Identification of GM1-Ganglioside Secondary Accumulation in Fibroblasts from Neuropathic Gaucher Patients and Effect of a Trivalent Trihydroxypiperidine Iminosugar Compound on Its Storage Reduction.

Gaucher disease (GD) is a rare genetic metabolic disorder characterized by a dysfunction of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) due to mutations in the gene GBA1, leading to the cellular accumulation of glucosylceramide (GlcCer). While most of the current research focuses on the primary accumulated material, lesser attention has been paid to secondary storage materials and their reciprocal intertwining. By using a novel approach based on flow cytometry and fluorescent labelling, we monitored changes in storage materials directly in fibroblasts derived from GD patients carrying N370S/RecNcil and homozygous L444P or R131C mutations with respect to wild type. In L444P and R131C fibroblasts, we detected not only the primary accumulation of GlcCer accumulation but also a considerable secondary increase in GM1 storage, comparable with the one observed in infantile patients affected by GM1 gangliosidosis. In addition, the ability of a trivalent trihydroxypiperidine iminosugar compound (CV82), which previously showed good pharmacological chaperone activity on GCase enzyme, to reduce the levels of storage materials in L444P and R131C fibroblasts was tested. Interestingly, treatment with different concentrations of CV82 led to a significant reduction in GM1 accumulation only in L444P fibroblasts, without significantly affecting GlcCer levels. The compound CV82 was selective against the GCase enzyme with respect to the ß-Galactosidase enzyme, which was responsible for the catabolism of GM1 ganglioside. The reduction in GM1-ganglioside level cannot be therefore ascribed to a direct action of CV82 on ß-Galactosidase enzyme, suggesting that GM1 decrease is rather related to other unknown mechanisms that follow the direct action of CV82 on GCase. In conclusion, this work indicates that the tracking of secondary storages can represent a key step for a better understanding of the pathways involved in the severity of GD, also underlying the importance of developing drugs able to reduce both primary and secondary storage-material accumulations in GD.

pH-Responsive Trihydroxylated Piperidines Rescue The Glucocerebrosidase Activity in Human Fibroblasts Bearing The Neuronopathic Gaucher-Related L444P/L444P Mutations in GBA1 Gene.

Engineering bioactive iminosugars with pH-responsive groups is an emerging approach to develop pharmacological chaperones (PCs) able to improve lysosomal trafficking and enzymatic activity rescue of mutated enzymes. The use of inexpensive l-malic acid allowed introduction of orthoester units into the lipophilic chain of an enantiomerically pure iminosugar affording only two diastereoisomers contrary to previous related studies. The iminosugar was prepared stereoselectively from the chiral pool (d-mannose) and chosen as the lead bioactive compound, to develop novel candidates for restoring the lysosomal enzyme glucocerebrosidase (GCase) activity. The stability of orthoester-appended iminosugars was studied by 1 H NMR spectroscopy both in neutral and acidic environments, and the loss of inhibitory activity with time in acid medium was demonstrated on cell lysates. Moreover, the ability to rescue GCase activity in the lysosomes as the result of a chaperoning effect was explored. A remarkable pharmacological chaperone activity was measured in fibroblasts hosting the homozygous L444P/L444P mutation, a cell line resistant to most PCs, besides the more commonly responding N370S mutation.

Gold nanoparticles decorated with monosaccharides and sulfated ligands as potential modulators of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS).

N-Acetylgalactosamine-6-sulfatase (GALNS) is an enzyme whose deficiency is related to the lysosomal storage disease Morquio A. For the development of effective therapeutic approaches against this disease, the design of suitable enzyme enhancers (i.e. pharmacological chaperones) is fundamental. The natural substrates of GALNS are the glycosaminoglycans keratan sulfate and chondroitin 6-sulfate, which mainly display repeating units of sulfated carbohydrates. With a biomimetic approach, gold nanoparticles (AuNPs) decorated with simple monosaccharides, sulfated ligands (homoligand AuNPs), or both monosaccharides and sulfated ligands (mixed-ligand AuNPs) were designed here as multivalent inhibitors of GALNS. Among the homoligand AuNPs, the most effective inhibitors of GALNS activity are the ß-D-galactoside-coated AuNPs. In the case of mixed-ligand AuNPs, ß-D-galactosides/sulfated ligands do not show better inhibition than the ß-D-galactoside-coated AuNPs. However, a synergistic effect is observed for α-D-mannosides in a mixed-ligand coating with sulfated ligands that reduced IC50 by one order of magnitude with respect to the homoligand α-D-mannoside-coated AuNPs. SAXS experiments corroborated the association of GALNS with ß-D-galactoside AuNPs. These AuNPs are able to restore the enzyme activity by almost 2-fold after thermal denaturation, indicating a potential chaperoning activity towards GALNS. This information could be exploited for future development of nanomedicines for Morquio A. The recent implications of GALNS in cancer and neuropathic pain make these kinds of multivalent bionanomaterials of great interest towards multiple therapies.

