Transferrin variants: Pitfalls in the diagnostics of Congenital disorders of glycosylation

https://doi.org/10.1016/j.clinbiochem.2014.09.022Get rights and content

Highlights

  • Transferrin variants impair the diagnostic procedures for suspected cases of Congenital disorders of glycosylation.

  • Known variants cause seemingly elevated Penta- and Trisialo-transferrin.

  • Neuraminidase digestion and Electrospray ionization time-of-flight mass spectrometry can identify transferrin variants.

Abstract

Objectives

Transferrin variants can hinder the diagnostic process in cases of suspected Congenital disorders of glycosylation which affect N-Glycosylation. In addition they can impair the use of Carbohydrate deficient Transferrin as a biomarker for chronic alcohol abuse, in which Asialo-Transferrin and Disialo-Transferrin are increased. We present a novel transferrin variant as well as an overview of transferrin mutations found at our laboratory.

Design and methods

Blood samples from patients with suspected CDG were analyzed using the standard diagnostic procedures of Isoelectric focusing and High-performance liquid chromatography as well as the additional procedures of neuraminidase digestion of glycans and Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS).

Results

Four known and one previously unreported transferrin variants were identified. Neuraminidase digestion and ESI-TOF MS revealed changes in charge of the transferrin molecules while the glycosylation status was found to be normal.

Conclusion

Transferrin variants are pitfalls in the diagnostics of CDG. The found variants change the charge of the transferrin molecule, thus affecting the standard diagnostic procedures. Neuraminidase digestion as well as ESI-TOF MS can identify variants and mutations in a laboratory context.

Introduction

Among the group of inborn errors of metabolism, Congenital disorders of glycosylation (CDG) represent a large and growing subgroup which expresses a great variety of symptoms. Many cases show multisystemic manifestations with often severe psychomotor retardation [1].

The initial diagnostics of CDG of N-Glycosylation rely on mainly two methods of analysis, which measure the glycosylation of the transferrin molecule: Isoelectric focusing, which remains the gold standard and the less time-consuming and more reproducible High-performance liquid chromatography (HPLC) [2]. In addition, Capillary zone electrophoresis has been established as another quick and reliable method of analysis of Carbohydrate deficient transferrin [3]. These methods have however been known to be hindered by variants of the transferrin gene which change the protein sequence of the transferrin molecule [4]. In addition, mutations compromising the glycosylation site of transferrin present an equally challenging task in the diagnostics of CDG [5].

Moreover, Carbohydrate-deficient transferrin (CDT) is used as a biomarker for excessive alcohol consumption [6] so that variants affecting the analytical methods can possibly lead to confusion in abstinence monitoring programs etc. The typical CDT pattern in chronic alcohol abuse consists of increase Asialo- and Disialo-Tf. Tf-variants have been reported to interfere in the correct application of monitoring tests in chronic alcohol abuse [7]. In this article, we present a novel, previously unknown transferrin mutation affecting the results of conventional screening methods. In addition, our analysis of transferrin using mass spectrometry elucidates the effects of the transferrin mutation c.829G > A (rs1799899) which has been described by Kutalik et al. [8]. Finally we give a brief overview of frequently found transferrin variants and their effects on CDG diagnostics.

One has to bear in mind however, that the described variants only affect the diagnostic process of CDG of N-Glycosylation and not the growing group of glycosylation disorders of O-Glycosylation [9].

Section snippets

Materials and methods

All samples were collected after informed consent was obtained either from the parents or the patients themselves.

Results

A graphic representation of our results can be found in Table 1, Table 2. The reported variants were all found among the samples analyzed in our laboratory, which processes approximately 1300 samples from numerous countries a year. In most cases, CDG is suspected.

Discussion

Transferrin mutations and variants impose diagnostic problems that can be relevant in the diagnostic process for suspected cases of CDG as well as for analysis of CDT for chronic alcohol consumption, which can be of great importance in legal contexts. In this study, we aimed to present an overview of the variants found in our laboratory in order to facilitate the interpretation of laboratory findings when testing for CDG or when analyzing CDT in general.

Seemingly increased Pentasialo-Tf

Conclusions

Various transferrin variants can hinder the diagnostic process for suspected CDG and for the evaluation of CDT. The said mutations can be identified using neuraminidase digestion, ESI-TOF MS and ultimately by sequencing of the transferrin gene.

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  • Analytical and diagnostic aspects of carbohydrate deficient transferrin (CDT): A critical review over years 2007–2017

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    In this latter type of cases the glycosylation defect is unknown by the subject and can cause false positive CDT results if the subject has to undergo CDT determination to investigate alcohol abuse. Indeed, in these cases of moderate glycosylation defect the CDT pattern can be similar to that associated to alcohol abuse [58–60]. This possible cause of alteration of transferrin glycosylation pattern should be always considered when a condition of alcohol misuse is investigated, being aware that the diagnosis of alcohol abuse can never be based only on CDT determination.

  • Two-dimensional electrophoresis highlights haptoglobin beta chain as an additional biomarker of congenital disorders of glycosylation

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    Notably, these two biomarkers could be potentially helpful in relatively frequent Trf polymorphism cases where accurate CDT determination is frequently disturbed [32,33]. Trf polymorphisms can also seriously complicate CDG screening since they frequently mimic abnormal IEF/HPLC/CE profiles [11,12]. Based on the analysis of 16 non-CDG samples with Trf polymorphism, we showed that associated normal AAT and Hpt 2-DE patterns allowed to unambiguously identify them.

  • A novel mutation on the transferrin gene abolishes one N-glycosylation site and alters the pattern of transferrin isoforms, mimicking that observed after excessive alcohol consumption

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    The combined mass shift caused by the two mutations is 37 Da, which explains the mass difference observed by LC–MS and indicates that the mutations are located in cis. Although CDT is still widely considered a specific marker of chronic alcohol consumption, false positives have been reported [7,8]. Helander et al. reported a healthy 32 year old woman who in a routine healthcare check-up showed elevated CDT levels (~ 17% disialo-TF and ~ 3% asialo-TF) which were initially mistaken for excessive alcohol consumption.

  • SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation

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    Due to the complex clinical phenotype, selective metabolic screening included investigation of glycosylation of serum transferrin, the common biomarker used to screen for congenital disorders of glycosylation (CDG).20–22 Glycosylation was investigated by isoelectric focusing (IEF), immunoprecipitation followed by SDS-PAGE, and high-performance liquid chromatography (HPLC), as described previously.23 Serum transferrin has two N-linked sugar side chains, each bearing two terminal, negatively charged sialic acid residues (tetrasialo-transferrin) (Figure 2A).

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1

These authors contributed equally to this work.

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