Jos Jansen

Chapter 1 16 Society for Human Genetics to publish strict guidelines for investigating causality. (53, 54) They recommend to provide firm functional evidence before a variant can be recognized as pathogenic. Another challenge is that patient clustering is difficult because of a variable phenotype. One single genetic mutation may be associated with severe disease but also with mild disease with different clinical symptoms. Another challenge is the lack of knowledge regarding metabolic pathways, especially when many proteins are involved, such as the secretory pathway which encompasses at least 1000 proteins. Methods to identify abnormal glycosylation Isoelectric focusing Diagnostics for CDG are based on the glycosylation profile of serum transferrin. Transferrin has two N-glycosylation sites at Asn432 and Asn630.(55) Isoelectric focusing (IEF) is an easily performed technique where protein isoforms are separated based on their isoelectric point. In most laboratory, it is the first step in CDG diagnostics. A glycan is negatively charged because of the two terminally located sialic acids. If both glycosylation sites of transferrin are occupied a total of four sialic acids are present. If glycosylation is incomplete, fewer sialic acids are present and this results in isoforms with a different isoelectric point. As already stated, type 1 CDG result in absence of complete glycans and thus accumulation of transferrin isoforms with two or zero sialic acids. Type 2 CDG, resulting in incomplete glycans, have an accumulation of isoforms with three, two, one or zero sialic acids. Advantages of IEF are low costs and high throughput. A disadvantage is the lack of information about other monosaccharides in the glycan, and about other glycan structural abnormalities, such as branching and fucosylation. A typical tIEF pattern is shown in Figure 3. Whole serum mass spectrometry glycomics Mass spectrometry of free serum glycans is an established method to analyze all glycans present in the serum.(56) For this, glycans are cleaved from the glycoprotein and the substrate injected into a MS. An advantage is the detailed information regarding the structure of the glycan, however due to the cleavage of the glycoprotein, it is not possible to attribute a glycan isoform to a specific