Light-Triggered Control of Glucocerebrosidase Inhibitors: Towards Photoswitchable Pharmacological Chaperones.

Piperidine-based photoswitchable derivatives have been developed as putative pharmacological chaperones for glucocerebrosidase (GCase), the defective enzyme in Gaucher disease (GD). The structure-activity study revealed that both the iminosugar and the light-sensitive azobenzene are essential features to exert inhibitory activity towards human GCase and a system with the correct inhibition trend (IC50 of the light-activated form lower than IC50 of the dark form) was identified. Kinetic analyses showed that all compounds are non-competitive inhibitors (mixed or pure) of GCase and the enzyme allosteric site involved in the interaction was identified by means of MD simulations. A moderate activity enhancement of mutant GCase assessed in GD patients' fibroblasts (ex vivo experiments) carrying the most common mutation was recorded. This promising observation paves the way for further studies to improve the benefit of the light-to-dark thermal conversion for chaperoning activity.

Higher frequency of TMEM199-CDG in the southern mediterranean area is associated with c.92G>C (p.Arg31Pro) mutation.

Congenital disorders of glycosylation (CDG) are genetic multisystem diseases, characterized by defective glycoconjugate synthesis. A small number of CDG with isolated liver damage have been described, such as TMEM199-CDG, a non-encephalopathic liver disorder with Wilson disease-like phenotype. Only eight patients with TMEM199-CDG have been described including seven Europeans (originating from Greece and Italy) and one Chinese. Three patients from southern Italy (Campania) shared the same known missense mutation pathogenetic variant NM_152464.3:c. 92G > C (p.Arg31Pro), also found in a Greek patient. Here we report a new patient from southern Italy (Sicily), with a homozygous c.92G > C p.(Arg31Pro) variant in TMEM199. The patient's phenotype is characterized by mild non-progressive hepatopathy with a normal hepatic echo structure. A persistent increase in serum transaminases, total and low-density lipoprotein cholesterol and low serum ceruloplasmin and copper levels and normal urinary copper excretion were observed. Matrix-assisted laser desorption/ionization mass spectrometry analyses showed abnormal N- and O- protein glycosylation, indicative of a Golgi processing defect and supporting the function of TMEM199 in maintaining Golgi homeostasis. TMEM199-CDG is an ultra-rare CDG relatively frequent in the southern Mediterranean area (7 in 9 patients, 77%). It is mainly associated with the c.92G > C (p.Arg31Pro) pathogenetic allele globally reported in 4 out of 7 families (57%), including one from Greece and three unrelated families from southern Italy. The almost uniform clinical phenotype described in patients with TMEM199-CDG appears to reflect a higher prevalence of the same variant in patients from the southern Mediterranean area.

Generation of human induced pluripotent stem cell line (AOUMEYi001-A) from a patient affected by Congenital disorders of glycosylation (ALG8-CDG) using self-replicating RNA vector.

Congenital Disorders of Glycosylation (CDG) are rare inherited metabolic diseases caused by genetic defects in the glycosylation of proteins and lipids. In this study, we describe the generation and characterization of one human induced pluripotent stem cell (hiPSC) line from a 15-year-old male patient with CDG. The patient carried three variants, one (c.122G > A; p.Arg41Gln) inherited from his father and two (c.445 T > G; p.Leu149Arg and the novel c.980C > G; p.Thr327Arg) inherited from his mother in the ALG8 gene (OMIM #608103). The generated hiPSC line shows a normal karyotype, expresses pluripotency markers, and is able to differentiate into the three germ layers.

