Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

Background

Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool.

Methods

We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3).

Results

Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel.

Conclusions

Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy.     

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

Background  

The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings.     

The External Aldimine Form of Serine Palmitoyltransferase: STRUCTURAL, KINETIC, AND SPECTROSCOPIC ANALYSIS OF THE WILD-TYPE ENZYME AND HSAN1 MUTANT MIMICS*

Sphingolipid biosynthesis begins with the condensation of l-serine and palmitoyl-CoA catalyzed by the PLP-dependent enzyme serine palmitoyltransferase (SPT). Mutations in human SPT cause hereditary sensory autonomic neuropathy type 1, a disease characterized by loss of feeling in extremities and severe pain. The human enzyme is a membrane-bound hetereodimer, and the most common mutations are located in the enzymatically incompetent monomer, suggesting a “dominant” or regulatory effect. The molecular basis of how these mutations perturb SPT activity is subtle and is not simply loss of activity. To further explore the structure and mechanism of SPT, we have studied the homodimeric bacterial enzyme from Sphingomonas paucimobilis. We have analyzed two mutants (N100Y and N100W) engineered to mimic the mutations seen in hereditary sensory autonomic neuropathy type 1 as well as a third mutant N100C designed to mimic the wild-type human SPT. The N100C mutant appears fully active, whereas both N100Y and N100W are significantly compromised. The structures of the holoenzymes reveal differences around the active site and in neighboring secondary structure that transmit across the dimeric interface in both N100Y and N100W. Comparison of the l-Ser external aldimine structures of both native and N100Y reveals significant differences that hinder the movement of a catalytically important Arg³⁷⁸ residue into the active site. Spectroscopic analysis confirms that both N100Y and N100W mutants subtly affect the chemistry of the PLP. Furthermore, the N100Y and R378A mutants appear less able to stabilize a quinonoid intermediate. These data provide the first experimental insight into how the most common disease-associated mutations of human SPT may lead to perturbation of enzyme activity.Sphingolipids are ubiquitous constituents of eukaryotic cells, where they play important roles in signaling, differentiation, and apoptosis (1–4). Defects in sphingolipid catabolism have been linked to several human diseases, such as hypertension, cancer, and disorders of the peripheral nervous system. The most common inherited peripheral neuropathy is hereditary sensory autonomic neuropathy type 1 (HSAN1).⁶ The disease leads to progressive loss of sensation in extremities and is often associated with searing pain (5–8). Genetic studies by two independent groups mapped the disease-associated mutations to the lcb1 (long chain base 1) gene on chromosome 9q22, which encodes the SPT1 subunit of serine palmitoyltransferase (SPT; EC 2.3.1.50) (9–11). SPT catalyzes the first and rate-limiting step of the sphingolipid biosynthetic pathway in all organisms studied to date (12). The reaction is a pyridoxal 5′-phosphate (PLP)-dependent, decarboxylative, Claisen condensation of the amino acid l-serine and the long chain (C16) fatty acid palmitoyl-CoA, which produces the sphingolipid precursor, 3-ketodihydrosphingosine (KDS).SPT belongs to the α-oxoamine synthase subfamily of PLP-dependent enzymes, which contains three other well characterized members: 8-amino-7-oxononanoate synthase (AONS) (13, 14), 5-aminolevulinate synthase (ALAS) (15–17), and 2-amino-3-ketobutyrate-CoA ligase (KBL) (18). These enzymes catalyze the Claisen-like condensation between an amino acid and an acyl-coenzyme A thioester (19). A common mechanism has been proposed (12, 14, 20–23) comprising the following steps: formation of an external aldimine via displacement of the lysine-PLP internal aldimine (holo-SPT) by the incoming amino acid substrate; formation of a quinonoid intermediate by abstraction of the α-proton from the PLP-amino acid external aldimine; a Claisen condensation with the fatty acid-CoA thioester substrate, followed by displacement of the CoASH to form a β-ketoacid intermediate; decarboxylation of this species to form a product quinonoid; protonation of this quinonoid to form the product external aldimine; and finally release of the α-oxoamine product and regeneration of the enzyme PLP-internal aldimine (Fig. 