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Technical Briefs |
1 Department of Clinical Neuroscience, Karolinska Institutet and University Hospital, Stockholm, Sweden2 Glycobiology and Carbohydrate Chemistry Program, The Burnham Institute, La Jolla, CA
aaddress correspondence to this author at: Alcohol Laboratory, L7:03, Karolinska University Hospital, SE-171 76 Stockholm, Sweden; fax 46-8-51771532, e-mail Anders.Helander{at}cns.ki.se
Congenital disorders of glycosylation (CDG), formerly named carbohydrate-deficient glycoprotein syndrome, are rare hereditary disorders caused by mutations in the genes coding for enzymes involved in the biosynthesis of glycoproteins and other glycoconjugates (1). The clinical characteristics are variable, but often include psychomotor, growth, and mental retardation from early childhood. CDG are the result of defects in the assembly and transfer (type I) or processing (type II) of the glycan moieties, and as a result, the carbohydrate chains are either completely missing (type I) or structurally abnormal (type II). CDG-Ia is the most frequent subtype, with 
500 patients reported, whereas <100 patients have been diagnosed with types Ib–Ik and only 10 with type II (IIa–IId) (1)(2)(3)(4)(5). However, it is assumed that the limited awareness of CDG, together with their variable and unspecific clinical symptoms, contribute to underdiagnosis of these disorders (6)(7)(8).
Another reason for underdiagnosis could be that methods for CDG testing are not generally available in clinical laboratories. Testing for CDG focuses mainly on the abnormal pattern of N-linked glycans of serum transferrin. The type I pattern is characterized by an increase of transferrin glycoforms missing one or both of the entire biantennary N-glycans (9)(10) (traditionally named disialo- and asialotransferrin, respectively, based on the number of terminal sialic acid residues), whereas type II is characterized by increased trisialo- and monosialotransferrin, indicating the presence of truncated glycans (11)(12)(13). A variety of laboratory techniques have been evaluated for this purpose, with isoelectric focusing being the most common and serving as the reference procedure to date. A sensitive HPLC method is widely used for detection of alcohol-induced changes in serum transferrin glycoforms [i.e., carbohydrate-deficient transferrin (CDT)] (14). This study examined the usefulness of this HPLC method for the detection and preliminary diagnosis of CDG.
Serum samples were obtained from nine patients with biochemically and/or genetically confirmed CDG type I (a, b, and g subtypes) and four with undefined CDG type IIx defects (The Burnham Institute). Serum samples used for comparison were obtained at the Karolinska University Hospital from 42 children and adolescents (random routine samples), 132 adults (60 female and 72 male social drinkers) (14), and 74 chronic alcohol misusers (17 females and 57 males) (14). The sera were stored at 4 °C when analyzed within 2 days or stored at –70 °C for longer times before analysis, conditions under which transferrin is stable (14). The procedures followed were approved by the ethics committee at the Karolinska University Hospital.
The HPLC system provides reproducible separation and relative quantification (limit of detection, 0.05%) of the iron-saturated transferrin glycoforms with only small volumes of serum or plasma (14). Transferrin was iron-saturated by mixing 10–50 µL of serum (depending on the volume available) at a volume ratio of 5:1 with FeNTA (final concentration, 1.7 mmol/L). Lipoproteins were precipitated by mixing the iron-saturated sample 6:1 (by volume) with dextran sulfate and CaCl2 (1.4 mg/L and 70 mmol/L, respectively). The samples were left at 5 °C for 30–60 min and then centrifuged at 3500g for 5 min. The clear supernatant was diluted fivefold with water and transferred to glass HPLC vials. Transferrin glycoforms were separated by use of a SOURCE® 15Q 4.6/100 PE anion-exchange column (Amersham Biosciences) with a linear salt gradient elution on an Agilent 1100 HPLC equipped with a quaternary pump. Quantification relied on the selective absorbance of the iron–transferrin complex at 470 nm.
The distribution of serum transferrin glycoforms in controls and alcohol-misusing patients is shown in Table 1
. In children up to 3 weeks of age, 6 of 13 (46%) showed a disialotransferrin concentration below the range in older children, adolescents, and adults. Trisialotransferrin concentrations were below the adult range in 9 of 11 (82%) children 1–9 months of age and 7 of 18 (39%) of those 1–18 years of age, and penta- + hexasialotransferrin concentrations were above the adult range in 50% of the children in both age groups. Otherwise, there were no marked age-related differences in the relative amounts of transferrin glycoforms, and as for adults (14), no gender differences were observed in children and adolescents (P = 0.45–1.0, Wilcoxon). Asialotransferrin was not detected in any control samples, but was detected in 50% of the alcohol-misusing patients.
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Sera from CDG-I patients showed increased relative amounts of disialo- and asialotransferrin and concomitant reductions in tetrasialotransferrin (Fig. 1
). Patients with the CDG-Ia subtype had the highest amounts of disialo- (range, 19–42%) and asialotransferrin (3.4–26%), whereas the patients with type Ib and Ig CDG had less asialotransferrin (<3%; Table 1
). This compares with disialotransferrin concentrations <2% and undetectable asialotransferrin in the controls. In heavy drinkers, disialo- and asialotransferrin concentrations were 0.8–16% and 0–4.0% of total transferrin, respectively (Table 1
). After the CDG-Ib patient was treated with low-dose mannose (15), the abnormal transferrin pattern improved, as indicated by marked reductions in disialo- (from 18% to 6%) and asialotransferrin (from 2.6% to 0.7%; Fig. 1C
).
