Analysis of exome data for 4293 trios suggests GPI-anchor biogenesis defects are a rare cause of developmental disorders

Abstract

Over 150 different proteins attach to the plasma membrane using glycosylphosphatidylinositol (GPI) anchors. Mutations in 18 genes that encode components of GPI-anchor biogenesis result in a phenotypic spectrum that includes learning disability, epilepsy, microcephaly, congenital malformations and mild dysmorphic features. To determine the incidence of GPI-anchor defects, we analysed the exome data from 4293 parent-child trios recruited to the Deciphering Developmental Disorders (DDD) study. All probands recruited had a neurodevelopmental disorder. We searched for variants in 31 genes linked to GPI-anchor biogenesis and detected rare biallelic variants in PGAP3, PIGN, PIGT (n=2), PIGO and PIGL, providing a likely diagnosis for six families. In five families, the variants were in a compound heterozygous configuration while in a consanguineous Afghani kindred, a homozygous c.709G>C; p.(E237Q) variant in PIGT was identified within 10-12 Mb of autozygosity. Validation and segregation analysis was performed using Sanger sequencing. Across the six families, five siblings were available for testing and in all cases variants co-segregated consistent with them being causative. In four families, abnormal alkaline phosphatase results were observed in the direction expected. FACS analysis of knockout HEK293 cells that had been transfected with wild-type or mutant cDNA constructs demonstrated that the variants in PIGN, PIGT and PIGO all led to reduced activity. Splicing assays, performed using leucocyte RNA, showed that a c.336-2A>G variant in PIGL resulted in exon skipping and p.D113fs*2. Our results strengthen recently reported disease associations, suggest that defective GPI-anchor biogenesis may explain ~0.15% of individuals with developmental disorders and highlight the benefits of data sharing.

Figure 1

Pedigrees and genetic data for six families harbouring rare biallelic variants in genes encoding components of the GPI-anchor biogenesis pathway. The Sanger sequencing traces shown are for the proband in each family and are shown in the coding direction, alongside the corresponding wild-type amino acid sequence. In the case of PIGT family 2 we show a trace from the father, where the variant is in the heterozygous state. For PIGT family 1 and the PIGL family, DNA was not available for the unaffected older siblings. Codon numbering is with respect to the following GenBank transcripts; PGAP3: NM_033419.4; PIGN: NM_176787.4; PIGT: NM_015937.5; PIGO: NM_032634.3; PIGL: NM_004278.3.

Figure 2

Follow-up studies on variants in PIGN and PIGT. (a) PIGN-knockout HEK293 cells were generated and transfected with human wild-type or p.(L311W) mutant PIGN cDNA cloned into pME or pTK expression vectors. Restoration of the cell surface expression of CD59 was evaluated by flow cytometry. The mutant construct using the pME promoter did not rescue CD59 surface expression as efficiently as the wild-type construct, indicating that the variant results in reduced PIGN activity. (b) Levels of expressed wild-type and p.(L311W) mutant HA-tagged PIGN in pME-vector transfected cells were analysed by western blotting using an anti-HA antibody. After normalisation with luciferase activity and GAPDH, expression of the mutant protein appeared to be reduced by only ~10% compared with the wild-type protein. (c) PIGT-knockout HEK293 cells were transfected with wild-type or mutant PIGT cDNA cloned into pME or pTK expression vectors. Restoration of the cell surface expression of CD59 was evaluated by flow cytometry. The mutant constructs using the pTK promoter did not rescue CD59 surface expression as efficiently as the wild-type construct, indicating that the variants result in reduced PIGT activity. (d) Levels of expressed wild-type and mutant FLAG-tagged PIGT in pME-vector transfected cells were analysed by western blotting. After normalisation, expression of the mutant protein appeared to be reduced only for the p.(L578fs*35) variant. (e) Allelic ratio plots along chromosome 20 (for high confidence SNVs only) showed that the PIGT variant shared in 270250 and 270306 lies within a large region of autozygosity.

Figure 3

Follow-up studies on variants in PIGO and PIGL. (a) PIGO-knockout HEK293 cells were transfected with wild-type, p.(R436W) or p.(G238D) PIGO cDNA. Restoration of the cell surface expression of CD59 was evaluated by flow cytometry. The p.(G238D) variant resulted in no detectable activity when using the pME promoter. For the p.(R436W) variant, reduced CD59 surface expression was only observed when using the pTK promoter. (b) Levels of expressed wild-type and mutant HA-tagged PIGO in pME-vector transfected cells were analysed by western blotting. After normalisation, expression of the mutant protein appeared to be mildly reduced for both missense variants. (c) 2100 Bioanalyser image showing PIGL RT-PCR amplicons using primers positioned in exons 2 and 5. A lower band was observed for 277013 and her father, consistent with skipping of exon 3. The expected sizes were calculated to be 280 bp and 189 bp if exon 3 is missing, which is consistent with the observed sizes given the margin for error reported by the manufacturer. Skipping of a 91 bp exon would lead to a frameshift and premature termination codon, as shown in Supplementary Figure S3.

Figure 4

Clinical images, shown with parental consent. (a) Photographs of individual 257982 aged 2 years and 8 months and her younger affected brother both showing thin upper lip and short nose with a broad nasal tip. Arrow indicates cleft palate, shown for younger sibling but also present in proband. (b) Photograph of 259633 showing thin tented upper lip and a short nose with a broad nasal tip. (c) Photographs of 258094 showing thin upper lip, nose with broad nasal tip and low-set ears; hands show tapering fingers. (d) Photograph of 263039 showing thin Cupid's-bow shaped upper lip, brachydactyly with absent fifth fingernail and dystrophic fourth and fifth toenails.