Azita Alavi
email: aalavi@sgul.ac.uk

Owen Fraser

Edward Tarelli

Martin J Bland

John S Axford

An open-label dosing study to evaluate the safety and effects of a dietary plant derived polysaccharide supplement on the N-glycosylation status of serum glycoproteins in healthy subjects

Keywords: Dietary plant polysaccharides, Serum glycosylation, N-glycans, Dietary fiber, Glycomodifications, Sialylation

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Introduction

Fuelled by the current interest in the link between diet and health [START_REF] Ma | Association between dietary fiber and markers of systemic inflammation in the Women's Health Initiative Observational Study[END_REF][START_REF] Estruch | Effects of dietary fibre intake on risk factors for cardiovascular disease in subjects at high risk[END_REF][START_REF] Lomax | Prebiotics, immune function, infection and inflammation: a review of the evidence[END_REF][START_REF] Schiffrin | The inflammatory status of the elderly: The intestinal contribution[END_REF] there is now a surge of research by various groups into the biological activities and potential beneficial effects of dietary saccharide biopolymers [START_REF] Courtois | Oligosaccharides from land plants and algae: production and applications in therapeutics and biotechnology[END_REF][START_REF] Rideout | Guar gum and similar soluble fibers in the regulation of cholesterol metabolism: current understandings and future research priorities[END_REF][START_REF] Graff | Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo[END_REF][START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF][START_REF] Cumashi | A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds[END_REF][START_REF] Vos | Immune-modulatory effects and potential working mechanisms of orally applied nondigestible carbohydrates[END_REF][START_REF] Bruzzese | A formula containing galacto-and fructo-oligosaccharides prevents intestinal and extra-intestinal infections: an observational study[END_REF][START_REF] Chan | The effects of beta-glucan on human immune and cancer cells[END_REF][START_REF] Jacobs | Non-digestible food ingredients, colonic microbiota and the impact on gut health and immunity: a role for metabolomics[END_REF][START_REF] Weickert | Metabolic effects of dietary fiber consumption and prevention of diabetes[END_REF][START_REF] Liu | Yeast-derived beta-glucan in combination with antitumor monoclonal antibody therapy in cancer[END_REF]). These biopolymers represent a broad range of structurally diverse [START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF], non-or low-digestible, dietary soluble fibers that have been derived from different species of flora including higher plants, fungi, lichens and algae, and have been demonstrated (in-vitro or in-vivo) to affect beneficially one or more target cellular or body functions [START_REF] Graff | Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo[END_REF][START_REF] Ma | Association between dietary fiber and markers of systemic inflammation in the Women's Health Initiative Observational Study[END_REF][START_REF] Bruzzese | A formula containing galacto-and fructo-oligosaccharides prevents intestinal and extra-intestinal infections: an observational study[END_REF][START_REF] Chan | The effects of beta-glucan on human immune and cancer cells[END_REF][START_REF] Lomax | Prebiotics, immune function, infection and inflammation: a review of the evidence[END_REF][START_REF] Van Den Heuvel | Short-chain fructooligosaccharides improve magnesium absorption in adolescent girls with a low calcium intake[END_REF][START_REF] Courtois | Oligosaccharides from land plants and algae: production and applications in therapeutics and biotechnology[END_REF][START_REF] Hua | Ganoderma lucidum polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expression[END_REF][START_REF] Ho | Ganoderma lucidum polysaccharide peptide reduced the production of proinflammatory cytokines in activated rheumatoid synovial fibroblast[END_REF][START_REF] Kim | Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cells[END_REF][START_REF] Cumashi | A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds[END_REF][START_REF] Vos | Immune-modulatory effects and potential working mechanisms of orally applied nondigestible carbohydrates[END_REF].

The physiological effects attributed to these "functional" polysaccharides can be divided into six main categories: 1) beneficial effects on colonic microflora [START_REF] Jacobs | Non-digestible food ingredients, colonic microbiota and the impact on gut health and immunity: a role for metabolomics[END_REF] and gastrointestinal physiology; i.e. acting as a prebiotic [START_REF] Lomax | Prebiotics, immune function, infection and inflammation: a review of the evidence[END_REF][START_REF] De Vrese | Probiotics, prebiotics, and synbiotics[END_REF][START_REF] Macfarlane | Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics[END_REF]; 2) immunomodulatory effects [START_REF] Leung | Polysaccharide biological response modifiers[END_REF][START_REF] Graff | Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo[END_REF][START_REF] Bruzzese | A formula containing galacto-and fructo-oligosaccharides prevents intestinal and extra-intestinal infections: an observational study[END_REF][START_REF] Benyacoub | Feeding a diet containing a fructooligosaccharide mix can enhance Salmonella vaccine efficacy in mice[END_REF][START_REF] Kim | Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cells[END_REF][START_REF] Vos | Immune-modulatory effects and potential working mechanisms of orally applied nondigestible carbohydrates[END_REF][START_REF] Cumashi | A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds[END_REF][START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF][START_REF] Mizuno | Different suppressive effects of fucoidan and lentinan on IL-8 mRNA expression in in vitro gut inflammation[END_REF][START_REF] Hua | Ganoderma lucidum polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expression[END_REF][START_REF] Burrows | Oral administration of PPC enhances antigen-specific CD8+ T cell responses while reducing IgE levels in sensitized mice[END_REF]; 3) anti-angiogenetic [START_REF] Cumashi | A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds[END_REF]) and anti-tumour effects [START_REF] Chan | The effects of beta-glucan on human immune and cancer cells[END_REF][START_REF] Liu | Yeast-derived beta-glucan in combination with antitumor monoclonal antibody therapy in cancer[END_REF]; 4) altered lipid metabolism (Chen andHuang 2009, Rideout et al. 2008); 5) improved bioavailability of essential minerals [START_REF] Van Den Heuvel | Short-chain fructooligosaccharides improve magnesium absorption in adolescent girls with a low calcium intake[END_REF][START_REF] Bo | Dietary magnesium and fiber intakes and inflammatory and metabolic indicators in middle-aged subjects from a population-based cohort[END_REF]); and 6) other beneficial health effects such as enhanced production of growth factors involved in re-epithelization and wound healing [START_REF] Jettanacheawchankit | Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing[END_REF].

In this respect, many of the dietary saccharide polymers studied (isolated from >35 species) [START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF], exhibiting multifunctional physicochemical and physiological characteristics, are now being redefined as secondary metabolites [START_REF] Courtois | Oligosaccharides from land plants and algae: production and applications in therapeutics and biotechnology[END_REF][START_REF] Macfarlane | Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics[END_REF][START_REF] Vos | Immune-modulatory effects and potential working mechanisms of orally applied nondigestible carbohydrates[END_REF][START_REF] Weickert | Metabolic effects of dietary fiber consumption and prevention of diabetes[END_REF], and or biological response modifiers (BRMs) [START_REF] Leung | Polysaccharide biological response modifiers[END_REF]).

These BRM polysaccharides (Table 1) have diverse structural complexity, and belong to a wide range of low or high MW, soluble or insoluble, acidic or neutral, homo-or hetero-polymers, which may or may not undergo varying degrees of degradation in the digestive tract either by bacterial enzymes or by specific conditions associated with the digestive process [START_REF] Courtois | Oligosaccharides from land plants and algae: production and applications in therapeutics and biotechnology[END_REF][START_REF] Arasaradnam | Colonic fermentation--more than meets the nose[END_REF][START_REF] Van Den Broek | Bifidobacterium carbohydrases-their role in breakdown and synthesis of (potential) prebiotics[END_REF][START_REF] Sinnott | Utilization of arabinogalactan, aloe vera gel polysaccharides, and a mixed saccharide dietary supplement by human colonic bacteria in vitro[END_REF][START_REF] Jacobs | Non-digestible food ingredients, colonic microbiota and the impact on gut health and immunity: a role for metabolomics[END_REF][START_REF] Macfarlane | Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics[END_REF][START_REF] Schiffrin | The inflammatory status of the elderly: The intestinal contribution[END_REF]. Recent evidence suggests that the potential for these polysaccharides to undergo some degree of degradation is highly dependent on the metabolic potential encoded by the combined genomes of the gut microbiota [START_REF] Tuohy | Studying the human gut microbiota in the trans-omics era--focus on metagenomics and metabonomics[END_REF][START_REF] Arasaradnam | Colonic fermentation--more than meets the nose[END_REF].

