Oral sialic acid supplementation in NANS‐CDG: Results of a single center, open‐label, observational pilot study

NANS‐CDG is a congenital disorder of glycosylation (CDG) caused by biallelic variants in NANS, encoding an essential enzyme in de novo sialic acid synthesis. It presents with intellectual developmental disorder (IDD), skeletal dysplasia, neurologic impairment, and gastrointestinal dysfunction. Some patients suffer progressive intellectual neurologic deterioration (PIND), emphasizing the need for a therapy. In a previous study, sialic acid supplementation in knockout nansa zebrafish partially rescued skeletal abnormalities. Here, we performed the first in‐human pre‐ and postnatal sialic‐acid study in NANS‐CDG. In this open‐label observational study, 5 patients with NANS‐CDG (range 0–28 years) were treated with oral sialic acid for 15 months. The primary outcome was safety. Secondary outcomes were psychomotor/cognitive testing, height and weight, seizure control, bone health, gastrointestinal symptoms, and biochemical and hematological parameters. Sialic acid was well tolerated. In postnatally treated patients, there was no significant improvement. For the prenatally treated patient, psychomotor and neurologic development was better than two other genotypically identical patients (one treated postnatally, one untreated). The effect of sialic acid treatment may depend on the timing, with prenatal treatment potentially benefiting neurodevelopmental outcomes. Evidence is limited, however, and longer‐term follow‐up in a larger number of prenatally treated patients is required.

patients (one treated postnatally, one untreated). The effect of sialic acid treatment may depend on the timing, with prenatal treatment potentially benefiting neurodevelopmental outcomes. Evidence is limited, however, and longer-term followup in a larger number of prenatally treated patients is required.

K E Y W O R D S
congenital disorder of, glycosylation, glycosylation, inherited metabolic disorder, intellectual developmental disorder, N-acetyl-D-neuraminic acid, personalized medicine, sialic acid biosynthesis

| INTRODUCTION
Congenital disorders of glycosylation (CDGs) are a group of inherited multisystem disorders characterized by abnormal glycosylation of proteins. 1 N-acetylneuraminic acid synthase congenital disorder of glycosylation (NANS-CDG; OMIM #605202) is a recessively inherited metabolic disorder (IMD) first reported by our group in 2016, a combined genomic and metabolomic discovery. 2 The classical phenotype includes intellectual developmental disorder (IDD), short stature with rhizomelia, skeletal dysplasia, facial dysmorphisms, and elevated N-acetylmannosamine (ManNAc). 3 The phenotype is caused by bi-allelic variants in NANS resulting in a defect of N-acetyl neuraminic acid synthase, which plays an important role in the de novo sialic acid synthesis pathway. 4 The upstream metabolite and biomarker ManNAc accumulates due to the block in this pathway. 2 Most importantly, the resulting shortage of sialic acid and cytidine-5 0 -monophospho-N-acetylneuraminic acid (CMP-sialic acid) causes hyposialylation of glycoproteins and glycolipids, known to be essential for normal brain, skeletal and muscle development. 5 Of note, sialic acid levels in urine and plasma are normal. 1 The demand for sialic acid is high during early development when organs, and especially the brain, undergo rapid growth. 5 This suggests that individuals with NANS-CDG are particularly vulnerable to deficiency of sialic acid biosynthesis during embryonic and fetal development. This is further supported by nans knockdown zebrafish models that recapitulate the human phenotype. 2 Since 2016, our group has reported 17 novel NANS-CDG patients referred for diagnosis based on their clinical phenotype, and confirmed by biochemical and genetic analysis. 2,3 The clinical spectrum was expanded to include shared ocular features, gastrointestinal symptoms, abnormal septum pellucidum, (progressive) cerebral atrophy, thrombocytopenia, and hypo-low-density lipoprotein cholesterol (LDL-cholesterol). 3 Also, a genotype-phenotype correlation was established: the pathogenic variants c.709C>T and c.562T>C were considered to be correlated with the severe end of the phenotypic spectrum. To date, management included symptomatic or supportive therapy such as antiepileptic drugs, laxatives, or enemas, 3 which is clearly insufficient especially given the worrisome finding of progressive intellectual deterioration (PIND) in patients with a severe genotype. The unmet medical need, that is, a treatment that halts or prevents neurodegeneration, motivated the current treatment trial.
Already in 2016, the possibility of sialic acid treatment was suggested based on studies in the knockdown nansa zebrafish; if supplemented in the embryonic phase partial rescue of abnormalities of the skeleton was observed. 2 The use of oral sialic acid supplementation in NANS-CDG aims to restore reduced sialic acid and CMP-sialic acid levels and is in analogy with the use of specific sugars for treatment of other glycosylation defects, such as mannose for MPI-CDG (OMIM#602579) 6 and galactose for PGM1-CDG (OMIM#171900). 7 Clinical trials with sialic acid in patients with GNE-myopathy (OMIM#605820), due to a defective enzyme proximal to NANS, showed an acceptable safety and tolerability profile. 8,9 It seemed the avenue towards a sialic acid treatment trial in NANS-CDG was paved, however, given its lack of effect in GNE-myopathy, slow-release quality grade sialic acid was pulled from the market. It took years to find another source of reliable sialic acid, hence the 4-year delay from zebrafish to human studies. Given the limited window of opportunity in zebrafish, that is, during the embryonic phase, we aimed to start the sialic acid therapy at the youngest possible age, thus both pre-and postnatally.
Here, we report the first-in-human observational study of sialic acid supplementation in NANS-CDG and outline the challenges of such investigator-driven trials along with possible solutions and future directions.