1). Structural studies have shown AONS, ALAS, and KBL isoforms from various species to be homodimers (13, 17, 18). In marked contrast, eukaryotic SPTs are heterodimeric, membrane-bound proteins consisting of two subunits, SPT1 and SPT2 (encoded by the lcb1 and lcb2 gene, respectively) (24).Open in a separate windowFIGURE 1.Proposed catalytic mechanism of SPT. a, the internal aldimine (holo-SPT) is displaced by l-serine to form the external adlimine; b, binding of second substrate palmitoyl-CoA causes conformational change to give the Dunathan intermediate; c, formation of the quinonoid by deprotonation of Cα hydrogen; d, thioester bond hydrolysis and release of CoASH to give β-keto acid intermediate; e, decarboxylation to form KDS product quinonoid; f, reprotonation to form KDS product external aldimine; g, KDS product release and reformation of the holo-form. The l-serine external aldimine has been trapped in the current study. The dotted line shows that steps e and f may be bypassed by an alternative mechanism that does not proceed via a product quinonoid (see Ref. 21).Analysis of a number of HSAN1 patients has revealed the four most common mutations to be C133W, C133Y, V144D, and G387A, with the cysteine mutations appearing to be the most prevalent in populations (10, 25). The impact that these mutations have on SPT activity and sphingolipid metabolism has been the focus of attention by a number of groups. Most surprisingly, these disease-associated mutations occur in the SPT1 monomer, a protein that must be inactive, since it lacks the key lysine, histidine, and aspartate residues necessary to bind and stabilize the PLP cofactor. It is the SPT2 protein that contains the conserved, active site lysine residue that forms a Schiff base internal aldimine with the PLP. However, both subunits are essential to produce functionally active SPT heterodimer (26–29), indicating that the inactive subunit has a crucial role in function. Dunn and co-workers (30) created several mutations in the yeast lcb1 and lcb2 genes, including those corresponding to the HSAN1 SPT1 mutations, and found that they dominantly inhibit SPT activity. A transgenic mouse model also revealed that the LCB1 C133W mutation led to mice with decreased tissue SPT activity and HSAN1 symptoms despite unaltered ceramide concentrations (31). A different study found that SPT activity was decreased in the tissues of HSAN1 patients, and in a Chinese hamster ovary model, hamster LCB1 C133Y and C133W mutations could not rescue cells lacking endogenous LCB1 (32). These combined studies revealed that the HSAN1 mutations act directly on the SPT enzyme but the pathological consequence on sphingolipid metabolism is unclear. Very recently, Dunn and colleagues discovered a small 80-amino acid protein (TSC3p) in yeast that stimulated yeast SPT activity, but its role is still unclear (33). Recently, a third eukaryotic subunit (SPT3) has been characterized and appears to be required for optimum SPT activity, although it is expressed most highly in placental tissue and human trophoblasts (34). It has been suggested that these three subunits could form a higher order SPT complex (35). The instability and hydrophobic nature of eukaryotic SPTs has made their isolation and therefore biophysical characterization particularly challenging (24, 36–38).We have targeted homodimeric SPT from Sphingomonas paucimobilis EY2395 to provide molecular insight into the enzymatic properties of SPT (39, 40). Recently, we reported the first high resolution x-ray crystal structure of the S. paucimobilis holo-SPT (41) and showed that the active site containing the PLP cofactor is at the dimer interface. We used this structure to model the human enzyme and map the human cysteine residue (Cys¹³³) of SPT1 onto Asn¹⁰⁰ of the bacterial SPT (41). We found that Asn¹⁰⁰ is proximal to the PLP binding site and lies at the dimer interface. To explore the effects of the HSAN1 mutations on human SPT activity and structure, we have studied mutants of the bacterial enzyme. We made the N100C mutant to better mimic native human SPT and N100W and N100Y to mimic the most common mutations in human SPT. We have characterized the mutations using kinetics, spectroscopy, and structural biology, including structures of the external l-Ser aldimine. These data provide molecular insights into the effects of HSAN1 mutations. Also, as part of these studies, we identify residues that play a role in the stabilization of intermediates in the SPT reaction and have been able to generate new insights into the substrate specificity and mechanism of the α-oxamine synthase family.     