Current studies into the link between the bioconversion of these dietary polysaccharides, their bioavailability and their downstream effects on the host metabolism and physiology are utilizing metabolomics and metagenomics approaches [START_REF] Jacobs | Non-digestible food ingredients, colonic microbiota and the impact on gut health and immunity: a role for metabolomics[END_REF]). These and other innovative approaches in the field of colonic fermentation [START_REF] Arasaradnam | Colonic fermentation--more than meets the nose[END_REF][START_REF] Chassard | Characterization of the xylan-degrading microbial community from human faeces[END_REF] are providing novel insights into gut microbial-human mutualism [START_REF] Possemiers | The intestinal environment in health and disease -recent insights on the potential of intestinal bacteria to influence human health[END_REF], its impact on regulating human health and disease, and the importance of dietary modulation [START_REF] Schiffrin | The inflammatory status of the elderly: The intestinal contribution[END_REF][START_REF] Leung | Polysaccharide biological response modifiers[END_REF][START_REF] Lomax | Prebiotics, immune function, infection and inflammation: a review of the evidence[END_REF][START_REF] Bruzzese | A formula containing galacto-and fructo-oligosaccharides prevents intestinal and extra-intestinal infections: an observational study[END_REF][START_REF] Macfarlane | Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics[END_REF][START_REF] Tuohy | Studying the human gut microbiota in the trans-omics era--focus on metagenomics and metabonomics[END_REF][START_REF] Liu | Yeast-derived beta-glucan in combination with antitumor monoclonal antibody therapy in cancer[END_REF].

Although many of the physiological traits attributed to dietary polysaccharides may be secondary, i.e. related to their effects on the gut microflora and its biochemical activities [START_REF] Schiffrin | The inflammatory status of the elderly: The intestinal contribution[END_REF], evidence from various pharmacodynamic and pharmacokinetic studies also indicate microflora-independent immunomodulatory effects [START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF][START_REF] Ho | Ganoderma lucidum polysaccharide peptide reduced the production of proinflammatory cytokines in activated rheumatoid synovial fibroblast[END_REF][START_REF] Hua | Ganoderma lucidum polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expression[END_REF].

This "direct" immunomodulatory effect is instigated by the binding of certain BRM polysaccharides (e.g. β-glucans) [START_REF] Dalmo | Beta-glucans as conductors of immune symphonies[END_REF] to specific receptors on immune cells in the gut associated lymphoid tissues, and results in the intracellular activation of signal transducers and transcription factors that are associated with various effector functions of the immune response [START_REF] Graff | Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo[END_REF][START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF][START_REF] Vos | Immune-modulatory effects and potential working mechanisms of orally applied nondigestible carbohydrates[END_REF][START_REF] Paur | Extracts of dietary plants are efficient modulators of nuclear factor kappa B[END_REF][START_REF] Mizuno | Different suppressive effects of fucoidan and lentinan on IL-8 mRNA expression in in vitro gut inflammation[END_REF][START_REF] Hua | Ganoderma lucidum polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expression[END_REF][START_REF] Vetvicka | Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells[END_REF][START_REF] Ho | Ganoderma lucidum polysaccharide peptide reduced the production of proinflammatory cytokines in activated rheumatoid synovial fibroblast[END_REF][START_REF] Burrows | Oral administration of PPC enhances antigen-specific CD8+ T cell responses while reducing IgE levels in sensitized mice[END_REF].

Although the potential mechanisms of the interaction and the subsequent signal transduction pathways are not fully understood, the evidence supports the direct binding of BRM polysaccharides (or their fragments) to pattern recognition receptors (PRRs). These receptors are key players in the immune response (receptors for recognition of microbial polysaccharides) and include toll like receptors [START_REF] Leung | Polysaccharide biological response modifiers[END_REF][START_REF] Graff | Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo[END_REF][START_REF] Hua | Ganoderma lucidum polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expression[END_REF][START_REF] Dalmo | Beta-glucans as conductors of immune symphonies[END_REF], non-Toll PRRs (e.g. β-glucan receptor or dectin-1), complement receptor CR3, and certain transmembrane lectins [START_REF] Gunning | Recognition of galactan components of pectin by galectin-3[END_REF][START_REF] Vetvicka | Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells[END_REF][START_REF] Vos | Immune-modulatory effects and potential working mechanisms of orally applied nondigestible carbohydrates[END_REF].

Recognition via these PRRs can result in intracellular signaling cascades e.g. activation of protein kinases or NF-kB. This can in turn result in subsequent activation or inactivation of a wide spectrum of target genes involved in the regulation of a variety of cellular responses, such as expression of various cell surface receptors [START_REF] Kim | Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cells[END_REF] and cytokine production [START_REF] Jettanacheawchankit | Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing[END_REF][START_REF] Paur | Extracts of dietary plants are efficient modulators of nuclear factor kappa B[END_REF][START_REF] Leung | Polysaccharide biological response modifiers[END_REF][START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF][START_REF] Cumashi | A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds[END_REF][START_REF] Dalmo | Beta-glucans as conductors of immune symphonies[END_REF].

One of the primary immunomodulatory effects of BRM polysaccharides is to promote or alter various leukocyte activities, in particular those of macrophages (Schepetkin andQuinn 2006, Leung et al. 2006) and immune-regulatory γδ T cells (a subpopulation of intraepithelial lymphocytes in the gastrointestinal tract) [START_REF] Graff | Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo[END_REF], via changes in cytokine expression [START_REF] Yoshino | Immunoregulatory effects of the antitumor polysaccharide lentinan on Th1/Th2 balance in patients with digestive cancers[END_REF][START_REF] Hua | Ganoderma lucidum polysaccharides enhance CD14 endocytosis of LPS and promote TLR4 signal transduction of cytokine expression[END_REF][START_REF] Benyacoub | Feeding a diet containing a fructooligosaccharide mix can enhance Salmonella vaccine efficacy in mice[END_REF][START_REF] Kim | Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cells[END_REF][START_REF] Schiffrin | The inflammatory status of the elderly: The intestinal contribution[END_REF][START_REF] Burrows | Oral administration of PPC enhances antigen-specific CD8+ T cell responses while reducing IgE levels in sensitized mice[END_REF]). This in turn can impact both the innate and adaptive arms of the immune response and thus result in activation or dampening of these responses [START_REF] Mizuno | Different suppressive effects of fucoidan and lentinan on IL-8 mRNA expression in in vitro gut inflammation[END_REF][START_REF] Ho | Ganoderma lucidum polysaccharide peptide reduced the production of proinflammatory cytokines in activated rheumatoid synovial fibroblast[END_REF][START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF][START_REF] Cumashi | A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds[END_REF][START_REF] Vos | Immune-modulatory effects and potential working mechanisms of orally applied nondigestible carbohydrates[END_REF][START_REF] Graff | Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo[END_REF].

Dietary polysaccharide induced immunomodulatory activities of note include: increased macrophage cytotoxic and phagocytic activities, altered pro-and anti-inflammatory [START_REF] Kim | Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cells[END_REF][START_REF] Benyacoub | Feeding a diet containing a fructooligosaccharide mix can enhance Salmonella vaccine efficacy in mice[END_REF][START_REF] Leung | Polysaccharide biological response modifiers[END_REF][START_REF] Ho | Ganoderma lucidum polysaccharide peptide reduced the production of proinflammatory cytokines in activated rheumatoid synovial fibroblast[END_REF][START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF][START_REF] Mizuno | Different suppressive effects of fucoidan and lentinan on IL-8 mRNA expression in in vitro gut inflammation[END_REF][START_REF] Schiffrin | The inflammatory status of the elderly: The intestinal contribution[END_REF]) and Th1-Th2 balance [START_REF] Yoshino | Immunoregulatory effects of the antitumor polysaccharide lentinan on Th1/Th2 balance in patients with digestive cancers[END_REF][START_REF] Vos | Immune-modulatory effects and potential working mechanisms of orally applied nondigestible carbohydrates[END_REF][START_REF] Burrows | Oral administration of PPC enhances antigen-specific CD8+ T cell responses while reducing IgE levels in sensitized mice[END_REF], as well as altered expression of certain adhesion molecules [START_REF] Graff | Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo[END_REF][START_REF] Ho | Ganoderma lucidum polysaccharide peptide reduced the production of proinflammatory cytokines in activated rheumatoid synovial fibroblast[END_REF][START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF][START_REF] Yoshino | Immunoregulatory effects of the antitumor polysaccharide lentinan on Th1/Th2 balance in patients with digestive cancers[END_REF][START_REF] Cumashi | A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds[END_REF].

