Deletion of CFHR3 and CFHR1 genes in age-related macular degeneration

Age-related macular degeneration (AMD) impairs vision for approximately 7.5 million Americans. Both susceptibility variants and protective haplotypes in the complement factor H (CFH) gene modulate risk for AMD. Recently, deletion of the 'CFH-related' genes CFHR1 and CFHR3 was found to be segregating with a particular CFH haplotype, which reduced the risk of AMD. We tested the deletion for association in a Caucasian population of 780 cases and 265 controls and examined its effect in the context of known AMD risk factors. The deletion did not segregate perfectly with any one SNP, as previously suggested. CFH haplotype P2 was the most frequent haplotype in deletion homozygotes (47%), and the majority (14/16) of these individuals were homozygous for the non-risk allele of Y402H. Overall, deletion homozygosity was significantly more frequent in controls than cases (2.6% controls, 0.8% cases, P = 0.025, OR = 0.29, 95% CI = 0.10-0.86). After controlling for age, Y402H, smoking and LOC387715 A69S, the protective effect of the deletion was no longer statistically significant (P = 0.27). However, using a CFH haplotype that all deletion homozygotes share as a surrogate for the deletion, this marker remained modestly associated with AMD after adjustment for known risk factors (OR = 0.63, 95% CI 0.39-1.04, P = 0.07). Therefore, deletion of CFHR1 and CFHR3 may account for a small portion of the protection from AMD associated with particular haplotypes in CFH. The presence of protective haplotypes in CFH that do not carry the deletion, suggests that other protective variants in this region have yet to be discovered.     

Cytokine Production Is Altered in Monocytes from Children with Hemolytic Uremic Syndrome

Purpose

The interaction of Shiga toxin (Stx) and/or lipopolysaccharide (LPS) with monocytes (Mo) may be central to the pathogenesis of hemolytic uremic syndrome (HUS), providing the cytokines necessary to sensitize endothelial cells to Stx action. We have previously demonstrated phenotypical alterations in Mo from HUS patients, including increased number of CD16+ Mo. Our aim was to investigate cytokine production in Mo from HUS patients.

Methods

We evaluated TNF-α and IL-10 intracellular contents and secretion in the different Mo subsets in mild (HUS 1) and moderate/severe (HUS 2?+?3) patients. As controls, we studied healthy (HC) and infected children (IC). We also studied Mo responsive capacity towards LPS, measuring the modulation of Mo surface molecules and cytokine production.

Results

In basal conditions, the intracellular measurement of TNF-α and IL-10 revealed that the highest number of cytokine-producing Mo was found in HUS 2?+?3 and IC, whereas LPS caused a similar increase in TNF-α and IL-10-producing Mo for all groups. However, when evaluating the release of TNF-α and IL-10, we found a diminished secretion capacity in the entire HUS group and IC compared to HC in basal and LPS conditions. Similarly, a lower Mo response to LPS in HUS 2?+?3 and IC groups was observed when surface markers were studied.

Conclusion

These results indicate that Mo from severe cases of HUS, similar to IC but different to mild HUS cases, present functional changes in Mo subpopulations and abnormal responses to LPS.     

Functional analysis in serum from atypical Hemolytic Uremic Syndrome patients reveals impaired protection of host cells associated with mutations in factor H

A subgroup of patients with the most severe form of the Hemolytic Uremic Syndrome (HUS) presents mutations in the complement regulatory protein factor H. The functional analyses of the factor H mutant proteins purified from some of these patients have shown a specific defect in the capacity to control complement activation on cellular surfaces. Here, we show that these factor H-related complement regulatory defects can be detected in the patients' serum with a simple hemolytic assay. Data obtained from HUS patients and control individuals indicate that this assay is a useful tool for the molecular diagnosis of factor H-related HUS.     

A Complement Factor B Mutation in a Large Kindred with Atypical Hemolytic Uremic Syndrome

Purpose

Gain-of-function mutations in complement factor B (CFB) were recently identified in patients with atypical hemolytic uremic syndrome (aHUS), but are extremely rare. Our purpose is to describe a large kindred with aHUS associated with a CFB mutation and to further understand CFB-mutated aHUS patients.

Methods and Results

We report a large kindred in which 3 members had aHUS. This kindred revealed that 9 of 12 members, including 2 affected patients, had persistent activation of the alternative pathway with low complement component 3 and that those 9 members showed a CFB mutation (c.1050G?>?C, p.Lys350Asn) in exon 8. This missense mutation was heterozygous in 8 of them and homozygous in only one. From structural studies, this mutation is shown to be located in close proximity to the Mg²-binding site within a von Willebrand factor type A domain of CFB, resulting in a gain-of-function effect of CFB and predisposition to aHUS. At present, 2 of the 3 members with aHUS have maintained normal renal function for a long-term period.

Conclusions

This kindred illustrates that a CFB mutation (c.1050G?>?C, p.Lys350Asn) can result in aHUS. In the future, phenotype-genotype correlations and outcome in CFB-mutated aHUS patients need to be further investigated by accumulation of a number of cases.     