Biocompatible and Printable Ionotronic Sensing Materials Based on Silk Fibroin and Soluble Plant-Derived Polyphenols.

The emergence of ionotronic materials has been recently exploited for interfacing electronics and biological tissues, improving sensing with the surrounding environment. In this paper, we investigated the synergistic effect of regenerated silk fibroin (RS) with a plant-derived polyphenol (i.e., chestnut tannin) on ionic conductivity and how water molecules play critical roles in regulating ion mobility in these materials. In particular, we observed that adding tannin to RS increases the ionic conductivity, and this phenomenon is accentuated by increasing the hydration. We also demonstrated how silk-based hybrids could be used as building materials for scaffolds where human fibroblast and neural progenitor cells can highly proliferate. Finally, after proving their biocompatibility, RS hybrids demonstrate excellent three-dimensional (3D) printability via extrusion-based 3D printing to fabricate a soft sensor that can detect charged objects by sensing the electric fields that originate from them. These findings pave the way for a viable option for cell culture and novel sensors, with the potential base for tissue engineering and health monitoring.

Expanded Newborn Screening in Italy Using Tandem Mass Spectrometry: Two Years of National Experience.

Newborn screening (NBS) for inborn errors of metabolism is one of the most advanced tools for secondary prevention in medicine, as it allows early diagnosis and prompt treatment initiation. The expanded newborn screening was introduced in Italy between 2016 and 2017 (Law 167/2016; DM 13 October 2016; DPCM 12-1-2017). A total of 1,586,578 infants born in Italy were screened between January 2017 and December 2020. For this survey, we collected data from 15 Italian screening laboratories, focusing on the metabolic disorders identified by tandem mass spectrometry (MS/MS) based analysis between January 2019 and December 2020. Aminoacidemias were the most common inborn errors in Italy, and an equal percentage was observed in detecting organic acidemias and mitochondrial fatty acids beta-oxidation defects. Second-tier tests are widely used in most laboratories to reduce false positives. For example, second-tier tests for methylmalonic acid and homocysteine considerably improved the screening of CblC without increasing unnecessary recalls. Finally, the newborn screening allowed us to identify conditions that are mainly secondary to a maternal deficiency. We describe the goals reached since the introduction of the screening in Italy by exchanging knowledge and experiences among the laboratories.

Fluorescent In Situ Staining and Flow Cytometric Procedures as New Pre-Diagnostic Tests for Sialidosis, GM1 Gangliosidosis and Niemann-Pick Type C.

BACKGROUND: Early diagnosis is essential in the field of lysosomal storage disorders for the proper management of patients and for starting therapies before irreversible damage occurs, particularly in neurodegenerative conditions. Currently, specific biomarkers for the diagnosis of lysosomal storage disorders are lacking in routine laboratory practice, except for enzymatic tests, which are available only in specialized metabolic centers. Recently, we established a method for measuring and verifying changes in GM1 ganglioside levels in peripheral blood lymphocytes in patients with GM1 gangliosidosis. However, fresh blood is not always available, and using frozen/thawed lymphocytes can lead to inaccurate results. METHODS: We used frozen/thawed fibroblasts obtained from stored biopsies to explore the feasibility of fluorescent imaging and flow-cytometric methods to track changes in storage materials in fibroblasts from patients with three lysosomal neurodegenerative conditions: GM1 gangliosidosis, Sialidosis, and Niemann-Pick type C. We used specific markers for each pathology. RESULTS AND CONCLUSIONS: We demonstrated that with our methods, it is possible to clearly distinguish the levels of accumulated metabolites in fibroblasts from affected and unaffected patients for all the three pathologies considered. Our methods proved to be rapid, sensitive, unbiased, and potentially applicable to other LSDs.

Case Report: Dramatic Cholestasis Responsive to Steroids in a Newborn Homozygous for H63D HFE Variant.

In a newborn with very precocious liver failure, cholestatic jaundice, and low γ-glutamyl transpeptidase, progressive hepatosplenomegaly induced a progressively worsening respiratory distress, that was successfully treated with steroids. Laboratory and genetic tests did not find any disease usually associated with neonatal cholestasis. However, the patient was positive for a homozygous mutation of the HFE gene, which is associated with hereditary hemochromatosis, a disease with typical onset in adulthood. Although no firm conclusions can be drawn from a single clinical case, this experience suggests that hereditary hemochromatosis could have played a role in the induction of this serious cholestasis, probably already arisen in the uterus. We suggest that hereditary hemochromatosis ought to be included in the panel of the possible causes of neonatal cholestasis and that steroids ought to be added to the pharmacological armamentarium for treating specific conditions which cause cholestasis in newborns.

