Epidemiological Approach to Identifying Genetic Predispositions for Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is caused by several susceptibility genes. A registry including analyses of susceptibility genes, familial occurrence and genotype-phenotype correlation should provide classification insights.
Registry data of 187 unrelated index patients included age at onset, gender, family history, relapse of aHUS and potentially triggering conditions. Mutation analyses were performed in the genes CFH , CD46 and CFI and in the six potential susceptibility genes, FHR1 to FHR5 and C4BP .
Germline mutations were identified in 17% of the index cases; 12% in CFH , 3% in CD46 and 2% in CFI . Twenty-nine patients had heterozygous mutations and one each had a homozygous and compound heterozygous mutation. Mutations were not found in the genes FHR1-5 and C4BP . In 40% of the patients with familial HUS a mutation was found. Penetrance by age 45 was 50% among carriers of any mutation including results of relatives of mutation-positive index cases. The only risk factor for a mutation was family history of HUS (p = 0.02).
P enetrance of aHUS in carriers of mutations is not complete. Occurrence of homo- and heterozygous mutations in the same gene suggests that the number of necessary DNA variants remains unclear. Among clinical information only familial occurrence predicts a mutation.     

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.     

Hemolytic Uremic Syndrome following Infection with O111 Shiga Toxin-Producing Escherichia coli Revealed through Molecular Diagnostics

We report a case of hemolytic uremic syndrome in a 69-year-old woman due to Shiga toxin-producing Escherichia coli, possibly serotype O111, to illustrate the potentially deleterious implications of a Campylobacter enzyme immunoassay (EIA) result and the increasing importance of molecular testing when conventional methods are limited.     

Functional Analysis of a De Novo ACTB Mutation in a Patient with Atypical Baraitser–Winter Syndrome

Exome sequence analysis can be instrumental in identifying the genetic etiology behind atypical disease. We report a patient presenting with microcephaly, dysmorphic features, and intellectual disability with a tentative diagnosis of Dubowitz syndrome. Exome analysis was performed on the patient and both parents. A de novo missense variant was identified in ACTB, c.349G>A, p.E117K. Recent work in Baraitser–Winter syndrome has identified ACTB and ACTG1 mutations in a cohort of individuals, and we rediagnosed the patient with atypical Baraitser–Winter syndrome. We performed functional characterization of the variant actin and show that it alters cell adhesion and polymer formation supporting its role in disease. We present the clinical findings in the patient, comparison of this patient to other patients with ACTB/ACTG1 mutations, and results from actin functional studies that demonstrate novel functional attributes of this mutant protein.     

A Family with Complement Factor I Deficiency

A family with inherited factor I deficiency is described. The proband was a 19-year-old Caucasian female with one episode of meningococcal meningitis and one episode of suspected septicaemia of unknown cause. Two obligate and two probable heterozyotes with factor I levels below the lower limit of the reference range were identified. None of these exhibited increased susceptibility to infectious diseases. The inheritance was autosomal codominant. In addition, molecular heterogeneity of factor H in plasma from the proband but not from any other family members was demonstrated by crossed Immunoelectrophoresis. The migration of factor H component of fast electrophoretic mobility was retarded by antibodies to C3eand C3d. suggesting the presence of a fluid-phase complex between factor H and excess C3h generated by the uncontrolled activity of the amplification loop.     

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...     

A Case of Congenital Central Hypoventilation Syndrome with PHOX2B Gene Mutation in a Korean Neonate

Congenital central hypoventilation syndrome (CCHS) is a life-threatening disorder with apnea and cyanosis during sleep requiring immediate endotracheal intubation during the first day of life. The PHOX2B gene has been identified as the major gene involved in CCHS. This is the first report of a Korean neonate with CCHS confirmed to have a PHOX2B mutation with expanded alleles containing 20 polyalanine repeats that is a relatively small number compared to previous cases. The patient required intermittent ventilator support during sleep only and did not suffer from any other disorders of the autonomic nerve system. He consistently needs ventilator support during sleep and remains alive. Analysis of PHOX2B gene is useful for diagnosis and appropriate therapeutic intervention of CCHS patients.     