| Patients
Five patients with confirmed NANS-CDG, meeting the diagnostic criteria of elevated ManNAc and bi-allelic variants in NANS and referred to the CDG Center of den HOLLANDER ET AL.
T A B L E 1 The baseline demographic, genetic, biochemical, and clinical data of all five patients. Expertise at the Radboud University Medical Center (Radboudumc Nijmegen NL), participated in the current study. Mean age at inclusion was 9.4 years (range 0-28 years) (for patient data and genetics see Table 1). Patients 1 and 2 are genotypically identical. Clinical features are detailed in Table 1 and described in our previous case series. 3

| Ethics and consent
The study was approved by the Medical Ethics Board of the Radboudumc, Nijmegen NL (CMO 2021-7373). For all patients, legal guardians provided informed oral and written consent for participation in this study as well as for publication.

| Oral sialic acid treatment
The PIND and neurodegeneration observed in patient 1 prompted us to pursue the prenatal therapy with sialic acid in patient 2 and to start the therapy in this patient and other patients. All patients received the food supplement Nacetylneuraminic acid dihydrate powder produced by Jennewein Biotechnologie GmbH for 15 months (patients 1, 3, 4, 5) or 24 months (patient 2). Sialic acid is "generally recognized as safe" (GRAS) 10 for use as a food ingredient for children above 10 years by The European Food Safety Authority (EFSA) 11 and also in term formulas by the Food and Drug Administration (FDA). 10,12 As a measure for its stability in the patients' home situation, the bottle containing sialic acid was left open for 1, 2, and 4 weeks and the amount in an aliquot was compared to an aliquot of a bottle that was left closed for 4 weeks, using a quantitative mass spectrometry method using a 13 C-Nacetylneuraminic acid internal standard. Recovery of these four conditions was 93%, 101%, 108%, and 107%, respectively, all within the 10% variation of the analytical test. Sialic acid intake was increased over the study period to avoid side effects; week 1 = 1000 mg/m 2 /day, weeks 2-4 = 2000 mg/m 2 /day, week 5-until end = 4000 mg/m 2 / day, in 4 doses. This dose selection was based on previous clinical trials with GNE-myopathy and in healthy individuals, 8,9,13 in which 12 g/day was considered as safe.
One patient (patient 2) was prenatally treated with sialic acid. The prenatal diagnosis in this patient provided the opportunity to intervene in the early embryonic phase, which was supported by the zebrafish study findings in which sialic acid rescue was observed only if administered during the embryonic phase. 2 Subsequently, prenatal treatment with sialic acid was started at 34 weeks gestation by oral administration to the mother. Sialic acid passes into breast milk 5 and therefore the patient's mother continued with sialic acid to treat him via breastfeeding. At age 12 days, maternal treatment was stopped, and sialic acid was added to the regular infant formula. There are no published studies providing direct evidence that sialic acid crosses the placenta. This is due in part to ethical and technological limitations. Despite this, Briese et al. measured the sialic acid concentration in maternal, retroplacental, and cord blood samples of 16 pregnant women. 14 They found significant correlations between maternal and retroplacental blood on the one side and between maternal and cord blood on the other sides. This suggests that the sialic acid synthesized by the mother crosses the placenta. The sialic acid is subsequently absorbed by the fetus and is capable of crossing the blood-brain barrier (BBB) and of being incorporated into the brain tissue. 5

