S Hackett

J Hamzah

T M E Davis

Professor Tim G St

Pierre
email: stpierre@physics.uwa.edu.au

Magnetic susceptibility of iron in malaria-infected red blood cells

Keywords: Phosphate Buffered Saline haemozoin, haemoglobin, magnetic susceptibility, malaria

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Summary

During intra-erythrocytic maturation, malaria parasites catabolize up to 80% of cellular haemoglobin. Haem is liberated inside the parasite and converted to haemozoin, preventing haem iron from participating in cell-damaging reactions. Several experimental techniques exploit the relatively large paramagnetic susceptibility of malaria-infected cells as a means of sorting cells or investigating haemoglobin degradation, but the source of the dramatic increase in cellular magnetic susceptibility during parasite growth has not been unequivocally determined. Plasmodium falciparum cultures were enriched using high-gradient magnetic fractionation columns and the magnetic susceptibility of cell contents was directly measured. The forms of haem iron in the erythrocytes were quantified spectroscopically. In the 3D7 laboratory strain, the parasites converted approximately 60% of host cell haemoglobin to haemozoin and this product was the primary source of the increase in cell magnetic susceptibility. Haemozoin iron was found to have a magnetic susceptibility of (11.0  0.9)  10 -3 mL.mol -1 . The calculated volumetric magnetic susceptibility (SI units) of the magnetically enriched cells was (1.88  0.60)  10 -6 relative to water while that of uninfected cells was not significantly different from water. Magnetic enrichment of parasitised cells can therefore be considered dependent primarily on the magnetic susceptibility of the parasitised cells.

Introduction

Measurements of the magnetic susceptibility of blood were performed first by Faraday in 1845 and later, with greater precision and care for sample preparation, by Pauling and

Coryell [START_REF] Pauling | The Magnetic Properties and Structure of Hemoglobin, Oxyhemoglobin and Carbonmonoxyhemoglobin[END_REF]. A diamagnetic susceptibility measured for the iron in oxyhaemoglobin molecules was interpreted as evidence for no unpaired electrons in oxyhaemoglobin, but

Pauling and Coryell also noted a paramagnetic contribution to the total magnetic susceptibility of deoxyhaemoglobin. The possibility of interaction between the haem groups in each globin molecule was rejected on principle and instead, an effective magnetic moment of 5.4  B for each iron atom in deoxyhaemoglobin was calculated, consistent with a model of a ferrous iron atom at the centre of each porphyrin ring, bonded to the globin molecule and four surrounding nitrogen atoms through ionic rather than covalent bonds. Further studies of the magnetic properties and structure of methaemoglobin found evidence for five unpaired electrons in each molecule, giving rise to an effective moment of 5.8  B per iron atom [START_REF] Coryell | The magnetic properties and structure of ferrihemoglobin (methemoglobin) and some of its compounds[END_REF]. Although these results were, and to some extent remain controversial [START_REF] Cerdonio | Magnetic properties of oxyhemoglobin[END_REF][START_REF] Cerdonio | Room-Temperature Magnetic-Properties of Oxyhemoglobin and Carbonmonoxy-Hemoglobin[END_REF][START_REF] Cerdonio | Magnetic susceptibility of hemoglobins[END_REF], many of the studies detecting net paramagnetic susceptibilities in samples of oxyhaemoglobin were later considered to be flawed [START_REF] Savicki | Magnetic susceptibility of oxy-and carbonmonoxyhemoglobins[END_REF] as small but significant amounts of deoxyhaemoglobin were present in solutions of concentrated haemoglobin, even after exposure to air.

Savicki et al also determined the magnetic susceptibility of the polypeptide constituent of haemoglobin [START_REF] Savicki | Magnetic susceptibility of oxy-and carbonmonoxyhemoglobins[END_REF]. Globin is more dense and thus more diamagnetic than water and so completely oxygenated erythrocytes are diamagnetic relative to water [START_REF] Spees | Water proton MR properties of human blood at 1.5 Tesla: Magnetic susceptibility, T-1, T-2, T-2* and non-Lorentzian signal behavior[END_REF]. However, cells containing entirely deoxygenated or oxidised haemoglobin are paramagnetic relative to

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 4
water. A model derived by Spees and co-workers [START_REF] Spees | Water proton MR properties of human blood at 1.5 Tesla: Magnetic susceptibility, T-1, T-2, T-2* and non-Lorentzian signal behavior[END_REF] predicts the magnetic susceptibility of cells containing varying fractions of oxygenated and deoxygenated haemoglobin. The model was later extended by Moore and co-workers, first to describe cells containing methaemoglobin and later for cells infected with malaria parasites [START_REF] Zborowski | Red blood cell magnetophoresis[END_REF][START_REF] Moore | Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis[END_REF].

Owen and co-workers first demonstrated the use of high-gradient magnetic columns to extract deoxygenated erythrocytes from a suspension of whole blood [START_REF] Owen | High Gradient Magnetic Separation of Erythrocytes[END_REF]. The experiment was later repeated with asynchronous cultures of P. falciparum [START_REF] Paul | Separation of Malaria-Infected Erythrocytes from Whole-Blood -Use of a Selective High-Gradient Magnetic Separation Technique[END_REF],

extracting cells containing mature stage parasites. Other experimental techniques use the relative motion of erythrocytes in an applied magnetic field as a surrogate measure of the magnetic susceptibility of the haem iron [START_REF] Zborowski | Red blood cell magnetophoresis[END_REF][START_REF] Moore | Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis[END_REF][START_REF] Marmur | Experimental substantiation of the possible use of magnetophoresis and magnetophonophoresis in ophthalmology[END_REF][START_REF] Vainshtein | Magnetophoresis and its experimental basis[END_REF][START_REF] Piruzian | Magnetophoresis and gravitational erythrocyte sedimentation[END_REF]. Moore and co-workers demonstrated that cells containing sufficient methaemoglobin concentrations [START_REF] Zborowski | Red blood cell magnetophoresis[END_REF] or containing mature parasites [START_REF] Moore | Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis[END_REF] will migrate in the direction of increasing field strength, indicating that these cells contain paramagnetic iron species.

Malaria parasites penetrate erythrocytes and use host cell haemoglobin as a principal source of amino acids [START_REF] Francis | Hemoglobin metabolism in the malaria parasite Plasmodium falciparum[END_REF]. As the polypeptide component of haemoglobin is digested, large quantities of free haem are liberated and immediately oxidised to haematin (ferriprotoporphyrin IX hydroxide). The ferric iron in haematin is potentially toxic to the parasites as the iron may catalyse cell-damaging reactions such as the Fenton reaction [16,[START_REF] Winterbourn | Reactions involving superoxide and normal and unstable haemoglobins[END_REF], so the haematin is detoxified by conversion to particulate haemozoin, the characteristic malaria pigment. The distinctive brown crystals, clearly visible in the

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 5
midgut of the mosquito and within infected erythrocytes [START_REF] Virchow | Zur pathologischen physiologie des blutes[END_REF], led to the identification of the Anopheles gambaei mosquito as the malaria vector [START_REF] Ross | On some peculiar pigmented cells found in two mosquitos fed on malarial blood[END_REF].

The structure of haemozoin was elucidated by Slater and co-workers [START_REF] Slater | An iron-carboxylate bond links the heme units of malaria pigment[END_REF], and later refined by Bohle and co-workers [START_REF] Bohle | Structural and Spectroscopic Studies of Beta-Hematin (the Heme Coordination Polymer in Malaria Pigment)[END_REF] and Pagola and co-workers [START_REF] Pagola | The structure of malaria pigment B-haematin[END_REF]. The current model

shows bonds between the central ferric iron of one haem group and a side-chain carboxylate group of another. These dimers are then formed into crystals by hydrogen bonds. The pathways of haemoglobin degradation and haemozoin synthesis are not yet established and reports of the fraction of cellular haemoglobin converted to haemozoin differ, with values ranging from 25% to 80% [START_REF] Ball | Studies on Malarial Parasites .9. Chemical and Metabolic Changes During Growth and Multiplication Invivo and Invitro[END_REF][START_REF] Goldberg | Hemoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle[END_REF][START_REF] Roth | Malarial Pigment-Dependent Error in the Estimation of Hemoglobin Content in Plasmodium-A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 26 Falciparum-Infected Red-Cells -Implications for Metabolic and Biochemical-Studies of the Erythrocytic Phases of Malaria[END_REF][START_REF] Egan | Fate of haem iron in the malaria parasite Plasmodium falciparum[END_REF].

The intrinsic relationship between the chemical and magnetic properties of iron provides a means of elucidating the form and structure of iron compounds. The paramagnetic susceptibilities of some chemical forms of iron are several orders of magnitude greater than the diamagnetic susceptibilities of organic materials [START_REF] Cerdonio | Reexamination of the evidence for paramagnetism in oxy-and carbonmonoxyhemoglobins[END_REF], so magnetic methodologies are potentially a highly sensitive and specific means of studying the forms of iron in biological systems. Previous experiments using magnetic fractionation [START_REF] Paul | Separation of Malaria-Infected Erythrocytes from Whole-Blood -Use of a Selective High-Gradient Magnetic Separation Technique[END_REF] and magnetophoresis [START_REF] Moore | Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis[END_REF] indicate a change of the chemical state of haem iron during parasite growth. However, the form(s) of iron responsible for the increase in cellular magnetic susceptibility have not been unequivocally determined.

