a significant decrease in pulmonary function until puberty and represented 8.1% of all asthmatic children and 19.1% of patients with asthma after infantile eczema. We found a strong synergistic interaction between the FLG-null alleles and early food sensitization in the disease transition from eczema to asthma (relative excess risk due to interaction, 2.64; 95% CI, 1.70-3.98; P = .00040). CONCLUSION: FLG mutations and food sensitization represent 2 distinct mechanisms interacting in the pathogenesis of asthma. In infants with eczema and food sensitization, genotyping of the FLG mutations allows the prediction of asthma before the onset of symptoms. Our findings might facilitate the development of early subgroup-specific interventions to prevent the progression from eczema to asthma.

Single-cell transcriptomics identifies an effectorness gradient shaping the response of CD4+ T cells to cytokines.

Naïve CD4+ T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. Here we use quantitative proteomics, bulk RNA-seq, and single-cell RNA-seq of over 40,000 human naïve and memory CD4+ T cells to show that responses to cytokines differ substantially between these cell types. Memory T cells are unable to differentiate into the Th2 phenotype, and acquire a Th17-like phenotype in response to iTreg polarization. Single-cell analyses show that T cells constitute a transcriptional continuum that progresses from naïve to central and effector memory T cells, forming an effectorness gradient accompanied by an increase in the expression of chemokines and cytokines. Finally, we show that T cell activation and cytokine responses are influenced by the effectorness gradient. Our results illustrate the heterogeneity of T cell responses, furthering our understanding of inflammation.

Chromatin activity at GWAS loci identifies T cell states driving complex immune diseases.

Immune-disease-associated variants are enriched in active chromatin regions of T cells and macrophages. However, whether these variants function in specific cell states is unknown. Here we stimulated T cells and macrophages in the presence of 13 cytokines and profiled active and open chromatin regions. T cell activation induced major chromatin remodeling, while the presence of cytokines fine-tuned the magnitude of changes. We developed a statistical method that accounts for subtle changes in the chromatin landscape to identify SNP enrichment across cell states. Our results point towards the role of immune-disease-associated variants in early rather than late activation of memory CD4+ T cells, with modest differences across cytokines. Furthermore, variants associated with inflammatory bowel disease are enriched in type 1 T helper (TH1) cells, whereas variants associated with Alzheimer's disease are enriched in different macrophage cell states. Our results represent an in-depth analysis of immune-disease-associated variants across a comprehensive panel of activation states of T cells and macrophages.

Identification of new therapeutic targets for osteoarthritis through genome-wide analyses of UK Biobank data.

Osteoarthritis is the most common musculoskeletal disease and the leading cause of disability globally. Here, we performed a genome-wide association study for osteoarthritis (77,052 cases and 378,169 controls), analyzing four phenotypes: knee osteoarthritis, hip osteoarthritis, knee and/or hip osteoarthritis, and any osteoarthritis. We discovered 64 signals, 52 of them novel, more than doubling the number of established disease loci. Six signals fine-mapped to a single variant. We identified putative effector genes by integrating expression quantitative trait loci (eQTL) colocalization, fine-mapping, and human rare-disease, animal-model, and osteoarthritis tissue expression data. We found enrichment for genes underlying monogenic forms of bone development diseases, and for the collagen formation and extracellular matrix organization biological pathways. Ten of the likely effector genes, including TGFB1 (transforming growth factor beta 1), FGF18 (fibroblast growth factor 18), CTSK (cathepsin K), and IL11 (interleukin 11), have therapeutics approved or in clinical trials, with mechanisms of action supportive of evaluation for efficacy in osteoarthritis.

A common haplotype of the IL-31 gene influencing gene expression is associated with nonatopic eczema.

BACKGROUND: IL-31 is a novel cytokine that, when overexpressed in transgenic mice, induces severe itching dermatitis resembling human eczema. OBJECTIVE: We aimed to evaluate the importance of polymorphisms in the human IL-31 gene (IL31) in the genetic susceptibility to eczema. METHODS: We sequenced the entire IL-31 gene, including the promoter region, and determined the haplotype structure. Single nucleotide polymorphisms tagging the main haplotypes were genotyped in 3 independent European populations comprising 690 affected families. An association analysis of IL31 gene variants with atopic and nonatopic eczema was performed. RESULTS: We found significant association of a common IL31 haplotype with the nonatopic type of eczema in all 3 study populations (combined P = 4.5 x 10(-5)). Analysis of PBMCs in healthy individuals revealed a strong induction IL31 mRNA expression on stimulation with anti-CD3 and anti-CD28 that was 3.8-fold higher in individuals homozygous for the risk haplotype (AA) in contrast to non-A haplotype carriers, suggesting that altered regulation of IL-31 gene expression is the disease-causing factor. CONCLUSION: Our results lend strong support to an important role of IL-31 in the pathogenesis of nonatopic eczema. CLINICAL IMPLICATIONS: This study presents the first genetic risk factor for the nonatopic type of eczema and indicates a primary role of IL-31-induced pruritus in the initiation of this disease, thus proposing a new target for the prevention and therapy of eczema.