| Study design
Patients with NANS-CDG were enrolled in a prospective pilot-study and were evaluated at the Radboudumc using the same protocol. The primary outcome was short-term safety and tolerability (treatment-related grade ≥3 and serious adverse events [AEs]) of oral sialic acid supplementation. The secondary endpoints were (1) Growth and weight evolution; (2) Psychomotor development, cognition, and behavior (motor function evaluated by the Gross Motor Function Measure [GMFM], 15 Scale for the Assessment and Rating of Ataxia [SARA], 6-minute walk test [6MWT], and the Childhood Myositis Assessment Scale [CMAS], behavioral changes reported by the parents). The outcome variables A (lying, rolling) and B (sitting) of the GMFM are used in three of five patients. The GMFM is not appropriate for the patient with a GMFM baseline score of 100%. The SARA sale, the 6MWT, and the CMAS were used in this patient; (3) Seizure control (according to a seizure log book and EEG); (4) Neurological response (by standard neurologic assessment and magnetic resonance imaging [MRI] of the brain); (5) Bone health (index response according to BoneExpert 16 measurements for patients of <4 years at baseline and changes in bone density and composition according to DEXA [Dual Energy X-ray Absorptiometry] scan evaluations for patients ≥4 years at baseline); (6) Gastrointestinal symptoms (by evaluating waist size, stomach ache, and bowel movement according to the Bristol stool scale); (7) Hematological (thrombocytes) and biochemical (LDL-cholesterol) response; (8) The metabolic fate of administered sialic acid by monitoring changes in biomarker levels (sialic acid, ManNAc, and Apolipoprotein C-III [apoC-III]) in plasma and urine.
(9) Parent-reported narratives. Assessments of the brain MRI scans and x-rays of the bones were performed by experts in neuroradiology and pediatric radiology.

| Study protocol
Clinical evaluation and laboratory studies were completed every 3 months (baseline, 3 months, 6 months, 9 months, 12 months, and 15 months). Parents completed the questionnaires (Bristol stool scale). The patients were seen by a pediatrician, biochemical geneticist specialized in metabolic disorders as well as a physician assistant in rehabilitation every 3 months. Patients 1 and 2 were also seen by a pediatric neurologist. Lab studies were performed by the central diagnostic laboratory of the hospital and the Translational Metabolic laboratory for genetic metabolic disorders.

| Data collection
Data were collected both retrospectively and prospectively from the electronic health records from the patients, from start until end of sialic acid treatment.

| Biochemical analysis
Quantitative proton nuclear magnetic resonance ( 1 H NMR) spectroscopy was performed to quantify excretion of ManNAc and sialic acid in patients' urinary samples.

| Statistics
SPSS 26 for Windows was used for statistical analysis. All results were expressed as means ± standard deviation and min-max. A p-value less than 0.05 was considered as statistically significant.

| Safety and compliance
AEs were monitored weekly; no clinical or metabolic AEs were reported to be associated with sialic acid intake.