Targeted Deep Resequencing Identifies MID2 Mutation for X‐Linked Intellectual Disability with Varied Disease Severity in a Large Kindred from India

We report a novel missense mutation (c.1040G>A, p.Arg347Gln) in MID2, which encodes ubiquitin ligase E3, as the likely cause of X‐linked mental retardation in a large kindred. The mutation was observed in all affected and obligate carriers but not in any unaffected males of the family or in population controls (n = 200). When transiently expressed in HEK293T cell line, the mutation was found to abolish the function of the COS domain in the protein. The GFP‐tagged mutant protein accumulated in the cytoplasm instead of binding to the cytoskeleton resulting in its altered subcellular localization. Screening of coding exons of this gene in additional 480 unrelated individuals with idiopathic intellectual disability identified another novel variation p.Asn343Ser. This study highlights the growing role of the ubiquitin pathway in intellectual disability and also, the difference in MID2 determined phenotype observed in this study compared with that of its paralogue MID1 reported in literature.     

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.     

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.     

Gerstmann‐Sträussler‐Scheinker Syndrome with Variable Phenotype in a New Kindred with PRNP‐P102L Mutation

Gerstmann‐Sträussler‐Scheinker syndrome (GSS) is a dominantly inherited disorder belonging to the group of transmissible human spongiform encephalopathies or prion diseases. Several families affected by GSS with patients carrying mutations in the prion protein gene have been described worldwide. We report clinical, genealogical, neuropathology and molecular study results from two members of the first Argentine kindred affected by GSS. Both family members presented a frontotemporal‐like syndrome, one with and the other without ataxia, with different lesions on neuropathology. A Pro to Leu point mutation at codon 102 (P102L) of the prion protein gene was detected in one of the subjects studied. The pathogenic basis of phenotypic variability observed in this family remains unclear, but resembles that observed in other P102L GSS patients from the same family.     

A Novel Mutation in the Complement Component 3 Gene in a Patient with Selective IgA Deficiency

Purpose

Immunological and molecular evaluation of a patient presenting with recurrent infections caused by Streptococcus pneumoniae and low complement component 3 (C3) levels.

Methods

Immunological evaluation included complement components and immunoglobulin level quantification as well as number and function of T cells, B cells and neutrophils. Serotype-specific immunoglobulin G antibodies against S. pneumoniae capsular polysaccharides were quantified by ELISA in serum samples before and after vaccination with unconjugated polysaccharide vaccine. For the molecular analysis, genomic DNA from the patient and parents were isolated and all exons as well as exon-intron boundaries of the C3 gene were sequenced by Sanger sequencing.

Results

A 16-year-old male, born to consanguineous parents, presented with recurrent episodes of pneumonia caused by S. pneumoniae and bronchiectasis. The patient showed severely reduced C3 and immunoglobulin A levels, while the parents showed moderately reduced levels of C3. Mutational analysis revealed a novel, homozygous missense mutation in the C3 gene (c. C4554G, p. Cys1518Trp), substituting a highly conserved amino acid in the C345C domain of C3 and interrupting one of its disulfide bonds. Both parents were found to be carriers of the affected allele. Vaccination against S. pneumoniae resulted in considerable clinical improvement.

Conclusions

We report a novel homozygous mutation in the C3 gene in a patient with concomitant selective IgA deficiency who presented with a marked clinical improvement after vaccination against S. pneumoniae. This observation underlines the notion that vaccination against this microorganism is an important strategy for treatment of PID patients, particularly those presenting with increased susceptibility to infections caused by this agent.     

Ankylosing Spondylitis in a Large Kindred: Clinical and Genetic Studies

In a kindred of 66 members spanning four generations, seven cases of ankylosing spondylitis (AS) have been found. Four of these were in a single sibship of 13. AS was associated with HL-A27 in three of the four involved siblings, but close linkage was shown to be unlikely. Knowledge of HL-A genotype has made possible informed counseling for younger members of the sibship of 13, some of whom, as teenagers, already have back pain.