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Sera from patients with undefined CDG-IIx defects had the type II pattern with typical increases in trisialo- (range, 7.1–32%) and monosialotransferrin (5.7–15%; Fig. 1E
), indicating the presence of truncated glycans. This compares with trisialotransferrin values typically <8% in controls and heavy drinkers, and monosialotransferrin was measurable only in individuals with high trisialotransferrin (Table 1
) (16). In all four CDG-II sera, two unidentified peaks were also observed (Fig. 1E
, peaks A and B).
Our results demonstrate that measurement of serum transferrin glycoforms by HPLC can be used for preliminary diagnosis of CDG and for assignment of cases to either type I or type II. This possibility was apparently independent of age, but it has been recommended that CDG testing should not be performed before 3 weeks of age to avoid false negatives (2). Moreover, all four CDG-II sera showed two unknown peaks, which probably represent other truncated variants (17)(18). This was further supported by the observation that they had retention times similar to those for two of the variants formed after control serum was treated with neuraminidase (Type II-a from Vibrio cholerae; Sigma-Aldrich; Fig. 1F
), which removes the terminal sialic acids of the N-glycans (19)(20). This indicates that the HPLC method allows for separation of transferrin glycoforms not only based on the net charge of the molecule (i.e., sialic acid content) but also on structural differences of the glycans. As demonstrated for the CDG-Ib patient, which is the only efficiently treatable subtype (2), the method could also be used for follow-up of mannose treatment.
The CDG type I pattern resembles that observed after chronic alcohol consumption (CDT), albeit the relative increases were much higher in CDG-Ia. In a previous study, four of six carriers of one CDG-Ia mutation (healthy parents of CDG patients) had increased asialo- and disialotransferrin concentrations indistinguishable from the values observed in alcohol abuse (16). However, because CDG testing is typically performed at young age, there is no risk for false-positive results attributable to alcohol misuse. Furthermore, the type I and type II patterns are clearly distinguishable from those observed with the most common genetic transferrin variants (16), which can cause false-positive and -negative results in the immunoassays for CDT.
In conclusion, if CDG testing could be combined with CDT testing for heavy alcohol consumption by HPLC, or possibly capillary electrophoresis (21), this could enable testing for the preliminary diagnosis of CDG to be carried out in many more clinical laboratories. All suspected cases detected in this way could be transferred to a specialized laboratory for confirmatory analysis by enzyme assays or molecular studies to identify the defective enzyme or the specific mutation involved and, hence, the CDG subtype.
Acknowledgments
This work was supported by grants from the Karolinska Institutet (A.H.) and the March of Dimes Foundation (H.H.F.).
References
The following articles in journals at HighWire Press have cited this article:
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  J. P. Bergstrom and A. Helander Clinical Characteristics of Carbohydrate-Deficient Transferrin (%Disialotransferrin) Measured by HPLC: Sensitivity, Specificity, Gender Effects, and Relationship with other Alcohol Biomarkers Alcohol Alcohol., April 24, 2008; (2008) agn017v2. [Abstract] [Full Text] [PDF]
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 P. R. Selvin, T. Lougheed, M. Tonks Hoffman, H. Park, H. Balci, B. H. Blehm, and E. Toprak Fluorescence Imaging with One-Nanometer Accuracy (FIONA) of Cy3-DNA under Deoxygenation Conditions CSH Protocols, October 1, 2007; 2007(20): pdb.prot4868 - pdb.prot4868. [Abstract] [Full Text]
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 R. K.T. Kam, T. C.W. Poon, H. L.Y. Chan, N. Wong, A. Y. Hui, and J. J.Y. Sung High-Throughput Quantitative Profiling of Serum N-Glycome by MALDI-TOF Mass Spectrometry and N-Glycomic Fingerprint of Liver Fibrosis Clin. Chem., July 1, 2007; 53(7): 1254 - 1263. [Abstract] [Full Text] [PDF]
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J. R. Delanghe, A. Helander, J. P.M. Wielders, J. M. Pekelharing, H. J. Roth, F. Schellenberg, C. Born, E. Yagmur, W. Gentzer, and H. Althaus Development and Multicenter Evaluation of the N Latex CDT Direct Immunonephelometric Assay for Serum Carbohydrate-Deficient Transferrin Clin. Chem., June 1, 2007; 53(6): 1115 - 1121. [Abstract] [Full Text] [PDF]
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 A. M. Ostrow, H. Freeze, and J. Rychik Protein-Losing Enteropathy After Fontan Operation: Investigations Into Possible Pathophysiologic Mechanisms Ann. Thorac. Surg., August 1, 2006; 82(2): 695 - 700. [Abstract] [Full Text] [PDF]
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H. A. Carchon, C. Nsibu Ndosimao, S. Van Aerschot, and J. Jaeken Use of serum on guthrie cards in screening for congenital disorders of glycosylation. Clin. Chem., April 1, 2006; 52(4): 774 - 775. [Full Text] [PDF]
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A. Helander, J. P.M. Wielders, R. te Stroet, and J. P. Bergstrom Comparison of HPLC and Capillary Electrophoresis for Confirmatory Testing of the Alcohol Misuse Marker Carbohydrate-Deficient Transferrin Clin. Chem., August 1, 2005; 51(8): 1528 - 1531. [Full Text] [PDF]
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