All of which supports the notion that certain dietary plant polysaccharides may have significant immunomodulatory potential [START_REF] Benyacoub | Feeding a diet containing a fructooligosaccharide mix can enhance Salmonella vaccine efficacy in mice[END_REF][START_REF] Trinchero | Antiretroviral activity of fucoidans extracted from the brown seaweed Adenocystis utricularis[END_REF][START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF][START_REF] Cumashi | A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds[END_REF][START_REF] Ho | Ganoderma lucidum polysaccharide peptide reduced the production of proinflammatory cytokines in activated rheumatoid synovial fibroblast[END_REF][START_REF] Tuohy | Studying the human gut microbiota in the trans-omics era--focus on metagenomics and metabonomics[END_REF][START_REF] Burrows | Oral administration of PPC enhances antigen-specific CD8+ T cell responses while reducing IgE levels in sensitized mice[END_REF].

In view of this and given the important link between changes in the immune response and the glycosylation of various effector glycoproteins [START_REF] Alavi | Sweet and sour: the impact of sugars on disease[END_REF], the aim of this study was to examine the possible affects, if any, that oral administration of dietary plant polysaccharides may have on the glycosylation status of serum glycoproteins in a cohort of healthy subjects. We chose a commercial, mixed saccharide dietary supplement (AA), the constituents of which have previously been shown to exhibit prebiotic [START_REF] Sinnott | Utilization of arabinogalactan, aloe vera gel polysaccharides, and a mixed saccharide dietary supplement by human colonic bacteria in vitro[END_REF]) as well as possible immunomodulatory activities [START_REF] Lefkowitz | Effects of a glyconutrient on macrophage functions[END_REF][START_REF] Koetzner | Plant-Derived Polysaccharide Supplements Inhibit Dextran Sulfate Sodium-Induced Colitis in the Rat[END_REF], and for the purposes of this study have no known reported toxicity or negative side effects associated with their use in human subjects [START_REF] Stancil | Glyconutrients and perception, cognition, and memory[END_REF].

Using AA we carried out an open-label dosing study to first evaluate safety of increased doses, and second to monitor the affect on the N-glycosylation status of serum glycoproteins in healthy subjects.

Materials and methods

Study Cohort

Healthy volunteers were recruited from the staff at St George's University of London. The study design was approved by the Ethics Committee and volunteers gave written informed consent.

Supplement

The supplement was Advanced Ambrotose TM (AA) powder (provided by Mannatech, Incorporated, USA). AA is a plant derived dietary supplement that contains a standardized mixture of partially purified polydisperse saccharide biopolymers: aloe vera gel powder (containing glucomannan based polysaccharides), arabinogalactan (high-M.wt soluble arabinose and galactose containing dietary fiber), gum ghatti and gum tragacanth (mainly soluble mixture of complex galactose polymers; with other saccharide constituents including arabinose, rhamnose, fucose, glucose, mannose, xylose, and uronic acids), glucosamine (plant derived supplemental glucosamine HCl), Undaria pinnatifida fucoidans (sulfated fucose polymer that contains other monosaccharides, galactose and mannose, derived from edible brown seaweed), and rice starch.

Study Design

An open-label dosing study was set up. The study was carried out in two phases.

Phase 1: Pilot study to establish safety and dose necessary to impact serum N-glycosylation profiles. 6 individuals (age range 28-55; 3 females), ingested increasing doses of AA supplement (increased by 1.3g/week) for seven weeks; starting with 1.3g/day for week 1, and reaching 9.1g/day at week 7). From week 7 to week 11, the group was divided into two arms; three stopped their supplement whilst the other three volunteers continued with supplementation at increasing doses (reaching 14.3g/day by week 11).

Phase 2: A larger study to evaluate glycosylation changes pre-and during-supplementation. 12 volunteers (age range 22-40; 6 females), were followed for 16 weeks. The first 7 weeks, designated as the pre-supplementation period (week -7 to 0), was to establish baseline serum Nglycosylation patterns and normal fluctuations. Following this period, the individuals ingested increasing doses of AA supplement (week 1-4 at 1.3g/day; week 5-6 at 2.6 g/day and week 7-8 at 5.2g/day). This was followed by a final week with no supplement intake (week 9).

Sample collection

Serum was extracted from blood samples and stored at -40°C. Samples were tested in a blind fashion. The codes were not revealed until the study was completed.

Glycosylation Analysis

Serum (40µl) glycoproteins were deglycosylated using a Peptide N-Glycosidase-F (New England BioLabs Inc, UK) according to the manufacturer's instructions. Released N-glycans were isolated by C18 (Supelco, UK), and graphitized carbon columns, freeze-dried, reconstituted in 100µL deionised water, ready for Glycan profiling by Matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MS; Kratos Analytical, Ltd, UK), set in positive linear mode and calibrated with a dextran ladder. Dihydroxy-benzoic acid was used as the matrix.

Statistical analysis

Where necessary the data were log transformed. To allow for time trends analysis of covariance with time as a continuous predictor and subject as a categorical factor was used. For each glycan structure, two regression models were fitted. The first ignored AA and fitted a straight line relationship with time. The second allowed for different regression lines in the pre-supplement and supplement periods. These two models were compared using an F test. The difference between the sums of squares was calculated. The ratio of this to the residual mean square for the larger, two period model = F ratio; used to test effect of AA.

Correction for multiple comparisons: The Bonferroni method was applied and significance was set at p≤0.006.

Dose-Effect:

Only the data following the start of AA were used to analyse regression of each glycan on dose and subject as a categorical factor. Time was included as a second covariate (to allow for possible time trends); such that if there was a significant dose trend even after adjustment for time, the effect of dose was fairly secure.

Results

Safety and compliance

Phase 1: All 6 subjects completed the trial. AA was well tolerated even at high doses, with no adverse reactions.

Phase 2: All six male and five female volunteers completed the trial. However, due to palatability issues at high doses, 4/5 female volunteers stopped supplementation earlier than planned; two at week 6 and two at week 7. One female volunteer withdrew at week 1 (due to palatability issues), and had data for pre-AA phase only, and was therefore omitted from the analysis.

Serum N-glycan analysis

Serum N-glycan profiles for each individual at weekly intervals were examined. Eight main biantennary serum glycoprotein derived N-glycan structures corresponding to different m/z values were identified (Figure 1). Peak intensities were analyzed.

Phase 1: analysis of the data from the pilot study demonstrated changes in the serum N-glycan profile in response to increasing doses of AA supplementation. Example of the observed changes for each of the 6 volunteers, at four time points, for four of the glycans, G0, G1, G1F and A2, are shown (Figure 2). The time points chosen were time =0 (representing baseline values), time = 1 (demonstrating that changes in serum protein glycosylation were evident within 1 week of supplementation at 1.3g/ day;), time = 7 (9.1g/day; time point at which the group were subdivided into 2 arms; 3 individuals continued with their oral supplementation at increasing doses, whilst the remaining 3 discontinued their supplementation), and time = 11 (the end of the study demonstrating possible differences between the serum glycosylation profile of the individuals who continued compared with those that discontinued with their AA supplementation).

Week 0-7: All apart from the monosialylated A1 glycans (not shown), showed some degree of change in response to AA supplementation.

Week 7-11: Comparisons of the glycan profiles in the 3 individuals that discontinued with those that continued AA supplementation showed differences. These differences were confined mainly to the neutral glycans (G0F, G1, G1F, G2 and G2F) and were particularly noticeable for G1F where the levels reverted (increased) towards baseline (week=0) when AA was discontinued (Figure 2).

Phase 2: The seven week pre-supplementation monitoring of serum protein glycan profiles allowed us to evaluate and monitor normal glycosylation fluctuations and to establish statistically meaningful baseline levels. Comparison of the glycan profiles during the supplementation period with the time trends observed during the pre-supplementation period detected distinct changes in the relative intensities of 7/8 glycans studied. These changes were particularly striking within the first 1-2 weeks of supplementation (Figure 3). G0 was the exception; showing major fluctuations in the pre-as well as supplementation period.

Analysis of covariance utilizing the two regression model, was used to assess the strength of evidence that serum glycosylation changes followed AA supplementation. A graphical example of the model as applied to the analysis of the data for G2 glycans is given (Figure 4). Using this analysis, AA supplementation was found to result in significant changes in the levels of all the serum protein derived N-linked glycans (G0F, G1, G1F, G2, G2F, A1 and A2; p<0.001) except G0 (p = 0.2).