Loss of autocrine endothelial-derived VEGF significantly reduces hemangiosarcoma development in conditional p53-deficient mice

Malignant transformation of the endothelium is rare, and hemangiosarcomas comprise only 1% of all sarcomas. For this reason and due to the lack of appropriate mouse models, the genetic mechanisms of malignant endothelial transformation are poorly understood. Here, we describe a hemangiosarcoma mouse model generated by deleting p53 specifically in the endothelial and hematopoietic lineages. This strategy led to a high incidence of hemangiosarcoma, with an average latency of 25 weeks. To study the in vivo roles of autocrine or endothelial cell autonomous VEGF signaling in the initiation and/or progression of hemangiosarcomas, we genetically deleted autocrine endothelial sources of VEGF in this mouse model. We found that loss of even a single conditional VEGF allele results in substantial rescue from endothelial cell transformation. These findings highlight the important role of threshold levels of autocrine VEGF signaling in endothelial malignancies and suggest a new approach for hemangiosarcoma treatment using targeted autocrine VEGF inhibition.     

A practical introduction to Random Forest for genetic association studies in ecology and evolution

Large genomic studies are becoming increasingly common with advances in sequencing technology, and our ability to understand how genomic variation influences phenotypic variation between individuals has never been greater. The exploration of such relationships first requires the identification of associations between molecular markers and phenotypes. Here, we explore the use of Random Forest (RF), a powerful machine‐learning algorithm, in genomic studies to discern loci underlying both discrete and quantitative traits, particularly when studying wild or nonmodel organisms. RF is becoming increasingly used in ecological and population genetics because, unlike traditional methods, it can efficiently analyse thousands of loci simultaneously and account for nonadditive interactions. However, understanding both the power and limitations of Random Forest is important for its proper implementation and the interpretation of results. We therefore provide a practical introduction to the algorithm and its use for identifying associations between molecular markers and phenotypes, discussing such topics as data limitations, algorithm initiation and optimization, as well as interpretation. We also provide short R tutorials as examples, with the aim of providing a guide to the implementation of the algorithm. Topics discussed here are intended to serve as an entry point for molecular ecologists interested in employing Random Forest to identify trait associations in genomic data sets.     

Modulation of Macrophage Activation State Protects Tissue from Necrosis during Critical Limb Ischemia in Thrombospondin-1-Deficient Mice

Background

Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI.

Methods and Findings

Using a genetic model of tsp-1 ^−/− mice subjected to femoral artery excision, we report that tsp-1 ^−/− mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1 ^−/− and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1 ^−/− mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1 ^−/− mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1 ^−/− mice, thereby demonstrating that macrophages mediated tissue protection in these mice.

Conclusion

This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.     

A highly standardized and characterized human platelet lysate for efficient and reproducible expansion of human bone marrow mesenchymal stromal cells

BackgroundHuman platelet lysate (hPL) represents a powerful alternative to fetal bovine serum (FBS) for human mesenchymal stromal cell (hMSC) expansion. However, the large variability in hPL sources and production protocols gives rise to discrepancies in product quality, characterization and poor batch-to-batch standardization.MethodshPL prepared with more than 200 donors (200+DhPL) or with five donors (5DhPL) were compared in terms of growth factor (GF) contents and biochemical analysis. A multiple protein assay and proteomic analysis were performed to further characterize 200+DhPL batches. We also compared the phenotypic and functional characteristics of bone marrow (BM)-hMSCs grown in 200+DhPL versus FBS+basic fibroblast growth factor (bFGF).ResultsBy contrast to 5DhPL, industrial 200+DhPL displayed a strong standardization of GF contents and biochemical characteristics. We identified specific plasmatic components and platelet-released factors as the most relevant markers for the evaluation of the standardization of hPL batches. We used a multiplex assay and proteomic analysis of 200+DhPL to establish a proteomic signature and demonstrated the robust standardization of batches. 200+DhPL was shown to improve and standardize BM-hMSC expansion compared with FBS+bFGF. The levels of expression of BM-hMSC membrane markers were found to be much more homogeneous between batches when cells were cultured in 200+DhPL. BM-hMSCs cultured in parallel under both conditions displayed similar adipogenic and osteogenic differentiation potential and immunosuppressive properties.ConclusionsWe report a standardization of hPL and the importance of such standardization for the efficient amplification of more homogeneous and reproducible cell therapy products.     

TNF-α is associated with loss of lean body mass only in already cachectic COPD patients

Background

Systemic inflammation may contribute to cachexia in patients with chronic obstructive pulmonary disease (COPD). In this longitudinal study we assessed the association between circulating C-reactive protein (CRP), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 levels and subsequent loss of fat free mass and fat mass in more than 400 COPD patients over three years.

Methods

The patients, aged 40–76, GOLD stage II-IV, were enrolled in 2006/07, and followed annually. Fat free mass and fat mass indexes (FFMI & FMI) were calculated using bioelectrical impedance, and CRP, TNF-α, IL-1ß, and IL-6 were measured using enzyme immunoassays. Associations with mean change in FFMI and FMI of the four inflammatory plasma markers, sex, age, smoking, FEV₁, inhaled steroids, arterial hypoxemia, and Charlson comorbidity score were analyzed with linear mixed models.

Results

At baseline, only CRP was significantly (but weakly) associated with FFMI (r = 0.18, p < 0.01) and FMI (r = 0.27, p < 0.01). Univariately, higher age, lower FEV₁, and use of beta2-agonists were the only significant predictors of decline in FFMI, whereas smoking, hypoxemia, Charlson score, and use of inhaled steroids predicted increased loss in FMI. Multivariately, high levels of TNF-α (but not CRP, IL-1ß or IL-6) significantly predicted loss of FFMI, however only in patients with established cachexia at entry.

Conclusion

This study does not support the hypothesis that systemic inflammation is the cause of accelerated loss of fat free mass in COPD patients, but suggests a role for TNF-α in already cachectic COPD patients.