血浆置换联合连续血液滤过救治小儿溶血尿毒综合征的机制探讨

血浆置换(PE)联合连续血液净化(CBP)对小儿溶血尿毒综合征(HUS)的疗效观察及机制探讨.回顾性分析21例重型HUS 患儿在内科综合治疗同时,采用序贯血浆置换(PE)加连续性静- 静脉血液滤过(CVVH)治疗前后生命体征、炎性细胞因子、生化指标等的变化.经序贯PE加CBP治疗后患儿全身状况迅速改善,溶血控制,尿量增...     

Factor H and CFHR1 polymorphisms associated with atypical Haemolytic Uraemic Syndrome (aHUS) are differently expressed in Tunisian and in Caucasian populations

Several polymorphisms in the complement components factor H and CFHR1 are associated with higher risk to develop atypical Haemolytic Uraemic Syndrome (aHUS) in Caucasians. We have determined the prevalence of these polymorphisms in Tunisian controls by using genetic and immunological techniques. No differences in the frequency of the factor H risk alleles c.-331C>T, c.2089A>G or c.2881G>T between Tunisian and Caucasians were found. On the contrary, the analysis of CFHR1 polymorphism revealed a higher frequency of Tunisian individuals homozygous for the CFHR1*Del (deleted) allele, and of individuals presenting the CFHR1*A phenotype. These results suggest distinct contributions of factor H and CFHR1 polymorphisms to aHUS in Tunisian and Caucasian populations.     

Distinct Renal Pathology and a Chemotactic Phenotype after Enterohemorrhagic Escherichia coli Shiga Toxins in Non-Human Primate Models of Hemolytic Uremic Syndrome

Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48 hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention.Contamination of food and water sources with Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) is a global cause of sporadic outbreaks of painful diarrhea and hemorrhagic colitis^1–3 with an estimated 176,000 cases in the United States annually and approximately one death for every 1000 infections.^4,5 Symptoms arise within 3 to 4 days after infection and most resolve, but 5% to approximately 10% of patients progress to develop hemolytic uremic syndrome (HUS).⁶ Postdiarrheal HUS is characterized by thrombocytopenia, nonimmune hemolytic anemia, and thrombotic microangiopathy, often progressing to acute renal injury with severe cases requiring renal dialysis.⁷ The most vulnerable to infection are the young and elderly,⁸ and EHEC infections are a leading cause of acute renal failure in otherwise healthy children in the United States.EHEC bacteria attach to the intestinal epithelium with characteristic attaching and effacing lesions, which allows type III secretion of bacterial effector proteins and the Shiga toxin type-1 and type-2 toxins (Stx1, Stx2) and several variants into the host.⁹ Bacteremia is rare, and these toxins are primary contributors to the development of HUS and organ damage.¹⁰ The strain often associated with greatest severity is the O157:H7 serotype,¹¹ although there are dozens of pathogenic strains. New strains are emerging with greater virulence as experienced in Germany during summer 2011 when a rare enteroaggregative E. coli O104:H4 strain that causes otherwise self-limiting diarrhea acquired both a stx2 gene and aggressive virulence.^12–14 This is a matter of considerable concern because antibiotics increase HUS risk,¹⁵ and no toxin-specific therapies are available.The relative contribution of the two toxins to organ injury is difficult to distinguish in patients because EHEC strains can secrete one or both toxins in differing ratios, and the EHEC strain may not be identified or reported. Organ injury is assumed to be roughly equivalent between the toxins, although postdiarrheal renal injury is more commonly associated with EHEC strains that secrete Stx2.¹⁰ There is suggestion that inhibition of only Stx2 is necessary for therapeutic relief,¹⁶ but no data are available that directly compares the toxins in an animal model that presents with full-spectrum HUS.In ongoing studies to develop clinically relevant EHEC and HUS animal models, we are characterizing the pathophysiology elicited by Stx1 or Stx2 in juvenile baboons (Papio). We previously showed that, when the toxins are administered intravenously, they elicit thrombocytopenia, hemolytic anemia, thrombotic microangiopathy, and acute kidney injury, consistent with HUS.¹⁷ Using this model, we demonstrated rescue of the animals from an otherwise lethal Stx2 challenge and preservation of kidney function, with a custom-designed anti-Stx2 synthetic peptide.¹⁸ When comparing effects of the two toxins, we observed substantial distinguishing features, including different proinflammatory responses and different timing with delayed organ injury after Stx2 challenge. We present here detailed pathology examinations of kidney tissue from the animals challenged with Stx1 or Stx2 and cytokine analyses that extend our prior characterizations of kidney injury. In the baboon model, as in humans, the glomeruli are a particular target of the toxins, but injury is not exclusive to that structure. Stx1 and Stx2 had distinct effects on the glomeruli, with endothelial injury predominating with Stx1 and mesangial injury a predominant feature with Stx2. Both toxins elicited a marked chemotactic environment in the kidneys and other organs that may contribute to the pathophysiology.     

