The study showed a close distribution of symptoms between boys and girls (Table 1) in the study group in contrast to previous studies that showed male predominance in many IEMs in the Egyptian community [7, 8]. The close distribution of the disease is homogenous with the autosomal recessive inheritance pattern of most types of CDGs.
The study also revealed the absence of a distinct age of presentation in the study group. The subjects of this study presented in a very wide range of ages (Table 1). The mean age of presentation was 3.59 years. It was also noted that the highest presentation was in the 2nd group (Table 2) with 54% of subjects presenting between 1 and 4 years of age while only 7 subjects (14%) presented in the first year of their lives, 3 of them already had affected siblings (6% of studied subjects, 42% of group I). It was noted from the history of the subjects that they remained undiagnosed or were previously misdiagnosed with other non-inheritable disorders.
Group II had the highest number of diagnosed cases (Table 4) while group I had only one diagnosed case who had 2 previously affected siblings. These data support the need for a CDG screening programme to decrease the age of presentation and diagnosis and therefore decrease the complications of these aggressive diseases.
The delayed diagnosis of these patients can be attributed to lack of specific symptoms of CDG, multi-system involvement, the limited number of cases reported for individual types of CDGs, lack of awareness by clinicians about the symptoms that raise suspicion of CDGs and/or the lack of screening programmes for IEMs in general and CDGs in particular .
A correlation with parental consanguinity was very strong in the study group (Table 1). The number of affected family members was much higher in consanguineous families than in non-consanguineous families. These findings point to the presence of a genetic disorder in patients born to consanguineous marriages especially those with diverse unexplained symptoms or with symptoms unassigned to a specific disease .
Subjects in this study presented with variable neurological and/or non-neurological signs and symptoms (Fig. 1). This was also noted by many other studies such as the study conducted by Goreta et al. , who recommended that CDGs should be suspected in any case with unexplained neurological manifestations, particularly in cases where the neurological manifestations are accompanied by other organ diseases. Additionally, CDGs should be suspected in cases with any unexplained syndrome without obvious neurological manifestation.
The diversity of clinical presentations can be explained in many ways, first by the complexity of various glycosylation pathways. Second, knowledge about CDGs started to develop in the 1980s and, as a result, experience with disease symptoms is still very limited. Moreover, the small number of patients diagnosed with different CDGs makes it very difficult to determine the phenotype of each type of disease .
In this study, we studied the N-glycosylation profile for subjects and controls using WB, while the O-glycosylation profile was studied by LC-MS/MS techniques.
Due to its convenience, WB was used to detect the hypoglycosylated form of 4 different serum proteins (transferrin, hepatoglobin, α-1-acid glycoprotein and α-1-antitrypsin) in all subjects and controls. The 4 proteins were analysed in parallel, and the results depended on the interpretation of the 4 profiles together. The study showed separation of molecular masses lower than those found in healthy individuals in at least one of the studied proteins in 8 of the studied subjects (16% of the study group).
We choose WB because it avoids some of the disadvantages of isoelectric focusing (IEF). WB separates glycans depending on their molecular weight; thus, any change will be due to defects in the synthesis of glycans, while in IEF, any change leading to alteration of sialic acid groups attached to glycans will result in a false-positive result. WB also provides a chance to analyse many N-glycosylated proteins not only transferrin as in the case of IEF .
Alpha 1-acid glycoprotein is an acute-phase plasma alpha-globulin that acts as a carrier for basic and neutral lipophilic compounds. Normally it has 5 N-glycan chains that are bi-, tri-, and tetra-antenna capped by terminal sialic acid residues. In our study, the separation of the affected subjects and positive controls showed separation of only 3 bands and not 6 corresponding to the normally present form with 5 glycan chains along with 5 abnormal forms with tetra-, tri-, di-, and mono-glycan chains as well as no glycan chains. In this study, the 2 most hypoglycosylated forms were not detectable. This is similar to the results obtained by Denecke et al.  and Seta et al. , who recommended further studies to detect the reasons for the absence of those 2 least glycosylated forms.
The study emphasized the importance of using more than one protein in WB analysis for patients suspected to have CDGs to avoid false results. The results of the 4 proteins were analysed in parallel for every sample, and the separation was repeated when the results were questionable. The selection of these proteins provided a range of glycosylation sites (transferrin: 2, α-1-antitrypsin: 3, hepatoglobin: 4 and α-1-acid glycoprotein: 5). The presence of only 2 glycan chains in transferrin guaranteed a very clean WB band compared to the more diffuse larger bands of hepatoglobin and α-1-acid glycoprotein; however, when the phenotypic expression of underglycosylation is low, the last 2 proteins could more easily give abnormal patterns than transferrin.