To address this question, P. falciparum parasites were magnetically separated and, rather than isolating the different iron-containing compounds, magnetic measurements were

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 6
performed on cell contents as a whole. The forms of iron present in the cells were identified using a combination of spectrophotometry and magnetometry. Standard samples of these forms were prepared and characterised both magnetically and spectroscopically. The physical processes underlying the enrichment of mature stage parasites in the magnetic fractionation columns were explored and several potential uses of magnetic fractionation processes in malaria research were considered.

Methods and Materials

Twenty-four unsynchronised cultures of P. falciparum 3D7 clones were maintained according to the method of Trager and Jensen [START_REF] Jensen | Plasmodium falciparum in culture: establishment of additional strains[END_REF]. Thin blood films, stained with 10% v/v Giemsa, were examined using a Zeiss optical microscope before and after magnetic fractionation. At least 1 000 cells per film were counted and cells were categorised as unparasitised, immature (ring and early trophozoite stages, i.e. the first 30 hours of parasite life cycle) and mature (late trophozoite and schizont stages i.e. 30 -48 hours of the parasite life cycle). The parasitaemia (defined as percentage of erythrocytes infected with parasites) and the mature parasite density (defined as percentage of erythrocytes infected with late trophozoites and schizonts) were measured. Cultures were selected for magnetic fractionation when parasitaemia ranged between 6 -10% and the mature parasite density ranged between 2.1 -5.4%.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

7

Magnetic Fractionation

Prior to fractionation, each culture was centrifuged at 2000 rpm for 10 minutes and the culture medium was removed. The pellet, containing approximately 1mL packed cells, was then resuspended in 5 mL phosphate-buffered saline (PBS) with foetal calf serum [START_REF] Miltenyi | High gradient magnetic cell separation with MACS[END_REF]. The cells were exposed to ambient oxygen to ensure complete oxygenation of functional haemoglobin.

Commercially available midiMACS columns and magnet were used to separate the cultures [START_REF] Miltenyi | High gradient magnetic cell separation with MACS[END_REF]. The magnetic field strength between the poles of the magnet was measured with a Hall probe. A sample of the magnetisable spheres forming the column matrix was taken and the magnetisation as a function of field strength was measured with a SQUID magnetometer. Sphere radii were measured with an optical microscope and 112 spheres were weighed to determine the mean sphere mass.

One fractionation column was used for each culture. The column was held between the poles of the magnet and the flow rate of cells through the column was regulated with an adjustable tap connected to the column with a length of silicone tubing. The average fluid velocity within the magnetisable length of the column was calculated from the flow rate, the length of the column containing the magnetisable spheres and the void volume. Flow rate was maintained at approximately 0.2 mL.min -1 so that the average fluid velocity was less than 340 m.s -1 . After the sample had passed through, the column was washed twice with 1 mL of fractionation buffer to flush out the nominally non-magnetic erythrocytes trapped within the matrix. The fraction of cells that passed through the column whilst it

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

8 was exposed to the magnetic field was classed as the non-magnetic fraction. The column was then removed from the magnet and flushed through with fractionation buffer, and the cells collected were classed as the magnetic fraction. Fractionation of each culture typically yielded 50 to 100 L of packed cells in the magnetic fraction, and so magnetic fractions from three or four fractionations were pooled. The total volume of cells in each pooled magnetic fraction was at least 200 L as this was the minimum volume suitable for magnetometry experiments. The non-magnetic and the pooled magnetic fractions were centrifuged and the pelleted cells were smeared for microscopy. The samples were then freeze-thawed to lyse the cells and ultra-centrifuged at 19 000 g for 30 minutes to remove the supernatant, containing PBS and water. Cell membranes formed a thin layer between the haemoglobin and the supernatant, and when possible were removed.

Magnetometry subsamples of at least 200 L and spectrophotometry subsamples of 50 L were taken from each individual non-magnetic fraction and each pooled magnetic fraction.

A magnetic fraction was diluted with uninfected red blood cells to create a sample with a mature parasite density intermediate to the non-magnetic and the remaining magnetic fractions. The final mature parasite density of the diluted sample was 13%. Owing to the small sample volume, haemozoin extraction and quantification were not performed.

Elemental Analysis

Following magnetic examination the magnetometry samples of a known volume were digested in 10 mL of 1:1 ratio of 10 M AR grade nitric / sulphuric acid to remove the

A C C E P T E D M A N U S C R I P T

ACCEPTED MANUSCRIPT 9 organic materials. The resulting aqueous solutions were then diluted up to a known volume for inductively coupled plasma atomic emission spectrometry and the iron and chlorine concentrations were measured.

Spectrophotometry

A standard solution of oxyhaemoglobin was prepared by bubbling pure oxygen gas through fresh red cells. Standard solutions of methaemoglobin were prepared by exposing 5 mL packed cells to a 5 mM sodium nitrite solution and incubating at 37C for 2 hours [START_REF] Zborowski | Red blood cell magnetophoresis[END_REF]. The solutions were frozen and thawed to lyse the cells, then centrifuged at 13 000 g and the cell membranes removed. A standard sample of haematin was prepared by dissolving 1 g of porcine haematin (commercially available from Sigma-Aldrich) in 1 M sodium hydroxide solution [START_REF] Orjih | Hemozoin Production by Plasmodium-Falciparum -Variation with Strain and Exposure to Chloroquine[END_REF], then diluting with 2.5% SDS buffered with 25mM Tris to pH 7.8, to a final concentration of 1.6 mM.

Dilutions of each standard sample were prepared in PBS and the absorption spectrum between 350 and 700 nm was recorded using a DU640 spectrometer (Beckman Coulter).

Dilutions of the haematin solution were used to form a standard curve for absorption versus concentration.

It is not possible to measure haemozoin concentration in situ with spectrophotometric techniques as biological matrices and free haem alter the absorption spectrum of haemozoin [START_REF] Munro | Heme-Peptide Models for Hemoproteins. 2. N-Acetylmicroperoxidase-8: Study of the pi-pi Dimers Formed at High Ionic Strength Using a Modified Version of Molecular Exciton Theory[END_REF][START_REF] Marques | Hemepeptide models for hemoproteins: the behavior of N-acetylmicroperoxidase-11 in aqueous solution[END_REF]. Likewise, the oxyhaemoglobin-to-methaemoglobin ratio cannot be measured in solutions containing significant concentrations of haemozoin. A 1:100 v/v

A C C E P T E D M A N U S C R I P T

ACCEPTED MANUSCRIPT 10 dilution of each non-magnetic sample with less than 5% parasitaemia was prepared in PBS in triplicate and the absorption coefficients at 400, 577 and 630 nm were measured.

Winterbourn's formula was used to determine the relative concentrations of oxyhaemoglobin and methaemoglobin, and thus the ratio of oxygenated haemoglobin to methaemoglobin [START_REF] Winterbourn | Reactions involving superoxide and normal and unstable haemoglobins[END_REF], as follows:

[Methaemoglobin]  279A 630  3.0A 577 [Oxyhaemoglobin]  66A 577  80A 630 (1)
where A 630 and A 577 are the absorption coefficients at 630 nm and 577 nm respectively.

Haemozoin was isolated by freeze-thawing cells to induce lysis, then centrifuging the sample and removing the supernatant. The pellets, containing haemozoin and other forms of non-protein bound haem, were treated with SDS and centrifuged. The SDS-insoluble haemozoin pellets were then solubilized with 0.1 M NaOH. Solutions were diluted with 2.5% SDS buffered with 25 mM Tris to pH 7.8 and the absorbance at 400 nm was measured [START_REF] Orjih | Hemozoin Production by Plasmodium-Falciparum -Variation with Strain and Exposure to Chloroquine[END_REF]. Haemozoin could not be isolated in sufficient quantities from the nonmagnetic fractions for accurate quantification and so the best estimate for the haemozoin concentration of these samples is zero with an uncertainty of approximately 5%.

Magnetometry

Magnetometry measurements were made using a Quantum Design MPMS-7 magnetic susceptometer with a superconducting quantum interference device based sensor.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

11 Samples for magnetometry consisted of approximately 200 L of cell lysate. Each sample was suspended in a plastic tube and held in place by a polycarbonate plug. After freezing the plug was removed and the sample was supported with ice made from deionised water. The plastic tubes and surrounding ice were much longer than the dimensions of the magnetometer sense coils, and so did not contribute to the measured signal. However, as the cell lysate was surrounded by ice, the magnetic susceptibility of the samples was measured relative to the volumetric susceptibility of ice. All cell lysate samples were less than 1 cm in length and hence could be approximated as point dipoles for the purpose of analysis of the MPMS-7 signal output.

Measurements of sample magnetic moment were corrected for changes in the density of water and of the sample constituents with temperature [START_REF] Cerdonio | Magnetic susceptibility of hemoglobins[END_REF]. Molar magnetic susceptibility values are quoted in the cgs units system to facilitate comparisons with literature data.

Conversion of susceptibility values to the SI unit system requires multiplication by a factor of 4.

Magnetic susceptibility, measured at 265 K and 10 K, was determined from the measurement of the magnetisation of the sample, relative to pure ice, in an applied field.