Synthesis of a New ß-Galactosidase Inhibitor Displaying Pharmacological Chaperone Properties for GM1 Gangliosidosis.

GM1 gangliosidosis is a rare lysosomal disease caused by the deficiency of the enzyme ß-galactosidase (ß-Gal; GLB1; E.C. 3.2.1.23), responsible for the hydrolysis of terminal ß-galactosyl residues from GM1 ganglioside, glycoproteins, and glycosaminoglycans, such as keratan-sulfate. With the aim of identifying new pharmacological chaperones for GM1 gangliosidosis, the synthesis of five new trihydroxypiperidine iminosugars is reported in this work. The target compounds feature a pentyl alkyl chain in different positions of the piperidine ring and different absolute configurations of the alkyl chain at C-2 and the hydroxy group at C-3. The organometallic addition of a Grignard reagent onto a carbohydrate-derived nitrone in the presence or absence of a suitable Lewis Acid was exploited, providing structural diversity at C-2, followed by the ring-closure reductive amination step. An oxidation-reduction process allowed access to a different configuration at C-3. The N-pentyl trihydroxypiperidine iminosugar was also synthesized for the purpose of comparison. The biological evaluation of the newly synthesized compounds was performed on leucocyte extracts from healthy donors and identified two suitable ß-Gal inhibitors, namely compounds 10 and 12. Among these, compound 12 showed chaperoning properties since it enhanced ß-Gal activity by 40% when tested on GM1 patients bearing the p.Ile51Asn/p.Arg201His mutations.

Liver transplantation in an infant with cerebrotendinous xanthomatosis, cholestasis, and rapid evolution of liver failure.

BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is a disorder of bile acid (BA) metabolism due to biallelic mutations in CYP27A1. The deposition of cholesterol and cholestanol in multiple tissues results, manifesting as neurologic disease in adults or older children. Neonatal cholestasis (NC) as a presentation of CTX is rare; it may self-resolve or persist, evolving to require liver transplantation (LT). METHODS: We present in the context of similar reports an instance of CTX manifest as NC and requiring LT. RESULTS: A girl aged 4mo was evaluated for NC with normal serum gamma-glutamyl transpeptidase activity. An extensive diagnostic work-up, including liver biopsy, identified no etiology. Rapid progression to end-stage liver disease required LT aged 5mo. The explanted liver showed hepatocyte loss and micronodular cirrhosis. Bile salt export pump (BSEP), encoded by ABCB11, was not demonstrable immunohistochemically. Both severe ABCB11 disease and NR1H4 disease-NR1H4 encodes farsenoid-X receptor, necessary for ABCB11 transcription-were considered. However, selected liver disorder panel sequencing and mass-spectrometry urinary BA profiling identified CTX, with homozygosity for the predictedly pathogenic CYP27A1 variant c.646G > C p.(Ala216Pro). Variation in other genes associated with intrahepatic cholestasis was not detected. Immunohistochemical study of the liver-biopsy specimen found marked deficiency of CYP27A1 expression; BSEP expression was unremarkable. Aged 2y, the girl is free from neurologic disease. CONCLUSIONS: Bile acid synthesis disorders should be routinely included in the NC/"neonatal hepatitis" work-up. The mutually supportive triple approach of BA profiling, immunohistochemical study, and genetic analysis may optimally address diagnosis in CTX, a treatable disease with widely varying presentation.

Generation of two hiPSC lines (UMILi027-A and UMILi028-A) from early and late-onset Congenital Central hypoventilation Syndrome (CCHS) patients carrying a polyalanine expansion mutation in the PHOX2B gene.

