A loss of function variant in AGPAT3 underlies intellectual disability and retinitis pigmentosa (IDRP) syndrome

Abstract

Intellectual disability (ID) and retinal dystrophy (RD) are the frequently found features of multiple syndromes involving additional systemic manifestations. Here, we studied a family with four members presenting severe ID and retinitis pigmentosa (RP). Using genome wide genotyping and exome sequencing, we identified a nonsense variant c.747 C > A (p.Tyr249Ter) in exon 7 of AGPAT3 which co-segregates with the disease phenotype. Western blot analysis of overexpressed WT and mutant AGPAT3 in HEK293T cells showed the absence of AGPAT3, suggesting instability of the truncated protein. Knockdown of Agpat3 in the embryonic mouse brain caused marked deficits in neuronal migration, strongly suggesting that reduced expression of AGPAT3 affects neuronal function. Altogether, our data indicates that AGPAT3 activity is essential for neuronal functioning and loss of its activity probably causes intellectual disability and retinitis pigmentosa (IDRP) syndrome.

Fig. 1. Pedigree and clinical information of family with IRDP syndrome.

a Five generation pedigree and haplotype of the family with IDRP syndrome. b Homozygosity mapper output showing homozygous by descent (HBD) regions. Red peaks represent homozygous regions detected across the chromosome. c Co-segregation of variant c.747 C > A with disease phenotype. d CT scan of V-5 individual and (e) MRI of individual V-1 and (f) V-5 generally show normal features. g, h OCT of right eye of individual V-5 showed poor retinal architecture. The retina appears thinned and without clear outer segment (Photoreceptor cells).

Fig. 2. Map of chromosome 21 showing location of AGPAT3 gene.

Schematic representation of gene and protein shows the position of identified variant. Domains and motifs of AGPAT3 protein are also shown.

Fig. 3. AGPAT3 overexpression and knockdown.

a Western blot of HEK293T cells (triplicate) transfected with vector (V), V5-tagged WT AGPAT3, V5-tagged mutant AGPAT3 Tyr249Ter (MU) and non-transfected (NT) samples. Antibodies against tdTomato, B-actin and V5 were used. b Quantification of Western blot (a) showing relative V5-AGPAT3 WT and mutant expression levels, normalized to tdTomato using two-tailed unpaired t test (p = 2.58 × 10^-8). c Images of in utero Electroporation (IUE), transfected with control vector and (d) Agpat3 shRNA in merged (upper) and black and white (lower) panels. Red color (tdTomato) indicates transfected cells and blue (DAPI) DNA-staining showing the general cortical structure. Arrow heads indicate II/III somatosensory cortical layers, and the arrow represents the subventricular zone (SVZ). Quantification of neuronal migration: e Percentage of neuronal cells migrated from intermediate zone (Bin10) to cortical layer 1 (Bin1) in mouse brain coronal sections (n = 3). f Statistical analysis of the percentage of cells in bins 1-4 corresponding to layers II/III of somatosensory cortex using two-tailed unpaired t test (p = 1.6 × 10^-2), numbers in the graph bar represent the number of analyzed pictures where n = 3.