The field was swept from 30 000 to 0 Oersteds (3 to 0 Tesla) and the magnetic susceptibility determined from the gradient of the magnetisation of the sample as a function of field. The volumetric magnetic susceptibility relative to air,  sample , was determined by subtracting the volumetric magnetic susceptibility of ice, i.e.

 sample   sample   H 2 0 , where  sample is the measured volumetric susceptibility of the

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

12 sample relative to ice and  H 2 0 is the volumetric susceptibility of ice (  H 2 0 = -0.72  10 -6 at 265 K and -0.75  10 -6 at 10 K) [START_REF] Weast | CRC Handbook of Chemistry and Physics[END_REF]. Some samples were found to contain small volumes of the magnetic fractionation buffer, as indicated by detectable concentrations of chlorine in the samples. At room temperature, the volumetric susceptibility of the buffer is similar to that of water, but the relative changes in density necessitated corrections for the diamagnetic volumetric susceptibility of the buffer at 265 K and 10 K. The globin contribution to the magnetic susceptibility of the sample,  globin , was calculated as

 globin  n globin M globin  (globin)
, where  (globin) is the mass susceptibility of globin in the haemoglobin molecule, measured by Savicki and co-workers as -0.58  10 -6 mL.g -1 [START_REF] Savicki | Magnetic susceptibility of oxy-and carbonmonoxyhemoglobins[END_REF],

M globin = 64 230 g.mol -1 is the molecular mass of globin and n globin , the concentration of globin, was calculated as n globin = 1/4 the molar concentration (mol.mL -1 ) of iron in the sample. The molar magnetic susceptibility of the iron,  Fe , was then calculated as

 Fe   sample   globin n Fe
, where n Fe is the molar concentration (mol.mL -1 ) of iron in the sample. Curie law-type dependence of magnetic susceptibility with temperature was assumed and so the value of the molar magnetic susceptibility of the iron at room temperature was calculated from each measurement using

 Fe (298K)   Fe (measured) T(measured) 298 .
The molar magnetic susceptibility of the iron at room temperature was taken to be the

Results

Optical Microscopy

Mature parasite densities in the unfractionated cultures ranged from 2.1 to 5.4%. After magnetic fractionation, the mature parasite density ranged from 34 to 84% in the magnetic fractions, and from 0.8 to 6.3% in the non-magnetic fractions. The process of magnetic fractionation increased the density of mature parasites between 6.3 and 31 fold, with a mean enrichment of 22 fold.

A sample with an initial mature parasite density of 4.2% was passed through a fractionation column, depleting the mature parasite density to 3.9% in the non-magnetic fraction. The non-magnetic fraction was then passed through a second fractionation column, further reducing the mature parasite density in the final non-magnetic fraction to 3.4%. The magnetic fractions extracted from the first and second fractionation process had mature parasite densities of 75% and 83% respectively.

Spectrophotometry

In the magnetic fractions the percentage of iron as haemozoin ranged from 25 to 52%, with a mean (and standard deviation) of 41  11%. Haemozoin concentration versus the mature parasite density is illustrated in Figure 1. As expected, the haemozoin

A C C E P T E D M A N U S C R I P T

ACCEPTED MANUSCRIPT 14 concentration correlates with mature parasite density. A linear model, constrained to pass through the origin, was fitted to the data and showed that the mature parasites converted on average 63  2% of cellular iron to haemozoin (Pearson's coefficient r = 0.99, p < 0.01).

The non-magnetic fractions appeared to contain little or no haemozoin and so the percentage of haemoglobin converted to methaemoglobin was measured using the Winterbourne formula. The methaemoglobin concentration ranged from 5.3 to 49% of cellular haemoglobin, with a mean (and standard deviation) concentration of 34  10%.

The absorption spectra of the oxygenated haemoglobin and methaemoglobin standard samples indicated 3% and 100% methaemoglobin concentrations respectively.

Magnetometry

The median molar magnetic susceptibility of the iron in the magnetic fractions was 5 600  10 -6 mL.mol -1 (range 2 200  10 -6 to 6 700  10 -6 mL.mol -1 ), almost six times greater than the median magnetic susceptibility of the iron in the non-magnetic fractions (median and range of 1 100  10 -6 mL.mol -1 and 670  10 -6 to 1 500  10 -6 mL.mol -1 respectively).

The magnetic susceptibilities of the iron in the standard samples of methaemoglobin, haematin and oxyhaemoglobin were measured as (14 000  800)  10 -6 , (13 000  650)

 10 -6 and (25  500)  10 -6 mL.mol -1 respectively. The susceptibilities corresponded to magnetic moments of 5.8  0.1, 5.6  0.1 and 0.2  0.1 Bohr magnetons per haem group for the methaemoglobin, haematin and oxyhaemoglobin samples respectively, in close

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

15 agreement with recent literature values [START_REF] Spees | Water proton MR properties of human blood at 1.5 Tesla: Magnetic susceptibility, T-1, T-2, T-2* and non-Lorentzian signal behavior[END_REF][START_REF] Zborowski | Red blood cell magnetophoresis[END_REF][START_REF] De Villiers | Speciation and structure of ferriprotoporphyrin IX in aqueous solution: spectroscopic and diffusion measurements demonstrate dimerization, but not mu-oxo dimer formation[END_REF]. After correcting the measurement of oxyhaemoglobin for a contribution from a 3% methaemoglobin component as measured via spectrophotometry, the magnetic susceptibility of the iron in oxyhaemoglobin was calculated to be -(350  500) 10 -6 mL.mol -1 .

Distributions of the magnetic susceptibilities of the iron in the magnetic and nonmagnetic fractions and the standard samples are displayed in Figure 2. It is worth noting that even a relatively large error on both the concentration and the magnetic susceptibility of globin and / or other protein constituents will have little effect on the magnetic susceptibility values calculated for cellular iron. Repeated measurements of magnetic susceptibility and iron concentration of methaemoglobin samples indicate that the final error of the magnetic susceptibility of the iron is approximately 5%.

A statistically significant correlation was identified between the magnetic susceptibility of the iron in the cell lysates and the mature parasite density. A linear model was fitted to the data, shown in Figure 3 (Pearson's coefficient r = 0.99, p < 0.01), and the parameters from the model are shown in the following equation:

 Fe (10 6 mL.mol 1 )  (830  60)  (67  21)  mature(%) , (2)
where mature(%) is the mature parasite density. The model indicates a net paramagnetic susceptibility of cellular iron in uninfected cells and / or cells infected with immature parasites. The magnetic susceptibility of (830  60)  10 -6 mL.mol -1 corresponds to approximately 5 -7% of iron atoms in the high-spin ferric state. Extrapolation of the model predicts a mean magnetic susceptibility of iron of (7 600  2 200)  10 -6 mL.mol -1

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

16 for mature parasitised cells, indicating that a further 59  16 % of cellular iron has been converted to high spin ferric iron by the parasites.

The magnetic susceptibility of iron versus spectrophotometric measurements of the percentage of cellular iron as haemozoin are shown in Figure 4 with a linear model fitted to the data (Pearson's coefficient r = 0.92, p < 0.01). The model yields the following equation:

 Fe (10 6 mL.mol 1 )  (1000 100)  [Hmz] 100  (110  9) (3)
where [Hmz] is the percentage of cellular iron as haemozoin. The gradient of the model corresponds to a magnetic susceptibility of haemozoin iron of (11 000  900)  10 -6 mL.mol -1 for haemozoin, which is not significantly different from the magnetic susceptibility of (13 000  600)  10 -6 mL.mol -1 evaluated for the iron in the haematin sample. The model also predicts a net paramagnetic susceptibility of (1 000  100)  10 -6 mL.mol -1 of cellular iron prior to haemozoin synthesis, implying that unparasitised cells and / or cells infected with immature parasites contained other forms of paramagnetic iron. The magnetic susceptibility of non-haemozoin paramagnetic iron,  Fe (non-Hmz), was calculated for each sample using the following equation,

 Fe (non  Hmz)(10 6 mL.mol 1 )   Fe (sample) [Hmz]  Fe (Hmz) (100 [Hmz]) (4)

A C C E P T E D M A N U S C R I P T

where  Fe (sample) is the magnetic susceptibility of the iron in the sample,  Fe (Hmz) = 11 000  10 -6 mL.mol -1 and [Hmz] is the percentage of cellular iron as haemozoin as measured via spectrophotometry and therefore (100-[Hmz]) is the percentage of nonhaemozoin cellular iron. No significant correlation between the magnetic susceptibility of the non-haemozoin iron and the mature parasite density was detected. A mean ( standard deviation) magnetic susceptibility of the iron of (710  200)  10 -6 mL.mol -1 was calculated for non-haemozoin iron.

