Previous reviews illustrated the relevance of an increased flux of Man-6-P (mannose-6-phosphate) into the glycosylation pathway, promoting individual or combined approaches of several pharmaceutical perspectives and mannose . Still, mannose found no further mention in the design of a potential therapy for PMM2-CDG in the past years. This first approach of a long-term mannose supplementation in PMM2-CDG provides valuable data, that mannose is not completely inert in PMM2-CDG and underscores, that the underrecognized role of mannose in PMM2-CDG should be reconsidered. Long-term oral mannose supplementation in PMM2-CDG was well tolerated and led to considerable biochemical improvements in the majority of patients and indicates possible clinical improvements. Mannose supplementation is currently the standard of care treatment for MPI-CDG resulting in favorable effects on the biochemistry and the clinical outcome [7, 9]. In PMM2-CDG patients, dietary supplementations with mannose at 100 mg/kg every 3 h over 9 days  or 0,17 g/kg every 3,5 h over a period of 6 months , as well as a continuous i.v. mannose infusion of 5,7 g/kg mannose in a PMM2 deficient patient over a period of 3 weeks , failed to show improvement in glycosylation patterns or clinical benefits. The most likely explanation for the failure of mannose therapy in vivo is, Man-6-P being catabolized by the fully operative MPI, transferring the surplus of mannose to glycolysis , as an unfavorable PMM2:MPI ratio does not favor flux into glycosylation pathways . However, the glycosylation deficiency in PMM2 fibroblasts can be restored completely by supplementing more than 250 µmol/l mannose. Assuming, that mutations in PMM2 are hypomorph and reduce enzyme affinity for Man-6-P , it is possible, that exogenous mannose supplementation in PMM2-CDG fibroblast cultures leads an increased intracellular Man-6-P concentration, that counters the increased Km requirements of deficient PMM2. This may lead to higher levels of Man-1-P, increasing the deficient GDP-mannose pools and culminating in the normalization of glycosylation . Another possibility may be cytosolic mannose being directly converted into Man-1-P by another enzyme or system (not detected yet) . Mannose therapy needs a long time to show effects in PMM2-CDG patients. Even in MPI-CDG, the first partial corrections in IEF- and SDS- patterns of serum transferrin occurred not until the first 6 months after initiation of a mannose therapy with a dose of 100 mg/kg three times a day , which cannot be explained by the half-life period of transferrin (CDT = ~ 14d; Not-CDT = ~ 8d) and other glycoproteins (AT III: 3d; Protein C: 6–8 h) . Effects on the IEF pattern would be expected not later than four weeks. Eleven months after initiation and increased mannose dosage, a funded decrease of the abnormal isoforms in MPI-CDG was observed . Thus, a biochemical correction in PMM2-CDG under mannose therapy may take a longer time and higher dosage to show positive effects, in the light of countering the Km- requirement of the attenuated PMM2. Ichikawa et al. found that the contribution of exogenous mannose is higher than previously thought and that other potential sources of mannose such as mannose salvaged from degraded glycoproteins, glycogen and gluconeogenesis do not make significant contributions to N-glycosylation . In fibroblasts increased exogenous mannose (1 mM) can completely replace glucose-derived mannose and become the sole source of mannose in N-glycans and also contribute to galactose and N-acetylglucosamine in N-glycans [10, 28]. Explanations for the higher contribution of mannose to N-glycans may be specific mannose transporters (GLUT-like mannose transporter, SGLT-5 mannose specific transporter) [29, 30]. About one third of mannose found in N-glycans takes detours as it is first converted to Frc-6-P and reconverted to Man-6-P again. Since the transient Frc-6-P derived from Man-6-P does not equilibrate with the total cellular pool of Frc-6-P, another suggestion may be the presence of separate Frc-6-P-pools (Frc-6-PGP/Frc-6-PG) like check-points for glycosylation, glycolysis and gluconeogenesis, generated by the anomeric selectivity of Glc-6-P and Man-6-P metabolizing enzymes (Fig. S1) [10, 28]. A preferable ratio of a- and ß- Man-6-P as well as of MPI (ß-Man-6-P anomer specific) and PMM2 (a-Man-6-P anomer specific) might result in a higher efficiency of exogenous mannose use in glycosylation . Substances enhancing the impact of check points of mannose flux to the glycosylation pathway may improve the effect of exogenous mannose supplementation. Which other undefined factors and check points of mannose metabolism  have an influence on the effect of mannose supplementation need to be further investigated.
It has to be considered, that the glycosylation of transferrin and other glycoproteins may improve in time, with age and the degree of liver involvement [31–33]. There are 2 major arguments against spontaneous improvement in this study. First, the patients in this study started their mannose therapy at very different ages from 1 year to 27 years (Table S1). The majority of responders showed a similar improvement with similar kinetics after a similar lag-time, suggesting that mannose supplementation, not age, was responsible for glycosylation improvement. Secondly the significant correction of the hypoglycosylated serum transferrin returned to approximately pretreatment patterns after long-term interruption of mannose supplementation (Fig. 1C). This clearly indicates the biochemical effect of mannose on these patients’ glycosylation and not an improvement with age. Crucial developmental steps during embryogenesis and infancy are negatively affected by hypoglycosylation. Thus, different organ manifestations of PMM2-CDG may have different responses to a mannose treatment .
Repetitive doses of orally ingested mannose at certain intervals can maintain elevated blood mannose levels in PMM2-CDG patients . Our patients showed fluctuations in blood mannose concentrations even when maintained on the same dose (Fig. S3). Healthy probands (40–80 µM baseline) reach and maintain blood-mannose levels of more than 200 µmol/l after 1 h by supplementing 0,2 g/kgBW of mannose.  . It must be assumed that, even when supplying ≥ 1 g/kg b.w. mannose, the blood mannose levels could not be maintained properly over the daily period and night time and were lower on average than the concentration shown to correct abnormal glycosylation in fibroblasts (≥ 250 µM ). Despite the positive responses in the majority of patients, these circumstances have certainly limited the effectiveness of mannose under clinical circumstances. Parenteral application, for instance by subcutaneous infusion, might be an approach to reach steady blood mannose levels during day and night time.
Since the collection of the clinical data in this study was done in everyday clinical practice without a specific protocol and without matching a control group, retrospective analysis necessarily introduces some bias, which affect the interpretation regarding the outcome and coherence negatively. Nonetheless, this study observed considerable data, that under mannose PMM2-CDG patients developed a normalization of their motor nerve conduction velocity and furthermore regained a redeemable knee jerk. In the literature peripheral neuropathy with reduced nerve conduction velocity as well as peripheral tendon reflexes stay stable or gradually deteriorate [17, 31, 35, 36]. These findings suggest a clinical effect of mannose therapy. For the respective patient, parents and caretakers uniformly reported improved reactivity, attention and better general state while supplementing mannose. Nonetheless, mannose therapy alone does not lead to the disappearance of the disease. There are crucial developmental steps during pregnancy and early childhood being disrupted and leading to evident, irreversible malformations and abnormalities (similar to MPI-patients with ductal plate malformations in the liver ). Therefore, prenatal application of mannose might be an issue to make a significant difference in improving these children’s health condition.  Detailed clinical efficacy should be tested in a controlled, double blinded, randomized study.