Risk of Atypical HUS Among Family Members of Patients Carrying Complement Regulatory Gene Abnormality.

INTRODUCTION: Atypical hemolytic uremic syndrome (aHUS) is mainly due to complement regulatory gene abnormalities with a dominant pattern but incomplete penetrance. Thus, healthy carriers can be identified in any family of aHUS patients, but it is unpredictable if they will eventually develop aHUS. METHODS: Patients are screened for 10 complement regulatory gene abnormalities and once a genetic alteration is identified, the search is extended to at-risk family members. The present cohort study includes 257 subjects from 71 families: 99 aHUS patients (71 index cases + 28 affected family members) and 158 healthy relatives with a documented complement gene abnormality. RESULTS: Fourteen families (19.7%) experienced multiple cases. Over a cumulative observation period of 7595 person-years, only 28 family members carrying gene mutations experienced aHUS (overall penetrance of 20%), leading to a disease rate of 3.69 events for 1000 person-years. The disease rate was 7.47 per 1000 person-years among siblings, 6.29 among offspring, 2.01 among parents, 1.84 among carriers of variants of uncertain significance, and 4.43 among carriers of causative variants. CONCLUSIONS: The penetrance of aHUS seems a lot lower than previously reported. Moreover, the disease risk is higher in carriers of causative variants and is not equally distributed among generations: siblings and the offspring of patients have a much greater disease risk than parents. However, risk calculation may depend on variant classification that could change over time.

Kidney transplant in patients with atypical hemolytic uremic syndrome in the anti-C5 era: single-center experience with tailored Eculizumab.

RATIONALE AND OBJECTIVE: Patients with atypical hemolytic uremic syndrome (aHUS) have long been considered ineligible for kidney transplantation (KTx) in several centers due to the high risk of disease recurrence, graft loss and life-threatening complications. The availability of Eculizumab (ECU) has now overcome this problem. However, the best approach towards timing, maintenance schedule, the possibility of discontinuation and patient monitoring has not yet been clearly established. STUDY DESIGN: This is a single center case series presenting our experience with KTx in aHUS. SETTING AND PARTICIPANTS: This study included 26 patients (16 females) with a diagnosis of aHUS, who spent a median of 5.5 years on kidney replacement therapy before undergoing KTx. We compared the aHUS relapse rate in three groups of patients who underwent KTx: patients who received no prophylaxis, patients who underwent plasma exchange, those who received Eculizumab prophylaxis. Complement factor H-related disease was by far the most frequent etiology (n = 19 patients). RESULTS: Untreated patients and patients undergoing pre-KTx plasma exchange prophylaxis had a relapse rate of 0.81 (CI 0.30-1.76) and 3.1 (CI 0.64-9.16) events per 10 years cumulative observation, respectively, as opposed to 0 events among patients receiving Eculizumab prophylaxis. The time between Eculizumab doses was tailored based on classic complement pathway activity (target to < 30%). Using this strategy, 12 patients are currently receiving  Eculizumab every 28 days, 5 every 24-25 days, and 3 every 21 days. CONCLUSION: Our experience supports the prophylactic use of Eculizumab in patients with a previous history of aHUS undergoing KTx, especially when complement dysregulation is well documented by molecular biology.

Functional Study of Novel Bartter's Syndrome Mutations in ClC-Kb and Rescue by the Accessory Subunit Barttin Toward Personalized Medicine.

Type III and IV Bartter syndromes (BS) are rare kidney tubulopathies caused by loss-of-function mutations in the CLCNKB and BSND genes coding respectively for the ClC-Kb chloride channels and accessory subunit barttin. ClC-K channels are expressed in the Henle's loop, distal convoluted tubule, and cortical collecting ducts of the kidney and contribute to chloride absorption and urine concentration. In our Italian cohort, we identified two new mutations in CLCNKB, G167V and G289R, in children affected by BS and previously reported genetic variants, A242E, a chimeric gene and the deletion of the whole CLCNKB. All the patients had hypokalemia and metabolic alkalosis, increased serum renin and aldosterone levels and were treated with a symptomatic therapy. In order to define the molecular mechanisms responsible for BS, we co-expressed ClC-Kb wild type and channels with point mutations with barttin in HEK 293 cells and characterized chloride currents through the patch-clamp technique. In addition, we attempted to revert the functional defect caused by BS mutations through barttin overexpression. G167V and A242E channels showed a drastic current reduction compared to wild type, likely suggesting compromised expression of mutant channels at the plasma membrane. Conversely, G289R channel was similar to wild type raising the doubt that an additional mutation in another gene or other mechanisms could account for the clinical phenotype. Interestingly, increasing ClC-K/barttin ratio augmented G167V and A242E mutants' chloride current amplitudes towards wild type levels. These results confirm a genotype-phenotype correlation in BS and represent a preliminary proof of concept that molecules functioning as molecular chaperones can restore channel function in expression-defective ClC-Kb mutants.