Congenital Central Hypoventilation Syndrome (CCHS) is a rare disorder of the autonomic nervous system (ANS), characterized by inadequate control of autonomic ventilation and global autonomic dysfunction. Heterozygous polyalanine repeat expansion mutations in exon 3 of the transcription factor Paired-like homeobox 2B (PHOX2B) gene occur in 90% of CCHS cases. In this study, we describe the generation and characterization of two human induced pluripotent stem cell (hiPSC) lines from female CCHS patients carrying a heterozygous + 5 alanine expansion mutation. The generated iPSC lines show a normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

3D Printing Silk-Based Bioresorbable Piezoelectric Self-Adhesive Holey Structures for In Vivo Monitoring on Soft Tissues.

Flexible and biocompatible adhesives with sensing capabilities can be integrated onto human body and organ surfaces, characterized by complex geometries, thus having the potential to sense their physiological stimuli offering monitoring and diagnosis of a wide spectrum of diseases. The challenges in this innovative field are the following: (i) the coupling method between the smart adhesive and the soft human substrates, (ii) the bioresorbable behavior of the material, and (iii) the electrical exchange with the substrate. Here, we introduce a multifunctional composite by mixing silk fibroin, featuring piezoelectric properties, with a soluble plant-derived polyphenol (i.e., chestnut tannin) modified with graphene nanoplatelets. This material behaves as a glue on different substrates and gives rise to high elongation at break, conformability, and adhesive performances to gastrointestinal tissues in a rat model and favors the printability via extrusion-based 3D printing. Exploiting these properties, we designed a bioresorbable 3D printed flexible and self-adhesive piezoelectric device that senses the motility once applied onto a phantom intestine and the hand gesture by signal translation. Experimental results also include the biocompatibility study using gastrointestinal cells. These findings could have applicability in animal model studies, and, thanks to the bioresorbable behavior of the materials, such an adhesive device could be used for monitoring the motility of the gastrointestinal tract and for the diagnosis of motility disorders.

Epimutations in both the TESK2 and MMACHC promoters in the Epi-cblC inherited disorder of intracellular metabolism of vitamin B12.

BACKGROUND: epi-cblC is a recently discovered inherited disorder of intracellular vitamin B12 metabolism associating hematological, neurological, and cardiometabolic outcomes. It is produced by an epimutation at the promoter common to CCDC163P and MMACHC, which results from an aberrant antisense transcription due to splicing mutations in the antisense PRDX1 gene neighboring MMACHC. We studied whether the aberrant transcription produced a second epimutation by encompassing the CpG island of the TESK2 gene neighboring CCDC163P. METHODS: We unraveled the methylome architecture of the CCDC163P-MMACHC CpG island (CpG:33) and the TESK2 CpG island (CpG:51) of 17 epi-cblC cases. We performed an integrative analysis of the DNA methylome profiling, transcriptome reconstruction of RNA-sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-Seq) of histone H3, and transcription expression of MMACHC and TESK2. RESULTS: The PRDX1 splice mutations and activation of numerous cryptic splice sites produced antisense readthrough transcripts encompassing the bidirectional MMACHC/CCDC163P promoter and the TESK2 promoter, resulting in the silencing of both the MMACHC and TESK2 genes through the deposition of SETD2-dependent H3K36me3 marks and the generation of epimutations in the CpG islands of the two promoters. CONCLUSIONS: The antisense readthrough transcription of the mutated PRDX1 produces an epigenetic silencing of MMACHC and TESK2. We propose using the term 'epi-digenism' to define this epigenetic disorder that affects two genes. Epi-cblC is an entity that differs from cblC. Indeed, the PRDX1 and TESK2 altered expressions are observed in epi-cblC but not in cblC, suggesting further evaluating the potential consequences on cancer risk and spermatogenesis.

3-Methylglutaconic Aciduria Type I Due to AUH Defect: The Case Report of a Diagnostic Odyssey and a Review of the Literature.