| Sialic acid intake
Sialic acid administration was followed by an increased excretion of sialic acid in urine samples, (Table S1), suggesting that oral doses are rapidly absorbed in the systemic circulation and excreted in urine. Additionally, this indicates that all the five patients were compliant with daily oral sialic acid intake.

| Case reports
The baseline demographic, genetic, biochemical, and clinical data of all five patients are presented in Table 1. All five patients have been described in our previous study, cross referencing is provided in Table 1. 3 For detailed follow-up data, see Table S1.
Four of the five patients had NANS-CDG-related symptoms at birth. At start of the treatment, the intellectual disability ranges from profoundly disabled to mild disabled. One patient suffered from epilepsy. Brain MRI revealed abnormalities, including (progressive) cerebral atrophy, in all the patients who underwent brain imaging. Bone growth and maturation were abnormal in four of the five patients. Gastrointestinal symptoms were present in three of the five patients. Three patients had thrombocytopenia (one required weekly platelet transfusions), one of whom had hypo-LDL-cholesterolemia. Two other patients also had hypo-LDL-cholesterolemia.

| Somatic growth
An increase in length was deemed unlikely for the adolescent (patient 3) and adult (patient 4). For the three remaining patients, height and weight percentiles did not improve ( Figure S1). Table 1 shows the baseline psychomotor and cognition levels of the patients at baseline. Table S1 shows detailed follow-up data. We observed no significant objective improvement in psychomotor development and cognition in any of the patients. Subjectively, patients 1 and 2 showed some progress during follow-up, especially in social interaction. However, regression of psychomotor development was reported in patient 1 at t = 9 months. The parents of patient 3 reported that he shows more initiative, and is more interested in school, family, and friends. He is more alert, energized, and less fatigued, also in school. No objective or subjective improvements were reported in patients 4 and 5.

| Motor function
The results of the motor function tests are shown in Table S1 and Figures S2 and S3. Because of wheelchair dependency, the GMFM was not measured in patient 4. No clinically significant improvement in motor function was observed.

| Epilepsy
One of five patients (20%) had confirmed epilepsy at baseline and did not improve during sialic acid treatment (see Table 1 and Table S1). Patient 1 developed drug-resistant West Syndrome during follow-up, with evolution to an epileptic encephalopathy. At t = 9 months, patient 2 showed non-epileptic myoclonus, with good levetiracetam response.

| EEG
A normal baseline EEG was reported in all patients who underwent EEG monitoring at baseline (see Table 1). In patient 4, an EEG was not indicated. Patient 1 developed West Syndrome, his EEG was abnormal throughout follow-up despite intensive anti-epileptic treatment (see Table S1). Patient 2 showed a mildly encephalopathic background EEG at t = 9 months, which normalized at t = 20 months (upon levetiracetam treatment).

| Brain MRI
Baseline neuroimaging (MRI) was available for 3 of 5 patients, 100% of whom showed abnormalities as illustrated in our previous study. 3 For detailed follow-up brain MRIs, see Table S1. In patient 4, a brain MRI was not performed. The brain abnormalities in patient 2, who was started on sialic acid prenatally, were relatively mild compared to the signs of disease progression on MRI in patient 1. Based on combined MRI and EEG results, we concluded that there was no convincing evidence of a positive clinical effect of postnatal sialic acid supplementation. There is a possible positive clinical effect of prenatal sialic acid supplementation on neurological outcomes and the progression of the brain abnormalities.

| Skeletal abnormalities and bone density
In one patient, baseline DEXA scan showed normal bone density, therefore no follow-up DEXA scans were performed. For detailed follow-up x-rays and DEXA scans see Table S1. No beneficial effect of sialic acid on bone development was observed. In all patients, a discrepancy between the ossification centers of the carpal bones and the phalanxes was reported (Figure 1): delayed development of the ossification centers of the phalanxes and accelerated development of the ossification centers of the carpal bones.