Although there was no significant difference in the number of patients with abnormal profiles of each individual protein (87.5% of patients with positive WB profiles showed hypoglycosylation of transferrin, alpha-1-antitrypsin and hepatoglobin compared to a 100% of the patients with positive WB profiles showing hypoglycosylation of alpha 1-acid glycoprotein) , not all patients with abnormal WB profiles showed separation of N-glycoproteins indicative of hypoglycosylation of the 4 proteins together. Only 56% of abnormal patients in our study group showed hypoglycosylation of all 4 proteins together.
In our study, α-1-acid glycoprotein had a higher sensitivity than the other tested proteins (100% compared to 87.5%). This is different from the work done by Seta et al. , which showed a relatively high sensitivity of hepatoglobin, and the work done by Yussa et al. , which showed similar sensitivity of both α-1-acid glycoprotein and transferrin.
The choice of a mass spectrometric method for the analysis of O-glycan profiles depends mainly on the size of the analysed glycan; the LC-MS/MS method is sensitive at m/z values less than 2000 and therefore was suitable to study the small T-antigen with an MRM transition of m/z 534/298 and ST-antigen with an MRM transition of m/z 895/520. LC-MS/MS is considered a “soft” ionization technique where the analysed compound is not subjected to in-source fragmentation that can disrupt its structure.
In any type of glycomic analysis, the preparation steps are as important as the detection method. Any mistake in sample preparation will severely affect the accuracy and efficiency of the analysis. Although the protein part of glycoprotein is important in the assignment of the N- or O-glycan to their specific glycosylation sites, the analysis of the glycopeptides is complicated for many reasons. According to various reports [14, 15], the number, microheterogeneity, macroheterogeneity, and protease resistance of O-glycoprotein can make it difficult to analyse these by MS/MS. They also added that peptide sequencing is very difficult to determine when the protein is heavily glycosylated; this peptide sequence is the main factor that assigns glycan chains to their glycosylation sites.
As an alternative, in this study, we chose to analyse the released O-glycans and not the whole set of O-glycopeptides. This was achieved by chemical cleavage using β-elimination in which the glycopeptide is subjected to alkali treatment, leading to breaking of the bond between GalNAc and Thr/Ser of the polypeptide chain with concomitant reduction of GalNAc to N-acetylgalctosaminitol by borohydride. The reduction of GalNAc prevented further degradation of the released glycan chain (peeling reaction) and facilitated fragmentation during MS/MS.
After the separation of the glycan chains from the polypeptide, derivatization of the glycan chain was performed to overcome the weakness of the glycosidic linkage and increase the glycan stability during ionization. We chose permethylation as a method for derivatization. After permethylation, all the free OH groups of the glycan are converted to methyl ethers, and the labile sialic acid terminals were esterified into more stable sialyl esters leading to successive separation of both the sialylated and nonsialylated T-antigen (T-antigen at m/z = 534 and ST-antigen at m/z = 895) (Table 3 and Fig. 5).
The reference ranges obtained from the analysis of O-glycans from 50 controls were close to the reference range of the study conducted by Xia et al. , who investigated 10 patients affected with different types of CDGs and compared their results to 150 normal healthy controls. Our reference ranges also matched those of another study conducted by Liu et al. , who investigated the glycosylation profiles of 19 galactosemia patients and compared them to 150 normal healthy controls.
The analysis of permethylated O-glycans for the study group by LC-MS/MS revealed only one patient (2%) with an abnormal O-glycan profile compared to the reference ranges of normal healthy controls (Fig. 5). The subject showed a normal T-antigen level and a low ST-antigen level with concomitant elevation of the T/ST ratio. The same patient showed a normal N-glycosylation pattern with WB separation, and this result suggests a disorder of O-glycosylation. However, the 2-dimensional electrophoresis of this sample showed an abnormal N-glycosylation pattern of the four proteins which is common in type II CDGs. This result raised our suspicion of an N-glycosylation disorder accompanied by disturbance of the O-glycosylation mechanism. These mixed findings highlight the importance of analysing both O-glycans and N-glycans together in any patient suspected to have a glycosylation defect, with 2-dimensional electrophoresis being necessary for the detection of CDGs II hypoglycosylation patterns. This conclusion matches the conclusion of Xia et al. , who recommended a combined N- and O-glycan profile analysis for better detection of different types of CDGs II as well as CDGs due to Golgi dysfunction or defects in nucleotide transfer. The molecular analysis of this subject showed a COG5 gene mutation.
This study added to the studies performed by Prien et al.  and Ahn et al.  that the analysis of N- and O-glycans can effectively detect changes in the glycan profile from the normal profile. However, they cannot determine the underlying cause of the change in this glycan profile. Molecular analysis to detect gene mutations is required to determine the specific type of CDG.
Similar to WB and mass spectrometric analysis, samples from the study group were sent for molecular analysis. The DNA results matched the biochemical results of our study. The 9 patients with abnormal N- and O-glycans, detected by WB and LC-MS/MS, showed mutation of genes involved in protein glycosylation (Table 4), with PMM2 deficiency having the highest incidence among the studied study groups (44.4% of the positive cases and 8% of total subjects).