Variations in the complement regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated with membranoproliferative glomerulonephritis type II (dense deposit disease)

Introduction

Membranoproliferative glomerulonephritis type II or dense deposit disease (MPGN II/DDD) causes chronic renal dysfunction that progresses to end stage renal disease in about half of patients within 10 years of diagnosis. Deficiency of and mutations in the complement factor H (CFH) gene are associated with the development of MPGN II/DDD, suggesting that dysregulation of the alternative pathway of the complement cascade is important in disease pathophysiology.

Subjects

Patients with MPGN II/DDD were studied to determine whether specific allele variants of CFH and CFHR5 segregate preferentially with the MPGN II/DDD disease phenotype. The control group was compromised of 131 people in whom age related macular degeneration had been excluded.

Results

Allele frequencies of four single nucleotide polymorphisms in CFH and three in CFHR5 were significantly different between MPGN II/DDD patients and controls.

Conclusion

We have identified specific allele variants of CFH and CFHR5 associated with the MPGN II/DDD disease phenotype. While our data can be interpreted to further implicate complement in the pathogenesis of MPGN II/DDD, these associations could also be unrelated to disease pathophysiology. Functional studies are required to resolve this question.     

Upregulation of Shiga Toxin Receptor CD77/Gb3 and Interleukin‐1β Expression in the Brain of EHEC Patients with Hemolytic Uremic Syndrome and Neurologic Symptoms

In 2011, a large outbreak of Shiga toxin‐producing enterohemorrhagic Escherichia coli (EHEC) infections occurred in northern Germany, which mainly affected adults. Out of 3842 patients, 104 experienced a complicated course comprising hemolytic uremic syndrome and neurological complications, including cognitive impairment, aphasia, seizures and coma. T2 hyperintensities on magnet resonance imaging (MRI) bilateral in the thalami and in the dorsal pons were found suggestive of a metabolic toxic effect. Five of the 104 patients died because of toxic heart failure. In the present study, the post‐mortem neuropathological findings of the five EHEC patients are described. Histological investigation of 13 brain regions (frontal, temporal, occipital cortex, corpora mammillaria, thalamus, frontal operculum, corona radiata, gyrus angularis, pons, medulla oblongata, cerebellar vermis and cerebellar hemisphere) showed no thrombosis, ischemic changes or fresh infarctions. Further, no changes were found in electron microscopy. In comparison with five age‐matched controls, slightly increased activation of microglia and a higher neuronal expression of interleukin‐1β and of Shiga toxin receptor CD77/globotriaosylceramide 3 was observed. The findings were confirmed by Western blot analyses. It is suggested that CD77/globotriaosylceramide upregulation may be a consequence to Shiga toxin exposure, whereas increased interleukin‐1β expression may point to activation of inflammatory cascades.     

High prevalence iron receptor genes of avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli are known to cause significant losses in the poultry industry worldwide. Although prophylactic measures based on vaccination are advisable, until now no full heterologous protection against colibacillosis has been achieved. Since iron is an essential nutrient to these bacteria, the aim of this study was to investigate the prevalence of 12 outer-membrane iron receptor genes in 239 pathogenic strains isolated from clinical cases of colibacillosis in chickens. Five multiplex polymerase chain reactions were developed as a tool for efficient screening. Among the 239 avian E. coli isolates, 100% were positive for fhuE and fepA, 96.2% for fiu, 92.9% for cir, 92.5% for iroN, 87.4% for iutA, 63.2% for fecA, 53.1% for fyuA, 46.9% for fhuA, 45.6% for ireA, 41.8% for chuA and 4.6% for iha.     

High prevalence of founder mutations of the succinate dehydrogenase genes in the Netherlands

Mutations in four genes encoding subunits or cofactors of succinate dehydrogenase (SDH) cause hereditary paraganglioma and pheochromocytoma syndromes. Mutations in SDHB and SDHD are generally the most common, whereas mutations in SDHC and SDHAF2 are far less frequently observed. A total of 1045 DNA samples from Dutch paraganglioma and pheochromocytoma patients and their relatives were analyzed for mutations of SDHB, SDHC, SDHD or SDHAF2. Mutations in these genes were identified in 690 cases, 239 of which were index cases. The vast majority of mutation carriers had a mutation in SDHD (87.1%). The second most commonly affected gene was SDHAF2 (6.7%). Mutations in SDHB were found in only 5.9% of samples, whereas SDHC mutations were found in 0.3% of samples. Remarkably, 69.1% of all carriers of a mutation in an SDH gene in the Netherlands can be attributed to a single founder mutation in SDHD, c.274G>T and p.Asp92Tyr. Moreover, 88.8% of all SDH mutation carriers carry one of just six Dutch founder mutations in SDHB, SDHD and SDHAF2. The dominance of SDHD mutations is unique to the Netherlands, contrasting with the higher prevalence of SDHB mutations found elsewhere. In addition, we found that most SDH mutation-related paragangliomas-pheochromocytomas in the Netherlands can be explained by only six founder mutations in SDHAF2, SDHB and SDHD. The findings underline the regional differences in the SDH mutation spectrum, differences that should be taken into account in the development of effective screening protocols. The results show the crucial role that demographic factors play in the frequency of gene mutations.