Dose effect

Regression analysis (with time as a second covariate) following the start of AA was used to assess the strength of evidence that change did occur following supplementation with AA and to examine the effect of dose. The results (Table 2) show dose effects for G1 and G2; the levels of which decreased significantly with increasing AA dose (coefficient factor by which mean glycan levels are multiplied /week = -0.130 and -0.690; p = 0.02 and 0.005 respectively), and for A2; the levels of which increased significantly with increasing AA dose (Coefficient factor =1.083; p=0.005).

Discussion

Our study reports novel findings showing that the ingestion of a standardized mixture of plant derived polysaccharides can induce significant changes in the N-glycosylation status of serum glycoproteins in normal healthy individuals. AA supplementation caused an overall, dose dependent, shift towards increased sialylation; resulting in significantly lower levels of neutral glycans (G1 and G2: -0.130 and -0.690 per week: respectively), and increased levels of fully processed, sialylated, glycans (A2: by a factor of 1.083 per week).

Given that glycosylation is a key post-translational modification that can significantly affect the overall biophysical and biochemical functions of proteins [START_REF] Alavi | Sweet and sour: the impact of sugars on disease[END_REF]Axford 2008, Biol-N'garagba and[START_REF] Biol-N'garagba | Regulation of the intestinal glycoprotein glycosylation during postnatal development: role of hormonal and nutritional factors[END_REF], the observed shift in the N-glycosylation status of serum glycoproteins in response to AA supplementation may be significant and could reflect changes in one or more physiological parameters.

Interpretation of the biological significance of the observed shift towards more silaylated serum glycoproteins is complex [START_REF] Alavi | Sweet and sour: the impact of sugars on disease[END_REF] and requires further more detailed structurefunction examination of given serum glycoproteins (e.g., IgG). However, what is known is that increased levels of sialic acid can affect absorption, serum half-life, and clearance from the serum, as well as the physical, chemical and immunogenic properties of various key serum glycoproteins [START_REF] Alavi | Sweet and sour: the impact of sugars on disease[END_REF][START_REF] Bork | Increasing the sialylation of therapeutic glycoproteins: the potential of the sialic acid biosynthetic pathway[END_REF][START_REF] Schauer | Sialic acids as regulators of molecular and cellular interactions[END_REF]. Indeed, sialylation changes can play a key role in many aspects of the immune response, in particular in relation to the regulation of inflammatory processes, as in the case of IgG, where sialylation has been shown to act as "switch". The presence of sialylation is associated with the steady state, normal, anti-inflammatory function of IgG, whilst its absence is associated with the pro-inflammatory effector function [START_REF] Alavi | Sweet and sour: the impact of sugars on disease[END_REF]Axford 2008, Anthony et al. 2008).

However, with this in mind, it is important to note that the elucidation of a specific physiological role for a given glycan modification, such as sialylation, poses formidable challenge. The presence or absence of a particular sugar residue, at a particular glycosylation site on a given protein, may have different roles in different biological settings. In the case of sialic acid, this is further complicated by the fact that sialylation is linkage-specific, and that the presence of the different forms of sialic acid may give rise to very different biological scenarios [START_REF] Alavi | Sweet and sour: the impact of sugars on disease[END_REF]Axford 2008, Schauer 2009). Further research is therefore needed to more clearly elucidate specific effects and their consequent biological significance.

Although the question of whether these serum glycosylation changes are directly linked to the constituents of AA per se, or the affect of these on gut microflora [START_REF] Sinnott | Utilization of arabinogalactan, aloe vera gel polysaccharides, and a mixed saccharide dietary supplement by human colonic bacteria in vitro[END_REF]) requires further investigation, the novel finding that small amounts of a polydisperse mixture of dietary polysaccharides can alter the glycosylation of serum proteins is unexpected and intriguing. This is especially true as it provides a potential mechanism through which dietary polysaccharides may alter the effector functions of circulating molecules. This notion is particularly relevant when one considers the fact that, time and again, cross-sectional [START_REF] Bo | Dietary magnesium and fiber intakes and inflammatory and metabolic indicators in middle-aged subjects from a population-based cohort[END_REF]) and longitudinal [START_REF] Estruch | Effects of dietary fibre intake on risk factors for cardiovascular disease in subjects at high risk[END_REF] studies indicate an association between dietary fiber intake and levels of inflammatory serum markers (e.g. an inverse correlation with IL-6 and TNF-α receptor-2) [START_REF] Ma | Association between dietary fiber and markers of systemic inflammation in the Women's Health Initiative Observational Study[END_REF]. Continued efficacy studies employing randomized placebo-controlled trials examining AA and its specific components are warranted to evaluate possible effect(s).

In conclusion, we have demonstrated that supplementation with dietary plant polysaccharides in AA can result in significant serum protein glycomodifications in normal healthy individuals.

These finding are interesting and merit further research, where it is hoped that an integrative approach using glycomics together with other -omics such as metabolomics and metagenomics; focusing on the gut associated microbe-host mutualism and its impact on physiology, can help unravel the exogenous and endogenous effects of dietary components and hence generate novel hypotheses for innovative dietary interventions that may impact health. Potent antioxidant and anticoagulant properties as well as immunopharmacological properties such as anti-inflammatory activity [START_REF] Cumashi | A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds[END_REF][START_REF] Kim | Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cells[END_REF][START_REF] Mizuno | Different suppressive effects of fucoidan and lentinan on IL-8 mRNA expression in in vitro gut inflammation[END_REF]). Glucomannan polysaccharides Acemannan -obtained from Aloe vera.

Figure legends

Potent immunostimmulants [START_REF] Leung | Polysaccharide biological response modifiers[END_REF]) that also exhibit anti tumour and wound healing properties via induction of fibroblast proliferation and increased type I collagen expression [START_REF] Jettanacheawchankit | Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing[END_REF][START_REF] Leung | Polysaccharide biological response modifiers[END_REF]. β glucans; Wide spread homo-polysaccharides (D-glucose monomers linked by glycosidic bonds) with different molecular weights (MW) and degree of branching.

They occur most commonly as the cell wall of yeast, certain fungi and mushrooms (e.g. lentinan).

These exhibit marked anti-tumour [START_REF] Chan | The effects of beta-glucan on human immune and cancer cells[END_REF][START_REF] Liu | Yeast-derived beta-glucan in combination with antitumor monoclonal antibody therapy in cancer[END_REF]) and immunostimulatory [START_REF] Mizuno | Different suppressive effects of fucoidan and lentinan on IL-8 mRNA expression in in vitro gut inflammation[END_REF]) activity, affecting both the innate as well as the adaptive arm (Th1 and Th2) of the immune response [START_REF] Dalmo | Beta-glucans as conductors of immune symphonies[END_REF], and may be effective in controlling blood lipids [START_REF] Chen | The effects of diets enriched in beta-glucans on blood lipoprotein concentrations[END_REF]. Generally insoluble (1,3/1,6) β glucan, has greater biological activity than the soluble (1,3/1,4) β glucans.

Inulin polysaccharides; Fructans

Fructose polymers ± terminal glucose. Found in many types of plants. Foods naturally high in inulin include garlic, onion and chicory. These are associated with immunomodulatory anti-inflammatory effects as well as selective mineral absorption. They have also been implicated in controlling blood lipids [START_REF] Bruzzese | A formula containing galacto-and fructo-oligosaccharides prevents intestinal and extra-intestinal infections: an observational study[END_REF][START_REF] Van Den Heuvel | Short-chain fructooligosaccharides improve magnesium absorption in adolescent girls with a low calcium intake[END_REF][START_REF] Courtois | Oligosaccharides from land plants and algae: production and applications in therapeutics and biotechnology[END_REF].

Arabinogalactan polysaccharides

Cconsisting of arabinose and galactose. Major constituents of many gums, including gum gutti and gum tragacanth.

A number of these exhibit immunomodulatory effects via induction of both pro-and anti-inflammatory cytokines [START_REF] Schepetkin | Botanical polysaccharides: macrophage immunomodulation and therapeutic potential[END_REF] and have been shown to exhibit potent complement-fixing activity and beneficial prebiotic properties, particularly in gastrointestinal functional disorders such as Irritable bowel syndrome, by increasing the colonic contents of short-chain fatty acids. Guar gum has also been implicated in improving blood lipids [START_REF] Rideout | Guar gum and similar soluble fibers in the regulation of cholesterol metabolism: current understandings and future research priorities[END_REF]. For the purposes of statistical analysis, log transformation was applied to those glycans that exhibited a skewed distribution (more variable at high levels than at low levels). † Analysed on natural scale; coefficient = increase in mean sugar per week.