Cell volumetric magnetic susceptibilities

Erythrocyte volumetric magnetic susceptibilities,  RBC , were evaluated using the measured molar magnetic susceptibilities of cellular iron together with a model based on that developed by Spees and co-workers [START_REF] Spees | Water proton MR properties of human blood at 1.5 Tesla: Magnetic susceptibility, T-1, T-2, T-2* and non-Lorentzian signal behavior[END_REF]. The red cell volumetric magnetic susceptibility is given by:

 RBC  (1 n Hb . Hb ) H 2 0  n Hb ( mol(globin)  4 Fe )
where  Hb = 48 000 mL.mol -1 is the molar volume of haemoglobin [START_REF] Cerdonio | Room-Temperature Magnetic-Properties of Oxyhemoglobin and Carbonmonoxy-Hemoglobin[END_REF],  mol(globin) = -37 800  10 -6 mL.mol -1 is the molar magnetic susceptibility of the globin [6],  H 2 0 = -0.72  10 -6 [START_REF] Weast | CRC Handbook of Chemistry and Physics[END_REF] and n Hb = 5.5  10 -6 mol.mL -1 is the erythrocytic haemoglobin concentration [START_REF] Hillman | The red cell manual[END_REF]. The erythrocyte magnetic susceptibilities for cells with different chemical species of iron are given in Table 1. To facilitate comparisons with literature values, all erythrocyte volumetric magnetic susceptibilities are quoted in SI units. Conversion to the cgs units system requires division by 4.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

18 Interestingly, the small paramagnetic susceptibility of cellular iron in the uninfected erythrocytes, or cells containing immature parasites, increases the erythrocyte susceptibility to coincide with the volumetric susceptibility of water. As (predominantly diamagnetic) haemoglobin is ingested and haemozoin produced, the relative susceptibility of the erythrocyte increases to approximately half the relative susceptibility of cells containing fully oxidised haemoglobin.

Discussion

The enrichment of mature stage parasites by magnetic fractionation techniques implies that, during the parasite development process, the magnetic properties of the cellular iron are altered and the magnetic susceptibility of the iron increases. The correlation between the average magnetic susceptibility of the cellular iron and the mature parasite density confirms that the ten-fold increase of the magnetic susceptibility of cellular iron is the primary reason for the magnetic enrichment of mature parasites. The magnetometry measurements of methaemoglobin and haematin confirm that both substances contain high-spin ferric iron, and therefore the conversion of diamagnetic oxyhaemoglobin to either paramagnetic form would increase the net magnetic susceptibility of the cellular Haemoglobin is oxidised to methaemoglobin immediately following ingestion into the food vacuole, converting the iron from the ferrous to the ferric form [START_REF] Francis | Hemoglobin metabolism in the malaria parasite Plasmodium falciparum[END_REF]. Therefore, the magnetic susceptibility of each haem iron increases as soon as the bond between the iron and the globin is denatured, rather than when the haem molecule is incorporated into haemozoin. Thus, the magnetic susceptibility of mature cells increases most likely because of digestion of haemoglobin rather than the detoxification of haem per se. The spectrophotometry measurements indicated that haemozoin was the predominant form of iron in the mature parasites studied here, accounting for 63  2% of cellular iron, and so haemozoin formation was the primary means of haem detoxification in the 3D7 parasite strain. If, as it has been hypothesised, alternative means of haem detoxification [START_REF] Atamna | Heme degradation in the presence of glutathione. A proposed mechanism to account for the high levels of non-heme iron found in the membranes of hemoglobinopathic red blood cells[END_REF] also exist, the net magnetic susceptibility of mature parasites will depend on the relative fractions of the final products of the detoxification pathways.

Spectrophotometry and magnetometry results indicate the presence of non-haemozoin paramagnetic iron, and the spectrophotometry measurements of the non-magnetic fractions show approximately 34% of cellular iron was oxidised to methaemoglobin.

Haemoglobin is oxidised during normal cell aging in vitro and so significant levels of methaemoglobin are expected in malaria cultures. However, this value is inconsistent with the magnetometry data, which indicates that less than 10% of cellular iron in uninfected cells or cells containing immature parasites is in the ferric state. Even The magnetometry results also show no correlation between the net magnetic susceptibility of non-haemozoin iron and mature parasite density. However, it is possible that the intracellular concentrations of chemical species of paramagnetic iron are continuously altered during parasite invasion and development. The parasites may preferentially ingest methaemoglobin, as it has been hypothesised that the net positive charge of the methaemoglobin complex may aid uptake into the food vacuole [START_REF] Akompong | In vitro activity of riboflavin against the human malaria parasite Plasmodium falciparum[END_REF]. There is likely a relatively small, transitory pool of non-haemozoin oxidised haem within the food vacuole as haem release in the food vacuole is carefully regulated to maintain supersaturated levels of haem [START_REF] Bray | Access to hematin: The basis of chloroquine resistance[END_REF].

The findings of this study indicate that caution should be exercised when interpreting magnetic susceptibility measurements performed on malarial-infected cells as significant levels of non-haemozoin high-spin iron(III), arising through natural cell aging or parasite metabolic activity, increase the mean magnetic susceptibility of cellular iron. Ursos and Roepe [START_REF] Ursos | Chloroquine resistance in the malarial parasite, Plasmodium falciparum[END_REF] discuss haem chemistry and emphasise the implications of non-haemozoin haem molecules within the food vacuole. Haem may exist, in unknown concentrations, as free haem or in dimeric forms. However, as both Egan and co-workers [START_REF] Egan | Fate of haem iron in the malaria parasite Plasmodium falciparum[END_REF] and Moore and co-workers [START_REF] Moore | Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis[END_REF] point out, the fraction of non-haemozoin iron in the food-vacuole accounts for less than 5% of cellular iron, consistent with the results of this study

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 21
showing no correlation of the non-haemozoin high spin iron(III) concentration with mature parasite density.

Oxidation of haem iron is associated with haemoglobin digestion and therefore with parasite growth, so as the parasite matures the volumetric susceptibility of the infected erythrocyte increases. Cells containing mature parasites are paramagnetic relative to the suspension buffer used in fractionations, whereas the magnetic susceptibility of unparasitised cells is indistinguishable from the buffer. The volumetric susceptibility of 1.89  0.63  10 -6 (SI units) of mature parasitised cells relative to water calculated here is consistent with the value measured experimentally by Moore et al [START_REF] Moore | Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis[END_REF] of 1.8  10 -6 . The magnetic fractionation procedure used in the present experiment was sufficiently sensitive to discriminate between cells with a volumetric susceptibility difference of 1.89  0.04  10 -6 (SI units). For a given set of fractionation conditions, i.e. flow rate, applied magnetic field environment and suspension buffer, a critical volumetric susceptibility exists that determines whether a cell will be retained in the 'magnetic fraction' of a fractionation. Adjusting the flow rate or changing the volumetric susceptibility of the suspension medium will change the critical volumetric susceptibility of erythrocytes, so that magnetic fractionation of cultures could be targeted to extract only parasites at specific stages of development. Alternatively, fractionation of erythrocytes under specific conditions could provide a surrogate measure of the magnetic susceptibility, and potentially the ferric iron content, of the cells.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Magnetic fractionation has been shown to yield mature parasite densities of up to 98% [START_REF] Trang | One-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells[END_REF], comparable to other methods such as Percoll density gradient centrifugation [START_REF] Tosta | Plasmodium yoelii and Plasmodium berghei: isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation[END_REF][START_REF] Kramer | Concentration of Plasmodium falciparum-infected erythrocytes by density gradient centrifugation in Percoll[END_REF]. Magnetic fractionation can also be a means of synchronising cultures as extracted parasites can be reintroduced into culture. However, the potential to target very specific stages of development through 'fine-tuning' of the fractionation conditions may allow for a greater degree of synchrony than can be achieved with conventional methods such as sorbitol lysis [START_REF] Aley | Identification of isolate-specific proteins on sorbitol-enriched Plasmodium falciparum infected erythrocytes from Gambian patients[END_REF]. Interestingly, mature parasites were detected in all non-magnetic fractions. Repeated fractionation of non-magnetic fractions did not change the enrichment of mature parasites, indicating that the magnetic susceptibility of mature parasites retained in the column is not necessarily greater than the magnetic susceptibility of the mature parasites that pass through into the non-magnetic fraction. The fractionation process is highly specific but only moderately sensitive and so repeating magnetic fractionations on cultures will increase the mature parasite yield.

  mean of the values obtained from the 10 K and the 265 K measurements. The standard deviation of the molar magnetic susceptibility at room temperature was calculated from the standard deviations of the 10 K and the 265 K measurements. It should be noted that A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 13 the molar magnetic susceptibility of the iron is expressed in units of mL per mole of iron. (To compare the  Fe values in this work with previous work measuring the magnetic susceptibility of iron per mole of haemoglobin, multiply the  Fe values by four.)

  iron. It is likely that multiple chemical species of iron coexist within the food vacuole of the parasite. The process of transforming globin-bound haem to haemozoin has intermediate steps and the presence of paramagnetic haemozoin precursors may explain A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 19 why the value of the magnetic susceptibility of haemozoin iron extrapolated from Equation 3 was slightly lower than the value measured for synthetic haematin.

  of haemozoin and / or haemozoin precursors may interfere with the absorption spectra of haemoglobin, causing an overestimation of the methaemoglobin concentration via spectrophotometry.

FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4

 1234 FIGURE 1 Percentage of cellular iron converted to haemozoin versus mature parasite

A C C E P T E D M A N U S C R I P T

 Fe (10 -6 mL.mol - susceptibilities relative to water. The volumetric susceptibilities of the cells have been evaluated using a model adapted from model developed by Spees and co-workers [START_REF] Spees | Water proton MR properties of human blood at 1.5 Tesla: Magnetic susceptibility, T-1, T-2, T-2* and non-Lorentzian signal behavior[END_REF] together with the molar magnetic susceptibilities of cellular iron measured in this study.

