NANS-CDG: Delineation of the Genetic, Biochemical, and Clinical Spectrum

Abstract

Background: NANS-CDG is a recently described congenital disorder of glycosylation caused by biallelic genetic variants in NANS, encoding an essential enzyme in de novo sialic acid synthesis. Sialic acid at the end of glycoconjugates plays a key role in biological processes such as brain and skeletal development. Here, we present an observational cohort study to delineate the genetic, biochemical, and clinical phenotype and assess possible correlations. Methods: Medical and laboratory records were reviewed with retrospective extraction and analysis of genetic, biochemical, and clinical data (2016-2020). Results: Nine NANS-CDG patients (nine families, six countries) referred to the Radboudumc CDG Center of Expertise were included. Phenotyping confirmed the hallmark features including intellectual developmental disorder (IDD) (n = 9/9; 100%), facial dysmorphisms (n = 9/9; 100%), neurologic impairment (n = 9/9; 100%), short stature (n = 8/9; 89%), skeletal dysplasia (n = 8/9; 89%), and short limbs (n = 8/9; 89%). Newly identified features include ophthalmological abnormalities (n = 6/9; 67%), an abnormal septum pellucidum (n = 6/9; 67%), (progressive) cerebral atrophy and ventricular dilatation (n = 5/9; 56%), gastrointestinal dysfunction (n = 5/9; 56%), thrombocytopenia (n = 5/9; 56%), and hypo-low-density lipoprotein cholesterol (n = 4/9; 44%). Biochemically, elevated urinary excretion of N-acetylmannosamine (ManNAc) is pathognomonic, the concentrations of which show a significant correlation with clinical severity. Genotypically, eight novel NANS variants were identified. Three severely affected patients harbored identical compound heterozygous pathogenic variants, one of whom was initiated on experimental prenatal and postnatal treatment with oral sialic acid. This patient showed markedly better psychomotor development than the other two genotypically identical males. Conclusions: ManNAc screening should be considered in all patients with IDD, short stature with short limbs, facial dysmorphisms, neurologic impairment, and an abnormal septum pellucidum +/- congenital and neurodegenerative lesions on brain imaging, to establish a precise diagnosis and contribute to prognostication. Personalized management includes accurate genetic counseling and access to proper supports and tailored care for gastrointestinal symptoms, thrombocytopenia, and epilepsy, as well as rehabilitation services for cognitive and physical impairments. Motivated by the short-term positive effects of experimental treatment with oral sialic, we have initiated this intervention with protocolized follow-up of neurologic, systemic, and growth outcomes in four patients. Research is ongoing to unravel pathophysiology and identify novel therapeutic targets.

Keywords: N-acetyl-D-neuraminic acid; congenital disorder of glycosylation; glycosylation; intellectual developmental disorder/IDD; metabolic disease; sialic acid biosynthesis; skeletal dysplasia; thrombocytopenia.

Figure 1

Gene and protein structure of NANS encoding NANS. The nature and position of the variants reported in this study (black) and the previous case study (gray) are indicated (5). (A) NANS is 1,080 bp in length and consists of six exons. In total, 17 variants associated with NANS-CDG are reported in both studies combined, of which 10 are newly described in this case study. (B) The NANS protein is 359 amino acids in length and contains two domains: the N-term, the N-acetylneuraminic acid synthase domain (green) and the C-term antifreeze, type III domain (orange). The majority of variants are located in a domain from which 10 are located in the N-acetylneuraminic acid synthase domain.

Figure 2

(A) Prenatal 3D ultrasound of patient 2 at 34 weeks 2 days' gestational age, frontal view showing carpe-shaped mouth with tenting of both upper and lower lip. (B) Prenatal 3D ultrasound of patient 2 at 34 weeks 2 days' gestational age, lateral view showing craniofacial features with depressed nasal bridge, upturned nasal tip and prominent upper lip. (C) Facial features of patient 4 at age 10 months, frontal view showing depressed midface with full cheeks and prominent philtral ridges. (D) Facial features of patient 4 at age 10 months, lateral view showing deep-set eyes and low-set ears. (E) Facial features of patient 1 at age 3 years, frontal view showing high forehead, depressed midface, full cheeks, and tented mouth. (F) Facial features of patient 1 at age 3 years, lateral view showing deep-set eyes, upturned nasal tip, and low-set and posteriorly rotated ears. (G) Facial features of patient 5 at age 7 years, frontal view showing minimal dysmorphic features with tented upper lip and widely spaced teeth. (H) Facial features of patient 5 at age 7 years, lateral view showing mild posterior rotated and low-set ear and prominent, short, philtrum.