| Gastrointestinal
The gastrointestinal symptoms did not improve during follow-up (for detailed follow-up data, see Table S1).

| Platelet count
No beneficial effect of sialic acid was found, as platelet count did not normalize by the end of the study in any of the initially thrombocytopenic patients (see Figure 2A, Table S1, and Figure S4).
F I G U R E 1 Radiography of the hand of patient 2 at age 22 months (15 months follow-up). The radiography shows a discrepancy between the ossification centers of the carpal bones (3.62 years) and the ossification centers of the phalanges (0.97 years).

| LDL-cholesterol
At the end of the follow-up, hypo-LDL cholesterolemia was observed in all patients (4 out 4) in whom LDL was measured (see Figure 2B, Table S1, and Figure S5), indicating that sialic acid was not effective for hypo-LDL cholesterolemia.

| N-acetyl-D-mannosamine and sialic acid
At t = 15 months, a decrease in ManNAc was observed in all patients in whom ManNAc was measured (see Figure 2C, Table S1, and Figure S6). Repeated measures analysis using mixed models revealed no significant relationship over the time periods between sialic acid treatment and ManNAc levels (p = 0.770).

| PRENATAL TREATMENT EFFECT
In patient 2, NANS-CDG was prenatally diagnosed based on abnormal growth velocity of both brain and skeleton, supported by other notable findings such polyhydramnios, facial dyspmorphisms, and rhizomelia. Subsequent prenatal genetic testing revealed bi-allelic NANS variants,   prompting the initiation of maternal sialic acid administration at 34 weeks of gestation. The patient was delivered at full term with a birth weight of 3000 g (P7). He was born with congenital brain abnormalities and skeletal dysplasia, which originate earlier in pregnancy, prior to treatment. After discharge, the patients' mother continued with oral sialic acid ingestion, with the aim of treating him via breastfeeding. At age 12 days, mother stopped breastfeeding and sialic acid was introduced into his regular infant formula. After 21 days, the dosage was increased to 200 mg/kg/day. During follow-up, he showed developmental progress, albeit slow: at age 15 months he is able to briefly lift his head in a prone position, maintain an upright sitting position with support, reaches for object in his surroundings such as toys, makes eye contact, smiles and makes booing sounds. Patients 1 (treated from age 2 years) and 2 (prenatally treated from gestational age 34 weeks until present) share the same genetic variants in NANS (Table 2). Both patients showed axial hypotonia, dystonia of limbs, and limited social interaction from the age of 0-30 months. Unaided sitting was not achieved at the age of 30 months (patient 2)/36 months (patient 1). The turning point of the disease course in patient 1 was around 32 months. He developed myoclonic epilepsy and West Syndrome with subsequent hospital admission for status epilepticus. Additionally, regression of his psychomotor development was reported from that time on; he was less alert, showed increased hypotonia, and very limited social interaction. In patient 2, non-epileptic myoclonus was reported at the age of 9 months, with good response to levetiracetam. At the time of writing this article, patient 2 is 30 months old. His non-epileptic myoclonus is under control with levetiracetam. He still shows hypotonia, however, muscle strength and tone as well milestones are better compared to patient 1 when he was of the same age. The brain abnormalities in patient 2 were mild compared to the progression of the brain abnormalities in patient 1. No other significant differences were observed in outcome measures between these two patients.
Longer follow-up of patient 2 as well as a larger cohort study including pre-and postnatal outcome data are required to gain more insight into the role of early sialic acid supplementation in early (neuro)development.