Values for oxyhaemoglobin and methaemoglobin are calculated from the standard samples.

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT


About 250 to 500 glycogenes (genes that are directly involved in glycan metabolism) are in the human genome representing about 1-2% of the total genome. Over 40 human congenital diseases associated with glycogene mutations have been described to date. It is almost certain that the causative glycogene mutations for many more congenital diseases remain to be discovered. Some glycogenes are involved in the synthesis of only a specific protein and/or a specific class of glycan whereas others play a role in the biosynthesis of more than one glycan class. Mutations in the latter type of glycogene result in complex clinical phenotypes that present difficult diagnostic problems to the clinician. In order to understand in biochemical terms the clinical signs and symptoms of a patient with a glycogene mutation, one must understand how the glycogene works. That requires, first of all, determination of the target protein or proteins of the glycogene followed by an understanding of the role, if any, of the glycogene-dependent glycan in the functions of the protein. Many glycogenes act on thousands of glycoproteins. There are unfortunately no general methods to identify all the potentially large number of glycogene target proteins and which of these proteins are responsible for the mutant phenotypes. Whereas biochemical methods have been highly successful in the discovery of glycogenes responsible for many congenital diseases, it has more recently been necessary to use other methods such as homozygosity mapping. Accurate diagnosis of many recently discovered diseases has become difficult and new diagnostic procedures must be developed. Last but not least is the lack of effective treatment for most of these children and of animal models that can be used to test new therapies.

Introduction

Glycans are significantly more complex than nucleic acids and proteins, the other two major information-bearing biological macromolecules. Nucleic acids and proteins are linear molecules in which the individual subunits (nucleotides and amino acids respectively) are linked respectively by identical phospho-diester and peptide bonds. In contrast, linkages between the individual carbohydrate subunits of glycans are not all the same and the glycan polymers are commonly branched rather than linear. The apparatus required for glycan assembly is unique. Whereas the biosynthesis of nucleic acids and proteins is carried out by relatively simple template mechanisms, glycans require a complex non-template assembly line along which many different enzymes and other proteins work together to make the final glycan product. The spectrum of all glycan structures -the glycome -is immense; in humans, the number of glycan structures is orders of magnitude greater than the number of proteins that are encoded by the genome [START_REF] Freeze | Genetic defects in the human glycome[END_REF]. Francis Crick' s original Central Dogma ignored post-translational modifications (such as protein glycosylation). It is now widely recognized that post-translational modifications can greatly magnify the functions of a single gene [START_REF] Krishna | Post-translational modification of proteins[END_REF][START_REF] Lowe | A Genetic Approach to Mammalian Glycan Function[END_REF]. This review discusses the nature of glycogenes involved in glycan metabolism, the extremely complex multi-systemic phenotypes that result from congenital defects in glycogenes, the discovery of new glycogenes, and the diagnosis and treatment of glycogene diseases.

Glycogenes

At least half of all the proteins in the human genome are probably N-glycosylated [START_REF] Apweiler | On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database[END_REF]. The number of genes directly involved in glycan metabolism (glycogenes [START_REF] Kikuchi | Bioinformatics for comprehensive finding and analysis of glycosyltransferases[END_REF][START_REF] Narimatsu | Human glycogene cloning: focus on beta 3-glycosyltransferase and beta 4-glycosyltransferase families[END_REF]) in the human genome is large (~ 250 to 500 genes representing ~ 1-2% of the total number of human genes) and encode many different types of protein: glycosyltransferases, sulfotransferases that act on glycans, oligosaccharyltransferases, nucleotide-sugar transporters and synthases, glycosidases, lectins, genes required for the synthesis or utilization of dolichol-phosphate-mannose and dolichol-phosphate-glucose, glycan flippases, Conserved Oligomeric Golgi (COG) complex proteins required for normal traffic in the Golgi apparatus, various kinases, mutases, isomerases and other enzymes that act on carbohydrates, chaperones and other proteins that ensure proper folding of glycogene-encoded proteins, and probably many others. Although patients with congenital abnormalities in glycan metabolism (glycan biosynthesis, catabolism or binding) are relatively rare, well over 40 human congenital diseases associated with glycogene mutations have been described to date: 16 Type I Congenital Disorders of Glycosylation (CDG-Ia to CDG-Ip) [START_REF] Freeze | Altered glycan structures: the molecular basis of congenital disorders of glycosylation[END_REF][START_REF] Freeze | Congenital Disorders of Glycosylation: CDG-I, CDG-II, and beyond[END_REF][START_REF] Kranz | A defect in dolichol phosphate biosynthesis causes a new inherited disorder with death in early infancy[END_REF][START_REF] Molinari | Oligosaccharyltransferase-subunit mutations in nonsyndromic mental retardation[END_REF][START_REF] Garshasbi | A defect in the TUSC3 gene is associated with autosomal recessive mental retardation[END_REF][START_REF] Frank | Does Rft1 flip an N-glycan lipid precursor?[END_REF][START_REF] Sanyal | Distinct flippases translocate glycerophospholipids and oligosaccharide diphosphate dolichols across the endoplasmic reticulum[END_REF][START_REF] Haeuptle | Human RFT1 deficiency leads to a disorder of N-linked glycosylation[END_REF], 8 Type II CDGs (CDG-IIa to CDG-IIh) [START_REF] Freeze | Altered glycan structures: the molecular basis of congenital disorders of glycosylation[END_REF][START_REF] Kranz | COG8 deficiency causes new congenital disorder of glycosylation type IIh[END_REF][START_REF] Foulquier | A new inborn error of glycosylation due to a Cog8 deficiency reveals a critical role for the Cog1-Cog8 interaction in COG complex formation[END_REF][START_REF] Foulquier | Conserved oligomeric Golgi complex subunit 1 deficiency reveals a previously uncharacterized congenital disorder of glycosylation type II[END_REF][START_REF] Zeevaert | Deficiencies in subunits of the Conserved Oligomeric Golgi (COG) complex define a novel group of Congenital Disorders of Glycosylation[END_REF][START_REF] Wu | Mutation of the COG complex subunit gene COG7 causes a lethal congenital disorder[END_REF], 6 Congenital Muscular Dystrophies (CMDs) [START_REF] Martin | Congenital muscular dystrophies involving the O-mannose pathway[END_REF], 2 involving GalNAcα1-O-Ser/Thr glycans [START_REF] Barbieri | Two novel nonsense mutations in GALNT3 gene are responsible for familial tumoral calcinosis[END_REF][START_REF] Garringer | Two novel GALNT3 mutations in familial tumoral calcinosis[END_REF][START_REF] Topaz | Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis[END_REF][START_REF] Ju | Protein glycosylation: chaperone mutation in Tn syndrome[END_REF], 2 involving Fucα1-O-Ser/Thr glycans [START_REF] Heinonen | Peters'-plus syndrome is a congenital disorder of glycosylation caused by a defect in the beta1,3-glucosyltransferase that modifies thrombospondin type 1 repeats[END_REF][START_REF] Rampal | Notch signaling in normal and disease States: possible therapies related to glycosylation[END_REF][START_REF] Sparrow | Mutation of the Lunatic Fringe gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype[END_REF][START_REF] Ladi | The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands[END_REF], 5 glycosaminoglycan-related diseases [START_REF] Furukawa | Galactosyltransferase I is a gene responsible for progeroid variant of Ehlers-Danlos syndrome: molecular cloning and identification of mutations[END_REF][START_REF] Zak | Hereditary multiple exostoses and heparan sulfate polymerization[END_REF][START_REF] Akama | Macular corneal dystrophy type I and type II are caused by distinct mutations in a new sulphotransferase gene[END_REF][START_REF] Liu | Mutations in corneal carbohydrate sulfotransferase 6 gene (CHST6) cause macular corneal dystrophy in Iceland[END_REF][START_REF] El-Ashry | Identification of novel mutations in the carbohydrate sulfotransferase gene (CHST6) causing macular corneal dystrophy[END_REF][START_REF] Van Roij | Spondyloepiphyseal dysplasia, Omani type: Further definition of the phenotype[END_REF][START_REF] Hermanns | Congenital joint dislocations caused by carbohydrate sulfotransferase 3 deficiency in recessive Larsen syndrome and humero-spinal dysostosis[END_REF][START_REF] Thiele | Loss of chondroitin 6-O-sulfotransferase-1 function results in severe human chondrodysplasia with progressive spinal involvement[END_REF], diseases involving defective glycosphingolipid [START_REF] Farukhi | Etiology of vision loss in ganglioside GM3 synthase deficiency[END_REF][START_REF] Simpson | Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase[END_REF] and glycosylphosphatidylinositol [START_REF] Kinoshita | Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria[END_REF] synthesis, galactosemia [START_REF] Sturiale | Multiplexed glycoproteomic analysis of glycosylation disorders by sequential yolk immunoglobulins immunoseparation and MALDI-TOF MS[END_REF][START_REF] Sturiale | Hypoglycosylation with increased fucosylation and branching of serum transferrin N-glycans in untreated galactosemia[END_REF][START_REF] Charlwood | Defective galactosylation of serum transferrin in galactosemia[END_REF], I-cell disease and pseudo-Hurler polydystrophy (lysosomal storage diseases) caused by mutations in the gene encoding UDP-N-acetylglucosamine:glycoprotein Nacetylglucosaminylphosphotransferase [START_REF] Reitman | Fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy are deficient in uridine 5'-diphosphate-Nacetylglucosamine: glycoprotein N-acetylglucosaminylphosphotransferase activity[END_REF][START_REF] Kornfeld | Trafficking of lysosomal enzymes in normal and disease states[END_REF], and hereditary inclusion body myopathy caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene [START_REF] Malicdan | Perspectives on distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy: contributions from an animal model. Lack of sialic acid, a central determinant in sugar chains, causes myopathy?[END_REF]. It is almost certain that the causative glycogene mutations for many more congenital diseases remain to be discovered. Some glycogenes are involved in the synthesis of only a specific protein and/or a specific class of glycan, e.g., the six genes involved in the CMDs appear to act primarily on αdystroglycan, and three of these genes act only on Manα1-O-Ser/Thr-protein, i.e., the genes encoding protein O-mannosyltransferases 1 and 2 (POMT1, POMT2) and protein O-mannosyl β1,2-N-acetylglucosaminyltransferase I (POMGNTI). Unlike the CDGs, these O-linked disorders possess distinctive muscle, eye, and brain phenotypes [START_REF] Martin | The dystroglycanopathies: the new disorders of O-linked glycosylation[END_REF][START_REF] Endo | Aberrant glycosylation of alpha-dystroglycan and congenital muscular dystrophies[END_REF]. Peters'-plus syndrome is a rare autosomal recessive disorder involving defects in multiple organ systems. The syndrome is a newly-discovered CDG due to mutations in the β1,3glucosyltransferase that adds glucose to Fucα1-O-Ser/Thr to form a disaccharide specific to thrombospondin type 1 repeats (TSRs). This disaccharide is only found on thrombospondin 1 and a few other other TSR-containing proteins [START_REF] Heinonen | Peters'-plus syndrome is a congenital disorder of glycosylation caused by a defect in the beta1,3-glucosyltransferase that modifies thrombospondin type 1 repeats[END_REF].