Figure 3

Brain MRI scans of patients 2, 3, 7, and 9. (A–C) Patient 2 at age 2 days; fetal gyral pattern with simplified sulcation, thin corpus callosum with hypoplastic splenium, widened ventricles and cisternae, subependymal pseudocysts, and a cavum septum pellucidum. (D,E) Patient 3 at age 4 days; moderate ventriculomegaly, absence of septum pellucidum, limited volume of the corpus callosum and periventricular white matter, suggestion of cortical malformation of the left temporoparietal region. (F,G) Patient 3 at age 25 months; ventriculomegaly (lateral and third ventricle) and enlarged subarachnoid space due to progressive loss of supratentorial white and gray matter volume; absence of septum pellucidum. (H) Patient 3 at age 28 months; severely enlarged lateral and third ventricle, narrow aqueduct and fourth ventricle, suggesting not only ex vacuo dilatation but dysfunction of cerebrospinal fluid. (I,J) Patient 7 at age 12 years; normal MRI but with persistent cavum septum pellucidum and vacuum vergae. (K,L) Patient 9 at age 15 years; marked ventriculomegaly (especially lateral ventricles).

Figure 4

(A) X-rays of the skeleton of patients 3, 4, and 6. (A–D) Patient 3 at age 4 days; born with multiple congenital abnormalities of the bones. (A) The pelvis shows flat acetabula and short femoral necks. (B) The right arm demonstrates metaphyseal widening and irregularity. (C) The knee in the lateral view demonstrates metaphyseal irregularity in detail. (D) The image of the right leg demonstrates irregularly widened metaphyses at the distal femur and proximal and distal tibia, with a dysplastic knee joint and varus deformity. (E) Patient 4 at age 4 months; total legs, demonstrating flat acetubular roofs, wide femoral head metaphyses with short femoral necks. There is slight bowing and varus in the knees. The metaphyses around the knee show widening and irregularity. (F–H) Patient 6 at age 7 years. (F,G) Total legs and pelvic showing small iliac wings, coxa vara with small femoral heads and necks. This is shown in detail in image (G). The metaphyses around the knee demonstrate the typical striated sclerosis. The knee joints are dysplastic, and there is a varus deformity. (H) Both hands with irregularly widened and sclerotic metaphyses in the distal radius and ulna. Irregular metaphyses of the phalanges. (I,J) Patient 6 at age 12 years; total spine with scoliosis. The vertebral plates seem to have a double layer, with abnormal sclerosis of the plates. Lateral view shows the sclerosing of the vertebral plates from dorsal to ventral, resulting in a double contour.

Figure 5

Abdominal X-rays of patients 1 and 3. (A,B) Patient 3 at age 2 years; severe dilatation of the stomach and bowel loops and fecal impaction especially in the right colon. (C) Patient 1 at age 8 years; a similar stomach and bowel loop dilatation.

Figure 6

(A) ManNAc excretion levels in urine (μmol/mmol creatinine, measured by ¹H NMR spectroscopy, reference value not detected) vs. the Nijmegen Pediatric CDG Rating Scale (NPCRS) for currently reported patients in whom a ManNAc excretion level was determined. (B) ManNAc excretion levels in urine (μmol/mmol creatinine, measured by ¹H NMR spectroscopy, reference value not detected) vs. the clinical severity classification for currently reported patients in whom a ManNAc excretion level was determined. (C) ManNAc excretion levels in urine (μmol/mmol creatinine, measured by ¹H NMR spectroscopy, reference value not detected) vs. thrombocyte count for currently reported patients in whom a ManNAc excretion level and thrombocyte counts were measured. If the thrombocyte counts were measured several times, we used the lowest value. (D) ManNAc excretion levels in urine (μmol/mmol creatinine, measured by ¹H NMR spectroscopy, reference value not detected) vs. LDL level for currently reported patients in whom a MaNAc excretion level and LDL were measured. If the LDL level was measured several times, we used the lowest value. (A–D) Label numbers indicate the patients. □, analyses excluding patient 2; *, significant at the 0.05 level; †, patients harbor the same mutation [c.709C>T p.(Arg237Cys); c.562T>C p.(Tyr188His)]; ~, in patient 2 (aged 3 months) the NPCRS score and clinical severity classification were low compared to his ManNAc excretion level. Important developmental milestones are not relevant at this young age, explaining why the clinical severity classification is lower than expected on the basis of ManNAc excretion level; ∧, patient is treated with prenatal and postnatal experimental sialic acid; CDG, congenital disorder of glycosylation; ManNAc, N-acetylmannosamine; ¹H NMR, quantitative proton nuclear magnetic resonance; NPCRS, Nijmegen Pediatric CDG Rating Scale; r, Pearson linear correlation coefficient.