Novel congenital disorder of O-linked glycosylation caused by GALNT2 loss of function

Abstract

Congenital disorders of glycosylation are a growing group of rare genetic disorders caused by deficient protein and lipid glycosylation. Here, we report the clinical, biochemical, and molecular features of seven patients from four families with GALNT2-congenital disorder of glycosylation (GALNT2-CDG), an O-linked glycosylation disorder. GALNT2 encodes the Golgi-localized polypeptide N-acetyl-d-galactosamine-transferase 2 isoenzyme. GALNT2 is widely expressed in most cell types and directs initiation of mucin-type protein O-glycosylation. All patients showed loss of O-glycosylation of apolipoprotein C-III, a non-redundant substrate for GALNT2. Patients with GALNT2-CDG generally exhibit a syndrome characterized by global developmental delay, intellectual disability with language deficit, autistic features, behavioural abnormalities, epilepsy, chronic insomnia, white matter changes on brain MRI, dysmorphic features, decreased stature, and decreased high density lipoprotein cholesterol levels. Rodent (mouse and rat) models of GALNT2-CDG recapitulated much of the human phenotype, including poor growth and neurodevelopmental abnormalities. In behavioural studies, GALNT2-CDG mice demonstrated cerebellar motor deficits, decreased sociability, and impaired sensory integration and processing. The multisystem nature of phenotypes in patients and rodent models of GALNT2-CDG suggest that there are multiple non-redundant protein substrates of GALNT2 in various tissues, including brain, which are critical to normal growth and development.

Keywords: O-glycosylation; GALNT2; HDL-cholesterol; apolipoprotein C-III glycosylation; congenital disorders of glycosylation.

Figure 1

Family pedigrees of GALNT2-CDG patients and immunoblots of plasma apoC-III. (A) Family pedigrees of GALNT2-CDG patients. (B) Immunoblots of plasma apoC-III for probands and family controls of the GALNT2 loss-of-function mutation. Migration positions of three major apoC-III isoforms: non-glycosylated apoC-III (apoC-III₀) lacking any O-glycan modification; monosialylated apoC-III (apoC-III₁) containing N-acetylgalactosamine, galactose, and one terminal sialic acid; and disialylated apoC-III (apoC-III₂) containing N-acetylgalactosamine, galactose, and two terminal sialic acids, are indicated on the left. Ctrl = analysis with healthy control plasma; M = marker lane with protein standard; Std = analysis with standard human apoC-III purified from plasma.

Figure 2

Front and side photographs of patients diagnosed with GALNT2-CDG. GALNT2-CDG patients shared dysmorphic facial features including elongated face, high forehead, almond-shaped eyes, protruding maxilla, short philtrum, low-set, posteriorly rotated ears and frequently full lips with a tented or curved upper lip. (A) Patient A additionally had down-slanting palpebral fissures, macrodontia, wide-spaced teeth, and broad chin. (B) Patient B shared down-slanting palpebral fissures with his brother (Patient A) and additionally had sparse medial and curved eyebrows, dense eyelashes, and hypertelorism. (C) Patient C additionally had sparse lateral eyebrows, hypertelorism, midface hypoplasia, depressed nasal bridge, anteverted nares, and downturned corners of the mouth. (D) Patient D additionally had arched eyebrows, dense eyelashes laterally, and high nasal bridge. (E) Patient E additionally had bushy eyebrows, dense eyelashes, and high nasal bridge. He also had pointed canines in upper jaw (not shown). (F) Patient F additionally had a broad nose, macrostomia, macrodontia, wide-spaced teeth, and a broad chin. (G) Patient G additionally had sparse lateral eyebrows, broad nose, hypoplastic nasal alae, macrostomia, macrodontia, wide-spaced teeth, and ears with fleshy lobules.

Figure 3

Brain MRI results obtained from seven GALNT2-CDG patients at different ages. MRI scans showed white matter lesions in scans from Patients A–F but not in scan from Patient G. Patient age at the time of the MRI scan is shown. Columns 1 and 2 depict axial FLAIR images (or coronal FLAIR for Patient A at 35 months, Patient B, and Patient F). Column 3 depicts T₂-weighted images in axial view (or coronal view for Patients C–E). Column 4 depicts sagittal T₁-weighted images (with intravenous contrast for Patient A at 35 months and Patient B).

Figure 4

Growth parameters of GALNT2-CDG rodent models. Bar plots of the mean body weight of wild-type (WT), Galnt2^+/− heterozygous (Het), and Galnt2^−/− (KO) rodents are shown for (A) female mice, (B) male mice, (C) female rats, and (D) male rats. Representative pictures of wild-type and knockout (E) mice and (F) rats are shown. Error bars represent standard deviation. Significant P-values from Tukey’s post-test are indicated. Number of animals is indicated in parenthesis.

Figure 5

Behavioural analysis of GALNT2-CDG mouse model. (A) Results of accelerating rotarod testing of coordination in wild-type (WT) and Galnt2 knockout (KO) mice. Data represented as mean values and error bars represent standard error of the mean (SEM). (B) Startle response was measured by displacement of a fixed platform in response to auditory stimulus (Supplemental material). Habituation was measured by comparing displacement from the first five 120 dB stimuli to the last five 120 dB stimuli. (C) Disruption of the normal startle response by initial smaller auditory prepulse stimuli of increasing intensities from 78 to 87 dB, followed by startle-evoking 120 dB stimulus. Data represented in box and whisker plots of mean values. Whiskers represent maximum and minimum values. (D) Time spent sniffing the area around a social cue (S, another mouse) or non-social inanimate cue (NS). Data represented with box plots of mean values. Error bars represent standard deviation. Significant P-values from (A) repeated measures ANOVA, (B and C) Student’s unpaired t-test, (D) Tukey’s post-test, or indicated as non-significant (ns). Number of mice indicated in parenthesis.