On the other hand, many glycogenes are involved in the biosynthesis of more than one glycan class because the biosynthetic pathways for glycosphingolipids and for proteinbound GlcNAcβ1-N-Asn, GalNAcα1-O-Ser/Thr, Manα1-O-Ser/Thr, Fucα1-O-Ser/Thr and Xylβ1-O-Ser (proteoglycans) glycans share glycogenes. For example, several sialyl- [START_REF] Hamamoto | ST3Gal-II (SAT-IV)[END_REF][START_REF] Hamamoto | ST6Gal-I[END_REF], galactosyl- [START_REF] Shaper | β4-Galactosyltransferase-I[END_REF][START_REF] Furukawa | β4-Galactosyltransferase-II, -III, -IV, -V, -VI, and -VII[END_REF][START_REF] Hennet | β3-Galactosyltransferase-I, -II, and -III[END_REF] and fucosyltransferases [START_REF] Oriol | α2-Fucosyltransferases (FUT1, FUT2, and Sec1)[END_REF][START_REF]Narimatsu H: α3/4-Fucosyltransferase (FUT3, Lewis enzyme)[END_REF][START_REF]Narimatsu H: α3-Fucosyltransferase-IV (FUT4)[END_REF][START_REF] Kannagi | α3-Fucosyltransferase-VI (FUT6)[END_REF] are common to two or more of the above pathways, as are various nucleotide-sugar transporters and synthases, members of the COG complex, and others.

Although the clinical presentations of "congenital glycosylation diseases" (this term is used in a very general way in this chapter to include CDGs, CMDs and other glycogenedeficient congenital diseases) may be quite distinct, clinical overlaps occur when mutations occur in glycogenes common to more than one biosynthetic pathway. The following CDGs clearly have the potential to involve both N-glycans and other glycan types. (i) Defective availability of dolichol-phospho-mannose (Dol-P-Man): CDG-Ia (phosphomannomutase), -Ib (phosphomannose isomerase), -Ie (Dol-P-Man synthase), -If (Dol-P-Man utilization), -Im (dolichol kinase). However, since complete absence of the Type I CDG pathway is lethal, these diseases are all due to "leaky" (hypomorphic) mutations; therefore clinical symptoms of diseases other than those of Type I CDG (e.g., CMD) are unlikely to be visible. (ii) Nucleotide-sugar transporter defects: CDG-IIc (GDP-Fuc transporter), -IId (UDP-Gal transporter), -IIf (CMP-sialic acid transporter). (iii) Conserved Oligomeric Golgi (COG) complex proteins: CDG-IIe (COG7), -IIg (COG1), -IIh (COG8). Defects in other COG subunits are also likely to be found. The COG complex consists of 8 Golgi-associated cytoplasmic proteins required for maintaining the structure and function of the Golgi apparatus and for the efficient trafficking of proteins within the Golgi and export out of the Golgi [START_REF] Freeze | Congenital Disorders of Glycosylation: CDG-I, CDG-II, and beyond[END_REF][START_REF] Kranz | COG8 deficiency causes new congenital disorder of glycosylation type IIh[END_REF][START_REF] Foulquier | A new inborn error of glycosylation due to a Cog8 deficiency reveals a critical role for the Cog1-Cog8 interaction in COG complex formation[END_REF][START_REF] Foulquier | Conserved oligomeric Golgi complex subunit 1 deficiency reveals a previously uncharacterized congenital disorder of glycosylation type II[END_REF][START_REF] Zeevaert | Deficiencies in subunits of the Conserved Oligomeric Golgi (COG) complex define a novel group of Congenital Disorders of Glycosylation[END_REF][START_REF] Wu | Mutation of the COG complex subunit gene COG7 causes a lethal congenital disorder[END_REF][START_REF] Ungar | Retrograde transport on the COG railway[END_REF]. Mutations in COG proteins therefore alter the activities of glycosyltransferases, glycosidases, nucleotide-sugar transporters and other Golgi-localized proteins with resultant defects in the biosynthesis of GlcNAcβ1-N-Asn, GalNAcα1-O-Ser/Thr, Manα1-O-Ser/Thr (defective in the CMDs) and other glycan types [START_REF] Wopereis | Mechanisms in protein O-glycan biosynthesis and clinical and molecular aspects of protein O-glycan biosynthesis defects: a review[END_REF][START_REF] Percival | Golgi complex organization in skeletal muscle: a role for Golgi-mediated glycosylation in muscular dystrophies[END_REF].

(iv) A group of mentally retarded dysmorphic patients with cutis laxa (a condition characterized by sagging skin) probably represents a novel CDG type with defects in either N-glycan or both Nand O-glycan synthesis and an unusual cobblestone-like cortical malformation over the frontal and parietal regions similar to the lesions found in muscle-eye-brain type CMD [START_REF] Van Maldergem | Cobblestone-like brain dysgenesis and altered glycosylation in congenital cutis laxa, Debre type[END_REF][START_REF] Wopereis | A combined defect in the biosynthesis of N-and Oglycans in patients with cutis laxa and neurological involvement: the biochemical characteristics[END_REF]. Loss-of-function mutations in the gene encoding a subunit of the V-type H+ ATPase in several of these patients result in abnormal glycosylation of serum proteins and cause an impairment of Golgi trafficking in fibroblasts from affected individuals [START_REF] Kornak | Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2[END_REF].

Proteins like the COG complex subunits and ATPases are not "glycogenes" in the strict sense since they may be involved in pathways other than glycosylation. Therefore the clinical presentations of such patients may contain elements that are not glycosylationdependent.

How do glycogenes work?

In order to understand in biochemical terms the clinical signs and symptoms of a patient with a glycogene mutation, one must understand how the glycogene works. That requires, first of all, determination of the target protein or proteins of the glycogene followed by an understanding of the role, if any, of the glycogene-dependent glycan in the functions of the protein. In the case of the CMDs (often called -dystroglycanopathies [START_REF] Toda | Fukuyama-type congenital muscular dystrophy (FCMD) and alpha-dystroglycanopathy[END_REF]), the target protein is known but the role of the Manα1-O-Ser/Thr glycan in α-dystroglycan function is not clear. The target protein problem becomes much more difficult in the CDGs because thousands of glycoproteins share the same glycan structures and a mutation in a particular glycogene affects the glycosylation of many proteins in most, if not all, cells in the body.

