Variant in the X-chromosome spliceosomal gene GPKOW causes male-lethal microcephaly with intrauterine growth restriction

Abstract

Congenital microcephaly, with or without additional developmental defects, is a heterogeneous disorder resulting from impaired brain development during early fetal life. The majority of causative genetic variants identified thus far are inherited in an autosomal recessive manner and impact key cellular pathways such as mitosis, DNA damage response and repair, apoptosis and splicing. Here, we report a novel donor splice site variant in the G-patch domain and KOW motifs (GPKOW) gene (NG_021310.2:g.6126G>A, NM_015698.4:c.331+5G>A) that segregates with affected and carrier status in a multigenerational family with an X-linked perinatal lethal condition characterized by severe microcephaly and intrauterine growth restriction (IUGR). GPKOW is a core member of the spliceosome that has been shown in numerous model organisms and in human cells to be essential for survival. By investigating GPKOW transcripts in lymphoblastoid cell lines (LCLs) of three carrier females, we show that the GPKOW c.331+5G>A variant disrupts normal splicing of its pre-mRNAs. In a clonal culture expressing only the c.331+5G>A allele isolated from one carrier female LCL, we observed an 80% reduction in wild type GPKOW mRNA, 70% reduction in the full length GPKOW protein and the presence of a truncated GPKOW protein with possible dominant negative effect. Based on our and published data we propose that the GPKOW gene is essential for fetal development and when disrupted, leads to a severe, male-lethal phenotype characterised by microcephaly and IUGR.

Figure 1

(a) Family pedigree. Genotyped individuals are indicated as WT +/Y or +/+ carrier −/+ affected -/Y. XCI ratios (peripheral blood DNA) for the tested females are shown (Supplementary Table 3). Foetus II-6 was a missed miscarriage at 25/40, III-3 died in the neonatal period at 36/40, III-5 was stillborn at 34/40, and pregnancies III-7 and IV-1 were terminated at 21/40 and 18.5/40, respectively, following diagnosis by ultrasound. IUGR, intrauterine growth restriction. (b) Schematic structure of GPKOW gene and protein. Arrow indicates location of +5G>A variant in the donor splice site of exon 2. One G-Patch domain, two KOW motifs and a predicted nuclear localisation sequence (NLS, aa 331–334) are shown. Exons are numbered as in NCBI Refseq NG_021310.2 and the amino acid locations of different domains are from the NCBI Refseq NP_056513.2. The shaded box indicates the aa 59–189 missing from the truncated protein, based on transcript D cDNA sequence. (c) Schematic representation of the four different transcripts identified in the cycloheximide (CHX) assay. Asterisks represent premature termination codons (PTC) introduced due to shifts in reading frame. (d) c.331+5G>A carrier female LCLs show additional aberrant processing of GPKOW pre-mRNA. Agarose gel showing GPKOW transcripts amplified from carrier and control female LCLs cultured in the presence (+) or absence (–) of CHX. Full-length (WT) and aberrantly-spliced (A–D) transcripts are indicated by arrows. XCI ratios assayed from LCL gDNA from three carrier females are also shown.

Figure 2

GPKOW full length WT mRNA and protein are significantly reduced in the c.331+5G>A LCL clone (MT). (a) Graph showing GPKOW mRNA levels relative to ACTB. RT-qPCR data from independent WT clones (n=3), c.331+5G>A clones (n=2; different passages of the same culture) from the carrier female II-2, and normal controls (n=3) was averaged and error bars show SD. (b) Western blot showing full length GPKOW from independent WT clones (n=3), the c.331+5G>A clone (n=2; different passages of the same culture; P1, P2), and normal controls (n=3). An additional band at around 40kDa in the c.331+5G>A (MT) clones P1 and P2 corresponds to the predicted size of a truncated protein translated from transcript D. (c) Graph showing the mean densitometry values of full length GPKOW protein relative to β-actin. Western blot signals in (b) were measured by ImageJ software and normalised to housekeeping β-actin protein. Error bars show SD.

Figure 3

Immunofluorescence showing a dramatic GPKOW reduction in c.331+5G>A clonal LCLs. Representative images of LCL immunofluorescence for GPKOW from normal controls from different individuals (n=3), independent WT clones (n=3) and c.331+5G>A clone (n=1, done in duplicate). Scale bars represent 20 μm.