Insights into hemolytic uremic syndrome: segregation of three independent predisposition factors in a large, multiple affected pedigree.

Mutations in the complement regulators factor H, membrane cofactor protein (MCP), and factor I are associated with atypical hemolytic uremic syndrome (aHUS, MIM 235400), suggesting that the disease develops as a consequence of the inefficient protection of the renal endothelium from damage by the complement system. Incomplete penetrance of the disease in individuals carrying these mutations is, however, relatively frequent. Here, we report the identification of a large, multiple affected aHUS pedigree in which there is independent segregation of three different aHUS risk factors: a MCP missense mutation (c.-598C>T; Pro165Ser) that decreases MCP expression on the cell surface, a dinucleotide insertion in the coding sequence of factor I (c.-1610insAT) that introduces a premature stop codon in the factor I protein, and the MCPggaac SNP haplotype block that was previously shown to decrease the transcription activity from the MCP promoter. Interestingly, individuals affected by aHUS in the pedigree are only those who have inherited the three aHUS risk factors. These data show an additive effect for mutations in MCP and factor I and provide definitive support to the conclusion that aHUS results from a defective protection of cellular surfaces from complement activation. Furthermore, they help to explain the incomplete penetrance of the disease, illustrating that concurrence of multiple hits in complement regulatory proteins may be necessary to significantly impair host tissue protection and to confer susceptibility to aHUS.

Shared genetic origin of asthma, hay fever and eczema elucidates allergic disease biology.

Asthma, hay fever (or allergic rhinitis) and eczema (or atopic dermatitis) often coexist in the same individuals, partly because of a shared genetic origin. To identify shared risk variants, we performed a genome-wide association study (GWAS; n = 360,838) of a broad allergic disease phenotype that considers the presence of any one of these three diseases. We identified 136 independent risk variants (P < 3 × 10-8), including 73 not previously reported, which implicate 132 nearby genes in allergic disease pathophysiology. Disease-specific effects were detected for only six variants, confirming that most represent shared risk factors. Tissue-specific heritability and biological process enrichment analyses suggest that shared risk variants influence lymphocyte-mediated immunity. Six target genes provide an opportunity for drug repositioning, while for 36 genes CpG methylation was found to influence transcription independently of genetic effects. Asthma, hay fever and eczema partly coexist because they share many genetic risk variants that dysregulate the expression of immune-related genes.

De novo gene conversion in the RCA gene cluster (1q32) causes mutations in complement factor H associated with atypical hemolytic uremic syndrome.

Many of the complement regulatory genes within the RCA cluster (1q32) have arisen through genomic duplication and the resulting high degree of sequence identity is likely to predispose to gene conversion events. The highest degree of identity is between the genes for factor H (CFH) and five factor H-related proteins--CFHL1, CFHL2, CFHL3, CFHL4, and CFHL5. CFH mutations are associated with atypical hemolytic uremic syndrome (aHUS). In the Newcastle cohort of 157 aHUS patients we have identified CFH mutations in 25 families or individuals. Eleven of these 25 independent mutations are either c.3226C>G,Q1076E; c.3572C>T,S1191L; c.3590T>C,V1197A or combined c.3572C>T,S1191L/c.3590T>C,V1197A. Sequence analysis shows that all four of these changes could have arisen as a result of gene conversion between CFH and CFHL1. Analysis of parental samples in two patients with S1191L/V1197A has shown that the changes are de novo thus providing conclusive evidence that gene conversion is the mutational mechanism in these two cases. To confirm that S1191L and V1197A are disease predisposing we examined their functional significance in three ways - analysis of the C3b/C3d binding characteristics of recombinant mutant S1191L/V1197A protein, heparin affinity chromatography and haemolytic assays of serum samples from aHUS patients carrying these changes. The results showed that these changes resulted in impaired C3b binding and a defective capacity to control complement activation on cellular surfaces. We, therefore, provide conclusive evidence that gene conversion is responsible for functionally significant CFH mutations in aHUS.