A classic approach to determination of gene target proteins and functions is the creation of animals with null mutations in the gene under study. Glycan function has been studied in many mutant organisms: yeast [START_REF] Freeze | Altered glycan structures: the molecular basis of congenital disorders of glycosylation[END_REF][START_REF] Lehle | Protein Glycosylation, Conserved from Yeast to Man: A Model Organism Helps Elucidate Congenital Human Diseases[END_REF], mice [START_REF] Lowe | A Genetic Approach to Mammalian Glycan Function[END_REF][START_REF] Angata | Cellular and molecular analysis of neural development of glycosyltransferase gene knockout mice[END_REF][START_REF] Ohtsubo | Glycosylation in cellular mechanisms of health and disease[END_REF], flies [START_REF] Leonard | The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing[END_REF][START_REF] Sarkar | Null mutations in Drosophila N-acetylglucosaminyltransferase I produce defects in locomotion and a reduced lifespan[END_REF][START_REF] Seppo | Induction of neuron-specific glycosylation by Tollo/Toll-8, a Drosophila Toll-like receptor expressed in nonneural cells[END_REF], worms [START_REF] Cipollo | srf-3, a mutant of Caenorhabditis elegans, resistant to bacterial infection and to biofilm binding, is deficient in glycoconjugates[END_REF][START_REF] Schachter | Protein glycosylation lessons from Caenorhabditis elegans[END_REF][START_REF] Shi | N-Glycans Are Involved in the Response of Caenorhabditis elegans to Bacterial Pathogens[END_REF], zebrafish [START_REF] Machingo | A beta1,4-galactosyltransferase is required for Bmp2-dependent patterning of the dorsoventral axis during zebrafish embryogenesis[END_REF][START_REF] Machingo | A beta1,4-galactosyltransferase is required for convergent extension movements in zebrafish[END_REF] and, of course, human "congenital glycosylation diseases". The identities of some of the target proteins of a mutant gene can be determined using mass spectrometry to compare glycopeptides isolated from wild type and mutant organisms [START_REF] Tan | Functional Glycoproteomic Analysis of Caenorhabditis elegans Interaction with Bacterial Pathogens[END_REF]. However, only a relatively small number of glycogene target proteins can be identified by current methods.

Taniguchi et al [START_REF] Taniguchi | Decoding sugar functions by identifying target glycoproteins[END_REF] have reviewed their approach to the identification of target proteins of three glycosyltransferases involved in N-glycan synthesis: β1,6-Nacetylglucosaminyltransferase V (GlcNAcTV; adds a branch to the N-glycan core), β1,4-N-acetylglucosaminyltransferase III (GlcNAcTIII; adds the bisecting GlcNAc to the Nglycan core), and α1,6-fucosyltransferase VIII (FucTVIII; adds a fucose residue to the Nglycan core). (i) GlcNAcTV has been shown to stimulate tumor metastasis in mice [START_REF] Granovsky | Suppression of tumor growth and metastasis in Mgat5-deficient mice[END_REF]. Mammary tumor growth and metastases induced by the polyomavirus middle T oncogene were considerably less in GlcNAcTV-null mice than in transgenic littermates expressing GlcNAcTV [START_REF] Granovsky | Suppression of tumor growth and metastasis in Mgat5-deficient mice[END_REF]. Matriptase (a metal-dependent serine protease) was identified as a GlcNAcTV target protein that enhances cancer invasion and metastasis if a specific Asn residue undergoes GlcNAcTV-dependent glycosylation [START_REF] Taniguchi | Decoding sugar functions by identifying target glycoproteins[END_REF]. (ii) E-cadherin, a homophilic adhesion molecule, has been identified as a GlcNAcTIII target protein [START_REF] Taniguchi | Decoding sugar functions by identifying target glycoproteins[END_REF][START_REF] Yoshimura | Aberrant glycosylation of Ecadherin enhances cell-cell binding to suppress metastasis[END_REF]. The addition of a bisecting GlcNAc by GlcNAcTIII inhibits the action of GlcNAcTV on the same N-glycan [START_REF] Schachter | Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides[END_REF][START_REF] Schachter | Coordination between enzyme specificity and intracellular compartmentation in the control of protein-bound oligosaccharide biosynthesis[END_REF]. GlcNAcTIII overexpression has an anti-metastatic effect that may be due, at least in part, to altered N-glycan structures on Ecadherin [START_REF] Taniguchi | Decoding sugar functions by identifying target glycoproteins[END_REF][START_REF] Yoshimura | Aberrant glycosylation of Ecadherin enhances cell-cell binding to suppress metastasis[END_REF]. (iii) The transforming growth factor-β (TGF-β) receptors are targets for FucTVIII [START_REF] Taniguchi | Decoding sugar functions by identifying target glycoproteins[END_REF]. FucTVIII down-regulation inhibits TGF-β signaling resulting in increased production of several metalloproteinases (MMPs). The increased levels of MMPs in FucTVIII-null mice causes emphysema-like degeneration of lung tissues [START_REF] Wang | Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice[END_REF].

While these results are of great interest, it is important to note that all three of the above enzymes also have many other target proteins that may also play a role in the respective phenotypes. There is unfortunately no general solution to identification of all the potentially large number of glycogene target proteins nor for the identification of which protein(s) are responsible for the mutant phenotypes. There is a very strong possibility that in many "congenital glycosylation diseases", it will not be possible to explain the phenotype on the basis of one or a small number of abnormally glycosylated proteins.

Another aspect of the problem is a possibly deleterious role of hypomorphic alleles and heterozygous mutations in the etiology of disease. Sub-optimal glycosylation may have its impact in a larger context and only under stressed conditions. Work with model organisms is usually carried out with null mutants; perhaps we also need to study the effects of hypomorphic and heterozygous mutations in animal models. Some pathology in glycosylation disorders is seen only when environmental insult(s) collide with the genetic insufficiency [START_REF] Bode | Applied glycoproteomics--approaches to study geneticenvironmental collisions causing protein-losing enteropathy[END_REF]. For instance, protein-losing enteropathy (PLE, loss of plasma proteins through the intestine) occurred in a CDG-Ic patient only during periods of intestinal rotavirus infections [START_REF] Westphal | Reduced heparan sulfate accumulation in enterocytes contributes to protein-losing enteropathy in a congenital disorder of glycosylation[END_REF]. CDG-Ib patients also develop PLE in inflammatory settings [START_REF] Niehues | Carbohydrate-deficient glycoprotein syndrome type Ib -Phosphomannose isomerase deficiency and mannose therapy[END_REF]. The effects of proinflammatory cytokines and portal hypertension caused by their abnormal liver architecture synergize with loss of heparan sulfate from the intestinal epithelial cells and cascade into a PLE avalanche [START_REF] Bode | Applied glycoproteomics--approaches to study geneticenvironmental collisions causing protein-losing enteropathy[END_REF][START_REF] Bode | Heparan sulfate plays a central role in a dynamic in vitro model of protein-losing enteropathy[END_REF][START_REF] Bode | Heparan sulfate depletion amplifies TNFalpha-induced protein leakage in an in vitro model of protein-losing enteropathy[END_REF]. Even small decreases in glycosylation can be deleterious. Mice with 50% normal phosphomannose isomerase have no obvious phenotype, but they show a significant increase in enteric protein loss, which is reversed by adding mannose to their drinking water. Increased pressure and exposure to proinflammatory cytokines further exacerbates protein leakage in these phosphomannose isomerase heterozygotes; mannose reverses the glycosylation-related contribution [START_REF] Bode | Applied glycoproteomics--approaches to study geneticenvironmental collisions causing protein-losing enteropathy[END_REF].

Gene discovery and diagnosis

Discovery of glycogenes for novel "congenital glycosylation diseases" and diagnosis of known diseases, especially of the Type 1 and some Type II Congenital Disorders of Glycosylation, have been carried out for many years by various well-established biochemical methods: (i) determination of protein-bound glycan structures, e.g., the GlcNAcβ1-N-Asn N-glycans present on serum transferrin and other proteins by isoelectric focusing and mass spectrometry [START_REF] Wada | Mass spectrometry for congenital disorders of glycosylation, CDG[END_REF], (ii) analysis of biochemical pathways in radio-labeled patient fibroblasts [START_REF] Kim | Dolichol phosphate mannose synthase (DPM1) mutations define congenital disorder of glycosylation Ie (CDG-Ie)[END_REF][START_REF] Wu | Deficiency of UDP-GlcNAc:Dolichol Phosphate N-Acetylglucosamine-1 Phosphate Transferase (DPAGT1) Causes a Novel Congenital Disorder of Glycosylation Type Ij[END_REF][START_REF] Rush | Mannose supplementation corrects GDP-mannose deficiency in cultured fibroblasts from some patients with Congenital Disorders of Glycosylation (CDG)[END_REF][START_REF] Panneerselvam | Human fibroblasts prefer mannose over glucose as a source of mannose for N-glycosylation. Evidence for the functional importance of transported mannose[END_REF], and (iii) functional assays (if available) of the enzymes and other proteins known to be involved in glycan synthesis [START_REF] Freeze | Congenital Disorders of Glycosylation: CDG-I, CDG-II, and beyond[END_REF].