* Analysed on logarithmic scale; coefficient = factor by which mean sugar is multiplied per week.

Figure 1 :

 1 Figure 1:

Figure 2 :

 2 Figure 2:

Figure 3 :

 3 Figure 3:

Figure 4 :

 4 Figure 4:

Table 1 :

 1 Examples of dietary polysaccharides that have been studied in relation to their biological activities

	Dietary Polysaccharides Features / examples	Biological Functions
	Sulphated	Heparin-like Fucoidans -
	polysaccharides;	obtained from edible
	glycosaminoglycans	seaweed.

Table 2 :

 2 Effect of increasing dose of AA supplement on serum protein N-glycosylation:Regression analysis of the data demonstrates significant dose effects for G1, G2 and A2 serum protein N-glycans.

		Dose alone	Dose adjusted for time
	Glycans
		Coefficient	p value	Coefficient	p value
	G0 *	1.124	0.1	0.993	0.09
	G0F *	0.996	0.9	1.020	0.6
	G1 †	-0.133	0.001	-0.1305	0.02
	G1F *	0.962	0.3	1.016	0.7
	G2 †	-0.976	<0.001	-0.690	0.005
	G2F †	-0.226	0.001	-0.170	0.2
	A1 †	0.532	0.007	0.669	0.1
	A2 *	1.082	0.002	1.083	0.005

AA and OF were supported by grant No: RKC0021 from the Research & Development Division of Mannatech, Incorporated, Texas, USA.

Abbreviations

Mannatech, Incorporated had no role in the conception or design of the study; collection, management, analysis and interpretation of the data; preparation, review or approval of the manuscript; or the decision to publish.

Contributions:

AA, JA and ET conceived and designed the study. AA carried out the study and performed the analysis and interpretation of the data, as well as coordinated and drafted the manuscript. OF and ET carried out the MS analysis. MB performed the statistical analysis. All authors have read and approved the final paper.


Lorena Fernández-Martínez

Stef Letteboer

Christian Y Mardin

Nicole Weisschuh

Eugen Gramer

Bernhard H F Weber

Bernd Rautenstrauss

Paulo Ferreira

Friedrich E Kruse

André Reis

Ronald Roepman

Dr Francesca Pasutto
email: francesca.pasutto@uk-erlangen.de

Evidence for RPGRIP1 gene as risk factor for primary open angle glaucoma

Keywords: glaucoma, RPGRIP1, mutation screening

ABSTRACT

Glaucoma is a genetically heterogeneous disorder and is the second cause of blindness worldwide owing to the progressive degeneration of retinal ganglion neurons. Very few genes causing glaucoma were identified to this date. In this study, we screened 10 candidate genes of glaucoma between the D14S261 and D14S121 markers of chromosome 14q11, a critical region previously linked to primary open angle glaucoma (POAG). Mutation analyses of two large cohorts of patients with POAG, normal tension glaucoma (NTG) and juvenile open angle glaucoma (JOAG), and control subjects, found only association of non-synonymous heterozygous variants of the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) with POAG, NTG and JOAG. The twenty non-synonymous variants identified in RPGRIP1 were all distinct from variants causing photoreceptor dystrophies and were found throughout all but one domain (RPGR-interacting domain, RID) of RPGRIP1. Among them, fourteen missense variants clustered within or around the C2 domains of RPGRIP1. Yeast twohybrid analyses of a subset of the missense mutations within the C2 domains of RPGRIP1 shows that five of them (p.R598Q, p.A635G, p.T806I, p.A837G and p.I838V) decrease the association of the C2 domains with nephrocystin-4 (NPHP4).

When considering only these five confirmed C2-domain mutations the association remains statistically significant (p= 0.001). Altogether, the data support that heterozygous non-synonymous variants of RPGRIP1 may cause or increase the susceptibility to various forms of glaucoma and that among other factors, physical impairment of the interaction of RPGRIP1with different proteins may contribute to the pathogenesis of forms of glaucoma.

INTRODUCTION

Glaucoma is the second most prevalent cause of blindness worldwide and comprises a heterogeneous group of complex neurodegenerative diseases of the retina. [START_REF] Kwon | Primary open-angle glaucoma[END_REF][START_REF] Thylefors | Global data on blindness[END_REF] Of the different forms of glaucoma, primary open angle glaucoma (POAG, OMIM 137760) is the most common affecting more than 35 million people worldwide. [START_REF] Quigley | The number of people with glaucoma worldwide in 2010 and 2020[END_REF] POAG is characterized by primary loss of retinal ganglion cells along with supporting glia and vasculature, specific atrophy of the optic nerve, progressive loss of neuroretinal rim of the optic disc, with corresponding progressive, peripheral visual field loss and, if untreated, eventual blindness. Elevated intraocular pressure (IOP) appears to be a major risk factor, although a number of patients develop glaucoma also in the absence of elevated IOP. [START_REF] Shields | Staging glaucoma for the twenty-first century: Sixth Chandler-Grant Lecture[END_REF] In this case, glaucoma is defined as normal tension glaucoma (NTG).

Based on age of onset before 40, a juvenile form of open angle glaucoma, JOAG, is differentiated from adult-onset POAG. [START_REF] Wiggs | The distinction between juvenile and adult-onset primary open-angle glaucoma[END_REF] A family history of POAG is a well established risk factor, indicating that specific genetic variants may contribute to pathogenesis, and a multifactorial aetiology is suggested. [START_REF] Wiggs | Genetic etiologies of glaucoma[END_REF] In some large pedigrees, though, glaucoma segregates as a Mendelian trait with reduced penetrance and variable expressivity, but most cases are sporadic.

Currently, through linkage studies, 14 chromosomal loci for POAG (GLC1A-N) have been identified (HUGO, http://www.genenames.org/index.html) and many more have been reported in the literature. [START_REF] Allingham | The genetics of primary open-angle glaucoma: a review[END_REF] However, only three causative genes have been described so far including: myocilin (MYOC/GLC1A) [START_REF] Stone | Identification of a gene that causes primary open angle glaucoma[END_REF] , optineurin (OPTN/GLC1E), [START_REF] Rezaie | Adult-onset primary open-angle glaucoma caused by mutations in optineurin[END_REF] and WD repeat domain 36 (WDR36/GLC1G). [START_REF] Monemi | Identification of a novel adult-onset primary open-angle glaucoma (POAG) gene on 5q22.1[END_REF] Altogether, mutations in these genes account for less than 10% of POAG cases. [START_REF] Fan | Gene mapping for primary open angle glaucoma[END_REF] In addition, association studies have implicated at least 20 other genes, [START_REF] Allingham | The genetics of primary open-angle glaucoma: a review[END_REF][START_REF] Fuse | Genetic bases for glaucoma[END_REF] among them cytochrome P450-1B (CYP1B1) [START_REF] Pasutto | Heterozygous loss of function variants in CYP1B1 predispose to primary open angle glaucoma[END_REF] and 12 neurotrophin 4 (NTF4), [START_REF] Pasutto | Heterozygous NTF4 mutations impairing neurotrophin-4 signaling in patients with primary open-angle glaucoma[END_REF] contributing to the pathogenesis of glaucoma, 7 but these are still insufficient to explain the most significant fraction of POAG cases. [START_REF] Van Koolwijk | Genetic contributions to glaucoma: heritability of intraocular pressure, retinal nerve fiber layer thickness, and optic disc morphology[END_REF] This may not come as a surprise since the POAG phenotype itself seems complex and to result from diverse pathological processes that involve, but are not limited to, the retina and optic nerve, the aqueous humor outflow pathways and even, as suggested recently, the cerebrospinal fluid dynamics. [START_REF] Berdahl | Cerebrospinal fluid pressure is decreased in primary open-angle glaucoma[END_REF] In 2000 Wiggs et al. published a first genome-wide linkage scan involving an initial set of 41 multiplex families and a second one of 33, where several loci, including regions on chromosome 2, 14, 17 and 19, were linked to POAG. [START_REF] Wiggs | Genome-wide scan for adult onset primary open angle glaucoma[END_REF] We therefore focused our efforts on chromosomal region 14q11. A further genome-wide scan comprising 1,327 individuals and 146 families of the Barbados family study, provided evidence for linkage to chromosomes 1, 2, 9, 11 and also gave weak support for the existence of a locus on chromosome 14. [START_REF] Nemesure | A genome-wide scan for primary open-angle glaucoma (POAG): the Barbados Family Study of Open-Angle Glaucoma[END_REF] To our knowledge, follow-up studies to refine the genetic intervals and to assess possible candidate genes located in these regions have been not yet reported.