| DISCUSSION
CDGs represent an ever-growing, complex family of disorders with a severe and heterogeneous presentation in virtually all organ systems. Management of CDG patients requires an approach that combines both nutritional and medical management, customized for the specific individual (e.g., individualized doses of therapy, sometimes in combination with other supplements and supportive therapy). We expect an increase in the number of available treatments in CDG. Although most forms of CDG only have symptomatic and supportive treatment options, recent years have seen substantial advances in the treatment of these ultra-rare diseases. 17 Even though a beneficial effect of sialic acid was lacking for GNE-myopathy 8,9 and oral sialic acid is rapidly excreted in the urine and does not change ManNAc levels, 13 we decided to investigate the effect of oral sialic acid in five NANS-CDG patients. The therapeutic rationale was based on several studies. First, nutritional intervention with oral sugars has shown beneficial effects in certain CDGs, including mannose in MPI-CDG, 18 D-galactose in PGM1-CDG, 19 and fucose in SLC35C1-CDG, 20 FUT8-CDG, 21 and GFUS-CDG. 22 These findings suggest that providing dietary sialic acid might resolve some of the hypo-glycosylation. Additionally, it has been proven that the human body is able to absorb and incorporate dietary sialic acid into biosynthetic pathways of sialylation in the cells. 23,24 Third, the skeletal phenotype of knockout nansa zebrafish was partially rescued by the administration of exogenous sialic acid during the embryonic phase. 2 Therefore, we were highly motivated to administer prenatal treatment. And most importantly, only symptomatic or supportive therapy is currently available for these severely affected patients. Both the PIND observed in these patients and the hope that sialic acid could be an effective symptomatic treatment, prompted us to start with sialic acid therapy. To our knowledge, this was the first study performing a long-term sialic acid intervention in NANS-CDG patients.
Ingestion of sialic acid was safe and well tolerated. Overall, in four of five patients, there was no significant biochemical or clinical improvement observed in the different outcome measures. Patients 4 and 5, both with profound central nervous system involvement, did not respond at all. This is probably related to the role of glycosylation in brain development and is as such unlikely to respond to improvement in glycosylation.
Despite the lack of measurable sialic acid effects, the parents of patients 1, 2, and 3 did report subjective improvement in different aspects of the clinical symptoms, particularly in psychomotor development. These parents reported improvement in social interaction, energy level, interest for surroundings, and initiative, especially at the beginning of the treatment. Although none of these improvements were captured in the outcome measures, this partly led to a subjective increase in the quality of life of these patients. Nevertheless, the expected natural evolution of the GMFM in children with NANS-CDG is unknown, which is essential to accurately interpret the therapeutic effect of sialic acid. Future research into sensitive outcome measurements may help to capture these subjective improvements.
The prenatally treated patient 2 seems to show increased improvement in development and social interaction compared to patient 1, with the same pathogenic variants. Moreover, his neurologic features and brain abnormalities (including size of ventricles and degree of atrophy) as measured by MRI give the impression of milder disease presentation. Sialic acid is an essential component of brain gangliosides and sialylated glycoproteins and therefore crucial for establishing neural structures and synaptic connections in the early stages of neurodevelopment in infants. 5 This finding suggests that an early (prenatal) treatment with sialic acid might be preferable. However, it should be emphasized that patient 2 has not reached the age of 32 months yet (the turning point age of patient 1). We will keep comparing patient 2, who continues to receive treatment, to patient 1 to gain more insight into the role of early sialic acid supplementation in early (neuro)development and to come to a definitive conclusion about the effect of prenatal sialic acid treatment. Nonetheless, we recognize the possibility of elective abortion in specific circumstances, and the administration of prenatal therapy should be thoroughly evaluated on a case-by-case basis.
