Clinical and molecular findings in three Japanese patients with N-acetylneuraminic acid synthetase-congenital disorder of glycosylation (NANS-CDG)

Abstract

We report clinical and molecular findings in three Japanese patients with N-acetylneuraminic acid synthetase-congenital disorder of glycosylation (NANS-CDG). Patient 1 exhibited a unique constellation of clinical features including marked hydrocephalus, spondyloepimetaphyseal dysplasia (SEMD), and thrombocytopenia which is comparable to that of an infant reported by Faye-Peterson et al., whereas patients 2 and 3 showed Camera-Genevieve type SMED with intellectual/developmental disability which is currently known as the sole disease name for NANS-CDG. Molecular studies revealed a maternally inherited likely pathogenic c.207del:p.(Arg69Serfs*57) variant and a paternally derived likely pathogenic c.979_981dup:p.(Ile327dup) variant in patient 1, a homozygous likely pathogenic c.979_981dup:p.(Ile327dup) variant caused by maternal segmental isodisomy involving NANS in patient 2, and a paternally inherited pathogenic c.133-12T>A variant leading to aberrant splicing and a maternally inherited likely pathogenic c.607T>C:p.(Tyr203His) variant in patient 3 (reference mRNA: NM_018946.4). The results, together with previously reported data, imply that (1) NANS plays an important role in postnatal growth and fetal brain development; (2) SMED is recognizable at birth and shows remarkable postnatal evolution; (3) NANS-CDG is associated with low-normal serum sialic acid, obviously elevated urine N-acetylmannosamine, and normal N- and O-glycosylation of serum proteins; and (4) NANS-CDG is divided into Camera-Genevieve type and more severe Faye-Peterson type.

Figure 1

Clinical findings in patients 1–3. y, year; m, month; and d, day. (A) Photographs of patients 1–3 and blood smear of patient 1 showing a megathrombocyte. (B–D) Radiological findings in patients 1–3, respectively.

Figure 2

Molecular findings in patients 1–3. (A) The position and the frequency and pathogenicity of the NANS variants identified in this study (see also Supplementary Tables 1–3). (B) Molecular data in patient 1, showing the c.207del and c.979_981dup variants. Red asterisks indicate the frameshifted direct sequences. Black asterisks denote a G/C SNP (rs1058446; allele frequency, G 81% and C 19%). (C) Molecular data in patient 2, showing the homozygous and heterozygous c.979_981dup variant in patient 2 and the mother, respectively. Red asterisks indicate the frameshifted direct sequences. CytoScan HD analysis reveals the normal copy number and segmental loss of heterozygosity for chromosome 9, indicating segmental isodisomic regions involving NANS (highlighted with light green). (D) Molecular data in patient 3, showing the c.133–12T>A and c.607T>C variants (indicated with red asterisks). The c.133–12T>A creates a novel splice acceptor site (SAS) which is predicted to be utilized more preferentially than the canonical SAS by SpliceAI and produce an aberrant mRNA subject to NMD. Consistent with this, experiments using mRNA samples reveal a variant mRNA containing 10 bp intronic sequence on the electrochromatogram for subcloned mRNAs, a roughly halved mRNA expression ratio between CHX-untreated and CHX-treated LCLs, and a reduced wildtype "T" peak for the c.607T>C variant on the electrochromatogram obtained by direct sequencing for the CHX untreated LCLs (indicated with red asterisks).

Figure 3

Glycosylation status of serum proteins in patients 1–3 and a control subject. The N-glycosylation status of transferrin (left) and the O-glycosylation status of apolipoprotein C-III (right) are normal in patients 1–3. Since the O-glycosylation status of apolipoprotein C-III in patient 1 was examined using serum sample stored at − 20 °C for 6 months, additional peaks due to oxidation of the core protein backbone (OX) are delineated. Other measurements were performed using fresh serum samples.