We screened the candidate chromosomal region, covering 6.3 Mb between markers D14S261 and D14S121, for putative functional candidate genes. Among more than 266 known and predicted genes annotated in the critical region, we selected 10 genes for mutational analysis because among other factors of their expression pattern in eyes tissues, especially in retina. These 10 genes were: adenylate cyclase type IV (ADCY4), B-cell/lymphoma 2-like 2 (BCL2L2), defender against cell death 1 (DAD1), interferonstimulated transcription factor 3 gamma (ISGF3G), matrix metalloproteinase 14 (MMP14), neural retina leucine zipper (NRL), oxidase cytochrome c assembly 1-like (OXA1L), salivary protein-like 2 (SALL2), zinc finger protein 219 (ZNF219) and retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1).

In this work we report the results of the mutation analysis of the 10 candidate genes in a small explorative collective of POAG patients and importantly, the mutation screen of the RPGRIP1 gene in two different cohorts of patients with POAG, NTG and JOAG from Germany and their respective control groups. We have found association of nonsynonymous variants predicted to alter the sequence of known structural and functional domains of RPGRIP1in patients with POAG, NTG and JOAG.

METHODS

STUDY POPULATION

The study was approved by the ethics review board of the Medical Faculty of the University of Erlangen-Nuremberg (Germany) and was in accordance with the tenets of the Declaration of Helsinki. All subjects gave informed consent before entering the study.

The first group of patients with glaucoma consisted of 399 subjects of German (European) origin: 270 had primary open-angle glaucoma (high-pressure POAG), 47 had juvenile open-angle glaucoma (JOAG), and 82 had normal-tension open-angle glaucoma (NTG). The 376 control subjects were all of German origin and were recruited from the same geographic region as the patients. All individuals underwent standardized clinical examinations for glaucoma at the Ophthalmologic Department of the University of Erlangen-Nuremberg, Erlangen. Further recruitment and clinical data have been previously reported. [START_REF] Pasutto | Heterozygous loss of function variants in CYP1B1 predispose to primary open angle glaucoma[END_REF][START_REF] Pasutto | Heterozygous NTF4 mutations impairing neurotrophin-4 signaling in patients with primary open-angle glaucoma[END_REF][START_REF] Pasutto | Profiling of WDR36 missense variants in German patients with glaucoma[END_REF] All subjects were also screened for MYOC, OPTN (data not shown), WDR36 [START_REF] Pasutto | Profiling of WDR36 missense variants in German patients with glaucoma[END_REF] , CYP1B1 [START_REF] Pasutto | Heterozygous loss of function variants in CYP1B1 predispose to primary open angle glaucoma[END_REF] and NTF4 14 mutations (to note that patients with mutations in one of these candidate genes were also included in the present screening study as the aetiology of glaucoma is still unknown).

The second group comprised 383 unrelated patients (304 NTG and 79 POAG) and 104 unrelated control subjects of German descent. The subjects of these groups were clinically investigated at the University Eye Hospital in Würzburg and Tübingen with the same clinical procedure as in Erlangen. Clinical details have been reported elsewhere. [START_REF] Weisschuh | Variations in the WDR36 gene in German patients with normal tension glaucoma[END_REF] Altogether, the age of patients range from 14 to 96 with a mean of 66.5±14.1 years, 61% were females and 37% had a family history of glaucoma. The age of controls subjects range from 34 to 97 with a mean of 72.1±6.9 years. 59.8% of control subjects were females.

MUTATIONAL SCREENING

Genomic DNA was extracted from peripheral blood leukocytes with automated techniques (AutoGenFlex 3000; Autogen, Holliston, MA) using DNA chemistry (Flexigene; Qiagen, Hilden, Germany). Individual coding exons of the RPGRIP1 gene including flanking intronic and untranslated region (UTR) sequences were amplified by polymerase chain reaction (PCR). Primer sequences were selected with Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi/) and are available on request. Purified PCR products (AMPur; Agencourt Bioscience, Beverly MA, purified on a Biomek NX96 platform; Beckman Instruments, Fullerton, CA) were sequenced using dye termination chemistry (Prism Fluorescent Dye Termination; ABI). Purified sequence reactions (CleanSEQ; Agencourt Bioscience) were resolved on a capillary automated sequencer (3730 Sequence Analyzer; ABI) and analyzed with genome assembly software (SEQMAN software; DNAStar, Madison, WI). Each variant was confirmed by a second independent analysis. GenBank Accession NM_020366 was used as cDNA reference sequence, NT_026437 as genomic reference sequence and NP_065099 as the reference protein sequence for RPGRIP1 (http://www.ncbi.nlm.nih.gov National Center for Biotechnology Information, Bethesda, MD). Evolutionary conservation of nonsynonymous variants was investigated with protein sequence alignment generated by ClustalW (http://www.ebi.ac.uk/clustalw/ European Molecular Biology Laboratory, Heidelberg, Germany) and compared with that presented by the Ensembl Database (http://www.ensembl.org).

DNA CONSTRUCTS

Gateway-adapted expression constructs were created using the Gateway cloning system (Invitrogen), and expression vectors used were described in ref. [START_REF] Roepman | Interaction of nephrocystin-4 and RPGRIP1 is disrupted by nephronophthisis or Leber congenital amaurosisassociated mutations[END_REF] . All PCR-generated fragments were verified by nucleotide sequencing. Mutations were introduced using the QuickChange site-directed mutagenesis kit (Stratagene).

ANALYSIS OF INTERACTIONS IN YEAST

The Saccharomyces cerevisiae strain PJ69-4A7alpha harbouring LacZ and HIS3 reporter genes was used to evaluate the interaction of the wild-type and mutant C2 domains of RPGRIP1with NPHP4 in the yeast two-hybrid assay (Clontech). [START_REF] Roepman | Interaction of nephrocystin-4 and RPGRIP1 is disrupted by nephronophthisis or Leber congenital amaurosisassociated mutations[END_REF] The transformants were selected by growth on SD medium lacking Trp at 30 °C for 3-4 d.

To test the expression of the LacZ gene, a colony lift filter assay was performed using X-GAL as a substrate. The transformants were also grown in SD medium minus His and minus Trp. The activities of reporters were analysed in 4 independent transformants.

The interactions of the wild-type and mutant RPGRIP1 and NPHP4 proteins were quantified by determining the β-galactosidase activity using the Yeast β-Galactosidase Assay Kit (Pierce) according to the manufacturer's instructions.

COIMMUNOPRECIPITATION ASSAYS

Constructs pcDNA3-HA-N4 FL /EXP (N-terminal hemagglutinin (HA) tag) and p3xFlag-RPGRIP1 C2-N+C2-C /EXP (N-terminal 3x Flag tag) were used to express epitope-tagged proteins for coimmunoprecipitation. As control plasmid, p3xFlag-LRRK2 LRR /EXP was used. COS-1 cells were cotransfected with plasmid DNA using Lipofectamine TM 2000 (Invitrogen). Proteins were expressed for 24 h, and cells were subsequently washed in PBS and lysed in ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X-100 and complete protease inhibitor cocktail (Roche). Lysates were cleared by centrifugation at 4°C for 10 min at 14,000g. Immunoprecipitation was performed with anti-HA monoclonal mouse antibody (Sigma) overnight at 4°C accomplished through interaction with protein A/G beads (Santa Cruz Biotechnology, NY). Beads were then washed 4 times with lysis buffer, mix with LDS-NuPAGE sample buffer (Invitrogen) and heated at 70°C for 10 min. The immunocomplexes were then analyzed with SDS-PAGE followed by western blot analysis using anti-Flag monoclonal mouse antibody (clone M2, Sigma) and anti-HA antibody. Proteins were detected by chemoluminiscence (Amersham).

RESULTS

Mutational analysis

Sequence analysis of the coding regions of the 10 selected positional and functional candidate genes (ADCY4, BCL2L2, DAD1, ISGF3G, MMP14, NRL, OXA1L, SALL2, ZNF219 and RPGRIP1) was performed in an explorative subset of 46 German patients with POAG chosen from the first group of patients and 46 control subjects. Overall 37 coding variants were found, 10 of which coded for non-synonymous amino acids. These 10 variants were further characterized, analyzing their presence in healthy subjects and their evolutionary conservation among different mammalian species (human, mouse, rat, cow and chimpanzee). Only 3 of these missense mutations, all located in RPGRIP1 gene, were both not present in healthy subjects and affecting evolutionary conserved amino acids. In line with these findings, RPGRIP1 arose as a potential promising candidate for POAG. Thus, we extended the screening for mutations in RPGRIP1 to the entire first group of patients and control subjects.