Research on the sialic acid biosynthesis pathway has shown that CMP-sialic acid as the final product of the pathway acts as a feedback inhibitor of GNE, the first committed step of the sialic acid pathway. 25 Hypothetically, sialic acid administration could increase CMP-sialic acid levels and thereby decrease GNE activity and production of ManNAc. Nevertheless, in our study, ManNAc concentrations were not reduced after sialic acid administration. This is in line with a recent study by Tran et al., showing that sialic acid does not affect plasma and urinary ManNAc and that ManNAc remained higher in NANS deficient subject than in controls. 13 The authors proposed that the free sialic acid may have been partially hydrolyzed to ManNAc by N-acetylneuraminate pyruvate lyase (NPL), an enzyme that catalyzes the cleavage of sialic acid to form pyruvate and ManNAc, 26 using the catabolic pathway. Nevertheless, the clinical significance of elevated ManNAc is unclear. A study in GNE-myopathy, evaluating the safety and pharmacokinetics of ManNAc administration, reported 3 and 6 g of oral ManNAc as safe, suggesting that elevated levels of ManNAc are not toxic. 27 A remarkable finding on the radiographies of the hands is the discrepancy between the ossification centers of the carpal bones and the phalanxes in three patients.
To our knowledge, this is never reported in the literature, and an explanation for this finding is currently lacking.
Clinical evaluation remains challenging in trials for rare disorders, especially in very heterogeneous groups and when a solid biomarker, for therapy response, is missing. Selection of meaningful and relevant endpoints is fundamental to rare disease clinical trials and requires an understanding of the natural disease history. However, rare diseases are heterogeneous and a detailed knowledge is often lacking, compounded by variable expression within disease and subtypes. Despite the potential hurdles for rare disease clinical trials, national and international collaboration is driving progress toward improved diagnosis and treatment for people living with rare disease. Regulatory bodies are starting to acknowledge that endpoint selection may not be straightforward and that biomarkers are not always available.
Limitations of this study include the small number of patients, inherent to the number of cases identified world-wide which are less than 20. Second, our study includes a small heterogeneous group with different genotypes, phenotypes, and ages. Outcome variables were based on the most common symptoms in NANS-CDG. However, during evaluation and processing data, it became apparent that patients differed substantially on these variables. In addition, some outcome measures were not sensitive and unable to detect (subtle) changes in a patient's progress. Personalized outcome measures, such as Goal Attainment Scaling and The Perceive, Recall, Plan an Perform system (PRPP), would be suitable in future studies to tackle this. 28,29 Fourth, slowrelease forms of sialic acid might allow better incorporation in tissues compared to the short half-life sialic acid used in this study. Furthermore, human data on oral sialic acid uptake in different tissues, especially in the brain, is lacking, which may require specific targeting strategies. Additionally, this study was an open-label uncontrolled study, which is at higher risk of bias. Lastly, more data on the natural history of NANS-CDG are needed to provide a control group. However, our recent study on the phenotype represents a beginning in this regard.
In summary, postnatal treatment with oral sialic acid supplementation over a period of 15 months neither led to a biochemical nor to a clinical response in our patients. Possible reasons for the negative outcome in these patients include: (1) The start of treatment was too late (except for the prenatally treated patient); (2) Other formulations, higher doses, or slow release of sialic acid may be necessary; (3) Sialic acid does not sufficiently cross the placenta; (4) Orally administered sialic acid is not incorporated into cells. This is in line with a recent study on the fate of orally administered sialic acid in a NANS-CDG patient, showing that free sialic acid was rapidly absorbed, but also rapidly excreted in urine. 13 Identification of blood-based biomarkers for NANS-CDG, either in plasma or blood cells, is required to facilitate studies on the effect of sialic acid or other future therapeutic strategies. In our previous study, we showed that ManNAc excretion levels significantly correlate with disease severity. 3 Hypothetically, the discovery of a new therapy-sensitive biomarker, that correlates with clinical severity (like ManNAc), could enable us to conduct placebo-controlled n-of-1 trials.
Nevertheless, our study suggests that prenatal treatment has possible beneficial effects on neurodevelopmental outcomes. Evidence is limited and longer-term follow-up of our and other prenatally treated patients is required. As mentioned above, our study has limitations and it is therefore essential that our results will be validated in the future using a larger sample size and controls and possibly a longer period of observation. Also, slow-release forms or other sialic formulations might well deserve further placebo-controlled trials including larger number of patients and a standardized protocol with sensitive outcome measures. In order to make progress on the path to a possible therapy, other targets and strategies for treatment of NANS-CDG (e.g. nutritional, small molecule, genetic) are under investigation and development in patientderived neuronal stem cells (induced pluripotent stem cells [iPSCs]).