3-Methylglutaconic aciduria type I (MGCA1) is an inborn error of the leucine degradation pathway caused by pathogenic variants in the AUH gene, which encodes 3-methylglutaconyl-coenzyme A hydratase (MGH). To date, MGCA1 has been diagnosed in 19 subjects and has been associated with a variable clinical picture, ranging from no symptoms to severe encephalopathy with basal ganglia involvement. We report the case of a 31-month-old female child referred to our center after the detection of increased 3-hydroxyisovalerylcarnitine levels at newborn screening, which were associated with increased urinary excretion of 3-methylglutaconic acid, 3-hydroxyisovaleric acid, and 3-methylglutaric acid. A next-generation sequencing (NGS) panel for 3-methylglutaconic aciduria failed to establish a definitive diagnosis. To further investigate the strong biochemical indication, we measured MGH activity, which was markedly decreased. Finally, single nucleotide polymorphism array analysis disclosed the presence of two microdeletions in compound heterozygosity encompassing the AUH gene, which confirmed the diagnosis. The patient was then supplemented with levocarnitine and protein intake was slowly decreased. At the last examination, the patient showed mild clumsiness and an expressive language disorder. This case exemplifies the importance of the biochemical phenotype in the differential diagnosis of metabolic diseases and the importance of collaboration between clinicians, biochemists, and geneticists for an accurate diagnosis.

3,4,5-Trihydroxypiperidine Based Multivalent Glucocerebrosidase (GCase) Enhancers.

The synthesis of five new multivalent derivatives of a trihydroxypiperidine iminosugar was accomplished through copper catalyzed alkyne-azide cycloaddition (CuAAC) reaction of an azido ending piperidine and several propargylated scaffolds. The resulting multivalent architectures were assayed as inhibitors of lysosomal GCase, the defective enzyme in Gaucher disease. The multivalent compounds resulted in much more potent inhibitors than a parent monovalent reference compound, thus showing a good multivalent effect. Biological investigation of these compounds as pharmacological chaperones revealed that the trivalent derivative (12) gives a 2-fold recovery of the GCase activity on Gaucher patient fibroblasts bearing the L444P/L444P mutations responsible for neuropathies. Additionally, a thermal denaturation experiment showed its ability to impart stability to the recombinant enzyme used in therapy.

Photoswitchable inhibitors of human ß-glucocerebrosidase.

Light-switchable inhibitors of the enzyme ß-glucocerebrosidase (GCase) have been developed by anchoring a specific azasugar to a dihydroazulene or an azobenzene responsive moiety. Their inhibitory effect towards human GCase, before and after irradiation are reported, and the effect on thermal denaturation of recombinant GCase and cytotoxicity were studied on selected candidates.

Defective IGF-1 prohormone N-glycosylation and reduced IGF-1 receptor signaling activation in congenital disorders of glycosylation.

The insulin-like growth factor-1 (IGF-1) signaling pathway is crucial for the regulation of growth and development. The correct processing of the IGF-1Ea prohormone (proIGF-1Ea) and the IGF-1 receptor (IGF-1R) peptide precursor requires proper N-glycosylation. Deficiencies of N-linked glycosylation lead to a clinically heterogeneous group of inherited diseases called Congenital Disorders of Glycosylation (CDG). The impact of N-glycosylation defects on IGF-1/IGF-1R signaling components is largely unknown. In this study, using dermal fibroblasts from patients with different CDG [PMM2-CDG (n = 7); ALG3-CDG (n = 2); ALG8-CDG (n = 1); GMPPB-CDG (n = 1)], we analyzed the glycosylation pattern of the proIGF-1Ea, IGF-1 secretion efficiency and IGF-1R signaling activity. ALG3-CDG, ALG8-CDG, GMPPB-CDG and some PMM2-CDG fibroblasts showed hypoglycosylation of the proIGF-1Ea and lower IGF-1 secretion when compared with control (CTR). Lower IGF-1 serum concentration was observed in ALG3-CDG, ALG8-CDG and in some patients with PMM2-CDG, supporting our in vitro data. Furthermore, reduced IGF-1R expression level was observed in ALG3-CDG, ALG8-CDG and in some PMM2-CDG fibroblasts. IGF-1-induced IGF-1R activation was lower in most PMM2-CDG fibroblasts and was associated with decreased ERK1/2 phosphorylation as compared to CTR. In general, CDG fibroblasts showed a slight upregulation of Endoplasmic Reticulum (ER) stress genes compared with CTR, uncovering mild ER stress in CDG cells. ER-stress-related gene expression negatively correlated with fibroblasts IGF-1 secretion. This study provides new evidence of a direct link between N-glycosylation defects found in CDG and the impairment of IGF-1/IGF-1R signaling components. Further studies are warranted to determine the clinical consequences of reduced systemic IGF-1 availability and local activity in patients with CDG.