The systematic mutation analysis in RPGRIP1 led to the identification of 14 amino acid substitutions (Table 1 and Figure 1). Altogether, the amino acid changes accounted for 6.5% of the patients' population (26/399). A total of 8 changes were detected in 376 healthy control subjects, accounting for only 2.1% (8/376) of the control group (p = 0.003, 2-Tail Fischer's exact test; OR=2.8, 95% CI = 1.4 -5.5).

As most of the patients carried a missense mutation located in the C2 domains of RPGRIP1 (16 patients (4%) against 3 control subjects (0.8%), p = 0.004, OR=5.2, 95% CI = 1.5 -18, Table 1 and Figure 1), we screened the complete coding region of the RPGRIP1 C2 domains in a further German cohort of 383 glaucoma patients (304 NTG and 79 POAG) and 104 control subjects to support the observed association data in these domains. We could identify 6 novel amino acid substitutions in 9 patients (2.3%) and in 2 control subjects (1.9%; Table1 and Figure 1). Altogether, the distribution of RPGRIP1 C2 domain variants found remained statistically significant between patients and controls despite the relatively small number of control individuals in the second studied cohort (p = 0.013, 2-Tail Fischer's exact test; OR = 2.5, 95% CI = 1.2 -5.3, Table1).

In addition, C2 domain missense variants resulted to be more or less common among the different phenotype groups, if we take into account the small number of JOAG patients analysed: 3.1% in POAG, 2.9% in NTG and 6.3% in JOAG. Moreover, these missense variants are almost equally distributed between the patients with high IOP (3.5%, combined POAG and JOAG) and patients with normal IOP value (2.9%, NTG).

The group of patients carrying RPGRIP1 missense mutations comprised both late juvenile and adult onset POAG at age of recruitment varying from 20 to 81 years (Table 2). Among these 35 identified patients, 22 had elevated maximum IOP ranging from 22 to 40 mmHg, while 13 had pressure measurements in the normal range. In addition, 2 of these patients presented missense mutations also in MYOC and CYP1B1 genes (Table 2).

RPGRIP1 yeast interaction and coimmunoprecipitation assays

Nephrocystin-4 (NPHP4) is one of the currently known partners of the C2 domains of RPGRIP1. [START_REF] Roepman | Interaction of nephrocystin-4 and RPGRIP1 is disrupted by nephronophthisis or Leber congenital amaurosisassociated mutations[END_REF] To compare the functional relevance of the non-synonymous variants found only in glaucoma patients with those found in glaucoma and healthy patients located within or around the C2 domains of RPGRIP1 (Table 1 and Figure1), we evaluated the effect of 8 of the 15 variants on the interaction activity between RPGRIP1 and NPHP4 with yeast two hybrid assay. As shown in Figure 2A, reduced interaction activity compared to the wild-type control was observed qualitatively by βgalactosidase staining of yeast transformants with the RPGRIP1 constructs p.R598Q, p.A635G, p.T806I, p.A837G, p.I838V , whereas no changes were noted with the p.Q589H, p.A764V and p.R812H variants. These observation were validated with semiquantitative β-galactosidase assays of yeast transformants harbouring identical constructs (Figure 2B), although the p.R812H construct had a higher binding activity than the wild-type protein. These results support that the variants p.A635G, p.T806I, p.A837G, p.I838V, which are found only in disease patients and within the C2 domains of RPGRIP1, impair the interaction of the C2 domains of RPGRIP1 with NPHP4, whereas the p.R598Q also found only in glaucoma patients may have an indirect (conformational) effect over the C2 domains of RPGRIP1 similarly to that observed with the truncation p. R890X (negative control). Finally, independent validation of the impairment of the interaction between the p.R598Q variant and NPHP4 was confirmed by coimmunoprecipitation assays of extracts of cells co-transfected with NPHP4 and wild-type or p.R598Q mutant constructs of the C2 domains of RPGRIP1(Figure 3).

Taking into account only the variants of the first group with impaired RPGRIP1nephrocystin-4 interaction (p.R598Q, p.A635G, p.T806I, p.A837G and p.I838V) we could count a total of 5 variants in10 patients (2.8%) and none in healthy subjects (Table1). This resulted in a significant increase of the effect over expectancy (p= 0.001, 2-Tail Fischer's exact test; OR=7.1, 95% CI = 2.2 -23.1, Table 1).

DISCUSSION

Our results suggest an implication of the RPGRIP1 gene as susceptibility gene in various forms of glaucoma.

Through alternative splicing, the RPGRIP1 gene expresses several isoforms in ocular tissues. [START_REF] Ferreira | Insights into X-linked retinitis pigmentosa type 3, allied diseases and underlying pathomechanisms[END_REF][START_REF] Roepman | The retinitis pigmentosa GTPase regulator (RPGR) interacts with novel transport-like proteins in the outer segments of rod photoreceptors[END_REF] These isoforms have different cellular and subcellular locations, biochemical properties, and species-specific expression among retinal neuron and also outside retina. [START_REF] Roepman | The retinitis pigmentosa GTPase regulator (RPGR) interacts with novel transport-like proteins in the outer segments of rod photoreceptors[END_REF][START_REF] Castagnet | RPGRIP1s with distinct neuronal localization and biochemical properties associate selectively with RanBP2 in amacrine neurons[END_REF][START_REF] Lu | Identification of novel murine-and human-specific RPGRIP1 splice variants with distinct expression profiles and subcellular localization[END_REF][START_REF] Mavlyutov | Species-specific subcellular localization of RPGR and RPGRIP isoforms: implications for the phenotypic variability of congenital retinopathies among species[END_REF] The RPGRIP1 protein contains three structurally different regions.

An N-terminal coiled-coil domains with homology to SMC proteins, [START_REF] Castagnet | RPGRIP1s with distinct neuronal localization and biochemical properties associate selectively with RanBP2 in amacrine neurons[END_REF][START_REF] Lu | Limited proteolysis differentially modulates the stability and subcellular localization of domains of RPGRIP1 that are distinctly affected by mutations in Leber's congenital amaurosis[END_REF] which possibly mediates homotypic and/or heterotypic interactions; 27 a C-terminal region that contains the RPGR-interacting domain (RID); [START_REF] Roepman | The retinitis pigmentosa GTPase regulator (RPGR) interacts with novel transport-like proteins in the outer segments of rod photoreceptors[END_REF] and a central region that contains a protein kinase C conserved region 2 (C2) motif mediating protein-protein interactions. [START_REF] Rizo | C2-domains, structure and function of a universal Ca2+binding domain[END_REF][START_REF] Nalefski | The C2 domain calcium-binding motif: structural and functional diversity[END_REF] A known interacting partner of the C2 domains is nephrocystin-4 (NPHP4). Mutations located in RPGRIP1 C2 domain disrupt the RPGRIP1-NPHP4 complex and cause Leber congenital amaurosis (LCA). [START_REF] Roepman | Interaction of nephrocystin-4 and RPGRIP1 is disrupted by nephronophthisis or Leber congenital amaurosisassociated mutations[END_REF] Another known interaction-partner of the RPGRIP1 C-terminal region (RID) is the X-linked retinitis pigmentosa GTPase regulator gene (RPGR), involved in X-linked retinitis pigmentosa type 3 (RP3). [START_REF] Ferreira | Insights into X-linked retinitis pigmentosa type 3, allied diseases and underlying pathomechanisms[END_REF] Furthermore RPGRIP1 has been detected in the retina as a complex with the basal body protein CEP290 [START_REF] Chang | In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse[END_REF] and in amacrine cells with the neuronal nucleoporin RANBP2 [START_REF] Castagnet | RPGRIP1s with distinct neuronal localization and biochemical properties associate selectively with RanBP2 in amacrine neurons[END_REF] .