A putative protein structurally related to zygote arrest 1 (Zar1), Zar1-like, is encoded by a novel gene conserved in the vertebrate lineage.

Identification and characterization of a bovine cDNA and the corresponding gene coding for a novel protein structurally related to Zar1, therefore called Zar1-like, are here reported for the first time. Structure of Zar1-like is similar to Zar1 gene, nevertheless they are located on distinct chromosomes. We demonstrated that the new gene as well as its genomic context are conserved along the whole vertebrate lineage. Analysis of the deduced protein primary structure showed a high conservation, among vertebrates, of the C-terminal region, where the putative presence of both zinc finger motifs and classical nuclear localization signals is also shared with Zar1. Bovine Zar1-like and the only two other available mRNA leader sequences (human and chicken) exhibit a number of upstream AUGs, suggesting that they are likely to be regulated at translational level. Expression patterns of the cattle transcripts show that Zar1-like is absent in early stages of embryo development, whereas Zar1 is expressed in matured oocytes and in in vitro produced pre-implantation embryos. In adult tissues Zar1-like transcript expression appears to be less restricted than Zar1, nevertheless, at least in bovine, both mRNAs are co-expressed in gonads, raising the question of a possible functional link.

Shadoo, a new protein highly conserved from fish to mammals and with similarity to prion protein.

We report evidence from cDNA isolation and expression analysis as well as analyses of genome, expressed sequence tag (EST), cDNA and expression databases for a new gene named SPRN (shadow of prion protein). SPRN comprises two exons, with the open reading frame (ORF) contained within exon 2, and codes for a protein of 130-150 amino acids named Shadoo (Japanese shadow), predicted to be extracellular and GPI-anchored. The SPRN gene was found in fish (zebrafish, Fugu) and mammals (mouse, rat, human). Conservation of order and transcription orientation of two proximal genes between fishes and mammals strongly indicates gene orthology. Sequence comparison shows: a highly conserved N-terminal signal sequence; Arg-rich basic region containing up to six tetrarepeats of consensus XXRG (where X is G, A or S); a hydrophobic region of 20 residues with strong homology to PrP; a less conserved C-terminal domain containing a conserved glycosylation motif; and a C-terminal peptide predicted to be a signal sequence for glycophosphotidylinositol (GPI)-anchor attachment. Fish Shadoos (Sho) show well conserved sequences (identity 54%) over 106 amino acids (zebrafish length), and conservation among the mammalian sequences is very high (identity 81-96%). The fish and mammalian sequences are also well conserved, particularly for zebrafish, to beyond the end of the hydrophobic sequence (identity 41-53%, 78 amino acids, zebrafish length). The overall structure appears closely related to prion proteins (PrPs), although the C-terminal domains of Shos are quite different from those of PrPs, for which conformational changes in mammals are implicated in disease. The structural similarity is particularly interesting given recent reports of three new genes with similarities to PrPs found in Fugu (PrP-like, PrP-461/stPrP-1 and stPrP-2) and other fish, but for which direct evolution to higher vertebrate PrPs is unlikely and for which no other mammalian homologues have been found. Database information indicates expression of SPRN in embryo, brain and retina of mouse and rat, hippocampus of human, and in embryo and retina of zebrafish, and we directly confirmed a strikingly specific expression of the mammalian (human, mouse, rat) transcripts in whole brain. This result together with some common structural features led to the suggestive hypothesis of a possible functional link between mammalian PrP and Sho proteins.

On the catalytic role of the active site residue E121 of E. coli L-aspartate oxidase.

L-aspartate oxidase (LASPO) is a flavoenzyme catalyzing the first step in the de novo biosynthesis of NAD+. The enzyme oxidizes L-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor. In accordance with its catalytic activities, LASPO displays strong primary and tertiary structure similarity with the flavin containing subunit of the proteins belonging to the succinate dehydrogenase/fumarate reductase family. The similarity extends to the active site residues, with LASPO differing from the other enzymes of the family only for the presence of a conserved glutamate (E121), which is substituted by apolar amino acids in the other enzymes. Three complementary approaches have been used to define the role of E121 in LASPO: characterization of mutants (E121A, E121Q, E121D and E121K), investigation of the catalytic activities of WT and mutants towards substrates and substrate analogues and molecular docking studies. All mutants retain fumarate reductase activity. On the contrary, all mutants lack L-aspartate oxidase activity, although retaining the ability to bind L-aspartate (except for E121K). These results and investigations on the oxidase activity towards substrate analogues suggest that the roles of E121 in catalysis include orienting L-aspartate in a productive binding mode and favouring proton abstraction from C2 by an active site base. Molecular docking studies of the substrate (L-aspartate), inhibitor (D-aspartate) and product (imino aspartate) in the active site of LASPO confirm that (a) the substrate/product energetically favoured orientation in the active site supports the conclusions reported above, (b) E121 interacts favourably with the charged amino group of the substrate and (c) different ligands might assume different orientations in the active site of the enzyme.