C-terminal frameshift variants in GPKOW are associated with a multisystemic X-linked disorder

Abstract

Purpose: GPKOW, a gene on the X-chromosome, encodes a nuclear RNA-binding protein important in messenger RNA (mRNA) processing as a spliceosome subunit. This work aims to establish GPKOW as a disease-associated gene.

Methods: We describe 3 males from 2 unrelated families with hemizygous frameshift variants affecting the last exon of GPKOW p.(Arg441SerfsTer30) and p.(Ser444GlufsTer28). The effect of p.(Ser444GlufsTer28) on gene expression was evaluated in patient's fibroblasts. In vivo studies in Drosophila melanogaster targeting the sole GPKOW fly ortholog, CG10324 (Gpkow) were performed.

Results: Clinical presentations included intrauterine growth restriction, microcephaly/microencephaly, and eye, brain, skin, and skeletal abnormalities. Heterozygote females presented with short stature, microcephaly, and vision problems. Sequencing of fibroblasts' mRNA confirmed that GPKOW mRNA escapes nonsense-mediated decay. Yet, reduced protein levels suggested protein instability. Studies in Drosophila showed that Gpkow is essential and broadly expressed. It is enriched in neurons and glia in eyes and head of developing and adult flies. Knockdown and overexpression of Gpkow in the fly eye cause eyeless/headless phenotype, suggesting that the gene is dosage sensitive. Importantly, overexpression of the p.(Ser444GlufsTer28) variant caused milder defects than the reference allele, indicating that the truncated protein behaves as a partial loss-of-function allele.

Conclusion: Rare variants in GPKOW cause a multisystemic X-linked syndrome.

Keywords: CG10324; Drosophila melanogaster; GPKOW; Minor Spliceosome; Neurodevelopment.

Figure 1.. GPKOW C-terminal frameshift variant escapes nonsense-mediated decay (NMD), leads to protein instability, and is associated with severe brain-eye phenotype and ichthyosis.

(A) Clinical presentation includes multiple brain anomalies, microphthalmia, and congenital ichthyosis in sibling I (Ia) and sibling II (II) of family 1 (individuals II.1 and II.3 in family 1 pedigree, respectively). Brain MRI done at 4 weeks of age (family 1, sibling I) shows absent septum pellucidum, ventriculomegaly, and right posterior diverticulum (Ib, axial view) as well as hypogenesis of the corpus callosum and aqueductal stenosis (Ic, sagittal view). Gyral pattern is appropriate for age. (B) Pedigrees of both families. (C) Graphic representation of GPKOW gene and protein. (D) RNAseq on fibroblasts from sibling 1, family 1 (F1), II.1, who carries p.(Ser444GlufsTer28) variant shows normal GPKOW mRNA expression suggesting that this allele escapes nonsense-mediated decay. (E-I) GPKOW expression is low in fibroblasts from sibling I (family 1) when compared to unrelated infant and adult males. (E-II) Quantification of (E-I). (E-III) GPKOW expression in fibroblasts from sibling I followed by treatment with DMSO or 10μM MG132 for 6 hours. Expression is compared to unrelated adult male and female. (E-IV) Quantification of western blot bands of (E-III) showing fold change increase in GPKOW expression in fibroblasts treated with MG132 relative to expression with DMSO (negative control). GPKOW protein levels are normalized to that of GAPDH. Western blot analysis was done with anti-GPKOW antibody against the full length GPKOW. F, female; M, male; P, proband (Individual I, family 1). (F) X-chromosome inactivation (XCI) ratios in genomic DNA extracted from lymphoblastoid cell lines and fibroblast cultures from heterozygote females in family 2 (mother II.2 and daughter III.1). PCR amplification of the polymorphic CAG repeat in the androgen receptor (AR) gene was carried out following reaction with the methylation-sensitive enzyme HpaII. Only methylated (correlated to inactive X-chromosome) is amplified. In all samples, the 285 bp allele persists following HpaII digestion, showing preferential inactivation of this same allele in both females’ samples.

Figure 2.. Gpkow, a fly ortholog of GPKOW, is an essential gene that is broadly expressed in Drosophila melanogaster.

(A,D) Wild type (Canton-S) adult fly and larvae. (B,E) Gpkow is strongly expressed in the adult flies and larvae. (C,F) Fluorescent imaging shows high expression of mCherry in Gpkow cells in adult and larvae (white arrow) compared to control adult and larvae (weak auto-fluorescent, marked as *). (G-G’”) Gpkow is expressed in most glial cells in the eye-antenna disc. (H) Gpkow is expressed in the subsets of cells in the larval brain. (I,J) Gpkow is expressed in both glial cells and neurons in the larval brain. (I’,J’) Magnified views from yellow dashed boxes in (I) and (J). (K) Complementation tests of Gpkow mutant alleles (Gpkow^KG, Gpkow^Df) and rescue experiments using a genomic duplication that covers this locus (Gpkow^DP). Scale bars: A,B: 500 μm. D,E: 200 μm. G-J: 100 μm.

Figure 3.. Downregulation of Gpkow in the eye-antenna disc causes severe eye developmental phenotypes.

(A) Control. Knockdown of Gpkow using an RNAi line (GD) causes eyeless (B, 18°C) to headless (C, 29°C, late pupal lethal) phenotypes. (D) Downregulation of Gpkow using an independent RNAi line (KK) results in similar eye phenotypes. (E) Knockdown of Gpkow with GMR-Gal4 causes a rough eye phenotype. (F) Control. Eye-antenna discs are attached to the larval brain via optic stalks. (G) Severely disrupted eye discs (yellow arrows) and elongated optic stalks (white arrows) are observed in Gpkow downregulated third instar larvae. (H) DCP-1 staining of control larval brain, labeling apoptotic cells. (I) Glia-specific knockdown of Gpkow causes massive apoptotic cell death in the larval brain. (J) Glial Gpkow knockdown significantly reduces larval brain lobe size. (K) Pan-neuronal Gpkow knockdown or (L) Neuroblast-specific Gpkow knockdown also induces reduced larval brain lobe size. Scale bars: A-E, H: 200 μm, F-G: 100 μm. The data shown are the mean with SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by unpaired T-test.

Figure 4.. GPKOW ^{p.S444EfsX28-3xHA} variant is a partial loss-of-function allele.

(A) Schematic representations of the GPKOW and Gpkow constructs. (B,C) Overexpression of either GPKOW reference or variant protein by GMR-Gal4 induces severely disrupted and smaller eyes. (D) The survival rate of GPKOW variant overexpression with GMR-Gal4 was 2-fold higher than that of GPKOW reference overexpression. (E) Eye size quantification result utilizing escapers from (D). (F) Both the GPKOW reference and variant proteins exhibited similar expression levels. (G-H’) Both GPKOW reference or variant proteins were overexpressed in developing photoreceptor cells by GMR-Gal4 and were labeled with an anti-GPKOW antibody. Elav (pan-neuronal marker) staining represents the nucleus of photoreceptor cells in the eye imaginal disc. (I) ERG (Electroretinogram) experiments show that GPKOW-expressing adult photoreceptor cells have defective photo-transduction activity. (J) Quantification of (I). GPKOW variant overexpression causes less reduction in receptor potential than GPKOW reference overexpression. Scale bars: B-C: 200 μm, G-H: 100 μm. The data shown are the mean with SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by unpaired T-test.