We identified biallelic mutations in NANS, the gene encoding the synthase for N-acetylneuraminic acid (NeuNAc; sialic acid), in nine individuals with infantile-onset severe developmental delay and skeletal dysplasia. Patient body fluids showed an elevation in N-acetyl-D-mannosamine levels, and patient-derived fibroblasts had reduced NANS activity and were unable to incorporate sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development, and exogenously added sialic acid partially rescued the skeletal phenotype. Thus, NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways for sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies to counteract NANS deficiency and to shed light on sialic acid metabolism and its implications for human nutrition.
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We thank M. Filocamo at the Gaslini Biobank (Genoa, Italy) for a fibroblast line for patient 1. We thank A. Reymond (CIG, FBM, Université de Lausanne) and his laboratory for lymphocyte immortalization. We thank C. Chiesa for Sanger sequencing and sample handling and shipment; S. de Boer for excellent technical assistance; B. Toh at the University of British Columbia for metabolic sample handling; X. Han for Sanger sequencing; B. Sayson for consenting and data management; M. Higginson for DNA extraction and sample handling; and A. Ghani for administrative assistance. We also thank R. Houben for skillfully preparing Figure 4 and A. Bandi for all other figures. We are grateful to our clinical colleagues in Dolo, Genoa, Lausanne, Manchester, Paris, Reggio Emilia, Tokyo, Treviso, and Vancouver for patient management. A.S.-F. dedicates this paper to the memory of Paolo Durand who pointed out the relationship between sialic acid metabolism and IDD to him in 1980. Finally, we wish to thank the patients reported here as well as their parents for the enthusiasm they showed for our research efforts, for their patience, which was challenged by the studies lasting many years, and for their repeated donation of biological samples. They have been the source of continuous motivation for us.
This work has been supported by funding from the Leenaards Foundation in Lausanne, Switzerland; the Faculty of Biology and Medicine of the University of Lausanne; the BC Children's Hospital Foundation (Treatable Intellectual Disability Endeavour in British Columbia: First Collaborative Area of Innovation); Genome BC (grant SOF-195); the Rare Diseases Foundation; the Rare Diseases Models and Mechanisms Network; the Canadian Institutes of Health Research (grant 301221); and the Dutch Organization for Scientific Research, ZONMW (Medium Investment Grant 40-00506-98-9001 and VIDI Grant 91713359 to D.J.L.). The zebrafish studies were supported by funding to X.-Y.W. from the Canadian Rare Disease Models and Mechanisms Network, the Brain Canada Foundation, the Natural Sciences and Engineering Research Council of Canada (NSERC), and the Canada Foundation for Innovation (CFI). The informatics infrastructures were supported by Genome BC and Genome Canada (ABC4DE Project) as well as by the Vital-IT Project of the Swiss Institute of Bioinformatics (SIB; Lausanne, Switzerland). C.J.R. is funded by a Canadian Institutes of Health Research New Investigator Award. C.D.M.v.K. is a recipient of the Michael Smith Foundation for Health Research Scholar Award (Vancouver, Canada). E.G. is supported by a Marie Skłodowska-Curie fellowship (MSCA-IF-661491).
The authors declare no competing financial interests.
Integrated supplementary information
(a–d) Axial T2-weighted images show a marked degree of ventriculomegaly. Note the diencephalic–mesencephalic junction dysplasia, characterized by a lack of separation between the hypothalamus and midbrain (arrowheads in a). In the nucleobasal region, bilaterally, there are dilated perivascular spaces (arrowheads in b) and an abnormal morphology of the basal nuclei, with prominent thalami (labeled T in c) and maloriented putamen and caudate heads (labeled P and C, respectively, in c). Note also the bilateral abnormal orientation of the posterior arms of the sylvian fissure (empty arrows in d). (e) An axial T1-weighted image obtained with a volumetric technique shows thickening of the left perisylvian cortex, consistent with polymicrogyria. Cortical abnormality is less prominent controlaterally, despite the abnormal orientation of the sylvian fissure. (f) A left parasagittal T1-weighted image shows abnormal orientation of the sylvian fissure (arrows), which is continuous with the sulci of the convexity without a normal posterior delimitation; this pattern, which was also visible controlaterally (data not shown), is typical of perisylvian polymicrogyria. (g) A midsagittal T1-weighted image shows shortened, hypoplastic corpus callosum with a thin splenium (thin arrows) and lacking a proper rostrum (thick arrow); also note the absence of a well-defined anterior commissure (arrowhead).
Supplementary Figure 2 Capillary electrophoresis analysis of NANS cDNA retrotranscribed and amplified from RNA extracted from cell cultures.
(a) Schematic of the exon–intron structure of the NANS gene. Asterisks indicate mutations with a putative effect on exon splicing (see Table 1 for details): EXins: c.385_386insT, exonic single-nucleotide insertion producing a frameshift and creating a premature termination codon; SPLko: c.488+1G>T, a canonical splice donor site mutation; INTindel: c.449–10delGATTACinsATGG, an intronic indel 5′ of exon 4. (b) Pedigrees of patients 1, 3, and 4, and corresponding capillary electrophoresis traces of semiquantitative RT-PCRs spanning all analyzed NANS mutations. We assessed unprocessed PCR products obtained with the same primer pairs and allowing the simultaneous ampli-fication of the wild-type and the mutant mRNA (cDNA) forms, indicated on the right, within the same reaction. See the main text for discussion of the results.
In brain, NANS is expressed at significantly higher levels in the thalamus than in other brain regions (top) (http://developinghumanbrain.org/). Expression seems to be highest in the dorsal thalamus (bottom), which is an important relay station for the limbic circuit (The Human Nervous System 439–468 Academic Press, 1990) and participates in learning and memory processes (J. Neurosci. 34, 15340–15346, 2014), which might contribute to the developmental delay in patients with NANS deficiency.
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van Karnebeek, C., Bonafé, L., Wen, XY. et al. NANS-mediated synthesis of sialic acid is required for brain and skeletal development. Nat Genet 48, 777–784 (2016). https://doi.org/10.1038/ng.3578
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