These results suggest that one of the main roles of one or more RPGRIP1 isoforms is to serve as a plastic and dynamic scaffold for proteins or protein modules acting in signalling pathways of different retinal cell subpopulations, thus linking one or more RPGRIP1 isoforms to a set of different clinical phenotypes. [START_REF] Arts | Focus on molecules: RPGRIP1[END_REF][START_REF] Oti | Phenome connections[END_REF] After analysis of the 10 candidate genes as reported in the results section, we sequenced the entire RPGRIP1 coding region in a large cohort of POAG patients as we expected to find association to rare variants rather than common polymorphisms (SNPs), in line with previous findings in other glaucoma genes such as MYOC [START_REF] Gong | Genetic dissection of myocilin glaucoma[END_REF] , WDR36 [START_REF] Pasutto | Profiling of WDR36 missense variants in German patients with glaucoma[END_REF] , CYP1B1 [START_REF] Pasutto | Heterozygous loss of function variants in CYP1B1 predispose to primary open angle glaucoma[END_REF] and NTF4 [START_REF] Pasutto | Heterozygous NTF4 mutations impairing neurotrophin-4 signaling in patients with primary open-angle glaucoma[END_REF] . In addition, previous data support the hypothesis that POAG is characterized by a high locus and allelic heterogeneity with different rare variants in numerous genes. [START_REF] Allingham | The genetics of primary open-angle glaucoma: a review[END_REF] Most rare variants are likely to be missense variants, whose functional effects may be expected to arise mainly from amino acid changes that affect protein-protein interactions and that can thus have mildly dominant or dominantnegative effects. Under these considerations, many variants with small effects, a smaller number with intermediate effects and relatively few with large effects are then expected to be found. [START_REF] Bodmer | Common and rare variants in multifactorial susceptibility to common diseases[END_REF] Indeed we identified several different missense variants in RPGRIP1 in almost 6.5% of cases, 4% of these are located in the C2-domains of the protein (p = 0.004). When we sequenced the second cohort of German patients, we focused our attention on variants located in the structurally and functionally important C2-domain regions. Also in this second cohort we detected different missense variants located in the C2-domains but at a lower frequency rate (2.3%). We hypothesize that this could be due to possible different characteristics of the second cohort composed mainly of NTG patients.

To determine whether the eight C2-domains variants identified in the first group are essential for the structure of the C2-domains, we performed yeast-two-hybrid experiments using NPHP4, the only currently known binding partner [START_REF] Roepman | Interaction of nephrocystin-4 and RPGRIP1 is disrupted by nephronophthisis or Leber congenital amaurosisassociated mutations[END_REF] . Five of them indeed showed impaired interaction (Table 1) suggesting that the variants are functionally relevant and not benign polymorphisms. When taking into account only these five variants (p.R598Q, p.A635G, p.T806I, p.A837G and p.I838V) detected in 10 patients and in none of the control subjects, the association remained significant (p= 0.001, Table 1). Since RPGRIP1 encodes scaffold protein isoforms, [START_REF] Ferreira | Insights into X-linked retinitis pigmentosa type 3, allied diseases and underlying pathomechanisms[END_REF] additional binding partners are likely to exist in the retina or tissues etiologically relevant to glaucoma (e.g. trabecular meshwork) and therefore we hypothesize that the other variants identified might affect the composition of RPGRIP1 complexes that might be relevant to the pathogenesis of glaucoma.

The detected effect of the C2-domain variants in protein-protein interaction however is mostly not pronounced and it might be compensated in cell subpopulations for many years. This is in line with glaucoma being a late on set disease with complex inheritance. In fact, age or other risk factors, such as elevated IOP, might be required to manifest the disease.

Like other glaucoma genes described to this date, the mutations found in RPGRIP1 are monoallelic. This is in contrast to all homozygous or compound heterozygous mutations in RPGRIP1 that are linked to inherited photoreceptor dystrophies, such as LCA. [START_REF] Dryja | Null RPGRIP1 alleles in patients with Leber congenital amaurosis[END_REF][START_REF] Booij | Identification of mutations in the AIPL1, CRB1, GUCY2D, RPE65, and RPGRIP1 genes in patients with juvenile retinitis pigmentosa[END_REF] Interestingly, the presence of clinical manifestations has also been reported in heterozygous carriers of LCA associated RPGRIP1 mutations characterized by reduced dark-/light-ERG responses and abnormal ERG amplitudes. [START_REF] Galvin | Clinical phenotypes in carriers of Leber congenital amaurosis mutations[END_REF] Similarly, delayed ERG responses and reduced amplitudes have been also shown in glaucoma. [START_REF] Raz | Functional damage to inner and outer retinal cells in experimental glaucoma[END_REF] Our clinical and genetic data support the hypothesis that retinal ganglion cell axon degeneration and loss of retina ganglion cells in POAG is not only due to elevated intraocular pressure alone but due to other concomitant factors compromising the survival of neuronal cell subpopulations in the inner retina as we recently reported. [START_REF] Pasutto | Heterozygous NTF4 mutations impairing neurotrophin-4 signaling in patients with primary open-angle glaucoma[END_REF] Therefore, the putative role of RPGRIP1 as a scaffold protein regulating different signal pathways among different neuronal cell populations of the retina and its association with glaucoma makes RPGRIP1 an interesting functional candidate gene to dissect pathobiological processes underlying POAG, JOAG and NTG. Interestingly, two patients, one with POAG and one with JOAG, carry also mutations in MYOC and CYP1B1 (Table 2). To date a digenic inheritance in open angle glaucoma has been reported in patients with mutations in both MYOC and CYP1B1. [START_REF] Pasutto | Heterozygous loss of function variants in CYP1B1 predispose to primary open angle glaucoma[END_REF][START_REF] Vincent | Digenic inheritance of early-onset glaucoma: CYP1B1, a potential modifier gene[END_REF] Thus, based on the present knowledge it is likely that the POAG in patient ID99168 (Table 2) is mainly due to the presence of mutations p.Q368X in MYOC and p.N203S in CYP1B1, as these have been previously confirmed to have a pathological function. [START_REF] Pasutto | Heterozygous loss of function variants in CYP1B1 predispose to primary open angle glaucoma[END_REF][START_REF] Aroca-Aguilar | Heterozygous expression of myocilin glaucoma mutants increases secretion of the mutant forms and reduces extracellular processed myocilin[END_REF] In fact, according to our experimental results, the p.Q589H in RPGRIP1 seems to be non pathologic (Table1). In patient ID10033 (Table 2) instead, the JOAG phenotype may be due mainly to the pathologic p.T806I mutation in RPGRIP1 (Table 1) as the MYOC mutation, p.Q352K, has been previously reported to be a probable benign sequence change. [START_REF] Zhou | A cellular assay distinguishes normal and mutant TIGR/myocilin protein[END_REF] Alternatively, it is possible that in patient ID10033 there is epistatic interaction between p.T806I mutation in RPGRIP1and the MYOC mutation, p.Q352K, because of the mild impairment effect of p.T806I mutation on the interaction of the C2 domains of RPGRIP1 with NPHP4.

Our study has some limitations, though. We focused our attention mainly on genes expressed in retina considering the process of retinal ganglion cell degeneration as a common etiologic denominator for these glaucoma forms. However, we cannot exclude from the chromosomal region analysed the presence of other candidate genes expressed in other tissues that might play a critical role in the pathogenesis of glaucoma, such as trabecular meshwork or ciliary body. We also could not prove segregation or de novo occurrence of these variants as no family members were available for study, a situation not uncommon for late onset diseases. Finally, the power of our study is limited and will require independent validation. In summary, our data suggest that rare heterozygous loss of function variants in RPGRIP1 are a risk factor for POAG and other forms of glaucoma, such as NTG and JOAG, and support the hypothesis that genetic predisposition to this disease is mainly caused by rare non-synonymous variants rather than common SNPs.

TITLES AND LEGENDS TO FIGURES

Figure 1 .

 1 Figure 1. RPGRIP1 protein structure and location of amino acids changes identified in

Figure 2 .

 2 Figure 2. Effect of RPGRIP1 mutations on interaction with nephrocystin-4. (A) Wildtype (wt) and mutated human RPGRIP1 C2-N+C2-C proteins (fused to GAL4 transcription

ACKNOWLEDGMENTS

We thank all patients and control individuals for their participation, Olga Zwenger for technical assistance and Juliane Niedzella for patients' recruitment support.

Grant SFB 539 from the German Research Foundation. P.A.F. was supported by grants from the National Institutes of Health (GM083165, EY019492 and 2P30-EY005722). P.A.F. is the Jules & Doris Stein Research to Prevent Blindness Professor.

WEB RESOURCES

The URLs for data presented herein are as follows: National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/ Genome Browser of the University of California Santa Cruz (UCSC), http://genome.ucsc.edu/cgi-bin/hgTracks (Reference sequences Used: NT_026437 and NM_020366 and NP_065099) Ensembl Database (http://www.ensembl.org). ClustalW, http://www.ebi.ac.uk/clustalw/ Online Mendelian Inheritance in Man (OMIM): http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM/ .

CONFLICT OF INTEREST

The authors declare no conflict of interest.