More recently, a candidate gene approach in combination with homozygosity mapping has been used to discover new disease-causing glycogenes when consanguineous families are available. An excellent example has been the discovery of some of the genes involved in the heterogeneous Walker-Warburg Syndrome (WWS), an autosomal recessive developmental disorder characterized by CMD and complex brain and eye abnormalities. A genomewide linkage analysis in 10 consanguineous families with WWS was reported in 2002 and indicated the existence of at least three WWS loci [START_REF] De Bernabe | Mutations in the O-mannosyltransferase gene POMT1 give rise to the severe neuronal migration disorder Walker-Warburg syndrome[END_REF]. A candidate-gene approach in combination with homozygosity mapping was subsequently carried out in 15 consanguineous families with WWS [START_REF] De Bernabe | Mutations in the O-mannosyltransferase gene POMT1 give rise to the severe neuronal migration disorder Walker-Warburg syndrome[END_REF]. On the basis of previous work on another CMD, the gene encoding protein O-mannosyltransferase 1 (POMT1) was chosen as a candidate gene. Analysis of the locus for POMT1 revealed homozygosity in 5 of 15 families. Sequencing of the POMT1 gene revealed mutations in 6 of the 30 unrelated patients with WWS. A similar homozygosity mapping approach reported in 2005 [START_REF] Van Reeuwijk | POMT2 mutations cause alpha-dystroglycan hypoglycosylation and Walker-Warburg syndrome[END_REF] identified WWS patients with mutations in the gene encoding POMT2. Homozygosity was found for the POMT2 locus at 14q24.3 in 4 of 11 consanguineous WWS families; homozygous POMT2 mutations were present in two of these families as well as in one patient from another cohort of six WWS families. Mutations in four glycogenes are currently known to cause WWS, i.e., the genes encoding POMT1, POMT2, fukutin and fukutin related protein (FKRP) [START_REF] Vajsar | Walker-Warburg syndrome[END_REF]. These four genes account for approximately one third of the WWS cases indicating that other WWS genes remain to be discovered [START_REF] Van Reeuwijk | POMT2 mutations cause alpha-dystroglycan hypoglycosylation and Walker-Warburg syndrome[END_REF].

Homozygosity mapping methods were also used in the discovery of the defective β1,3glucosyltransferase glycogene in Peters'-plus syndrome (see above) [START_REF] Heinonen | Peters'-plus syndrome is a congenital disorder of glycosylation caused by a defect in the beta1,3-glucosyltransferase that modifies thrombospondin type 1 repeats[END_REF], of the V-type H+ ATPase-dependent CDG (see above) [START_REF] Van Maldergem | Cobblestone-like brain dysgenesis and altered glycosylation in congenital cutis laxa, Debre type[END_REF][START_REF] Kornak | Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2[END_REF], and of the role of a mutant oligosaccharyltransferase subunit in autosomal-recessive nonsyndromic mental retardation [START_REF] Molinari | Oligosaccharyltransferase-subunit mutations in nonsyndromic mental retardation[END_REF][START_REF] Garshasbi | A defect in the TUSC3 gene is associated with autosomal recessive mental retardation[END_REF]. Homozygosity mapping is a powerful method of gene discovery if consanguineous families are available. The biochemical methods that served so well in the early days of CDG discovery are often not successful in the more complex "congenital glycosylation diseases" but will remain an important part of the tool box to understand the mechanisms of action and biology of the glycogenes being discovered by other methods.

Many patients with "congenital glycosylation diseases" have been reported in which the function of the mutant gene is not known or in which no gene locus has as yet been identified [START_REF] Freeze | Altered glycan structures: the molecular basis of congenital disorders of glycosylation[END_REF][START_REF] Freeze | Congenital Disorders of Glycosylation: CDG-I, CDG-II, and beyond[END_REF][START_REF] Freeze | Novel perspectives on glycosylation and human disease[END_REF][START_REF] Jaeken | Congenital Disorders of Glycosylation: A Rapidly Expanding Disease Family[END_REF][START_REF] Morava | Congenital disorder of glycosylation type Ix: review of clinical spectrum and diagnostic steps[END_REF]. For example, three CMD genes have no known function: fukutin [START_REF] Toda | Fukutin and alpha-dystroglycanopathies[END_REF], fukutin-related protein (FKRP) [START_REF] Schachter | The role of defective glycosylation in congenital muscular dystrophy[END_REF] and LARGE [100]. A long-standing puzzle is hereditary erythroblastic multinuclearity with a positive acidified-serum lysis test (HEMPAS; congenital dyserythropoietic anemia type II). HEMPAS presents as a relatively mild genetic dyserythropoietic anemia caused by an unknown glycosylation defect [101-103]. Erythrocyte membrane glycoproteins, such as band 3 and band 4.5, which are normally glycosylated with polylactosamines lack these carbohydrates in The problem of accurate diagnosis of "congenital glycosylation diseases" has become more difficult due to the rapid rate of discovery of new glycogenes that do not fit neatly into the pattern of genes involved primarily in N-glycan biosynthesis. Some patients do not show an abnormal serum transferrin, the classic screening test for CDGs, e.g., autosomal-recessive nonsyndromic mental retardation associated with a mutant oligosaccharyltransferase, defects in the GDP-fucose and CMP-Sia transporters and in glucosidase I, and in some cases of phosphomannomutase deficiency (CDG-Ia).

A C C E P T E D M A N U S C
The diversity of proteins that can be affected by a glycogene mutation results in defective development of multiple organ systems (brain, gastrointestinal tract, liver, vision, and the endocrine and immune systems). The biochemically and clinically heterogeneous pictures of these patients indicate the importance of normal glycosylation in these organs and present difficult diagnostic problems to the clinician. It is clear that the current diagnostic approaches have to be expanded and that some of these methods may have to target only one or a few glycogenes.

There are many surprises waiting for us and we should consider a "congenital glycosylation disease" in every new clinical presentation and also in all known clinical syndromes in which the basic defect remains unknown.

Treatment

A serious problem for CDG and other glycogene-deficient patients, many of whom are desperately non-functional, is the lack of effective treatments. Only two CDG subtypes, CDG-Ib and CDG-IIc, have been treated successfully [107]. In CDG-Ib, alimentary addition of mannose bypasses the defective step (conversion of fructose-6-phosphate to mannose-6-phosphate) by allowing for formation of mannose-6-phosphate via the action of hexokinase. These children show remarkable improvement with therapy, probably because there are no detectable neuronal abnormalities in this disease. CDG-IIc, also known as leukocyte adhesion deficiency type II (LAD-II), stems from a defective membrane transport of GDP-L-Fuc. Alimentary supplementation with L-fucose in one CDG-IIc patient corrected the immunological defect and, surprisingly, also improved the neurological symptoms. However, treatment was ineffective in three other CDG-IIc patients probably due to differences in mutations between the patients.

Although mannose treatment of fibroblasts from CDG-Ia patients, the most common form of CDG, corrects altered N-glycosylation due to the absence of phosphomannomutase (the enzyme that converts Man-6-phosphate to Man-1-phosphate) [START_REF] Rush | Mannose supplementation corrects GDP-mannose deficiency in cultured fibroblasts from some patients with Congenital Disorders of Glycosylation (CDG)[END_REF][START_REF] Panneerselvam | Human fibroblasts prefer mannose over glucose as a source of mannose for N-glycosylation. Evidence for the functional importance of transported mannose[END_REF]108], oral administration of Man to these patents is unfortunately not effective

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 9
[107]. Oral administration of Man-1-phosphate is also not effective because this material is not transported across the cell membrane. Attempts have been made to synthesize cellpermeable compounds that would be converted to free Man-1-phosphate upon entry into the cell [107]. Although such compounds correct the biochemical phenotype of CDG-Ia fibroblasts, the compounds synthesized to date are too unstable and too toxic to be useful in the clinic. Another approach to therapy is enzyme replacement, i.e., systemic delivery of normal versions of the mutant enzyme (e.g., phosphomannomutase) [107].

There is currently a lack of Type I CDG animal models that can be used to test putative therapeutic agents. Complete lack of N-glycosylation is lethal in all eukaryotes and null mutations in the glycogenes defective in Type I CDGs are embryonic lethal in humans and mice. All Type I CDG patients have hypomorphic mutations and retain the ability to synthesize reduced amounts of normal N-glycans. Animal models for the Type I CDGs would therefore need to be hypomorphic mutants.

Therapy of CDG-Ib patients is effective partly because these patients do not have the severe abnormalities present in most other forms of Type I CDG (psychomotor retardation, retinopathy, and muscular hypotonia). This damage is probably established during fetal development and therefore may not be reversible with postpartum treatment. However, recent studies on neuroplasticity suggest that the brain and nervous system are capable of considerable repair in both children and adults.

Hyperglycosylation strategies have been proposed as therapeutic interventions in the CMDs (α-dystroglycanopathies) [START_REF] Martin | Congenital muscular dystrophies involving the O-mannose pathway[END_REF][109][110][111]. Recent work has demonstrated that the overexpression of either one of two related glycogenes, LARGE and LARGE2, results in the hyperglycosylation of α-dystroglycan and resultant restoration of normal αdystroglycan ligand binding. The mechanism of this important therapeutic effect has not been determined.

Conclusions

We have attempted in this relatively brief review to present a picture of the highly complicated nature of glycan structure and assembly and the devastating effects of glycogene malfunctions on the human body. Whereas spectacular advances have been made in recent years in the discovery of new glycogenes and the phenotypes of the diseases resulting from defects in these genes, a great deal of work remains to be done especially in the areas of diagnosis and treatment.

A

  C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

  HEMPAS. Polylactosamines accumulate as glycolipids in HEMPAS erythrocytes. Analysis of N-glycans from HEMPAS erythrocyte membranes revealed a series of incompletely processed N-glycan structures. Linkage analysis of HEMPAS cases from southern Italy have excluded two long-suspected candidate genes (encoding αmannosidase II and GlcNAc-transferase II) as the causative gene, but identified a gene on chromosome 20q11 [104-106].

	ACCEPTED MANUSCRIPT
	R I P T
	8

Acknowledgments

Supported by the Canadian Institutes for Health Research (CIHR) to HS and by NIH grant R01 DK55615 and the Children's Hearts Fund to HF.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T